**Meet the editor**

Dr. Uday Kishore is the Director of the Centre for Infection, Immunity and Disease Mechanisms, Brunel University, London. He earned his MSc and PhD from the University of Delhi, India, followed by post-doctoral training at the Salk Institute, California, USA and University of Oxford, UK (MRC Immunochemistry Unit and Weatherall Institute of Molecular Medicine, John Rad-

cliffe Hospital). He has been a recipient of fellowships awarded by NASA, Wellcome Trust, Humboldt Foundation and Medical Research Council. Dr Kishore has authored over 80 peer-reviewed research papers, edited two books, and is currently writing a text book on host pathogen interaction. In addition to innate immunity, complement system and host-pathogen interaction, Dr Kishore's research interests include dissecting role of complement system in neuroinflammation and neurodegeneration.

Contents

**Preface IX**

**Section 1 Disease Mechanisms in Alzheimer's Disease 1**

Chapter 1 **Alzheimer's Disease: A Clinical Perspective 3**

**and the Search for Biomarkers 35**

Chapter 3 **Role of Protein Aggregation in Neurodegenerative**

Yusuf Tutar, Aykut Özgür and Lütfi Tutar

Chapter 4 **Role of Oxidative Stress in Aβ Animal Model of Alzheimer's**

Chapter 5 **Alterations of Mitochondria and Golgi Apparatus Are Related to Synaptic Pathology in Alzheimer's Disease 101**

Chapter 7 **Brain Reserve Regulators in Alzheimer's Disease 151**

**Disease: Vicious Circle of Apoptosis, Nitric Oxide and Age 77**

Marisol Herrera-Rivero

**Diseases 55**

Ferihan Cetin

Yan Zhang

Ivana Delalle

Stavros J. Baloyannis

Chapter 6 **Caspases in Alzheimer's Disease 125**

Chapter 8 **Cholesterol and Alzheimer's Disease 165**

Iuliana Nicola-Antoniu

Weili Xu, Anna Marseglia, Camilla Ferrari and Hui-Xin Wang

Chapter 2 **Late-Onset Alzheimer's Disease: Risk Factors, Clinical Diagnosis**

### Contents

#### **Preface XIII**


Iuliana Nicola-Antoniu


Chapter 9 **Emerging Therapeutic Strategies in Alzheimer's Disease 181** Tomohiro Chiba

Chapter 18 **Oxidative Changes and Possible Effects of Polymorphism of**

Chapter 19 **Intermediate Filaments in Neurodegenerative Diseases 457**

Chapter 20 **Astrocytes Role in Parkinson: A Double-Edged Sword 491**

Chapter 22 **Oligodendrocyte Metabolic Stress in Neurodegeneration 535**

Jana Jurecekova

**Section 4 Miscellaneous 559**

**Therapeutics 561**

Antonio Ibarra

Rodolphe Perrot and Joel Eyer

Chapter 21 **Zinc and Neurodegenerative Diseases 519**

Ohkawara and Yutaka Sadakane

Daniel Radecki and Alexander Gow

Chapter 23 **Spinal Muscular Atrophy: Classification, Diagnosis,**

**Antioxidant Enzymes in Neurodegenerative Disease 421** Eva Babusikova, Andrea Evinova, Jozef Hatok, Dusan Dobrota and

Contents **VII**

Ricardo Cabezas, Marco Fidel Avila, Daniel Torrente, Ramon Santos El-Bachá, Ludis Morales, Janneth Gonzalez and George E. Barreto

Masahiro Kawahara, Keiko Konoha, Hironari Koyama, Susumu

**Background, Molecular Mechanism and Development of**

Humberto Mestre, Yael Cohen-Minian, Daniel Zajarias-Fainsod and

Faraz Tariq Farooq, Martin Holcik and Alex MacKenzie

Camilo Orozco Sanabria, Francisco Olea and Manuel Rojas

Chapter 24 **Pharmacological Treatment of Acute Ischemic Stroke 581**

Chapter 25 **Cognitive Dysfunction Syndrome in Senior Dogs 615**

Chapter 10 **Dropping the BACE: Beta Secretase (BACE1) as an Alzheimer's Disease Intervention Target 227** Justin Read and Cenk Suphioglu


**Section 2 Therapeutic Aspects of Alzheimer 179**

**Disease Intervention Target 227** Justin Read and Cenk Suphioglu

Chapter 11 **QSAR Analysis of Purine-Type and Propafenone-Type**

Chapter 12 **Therapeutic Interventions in Alzheimer Disease 291**

**Section 3 Pathopysiological Aspects of Other Neurodegenerative**

Chapter 14 **The Role of Epigenetics in Neurodegenerative Diseases 345** Luca Lovrečić, Aleš Maver, Maja Zadel and Borut Peterlin

Chapter 15 **Plasma Membrane Channels Formed by Connexins or Pannexins in Microglia: Possible Role in the**

Chapter 16 **Influence of Obesity on Neurodegenerative Diseases 381**

Chapter 17 **Electro-Physiological Approaches to Monitoring Neuro-**

Manuel J. Rojas, Camilo Orozco and Francisco Olea

**Degenerative Diseases 403**

Rana Awada , Avinash Parimisetty and Christian Lefebvre

Abhishek Shastri, Domenico Marco Bonifati and Uday Kishore

Analava Mitra and Baishakhi Dey

Tomohiro Chiba

**VI** Contents

**Clearance 257** Jie Yang and Jie Chen

**Diseases 319**

**Inflamed Brain 367** Juan A. Orellana

d'Hellencourt

Chapter 13 **Other Dementias 321**

Chapter 9 **Emerging Therapeutic Strategies in Alzheimer's Disease 181**

Chapter 10 **Dropping the BACE: Beta Secretase (BACE1) as an Alzheimer's**

**Substrates of P-Glycoprotein Targeting β-Amyloid**


Preface

It is my pleasure to present to you this book that I edited with great interest and anticipation. My own research interests include examining the role of complement proteins in neuroinflam‐ mation and neurodegeneration using the models of Alzheimer's disease as well as British and Danish familial dementia. However, through my own studies and during the course of editing this book, it became evident that one needs to have a holistic view about the pathophysiology of various neurodegenerative diseases. These are multi-factorial and complex disease process‐ es that require a range of multi-disciplinary approaches from various angles and directions in order to achieve diagnostic and therapeutic outcomes. This book is a small but significant step in highlighting the complexities of issues relevant for basic and translational research in the

The book has three major sections addressing disease mechanisms and therapeutic interven‐ tions in Alzheimer's disease, followed by generic mechanisms in other forms of the neurode‐ generative diseases. The book ends with its last miscellaneous section that is a small conglomeration of chapters on Spinal Muscular Atrophy, Acute Ischemic Stroke, and Cogni‐ tive dysfunction syndrome. I sincerely hope that this book is an exciting point of reference for

I am ever so indebted to all the authors who have contributed various chapters for this book. I am also grateful to Daria Nahtigal and Iva Simcic of the InTech Europe for inviting me to edit this book. I would like to thank Dr Annapurna Nayak, Dr Patrick Waters, Dr Marco Bonifati, Dr Abhishek Shastri and Dr Janez Ferluga for stimulating discussions during the course of editing this book. Many thanks are also due to Prof Bob Sim, Prof Nick Willcox and Prof An‐

**Dr Uday Kishore**

Brunel University London, UK

Centre for Infection, Immunity and Disease Mechanisms

Director

area of neuroinflammation and neurodegeneration.

many clinical and non-clinical neuroscientists.

gela Vincent for priming me into the field of neuroimmunology.

### Preface

It is my pleasure to present to you this book that I edited with great interest and anticipation. My own research interests include examining the role of complement proteins in neuroinflam‐ mation and neurodegeneration using the models of Alzheimer's disease as well as British and Danish familial dementia. However, through my own studies and during the course of editing this book, it became evident that one needs to have a holistic view about the pathophysiology of various neurodegenerative diseases. These are multi-factorial and complex disease process‐ es that require a range of multi-disciplinary approaches from various angles and directions in order to achieve diagnostic and therapeutic outcomes. This book is a small but significant step in highlighting the complexities of issues relevant for basic and translational research in the area of neuroinflammation and neurodegeneration.

The book has three major sections addressing disease mechanisms and therapeutic interven‐ tions in Alzheimer's disease, followed by generic mechanisms in other forms of the neurode‐ generative diseases. The book ends with its last miscellaneous section that is a small conglomeration of chapters on Spinal Muscular Atrophy, Acute Ischemic Stroke, and Cogni‐ tive dysfunction syndrome. I sincerely hope that this book is an exciting point of reference for many clinical and non-clinical neuroscientists.

I am ever so indebted to all the authors who have contributed various chapters for this book. I am also grateful to Daria Nahtigal and Iva Simcic of the InTech Europe for inviting me to edit this book. I would like to thank Dr Annapurna Nayak, Dr Patrick Waters, Dr Marco Bonifati, Dr Abhishek Shastri and Dr Janez Ferluga for stimulating discussions during the course of editing this book. Many thanks are also due to Prof Bob Sim, Prof Nick Willcox and Prof An‐ gela Vincent for priming me into the field of neuroimmunology.

> **Dr Uday Kishore** Director Centre for Infection, Immunity and Disease Mechanisms Brunel University London, UK

**Section 1**

**Disease Mechanisms in Alzheimer's Disease**

**Disease Mechanisms in Alzheimer's Disease**

**Chapter 1**

**Alzheimer's Disease: A Clinical Perspective**

Dementia is defined as a clinical syndrome characterized by progressive deterioration in multiple cognitive domains that are severe enough to interfere with daily functioning, in‐ cluding social and professional functioning. Alzheimer disease (AD) is the most common form of dementia often diagnosed in people over 65 years old, even though the early-onset AD can occur much earlier since 40 years of age. AD is a multifactorial disorder in which the causes and the progression are still not well-understood. Aging is the most common nonmodifiable cause of dementia in the elderly, but it accounts only for approximately half of all cause. Research identified other potential causes among the interaction between modifia‐ ble environmental factors, such as vascular disease and genetic susceptibility. The recent ge‐ netic discoveries have shown that mutation of the β-amyloid precursor protein on chromosome 21, and the mutations of presenilin 1 and presenilin 2 on chromosome 14 and 1, were associated with increased susceptibility of AD. Finally, the presence of the ε4 allele of Apolipoprotein E (APOE) is considered as a risk factor for late-onset of AD. The Diagnos‐ tic and Statistical Manual on Mental Disorders, fourth edition text revised (DSM-IV-TR), de‐ fines dementia as an acquired disease characterized by decline in memory and at least one other cognitive function such as attention, visuo-spatial skills, language, or executive func‐ tions. Beside the cognition, the disease affects the emotional abilities and interferes signifi‐ cantly with work and daily-life activities. Dementia can be defined as either possible, or probable based on the recent published diagnostic criteria [1]. Since 1980s, numerous com‐ munity-based prospective studies of aging and health have been implemented in the world; many of which have focused on dementia and its main subtypes of AD and vascular demen‐ tia (VaD). In this Chapter, we review the literature of clinical and epidemiological research

> © 2013 Xu et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Weili Xu, Anna Marseglia, Camilla Ferrari and

Additional information is available at the end of the chapter

in the dementias by focusing on most recent studies.

Hui-Xin Wang

**1. Introduction**

http://dx.doi.org/10.5772/54539

### **Alzheimer's Disease: A Clinical Perspective**

Weili Xu, Anna Marseglia, Camilla Ferrari and Hui-Xin Wang

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54539

#### **1. Introduction**

Dementia is defined as a clinical syndrome characterized by progressive deterioration in multiple cognitive domains that are severe enough to interfere with daily functioning, in‐ cluding social and professional functioning. Alzheimer disease (AD) is the most common form of dementia often diagnosed in people over 65 years old, even though the early-onset AD can occur much earlier since 40 years of age. AD is a multifactorial disorder in which the causes and the progression are still not well-understood. Aging is the most common nonmodifiable cause of dementia in the elderly, but it accounts only for approximately half of all cause. Research identified other potential causes among the interaction between modifia‐ ble environmental factors, such as vascular disease and genetic susceptibility. The recent ge‐ netic discoveries have shown that mutation of the β-amyloid precursor protein on chromosome 21, and the mutations of presenilin 1 and presenilin 2 on chromosome 14 and 1, were associated with increased susceptibility of AD. Finally, the presence of the ε4 allele of Apolipoprotein E (APOE) is considered as a risk factor for late-onset of AD. The Diagnos‐ tic and Statistical Manual on Mental Disorders, fourth edition text revised (DSM-IV-TR), de‐ fines dementia as an acquired disease characterized by decline in memory and at least one other cognitive function such as attention, visuo-spatial skills, language, or executive func‐ tions. Beside the cognition, the disease affects the emotional abilities and interferes signifi‐ cantly with work and daily-life activities. Dementia can be defined as either possible, or probable based on the recent published diagnostic criteria [1]. Since 1980s, numerous com‐ munity-based prospective studies of aging and health have been implemented in the world; many of which have focused on dementia and its main subtypes of AD and vascular demen‐ tia (VaD). In this Chapter, we review the literature of clinical and epidemiological research in the dementias by focusing on most recent studies.

© 2013 Xu et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

AD is an age-related phenomenon and is the most common cause of dementia, but increas‐ ing evidence from population-based neuropathological and neuroimaging studies shows that mixed brain pathology (neurodegenerative and vascular) account for a large number of dementia cases, especially in very old people [2]. According to the World Alzheimer Report, there were 35.6 million people living with dementia worldwide in 2010, a number that will increase to 65.7 million by 2030 and 115.4 million by 2050 unless effective means of reducing disease incidence are introduced. The total estimated worldwide costs of dementia were US \$604 billion in 2010, including the costs of informal care, direct costs of social care, and the direct costs of medical care [3]. Increasing age is a well-established risk factor for dementia and AD. Both prevalence and incidence of AD increases exponentially with advancing age, and 70% of all dementia cases occur in people aged 75+ years [4]. Notwithstanding, despite the incidence rate of AD increases almost exponentially until 85 years of age, it remains un‐ certain whether the incidence continues to increase even at more advances ages or reaches a plateau [5]. In Europe, the age-adjusted prevalence is 6.4% for dementia in general, and 4.4% for AD among people 65 years and older [6]. In the US, has been estimated that the 9.7% of people aged 70+ years has AD [7]. More than 25 million people in the world are affected by dementia; most of them suffer from AD, with about 5 million new cases every year [8, 9]. The estimated global annual incidence is around 7.5 per 1000 people [8]. The incident rate increases from approximately one per 1000 person-year in people aged 60-64 to more than 70 per 1000 person-year in 90+ years-old. In Europe, the pooled incidence rate of AD in peo‐ ple aged 65 years and older was 19.4 per 1000 person-year. The incidence rates of AD across different regions are quite similar in the younger-old, but greater variations have been seen among the older ages, but this is probably because of differences in methodology such as study designs and case ascertainment [5]. In conclusion, the worldwide population aging explains the epidemic proportions for dementia making the disease an important issue for the public health.

cleavage of APP by β, and γ–secretases creates Aβ42 peptide, while the cleavage by α-secre‐ tase produce Aβ40. Aβ42 peptide aggregates more readily than Aβ 40, and the ratio of these two isoforms influence the formation of the senile plaques [12]. Genetic studies [13] have identified mutations in APP and presenilin 1 and 2 (components of the γ-secretase) that cause rare, dominantly inherited familial AD. These findings strongly supported the amy‐ loid hypothesis [14], which posits that β-amyloid peptides play a pivotal role in AD patho‐ genesis. The amyloid cascade hypothesis suggests that deposition of Aβ triggers neuronal dysfunction and death in the brain. In the original hypothesis, this neuronal dysfunction

Alzheimer's Disease: A Clinical Perspective http://dx.doi.org/10.5772/54539 5

As knowledge of pathological changes in Alzheimer's disease increased, research identified Aβ oligomers as the principle players of the toxic effect [10]. Changes in tau phosphoryla‐ tion status and consequent neurofibrillary tangles formation are also triggered by toxic con‐ centrations of Aβ [14]. All the factors mentioned above (aging, genes, inflammation, and vascular pathology) can increase the production of Aβ. Despite genetic and cell biological evidence support the amyloid hypothesis [15], which is also the target of the new immuno‐ therapies for AD, it is becoming clear that AD etiology is complex and that Aβ alone is un‐ able to account for all the aspects of the disease. Others amyloid- independent hypothesis have been proposed [16]. The inflammatory hypothesis is based on the presence of activated microglia in AD brain. These cells, which have been shown to cluster around senile plaques, produce massive amounts of oxygen radicals and inflammatory mediators that are toxic to brain cells ultimately destroying them [17]. There is general agreement that the overproduc‐ tion of free radicals generated from oxidative stress has a major role in neurodegeneration [18] and, as a reactive process, it may be involved in cell cycle regulation contributing to cell death [19]. In brain, a variety of stressants can induce oxidative stress as cerebral hypoperfu‐ sion, inflammation, aging, hypoxia, cigarette smoking, excess alcohol, or cardiovascular dis‐ ease. There is also a vascular hypothesis that suggests that cerebral hypoperfusion in the presence of vascular risk factors can further lower cerebral blood flow to a critical level that threatens neuronal survival [20, 21]. Many other mechanisms have been suggested and can be involved in AD neurodegeneration, anyhow none hypothesis alone can explain the

The aetiology of dementia and AD has been extensively studied trying to find efficacious prevention and treatment strategies. As said, dementia is a multifactorial disorder caused by complex interaction between environmental and genetic factors. It has been estimated that 1-5% of AD cases are due to genetic mutations, while the most part are ascribable to modifi‐ able environmental factors and their interaction with genetic susceptibility [10]. Age is the most powerful determinant of dementia, suggesting that aging-related biological process may be involved in the pathogenesis of AD [23]. In actuality, the association between age and AD is mediated by the cumulative effect of other risk and protective factors over the lifespan. The major risk and protective factors for AD can be summarized basing on the dif‐

and death was thought to be a toxic effect of the total amyloid load.

pathogenesis of AD [22].

**3. Risk factors**

#### **2. Pathogenesis and mechanisms of AD**

Alzheimer's disease has not a single cause but is the results of the interaction of multiple mechanisms that can be grouped into aging, genetic influence, vascular pathology, inflam‐ mation and environmental influence such as toxic exposure. However, currently, the precise pathogenesis of AD is not known. One of the most important pathologic features character‐ ising AD is the brain atrophy which results by loss of neurons, synapses and dendritic arbo‐ rization in the cerebral cortex and subcortical regions. Cerebral atrophy is associated with the presence of neurofibrillary tangles and amyloid plaques, two hallmarks over-expressed in AD brain [10]. Neurofibrillary tangles are insoluble aggregates of hyperphoshorylated microtubule-associated tau protein that become accumulate inside the cells themselves. Changes in tau protein lead to the disintegration of the brain microtubules, the main neu‐ ron's transport system [11]. Amyloid plaques are dense and insoluble extracellular deposits of β-amyloid peptide (Aβ). Aβ derived from APP protheolisis. This transmembrane protein is divided into smaller fragments by three different enzymes: α, β, and γ–secretases. The cleavage of APP by β, and γ–secretases creates Aβ42 peptide, while the cleavage by α-secre‐ tase produce Aβ40. Aβ42 peptide aggregates more readily than Aβ 40, and the ratio of these two isoforms influence the formation of the senile plaques [12]. Genetic studies [13] have identified mutations in APP and presenilin 1 and 2 (components of the γ-secretase) that cause rare, dominantly inherited familial AD. These findings strongly supported the amy‐ loid hypothesis [14], which posits that β-amyloid peptides play a pivotal role in AD patho‐ genesis. The amyloid cascade hypothesis suggests that deposition of Aβ triggers neuronal dysfunction and death in the brain. In the original hypothesis, this neuronal dysfunction and death was thought to be a toxic effect of the total amyloid load.

As knowledge of pathological changes in Alzheimer's disease increased, research identified Aβ oligomers as the principle players of the toxic effect [10]. Changes in tau phosphoryla‐ tion status and consequent neurofibrillary tangles formation are also triggered by toxic con‐ centrations of Aβ [14]. All the factors mentioned above (aging, genes, inflammation, and vascular pathology) can increase the production of Aβ. Despite genetic and cell biological evidence support the amyloid hypothesis [15], which is also the target of the new immuno‐ therapies for AD, it is becoming clear that AD etiology is complex and that Aβ alone is un‐ able to account for all the aspects of the disease. Others amyloid- independent hypothesis have been proposed [16]. The inflammatory hypothesis is based on the presence of activated microglia in AD brain. These cells, which have been shown to cluster around senile plaques, produce massive amounts of oxygen radicals and inflammatory mediators that are toxic to brain cells ultimately destroying them [17]. There is general agreement that the overproduc‐ tion of free radicals generated from oxidative stress has a major role in neurodegeneration [18] and, as a reactive process, it may be involved in cell cycle regulation contributing to cell death [19]. In brain, a variety of stressants can induce oxidative stress as cerebral hypoperfu‐ sion, inflammation, aging, hypoxia, cigarette smoking, excess alcohol, or cardiovascular dis‐ ease. There is also a vascular hypothesis that suggests that cerebral hypoperfusion in the presence of vascular risk factors can further lower cerebral blood flow to a critical level that threatens neuronal survival [20, 21]. Many other mechanisms have been suggested and can be involved in AD neurodegeneration, anyhow none hypothesis alone can explain the pathogenesis of AD [22].

#### **3. Risk factors**

AD is an age-related phenomenon and is the most common cause of dementia, but increas‐ ing evidence from population-based neuropathological and neuroimaging studies shows that mixed brain pathology (neurodegenerative and vascular) account for a large number of dementia cases, especially in very old people [2]. According to the World Alzheimer Report, there were 35.6 million people living with dementia worldwide in 2010, a number that will increase to 65.7 million by 2030 and 115.4 million by 2050 unless effective means of reducing disease incidence are introduced. The total estimated worldwide costs of dementia were US \$604 billion in 2010, including the costs of informal care, direct costs of social care, and the direct costs of medical care [3]. Increasing age is a well-established risk factor for dementia and AD. Both prevalence and incidence of AD increases exponentially with advancing age, and 70% of all dementia cases occur in people aged 75+ years [4]. Notwithstanding, despite the incidence rate of AD increases almost exponentially until 85 years of age, it remains un‐ certain whether the incidence continues to increase even at more advances ages or reaches a plateau [5]. In Europe, the age-adjusted prevalence is 6.4% for dementia in general, and 4.4% for AD among people 65 years and older [6]. In the US, has been estimated that the 9.7% of people aged 70+ years has AD [7]. More than 25 million people in the world are affected by dementia; most of them suffer from AD, with about 5 million new cases every year [8, 9]. The estimated global annual incidence is around 7.5 per 1000 people [8]. The incident rate increases from approximately one per 1000 person-year in people aged 60-64 to more than 70 per 1000 person-year in 90+ years-old. In Europe, the pooled incidence rate of AD in peo‐ ple aged 65 years and older was 19.4 per 1000 person-year. The incidence rates of AD across different regions are quite similar in the younger-old, but greater variations have been seen among the older ages, but this is probably because of differences in methodology such as study designs and case ascertainment [5]. In conclusion, the worldwide population aging explains the epidemic proportions for dementia making the disease an important issue for

Alzheimer's disease has not a single cause but is the results of the interaction of multiple mechanisms that can be grouped into aging, genetic influence, vascular pathology, inflam‐ mation and environmental influence such as toxic exposure. However, currently, the precise pathogenesis of AD is not known. One of the most important pathologic features character‐ ising AD is the brain atrophy which results by loss of neurons, synapses and dendritic arbo‐ rization in the cerebral cortex and subcortical regions. Cerebral atrophy is associated with the presence of neurofibrillary tangles and amyloid plaques, two hallmarks over-expressed in AD brain [10]. Neurofibrillary tangles are insoluble aggregates of hyperphoshorylated microtubule-associated tau protein that become accumulate inside the cells themselves. Changes in tau protein lead to the disintegration of the brain microtubules, the main neu‐ ron's transport system [11]. Amyloid plaques are dense and insoluble extracellular deposits of β-amyloid peptide (Aβ). Aβ derived from APP protheolisis. This transmembrane protein is divided into smaller fragments by three different enzymes: α, β, and γ–secretases. The

the public health.

4 Neurodegenerative Diseases

**2. Pathogenesis and mechanisms of AD**

The aetiology of dementia and AD has been extensively studied trying to find efficacious prevention and treatment strategies. As said, dementia is a multifactorial disorder caused by complex interaction between environmental and genetic factors. It has been estimated that 1-5% of AD cases are due to genetic mutations, while the most part are ascribable to modifi‐ able environmental factors and their interaction with genetic susceptibility [10]. Age is the most powerful determinant of dementia, suggesting that aging-related biological process may be involved in the pathogenesis of AD [23]. In actuality, the association between age and AD is mediated by the cumulative effect of other risk and protective factors over the lifespan. The major risk and protective factors for AD can be summarized basing on the dif‐ ferent etiological hypotheses including genetic susceptibility hypothesis, vascular pathway hypothesis, psychosocial hypothesis, nutrition and dietary hypothesis, and others (e.g., toxic or inflammatory factors). While the role of genetic, vascular and psychosocial factors in the AD onset is supported by strong to moderate epidemiological, neuroimaging and neuropa‐ thological researches, the evidences for the other factors are controversial and insufficient [5]. Following age, the presence of Apolipoprotein E ε4 allele (APOE ε4) is the most estab‐ lished genetic risk factor for developing late-onset AD. There are three forms of APOE al‐ leles, ε2, ε3, and ε4, APOE ε4 increased the risk of AD by three times in heterozygote and more in homozygote, while ε2 decreases the risk [24, 25]. APOE allele ε4 is a susceptibility gene, being neither necessary nor sufficient for the development of AD.

**4. Clinical features**

trol" functions and rise of neuropsychiatric disorders.

A peculiar feature of AD is the progressive and multi-focal cognitive deterioration charac‐ terized by the insidious onset, the absence of focal neurological signs, and memory disor‐ ders. It develops slowly and gets worse overtime. Progressing, the disease damages the most areas of the brain, and this is manifested through the gradually deterioration of memo‐ ry, attention, executive functions, language, praxia, movement and personality. AD should be suspected when any individual, without alterations of awareness, refers generalized epi‐ sodic memory disturbances with insidious onset that escalate up to interfere with daily liv‐ ing, social and/or occupational activities. In an attempt to help clinicians in recognizing the severity of disease, the Mayo Clinic group proposes three main stages in the natural history of AD, each characterized by distinctive symptoms and duration. The classification has been made based on clinical experience and generalization. It is not intended to be in an inflexible and taxonomic way, but it is important to realize that AD is the neurodegenerative process of the single person, and thus the duration and the type of symptoms may change from one patients to another. Considering that, it is helpful divide the AD progression into following stages: 1) "Early stage" characterized mainly by memory disorders; 2) "Moderate stage" where appears progressive cortical dysfunction (apraxia, aphasia, visuo-spatial disturbance) and disorders of instrumental functions; 3) "Advanced stage" with disorders of the "Con‐

Alzheimer's Disease: A Clinical Perspective http://dx.doi.org/10.5772/54539 7

People may manifest mild symptoms long time before the clinical diagnosis of AD and often they are underestimated and mistakenly ascribe to either aging or stress. The early detection of symptoms is difficult because of the absence of a definite time of disease onset, so in the clinical practice patients with dementia are often first diagnosed when the disease is ad‐ vanced to the early stage with the clear manifestation of cognitive and behavioural disorders [53]. The prodromal stage of the disease is known as "Mild cognitive impairment" (MCI) in‐ dicating those people likely to be in the earliest stage of dementia, but with so mild symp‐ toms that cannot be formulate a formal diagnosis of dementia. Patients with MCI typically present forgetfulness due to the episodic memory impairment that leads to difficulties of re‐ call and learning new information, but they do not have a clear deficit in daily functioning, being able to live independently with a minimal help [54]. Not all the subjects with MCI de‐ velop dementia, but all subjects affected by AD had first presented this mild stage. It is ac‐ cepted that AD pathogenesis starts decades before clinical manifestation and that subjects decline slowly in cognition for years before meeting the diagnostic criteria for dementia [55, 56]. Longitudinal studies have shown that people with MCI have a high probability to de‐ velop dementia within 1-3 years after diagnosis [54]. When the progression of memory im‐ pairment and the decline in other cognitive domains (executive function, visuo-spatial, language, behaviour or personality) significantly interfere with the ability to function at work or at usual activities, the criteria for diagnosis of dementia are met [1]. The AD's "Mild stage" can last from 2 to 4 years. The typical scenario includes memory impairment for re‐ cent events with relative sparing for remote events (autobiographical memory), prospective memory disorders (remembering to perform a planned action or intention at the appropri‐ ate moment), difficulty with problem solving, complex task and sound judgment, difficulty

In the last decade many others AD susceptibility genes have been identified, highlighting the importance of a genetic susceptibility for AD development [26]. Over the last decade, great attention has been paid to figure out which AD-related factors may be modified to de‐ crease the risk of AD. Two groups of modifiable factors for late-life dementias have been identified as "vascular risk factors" that have been strongly associated with an increased risk of dementia; and the "psychosocial factors" that may contribute to the delay of demen‐ tia onset. Strong epidemiological evidences suggested that cardiovascular risk factors and vascular disease are associated with an increased risk of symptomatic AD [27].Thus, studies revealed age-dependents associations with AD for several aging-related conditions. The most important cardiovascular risk factors for subsequent AD include cigarette smoking [28, 29], heavy alcohol consumption [30], midlife high blood pressure [31], atrial fibrillation and heart failure [32], spontaneous cerebral emboli [33], midlife obesity or central adiposity as well as low BMI in late-life [34-36], midlife high cholesterol levels [37], diabetes mellitus and impaired glucose regulation [38-40], neuroinflammation [41, 42], and elevated plasma and total homocysteine levels [43].

Other risk factors for AD may include traumatic brain injury, late-life metabolic syndrome and depression, but their role is not clear and studies with long-term follow-up need to sup‐ port the risk factors hypothesis [44, 45]. About psychological factors epidemiological re‐ search has been accumulating that some psychosocial factors and healthy lifestyle such as the social network and social engagement, weekly-to-daily physical activity, higher educa‐ tional and socio-economic status and mentally stimulating activity, may postpone the onset of dementia by enhancing cognitive reserve [3, 46-48]. In addition, several studies reported a decreased risk of AD and dementia associated with a diet rich in both high polyunsaturated and fish-related fatty acids, such as the Mediterranean diet [49], and elevated levels of vita‐ min B12 and folate (50). Finally, controversy exists about the role of hormone replacement therapy with estrogens and progestin and subsequent development of AD. Several studies suggest that normal age-related depletion of estrogens in women and testosterone in men may represent potential risk factors for AD's onset [51], suggesting hormone replacement therapy (HRT) as method to reduce risk of late-life AD in postmenopausal women. Never‐ theless, the therapeutic effects of HRT is not supported by the Cochrane's review, which found that HRT or estrogens for improving or maintaining cognition was not indicated for women with AD [52].

#### **4. Clinical features**

ferent etiological hypotheses including genetic susceptibility hypothesis, vascular pathway hypothesis, psychosocial hypothesis, nutrition and dietary hypothesis, and others (e.g., toxic or inflammatory factors). While the role of genetic, vascular and psychosocial factors in the AD onset is supported by strong to moderate epidemiological, neuroimaging and neuropa‐ thological researches, the evidences for the other factors are controversial and insufficient [5]. Following age, the presence of Apolipoprotein E ε4 allele (APOE ε4) is the most estab‐ lished genetic risk factor for developing late-onset AD. There are three forms of APOE al‐ leles, ε2, ε3, and ε4, APOE ε4 increased the risk of AD by three times in heterozygote and more in homozygote, while ε2 decreases the risk [24, 25]. APOE allele ε4 is a susceptibility

In the last decade many others AD susceptibility genes have been identified, highlighting the importance of a genetic susceptibility for AD development [26]. Over the last decade, great attention has been paid to figure out which AD-related factors may be modified to de‐ crease the risk of AD. Two groups of modifiable factors for late-life dementias have been identified as "vascular risk factors" that have been strongly associated with an increased risk of dementia; and the "psychosocial factors" that may contribute to the delay of demen‐ tia onset. Strong epidemiological evidences suggested that cardiovascular risk factors and vascular disease are associated with an increased risk of symptomatic AD [27].Thus, studies revealed age-dependents associations with AD for several aging-related conditions. The most important cardiovascular risk factors for subsequent AD include cigarette smoking [28, 29], heavy alcohol consumption [30], midlife high blood pressure [31], atrial fibrillation and heart failure [32], spontaneous cerebral emboli [33], midlife obesity or central adiposity as well as low BMI in late-life [34-36], midlife high cholesterol levels [37], diabetes mellitus and impaired glucose regulation [38-40], neuroinflammation [41, 42], and elevated plasma and

Other risk factors for AD may include traumatic brain injury, late-life metabolic syndrome and depression, but their role is not clear and studies with long-term follow-up need to sup‐ port the risk factors hypothesis [44, 45]. About psychological factors epidemiological re‐ search has been accumulating that some psychosocial factors and healthy lifestyle such as the social network and social engagement, weekly-to-daily physical activity, higher educa‐ tional and socio-economic status and mentally stimulating activity, may postpone the onset of dementia by enhancing cognitive reserve [3, 46-48]. In addition, several studies reported a decreased risk of AD and dementia associated with a diet rich in both high polyunsaturated and fish-related fatty acids, such as the Mediterranean diet [49], and elevated levels of vita‐ min B12 and folate (50). Finally, controversy exists about the role of hormone replacement therapy with estrogens and progestin and subsequent development of AD. Several studies suggest that normal age-related depletion of estrogens in women and testosterone in men may represent potential risk factors for AD's onset [51], suggesting hormone replacement therapy (HRT) as method to reduce risk of late-life AD in postmenopausal women. Never‐ theless, the therapeutic effects of HRT is not supported by the Cochrane's review, which found that HRT or estrogens for improving or maintaining cognition was not indicated for

gene, being neither necessary nor sufficient for the development of AD.

total homocysteine levels [43].

6 Neurodegenerative Diseases

women with AD [52].

A peculiar feature of AD is the progressive and multi-focal cognitive deterioration charac‐ terized by the insidious onset, the absence of focal neurological signs, and memory disor‐ ders. It develops slowly and gets worse overtime. Progressing, the disease damages the most areas of the brain, and this is manifested through the gradually deterioration of memo‐ ry, attention, executive functions, language, praxia, movement and personality. AD should be suspected when any individual, without alterations of awareness, refers generalized epi‐ sodic memory disturbances with insidious onset that escalate up to interfere with daily liv‐ ing, social and/or occupational activities. In an attempt to help clinicians in recognizing the severity of disease, the Mayo Clinic group proposes three main stages in the natural history of AD, each characterized by distinctive symptoms and duration. The classification has been made based on clinical experience and generalization. It is not intended to be in an inflexible and taxonomic way, but it is important to realize that AD is the neurodegenerative process of the single person, and thus the duration and the type of symptoms may change from one patients to another. Considering that, it is helpful divide the AD progression into following stages: 1) "Early stage" characterized mainly by memory disorders; 2) "Moderate stage" where appears progressive cortical dysfunction (apraxia, aphasia, visuo-spatial disturbance) and disorders of instrumental functions; 3) "Advanced stage" with disorders of the "Con‐ trol" functions and rise of neuropsychiatric disorders.

People may manifest mild symptoms long time before the clinical diagnosis of AD and often they are underestimated and mistakenly ascribe to either aging or stress. The early detection of symptoms is difficult because of the absence of a definite time of disease onset, so in the clinical practice patients with dementia are often first diagnosed when the disease is ad‐ vanced to the early stage with the clear manifestation of cognitive and behavioural disorders [53]. The prodromal stage of the disease is known as "Mild cognitive impairment" (MCI) in‐ dicating those people likely to be in the earliest stage of dementia, but with so mild symp‐ toms that cannot be formulate a formal diagnosis of dementia. Patients with MCI typically present forgetfulness due to the episodic memory impairment that leads to difficulties of re‐ call and learning new information, but they do not have a clear deficit in daily functioning, being able to live independently with a minimal help [54]. Not all the subjects with MCI de‐ velop dementia, but all subjects affected by AD had first presented this mild stage. It is ac‐ cepted that AD pathogenesis starts decades before clinical manifestation and that subjects decline slowly in cognition for years before meeting the diagnostic criteria for dementia [55, 56]. Longitudinal studies have shown that people with MCI have a high probability to de‐ velop dementia within 1-3 years after diagnosis [54]. When the progression of memory im‐ pairment and the decline in other cognitive domains (executive function, visuo-spatial, language, behaviour or personality) significantly interfere with the ability to function at work or at usual activities, the criteria for diagnosis of dementia are met [1]. The AD's "Mild stage" can last from 2 to 4 years. The typical scenario includes memory impairment for re‐ cent events with relative sparing for remote events (autobiographical memory), prospective memory disorders (remembering to perform a planned action or intention at the appropri‐ ate moment), difficulty with problem solving, complex task and sound judgment, difficulty to organize and express thoughts, deficit in the ability to plan and execute actions in a cor‐ rect sequence (executive function and apraxia), slowing in the ability to switch from one ac‐ tivity to another [54]. In addition, patients in the mild stage may start to present sporadic language disorders mainly characterized by decreased vocabulary and word fluency with a subsequent impoverishment of the speaking and writing [57].

rological disorders (tonic grasping, echolalia, oral grasping, Kluver-Bucy syndrome and bilateral apraxia), and motor abnormalities can develop. Muscle mass and mobility deteri‐ orate until the patient is completely bedridden and not self-sufficient. People with AD typically die from medical complication as bronchitis, pneumonia, or pressure ulcers, and

Alzheimer's Disease: A Clinical Perspective http://dx.doi.org/10.5772/54539 9

The 1984 criteria made by the National Institute of Neurologic, Communicative Disorders and Stroke/Alzheimer's disease and Related Disorders Association (NINCDS-ADRDA) work group [61] were based on doctor's clinical judgment about the cause of patient's symp‐ toms, taking into account reports from the patient and family members, and results of cogni‐ tive tests and general neurological assessment. These criteria have been quite successful and they have been widely used in clinical trials and clinical research, showing a sensitivity of 81% and specificity of 70% [62]. However, the increasing knowledge of the clinical manifes‐ tations and biology of AD determined the need of criteria revision. After 27 years, in 2011 the National Institute on Aging (NIA) and the Alzheimer's Association recommended new diagnostic criteria and guidelines for Alzheimer's disease [1,55,56]. The notable changes of the 2011 criteria are mainly due to the concept that the pathophysiological process of AD begins years, if not decades, before the diagnosis of clinical dementia, and to the incorpora‐ tion of biomarkers that can indicate the presence or absence of AD pathology. The new crite‐ ria propose three different stages of Alzheimer's disease: preclinical AD, MCI due to AD, dementia due to AD. The preclinical stage occurs before symptoms development and indi‐ cates cognitive intact subjects with positive biomarkers of AD-related brain changes [55]. Ta‐ ble 1 shows diagnostic criteria of the different stages of preclinical AD phase. The investigation of the presence of this stage is strictly for research purposes only and usually used for individuals with high genetic risk of AD. Individuals with MCI have mild but measurable changes in cognition that are noticeable to the person affected and to family and friends but that do not affect the individual's ability to carry out everyday activities. Not all the subjects with MCI develop dementia, it is estimated that the rate of progression can be 10% per year. It is unclear why some individual progress into dementia and some others not, however it is believed that MCI can be an early stage of dementia. In the new criteria (Table 2) the use of biomarkers is suggested in order to investigate whether subjects have brain changes that put them at higher risk of developing AD. If biomarkers of AD-pathology

The new criteria proposed to classify people with AD dementia in the following groups af‐ ter meet general criteria for dementia [1]: 1) Probable AD dementia, 2) Possible AD demen‐ tia, and 3) Probable or Possible AD dementia with evidence of the AD pathophysiological process. Table 3 shows the revised criteria for AD. The first two have been planned to use in a clinical setting and are very much similar to the previous NINCS-ADRDA criteria of possi‐

ble and probable AD. The third group has been suggested only for research field.

not because of disease itself [59, 60].

**5. Alzheimer's disease diagnosis**

result positive the diagnosis is MCI due to AD [56].

Neurocognitive studies show that the episodic memory impairment is due to a deficit of storage newly acquired information in long-term memory [58] reflecting an early impair‐ ment of the "central executive" component of the working memory with relative sparing of the "slave systems". In this stage it is also likely that personality changes appear and the most prominent is apathy with symptoms of diminished interests and concerns and may be associated with depression. Social withdrawal, fluctuating mood, irritability or anxiety are less frequent. In some cases the non-cognitive symptoms may be more prominent than the cognitive impairment complicating the care of patients with AD. Despite that, they are not present in all patients and are not constantly progressive as the cognitive deficits. Decreased attention and motivation to complete task as well as dresses inappropriately are also com‐ mon. Towards the end of the mild stage, patients may start to be confused especially in un‐ familiar place, reflecting the onset of orientation defects. The "Moderate stage" of AD is the longest stage lasting from 2 to 10 years. The cognitive and behavioural symptoms increase in the severity, people get more confused and forgetful and the progressive deterioration in‐ terfere with individual independence, with person being unable to perform most common daily living activities and self-care. On cognition, the middle stage is characterized by a pro‐ gressive cortical dysfunction with prominent language, praxis, visuo-spatial, executive func‐ tion and abstract reasoning disorders. Progressive deterioration of oral and written communication includes anomias of aphasic lexico-semantic origin that progress into a flu‐ ent aphasia, speech planning defects and "empty speech" because of inability to recall vo‐ cabulary which leads to semantic paraphasias, progressive loss of reading and writing. Ideational and ideo-motor apraxia is responsible for difficulties in number processing and calculation. Memory impairment worsen may involve also the autobiographical memory.

Disturbance of visuo-perceptual contour processing and spatial processing evolve leading to deficit in recognizing familiar faces (prosopagnosia) and person, geographical and envi‐ ronmental disorientation, difficulty in coping figure, and visual imagery deficits. Patients become less able to succeed the more demanding tasks of daily living, such as manage fi‐ nances and driving. The personality and behavioural changes worsen. Psychotic behav‐ iour, paranoia, delusions, auditory or visual hallucinations are not unusual in this stage. Common manifestations are labile affect, irritability, wandering, aggression or resistance to caregiver. Sleep disorders including disruption in the sleep/wake cycle, sundowning, and urinary incontinence can develop. Patients lose awareness of their disease process and limitation (anosognosia). In the AD's "Severe stage" (1-3+ years), patients generally lose the ability to communicate coherently, and experience a great decline in physical abil‐ ities becoming mostly dependent on caregiver for feeding and hygiene. Language is re‐ duced to single simple phrases or words. Despite the severity of communication problem, people can understand and return emotional signal. On the behavioural level, aggressive‐ ness can be still present, but severe apathy and exhaustion are common symptoms. Neu‐ rological disorders (tonic grasping, echolalia, oral grasping, Kluver-Bucy syndrome and bilateral apraxia), and motor abnormalities can develop. Muscle mass and mobility deteri‐ orate until the patient is completely bedridden and not self-sufficient. People with AD typically die from medical complication as bronchitis, pneumonia, or pressure ulcers, and not because of disease itself [59, 60].

#### **5. Alzheimer's disease diagnosis**

to organize and express thoughts, deficit in the ability to plan and execute actions in a cor‐ rect sequence (executive function and apraxia), slowing in the ability to switch from one ac‐ tivity to another [54]. In addition, patients in the mild stage may start to present sporadic language disorders mainly characterized by decreased vocabulary and word fluency with a

Neurocognitive studies show that the episodic memory impairment is due to a deficit of storage newly acquired information in long-term memory [58] reflecting an early impair‐ ment of the "central executive" component of the working memory with relative sparing of the "slave systems". In this stage it is also likely that personality changes appear and the most prominent is apathy with symptoms of diminished interests and concerns and may be associated with depression. Social withdrawal, fluctuating mood, irritability or anxiety are less frequent. In some cases the non-cognitive symptoms may be more prominent than the cognitive impairment complicating the care of patients with AD. Despite that, they are not present in all patients and are not constantly progressive as the cognitive deficits. Decreased attention and motivation to complete task as well as dresses inappropriately are also com‐ mon. Towards the end of the mild stage, patients may start to be confused especially in un‐ familiar place, reflecting the onset of orientation defects. The "Moderate stage" of AD is the longest stage lasting from 2 to 10 years. The cognitive and behavioural symptoms increase in the severity, people get more confused and forgetful and the progressive deterioration in‐ terfere with individual independence, with person being unable to perform most common daily living activities and self-care. On cognition, the middle stage is characterized by a pro‐ gressive cortical dysfunction with prominent language, praxis, visuo-spatial, executive func‐ tion and abstract reasoning disorders. Progressive deterioration of oral and written communication includes anomias of aphasic lexico-semantic origin that progress into a flu‐ ent aphasia, speech planning defects and "empty speech" because of inability to recall vo‐ cabulary which leads to semantic paraphasias, progressive loss of reading and writing. Ideational and ideo-motor apraxia is responsible for difficulties in number processing and calculation. Memory impairment worsen may involve also the autobiographical memory.

Disturbance of visuo-perceptual contour processing and spatial processing evolve leading to deficit in recognizing familiar faces (prosopagnosia) and person, geographical and envi‐ ronmental disorientation, difficulty in coping figure, and visual imagery deficits. Patients become less able to succeed the more demanding tasks of daily living, such as manage fi‐ nances and driving. The personality and behavioural changes worsen. Psychotic behav‐ iour, paranoia, delusions, auditory or visual hallucinations are not unusual in this stage. Common manifestations are labile affect, irritability, wandering, aggression or resistance to caregiver. Sleep disorders including disruption in the sleep/wake cycle, sundowning, and urinary incontinence can develop. Patients lose awareness of their disease process and limitation (anosognosia). In the AD's "Severe stage" (1-3+ years), patients generally lose the ability to communicate coherently, and experience a great decline in physical abil‐ ities becoming mostly dependent on caregiver for feeding and hygiene. Language is re‐ duced to single simple phrases or words. Despite the severity of communication problem, people can understand and return emotional signal. On the behavioural level, aggressive‐ ness can be still present, but severe apathy and exhaustion are common symptoms. Neu‐

subsequent impoverishment of the speaking and writing [57].

8 Neurodegenerative Diseases

The 1984 criteria made by the National Institute of Neurologic, Communicative Disorders and Stroke/Alzheimer's disease and Related Disorders Association (NINCDS-ADRDA) work group [61] were based on doctor's clinical judgment about the cause of patient's symp‐ toms, taking into account reports from the patient and family members, and results of cogni‐ tive tests and general neurological assessment. These criteria have been quite successful and they have been widely used in clinical trials and clinical research, showing a sensitivity of 81% and specificity of 70% [62]. However, the increasing knowledge of the clinical manifes‐ tations and biology of AD determined the need of criteria revision. After 27 years, in 2011 the National Institute on Aging (NIA) and the Alzheimer's Association recommended new diagnostic criteria and guidelines for Alzheimer's disease [1,55,56]. The notable changes of the 2011 criteria are mainly due to the concept that the pathophysiological process of AD begins years, if not decades, before the diagnosis of clinical dementia, and to the incorpora‐ tion of biomarkers that can indicate the presence or absence of AD pathology. The new crite‐ ria propose three different stages of Alzheimer's disease: preclinical AD, MCI due to AD, dementia due to AD. The preclinical stage occurs before symptoms development and indi‐ cates cognitive intact subjects with positive biomarkers of AD-related brain changes [55]. Ta‐ ble 1 shows diagnostic criteria of the different stages of preclinical AD phase. The investigation of the presence of this stage is strictly for research purposes only and usually used for individuals with high genetic risk of AD. Individuals with MCI have mild but measurable changes in cognition that are noticeable to the person affected and to family and friends but that do not affect the individual's ability to carry out everyday activities. Not all the subjects with MCI develop dementia, it is estimated that the rate of progression can be 10% per year. It is unclear why some individual progress into dementia and some others not, however it is believed that MCI can be an early stage of dementia. In the new criteria (Table 2) the use of biomarkers is suggested in order to investigate whether subjects have brain changes that put them at higher risk of developing AD. If biomarkers of AD-pathology result positive the diagnosis is MCI due to AD [56].

The new criteria proposed to classify people with AD dementia in the following groups af‐ ter meet general criteria for dementia [1]: 1) Probable AD dementia, 2) Possible AD demen‐ tia, and 3) Probable or Possible AD dementia with evidence of the AD pathophysiological process. Table 3 shows the revised criteria for AD. The first two have been planned to use in a clinical setting and are very much similar to the previous NINCS-ADRDA criteria of possi‐ ble and probable AD. The third group has been suggested only for research field.


**Probable AD** A. Insidious onset. Symptoms have a gradual onset over months to years, not sudden over hours or days B. Clear-cut history of worsening of cognition by report or observation; and

cognitve dysfunction in at least one other cognitive domain

Deficits in other cognitive domains should be present

cognitive documentation of progressive decline

that could have a substantial effect on cognition

Neuronal injury unavailable or indeterminate

High but does not rule out second etiology

Etiologically mixed presentation

hyperintensity burden; or

Aβ unavailable or indeterminate Neuronal injury positive Intermediate Aβ positive

Neuronal injury positive

Neuronal injury positive

following categories

b. Nonamnestic presentations:

domains should be present

**Possible AD** Atypical course

or

or

High Aβ positive

Aβ positive

Intermediate

**Probable AD with three levels of evidence of AD pathophysiology**

**Possible AD (atypical presentation) with evidence of AD pathophysiology**

following:

C. The initial and most prominent cognitive deficit are evident on history and examination in one of the

Alzheimer's Disease: A Clinical Perspective http://dx.doi.org/10.5772/54539 11

a. Amnestic presentation is the most common syndromic presentation of AD dementia. The deficits should include impairment in lerning and recall of recently learned information. There should also be evidence of

• Language presentation: The most prominent deficits are in word-finding, but deficits in other cognitive

• Visuospatial presentation: The most prominent deficits are in spatial cognition, including object agnosia, impaired face recognition, simultagnosia, and alexia. Deficits in other cognitive domains should be present • Executive dysfunction: The most prominent deficits are impaired reasoning, judgment, and problem solving.

A. Atypical course meets the core clinical criteria in terms of the cognitive deficits for AD dementia, but either has a sudden onset of cognitive impairment or demonstrates insufficient historical detail or objective

B. Etiologically mixed presentation meets all core clinical criteria for AD dementia but has evidence as the

a. Concomitant cerebrovascular disease, defined by a history of stroke temporally related to the onset or worsening of cognitive impairment; or the presence of multiple or extensive infarcts or severe white matter

c. Evidence for another neurological diseases or a non-neurological medical comorbidity or medication use

b. Features of Dementia with Lewy bodies other than the dementia itself;

**Table 1.** Pre-clinical Alzheimer´s Disease stages: National Institute on Aging (NIA) and the Alzheimer's Association diagnostic criteria 2011.

MCI- core clinical criteria

• Cognitive concern reflecting a change in cognition reported by patient or informant or clinician (i.e., historical or observed evidence of decline over time)

• Objective evidence of impairment in one or more cognitive domains, typically including memory (i.e., formal or bedside testing to establish level of cognitive function in multiple domains)

• Preservation of independence in functional abilities

• Not demented

Examine etiology of MCI consistent with AD pathophysiological process

• Rule out vascular, traumatic, medical causes of cognitive decline, where possible

• Provide evidence of longitudinal decline in cognition, when feasible

• Report history consistent with AD genetic factors, where relevant

MCI due to AD:

Intermediate likelihood Clinical criteria + positive Aβ biomarkers and untested neuronal injury biomarkers Clinical criteria + untested Aβ biomarkers and positive neuronal injury biomarkers High likelihood Clinical criteria+ positive Aβ and neuronal injury biomarkers MCI- unlikely due to AD Clinical criteria + negative Aβ and neuronal injury biomarkers

(Albert SA et al. 2011) [56]

**Table 2.** Mild Cognitive Impairment due to AD: National Institute on Aging (NIA) and the Alzheimer's Association diagnostic criteria 2011.


**Aβ (PET or CSF) Markers of neuronal injury**

**Asymptomatic cerebral amyloidosis** Positive Negative Negative

**Table 1.** Pre-clinical Alzheimer´s Disease stages: National Institute on Aging (NIA) and the Alzheimer's Association

• Cognitive concern reflecting a change in cognition reported by patient or informant or clinician (i.e., historical or

• Objective evidence of impairment in one or more cognitive domains, typically including memory (i.e., formal or

**Table 2.** Mild Cognitive Impairment due to AD: National Institute on Aging (NIA) and the Alzheimer's Association

**Asymptomatic cerebral amyloidosis +**

**Amyloidosis+ neuronal injury+ subtle cognitive/ behavioral decline**

observed evidence of decline over time)

• Preservation of independence in functional abilities

bedside testing to establish level of cognitive function in multiple domains)

Examine etiology of MCI consistent with AD pathophysiological process

• Provide evidence of longitudinal decline in cognition, when feasible • Report history consistent with AD genetic factors, where relevant

Clinical criteria+ positive Aβ and neuronal injury biomarkers

Clinical criteria + negative Aβ and neuronal injury biomarkers

• Rule out vascular, traumatic, medical causes of cognitive decline, where possible

Clinical criteria + positive Aβ biomarkers and untested neuronal injury biomarkers Clinical criteria + untested Aβ biomarkers and positive neuronal injury biomarkers

**neurodegeneration**

10 Neurodegenerative Diseases

(Sperling RA et al. 2011) [55]

diagnostic criteria 2011.

MCI- core clinical criteria

• Not demented

MCI due to AD: Intermediate likelihood

High likelihood

MCI- unlikely due to AD

(Albert SA et al. 2011) [56]

diagnostic criteria 2011.

**(tau CZ-F, PET-FDG, MRI)**

Positive Positive Negative

Positive Positive Positive

**Evidence of subtle cognitive change**


sidered in individuals at high risk [71]. Structural neuroimaging, computed tomography (CT) or magnetic resonance imaging (MRI) is recommended as level B in the Dementia Guideline [71] at least once in each patient in order to exclude other condition as neoplasms, subdural hematomas. The lumbar puncture is not recommended unless the suspicion of

Alzheimer's Disease: A Clinical Perspective http://dx.doi.org/10.5772/54539 13

A biomarker is any characteristic that is objectively measured and evaluated as an indicator of biologic or pathogenic process and it should also be reliable, non-invasive and simple to perform [72]. Regarding AD, biomarkers included in the new diagnostic criteria are a meas‐ urement of the underline pathology and can be divided in markers of amyloid accumulation (CSF Aβ level, PET with amyloid-tracers) and markers of neuronal injury (atrophy of MTL

Bilateral atrophy of medial temporal lobe structures, including hippocampus, has been found in patients with AD (Figure 1). Moreover it has been reported that the brain atrophy detected with neuroimaging reflects the typical pattern of progression of neuropathology, spreading from entorhinal cortex and hippocampus to the association cortices, as describe by Braak and Braak [73, 74]. In a meta-analysis of studies using visual and linear measure‐ ments of medial temporal lobe atrophy (MTA) on MRI, the overall sensitivity and specificity for detection of AD compared with controls was estimated to be 85% and 88%, respectively [75]. A yearly decline in hippocampal volume is approximately 2.5 times greater in patients with AD than in normal aged subjects. In clinical practice simple visual rating scales esti‐ mating hippocampal atrophy has proven to be useful to support the diagnosis in the first stage of the disease [74]. Although MTL is a biomarker of neurodegeneration and a good surrogate of disease progression, it has low sensitivity and specificity (51-70% and 68-69%,

**Figure 1.** Coronal T1-weighted MRI scans of control (left) and patient with AD (right). The patient with AD shows atro‐

at MRI, tau level in CSF, metabolic PET) [55]. Table 4 shows all the biomarkers.

prion disease or viral encephalitis.

**6.1. Medial temporal lobe atrophy**

phy of the hippocampus (arrow) [74].

respectively) in identifying prodromal AD stage [76].

**6. Biomarkers**

**Table 3.** Dementia due to AD: National Institute on Aging (NIA) and the Alzheimer's Association diagnostic criteria

The diagnostic procedure in clinical setting is usual divided into two phases. Screening is used to formulate the diagnostic hypothesis and is followed by the diagnostic confirmation. During the screening phase, the main point is to collect detailed information on history not only from the patient itself, but also from the people who take care of patients such as fami‐ lial or other type of caregiver. Information about history includes medical history (presence of severe medical disease that may cause encephalopathy, psychiatric disease, traumatic brain injury or other neurological disease), medications, family history of dementia, and changes in both basic activities of daily living (such as self-feeding, dressing and bathing, ambulation) and instrumental activities of daily living (such as grooming, homework, man‐ age finances, driving, and leisure). To obtain information on activities daily living could be particularly helpful to use standardized evaluation instruments such as Katz ADL scale [63] Lawton-Brody IADL scale [64], and the Bristol Activities of Daily Living Scale [65]. In addi‐ tion to the history, a systematic assessment of general cognitive functioning is required through instruments designed for this purpose. There are no screening tools that can quick‐ ly assess different levels of cognitive impairment. The American Academy of Neurology guidelines suggested to use the Mini-Mental Status Examination [66], and the Memory Im‐ pairment Screen (MIS) [67]. In recent times, the Montreal Cognitive Assessment (MoCA) was developed as a tool to screen patients in who has been hypothesized a mild cognitive decline and usually performed in the normal range on the MMSE [68, 69]. Studies have shown that MoCa is sensitive for the mild stages of AD dementia, whereas MMSE is superi‐ or for more advanced stages with the functional impairment. A complete summary of neu‐ ropsychological tests has been proposed [70].

After the screening, the second phase consists of a neurological examination, a neuropsycho‐ logical assessment, and a behavioural disease evaluation. A complete general neurological examination has been recommended as well as accurate neuropsychological evaluation in order to test possible differential diagnosis. The presence of Parkinsonism can suggest a Lewy Body's dementia, while asymmetric tendon reflex or other lateralizing signs can sug‐ gest a vascular component. Other neurological signs, for example peripheral neuropathy may indicate toxic or metabolic problems. There is no evidence-based data to support the usefulness of specific routine blood tests for evaluation of those with dementia but these are useful in excluding co-morbidities. Most expert opinion advises to screen for vitamin B12, folate, thyroid stimulating hormone, calcium, glucose, complete blood cell count, renal and liver function abnormalities. Serological tests for syphilis, Borrelia and HIV should be con‐ sidered in individuals at high risk [71]. Structural neuroimaging, computed tomography (CT) or magnetic resonance imaging (MRI) is recommended as level B in the Dementia Guideline [71] at least once in each patient in order to exclude other condition as neoplasms, subdural hematomas. The lumbar puncture is not recommended unless the suspicion of prion disease or viral encephalitis.

### **6. Biomarkers**

**Dementia-unlikely due to AD**

12 Neurodegenerative Diseases

(McKhann GM et al. 2011) [1].

1. Does not meet clinical criteria for AD dementia.

ever, overlap with AD.

are negative

ropsychological tests has been proposed [70].

2. a. Regardless of meeting clinical criteria for probable or possible AD dementia, there is sufficient evidence for an alternative diagnosis such as HIV dementia, dementia of Huntington's disease, or others that rarely, if

b. Regardless of meeting clinical criteria for possible AD dementia, both Aβ and neuronal injury biomarkers

**Table 3.** Dementia due to AD: National Institute on Aging (NIA) and the Alzheimer's Association diagnostic criteria

The diagnostic procedure in clinical setting is usual divided into two phases. Screening is used to formulate the diagnostic hypothesis and is followed by the diagnostic confirmation. During the screening phase, the main point is to collect detailed information on history not only from the patient itself, but also from the people who take care of patients such as fami‐ lial or other type of caregiver. Information about history includes medical history (presence of severe medical disease that may cause encephalopathy, psychiatric disease, traumatic brain injury or other neurological disease), medications, family history of dementia, and changes in both basic activities of daily living (such as self-feeding, dressing and bathing, ambulation) and instrumental activities of daily living (such as grooming, homework, man‐ age finances, driving, and leisure). To obtain information on activities daily living could be particularly helpful to use standardized evaluation instruments such as Katz ADL scale [63] Lawton-Brody IADL scale [64], and the Bristol Activities of Daily Living Scale [65]. In addi‐ tion to the history, a systematic assessment of general cognitive functioning is required through instruments designed for this purpose. There are no screening tools that can quick‐ ly assess different levels of cognitive impairment. The American Academy of Neurology guidelines suggested to use the Mini-Mental Status Examination [66], and the Memory Im‐ pairment Screen (MIS) [67]. In recent times, the Montreal Cognitive Assessment (MoCA) was developed as a tool to screen patients in who has been hypothesized a mild cognitive decline and usually performed in the normal range on the MMSE [68, 69]. Studies have shown that MoCa is sensitive for the mild stages of AD dementia, whereas MMSE is superi‐ or for more advanced stages with the functional impairment. A complete summary of neu‐

After the screening, the second phase consists of a neurological examination, a neuropsycho‐ logical assessment, and a behavioural disease evaluation. A complete general neurological examination has been recommended as well as accurate neuropsychological evaluation in order to test possible differential diagnosis. The presence of Parkinsonism can suggest a Lewy Body's dementia, while asymmetric tendon reflex or other lateralizing signs can sug‐ gest a vascular component. Other neurological signs, for example peripheral neuropathy may indicate toxic or metabolic problems. There is no evidence-based data to support the usefulness of specific routine blood tests for evaluation of those with dementia but these are useful in excluding co-morbidities. Most expert opinion advises to screen for vitamin B12, folate, thyroid stimulating hormone, calcium, glucose, complete blood cell count, renal and liver function abnormalities. Serological tests for syphilis, Borrelia and HIV should be con‐

A biomarker is any characteristic that is objectively measured and evaluated as an indicator of biologic or pathogenic process and it should also be reliable, non-invasive and simple to perform [72]. Regarding AD, biomarkers included in the new diagnostic criteria are a meas‐ urement of the underline pathology and can be divided in markers of amyloid accumulation (CSF Aβ level, PET with amyloid-tracers) and markers of neuronal injury (atrophy of MTL at MRI, tau level in CSF, metabolic PET) [55]. Table 4 shows all the biomarkers.

#### **6.1. Medial temporal lobe atrophy**

Bilateral atrophy of medial temporal lobe structures, including hippocampus, has been found in patients with AD (Figure 1). Moreover it has been reported that the brain atrophy detected with neuroimaging reflects the typical pattern of progression of neuropathology, spreading from entorhinal cortex and hippocampus to the association cortices, as describe by Braak and Braak [73, 74]. In a meta-analysis of studies using visual and linear measure‐ ments of medial temporal lobe atrophy (MTA) on MRI, the overall sensitivity and specificity for detection of AD compared with controls was estimated to be 85% and 88%, respectively [75]. A yearly decline in hippocampal volume is approximately 2.5 times greater in patients with AD than in normal aged subjects. In clinical practice simple visual rating scales esti‐ mating hippocampal atrophy has proven to be useful to support the diagnosis in the first stage of the disease [74]. Although MTL is a biomarker of neurodegeneration and a good surrogate of disease progression, it has low sensitivity and specificity (51-70% and 68-69%, respectively) in identifying prodromal AD stage [76].

**Figure 1.** Coronal T1-weighted MRI scans of control (left) and patient with AD (right). The patient with AD shows atro‐ phy of the hippocampus (arrow) [74].

#### **6.2. Metabolic PET**

PET with traced glucose (FDG-PET) shows brain metabolism and reflects pattern of neuro‐ degeneration. Metabolic reduction in bilateral temporal- parietal regions and in posterior cingulate is the most commonly described diagnostic criterion for AD [77].This specific pat‐ tern significantly predicts decline to AD with an average overall accuracy of 86%, and with sensitivity and specificity about 75-80%. Moreover some studies on pre-symptomatic carri‐ ers of genetic mutations for AD revealed FDG-PET hypometabolism many years before the clinical onset of the disease [78,79].

racy, there is limited standardization of the biomarkers and the access is limited to

APP, amyloid precursor protein, is sequentially cleaved by α-or β-secretase (BACE1), fol‐ lowed by γ-secretase enzyme. The cleavage by BACE1 and γ-secretase generates Aβ pep‐ tide, likely to aggregate in plaques, and the N-terminal secreted fragment of APPβ (sAPPβ). In contrast, APP cleavage by α-and γ-secretase generates non-amyloidogenic fragments and secreted fragment of APPα (sAPPα). Thus, CSF BACE1 activity and sAPPβ and sAPPα proteins have been testing to provide information about amyloidogenic vs. nonamyloidogenic processing in the brain. Although some reports have shown higher lev‐ els of CSF BACE1 activity in AD compared with healthy controls and higher levels in subjects with MCI who progress to AD, others have not observed these, or have shown a decline of BACE1 activity in AD [86]. It is possible that CSF BACE activity is elevated in incipient AD and subsequently decline with disease progression. Several groups have measured CSF sAPPβ and sAPPα levels from AD and control subjects to understand brain APP metabolism better. Some studies reported higher levels of sAPPβ and a reduc‐

tion in sAPPα levels in AD, however, these results need further confirmation [86].

Several studies investigated plasma level of Aβ in AD. One group of researchers reported that in patients with newly acquired Alzheimer's disease, the plasma Aβ levels decline sig‐ nificantly compared with controls or participants with prevalent Alzheimer's disease during an average follow-up period of 3 years [87]. Another study reported that higher baseline plasma Aβ concentrations and greater reductions in plasma Aβ concentrations were associ‐ ated with cognitive decline in non-demented elderly people over 4 years follow-up [88].

CSF Aβ [83] 90% 86% PET amyloid imaging [78] 56.2% 93.5%

CSF tau [83] 90% 81% Medial temporal lobe atrophy on MRI [75] 88% 85% FDG-PET imaging [78] 74% 78.7%

**Specificity Sensitivity**

Alzheimer's Disease: A Clinical Perspective http://dx.doi.org/10.5772/54539 15

university hospitals.

**Biomarkers of Aβ deposition**

**Biomarkers of neuronal injury**

**7. Others biomarkers**

**7.1. CSF BACE1 and sAPP**

**7.2. Plasma Aβ**

**Table 4.** Alzheimer´s disease biomarkers: diagnostic accuracy

#### **6.3. PET with amyloid-tracers**

Interestingly recently has been developed a technique to detect amyloid in vivo using PET. [18F]-FDDNP and [11C] Pittsburgh compound-B (PIB) were the first amyloid PET tracers developed. Both tracers bind with nanomolar affinity to amyloid and enter the brain in amounts sufficient for imaging with PET. Retention of the tracers in neocortical and subcort‐ ical brain regions was significantly higher in AD patients than in controls. In subjects with MCI and positive retention the rate of progression to AD is estimated 25% per year [80]. A recent meta-analysis estimated a sensitivity of 93% and specificity of 56.2% [78]. Similarly, genetic at-risk cohorts demonstrate evidence of Aβ accumulation many years before detecta‐ ble cognitive impairment [79]. This method could be used to follow the therapeutic efficacy of the new AD immunotherapies.

#### **6.4. CSF Aβ and tau**

Many reports have demonstrated a decline in CSF Aβ and elevation of total tau, phosphotau, and tau/Aβ ratio in AD subjects. The reduction of Aβ could be by about 50% in subjects with AD compared with age-matched controls [81] and this phenomenon is thought to re‐ sult from deposition of Aβ into plaques, leaving less Aβ being available to diffuse into the CSF. CSF total tau reflects the intensity of the neuronal and axonal damage, and it is in‐ creased in AD subjects by 2-3 folds compare with controls. However, tau, as a marker of neuronal injury, can be transiently increased after any acute brain injury (such as stroke or trauma) [82]. A comprehensive review [83] reports that Aβ shows a sensitivity and specifici‐ ty of 86% and 90%, respectively, in differentiating AD from controls. For tau, the sensitivity is 81% and the specificity 90%, and p-tau has a mean sensitivity of 80% when specificity is set at 92%. By use of a combination of concentrations of Aβ42 and t-tau for AD versus con‐ trols, high sensitivities (85–94%) and specificities (83–100%) can be reached. The reliability of CSF biomarker has been tested by the comparison with autopsy results, showing high sensi‐ tivity and specificity in discriminating AD from both the cognitively normal elderly and from patients with other dementias. These CSF markers have also been shown to predict AD in patients with MCI [84], and to precede symptoms in familial AD [85]. However CSF bio‐ markers are not related with dementia severity.

Biomarkers, despite their great potential especially in the research field, are not recommend‐ ed for the routine use in clinical diagnostic setting. Clinical criteria provide very good accu‐


racy, there is limited standardization of the biomarkers and the access is limited to university hospitals.

**Table 4.** Alzheimer´s disease biomarkers: diagnostic accuracy

### **7. Others biomarkers**

**6.2. Metabolic PET**

14 Neurodegenerative Diseases

clinical onset of the disease [78,79].

**6.3. PET with amyloid-tracers**

of the new AD immunotherapies.

markers are not related with dementia severity.

**6.4. CSF Aβ and tau**

PET with traced glucose (FDG-PET) shows brain metabolism and reflects pattern of neuro‐ degeneration. Metabolic reduction in bilateral temporal- parietal regions and in posterior cingulate is the most commonly described diagnostic criterion for AD [77].This specific pat‐ tern significantly predicts decline to AD with an average overall accuracy of 86%, and with sensitivity and specificity about 75-80%. Moreover some studies on pre-symptomatic carri‐ ers of genetic mutations for AD revealed FDG-PET hypometabolism many years before the

Interestingly recently has been developed a technique to detect amyloid in vivo using PET. [18F]-FDDNP and [11C] Pittsburgh compound-B (PIB) were the first amyloid PET tracers developed. Both tracers bind with nanomolar affinity to amyloid and enter the brain in amounts sufficient for imaging with PET. Retention of the tracers in neocortical and subcort‐ ical brain regions was significantly higher in AD patients than in controls. In subjects with MCI and positive retention the rate of progression to AD is estimated 25% per year [80]. A recent meta-analysis estimated a sensitivity of 93% and specificity of 56.2% [78]. Similarly, genetic at-risk cohorts demonstrate evidence of Aβ accumulation many years before detecta‐ ble cognitive impairment [79]. This method could be used to follow the therapeutic efficacy

Many reports have demonstrated a decline in CSF Aβ and elevation of total tau, phosphotau, and tau/Aβ ratio in AD subjects. The reduction of Aβ could be by about 50% in subjects with AD compared with age-matched controls [81] and this phenomenon is thought to re‐ sult from deposition of Aβ into plaques, leaving less Aβ being available to diffuse into the CSF. CSF total tau reflects the intensity of the neuronal and axonal damage, and it is in‐ creased in AD subjects by 2-3 folds compare with controls. However, tau, as a marker of neuronal injury, can be transiently increased after any acute brain injury (such as stroke or trauma) [82]. A comprehensive review [83] reports that Aβ shows a sensitivity and specifici‐ ty of 86% and 90%, respectively, in differentiating AD from controls. For tau, the sensitivity is 81% and the specificity 90%, and p-tau has a mean sensitivity of 80% when specificity is set at 92%. By use of a combination of concentrations of Aβ42 and t-tau for AD versus con‐ trols, high sensitivities (85–94%) and specificities (83–100%) can be reached. The reliability of CSF biomarker has been tested by the comparison with autopsy results, showing high sensi‐ tivity and specificity in discriminating AD from both the cognitively normal elderly and from patients with other dementias. These CSF markers have also been shown to predict AD in patients with MCI [84], and to precede symptoms in familial AD [85]. However CSF bio‐

Biomarkers, despite their great potential especially in the research field, are not recommend‐ ed for the routine use in clinical diagnostic setting. Clinical criteria provide very good accu‐

#### **7.1. CSF BACE1 and sAPP**

APP, amyloid precursor protein, is sequentially cleaved by α-or β-secretase (BACE1), fol‐ lowed by γ-secretase enzyme. The cleavage by BACE1 and γ-secretase generates Aβ pep‐ tide, likely to aggregate in plaques, and the N-terminal secreted fragment of APPβ (sAPPβ). In contrast, APP cleavage by α-and γ-secretase generates non-amyloidogenic fragments and secreted fragment of APPα (sAPPα). Thus, CSF BACE1 activity and sAPPβ and sAPPα proteins have been testing to provide information about amyloidogenic vs. nonamyloidogenic processing in the brain. Although some reports have shown higher lev‐ els of CSF BACE1 activity in AD compared with healthy controls and higher levels in subjects with MCI who progress to AD, others have not observed these, or have shown a decline of BACE1 activity in AD [86]. It is possible that CSF BACE activity is elevated in incipient AD and subsequently decline with disease progression. Several groups have measured CSF sAPPβ and sAPPα levels from AD and control subjects to understand brain APP metabolism better. Some studies reported higher levels of sAPPβ and a reduc‐ tion in sAPPα levels in AD, however, these results need further confirmation [86].

#### **7.2. Plasma Aβ**

Several studies investigated plasma level of Aβ in AD. One group of researchers reported that in patients with newly acquired Alzheimer's disease, the plasma Aβ levels decline sig‐ nificantly compared with controls or participants with prevalent Alzheimer's disease during an average follow-up period of 3 years [87]. Another study reported that higher baseline plasma Aβ concentrations and greater reductions in plasma Aβ concentrations were associ‐ ated with cognitive decline in non-demented elderly people over 4 years follow-up [88]. This study indicated that plasma Aβ level is elevated during the pre-symptomatic stage in at-risk individuals, but subsequently start falling with the development of Alzheimer's dis‐ ease/MCI. Anyhow most groups have not found any significant differences between pa‐ tients and controls. Wu and colleagues [86] recently tried to measure, through specific antibody, plasma level of BACE1, sAPPβ and sAPPα. They reported significant increase in plasma BACE activity, sAPPβ, and sAPPα in a small sample of AD patients (n=20) com‐ pared with age-matched controls (n=30).

**7.5. Visual variant**

**7.6. Progressive apraxic syndrome**

**7.7. Frontal variant**

stricted to the frontal lobes for very long.

Posterior cortical atrophy (PCA) or visual variant of AD is characterized by early impair‐ ment of visuo-spatial skills with less prominent memory loss and is associated with atro‐ phy in parieto-occipital and posterior temporal cortices with right predominance [98]. Clinical presentation includes difficulties in reading lines of text, in judging distances, in identifying static objects within the visual field, alexia, and features of Balint's syndrome (simultanagnosia, oculomotor apraxia, optic ataxia, environmental agnosia) and Gerst‐ mann's syndrome (acalculia, agraphia, finger agnosia, left–right disorientation) [99]. Defi‐ cits in working memory and limb apraxia have also been noted [100]. By the time PCA has run its course, many patients develop also memory and language deficits. Findings of pathological studies all show that Alzheimer's disease is the most common underlying cause of PCA [101]. Some studies have shown that PCA cases have the greatest density of both plaques and neurofibrillary tangles in visual and visual-association cortices and few‐ er tangles and senile plaques in the hippocampus and subiculum [101]. CSF biomarkers (Aβ, tau, and P-tau) show similar pattern in patients with PCA compared with AD sub‐ jects, supporting the hypothesis that PCA is associated typically with underlying Alz‐ heimer's disease pathology. However, some cases are attributable to other causes, such as

Alzheimer's Disease: A Clinical Perspective http://dx.doi.org/10.5772/54539 17

corticobasal degeneration, dementia with Lewy bodies, or prion disease [98].

less involvement of pre-frontal regions compare with FTD and CBD [103].

Autopsy proven AD cases can be related to an apraxis clinical syndrome. Patients with these phenotype present progressive loss of use of the limbs which compromises performance on manual tasks such as dressing, handling a knife or a fork and writing. Cognitive assessment reveals apraxia and in less extends deficits in spatial function, with initially preserved mem‐ ory [102]. The clinical spectrum of that phenotype may also include others symptoms of the corticobasal syndrome (CBS), as asymmetric parkinsonism, ideomotor apraxia and alien limb phenomena. CBS is an unusual clinical manifestation of various neurodegenerative pathologies, AD, FTD, corticobasal degeneration (CBD). The one related to AD presents a temporo-parietal atrophy prevalent on the left side and hypoperfusion of parietal lobe, with

Sometimes AD patients presents a prevalent impairment of the executive function in the early stages of the disease, but there is also a multidomain deficit [90, 102]. Two reports, [91, 94] have claimed that a behavioural onset of cognitive dysfunction, with disinhibi‐ tion, apathy and personality change could be also an atypical presentation of AD patholo‐ gy. Alladi and colleagues [90] after the examination of 28 cases of behavioral variant - FTD found AD pathology in two cases. None of the two patients had amnesia at the onset of the disease, but in both cases diffuse cognitive dysfunction developed early in the course of the disease. Authors concluded that a behavioural variant of AD exists, but in contrast to patients with non-AD pathology, the disease does not appear to remain re‐

#### **7.3. Clinical variants of Alzheimer´s disease**

The common conception of Alzheimer's disease (AD) is a disorder that initially affects memory function, associated with early pathological changes in medial temporal lobes, and progresses to involve language, visuospatial skills and other cognitive abilities, re‐ flecting progressive involvement of association neocortices [61]. It is recognised, however, that the clinical presentation of AD is variable and in some cases the presenting domi‐ nant symptom is not memory [89-91]. Non-amnestic presentations are frequently referred to as "focal" presentations of AD. It is well established that most patients with a progres‐ sive disturbance of aspects of visuo-perceptual and spatial abilities, often referred to as posterior cortical atrophy, have underlying AD pathology. In addition, it is now clear that a proportion of patients with progressive aphasia, both fluent and non-fluent type, can have AD as the primary pathology. Recently, cases of corticobasal syndrome (CBS) secon‐ dary to AD pathology have also been reported [90]. The existence of a frontal presenta‐ tion is more controversial. Patients with familial AD secondary to presenilin 1 mutations may have a behavioural onset [92, 93] and there are isolated reports of sporadic AD re‐ sembling fronto-temporal dementia (FTD) [91, 94].

#### **7.4. Progressive aphasia**

Primary progressive aphasia (PPA) is a clinical syndrome in which cognitive decline is limited to one or more components of the language system. Since Mesulam's first descrip‐ tion of the phenomenon [95] clinical, neuropsychological and imaging studies have con‐ verged on the existence of three distinct clinical subtypes: semantic dementia (SD), characterized by fluent but empty speech, impaired comprehension and high incidence of dyslexic errors, in association with selective atrophy in anterior temporal regions; non flu‐ ent/agrammatic aphasia (PNFA) with phonologically and/or grammatically distorted speech output, preserved single word comprehension, and atrophy focused on the left in‐ ferior frontal and insular regions; logopenic variant ( LPA), characterized by a slow pro‐ duction rate, long word finding pauses, sparse phonological paraphasias and difficulty with sentence (but not single word) repetition. MRI reveals abnormalities in more posteri‐ or brain regions [96]. Pathologically, PNFA and SD are more likely to present an FTD pat‐ tern, although in some cases they can be the clinical presentation of atypical AD pathology. In contrast, biochemical, amyloid imaging and post mortem findings in LPA support the idea that the syndrome is a clinical marker of AD pathology [97]. Clinical evolution of that variant leads to mutism and memory impairment.

#### **7.5. Visual variant**

This study indicated that plasma Aβ level is elevated during the pre-symptomatic stage in at-risk individuals, but subsequently start falling with the development of Alzheimer's dis‐ ease/MCI. Anyhow most groups have not found any significant differences between pa‐ tients and controls. Wu and colleagues [86] recently tried to measure, through specific antibody, plasma level of BACE1, sAPPβ and sAPPα. They reported significant increase in plasma BACE activity, sAPPβ, and sAPPα in a small sample of AD patients (n=20) com‐

The common conception of Alzheimer's disease (AD) is a disorder that initially affects memory function, associated with early pathological changes in medial temporal lobes, and progresses to involve language, visuospatial skills and other cognitive abilities, re‐ flecting progressive involvement of association neocortices [61]. It is recognised, however, that the clinical presentation of AD is variable and in some cases the presenting domi‐ nant symptom is not memory [89-91]. Non-amnestic presentations are frequently referred to as "focal" presentations of AD. It is well established that most patients with a progres‐ sive disturbance of aspects of visuo-perceptual and spatial abilities, often referred to as posterior cortical atrophy, have underlying AD pathology. In addition, it is now clear that a proportion of patients with progressive aphasia, both fluent and non-fluent type, can have AD as the primary pathology. Recently, cases of corticobasal syndrome (CBS) secon‐ dary to AD pathology have also been reported [90]. The existence of a frontal presenta‐ tion is more controversial. Patients with familial AD secondary to presenilin 1 mutations may have a behavioural onset [92, 93] and there are isolated reports of sporadic AD re‐

Primary progressive aphasia (PPA) is a clinical syndrome in which cognitive decline is limited to one or more components of the language system. Since Mesulam's first descrip‐ tion of the phenomenon [95] clinical, neuropsychological and imaging studies have con‐ verged on the existence of three distinct clinical subtypes: semantic dementia (SD), characterized by fluent but empty speech, impaired comprehension and high incidence of dyslexic errors, in association with selective atrophy in anterior temporal regions; non flu‐ ent/agrammatic aphasia (PNFA) with phonologically and/or grammatically distorted speech output, preserved single word comprehension, and atrophy focused on the left in‐ ferior frontal and insular regions; logopenic variant ( LPA), characterized by a slow pro‐ duction rate, long word finding pauses, sparse phonological paraphasias and difficulty with sentence (but not single word) repetition. MRI reveals abnormalities in more posteri‐ or brain regions [96]. Pathologically, PNFA and SD are more likely to present an FTD pat‐ tern, although in some cases they can be the clinical presentation of atypical AD pathology. In contrast, biochemical, amyloid imaging and post mortem findings in LPA support the idea that the syndrome is a clinical marker of AD pathology [97]. Clinical

pared with age-matched controls (n=30).

16 Neurodegenerative Diseases

**7.3. Clinical variants of Alzheimer´s disease**

sembling fronto-temporal dementia (FTD) [91, 94].

evolution of that variant leads to mutism and memory impairment.

**7.4. Progressive aphasia**

Posterior cortical atrophy (PCA) or visual variant of AD is characterized by early impair‐ ment of visuo-spatial skills with less prominent memory loss and is associated with atro‐ phy in parieto-occipital and posterior temporal cortices with right predominance [98]. Clinical presentation includes difficulties in reading lines of text, in judging distances, in identifying static objects within the visual field, alexia, and features of Balint's syndrome (simultanagnosia, oculomotor apraxia, optic ataxia, environmental agnosia) and Gerst‐ mann's syndrome (acalculia, agraphia, finger agnosia, left–right disorientation) [99]. Defi‐ cits in working memory and limb apraxia have also been noted [100]. By the time PCA has run its course, many patients develop also memory and language deficits. Findings of pathological studies all show that Alzheimer's disease is the most common underlying cause of PCA [101]. Some studies have shown that PCA cases have the greatest density of both plaques and neurofibrillary tangles in visual and visual-association cortices and few‐ er tangles and senile plaques in the hippocampus and subiculum [101]. CSF biomarkers (Aβ, tau, and P-tau) show similar pattern in patients with PCA compared with AD sub‐ jects, supporting the hypothesis that PCA is associated typically with underlying Alz‐ heimer's disease pathology. However, some cases are attributable to other causes, such as corticobasal degeneration, dementia with Lewy bodies, or prion disease [98].

#### **7.6. Progressive apraxic syndrome**

Autopsy proven AD cases can be related to an apraxis clinical syndrome. Patients with these phenotype present progressive loss of use of the limbs which compromises performance on manual tasks such as dressing, handling a knife or a fork and writing. Cognitive assessment reveals apraxia and in less extends deficits in spatial function, with initially preserved mem‐ ory [102]. The clinical spectrum of that phenotype may also include others symptoms of the corticobasal syndrome (CBS), as asymmetric parkinsonism, ideomotor apraxia and alien limb phenomena. CBS is an unusual clinical manifestation of various neurodegenerative pathologies, AD, FTD, corticobasal degeneration (CBD). The one related to AD presents a temporo-parietal atrophy prevalent on the left side and hypoperfusion of parietal lobe, with less involvement of pre-frontal regions compare with FTD and CBD [103].

#### **7.7. Frontal variant**

Sometimes AD patients presents a prevalent impairment of the executive function in the early stages of the disease, but there is also a multidomain deficit [90, 102]. Two reports, [91, 94] have claimed that a behavioural onset of cognitive dysfunction, with disinhibi‐ tion, apathy and personality change could be also an atypical presentation of AD patholo‐ gy. Alladi and colleagues [90] after the examination of 28 cases of behavioral variant - FTD found AD pathology in two cases. None of the two patients had amnesia at the onset of the disease, but in both cases diffuse cognitive dysfunction developed early in the course of the disease. Authors concluded that a behavioural variant of AD exists, but in contrast to patients with non-AD pathology, the disease does not appear to remain re‐ stricted to the frontal lobes for very long.

#### **8. Treatment and management**

Regarding the therapeutic management of a disease generally there are at least three possi‐ bilities: (i) prevention strategy, (ii) symptomatic treatment, and (iii) disease modifying thera‐ pies. Currently, a long list of factors that can reduce or delay the risk of AD onset has been reported, but so far there is no certain evidence supporting the prevention efficacy in AD. In Europe, there are three ongoing multidomain interventional random clinical trials (RTCs) that focus on the optimal management of vascular risk factors and vascular diseases and in‐ clude also medical and lifestyle interventions. The results of the RTCs might help in improv‐ ing strategies of dementia prevention [3]. This indicates the principle type of AD treatment is based on symptomatic drugs. There is no cure for AD, but new types of disease modifying treatments are under investigation. Non-pharmacological interventions have been also re‐ cently added in AD patient management.

yl) starts with 4 mg twice daily and increases in increments of 4 mg per dose twice a day to a maximum of 12 mg twice daily if tolerated. Currently it is also available in an extended-re‐ lease formulation that can be taken once daily. Galantamine has some nicotinic receptor ac‐ tivity. All AChEIs can influence cardiac rhytm, but is not common unless a person has an underlying disturbance in cardiac conduction. An electrocardiogram prior to initiating the treatment is recommended. AChEIs may also have an effect on respiratory conditions, such as chronic obstructive pulmonary disease or asthma, or gastrointestinal disease, such as gas‐ tric ulcer. The absorption of AChEI is not influenced by food intake. These agents are recom‐

Alzheimer's Disease: A Clinical Perspective http://dx.doi.org/10.5772/54539 19

Memantine is an NMDA (N-methyl-D-aspartate) receptor antagonist that reduces glutama‐ tergic excitotoxicity. Based on the glutammatergic hypothesis of AD, Memantine has been claimed to be a disease modifying therapy. Clinical trial with Memantine reports a mild effi‐ cacy in maintaning functional level in patients with severe dementia [107]. Memantine is li‐ censed for the treatment of people with moderate-to-severe AD. The starting dose is 5 mg that can be increased of 5 mg every week up to the dose of 20 mg. Side effects are very un‐ usual and include restlessness, hyperexitation and fatigue. There are good evidence of clini‐ cal benefit in patients moving into severe stages of AD from a combination therapy with

Since the role of beta amyloid (Aβ) is considered to be paramount in the development of AD, several research strategies have been undertaken to alter the biochemistry of Aβ in the brain through interference of either the formation or the deposition of Aβ. The amyloid pre‐ cursor protein can be processed in two different pathways, non-amyloidogenic by α-secre‐ tase and amyloidogenic ones by β-secretase followed by γ-secretase [12]. Thus, the inhibition of β or γ-secretase is the target of therapies that aim to reduce the production of Aβ, while new immunotherapeutic strategies promote removal of Aβ from the brain. Drugs that can act as β-secretase inhibitors belong to a group of type 2 diabetes therapies, thiazoli‐ dinediones (rosiglitazone and pioglitazone). Despite the promising biological plausibility of these compounds, the results of randomized clinical trials (RCTs) have been disappointing [111-112]. A number of γ-secretase modulators (semagacestat and tarenflurbil) have also failed to provide benefits in the treatment of AD [113,114]. Immunotherapies or "vaccines" are based on both active and passive immunization. Initial approaches based on immuniza‐ tion with Aβ fragments performed extremely well in transgenic mouse models but showed less promise in humans [51]. The most promising of these, AN-1792 (QS-21) resulted in sig‐ nificant Aβ-antibody titers in patients with mild-to-moderate AD in Phase II trials. Postmor‐ tem analysis on long-term follow-up also confirmed that the therapy had resulted in a significant reduction in Aβ burden in the brain. However, there was no evidence of any clin‐ ical benefit and the trial was halted owing to patients developing aseptic meningoencephali‐ tis, thought to have been induced by cytotoxic T-cell activation. Immunotherapies have

mended for the treatment for patient only in the mild and moderate stages [71].

Memantine and an acetylcholinesterase inhibitor [108-110].

**8.3. Disease-modifying treatments**

**8.2. Memantine**

#### **8.1. Symptomatic treatments: acetylcholinesterase inhibitors**

The neuropathology of Alzheimer's disease is characterized by early loss of basal forebrain cholinergic neurons, leading to decreased cholinergic transmission which is involved in many aspects of cognition, including memory and attention. Inhibitors of the acetylcholines‐ terase enzyme (AChEIs) increase acetylcoline level in brain, which leads to memory im‐ provement. Since the introduction of the first ChEI in 1997, these agents are considered firstline pharmacotherapy for mild to moderate AD stages [71]. Four ChEIs are currently available: tacrine, donepezil, rivastigmine and galantamine. Tacrine (Cognex), the first ap‐ proved, is not commonly used because of a poor tolerability profile and low oral bioavaila‐ bility [104]. The Cochrane's Review [105] of placebo controlled trials of ChEIs demonstrated that the treatment determine an improvement of 1.4- to 3.9-point in the ADAS-Cog scale at 6 months and 1 year. In clinical trials, a change of 4 points is considered clinically significant for patients with mild to moderate dementia. In addition to their effects on cognition, these agents also have demonstrated beneficial effects on measures of behavior, activities of daily living (ADLs), and global patient function as reported in a recent meta-analysis [106]. Done‐ pezil (Aricept) was approved in the mid-1990s. The starting dose is 5 mg once daily which can be increased after 4 weeks to 10 mg, if well tolerated. The common side effects are nau‐ sea, vomiting, gastritis and diarrhea. The length of the response has been documented up to 52 weeks. When donepezil is discontinued, performance of the subject returns to the same as in the untreated state. Rivastigmine (Exelon) is a pseudo-irreversible inhibitor as it dissoci‐ ates from the enzyme slowly. Two type of administration are available: oral and transder‐ mal patch. The oral starting dose is 1,5 mg twice daily that can be weekly increase of 1,5 mg until a total amount of 12 mg per day (6 mg twice daily). The transdermal patch last 24 hours and has two dosages: 4,6 mg and 9,5 mg. The target dose, 9,5 mg/24 h, can be reached after 4 weeks if the low dosage is well tolerated. Side effects of oral Rivastigmine are ap‐ proximately the same as donepezil, while gastrointestinal symptoms are at least three times less prominent with the patch [104]. Rivastigmine is also an inhibitor of butyrylcholinester‐ ase that facilitate cholinergic neurotransmission by slowing the degradation of acetylcholine released by functionally intact cholinergic neurons. The therapy with Galantamine (Remin‐ yl) starts with 4 mg twice daily and increases in increments of 4 mg per dose twice a day to a maximum of 12 mg twice daily if tolerated. Currently it is also available in an extended-re‐ lease formulation that can be taken once daily. Galantamine has some nicotinic receptor ac‐ tivity. All AChEIs can influence cardiac rhytm, but is not common unless a person has an underlying disturbance in cardiac conduction. An electrocardiogram prior to initiating the treatment is recommended. AChEIs may also have an effect on respiratory conditions, such as chronic obstructive pulmonary disease or asthma, or gastrointestinal disease, such as gas‐ tric ulcer. The absorption of AChEI is not influenced by food intake. These agents are recom‐ mended for the treatment for patient only in the mild and moderate stages [71].

#### **8.2. Memantine**

**8. Treatment and management**

18 Neurodegenerative Diseases

cently added in AD patient management.

**8.1. Symptomatic treatments: acetylcholinesterase inhibitors**

Regarding the therapeutic management of a disease generally there are at least three possi‐ bilities: (i) prevention strategy, (ii) symptomatic treatment, and (iii) disease modifying thera‐ pies. Currently, a long list of factors that can reduce or delay the risk of AD onset has been reported, but so far there is no certain evidence supporting the prevention efficacy in AD. In Europe, there are three ongoing multidomain interventional random clinical trials (RTCs) that focus on the optimal management of vascular risk factors and vascular diseases and in‐ clude also medical and lifestyle interventions. The results of the RTCs might help in improv‐ ing strategies of dementia prevention [3]. This indicates the principle type of AD treatment is based on symptomatic drugs. There is no cure for AD, but new types of disease modifying treatments are under investigation. Non-pharmacological interventions have been also re‐

The neuropathology of Alzheimer's disease is characterized by early loss of basal forebrain cholinergic neurons, leading to decreased cholinergic transmission which is involved in many aspects of cognition, including memory and attention. Inhibitors of the acetylcholines‐ terase enzyme (AChEIs) increase acetylcoline level in brain, which leads to memory im‐ provement. Since the introduction of the first ChEI in 1997, these agents are considered firstline pharmacotherapy for mild to moderate AD stages [71]. Four ChEIs are currently available: tacrine, donepezil, rivastigmine and galantamine. Tacrine (Cognex), the first ap‐ proved, is not commonly used because of a poor tolerability profile and low oral bioavaila‐ bility [104]. The Cochrane's Review [105] of placebo controlled trials of ChEIs demonstrated that the treatment determine an improvement of 1.4- to 3.9-point in the ADAS-Cog scale at 6 months and 1 year. In clinical trials, a change of 4 points is considered clinically significant for patients with mild to moderate dementia. In addition to their effects on cognition, these agents also have demonstrated beneficial effects on measures of behavior, activities of daily living (ADLs), and global patient function as reported in a recent meta-analysis [106]. Done‐ pezil (Aricept) was approved in the mid-1990s. The starting dose is 5 mg once daily which can be increased after 4 weeks to 10 mg, if well tolerated. The common side effects are nau‐ sea, vomiting, gastritis and diarrhea. The length of the response has been documented up to 52 weeks. When donepezil is discontinued, performance of the subject returns to the same as in the untreated state. Rivastigmine (Exelon) is a pseudo-irreversible inhibitor as it dissoci‐ ates from the enzyme slowly. Two type of administration are available: oral and transder‐ mal patch. The oral starting dose is 1,5 mg twice daily that can be weekly increase of 1,5 mg until a total amount of 12 mg per day (6 mg twice daily). The transdermal patch last 24 hours and has two dosages: 4,6 mg and 9,5 mg. The target dose, 9,5 mg/24 h, can be reached after 4 weeks if the low dosage is well tolerated. Side effects of oral Rivastigmine are ap‐ proximately the same as donepezil, while gastrointestinal symptoms are at least three times less prominent with the patch [104]. Rivastigmine is also an inhibitor of butyrylcholinester‐ ase that facilitate cholinergic neurotransmission by slowing the degradation of acetylcholine released by functionally intact cholinergic neurons. The therapy with Galantamine (Remin‐ Memantine is an NMDA (N-methyl-D-aspartate) receptor antagonist that reduces glutama‐ tergic excitotoxicity. Based on the glutammatergic hypothesis of AD, Memantine has been claimed to be a disease modifying therapy. Clinical trial with Memantine reports a mild effi‐ cacy in maintaning functional level in patients with severe dementia [107]. Memantine is li‐ censed for the treatment of people with moderate-to-severe AD. The starting dose is 5 mg that can be increased of 5 mg every week up to the dose of 20 mg. Side effects are very un‐ usual and include restlessness, hyperexitation and fatigue. There are good evidence of clini‐ cal benefit in patients moving into severe stages of AD from a combination therapy with Memantine and an acetylcholinesterase inhibitor [108-110].

#### **8.3. Disease-modifying treatments**

Since the role of beta amyloid (Aβ) is considered to be paramount in the development of AD, several research strategies have been undertaken to alter the biochemistry of Aβ in the brain through interference of either the formation or the deposition of Aβ. The amyloid pre‐ cursor protein can be processed in two different pathways, non-amyloidogenic by α-secre‐ tase and amyloidogenic ones by β-secretase followed by γ-secretase [12]. Thus, the inhibition of β or γ-secretase is the target of therapies that aim to reduce the production of Aβ, while new immunotherapeutic strategies promote removal of Aβ from the brain. Drugs that can act as β-secretase inhibitors belong to a group of type 2 diabetes therapies, thiazoli‐ dinediones (rosiglitazone and pioglitazone). Despite the promising biological plausibility of these compounds, the results of randomized clinical trials (RCTs) have been disappointing [111-112]. A number of γ-secretase modulators (semagacestat and tarenflurbil) have also failed to provide benefits in the treatment of AD [113,114]. Immunotherapies or "vaccines" are based on both active and passive immunization. Initial approaches based on immuniza‐ tion with Aβ fragments performed extremely well in transgenic mouse models but showed less promise in humans [51]. The most promising of these, AN-1792 (QS-21) resulted in sig‐ nificant Aβ-antibody titers in patients with mild-to-moderate AD in Phase II trials. Postmor‐ tem analysis on long-term follow-up also confirmed that the therapy had resulted in a significant reduction in Aβ burden in the brain. However, there was no evidence of any clin‐ ical benefit and the trial was halted owing to patients developing aseptic meningoencephali‐ tis, thought to have been induced by cytotoxic T-cell activation. Immunotherapies have since been designed using shorter peptides designed to mimic immunoreactive sections of Aβ, in an effort to avoid severe inflammatory response. There are various immunotherapies taking these approaches. For example, CAD-106, which targets Aβ1–6, resulted in Aβ clear‐ ance without collateral immunoreactivity in Phase I trials, and is now in a Phase II RCT. Pas‐ sive immunotherapy for AD has met with some criticism owing to the challenge of designing an approach that can achieve significant antibody concentrations in the brain. Al‐ though some of the data from animal studies do suggest a possible impact on oligomer for‐ mation and brain amyloid load. Currently, monoclonal antibody therapies include bapineuzumab (AAB-001) and solanezumab (LY-2062430) that are now in a phase III RCT [115]. The results of these trials are eagerly awaited, but experts' consensus is not anticipat‐ ing positive outcomes. Despite the facts that vaccines can remove Aβ from the brain, a fun‐ damental debate continues around the clinical benefit of Aβ clearance. Neurofibrillary tangles are another hallmark of AD pathology, however treatments to target tauopathy have received far less attention than amyloid therapies. Very preliminary results in animal mod‐ els have shown that a tau immunotherapy might be a valuable approach [116].

#### **8.4. Non-pharmacological treatment**

In the last ten years there has been a great public interest in possible non-pharmacological therapies to delay disease progression and functional decline. The psychosocial interven‐ tions fitted to this goal and they were developed based on the concept of "cognitive re‐ serve". Evidence from meta-analyses and systematic reviews has shown that a higher cognitive reserve is associated with a significantly reduced risk to develop dementia [117]. Generally "cognitive reserve" describes the mind's resistance to damage of the brain. There has been proposed two models to explore the reserve, a passive model called "brain re‐ serve" and an active model knows as "cognitive reserve" [118]. There are several different approaches to neuropsychological and training interventions focusing on cognition with dif‐ ferent evidence for efficacy in people with AD. In large part, the psychosocial interventions have shown significant, but modest effect-size when used alone. The American Association for Gertiatric Psychiatry (AAGP) proposed a care/treatment model that combines pharmaco‐ logical therapies with psychosocial intervention for people with AD [119]. To date, the liter‐ ature about psychosocial intervention is wide [120-123]. For this reason we decide to illustrate briefly the most important intervention below. The psychosocial interventions can be classified according to the treatment goal and include behaviour, emotion-oriented and stimulation-oriented treatment, and cognitive training [124].

haviours and mood, but the main effect is to change the daily-life routine [124]. Emotionoriented approaches include supportive psychotherapy, reminiscence therapy, validation therapy, sensory integration (snoezelen), and simulated presence therapy (SPT). Supportive psychotherapy has not received formal scientific studies, but can be used to address issue of loss in the early stage of AD and help mildly impaired patients to adapt themselves to a new lifestyle imposed by the disease. Reminiscence therapy remains controversial, individually or in group, on past life events of the patient helped by using external aids such as photo‐ graphs, household items, music and sound recordings, or other familiar items from the past. The final goal is to improve the psychological well-being, mood, and coping skills of pa‐ tients with AD [128]. Researches have shown that reminiscence is useful to improve directly emotions in overall mood, thus can improve cognitive functioning [129-132]. Validation therapy [133] is based on the empathic relationship between the patient and the therapist.

**Drug Suggested Dosage Approved indication**

can be increased up to 6 mg twice daily in 6

Transdermal patch: once daily, 4,6 mg/24 h,

increase after 4 weeks to 8 mg twice daily. A dosage of 12 mg twice daily can also be reached after a medical examination Availabe a new extended-release formulation that can be taken once daily

up to 10 mg twice daily in 4 weeks Available a dosage of 20 mg that can be Mild to moderate AD Sever AD in add-on with

Alzheimer's Disease: A Clinical Perspective http://dx.doi.org/10.5772/54539 21

Mild to moderae AD

Mild to moderate AD

Moderate to severe AD

Pashe II RCT\* Phase III RCT\* Phase III RCT\*

Memantine

10 mg/day after 4 weeks

*Donepezil (Aricept)* 5 mg once daily, which can be increased to

*Rivastigmine (Exelon)* Oral: Twice daily starting with 1.5 mg which

or 9,5 mg/24 h.

weeks

*Galantamine (Remynil)* Twice daily, beginning with 4 mg and

*Mematine (Ebixa)* Twice daily beginning with 5 mg increase

taken once daily

**Cholinesterase inhibitors**

**NMDA atagonist**

**Immunotherapies** Active immunization

Passive immunization Bapineuzumab (AAB-001) Solanezumab (LY-2062430)

\*RCT= randomize clinical trial

**Table 5.** Alzheimer's disease pharmacological treatments

CAD-106

Behaviour-oriented therapy is used to modify dysfunctional behaviour employing behav‐ iour change techniques which increase or decrease the frequency of behaviour through the use of reinforcement, punishment, and extinction following the Experimental Analysis of Behaviour (B.F. Skinner). Behaviour therapy is helpful to reduce typical behaviour's prob‐ lems such as incontinence and wondering [125-127]. Stimulation-oriented interventions in‐ clude recreational activities such as creative arts (such as craft, music, dance, andtheatre) and leisure education, art therapy, music therapy, pet therapy, and other formal activities aim to maximize pleasurable activities for the patients. Stimulation improves modestly be‐


**Table 5.** Alzheimer's disease pharmacological treatments

since been designed using shorter peptides designed to mimic immunoreactive sections of Aβ, in an effort to avoid severe inflammatory response. There are various immunotherapies taking these approaches. For example, CAD-106, which targets Aβ1–6, resulted in Aβ clear‐ ance without collateral immunoreactivity in Phase I trials, and is now in a Phase II RCT. Pas‐ sive immunotherapy for AD has met with some criticism owing to the challenge of designing an approach that can achieve significant antibody concentrations in the brain. Al‐ though some of the data from animal studies do suggest a possible impact on oligomer for‐ mation and brain amyloid load. Currently, monoclonal antibody therapies include bapineuzumab (AAB-001) and solanezumab (LY-2062430) that are now in a phase III RCT [115]. The results of these trials are eagerly awaited, but experts' consensus is not anticipat‐ ing positive outcomes. Despite the facts that vaccines can remove Aβ from the brain, a fun‐ damental debate continues around the clinical benefit of Aβ clearance. Neurofibrillary tangles are another hallmark of AD pathology, however treatments to target tauopathy have received far less attention than amyloid therapies. Very preliminary results in animal mod‐

els have shown that a tau immunotherapy might be a valuable approach [116].

stimulation-oriented treatment, and cognitive training [124].

In the last ten years there has been a great public interest in possible non-pharmacological therapies to delay disease progression and functional decline. The psychosocial interven‐ tions fitted to this goal and they were developed based on the concept of "cognitive re‐ serve". Evidence from meta-analyses and systematic reviews has shown that a higher cognitive reserve is associated with a significantly reduced risk to develop dementia [117]. Generally "cognitive reserve" describes the mind's resistance to damage of the brain. There has been proposed two models to explore the reserve, a passive model called "brain re‐ serve" and an active model knows as "cognitive reserve" [118]. There are several different approaches to neuropsychological and training interventions focusing on cognition with dif‐ ferent evidence for efficacy in people with AD. In large part, the psychosocial interventions have shown significant, but modest effect-size when used alone. The American Association for Gertiatric Psychiatry (AAGP) proposed a care/treatment model that combines pharmaco‐ logical therapies with psychosocial intervention for people with AD [119]. To date, the liter‐ ature about psychosocial intervention is wide [120-123]. For this reason we decide to illustrate briefly the most important intervention below. The psychosocial interventions can be classified according to the treatment goal and include behaviour, emotion-oriented and

Behaviour-oriented therapy is used to modify dysfunctional behaviour employing behav‐ iour change techniques which increase or decrease the frequency of behaviour through the use of reinforcement, punishment, and extinction following the Experimental Analysis of Behaviour (B.F. Skinner). Behaviour therapy is helpful to reduce typical behaviour's prob‐ lems such as incontinence and wondering [125-127]. Stimulation-oriented interventions in‐ clude recreational activities such as creative arts (such as craft, music, dance, andtheatre) and leisure education, art therapy, music therapy, pet therapy, and other formal activities aim to maximize pleasurable activities for the patients. Stimulation improves modestly be‐

**8.4. Non-pharmacological treatment**

20 Neurodegenerative Diseases

haviours and mood, but the main effect is to change the daily-life routine [124]. Emotionoriented approaches include supportive psychotherapy, reminiscence therapy, validation therapy, sensory integration (snoezelen), and simulated presence therapy (SPT). Supportive psychotherapy has not received formal scientific studies, but can be used to address issue of loss in the early stage of AD and help mildly impaired patients to adapt themselves to a new lifestyle imposed by the disease. Reminiscence therapy remains controversial, individually or in group, on past life events of the patient helped by using external aids such as photo‐ graphs, household items, music and sound recordings, or other familiar items from the past. The final goal is to improve the psychological well-being, mood, and coping skills of pa‐ tients with AD [128]. Researches have shown that reminiscence is useful to improve directly emotions in overall mood, thus can improve cognitive functioning [129-132]. Validation therapy [133] is based on the empathic relationship between the patient and the therapist. Through listening, the therapist examines the reality's perception of the patient in order to create significant emotional and relational contacts. The objectives are to stimulate the pa‐ tient to take social role, stimulate verbal communication, and encourage social interaction. Validation is intended for patients with severe to moderate dementia. Cognitive training in‐ volves guided practice on a set of standard tasks designed to stimulate specific cognitive functions (memory, attention, or problem-solving). The underlying assumption is that prac‐ tice may improve or maintain functioning in a given domain, generalizing the effects of practice beyond the clinical context to everyday life [121].The aim is to reduce cognitive defi‐ cits. The Reality Orientation Therapy (ROT) is a technique widely used in treatment of AD [134-136]. The objective is to stimulate the personal, time, and space orientation in the pa‐ tient through repeated multimodal stimulation (verbal, visual, and musical), strengthen the basic information with respect to space and time coordinates, and his personal history. The level of stimulation is modulated agree with residual cognitive resource of patient. Other types of cognitive training include skills training and cognitive retraining focusing on cogni‐ tive deficits. There has been demonstrated that cognitive training improve cognitive func‐ tioning, but effects were transient and often accompanied by negative effects linked to frustration [137]. Finally, the caregivers are also part of the treatment and should be careful‐ ly managed overtime.

was also supported in part by funds from the Loo and Hans Ostermans Foundation and the Foundation for Geriatric Diseases at Karolinska Institutet, the Gamla Tjänarinnor Founda‐

Alzheimer's Disease: A Clinical Perspective http://dx.doi.org/10.5772/54539 23

tion, Demens fonden and the Bertil Stohnes Foundation.

\*Address all correspondence to: weili.xu@ki.se

Weili Xu1,2\*, Anna Marseglia2,3, Camilla Ferrari2,4 and Hui-Xin Wang2

4 Department of Neuroscience, University of Florence, Italy

1 Department of Epidemiology, Tianjin Medical University, Tianjin, P.R. China

2 Aging Research Center, Karolinska Institutet-Stockholm University, Stockholm, Sweden

3 Institute of Neuroscience - Aging Branch, National Council of Research (CNR), Padua, Italy

[1] McKhann GM, Knopman DS, Chertkow H, Hyman BT, Jack CR, Jr., Kawas CH, et al. The diagnosis of dementia due to Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic

[2] Schneider JA, Arvanitakis Z, Bang W, Bennett DA. Mixed brain pathologies account for most dementia cases in community-dwelling older persons. Neurology.

[3] Mangialasche F, Kivipelto M, Solomon A, Fratiglioni L. Dementia prevention: cur‐ rent epidemiological evidence and future perspective. Alzheimers Res Ther.

[4] Fratiglioni L, Launer LJ, Andersen K, Breteler MM, Copeland JR, Dartigues JF, et al. Incidence of dementia and major subtypes in Europe: A collaborative study of popu‐ lation-based cohorts. Neurologic Diseases in the Elderly Research Group. Neurology.

[5] Qiu C, Kivipelto M, von Strauss E. Epidemiology of Alzheimer's disease: occurrence, determinants, and strategies toward intervention. Dialogues Clin Neurosci.

[6] Lobo A, Launer LJ, Fratiglioni L, Andersen K, Di Carlo A, Breteler MM, et al. Preva‐ lence of dementia and major subtypes in Europe: A collaborative study of popula‐

guidelines for Alzheimer's disease. Alzheimers Dement. 2011;7(3):263-9.

**Author details**

**References**

2007;69(24):2197-204.

2000;54(11 Suppl 5):S10-5.

2009;11(2):111-28.

2012;4(1):6.

### **9. Conclusions**

Alzheimer´s disease is a common disorder of aging, and a major cause of dependence and mortality among elderly. Substantial progress has been made over the past few decades in understanding AD. Nevertheless, our knowledge of this disease is still profoundly imper‐ fect, as demonstrated by the failure of all but symptomatic treatments for clinically diag‐ nosed AD. We know that in people aged >85 years, dementia and cognitive impairment are common, reaching a combined prevalence >50% in the oldest old, and that the incidence of dementia continues to rise in the oldest age groups. Thus, screening is essential to identify cognitively normal individuals in midlife or old age who have a high risk of developing MCI and AD, so that interventions, when available, can be administered to stop the devel‐ opment of specific disease-related pathologies. Although the exact pathogenetic mechanism of AD is still unclear, thanks to new technologies, we are now able to detect in vivo subjects with AD-related brain pathological changes. Many studies have provided evidence that AD pathology begins as many as 20 years before symptoms appear. These findings determined a new concept of AD, where the symptom of dementia represents the final part of the "con‐ tinuum" of AD. Recently, based on the new knowledge about AD, some disease modifying therapies have been developed and their results are eagerly awaited.

#### **Acknowledgements**

Research grants were received from the Swedish council for working life and social re‐ search, the Swedish Research Council in Medicine and the Swedish Brain Power. This study was also supported in part by funds from the Loo and Hans Ostermans Foundation and the Foundation for Geriatric Diseases at Karolinska Institutet, the Gamla Tjänarinnor Founda‐ tion, Demens fonden and the Bertil Stohnes Foundation.

### **Author details**

Through listening, the therapist examines the reality's perception of the patient in order to create significant emotional and relational contacts. The objectives are to stimulate the pa‐ tient to take social role, stimulate verbal communication, and encourage social interaction. Validation is intended for patients with severe to moderate dementia. Cognitive training in‐ volves guided practice on a set of standard tasks designed to stimulate specific cognitive functions (memory, attention, or problem-solving). The underlying assumption is that prac‐ tice may improve or maintain functioning in a given domain, generalizing the effects of practice beyond the clinical context to everyday life [121].The aim is to reduce cognitive defi‐ cits. The Reality Orientation Therapy (ROT) is a technique widely used in treatment of AD [134-136]. The objective is to stimulate the personal, time, and space orientation in the pa‐ tient through repeated multimodal stimulation (verbal, visual, and musical), strengthen the basic information with respect to space and time coordinates, and his personal history. The level of stimulation is modulated agree with residual cognitive resource of patient. Other types of cognitive training include skills training and cognitive retraining focusing on cogni‐ tive deficits. There has been demonstrated that cognitive training improve cognitive func‐ tioning, but effects were transient and often accompanied by negative effects linked to frustration [137]. Finally, the caregivers are also part of the treatment and should be careful‐

Alzheimer´s disease is a common disorder of aging, and a major cause of dependence and mortality among elderly. Substantial progress has been made over the past few decades in understanding AD. Nevertheless, our knowledge of this disease is still profoundly imper‐ fect, as demonstrated by the failure of all but symptomatic treatments for clinically diag‐ nosed AD. We know that in people aged >85 years, dementia and cognitive impairment are common, reaching a combined prevalence >50% in the oldest old, and that the incidence of dementia continues to rise in the oldest age groups. Thus, screening is essential to identify cognitively normal individuals in midlife or old age who have a high risk of developing MCI and AD, so that interventions, when available, can be administered to stop the devel‐ opment of specific disease-related pathologies. Although the exact pathogenetic mechanism of AD is still unclear, thanks to new technologies, we are now able to detect in vivo subjects with AD-related brain pathological changes. Many studies have provided evidence that AD pathology begins as many as 20 years before symptoms appear. These findings determined a new concept of AD, where the symptom of dementia represents the final part of the "con‐ tinuum" of AD. Recently, based on the new knowledge about AD, some disease modifying

Research grants were received from the Swedish council for working life and social re‐ search, the Swedish Research Council in Medicine and the Swedish Brain Power. This study

therapies have been developed and their results are eagerly awaited.

ly managed overtime.

22 Neurodegenerative Diseases

**9. Conclusions**

**Acknowledgements**

Weili Xu1,2\*, Anna Marseglia2,3, Camilla Ferrari2,4 and Hui-Xin Wang2

\*Address all correspondence to: weili.xu@ki.se

1 Department of Epidemiology, Tianjin Medical University, Tianjin, P.R. China

2 Aging Research Center, Karolinska Institutet-Stockholm University, Stockholm, Sweden

3 Institute of Neuroscience - Aging Branch, National Council of Research (CNR), Padua, Italy

4 Department of Neuroscience, University of Florence, Italy

#### **References**


tion-based cohorts. Neurologic Diseases in the Elderly Research Group. Neurology. 2000;54(11 Suppl 5):S4-9.

[23] Herrup K. Re-imagining Alzheimer's disease – an age-based hypothesis. J Neurosci.

Alzheimer's Disease: A Clinical Perspective http://dx.doi.org/10.5772/54539 25

[24] Huang Y, Weisgraber HK, Mucke L et al. Apolipoprotein E. Diversity of Cellular Ori‐ gins, Structural and Biophysical Properties, and Effects in Alzheimer's Disease. J Mol

[25] Ahn Jo S, Ahn K, Kim JH, Kang BH, Kim E, Jo I, et al. ApoE-epsilon 4-dependent as‐ sociation of the choline acetyltransferase gene polymorphisms (2384G>A and

[26] Cohn-Hokke PE, Elting MW, Pijnenburg YAL, van Swieten JC. Genetics of Dementia: Update and Guidelines for the Clinician. Am J Med Genet Part B. 2012; 159B:628–643

[27] Qiu C. Epidemiological findings of vascular risk factors in Alzheimer's disease: im‐ plications for therapeutic and preventive intervention. Expert Rev Neurother.

[28] Barnes DE, Haight TJ, Mehta KM et al. Secondhand smoke, vascular disease, and de‐ mentia incidence: findings from the cardiovascular health cognition study. Am J Epi‐

[29] Rusanen M, Kivipelto M, Quensenberry CP et al. Heavy smoking in midlife and long-term risk of Alzheimer disease and vascular dementia. Arch Intern Med.2011;

[30] Anttila T, Helkala EL, Viitanen M, Kareholt I, Fratiglioni L, Winblad B, et al. Alcohol drinking in middle age and subsequent risk of mild cognitive impairment and de‐ mentia in old age: a prospective population based study. BMJ. 2004; 329(7465):539.

[31] Qiu C, Winblad B, Fratiglioni L. The age-dependent relation of blood pressure to cog‐

[32] Qiu C, Winblad B, Marengoni A, Klarin I, Fastbom J, Fratiglioni L. Heart failure and risk of dementia and Alzheimer disease: a population-based cohort study. Arch In‐

[33] Purandare N, Burns A, Daly KJ, Hardicre J, Morris J, Macfarlane G, et al. Cerebral emboli as a potential cause of Alzheimer's disease and vascular dementia: case-con‐

[34] Beydoun MA, Lhotsky A, Wang Y, Dal Forno G, An Y, Metter EJ, et al. Association of adiposity status and changes in early to mid-adulthood with incidence of Alzheim‐

[35] Atti AR, Palmer K, Volpato S, Winblad B, De Ronchi D, Fratiglioni L. Late-life body mass index and dementia incidence: nine-year follow-up data from the Kungshol‐

nitive function and dementia. Lancet Neurol. 2005;4(8):487-99.

1882G>A) with Alzheimer's disease. Clin Chim Acta. 2006;368(1-2):179-82

2010; 30(50):16755-62

2011;11(11):1593-607.

171(4):333-339

demiol. 2010; 1: 171 (3): 292-302

tern Med. 2006;166(9):1003-8.

trol study. BMJ. 2006; 332(7550):1119-24.

er's disease. Am J Epidemiol. 2008;168(10):1179-89.

men Project. J Am Geriatr Soc. 2008; 56(1):111-6.

Neurosci 2004; 23(3):189-204


[23] Herrup K. Re-imagining Alzheimer's disease – an age-based hypothesis. J Neurosci. 2010; 30(50):16755-62

tion-based cohorts. Neurologic Diseases in the Elderly Research Group. Neurology.

[7] Plassman BL, Langa KM, Fisher GG, Heeringa SG, Weir DR, Ofstedal MB, et al. Prev‐ alence of dementia in the United States: the aging, demographics, and memory

[8] Ferri CP, Prince M, Brayne C, Brodaty H, Fratiglioni L, Ganguli M, et al. Global prev‐ alence of dementia: a Delphi consensus study. Lancet. 2005;366(9503):2112-7.

[9] Brookmeyer R, Johnson E, Ziegler-Graham K, Arrighi HM. Forecasting the global

[10] Ballard C, Gauthier S, Corbett A et al. Alzheimer's disease. Lancet 2011; 377:

[12] Schaeffer EL, Figueiro´ IM, Wagner F, et al. Insights into Alzheimer disease patho‐ genesis from studies in transgenic animal models. CLINICS 2011;66(S1):45-54

[13] Price DL, Tanzi RE, Borchelt DR, Sisodia SS et al. Alzheimer's disease: genetic studies

[14] Hardy JA, Higgins GA. Alzheimer's disease: the amyloid cascade hypothesis. Sci‐

[15] Bettens K, Sleegers K, Van Broeckhoven C. Current status on Alzheimer disease mo‐ lecular genetics; from past, to present, to future. Hum Mol Genet 2010; 19: R4–R11

[16] Pimplikar SW, Nixon RA, Robakis NK. Et al. Amyloid-independent mechanisms in

[17] McGeer PL, McGeer EG, Yasojima K et al. Alzheimerndisease and neuroinflamma‐

[18] Guglielmotto M, Tamagno E, Danni O et al. Oxidative stress and hypoxia contribute to Alzheimer's disease pathogenesis: two sides of the same coin. Scientific World

[19] Yang Y. Mufson EJ, Herrup K et al. Neuronal cell death is preceded by cell cycle events at all stages of Alzheimer's disease. J Neurosci. 2003; 23: 2557-2563.

[20] de la Torre JC. Critically attained threshold of cerebral hypoperfusion: the CATCH hypothesis of Alzheimer's pathogenesis. Neurobiol Aging 2000; 21: 331-342.

[21] de la Torre JC, Mussivand T. Can disturbed brain microcirculation cause Alzheimer's

[22] de la Torre JC. Three Postulates to Help Identify the Cause of Alzheimer's Disease.

Alzheimer's disease pathogenesis. J Neurosci. 2010 ;30(45):14946-54

tion. J Neural Transm Suppl. 2000; 59: 53-57

disease? Neurol Res 1993; 15:146-153.

Journal of Alzheimer's Disease. 2011; 24: 657–668

burden of Alzheimer's disease. Alzheimers Dement. 2007;3(3):186-91.

[11] Hernandez F, Avila J. Tauopathies. Cell Mol Life Sci. 2007;64(17):2219-33.

and transgenic models. Annu Rev Genet.19998; 32:461– 493.

2000;54(11 Suppl 5):S4-9.

1019-1031

24 Neurodegenerative Diseases

ence. 1992;256:184-185

Journal. 2009; 9:781-791.

study. Neuroepidemiology. 2007;29(1-2):125-32.


[36] Hassing LB, Dahl AK, Thorvaldsson V, Berg S, Gatz M, Pedersen NL, et al. Over‐ weight in midlife and risk of dementia: a 40-year follow-up study. Int J Obes (Lond). 2009; 33(8):893-8.

[50] Hooshmand B, Solomon A, Kareholt I, Leiviska J, Rusanen M, Ahtiluoto S, et al. Ho‐ mocysteine and holotranscobalamin and the risk of Alzheimer disease: a longitudinal

Alzheimer's Disease: A Clinical Perspective http://dx.doi.org/10.5772/54539 27

[51] Barron AM, Pike CJ. Sex hormones, aging, and Alzheimer's disease. Front Biosci

[52] Hogervorst E, Yaffe K, Richards M, Huppert FA. Hormone replacement therapy to maintain cognitive function in women with dementia. Cochrane Database Syst Rev.

[53] Callahan CM, Hendrie HC, Tierney WM. Documentation and evaluation of cognitive impairment in elderly primary care patients. Ann Intern Med. 1995; 122(6):422-9. [54] Petersen RC, Doody R, Kurz A, Mohs RC, Morris JC, Rabins PV, et al. Current con‐

[55] Sperling RA, Aisen PS, Beckett LA., et al. Toward defining the preclinical stages of Alzheimer's disease: Recommendations from the National Institute on Aging-Alz‐ heimer's Association workgroups on diagnostic guidelines for Alzheimer's disease.

[56] Albert MS, DeKosky ST, Dickson D, et al. The diagnosis of mild cognitive impair‐ ment due to Alzheimer's disease: Recommendations from the National Institute on Aging- Alzheimer's Association workgroups on diagnostic guidelines for Alzheim‐

[57] Taler V, Phillips NA. Language performance in Alzheimer's disease and mild cogni‐ tive impairment: a comparative review. J Clin Exp Neuropsychol. 2008; 30(5):501-56.

[58] Mohs RC. The clinical syndrome of Alzheimer's disease: aspects particularly relevant

[59] The Swedish Council on Technology Assessment in Health Care. Dementia- Etiology

[60] Vellas B, Hausner L, Frölich L et al. Progression of Alzheimer disease in Europe: Da‐

[61] McKhann, G., Drachman, D., Folstein, et al. Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease. Neurology. 1984; 34,

[62] Knopman DS, DeKosky ST, Cummings JL, Chuit H, Corey-Bloom J, Relkin N, et al. Practice parameter: diagnosis of dementia (an evidence-based review). Neurology.

ta from the European ICTUS study. Curr Alzheimer Res. 2012 Jun 26

cepts in mild cognitive impairment. Arch Neurol. 2001;58(12):1985-92.

study. Neurology. 2010; 75(16):1408-14.

Alzheimers Dement. 2011; 7(3): 280–292.

er's disease. Alzheimers Dement. 2011; 7(3): 270–279.

to clinical trials. Genes Brain Behav. 2005;4(3):129-33.

and Epidemiology: A Systematic Review. 2008

(Elite Ed). 2012; 4:976-97.

2009(1):CD003799.

939-944.

2001; 56:1143–53.


[50] Hooshmand B, Solomon A, Kareholt I, Leiviska J, Rusanen M, Ahtiluoto S, et al. Ho‐ mocysteine and holotranscobalamin and the risk of Alzheimer disease: a longitudinal study. Neurology. 2010; 75(16):1408-14.

[36] Hassing LB, Dahl AK, Thorvaldsson V, Berg S, Gatz M, Pedersen NL, et al. Over‐ weight in midlife and risk of dementia: a 40-year follow-up study. Int J Obes (Lond).

[37] Solomon A, Kivipelto M. Cholesterol-modifying strategies for Alzheimer's disease.

[38] Xu W, Qiu C, Winblad B, Fratiglioni L. The effect of borderline diabetes on the risk of

[39] Lu FP, Lin KP, Kuo HK. Diabetes and the risk of multi-system aging phenotypes: a

[40] Xu W, Caracciolo B, Wang HX, Winblad B, Backman L, Qiu C, et al. Accelerated pro‐ gression from mild cognitive impairment to dementia in people with diabetes. Dia‐

[41] Tan ZS, Beiser AS, Vasan RS, Roubenoff R, Dinarello CA, Harris TB, et al. Inflamma‐ tory markers and the risk of Alzheimer disease: the Framingham Study. Neurology.

[42] Sundelof J, Kilander L, Helmersson J, Larsson A, Ronnemaa E, Degerman-Gunnars‐ son M, et al. Systemic inflammation and the risk of Alzheimer's disease and demen‐

[43] Van Dam F, Van Gool WA. Hyperhomocysteinemia and Alzheimer's disease: A sys‐

[44] Ownby RL, Crocco E, Acevedo A, John V, Loewenstein D. Depression and risk for Alzheimer disease: systematic review, meta-analysis, and metaregression analysis.

[45] Raffaitin C, Gin H, Empana JP, Helmer C, Berr C, Tzourio C, et al. Metabolic syn‐ drome and risk for incident Alzheimer's disease or vascular dementia: the Three-City

[46] Rovio S, Kareholt I, Helkala EL, Viitanen M, Winblad B, Tuomilehto J, et al. Leisuretime physical activity at midlife and the risk of dementia and Alzheimer's disease.

[47] Karp A, Andel R, Parker MG, Wang HX, Winblad B, Fratiglioni L. Mentally stimulat‐ ing activities at work during midlife and dementia risk after age 75: follow-up study

[48] Paillard-Borg S, Fratiglioni L, Winblad B, Wang HX. Leisure activities in late life in relation to dementia risk: principal component analysis. Dement Geriatr Cogn Dis‐

[49] Scarmeas N, Stern Y, Tang MX, Mayeux R, Luchsinger JA. Mediterranean diet and

risk for Alzheimer's disease. Ann Neurol. 2006 Jun;59(6):912-21

from the Kungsholmen Project. Am J Geriatr Psychiatry. 2009;17(3):227-36.

tia: a prospective population-based study. J Alzheimers Dis. 2009; 18(1):79-87.

tematic review. Arch Gerontol Geriatr. 2009;48(3):425-30.

Arch Gen Psychiatry. 2006; 63(5):530-8.

Study. Diabetes Care. 2009; 32(1):169-74.

Lancet Neurol. 2005;4(11):705-11.

ord. 2009;28(2):136-44

dementia and Alzheimer's disease. Diabetes. 2007; 56(1):211-6.

systematic review and meta-analysis. PLoS One. 2009;4(1):e4144.

2009; 33(8):893-8.

26 Neurodegenerative Diseases

betes. 2010; 59(11):2928-35.

2007; 68(22):1902-8.

Expert Rev Neurother. 2009; 9(5):695-709.


[63] Katz S, Ford AB, Moskowitz RW, Jackson BA, Jaffe MW. Studies of Illness in the Aged. The Index of Adl: A Standardized Measure of Biological and Psychosocial Function. JAMA. 1963; 185:914-9.

[76] Bruno Dubois B, Feldman HH, Jacova C, DeKosky ST et al. Research criteria for the diagnosis of Alzheimer's disease: revising the NINCDS–ADRDA criteria. Lancet

Alzheimer's Disease: A Clinical Perspective http://dx.doi.org/10.5772/54539 29

[77] Coleman RE. Positron emission tomography diagnosis of Alzheimer's disease. Neu‐

[78] Zhang. S, Han D, Tan X et al. Diagnostci accuracy of 18F-FDG and 11C-PIB-PET for predict of short-term conversion to Alzheimer´s disease in subjects with mild cogni‐

[79] Ringman JM, Younkin SG, Pratico D, Seltzer W, Cole GM, Geschwind DH, et al. Bio‐ chemical markers in persons with preclinical familial Alzheimer disease. Neurology.

[80] Nordberg A, Carter SF, Rinne J, Drzezga A, Brook DJ et al. A European multicentre PET study of fibrillar amyloid in Alzheimer's disease. Eur J Nucl Med Mol Imaging.

[81] Sunderland, T., Linker, G., Mirza, N., Putnam, K.T., Friedman, D.L.,Kimmel, L.H., Bergeson, J., Manetti, G.J., Zimmermann, M., Tang, B., Bartko, J.J., Cohen, R.M. De‐ creased beta-amyloid1-42 and increased tau levels in cerebrospinal fluid of patients

[82] Hesse, C., Rosengren, L., Andreasen, N., Davidsson, P., Vanderstichele,H., Vanme‐ chelen, E., Blennow, K. et al. Transient increase in total tau but not phospho-tau in human cerebrospinal fluid after acute stroke.Neurosci. Lett. 2001; 297:187–190. [83] Blennow K, Zetterberg H and Fagan AM. Fluid Biomarkers in Alzheimer Disease.

[84] Snider BJ, Fagan AM, Roe C, Shah AR, Grant EA, Xiong C, Morris JC, Holtzman DM. Cerebrospinal fluid biomarkers and rate of cognitive decline in very mild dementia

[85] Moonis M, Swearer JM, Dayaw MP, St George-Hyslop P, Rogaeva E, Kawarai T, et al. Familial Alzheimer disease: decreases in CSF Abeta42 levels precede cognitive de‐

[86] Wu G, Sankaranarayanan S, Wong J, Tugusheva K et al. Characterization of Plasma b-Secretase (BACE1) Activity and Soluble Amyloid Precursor Proteins as Potential Biomarkers for Alzheimer's Disease . Journal of Neuroscience Research. 2012. Jul 16.

[87] Mayeux R, Honig LS, Tang MX, Manly J, Stern Y, Schupf N, Mehta PD. Plasma Ab40 and Ab42 and Alzheimer's disease: relation to age, mortality, and risk. Neurology

Neurol 2007; 6: 734–46

2008; 71:85–92.

2012. Sept 8.

roimaging Clin N Am. 2005; 15: 837–46.

tive impairment. Int J Clin Pract. 2012; 66 (2): 185-198

with Alzheimer disease. JAMA.2003; 289: 2094–2103

Cold Spring Harb Perspect Med 2012; 2:a006221

cline. Neurology. 2005; 65:323–5.

2003; 61:1185–1190.

of the Alzheimer type. Arch Neurol. 2009; 66: 638–645.


[76] Bruno Dubois B, Feldman HH, Jacova C, DeKosky ST et al. Research criteria for the diagnosis of Alzheimer's disease: revising the NINCDS–ADRDA criteria. Lancet Neurol 2007; 6: 734–46

[63] Katz S, Ford AB, Moskowitz RW, Jackson BA, Jaffe MW. Studies of Illness in the Aged. The Index of Adl: A Standardized Measure of Biological and Psychosocial

[64] Lawton MP, Brody EM. Assessment of older people: self-maintaining and instrumen‐

[65] Bucks RS, Ashworth DL, Wilcock GK, Siegfried K. Assessment of activities of daily living in dementia: development of the Bristol Activities of Daily Living Scale. Age

[66] Folstein MF, Folstein SE, McHugh PR. "Mini-mental state". A practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res. 1975; 12(3):

[67] Buschke H, Kuslansky G, Katz M, Stewart WF, Sliwinski MJ, Eckholdt HM, et al. Screening for dementia with the memory impairment screen. Neurology. 1999; 52(2):

[68] Price CC, Cunningham H, Coronado N, Freedland A, Cosentino S, Penney DL, et al. Clock drawing in the Montreal Cognitive Assessment: recommendations for demen‐

[69] Damian AM, Jacobson SA, Hentz JG, Belden CM, Shill HA, Sabbagh MN, et al. The Montreal Cognitive Assessment and the mini-mental state examination as screening instruments for cognitive impairment: item analyses and threshold scores. Dement

[70] Lezack M, Howieson DB, Bigler ED, Tranel D. Neuropsychological assessment. 5th

[71] Hort J, O´Brien JT, Gainotti G, Pirttila T, Popescu BO et al. EFNS guidelines for the diagnosis and managment f Azlehimer´s disease. European Journal of Neurology.

[72] Wright CF, Hall A, Matthews FE and Brayne C. biomarkers, dementia and Public

[73] Braak H, Braak E. Morphological criteria for recognition of Alzheimer´s disease and the distribution pattern of cortical changes related to this disorder. Neurobiol Aging.

[74] Scheltens P. Imaging in Alzheimer´s disease. Dialogues Clin Neurosci. 2009; 11: 191–

[75] Sluimer JD, van der Flier WM, Karas GB, et al. Whole-brain atrophy rate and cogni‐ tive decline: longitudinal MR study of memory clinic patients. Radiology. 2008;

tia assessment. Dement Geriatr Cogn Disord. 2011; 31(3):179-87.

Geriatr Cogn Disord. 2011; 31(2):126-31.

2010; 17:1236-1248

1994; 15:355-356

248:590-598.

199.

ed. New York: Oxford University press; 2012.

Health. Ann. N.Y. Acad. Sci. 2009; 11880:11-19

tal activities of daily living. Gerontologist. 1969; 9(3):179-86.

Function. JAMA. 1963; 185:914-9.

Ageing. 1996; 25(2):113-20.

189-98.

28 Neurodegenerative Diseases

231-8.


[88] Pomara N, Willoughby LM, Sidtis JJ, Mehta PD. Selective reductions in plasma Ab1– 42 in healthy elderly subjects during longitudinal followup: a preliminary report. Am J Geriatr Psychiatry 2005;13:914–917

[103] Hassan A, Whitwell JL, and Josephs KA. The corticobasal syndrome–Alzheimer's

Alzheimer's Disease: A Clinical Perspective http://dx.doi.org/10.5772/54539 31

[104] Sadowsky CH, and Galvin JE. Guidelines for the Management of Cognitive and Be‐ havioral Problems in Dementia J Am Board Fam Med. 2012; 25:350 –366.

[105] Birks J. Cholinesterase inhibitors for Alzheimer's disease. Cochrane Database Syst

[106] Trinh NH, Hoblyn J, Mohanty S, Yaffe K. et al. Efficacy of cholinesterase inhibitors in the treatment of neuropsychiatric symptoms and functional impairment in Alzheim‐

[107] Winblad B and Poritis N. Memantine in severe dementia: results of the 9M-BEST study (benefit and efficacy in severely demented patients during treatment with

[108] Tariot PN, Farlow MR, Grossberg GT, Graham SM, McDonald S, Gergel I. Meman‐ tine treatment in patients with moderate to severe Alzheimer disease already receiv‐

[109] Atri A, Shaughnessy LW, Locascio JJ, Growdon JH. Long-term course and effective‐ ness of combination therapy in Alzheimer disease. Alzheimer Dis. Assoc. Disord.

[110] Howard R, McShane R, Lindesay J et al. Donepezil and memantine for moderate-to-

[111] Landreth G, Jiang Q, Mandrekar S, Heneka M. PPARgamma agonists as therapeutics for the treatment of Alzheimer's disease. Neurotherapeutics . 2008; 5(3), 481–489 [112] Gold M, Alderton C, Zvartau-Hind ME et al. Effects of rosiglitazone as monotherapy in apoE4-stratified subjects with mild-to-moderate Alzheimer's disease. Alzheimers

[113] Henley DB, May PC, Dean RA, Siemers ER. Development of semagacestat (LY450139), a functional gamma-secretase inhibitor, for the treatment of Alzheimer's

[114] Green RC, Schneider LS, Amato DA et al. Effect of tarenflurbil on cognitive decline and activities of daily living in patients with mild Alzheimer disease: a randomized

[115] Anne Corbett1, Jessica Smith1 and Clive Ballard. New and emerging treatments for

[116] Wilcock DM, Colton CA. Anti-Ab immunotherapy in Alzheimer's disease; relevance of transgenic mouse studies to clinical trials J. Alzheimers Dis. 2008;15(4): 555–569.

severe Alzheimer's disease. N. Engl. J. Med. 2012; 366, 893–903.

disease. Expert Opin. Pharmacother. 2009; 10(10): 1657–1664.

Alzheimer's disease Expert Rev. Neurother. 2012; 12(5):535–543.

controlled trial. JAMA. 2009; 302(23): 2557–2564.

ing donepezil: a randomized controlled trial. JAMA 2004; 291(3), 317–324.

disease conundrum Expert Rev Neurother. 2011; 11(11): 1569–1578.

er disease: a meta-analysis. JAMA 2003; 289:210–6.

memantine). Int. J. Geriat. Psychiatry, 1999; 14:135-146

Rev 2006;(1): CD005593

2008; 22(3), 209–221.

Dement. 2009; 5(4), 86


[103] Hassan A, Whitwell JL, and Josephs KA. The corticobasal syndrome–Alzheimer's disease conundrum Expert Rev Neurother. 2011; 11(11): 1569–1578.

[88] Pomara N, Willoughby LM, Sidtis JJ, Mehta PD. Selective reductions in plasma Ab1– 42 in healthy elderly subjects during longitudinal followup: a preliminary report.

[89] Galton CJ, Patterson K, Xuereb JH and Hodges JR. Atypical and typical presentations of Alzheimer's disease: A clinical, neuropsychological, neuroimaging and pathologi‐

[90] Alladi S, Xuereb J, Bak T, et al. Focal cortical presentations of Alzheimer's disease.

[91] von Gunten A, Bouras C, Kovari E, Giannakopoulos P, Hof PR. Neural substrates of cognitive and behavioral deficits in atypical Alzheimer's disease. Brain Res Rev 2006;

[92] Portet F, Dauvilliers Y, Campion D, Raux G, Hauw JJ, Lyon-Caen O, et al. Very early onset AD with a de novo mutation in the presenilin 1 gene (Met 233 Leu). Neurology

[93] Mendez MF, McMurtray A. Frontotemporal dementia-like phenotypes associated with presenilin-1 mutations. Am J Alzheimer's Dis Other Demen 2006; 214: 281–6.

[94] Johnson JK, Head E, Kim R, Starr A, Cotman CW. Clinical and pathological evidence

[95] Mesulam MM. Slowly progressive aphasia without generalized dementia. Ann Neu‐

[96] Gorno-Tempini ML, Hillis AE, Weintraub S, et al. Classification of primary progres‐

[97] Ahmed S, de Jager CA, Haigh AF, Garrard P et al. Logopenic aphasia in Alzheimer's disease: clinical variant or clinical feature? J Neurol Neurosurg Psychiatry. 2012;

[98] Renner JA, Burns JM, Hou CE, McKeel DW Jr, Storandt M, Morris JC. Progressive posterior cortical dysfunction: a clinicopathologic series. Neurology 2004; 63: 1175–

[99] McMonagle P, Deering F, Berliner Y, Kertesz A. The cognitive profile of posterior

[100] Kas A, de Souza LC, Samri D, et al. Neural correlates of cognitive impairment in pos‐

[101] Crutch SJ, Lehmann M, Schott JM, et al. Posterior cortical atrophy. Lancet Neurol.

[102] Snowden JS, Stopford CL, Julien CL et al.Cognitive phenotypes in Alzheimer´disease

for a frontal variant of Alzheimer disease. Arch Neurol 1999; 56: 1233–9.

sive aphasia and its variants. Neurology 2011; 76:1006e14.

cortical atrophy. Neurology 2006; 66: 331–38.

terior cortical atrophy. Brain 2011; 134: 1464–78.

and genetic risk. Cortex. 2007; 43:835-845

Am J Geriatr Psychiatry 2005;13:914–917

Brain 2007; 130 (10):2636–2645.

51: 176–211

30 Neurodegenerative Diseases

2003; 61: 1136–7.

rol 1982;11:592e8.

80.

10.1136/jnnp-2012-302798

2012 ; 11(2):170-8. Review

cal study of 13 cases. Brain. 2000; 123: 484-498.


[117] Valenzuela MJ, Sachdev P. Brain reserve and dementia: a systematic review. Psychol Med. 2006;36(4):441-54.

[130] Bohlmeijer E, Valenkamp M, Westerhof G, Smit F, Cuijpers P. Creative reminiscence as an early intervention for depression: results of a pilot project. Aging Ment Health.

Alzheimer's Disease: A Clinical Perspective http://dx.doi.org/10.5772/54539 33

[131] Katsuaki T, Yukiko Y, Yoshio K, Kazuki S, Ayako M, Ryuhei N, et al. Improved cog‐ nitive function, mood and brain blood flow in single photon emission computed to‐ mography following individual reminiscence therapy in an elderly patient with

Alzheimer's disease. Geriatrics & Gerontology International. 2007; 7(3):305-9.

[132] Afonso R, Bueno B. [Reminiscence with different types of autobiographical memo‐ ries: Effects on the reduction of depressive symptomatology in old age]. Psicothema.

[133] Neal M, Briggs M. Validation therapy for dementia. Cochrane Database Syst Rev.

[134] Spector A, Orrell M, Davies S, Woods B. Reality orientation for dementia. Cochrane

[135] Spector A, Orrell M, Davies S, Woods B. WITHDRAWN: Reality orientation for de‐

[136] Savorani G, Chattat R, Capelli E, Vaienti F, Giannini R, Bacci M, et al. Immediate ef‐ fectiveness of the "new identity" reality orientation therapy (ROT) for people with dementia in a geriatric day hospital. Arch Gerontol Geriatr Suppl. 2004; 9:359-64. [137] Spector A, Thorgrimsen L, Woods B, Royan L, Davies S, Butterworth M, et al. Effica‐ cy of an evidence-based cognitive stimulation therapy programme for people with

dementia: randomised controlled trial. Br J Psychiatry 2003; 183:248-54.

2005; 9(4):302-4.

2010; 22(2):213-20.

2003; 3:CD001394.

Database Syst Rev. 2000; 4:CD001119.

mentia. Cochrane Database Syst Rev. 2000; 3:CD001119.


[130] Bohlmeijer E, Valenkamp M, Westerhof G, Smit F, Cuijpers P. Creative reminiscence as an early intervention for depression: results of a pilot project. Aging Ment Health. 2005; 9(4):302-4.

[117] Valenzuela MJ, Sachdev P. Brain reserve and dementia: a systematic review. Psychol

[118] Stern Y. What is cognitive reserve? Theory and research application of the reserve

[119] Lyketsos CG, Colenda CC, Beck C, Blank K, Doraiswamy MP, Kalunian DA, et al. Position statement of the American Association for Geriatric Psychiatry regarding principles of care for patients with dementia resulting from Alzheimer disease. Am J

[120] De Vreese LP, Neri M, Fioravanti M, Belloi L, Zanetti O. Memory rehabilitation in Alzheimer's disease: a review of progress. Int J Geriatr Psychiatry. 2001; 8:794-809.

[121] Clare L, Woods RT, Moniz Cook ED, Orrell M, Spector A. Cognitive rehabilitation and cognitive training for early-stage Alzheimer's disease and vascular dementia.

[122] Wilson BA. Neuropsychological rehabilitation. Annu Rev Clin Psychol. 2008;

[123] Yamaguchi H, Maki Y, Yamagami T. Overview of non-pharmacological intervention for dementia and principles of brain-activating rehabilitation. Psychogeriatrics.

[124] Rabins PV, Blacker D, Rovner BW, Rummans T, Schneider LS, Tariot PN, et al. Amer‐ ican Psychiatric Association practice guideline for the treatment of patients with Alz‐ heimer's disease and other dementias. Second edition. Am J Psychiatry. 2007; 164(12

[125] Doody RS, Stevens JC, Beck C, Dubinsky RM, Kaye JA, Gwyther L, et al. Practice pa‐ rameter: management of dementia (an evidence-based review). Report of the Quality Standards Subcommittee of the American Academy of Neurology. Neurology. 2001;

[126] Hermans DG, Htay UH, McShane R. Non-pharmacological interventions for wander‐ ing of people with dementia in the domestic setting. Cochrane Database Syst Rev.

[127] Robinson L, Hutchings D, Dickinson HO, Corner L, Beyer F, Finch T, et al. Effective‐ ness and acceptability of non-pharmacological interventions to reduce wandering in

[128] Tadaka E, Kanagawa K. Effects of reminiscence group in elderly people with Alz‐ heimer disease and vascular dementia in a community setting. Geriatrics & Gerontol‐

[129] Bohlmeijer E, Smit F, Cuijpers P. Effects of reminiscence and life review on late-life depression: a meta-analysis. Int J Geriatr Psychiatry. 2003; 18(12):1088-94.

dementia: a systematic review. Int J Geriatr Psychiatry. 2007; 22(1):9-22.

Med. 2006;36(4):441-54.

32 Neurodegenerative Diseases

4:141-62.

2010;10(4):206-13.

Suppl):5-56.

56(9):1154-66.

2007(1):CD005994.

ogy International 2007; 7(2):167-73.

concept. J Int Neuropsychol Soc. 2002;8(3):448-60.

Cochrane Database Syst Rev. 2003; 4:CD003260.

Geriatr Psychiatry. 2006;14(7):561-72.


**Chapter 2**

**Late-Onset Alzheimer's Disease: Risk Factors,**

Marisol Herrera-Rivero

http://dx.doi.org/10.5772/53775

**1. Introduction**

Additional information is available at the end of the chapter

outcomes as well as the implications for quality of life.

**Clinical Diagnosis and the Search for Biomarkers**

Even with the progress that has been made in the past years on our understanding of Alz‐ heimer's disease (AD) we don't seem to be closer to finding a cure than we were before. AD is a complex disorder wherein the pathophysiology is influenced by a great number of environmental and genetic factors, thus making it difficult to uncover the triggering events underlying disease's onset. In this chapter, we will first discuss some common conditions within the general population that have been associated with an increased risk to develop lateonset Alzheimer's disease (LOAD), such as hypertension, type 2 diabetes mellitus and high serum cholesterol and triglycerides levels, and the way they might be contributing to cognitive decline; depression and traumatic brain injuries, amongst others, will also be boarded.

Misdiagnosis is a frequent issue with important repercussions not only for the patient's condition but also for family members. For this reason, in the second part we will review the main basic aspects that should be involved in AD diagnosis, from laboratory tests to neuroi‐ maging technologies, highlighting the importance of seeking a differential diagnosis of dementias in the elderly and the crucial role an accurate diagnosis may play in therapeutic

Early diagnosis of Alzheimer's may be the best tool we could find, as for now, to improve treatment outcomes by slowing the disease progression rate. This idea has led an important number of scientists around the globe to the search for biomarkers using a wide variety of approaches in the brain, cerebrospinal fluid (CSF) and blood. On this matter, we will briefly comment on our preliminary study using lymphocytes from cognitively healthy people and neuropsychological patients affected not only with LOAD but other types of neuropathologies as well to analyse in blood cells the expression of the main genes directly related to AD in brain cells. This study included 72 subjects in whom the expression of the microtubule-associated

and reproduction in any medium, provided the original work is properly cited.

© 2013 Herrera-Rivero; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

### **Late-Onset Alzheimer's Disease: Risk Factors, Clinical Diagnosis and the Search for Biomarkers**

Marisol Herrera-Rivero

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53775

### **1. Introduction**

Even with the progress that has been made in the past years on our understanding of Alz‐ heimer's disease (AD) we don't seem to be closer to finding a cure than we were before. AD is a complex disorder wherein the pathophysiology is influenced by a great number of environmental and genetic factors, thus making it difficult to uncover the triggering events underlying disease's onset. In this chapter, we will first discuss some common conditions within the general population that have been associated with an increased risk to develop lateonset Alzheimer's disease (LOAD), such as hypertension, type 2 diabetes mellitus and high serum cholesterol and triglycerides levels, and the way they might be contributing to cognitive decline; depression and traumatic brain injuries, amongst others, will also be boarded.

Misdiagnosis is a frequent issue with important repercussions not only for the patient's condition but also for family members. For this reason, in the second part we will review the main basic aspects that should be involved in AD diagnosis, from laboratory tests to neuroi‐ maging technologies, highlighting the importance of seeking a differential diagnosis of dementias in the elderly and the crucial role an accurate diagnosis may play in therapeutic outcomes as well as the implications for quality of life.

Early diagnosis of Alzheimer's may be the best tool we could find, as for now, to improve treatment outcomes by slowing the disease progression rate. This idea has led an important number of scientists around the globe to the search for biomarkers using a wide variety of approaches in the brain, cerebrospinal fluid (CSF) and blood. On this matter, we will briefly comment on our preliminary study using lymphocytes from cognitively healthy people and neuropsychological patients affected not only with LOAD but other types of neuropathologies as well to analyse in blood cells the expression of the main genes directly related to AD in brain cells. This study included 72 subjects in whom the expression of the microtubule-associated

© 2013 Herrera-Rivero; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

protein tau (MAPT), the amyloid precursor protein (APP), nicastrin (NCSTN), a component of the γ-secretase, and β-secretase (beta-site APP cleaving enzyme 1, BACE1) were analysed through 4 groups of cognitively healthy individuals ranging from 25 to 92 years of age and in conditions such as LOAD, vascular dementia (VaD) and Parkinson's disease (PD) in elderly individuals. We also aimed to discover the manner in which the expression of these genes might be affected by conditions associated with increased risk for LOAD such as hypertension and glucose, cholesterol and triglycerides serum levels.

**d.** Depression.

**e.** Sleep.

AD patients.

**g.** Diet.

**2.2. Genetic**

**h.** Alcohol and smoking.

**f.** Education.

risk to develop LOAD [8].

It has been subject for debate whether depression might be an early manifestation of AD or a contributing factor for development of the disease. If one thing is sure is that depression contributes to cognitive decline and may by itself cause a condition known as "depressive pseudo-dementia" which can frequently evolve to a true dementia (mainly AD) when not treated, particularly in the elderly [7]. A history of major depressive disorder or susceptibility to depression during an individual's lifetime has therefore been associated with an increased

Late–Onset Alzheimer's Disease: Risk Factors, Clinical Diagnosis and the Search for Biomarkers

http://dx.doi.org/10.5772/53775

37

Chronic sleep deprivation and other sleep disorders have a negative effect on cognitive function primarily due to the role sleep plays in memory and learning processes and possibly in synaptic plasticity, although it is also believed there is an increase in Aβ during the waking hours [9]. History of sleep disorders has thus been linked to an increase in the risk to develop LOAD; furthermore, sleep disorders occurring as part of the aging process and those related to coexisting medical conditions contribute to cognitive decline and behavioural problems in

People with a lower level of education show an increased risk to develop LOAD. This might be due to the relative lack of constant stimulation of cognitive processes (such as learning and memory) compared to individuals with a higher education degree. In fact, cognitive stimula‐ tion therapies are widely used for helping the treatment of people with dementia [10].

It is well known that diet affects every aspect of an individual's health. A number of medical conditions associated with an increased risk for developing AD have a strong nutritional background as cholesterol, glucose and vitamin B12 levels, just to mention a few, importantly

It remains unclear whether alcohol and smoking increase the risk for AD as their association has been inconsistent. Although smoking is a strong risk factor for vascular disease, and the association of the latter with AD has been established, the possibility of a neuroprotective effect of nicotine by a smoking-induced increase in nicotinic receptors exists [3]. Similar is the case of alcohol where wine presents protective effects because of its antioxidant contents while

Medical conditions and lifestyle in midlife, especially when a group of these factors coexist in an individual creating a synergistic effect, can contribute to cellular and molecular alterations ultimately leading to the hallmark pathological processes of AD later in life. Although

associations between other types of alcohol and risk of AD remain controversial.

influence the risk for dementia [11], particularly in old age.

### **2. Risk factors for AD**

A wide variety of factors have been associated with an increased risk to develop LOAD; nevertheless, a number of these associations remain controversial. Age is the main risk factor to develop AD of the sporadic type and is only followed by apolipoprotein E (ApoE) genotype. However, other genetic and environmental conditions have also been proven to influence the risk of developing the disease and the rate of cognitive decline which affects disease progres‐ sion. Amongst these are found cardiovascular risk factors, type 2 diabetes mellitus, sleep disorders, depression, education, smoking, alcohol, traumatic brain injury (TBI) and several single nucleotide polymorphisms (SNPs) in a growing list of candidate genes.

#### **2.1. Environmental**

**a.** Cardiovascular risk factors.

Within the risk factors for vascular disease hypertension and high cholesterol levels are most importantly associated with LOAD. Various longitudinal studies have shown an association between a diagnosis of hypertension in midlife and the development of AD in late life [1,2]. On this regard, hypertension may cause cerebrovascular disease which would contribute to accelerate AD processes, even when there are reports on a decrease in blood pressure during the disease course probably due to affection of the brain regions implicated in its regulation [3]. Cholesterol and its metabolism is associated with AD by several proposed mechanisms including regulation of beta-amyloid (Aβ) generation by an increase in β-secretase activity, APP membrane localization and cleavage variations by changes in cholesterol rich lipid rafts and ApoE-regulated interactions between cholesterol and Aβ.

**b.** Type 2 diabetes mellitus.

Diabetes and even high glucose levels in the absence of a diabetes diagnosis are associated with an increased risk to develop AD involving various mechanisms, primarily being changes in the blood brain barrier (BBB) and the transport within cerebral small vessels [4].

**c.** Traumatic brain injury.

TBI has been associated with an increased risk to develop AD by longitudinal and retrospective studies, suggesting an effect between severity and repetitive episodes of TBI particularly by an increase in amyloid deposition [5, 6].

#### **d.** Depression.

protein tau (MAPT), the amyloid precursor protein (APP), nicastrin (NCSTN), a component of the γ-secretase, and β-secretase (beta-site APP cleaving enzyme 1, BACE1) were analysed through 4 groups of cognitively healthy individuals ranging from 25 to 92 years of age and in conditions such as LOAD, vascular dementia (VaD) and Parkinson's disease (PD) in elderly individuals. We also aimed to discover the manner in which the expression of these genes might be affected by conditions associated with increased risk for LOAD such as hypertension

A wide variety of factors have been associated with an increased risk to develop LOAD; nevertheless, a number of these associations remain controversial. Age is the main risk factor to develop AD of the sporadic type and is only followed by apolipoprotein E (ApoE) genotype. However, other genetic and environmental conditions have also been proven to influence the risk of developing the disease and the rate of cognitive decline which affects disease progres‐ sion. Amongst these are found cardiovascular risk factors, type 2 diabetes mellitus, sleep disorders, depression, education, smoking, alcohol, traumatic brain injury (TBI) and several

Within the risk factors for vascular disease hypertension and high cholesterol levels are most importantly associated with LOAD. Various longitudinal studies have shown an association between a diagnosis of hypertension in midlife and the development of AD in late life [1,2]. On this regard, hypertension may cause cerebrovascular disease which would contribute to accelerate AD processes, even when there are reports on a decrease in blood pressure during the disease course probably due to affection of the brain regions implicated in its regulation [3]. Cholesterol and its metabolism is associated with AD by several proposed mechanisms including regulation of beta-amyloid (Aβ) generation by an increase in β-secretase activity, APP membrane localization and cleavage variations by changes in cholesterol rich lipid rafts

Diabetes and even high glucose levels in the absence of a diabetes diagnosis are associated with an increased risk to develop AD involving various mechanisms, primarily being changes

TBI has been associated with an increased risk to develop AD by longitudinal and retrospective studies, suggesting an effect between severity and repetitive episodes of TBI particularly by

in the blood brain barrier (BBB) and the transport within cerebral small vessels [4].

single nucleotide polymorphisms (SNPs) in a growing list of candidate genes.

and ApoE-regulated interactions between cholesterol and Aβ.

and glucose, cholesterol and triglycerides serum levels.

**2. Risk factors for AD**

36 Neurodegenerative Diseases

**2.1. Environmental**

**a.** Cardiovascular risk factors.

**b.** Type 2 diabetes mellitus.

**c.** Traumatic brain injury.

an increase in amyloid deposition [5, 6].

It has been subject for debate whether depression might be an early manifestation of AD or a contributing factor for development of the disease. If one thing is sure is that depression contributes to cognitive decline and may by itself cause a condition known as "depressive pseudo-dementia" which can frequently evolve to a true dementia (mainly AD) when not treated, particularly in the elderly [7]. A history of major depressive disorder or susceptibility to depression during an individual's lifetime has therefore been associated with an increased risk to develop LOAD [8].

#### **e.** Sleep.

Chronic sleep deprivation and other sleep disorders have a negative effect on cognitive function primarily due to the role sleep plays in memory and learning processes and possibly in synaptic plasticity, although it is also believed there is an increase in Aβ during the waking hours [9]. History of sleep disorders has thus been linked to an increase in the risk to develop LOAD; furthermore, sleep disorders occurring as part of the aging process and those related to coexisting medical conditions contribute to cognitive decline and behavioural problems in AD patients.

#### **f.** Education.

People with a lower level of education show an increased risk to develop LOAD. This might be due to the relative lack of constant stimulation of cognitive processes (such as learning and memory) compared to individuals with a higher education degree. In fact, cognitive stimula‐ tion therapies are widely used for helping the treatment of people with dementia [10].

**g.** Diet.

It is well known that diet affects every aspect of an individual's health. A number of medical conditions associated with an increased risk for developing AD have a strong nutritional background as cholesterol, glucose and vitamin B12 levels, just to mention a few, importantly influence the risk for dementia [11], particularly in old age.

**h.** Alcohol and smoking.

It remains unclear whether alcohol and smoking increase the risk for AD as their association has been inconsistent. Although smoking is a strong risk factor for vascular disease, and the association of the latter with AD has been established, the possibility of a neuroprotective effect of nicotine by a smoking-induced increase in nicotinic receptors exists [3]. Similar is the case of alcohol where wine presents protective effects because of its antioxidant contents while associations between other types of alcohol and risk of AD remain controversial.

#### **2.2. Genetic**

Medical conditions and lifestyle in midlife, especially when a group of these factors coexist in an individual creating a synergistic effect, can contribute to cellular and molecular alterations ultimately leading to the hallmark pathological processes of AD later in life. Although environmental factors appear to play an important role in the development of LOAD, a number of them may depend on a genetic-influenced predisposition. Genetic variations in candidate genes have been widely investigated for associations with an increased risk of development, age of onset and progression of AD and, while results on several studies are controversial or inconclusive, it remains clear that common genetic variations do associate with LOAD whether in a general or population-specific manner. The AlzGene database holds information on 2973 polymorphisms in 695 genes reported in 1395 studies. We summarize in Table 1 some of the main findings on this field.

**3. Clinical diagnosis of LOAD**

**3.1. Initial evaluation**

**3.2. Laboratory tests**

in CSF are found in AD.

**3.3. Neuropsychological evaluation**

To perform a clinical diagnosis of AD or other dementias may not be challenging when symptoms are from moderate to severe and well differentiated; however, it might be more difficult to achieve an accurate diagnosis in the early stages of the disease, especially in elderly individuals where initial manifestations of AD can be taken as an effect of aging. Early detection of cognitive decline in primary care thus becomes crucial for canalization of the patient to an adequate specialist and a subsequent early accurate diagnosis which allows a better management of disease progression. In the first stages of AD, memory processes are the most affected but since a number of other treatable medical conditions also present with deficiencies in memory performance, a differential diagnosis based on all available tools must always be pursued. Clinical diagnosis of LOAD should include laboratory and neuropsychological tests as well as structural and functional imaging of the brain which, together with the clinical history and interviews with those living closest to

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Clinical history and, importantly, interviews with close family members and friends provide valuable information on patient's medical history, lifestyle and disease onset and progression. Several issues may be considered though when evaluating an aged individual: clinical manifestations of psychiatric disorders differ from younger patients; life events, social and financial situations as well as physical status are also relevant aspects to be taken into account.

Running laboratory tests is important to identify secondary causes of dementia and medical conditions common in the aged population. It is advisable to perform a complete blood count, serum electrolytes, glucose, vitamin B12, BUN/creatinine ratio and thyroid and hepatic function panels. Electrocardiography (ECG), electroencephalography (EEG) and thorax x-rays can also be included. Increase in total tau protein (t-tau) and decrease in Aβ<sup>42</sup>

Neuropsychological evaluation is fundamental for dementia diagnosis providing evidence of cognitive dysfunction and specific patterns helping to uncover the cause. AD diagnosis is based on NINCDS-ADRDA (National Institute of Neurological and Communicative Disorders and the Alzheimer's Disease and Related Disorders Association) criteria and is classified as definitive (clinical diagnosis with histological confirmation), probable (typical clinical features without histological confirmation) or possible (atypical clinical features, no apparent alternative diagnosis and no histological confirmation). Diagnosis of probable AD can only be made when objective evidence of significant memory deterioration exists by neuropsychological evaluation together with at least one other biological feature such as

the patient, would provide a better idea of the patient's overall condition.


Source: Meta-analysis for all studies from AlzGene-field synopsis of genetic association studies in AD (www.alzgene.org)

**Table 1.** Some widely studied genetic variations associated with an increased risk of AD.

At this point there is a need to highlight that prevention of LOAD may only be achieved by a healthy lifestyle implemented early in life, most importantly by those individuals with a genetic susceptibility.

### **3. Clinical diagnosis of LOAD**

environmental factors appear to play an important role in the development of LOAD, a number of them may depend on a genetic-influenced predisposition. Genetic variations in candidate genes have been widely investigated for associations with an increased risk of development, age of onset and progression of AD and, while results on several studies are controversial or inconclusive, it remains clear that common genetic variations do associate with LOAD whether in a general or population-specific manner. The AlzGene database holds information on 2973 polymorphisms in 695 genes reported in 1395 studies. We summarize in Table 1 some of the

*Gene Chromosome SNP Odds ratio Studies* APOE 19 e2/3/4 3.68 (4 vs. 3) 37 BIN1 2 rs744373 1.17 21 CLU 8 rs11136000 0.89 33 ABCA7 19 rs3764650 1.23 10 CR1 1 rs3818361 1.15 27 PICALM 11 rs3851179 0.88 27 MS4A6A 11 rs610932 0.90 11 CD33 19 rs3865444 0.89 5 MS4A4E 11 rs670139 1.08 11 CD2AP 6 rs9349407 1.12 5 EPHA1 7 rs11767557 0.89 5 GSTO1 10 rs4925 0.97 7 TOMM40 19 rs8106922 0.66 7 SORL1 11 rs2282649 1.10 23 PVRL2 19 rs6859 1.50 8 NCSTN 1 rs2274185 0.90 5 BDNF 11 270C/T 1.09 20 GAB2 11 rs2373115 0.85 13

Source: Meta-analysis for all studies from AlzGene-field synopsis of genetic association studies in AD (www.alzgene.org)

At this point there is a need to highlight that prevention of LOAD may only be achieved by a healthy lifestyle implemented early in life, most importantly by those individuals with a

**Table 1.** Some widely studied genetic variations associated with an increased risk of AD.

main findings on this field.

38 Neurodegenerative Diseases

genetic susceptibility.

To perform a clinical diagnosis of AD or other dementias may not be challenging when symptoms are from moderate to severe and well differentiated; however, it might be more difficult to achieve an accurate diagnosis in the early stages of the disease, especially in elderly individuals where initial manifestations of AD can be taken as an effect of aging. Early detection of cognitive decline in primary care thus becomes crucial for canalization of the patient to an adequate specialist and a subsequent early accurate diagnosis which allows a better management of disease progression. In the first stages of AD, memory processes are the most affected but since a number of other treatable medical conditions also present with deficiencies in memory performance, a differential diagnosis based on all available tools must always be pursued. Clinical diagnosis of LOAD should include laboratory and neuropsychological tests as well as structural and functional imaging of the brain which, together with the clinical history and interviews with those living closest to the patient, would provide a better idea of the patient's overall condition.

#### **3.1. Initial evaluation**

Clinical history and, importantly, interviews with close family members and friends provide valuable information on patient's medical history, lifestyle and disease onset and progression. Several issues may be considered though when evaluating an aged individual: clinical manifestations of psychiatric disorders differ from younger patients; life events, social and financial situations as well as physical status are also relevant aspects to be taken into account.

#### **3.2. Laboratory tests**

Running laboratory tests is important to identify secondary causes of dementia and medical conditions common in the aged population. It is advisable to perform a complete blood count, serum electrolytes, glucose, vitamin B12, BUN/creatinine ratio and thyroid and hepatic function panels. Electrocardiography (ECG), electroencephalography (EEG) and thorax x-rays can also be included. Increase in total tau protein (t-tau) and decrease in Aβ<sup>42</sup> in CSF are found in AD.

#### **3.3. Neuropsychological evaluation**

Neuropsychological evaluation is fundamental for dementia diagnosis providing evidence of cognitive dysfunction and specific patterns helping to uncover the cause. AD diagnosis is based on NINCDS-ADRDA (National Institute of Neurological and Communicative Disorders and the Alzheimer's Disease and Related Disorders Association) criteria and is classified as definitive (clinical diagnosis with histological confirmation), probable (typical clinical features without histological confirmation) or possible (atypical clinical features, no apparent alternative diagnosis and no histological confirmation). Diagnosis of probable AD can only be made when objective evidence of significant memory deterioration exists by neuropsychological evaluation together with at least one other biological feature such as abnormal CSF biomarkers and specific positron emission tomography (PET) patterns. Patients are evaluated using structured/semi-structured interviews and neuropsychologi‐ cal batteries with a variety of available tests to explore different aspects of cognition and behaviour. It may be advisable to apply a quick neuropsychological test to patients with apparent cognitive decline at primary care level; this would facilitate a proper canaliza‐ tion and management of the patient. Initial evaluations of general cognitive status can be performed using the popular Mini-Mental State Examination (MMSE) which is quick and easy to apply and provides information on global cognitive efficiency and dementia severity, although is not recommended for a definitive diagnosis.

Non-cognitive, psychiatric and behavioural alterations common to dementia such as apathy, aggression, depression, psychosis and sleep disorders vary according to disease severity and may fluctuate; they can also present as initial manifestations of dementia. It is believed that neuropsychiatric alterations may even serve as clinical indicators of MCI conversion to AD. Scales for mood and behaviour not only evaluate the presence/absence of symptoms but their frequency, severity and impact; they can be applied to family and caregivers. The Neuro‐ psychiatric Inventory can evaluate up to 10 behavioural alterations and is considered a standard tool, although a number of different batteries are available for this purpose. In Table 2 we provide a list of neuropsychological tests and batteries available to evaluate different

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aspects of cognitive and non-cognitive alterations.

**General cognitive function** Cognitive Assessment System (CAS)

Dementia Rating Scale—2 (DRS-2)

Kaufman Brief Intelligence Test (K-BIT) Mini-Mental State Examination (MMSE) National Adult Reading Test (NART)

Raven's Progressive Matrices (RPM)

**Executive function** Behavioural Assessment of the Dysexecutive Syndrome (BADS)

Cognitive Estimation Test (CET)

CANTAB

Category Test (CT)

Design Fluency Test Five Point Test

Stroop Test Verbal Fluency

**Attention** Brief Test of Attention (BTA)

Hayling and Brixton Tests Ruff Figural Fluency Test (RFFT) Self-Ordered Pointing Test (SOPT)

Colour Trails Test (CTT)

Wisconsin Card Sorting Test (WCST)

Comprehensive Trail Making Test (CTMT) Conners' Continuous Performance Test II (CPT-II)

Kaplan Baycrest Neurocognitive Assessment (KBNA)

Neuropsychological Assessment Battery (NAB)

Stanford-Binet Intelligence Scales—5th edition (SB5) The Test of Nonverbal Intelligence—3 (TONI-3)

Wechsler Abbreviated Scale of Intelligence (WASI) Wechsler Adult Intelligence Scale—III (WAIS-III)

Delis-Kaplan Executive Function System (D-KEFS)

The Speed and Capacity of Language Processing Test (SCOLP)

Woodcock-Johnson III Tests of Cognitive Abilities (WJ III COG)

Repeatable Battery for the Assessment of Neuropsychological Status (RBANS)

The issue of neuropsychiatric syndromes should also be addressed during dementia evaluation as their presence is associated with a rapid deterioration of cognition. It is important to notice that AD and cerebrovascular disease frequently coexist and the latter strongly determines presence and severity of clinical symptoms; thus, taking into account vascular risk factors and focal neurological signs as well as Hachinski Ischemic Scale results, combined with neuroimaging, serve as powerful tools to uncover a mixed dementia, especially in elderly individuals.

The most prominent feature of AD is a decline in cognitive function initially characterized by deficient memory for recent events, unusually repetitive omissions and difficulty to learn new information. The Free and Cued Selective Reminding Test (FCSRT) is a useful tool to explore these initial deficiencies in suspected AD patients. Temporo-spatial disorientation appears in early stages of the disease and progresses towards intermediate stages, where the patient can be disoriented in familiar places and aphasia appears with a decrease in verbal comprehension and nominal difficulty. To evaluate the fluency and coherence of language simple and complex orders and naming tests are used.

Confident neuropsychological markers of AD in early stages are deficiencies in episodic memory. Neuropsychological evaluation of AD patients also finds a loss of autonomy with disease progression from higher level to basic daily activities, for which daily living activities tests are applied. Instrumental functions such as language, praxis and visuospatial skills start being affected in intermediate stages of AD. Visuospatial dysfunction is a common feature in this stage of disease and can be evaluated by drawing and copying tests. In moderate to severe stages, difficulty to use objects and dressing apraxia can be observed as well as visual agnosia and a visual processing dysfunction recognized by facial and object recognition tests. Working memory and attention are usually affected by the time of diagnosis.

Deficiency in activities of daily living due to cognitive decline is essential diagnostic criteria for dementia and has great impact on quality of life. Neuropsychological tests to evaluate this feature allow differentiation of dementia from mild cognitive impairment (MCI). These tests measure basic (e.g. dressing, hygiene, feeding) and instrumental (e.g. cooking, cleaning, money management) activities. Activities of the Daily Living (ADL) and Instrumental Activities of the Daily Living (IADL) are widely used scales and provide the advantage of being easily applicable in primary care.

Non-cognitive, psychiatric and behavioural alterations common to dementia such as apathy, aggression, depression, psychosis and sleep disorders vary according to disease severity and may fluctuate; they can also present as initial manifestations of dementia. It is believed that neuropsychiatric alterations may even serve as clinical indicators of MCI conversion to AD. Scales for mood and behaviour not only evaluate the presence/absence of symptoms but their frequency, severity and impact; they can be applied to family and caregivers. The Neuro‐ psychiatric Inventory can evaluate up to 10 behavioural alterations and is considered a standard tool, although a number of different batteries are available for this purpose. In Table 2 we provide a list of neuropsychological tests and batteries available to evaluate different aspects of cognitive and non-cognitive alterations.

abnormal CSF biomarkers and specific positron emission tomography (PET) patterns. Patients are evaluated using structured/semi-structured interviews and neuropsychologi‐ cal batteries with a variety of available tests to explore different aspects of cognition and behaviour. It may be advisable to apply a quick neuropsychological test to patients with apparent cognitive decline at primary care level; this would facilitate a proper canaliza‐ tion and management of the patient. Initial evaluations of general cognitive status can be performed using the popular Mini-Mental State Examination (MMSE) which is quick and easy to apply and provides information on global cognitive efficiency and dementia severity,

The issue of neuropsychiatric syndromes should also be addressed during dementia evaluation as their presence is associated with a rapid deterioration of cognition. It is important to notice that AD and cerebrovascular disease frequently coexist and the latter strongly determines presence and severity of clinical symptoms; thus, taking into account vascular risk factors and focal neurological signs as well as Hachinski Ischemic Scale results, combined with neuroimaging, serve as powerful tools to uncover a mixed dementia,

The most prominent feature of AD is a decline in cognitive function initially characterized by deficient memory for recent events, unusually repetitive omissions and difficulty to learn new information. The Free and Cued Selective Reminding Test (FCSRT) is a useful tool to explore these initial deficiencies in suspected AD patients. Temporo-spatial disorientation appears in early stages of the disease and progresses towards intermediate stages, where the patient can be disoriented in familiar places and aphasia appears with a decrease in verbal comprehension and nominal difficulty. To evaluate the fluency and coherence of language simple and complex

Confident neuropsychological markers of AD in early stages are deficiencies in episodic memory. Neuropsychological evaluation of AD patients also finds a loss of autonomy with disease progression from higher level to basic daily activities, for which daily living activities tests are applied. Instrumental functions such as language, praxis and visuospatial skills start being affected in intermediate stages of AD. Visuospatial dysfunction is a common feature in this stage of disease and can be evaluated by drawing and copying tests. In moderate to severe stages, difficulty to use objects and dressing apraxia can be observed as well as visual agnosia and a visual processing dysfunction recognized by facial and object recognition tests. Working

Deficiency in activities of daily living due to cognitive decline is essential diagnostic criteria for dementia and has great impact on quality of life. Neuropsychological tests to evaluate this feature allow differentiation of dementia from mild cognitive impairment (MCI). These tests measure basic (e.g. dressing, hygiene, feeding) and instrumental (e.g. cooking, cleaning, money management) activities. Activities of the Daily Living (ADL) and Instrumental Activities of the Daily Living (IADL) are widely used scales and provide the advantage of

memory and attention are usually affected by the time of diagnosis.

although is not recommended for a definitive diagnosis.

especially in elderly individuals.

40 Neurodegenerative Diseases

orders and naming tests are used.

being easily applicable in primary care.



**Motor function** Finger Tapping Test (FTT)

commentary, 3rd ed. New York: Oxford University Press; 2006.

**Table 2.** Tests and batteries for neuropsychological evaluation.

found in patients with clear dementia symptoms.

**Mood, personality and adaptive functions**

**Activities of the daily**

**3.4. Neuroimaging**

**living**

Grip Strength Grooved Pegboard Purdue Pegboard Test

Beck Depression Inventory—2th edition (BDI-II)

Instrumental Activities of Daily Living (IADL)

Personality Assessment Inventory (PAI)

Geriatric Depression Scale (GDS)

Trauma Symptom Inventory (TSI)

Activities of the Daily Living (ADL)

Functional Activities Questionnaire Progressive Deterioration Scale

Behaviour Rating Inventory of Executive Function (BRIEF)

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Minnesota Multiphasic Personality Inventory-2 (MMPI-2)

Scales of Independent Behaviour—Revised (SIB-R)

Instrumental Activities of the Daily Living (IADL) Disability Assessment for Dementia Scale Alzheimer Disease Cooperative Study ADL Scale

Source: Strauss E, Sherman EMS, Spreen O. A compendium of neuropsychological tests: administration, norms, and

Imaging of the brain can reflect anatomical and physiological changes related to specific pathological processes. Structural neuroimaging technologies such as computed tomography (CT) and magnetic resonance imaging (MRI) provide relevant information on brain structures and help to exclude treatable conditions while functional neuroimaging including single photon emission computed tomography (SPECT), PET and functional magnetic resonance imaging (fMRI) informs about brain activity status. CT and MRI on AD evaluation are used to exclude neurosurgical lesions (tumors, subdural hematomas), search for evidence of cerebro‐ vascular lesions (stroke, white matter lesions) and identify medial temporal lobe atrophy. The overlap of whole-brain atrophy in AD with normal aging and other dementias is considerable and therefore lacks diagnostic value in clinical practice. Absolute values of glucose metabolism in the hippocampus are normal in early stages of AD but decrease progressively during disease course as detected by PET. Hypometabolism in the associative parietal cortex, external temporal area, precuneus, posterior cingulate cortex and dorsolateral frontal cortex can be

AD is characterized by changes in neurotransmission correlating with cognitive decline, particularly acetylcholine. A few PET tracers to measure acetylcholinesterase (AChE) and ligands to muscarinic and nicotinic receptors have been developed on the basis of the role of the cholinergic system in cognition and AD. AChE activity can be measured by its radioactively


Source: Strauss E, Sherman EMS, Spreen O. A compendium of neuropsychological tests: administration, norms, and commentary, 3rd ed. New York: Oxford University Press; 2006.

**Table 2.** Tests and batteries for neuropsychological evaluation.

#### **3.4. Neuroimaging**

Integrated Visual and Auditory Continuous Performance Test (IVA + Plus)

Paced Auditory Serial Addition Test (PASAT) Ruff 2 & 7 Selective Attention Test (2 & 7 Test) Symbol Digit Modalities Test (SDMT), Test of Everyday Attention (TEA) Test of Variables of Attention (T.O.V.A.)

Benton Visual Retention Test (BVRT-5)

Buschke Selective Reminding Test (SRT) California Verbal Learning Test-II (CVLT-II)

Brief Visuospatial Memory Test—Revised (BVMT-R)

Hopkins Verbal Learning Test—Revised (HVLT-R)

Wechsler Memory Scale—3th edition (WMS-III)

Rey-Osterrieth Auditory Verbal Learning Test (RAVLT)

Rivermead Behavioural Memory Test—2th edition (RBMT-II)

Wide Range Assessment of Memory and Learning—2th edition (WRAML2)

Expressive One-Word Picture Vocabulary Test—3th edition (EOWPVT3)

Peabody Picture Vocabulary Test—3th edition (PPVT-III)

Trail Making Test (TMT)

Brown-Peterson Task

Doors and People Test (DPT)

Recognition Memory Test (RMT)

Rey Complex Figure Test (ROCF)

**Language** Boston Diagnostic Aphasia Examination—3th edition (BDAE-3) Boston Naming Test—2 (BNT-2) Dichotic listening – Words

> Expressive Vocabulary Test (EVT) Multilingual Aphasia Examination (MAE)

Hooper Visual Organization Test (VOT) Judgement of Line Orientation (JLO)

Visual Object and Space Perception Battery (VOSP)

Rivermead Assessment of Somatosensory Performance (RASP)

Token Test (TT)

Bells Cancellation Test Clock Drawing Test (CDT) Facial Recognition Test (FRT)

Finger localization

Right-Left Orientation (RLO)

Smell Identification Test (SIT) Tactual Performance Test (TPT)

**Visual perception** Balloons Test

**Somatosensory and olfactory function, body**

42 Neurodegenerative Diseases

**orientation**

Sentence Repetition Test

Ruff-Light Trail Learning Test (RULIT)

**Memory** Autobiographical Memory Interview (AMI)

Imaging of the brain can reflect anatomical and physiological changes related to specific pathological processes. Structural neuroimaging technologies such as computed tomography (CT) and magnetic resonance imaging (MRI) provide relevant information on brain structures and help to exclude treatable conditions while functional neuroimaging including single photon emission computed tomography (SPECT), PET and functional magnetic resonance imaging (fMRI) informs about brain activity status. CT and MRI on AD evaluation are used to exclude neurosurgical lesions (tumors, subdural hematomas), search for evidence of cerebro‐ vascular lesions (stroke, white matter lesions) and identify medial temporal lobe atrophy. The overlap of whole-brain atrophy in AD with normal aging and other dementias is considerable and therefore lacks diagnostic value in clinical practice. Absolute values of glucose metabolism in the hippocampus are normal in early stages of AD but decrease progressively during disease course as detected by PET. Hypometabolism in the associative parietal cortex, external temporal area, precuneus, posterior cingulate cortex and dorsolateral frontal cortex can be found in patients with clear dementia symptoms.

AD is characterized by changes in neurotransmission correlating with cognitive decline, particularly acetylcholine. A few PET tracers to measure acetylcholinesterase (AChE) and ligands to muscarinic and nicotinic receptors have been developed on the basis of the role of the cholinergic system in cognition and AD. AChE activity can be measured by its radioactively tagged analogues N-[11C]-methyl-piperidine-4-yl-propionate (11C-PMP) and N-[11C]-methylpiperidine-4-yl-acetate (11C-MP4A); nicotinic receptors can be measured by 11C-nicotine. A decrease in 11C-nicotine correlates with cognition measured by MMSE [12]. Visualization of amyloid plaques started some 15 years ago initially using Aβ monoclonal antibodies and peptidic fragments and later small radioactively tagged Congo red, chrysamine G and thioflavin analogues for PET and SPECT. 18F tagging may present some advantages for clinical applications compared to 11C. Studies have shown there is less correlation between cognitive decline and the amount of amyloid plaques in the AD brain than between cognitive decline and the amount of neurofibrillary tangles and/or neurotransmitter activity as amyloid deposits are observed in up to 30% of cognitively healthy aged individuals [13]. An increase in micro‐ glial activation has also been reported in AD patients using neuroimaging technologies [14].

**4. The search for AD biomarkers**

**4.1. Brain biomarkers**

in early stages [15].

has become a very active field in AD research.

**4.2. Peripheral biomarkers**

Biomarkers for LOAD could help predict and diagnose the disease as well as follow its progression, evaluate treatments and find new therapeutic targets. Neuroimaging technolo‐ gies, genomics, transcriptomics and proteomics approaches are being extensively used globally to search for novel biomarkers for AD capable of detecting changes in the brain and peripheral tissues occurring early in the disease. Nevertheless, the finding of biomarkers with good sensitivity and specificity for AD, and furthermore, their validation, is challenging, reason why different combinations of biomarkers, cognitive markers and risk factors may represent a more suitable tool to pursue diagnostic sensitivity and specificity for LOAD.

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Markers of AD pathology have been extensively investigated in the brain of these patients; unfortunately, the task has been challenging due to overlap of a number of these findings with other brain disorders and even with normal ageing, as is the case of whole-brain atrophy. Although some findings might still represent specific biomarkers for AD as shown by a recent study where a decrease in grey matter diffusion values observed mainly in the posterior cingulate gyrus and precuneus area has been suggested as a potential new biomarker for AD

However, abnormalities in brain imaging (structural and functional), cognitive markers (neuropsychological scores) and molecules (mRNA, proteins) measured in specific brain regions affected by AD are currently demonstrating a better potential to identify AD and differentiate it from other disorders in a number of different combinations. Recent studies on the Alzheimer's Disease Neuroimaging Initiative (ADNI) subjects have shown correlations between temporal lobe atrophy measured by serial MRI scans with CSF p-tau and tau/Aβ<sup>42</sup> ratio as well as with cognitive markers which apparently may also be able to predict MCI-AD conversion. Hippocampal volume loss showed to correlate with ApoE4 genotype. PET imaging using 18F-fludeoxyglucose (FDG) showed correlations with Aβ42, but using 11Clabelled Pittsburgh compound B (11C-PIB) to specifically bind fibrillar Aβ plaques showed correlations not only with Aβ<sup>42</sup> but t-tau and p-tau181 [16]. However, recently, a comparative study suggested ADNI subjects appear to have a more aggressive pathology than populationbased samples, as observed by the rates of decline in hippocampal volume measured by MRI, which may raise concerns about ADNI subjects not being representative of the general population [17]. In any case, we must remember all findings should be validated in different cohorts and here may be the point where reproducibility cannot be achieved, reason why this

The search for easily available biomarkers for AD has lead scientists in the past years to investigate a number of molecules in CSF and blood components. From a variety of these studies several molecules were proposed as potential blood biomarkers for the disease (Table 4), although some results remained controversial. Until now, the best biomarker validated for

#### **3.5. Differential diagnosis**

Even when AD is the most common dementing syndrome in the elderly, one shall not forget that AD can share symptoms with other disorders. Medical, neurological and psychiatric conditions to which memory loss, depression, disorientation and other symptoms can be attributed must be also investigated for a differential diagnosis of dementia. When performing a diagnosis, it is important to be aware of features found during the patient evaluation providing doubt for the AD diagnosis and bear in mind that the more accurate the diagnosis is, the better the disease can be managed. AD diagnosis can be questioned when the conditions presented in Table 3 exist.


**Table 3.** Evaluation findings suggesting a diagnosis different from AD.

### **4. The search for AD biomarkers**

Biomarkers for LOAD could help predict and diagnose the disease as well as follow its progression, evaluate treatments and find new therapeutic targets. Neuroimaging technolo‐ gies, genomics, transcriptomics and proteomics approaches are being extensively used globally to search for novel biomarkers for AD capable of detecting changes in the brain and peripheral tissues occurring early in the disease. Nevertheless, the finding of biomarkers with good sensitivity and specificity for AD, and furthermore, their validation, is challenging, reason why different combinations of biomarkers, cognitive markers and risk factors may represent a more suitable tool to pursue diagnostic sensitivity and specificity for LOAD.

#### **4.1. Brain biomarkers**

tagged analogues N-[11C]-methyl-piperidine-4-yl-propionate (11C-PMP) and N-[11C]-methylpiperidine-4-yl-acetate (11C-MP4A); nicotinic receptors can be measured by 11C-nicotine. A decrease in 11C-nicotine correlates with cognition measured by MMSE [12]. Visualization of amyloid plaques started some 15 years ago initially using Aβ monoclonal antibodies and peptidic fragments and later small radioactively tagged Congo red, chrysamine G and thioflavin analogues for PET and SPECT. 18F tagging may present some advantages for clinical applications compared to 11C. Studies have shown there is less correlation between cognitive decline and the amount of amyloid plaques in the AD brain than between cognitive decline and the amount of neurofibrillary tangles and/or neurotransmitter activity as amyloid deposits are observed in up to 30% of cognitively healthy aged individuals [13]. An increase in micro‐ glial activation has also been reported in AD patients using neuroimaging technologies [14].

Even when AD is the most common dementing syndrome in the elderly, one shall not forget that AD can share symptoms with other disorders. Medical, neurological and psychiatric conditions to which memory loss, depression, disorientation and other symptoms can be attributed must be also investigated for a differential diagnosis of dementia. When performing a diagnosis, it is important to be aware of features found during the patient evaluation providing doubt for the AD diagnosis and bear in mind that the more accurate the diagnosis is, the better the disease can be managed. AD diagnosis can be questioned when the conditions

Fronto-temporal dementia (FTD)

Vascular dementia (VaD)

Semantic dementia

Non-fluent progressive aphasia

Logopenic progressive aphasia

Progressive posterior cortical atrophy

**Feature Possible cause**

Early extrapyramidal signs Dementia with Lewy Bodies

Early alterations of behaviour: inappropriate social behaviour/feeding


Onset with high order visuospatial dysfunction/Balint syndrome: ocular

**Table 3.** Evaluation findings suggesting a diagnosis different from AD.

Early visual hallucinations (DLB)

**3.5. Differential diagnosis**

44 Neurodegenerative Diseases

presented in Table 3 exist.

Fluctuation of symptoms Vascular lesions in neuroimaging Sudden onset of symptoms Focal neurological signs

Early language alterations:

decline of speech with anomia

apraxia, optic ataxia, simultagnosia


Early visuospatial and attention deterioration Behavioural disturbances during REM sleep

alterations

Markers of AD pathology have been extensively investigated in the brain of these patients; unfortunately, the task has been challenging due to overlap of a number of these findings with other brain disorders and even with normal ageing, as is the case of whole-brain atrophy. Although some findings might still represent specific biomarkers for AD as shown by a recent study where a decrease in grey matter diffusion values observed mainly in the posterior cingulate gyrus and precuneus area has been suggested as a potential new biomarker for AD in early stages [15].

However, abnormalities in brain imaging (structural and functional), cognitive markers (neuropsychological scores) and molecules (mRNA, proteins) measured in specific brain regions affected by AD are currently demonstrating a better potential to identify AD and differentiate it from other disorders in a number of different combinations. Recent studies on the Alzheimer's Disease Neuroimaging Initiative (ADNI) subjects have shown correlations between temporal lobe atrophy measured by serial MRI scans with CSF p-tau and tau/Aβ<sup>42</sup> ratio as well as with cognitive markers which apparently may also be able to predict MCI-AD conversion. Hippocampal volume loss showed to correlate with ApoE4 genotype. PET imaging using 18F-fludeoxyglucose (FDG) showed correlations with Aβ42, but using 11Clabelled Pittsburgh compound B (11C-PIB) to specifically bind fibrillar Aβ plaques showed correlations not only with Aβ<sup>42</sup> but t-tau and p-tau181 [16]. However, recently, a comparative study suggested ADNI subjects appear to have a more aggressive pathology than populationbased samples, as observed by the rates of decline in hippocampal volume measured by MRI, which may raise concerns about ADNI subjects not being representative of the general population [17]. In any case, we must remember all findings should be validated in different cohorts and here may be the point where reproducibility cannot be achieved, reason why this has become a very active field in AD research.

#### **4.2. Peripheral biomarkers**

The search for easily available biomarkers for AD has lead scientists in the past years to investigate a number of molecules in CSF and blood components. From a variety of these studies several molecules were proposed as potential blood biomarkers for the disease (Table 4), although some results remained controversial. Until now, the best biomarker validated for AD diagnosis is the CSF tau/Aβ42 ratio. Other biomarkers in CSF have been investigated though and, recently, protein markers of DNA damage have shown potential as biomarkers for AD and other dementias [18].

of technologies. We shall not forget serum and blood cells are also a potential source of

**Sample Cohort Panel Change Ref.**

cortisol, pancreatic polypeptide, insulin like growth factor binding protein 2, β2

apolipoprotein E, epidermal growth factor receptor, hemoglobin, calcium, zinc, interleukin 17, and albumin

eotaxin 3, pancreatic polypeptide, N-terminal protein B-type brain natriuretic peptide,

expression, protein levels and activity

α2-macroglobulin, angiotensinogen, apolipoprotein A-II (ApoA-II), ApoE, betacellulin (BTC), Fas ligand (FasL), heparinbinding EGF-like growth factor (HB-EGF), macrophage inflammatory protein-1α (MIP-1α), peptide YY (PYY), m glutamic oxaloacetic transaminase (SGOT), transthyretin

EGF, G-CSF, GDNF, ICAM-1, IGFBP-6, IL-1α, IL-3, IL-11, M-CSF, PDGF-BB, TNF-α, TRAIL-R4

Plasma AD and controls EGF, PDG-BB and MIP-1δ Not stated [24]

complement components C3 and C3a, complement factor-I, γ-fibrinogen and

IgM and ApoE Decrease

1, carcinoembryonic antigen, matrix metalloprotein 2, CD40, macrophage inflammatory protein 1α, superoxide dismutase, and homocysteine

microglobulin, vascular cell adhesion molecule

Late–Onset Alzheimer's Disease: Risk Factors, Clinical Diagnosis and the Search for Biomarkers

Increase [19]

http://dx.doi.org/10.5772/53775

47

Increase [20]

Increase [21]

Signature [22]

Signature [23]

[25]

[26]

regulated

Associate with whole-brain volume

Decrease

pathologic alterations reflecting disease.

from the Australian Imaging Biomarker and Lifestyle study

controls from ADNI

and controls from

AD and other dementias

control from AddNeuroMed

**Table 5.** Recent findings on blood biomarkers for AD.

Plasma MCI, AD and

tenascin C

(TTR)

TRAIL-R4

Serum AD and controls miR-137, -181c, -9, -29a/b Down-

alpha-1-microglobulin

Plasma AD and controls ANG-2, CCL5, CCL7, CCL15, CCL18, CXCL8,

LOAD and controls fatty acid amide hydrolase (FAAH) gene

Plasma AD and controls

Plasma MCI, AD and

Plasma MCI-AD converters

ADNI

Peripheral blood mononuclear cells (PBMCs)

(AIBL)


**Table 4.** Proposed blood biomarkers for AD.

In recent years, the approach for biomarker discovery has changed, now focusing on pattern recognition and combinations of different markers to identify panels capable of differentiating AD from healthy control subjects and, in some cases, from other brain disorders. As the work on this field has been so extensive we will limit ourselves now to briefly mention some of the recent biomarker findings reported principally in blood plasma (Table 5), where protein profiles have been extensively analysed in AD, MCI and control subjects using a wide range of technologies. We shall not forget serum and blood cells are also a potential source of pathologic alterations reflecting disease.

AD diagnosis is the CSF tau/Aβ42 ratio. Other biomarkers in CSF have been investigated though and, recently, protein markers of DNA damage have shown potential as biomarkers for AD

Antioxidant levels (carotene, lycopene, vitamins A, C and E, urate, bilirubin)

In recent years, the approach for biomarker discovery has changed, now focusing on pattern recognition and combinations of different markers to identify panels capable of differentiating AD from healthy control subjects and, in some cases, from other brain disorders. As the work on this field has been so extensive we will limit ourselves now to briefly mention some of the recent biomarker findings reported principally in blood plasma (Table 5), where protein profiles have been extensively analysed in AD, MCI and control subjects using a wide range

Lipid peroxidation (F2-isoprostanes, 4-hydroxynonenal)

Total tau (t-tau) and phosphorilated tau (p-tau)

and other dementias [18].

46 Neurodegenerative Diseases

**Markers involved in APP and Aβ metabolisms**

Brain-plasma Aβ flux Aβ autoantibodies APP platelet isoforms BACE1 activity

24S-hydroxycholesterol

Cholesterol

Homocysteine

C reactive protein

IL-6 and its receptor α1-antichymotrypsin

Phospholipase-A2 α2-macroglobulin

Desmosterol

**Table 4.** Proposed blood biomarkers for AD.

Complement factor H (CFH)

ApoE Lp(a)

**Markers of oxidation**

**Markers of inflammation**

**Other proposed markers**

p97 GSK3 MAO-B

IL-1β TNF-α

Aβ peptides (total Aβ, Aβ40 and Aβ42)

**Markers related to cholesterol metabolism and vascular disease**


**Table 5.** Recent findings on blood biomarkers for AD.

#### *4.2.1. Expression of MAPT, APP, NCSTN and BACE1 in lymphocytes*

Neurofibrillary tangles and amyloid plaques are hallmarks in the AD brain involving the microtubule-associated protein tau (MAPT) and Aβ peptides generated from the amyloid precursor protein (APP) by sequential cleavage of the β- and γ-secretases, nicastrin (NCSTN) being a major component of the latter. These molecules have been widely investigated in the brain; nevertheless, little is known about their expression in peripheral cells. The established role of inflammation in the AD pathology lead us to question how these major genes express in peripheral lymphocytes and, furthermore, whether their expression may correlate with common medical conditions in the elderly that have been identified as risk factors for LOAD. **c.** Data analysis

**d.** Main findings

Gene expression values were compared between study groups using Student's *t* and Mann-Whitney's *U* tests. Associations between gene expression levels and between expression with raw values of the analysed risk factor conditions were investigated using Spearman's corre‐

Late–Onset Alzheimer's Disease: Risk Factors, Clinical Diagnosis and the Search for Biomarkers

http://dx.doi.org/10.5772/53775

49

As the full report of this exploratory study is currently under review for publication, we will limit ourselves here to share only the most relevant of our results. From the four genes analysed, we were not able to detect NCSTN in any AD subjects, therefore showing significant differences between the AD group and all the others, but particularly differing from the group of other brain disorders which showed the highest expression levels for this study (Figure 1). We also investigated the prevalence of commonly elevated variables increasing the risk for AD: blood pressure (taken as systolic/diastolic pressure and pulse), glucose, total cholesterol and triglycerides. High serum triglycerides and cholesterol prevailed in the whole study population whereas the AD group showed no greater prevalence of any of these conditions. Correlation analyses associated NCSTN expression in lymphocytes with that observed for APP and BACE1, as well as with serum total cholesterol levels in the whole study population but, when separated by groups, NCSTN

lation coefficients. Differences were considered significant at p<0.05.

expression only correlated with cholesterol levels in the group NP.

**Figure 1.** NCSTN relative expression in lymphocytes.

#### **a.** Samples

To address this question, we collected blood samples from a total of 72 subjects, including 48 healthy individuals divided into 4 groups by age (25 to 92 years of age), 12 clinically diagnosed AD patients and 12 patients suffering from other brain disorders (vascular dementia-VaD-, Parkinson's disease-PD-, traumatic brain injury-TBI-, cerebrovascular events-CVE-, psychotic disorder) as a comparative group named NP (Table 6). All patients had been previously diagnosed by appropriate professionals in the private practice and healthy subjects volun‐ teered, making this a small population-based cohort with exploratory purposes. For all subjects, blood pressure was measured and history was briefly collected; for all patients, interviews with family members were also performed. All patients are participants in our neuro-rehabilitation program [27] and samples were collected at the time of enrolment.


**Table 6.** Basic characteristics of the study groups.

#### **b.** Methodology

After collection, serum was separated from whole blood to quantify total cholesterol, glucose and triglycerides. Lymphocytes were isolated from EDTA whole blood using Lymphoprep (Nycomed Pharma). After washing the pellet, total RNA was extracted from lymphocytes by the TRIzol method (Invitrogen). Endpoint RT-PCR was selected to semiquantify MAPT, APP, NCSTN and the β-secretase BACE1 expression levels because of our strong interest in the use of the most easily available technologies for small research and clinical laboratories. Glycer‐ aldehyde-3-phosphate-dehydrogenase (GAPDH) was the housekeeping gene of choice.

#### **c.** Data analysis

*4.2.1. Expression of MAPT, APP, NCSTN and BACE1 in lymphocytes*

*Group n Mean age Diagnosis*

**AD** 12 80 LOAD

**Table 6.** Basic characteristics of the study groups.

**b.** Methodology

**I** 12 30

**II** 12 44 **III** 12 58 **IV** 12 80

**a.** Samples

48 Neurodegenerative Diseases

Neurofibrillary tangles and amyloid plaques are hallmarks in the AD brain involving the microtubule-associated protein tau (MAPT) and Aβ peptides generated from the amyloid precursor protein (APP) by sequential cleavage of the β- and γ-secretases, nicastrin (NCSTN) being a major component of the latter. These molecules have been widely investigated in the brain; nevertheless, little is known about their expression in peripheral cells. The established role of inflammation in the AD pathology lead us to question how these major genes express in peripheral lymphocytes and, furthermore, whether their expression may correlate with common medical conditions in the elderly that have been identified as risk factors for LOAD.

To address this question, we collected blood samples from a total of 72 subjects, including 48 healthy individuals divided into 4 groups by age (25 to 92 years of age), 12 clinically diagnosed AD patients and 12 patients suffering from other brain disorders (vascular dementia-VaD-, Parkinson's disease-PD-, traumatic brain injury-TBI-, cerebrovascular events-CVE-, psychotic disorder) as a comparative group named NP (Table 6). All patients had been previously diagnosed by appropriate professionals in the private practice and healthy subjects volun‐ teered, making this a small population-based cohort with exploratory purposes. For all subjects, blood pressure was measured and history was briefly collected; for all patients, interviews with family members were also performed. All patients are participants in our neuro-rehabilitation program [27] and samples were collected at the time of enrolment.

Cognitively healthy

After collection, serum was separated from whole blood to quantify total cholesterol, glucose and triglycerides. Lymphocytes were isolated from EDTA whole blood using Lymphoprep (Nycomed Pharma). After washing the pellet, total RNA was extracted from lymphocytes by the TRIzol method (Invitrogen). Endpoint RT-PCR was selected to semiquantify MAPT, APP, NCSTN and the β-secretase BACE1 expression levels because of our strong interest in the use of the most easily available technologies for small research and clinical laboratories. Glycer‐ aldehyde-3-phosphate-dehydrogenase (GAPDH) was the housekeeping gene of choice.

**NP** 12 78 PD, MCI, VaD, CVE, TBI, psychotic disorder/ schizophrenia

Gene expression values were compared between study groups using Student's *t* and Mann-Whitney's *U* tests. Associations between gene expression levels and between expression with raw values of the analysed risk factor conditions were investigated using Spearman's corre‐ lation coefficients. Differences were considered significant at p<0.05.

#### **d.** Main findings

As the full report of this exploratory study is currently under review for publication, we will limit ourselves here to share only the most relevant of our results. From the four genes analysed, we were not able to detect NCSTN in any AD subjects, therefore showing significant differences between the AD group and all the others, but particularly differing from the group of other brain disorders which showed the highest expression levels for this study (Figure 1). We also investigated the prevalence of commonly elevated variables increasing the risk for AD: blood pressure (taken as systolic/diastolic pressure and pulse), glucose, total cholesterol and triglycerides. High serum triglycerides and cholesterol prevailed in the whole study population whereas the AD group showed no greater prevalence of any of these conditions. Correlation analyses associated NCSTN expression in lymphocytes with that observed for APP and BACE1, as well as with serum total cholesterol levels in the whole study population but, when separated by groups, NCSTN expression only correlated with cholesterol levels in the group NP.

**Figure 1.** NCSTN relative expression in lymphocytes.

#### **5. Conclusions**

Late-onset Alzheimer's disease (LOAD) is the most common dementia in the aged population and therefore great efforts to discover the factors conferring an increased risk for this disease and biomarkers able to help in its diagnosis and prognosis are made worldwide. These intensively active fields of research have produced huge amounts of information, some of which have been controversial but also a good volume showing real potential and waiting to seek further replication and validation. However, while research on biomarkers for AD struggles with the challenge of sensibility, specificity and reprodu‐ cibility in different cohorts, it is of major importance to assure the best possible perform‐ ance when clinically diagnosing a patient with LOAD. Clinical diagnosis of dementias may be a bit of a long process which requires some expertise and must include specific laboratory and neuropsychological tests as well as imaging of the brain and family interviews. An accurate diagnosis has implications on therapeutic approaches, disease progression and costs, quality of life of patients, family and caregivers and other impor‐ tant aspects for disease management.

**References**

[1] Shah NS, Vidal JS, Masaki K, Petrovitch H, Ross GW, Tilley C et al. Midlife blood pressure, plasma beta-amyloid, and the risk for Alzheimer disease. *Hypertension*

Late–Onset Alzheimer's Disease: Risk Factors, Clinical Diagnosis and the Search for Biomarkers

http://dx.doi.org/10.5772/53775

51

[2] Yang YH, Roe CM, Morris JC. Relationship between late-life hypertension, blood pressure, and Alzheimer's disease. *An J Alzheimers Dis Other Demen* 2011; 26(6):

[3] Rodríguez-Valdés R, Álvarez-Amador A, Aguilar-Fabré L. Vascular factors and Alz‐ heimer's disease (in Spanish). *Rev Mex Neuroci* 2006; 7(3): 225-230. http://www.medi‐

[4] Kalaria RN. Cerebral vessels in aging and Alzheimer's disease. *Pharmacol Ther* 1996;

[5] Jellinger KA, Paulus W, Wrocklage C, Litvan I. Traumatic brain injury as a risk factor for Alzheimer's disease: comparison of two retrospective autopsy cohorts with eval‐

[6] Uryu K, Laurer H, McIntosh T, Praticò D, Martínez D, Leight S et al. Repetitive mild brain trauma accelerates Abeta deposition, lipid peroxidation, and cognitive impair‐ ment in a transgenic mouse model of Alzheimer amyloidosis. *J Neurosci* 2002; 22(2):

[7] Sáez-Fonseca JA, Lee L, Walker Z. Long-term outcome of depressive pseudodemen‐ tia in the elderly*. J Affect Disord* 2007; 101(1-3): 123-9. doi: 10.1016/j.jad.2006.11.004 [8] Barnes DE, Yaffe K, Byers AL, McCormick M, Schaefer C, Whitmer RA. Midlife vs late-life depressive symptoms and risk of dementia: differential effects for Alzheimer disease and vascular dementia. *Arch Gen Psychiatry* 2012; 69(5): 493-8. doi: 10.1001/

[9] Kang JE, Lim MM, Bateman RJ, Lee JJ, Smyth LP, Cirrito JR et al. Amyloid-beta dy‐ namics are regulated by orexin and the sleep-wake cycle. *Science* 2009; 326(5955):

[10] Liberati G, Raffone A, Olivetti Belardinelli M. Cognitive reserve and its implications for rehabilitation and Alzheimer's disease. *Cogn Process* 2012; 13(1): 1-12. doi: 10.1007/

[11] Lanyau Domínguez Y. The diet in Alzheimer's disease (in Spanish). *Revista Cubana de Salud Pública* 2009; 35(4): 55-64. http://redalyc.uaemex.mx/redalyc/pdf/

uation of ApoE genotype. *MNC Neurol* 2001; 1:3. doi: 10.1186/1471-2377-1-3

2012; 59: 780-86. doi: 10.1161/hypertensionaha.111.178962

graphic.com/pdfs/revmexneu/rmn-2006/rmn063h.pdf

446-54. http://www.jneurosci.org/content/22/2/446.long

archgenpsychiatry.2011.1481

s10339-011-0410-3

214/21418848007.pdf

1005-7. doi: 10.1126/science.1180962

72(3): 193-214. tp://dx.doi.org/10.1016/S0163-7258(96)00116-7

457-62. doi: 10.1177/1533317511421779

We previously reported the establishment of a neuro-rehabilitation program for AD and other neuropsychological disorders in our locality [27] and made brief mention of a biomarker study for which now we reported results on this occasion. Our major interest resides in using the most easily and widely available technologies for biomarker discov‐ ery and therapeutic approaches which could be directed to the general population around the globe in a cost effective manner. Even when our work is in the very first stages of this research, we seek to continue our studies in larger cohorts, including more variables and using better technologies.

### **Acknowledgements**

The author would like to thank to Gonzalo E. Aranda-Abreu, PhD, leading researcher of the original work commented herein, and María E. Hernández Aguilar, PhD, both from the Center of Cerebral Investigations, Universidad Veracruzana. Also thanks to Consejo Nacional de Ciencia y Tecnología (CONACyT, Mexico) for the doctoral scholarship number 223277 in biomedical sciences granted to M.H.R., and the nursing home Emperatríz de las Américas.

### **Author details**

Marisol Herrera-Rivero

Doctorate in Biomedical Sciences, Centre of Biomedical Investigations, Universidad Ve‐ racruzana, Xalapa, Veracruz, Mexico

### **References**

**5. Conclusions**

50 Neurodegenerative Diseases

tant aspects for disease management.

using better technologies.

**Acknowledgements**

**Author details**

Marisol Herrera-Rivero

racruzana, Xalapa, Veracruz, Mexico

Late-onset Alzheimer's disease (LOAD) is the most common dementia in the aged population and therefore great efforts to discover the factors conferring an increased risk for this disease and biomarkers able to help in its diagnosis and prognosis are made worldwide. These intensively active fields of research have produced huge amounts of information, some of which have been controversial but also a good volume showing real potential and waiting to seek further replication and validation. However, while research on biomarkers for AD struggles with the challenge of sensibility, specificity and reprodu‐ cibility in different cohorts, it is of major importance to assure the best possible perform‐ ance when clinically diagnosing a patient with LOAD. Clinical diagnosis of dementias may be a bit of a long process which requires some expertise and must include specific laboratory and neuropsychological tests as well as imaging of the brain and family interviews. An accurate diagnosis has implications on therapeutic approaches, disease progression and costs, quality of life of patients, family and caregivers and other impor‐

We previously reported the establishment of a neuro-rehabilitation program for AD and other neuropsychological disorders in our locality [27] and made brief mention of a biomarker study for which now we reported results on this occasion. Our major interest resides in using the most easily and widely available technologies for biomarker discov‐ ery and therapeutic approaches which could be directed to the general population around the globe in a cost effective manner. Even when our work is in the very first stages of this research, we seek to continue our studies in larger cohorts, including more variables and

The author would like to thank to Gonzalo E. Aranda-Abreu, PhD, leading researcher of the original work commented herein, and María E. Hernández Aguilar, PhD, both from the Center of Cerebral Investigations, Universidad Veracruzana. Also thanks to Consejo Nacional de Ciencia y Tecnología (CONACyT, Mexico) for the doctoral scholarship number 223277 in biomedical sciences granted to M.H.R., and the nursing home Emperatríz de las Américas.

Doctorate in Biomedical Sciences, Centre of Biomedical Investigations, Universidad Ve‐


[12] Nordberg A, Lundqvist H, Hartvig P, Lilja A, Langstrom B. Kinetic analysis of re‐ gional (S)(-)11C-nicotine binding in normal and Alzheimer brains. In vivo assess‐ ments using positron emission tomography. *Alzh Dis Assoc Disord* 1995; 9(1):21-7.

[24] Björkqvist M, Ohlsson M, Minthon L, Hansson O. Evaluation of a previously sug‐ gested plasma biomarker panel to identify Alzheimer's disease. *PLoS One* 2012; 7(1):

Late–Onset Alzheimer's Disease: Risk Factors, Clinical Diagnosis and the Search for Biomarkers

http://dx.doi.org/10.5772/53775

53

[25] Geekiyanage H, Jicha GA, Nelson PT, Chan C. Blood serum miRNA: non-invasive bi‐ omarkers for Alzheimer's disease. *Exp Neurol* 2012; 235(2): 491-6. doi: 10.1016/

[26] Thambisetty M, Simmons A, Hye A, Campbell J, Westman E, Zhang Y et al. Plasma biomarkers of brain atrophy in Alzheimer's disease. *PLoS One* 2011; 6(12): e28527.

[27] Herrera-Rivero M, Aranda-Abreu GE. Therapeutics of Alzheimer's Disease. In: *Ad‐ vanced Understanding of Neurodegenerative Diseases,* Raymond Chuen-Chung Chang (Ed.), 2011. ISBN: 978-953-307-529-7, InTech. Available from: http://www.intechop‐ en.com/books/advanced-understanding-of-neurodegenerative-diseases/therapeutics-

e29868. doi: 10.1371/journal.pone.0029868

j.expneurol.2011.11.026

of-alzheimer-s-disease

doi: 10.1371/journal.pone.0028527


[24] Björkqvist M, Ohlsson M, Minthon L, Hansson O. Evaluation of a previously sug‐ gested plasma biomarker panel to identify Alzheimer's disease. *PLoS One* 2012; 7(1): e29868. doi: 10.1371/journal.pone.0029868

[12] Nordberg A, Lundqvist H, Hartvig P, Lilja A, Langstrom B. Kinetic analysis of re‐ gional (S)(-)11C-nicotine binding in normal and Alzheimer brains. In vivo assess‐ ments using positron emission tomography. *Alzh Dis Assoc Disord* 1995; 9(1):21-7. [13] Villemagne VL, Pike KE, Darby D, Maruff P, Savage G, Ng S et al. Abeta deposition in older non-demented individuals with cognitive decline are indicative of preclini‐ cal Alzheimer's disease. *Neuropsychologia* 2008; 46(6):1688-97. http://dx.doi.org/

[14] Cagnin A, Brooks DJ, Kennedy AM, Gunn RN, Myers R, Turkheimer FE et al. In vivo measurement of activated microglia in dementia*. Lancet* 2001; 358(9280):461–7. doi:

[15] Jacobs HI, van Boxtel MP, Gronenschild EH, Uylings HB, Jolles J, Verhey FR. *Alz‐*

[16] Trojanowski JQ, Trojanowski JQ, Vandeerstichele H, Korecka M, Clark CM, Aisen PS et al. Update on the biomarker core of the Alzheimer's Disease Neuroimaging Initia‐

tive subjects. *Alzheimers Dement* 2010; 6(3): 230-8. doi: 10.1016/j.jalz.2010.03.008

[17] Whitwell JL, Wiste HJ, Weigand SD, Rocca WA, Knopman DS, Roberts RO et al. Comparison of imaging biomarkers in the Alzheimer Disease Neuroimaging Initia‐ tive and the Mayo Clinic Study of Aging. *Arch Neurol* 2012; 69(5): 614-44. doi:

[18] Watabe-Rudolph M, Song Z, Lausser L, Schnack C, Begus-Nahrmann Y, Scheithauer MO et al. Chitinase enzyme activity in CSF is a powerful biomarker of Alzheimer

[19] Doecke JD, Laws SM, Faux NG, Wilson W, Burnham SC, Lam CP et al. Blood-based protein biomarkers for diagnosis of Alzheimer Disease. *Arch Neurol* 2012. doi:

[20] Soares HD, Potter WZ, Pickering E, Kuhn M, Immermann FW, Shera DM et al. Plas‐ ma biomarkers associated with the Apolipoprotein E genotype and Alzheimer dis‐

[21] D'Addario C, Di Francesco A, Arosio B, Gussago C, Dell'osso B, Bari M et al. Epige‐ netic regulation of fatty acid amide hydrolase in Alzheimer disease. *PLoS One* 2012;

[22] Johnstone D, Milward EA, Berretta R, Moscato P. Multivariate protein signatures of pre-clinical Alzheimer's disease in the Alzheimer's disease neuroimaging initiative (ADNI) plasma proteome dataset. *PLoS One* 2012; 7(4): e34341. doi: 10.1371/jour‐

[23] Ray S, Britschgi M, Herbert C, Takeda-Uchimura Y, Boxer A, Blennow K et al. Classi‐ fication and prediction of clinical Alzheimer's diagnosis based on plasma signaling

ease. *Arch Neurol* 2012. doi: 10.1001/archneurol.2012.1070

proteins. Nat Med 2007; 13(11): 1359-62. doi: 10.1038/nm1653

7(6): e39186. doi: 10.1371/journal.pone.0039186

disease. *Neurology* 2012; 78(8): 569-77. doi: 10.1212/WNL.0b013e318247caa1

10.1016/j.neuropsychologia.2008.02.008

*heimers Dement* 2012. doi: 10.1016/j.jalz.2011.11.004

10.1016/S0140-6736(01)05625-2

52 Neurodegenerative Diseases

10.1001/archneurol.2011.3029

10.1001/archneurol.2012.1282

nal.pone.0034341


**Chapter 3**

**Role of Protein Aggregation in**

**Neurodegenerative Diseases**

Yusuf Tutar, Aykut Özgür and Lütfi Tutar

http://dx.doi.org/10.5772/54487

tion of native protein conformation [1].

tein aggregation [1, 4, 5].

cellular mechanisms [6-10].

**1. Introduction**

Additional information is available at the end of the chapter

Proteins and peptides are essential complex macromolecules of organisms and participate in actually every process within cells. Three dimensional structures of proteins play a critical role for biological functions. Therefore, they must be properly folded for performing these functions. Three dimensional structures are determined by composition of amino acid se‐ quence. In addition to hydrophobic forces, covalent and weak interactions direct the forma‐

Proteins can be exposed to internal and external forces such as protein-protein interac‐ tions, various stresses, mutations etc. Since these forces alternates protein conformation, the biological activity of the protein decreases. However, newly synthesized proteins may not fold correctly, or properly folded proteins cannot spontaneously fold. In this case, proteins have a strong tendency to aggregate [1-3]. Especially, heat shock pro‐ teins (chaperones) play a key role in correcting protein folding and prevention of pro‐

Protein aggregates has toxic effects when accumulated over a certain amount in the cell. The accumulation of abnormal proteins leads to progressive loss of structure and/or function of neurons, including the death of neurons. Many diseases associate with protein aggregation such as prion, Alzheimer's (AD), Parkinson's (PD), and Hun‐ tington's diseases (HD). Thus, dysmnesia, mental retardation, and also cancer are seen in these diseases. Many of the neurodegenerative disorders likely occur due to envi‐ ronmental and genetic factors. Especially, probability of AD and PD occurrence rise with increasing age. Briefly, neurodegenerative diseases have similar pathogenesis and

> © 2013 Tutar et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**Chapter 3**

### **Role of Protein Aggregation in Neurodegenerative Diseases**

Yusuf Tutar, Aykut Özgür and Lütfi Tutar

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54487

### **1. Introduction**

Proteins and peptides are essential complex macromolecules of organisms and participate in actually every process within cells. Three dimensional structures of proteins play a critical role for biological functions. Therefore, they must be properly folded for performing these functions. Three dimensional structures are determined by composition of amino acid se‐ quence. In addition to hydrophobic forces, covalent and weak interactions direct the forma‐ tion of native protein conformation [1].

Proteins can be exposed to internal and external forces such as protein-protein interac‐ tions, various stresses, mutations etc. Since these forces alternates protein conformation, the biological activity of the protein decreases. However, newly synthesized proteins may not fold correctly, or properly folded proteins cannot spontaneously fold. In this case, proteins have a strong tendency to aggregate [1-3]. Especially, heat shock pro‐ teins (chaperones) play a key role in correcting protein folding and prevention of pro‐ tein aggregation [1, 4, 5].

Protein aggregates has toxic effects when accumulated over a certain amount in the cell. The accumulation of abnormal proteins leads to progressive loss of structure and/or function of neurons, including the death of neurons. Many diseases associate with protein aggregation such as prion, Alzheimer's (AD), Parkinson's (PD), and Hun‐ tington's diseases (HD). Thus, dysmnesia, mental retardation, and also cancer are seen in these diseases. Many of the neurodegenerative disorders likely occur due to envi‐ ronmental and genetic factors. Especially, probability of AD and PD occurrence rise with increasing age. Briefly, neurodegenerative diseases have similar pathogenesis and cellular mechanisms [6-10].

© 2013 Tutar et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nowadays, mechanisms of protein aggregations and generation of neurodegenerative dis‐ eases with misfolded proteins are not clear at the molecular level. Thus, protein aggregation is a bone to pick for biotechnology and pharmaceutical industries. The aim of this chapter is to bring a perspective to the role of misfolded proteins in neurodegenerative diseases in terms of molecular and cellular basis.

Recently, scientists have been suggested a new protein for understanding of ALS (amyotro‐ phic lateral sclerosis), AD, cystic fibrosis (CF) and frontotemporal lobar degeneration (FTLD) mechanisms. The transactive response DNA binding protein 43 (TDP-43) is ex‐ pressed by all mammalian tissues, conformational changes in this protein cause aggregation and loss of function. TDP-43 has been shown to bind to DNA and mRNA and participate in regulation of transcription and translation. TDP-43 has a glycine rich C-terminal tail and mutation occurs from this region. Consequently, TDP-43 is converted to aggregated form

Role of Protein Aggregation in Neurodegenerative Diseases

http://dx.doi.org/10.5772/54487

57

After a protein is synthesized, the posttranslational modifications (PTM) of amino acids may increase the diversity of proteins by additional functional groups (acetate, phosphate, vari‐ ous proteins etc.) and structural changes [15]. In particular, phosphorylation plays a signifi‐ cant role in neurodegenerative diseases. It is also known that, occurrence of AD is associated with tauopathy due to aggregation of the tau protein. In brain, tau protein is found in neu‐ rons and it can be phosphorylated with kinase enzymes. Thus, aberrant tau aggregates are formed and they can be accumulated in neurons, thereby their toxic effects are caused neu‐

Glycosylation is an important PTM for protein stability and aggregation potential. Human prion protein has two potential *N*-glycosylation sites (Asn181 and Asn197). However, in prion pathology, conversion of PrPc to PrPSc occurs easily if the PrPc is glycosylated. In AD

Hyperphosphorylation and hyperglycosylation are seemed to be required for protein aggregation and misfolding in neurodegenerative diseases. Moreover, the other PTMs such as glycation, nitration, truncation, polyamination etc. involve in protein misfold‐

Oxidative stress leads to protein oxidation which is a biomarker for many neurodege‐ nerative diseases. In particular, free radicals and ROS (reactive oxygen species) cause protein oxidation. A variety of oxidants can be occurred in normal aerobic metabolism [20]. Also, lack of antioxidants, excess of oxygen and lipid and metal ions can gener‐

The oxidation of proteins extremely depends on their amino acid compositions. Generally; lysine, histidine, arginine, methionine, cysteine, phenylalanine, tryptophan, threonine, glu‐ tamic acid, and proline residues incline oxidation. Some proteins have metal binding re‐ gions on its own structure. Metal ions such as copper, zinc, and iron, are capable of redox reactions and electrons are transferred from ions to oxidizing compounds. Therefore, toxic free radicals are formed and proteins can be converted into aggregation forms or proteins

which is accumulated in tissues [14].

*2.1.3. Post-translational modifications*

ronal loss and synaptic alteration [16, 17].

ing diseases [17].

ate free radicals.

*2.1.4. Oxidative stress*

patients, hyperglycosylated tau protein is found in brains [18, 19].

can be aggregated by conformational changes [20, 22].

### **2. Overview of protein aggregation**

Formation of neurodegenerative diseases has not been elucidated for many years. To date, a variety of mechanisms have been suggested for explaining protein misfolding and protein aggregation, however we cannot understand the mechanism clearly at the molecular and cellular basis.

Functional proteins must pass a quality control process in terms of folding to perform catal‐ ysis, cellular transport, signal transmission and regulation. However, variety of structural and environmental factors influence this process negatively [1-3]. In this section, we will fo‐ cus on aggregation behavior of proteins and these factors.

#### **2.1. Factors affecting protein aggregation in neurodegenerative diseases**

#### *2.1.1. Protein structures*

The first critical factor is protein structure. Especially, primary and secondary structures of a protein are two of the most important factors for physical and chemical features.

Encoded information in amino acid sequence of a protein determines the three dimensional structure. Position and number of different characteristic amino acid residues in primary structure may lead to an increase or a decrease in aggregation behavior. Number of hydro‐ phobic amino acids in proteins is proportional to tendency of aggregation [2].

Secondary structures of proteins involve in protein misfolding as well as stability. Proteins often fold locally into stable structures that include α-helix and β-sheet. Generally, some βsheet-rich proteins (such as scrapie infected prion protein) associate with pathological states. During protein aggregation, the secondary structure is converted from α-helix to β-sheet. Thus, protein gets strictness and wide surface area [1, 11, 12].

#### *2.1.2. Mutations*

Mutations play determinative role in protein aggregation and they may dramatically alter solubility, stability, and aggregation tendency of proteins [13].

Thermally stable proteins may change its stability even with a point mutation in its struc‐ ture. For example, a human lysozyme I56T and D67H mutants greatly decreases the lyso‐ zyme stability and as a result the lysozyme aggregates easily upon heating. Further aggregation cause amyloid fibrils and these fibrils are deposited in tissues and are associat‐ ed with neurodegenerative diseases [2].

Recently, scientists have been suggested a new protein for understanding of ALS (amyotro‐ phic lateral sclerosis), AD, cystic fibrosis (CF) and frontotemporal lobar degeneration (FTLD) mechanisms. The transactive response DNA binding protein 43 (TDP-43) is ex‐ pressed by all mammalian tissues, conformational changes in this protein cause aggregation and loss of function. TDP-43 has been shown to bind to DNA and mRNA and participate in regulation of transcription and translation. TDP-43 has a glycine rich C-terminal tail and mutation occurs from this region. Consequently, TDP-43 is converted to aggregated form which is accumulated in tissues [14].

#### *2.1.3. Post-translational modifications*

Nowadays, mechanisms of protein aggregations and generation of neurodegenerative dis‐ eases with misfolded proteins are not clear at the molecular level. Thus, protein aggregation is a bone to pick for biotechnology and pharmaceutical industries. The aim of this chapter is to bring a perspective to the role of misfolded proteins in neurodegenerative diseases in

Formation of neurodegenerative diseases has not been elucidated for many years. To date, a variety of mechanisms have been suggested for explaining protein misfolding and protein aggregation, however we cannot understand the mechanism clearly at the molecular and

Functional proteins must pass a quality control process in terms of folding to perform catal‐ ysis, cellular transport, signal transmission and regulation. However, variety of structural and environmental factors influence this process negatively [1-3]. In this section, we will fo‐

The first critical factor is protein structure. Especially, primary and secondary structures of a

Encoded information in amino acid sequence of a protein determines the three dimensional structure. Position and number of different characteristic amino acid residues in primary structure may lead to an increase or a decrease in aggregation behavior. Number of hydro‐

Secondary structures of proteins involve in protein misfolding as well as stability. Proteins often fold locally into stable structures that include α-helix and β-sheet. Generally, some βsheet-rich proteins (such as scrapie infected prion protein) associate with pathological states. During protein aggregation, the secondary structure is converted from α-helix to β-sheet.

Mutations play determinative role in protein aggregation and they may dramatically alter

Thermally stable proteins may change its stability even with a point mutation in its struc‐ ture. For example, a human lysozyme I56T and D67H mutants greatly decreases the lyso‐ zyme stability and as a result the lysozyme aggregates easily upon heating. Further aggregation cause amyloid fibrils and these fibrils are deposited in tissues and are associat‐

terms of molecular and cellular basis.

cellular basis.

56 Neurodegenerative Diseases

*2.1.1. Protein structures*

*2.1.2. Mutations*

**2. Overview of protein aggregation**

cus on aggregation behavior of proteins and these factors.

**2.1. Factors affecting protein aggregation in neurodegenerative diseases**

protein are two of the most important factors for physical and chemical features.

phobic amino acids in proteins is proportional to tendency of aggregation [2].

Thus, protein gets strictness and wide surface area [1, 11, 12].

solubility, stability, and aggregation tendency of proteins [13].

ed with neurodegenerative diseases [2].

After a protein is synthesized, the posttranslational modifications (PTM) of amino acids may increase the diversity of proteins by additional functional groups (acetate, phosphate, vari‐ ous proteins etc.) and structural changes [15]. In particular, phosphorylation plays a signifi‐ cant role in neurodegenerative diseases. It is also known that, occurrence of AD is associated with tauopathy due to aggregation of the tau protein. In brain, tau protein is found in neu‐ rons and it can be phosphorylated with kinase enzymes. Thus, aberrant tau aggregates are formed and they can be accumulated in neurons, thereby their toxic effects are caused neu‐ ronal loss and synaptic alteration [16, 17].

Glycosylation is an important PTM for protein stability and aggregation potential. Human prion protein has two potential *N*-glycosylation sites (Asn181 and Asn197). However, in prion pathology, conversion of PrPc to PrPSc occurs easily if the PrPc is glycosylated. In AD patients, hyperglycosylated tau protein is found in brains [18, 19].

Hyperphosphorylation and hyperglycosylation are seemed to be required for protein aggregation and misfolding in neurodegenerative diseases. Moreover, the other PTMs such as glycation, nitration, truncation, polyamination etc. involve in protein misfold‐ ing diseases [17].

#### *2.1.4. Oxidative stress*

Oxidative stress leads to protein oxidation which is a biomarker for many neurodege‐ nerative diseases. In particular, free radicals and ROS (reactive oxygen species) cause protein oxidation. A variety of oxidants can be occurred in normal aerobic metabolism [20]. Also, lack of antioxidants, excess of oxygen and lipid and metal ions can gener‐ ate free radicals.

The oxidation of proteins extremely depends on their amino acid compositions. Generally; lysine, histidine, arginine, methionine, cysteine, phenylalanine, tryptophan, threonine, glu‐ tamic acid, and proline residues incline oxidation. Some proteins have metal binding re‐ gions on its own structure. Metal ions such as copper, zinc, and iron, are capable of redox reactions and electrons are transferred from ions to oxidizing compounds. Therefore, toxic free radicals are formed and proteins can be converted into aggregation forms or proteins can be aggregated by conformational changes [20, 22].

#### *2.1.5. Protein concentration*

Protein concentration is an important parameter in protein aggregation. High protein con‐ centration can increase the likelihood of aggregation. Protein-protein interactions and inter‐ molecular interactions (especially interactions among hydrophobic amino acids) may generate abnormal protein structures. Some misfolded protein aggregates can be constituted neurodegenerative diseases above a certain concentration. Moreover, proteins are refolded at low concentrations spontaneously. For example, lysozyme and immunoglobulin G refold itself at low protein concentration however, refolding yield decreases with increasing pro‐ tein concentration. Therefore, the optimum spontaneous protein concentration range is ac‐ cepted as 10-50 µg/ml. [2, 23].

**Neurodegenerative**

**3.1. Prion diseases**

*3.1.1. Structure of PrPc*

(PrPc

The PrPc

**Respective Proteins**

Prion Diseases Prion Protein

Alzheimer's Diseases Tau β-amyloid Protein

Parkinson's Diseases α-Synuclein Protein

Huntington's Diseases Huntington Protein

 *and PrPSc*

while misfolded form is denoted as PrPSc [27].

lism, and protective antioxidant activity [8, 12, 28].

His96 and His111 are found in metal binding domains of PrPc

Cys214 play a significant role for proper folding of PrPc

ine the primary structure of the protein, PrPc

**Table 1.** Properties of neurodegenerative diseases.

**Mechanism Conformation in**

aggregation

aggregation

aggregation

aggregation

The group of prion diseases, including Creutzfeldt-Jakob (CJD), fatal familial insomnia (FFI), Kuru, Gerstmann-Sträussler-Scheinker syndrome (GSS) are seen in humans, and in similar fashion scrapie, bovine spongiform encephalopathy (mad cow disease), chronic wasting diseases (CWD), transmissible mink encephalopathy (TME), feline spongiform en‐ cephalopathy (FSE) diseases are observed in animals. All of these diseases give similar neu‐ rological symptoms such as dysmnesia, depression, sense disturbances, and psychosis [26].

In 1982, prion term coined by Stanley Prusiner and co-workers from "proteinaceous infec‐ tious particle". Prion protein is found in two different forms: a cellular form of prion protein

monly found on neuronal cell membrane by a glycosyl phosphatidylinositol (GPI), however it is also expressed on other cells such as leukocytes and dendritic cells. PrPc has been as‐ sumed a variety of functions including cell adhesion, intracellular signaling, copper metabo‐

PrPc is highly conserved protein among mammals during evolution (Fig.1.). When we exam‐

tide repeats (PHGGGWGQ) (51-91), a highly conserved hydrophobic domain (106-126), and a GPI (glyco-sylphosphatidylinositol) anchor. Furthermore, PrPc contains two N-linked gly‐ cosylation sites (181Asn-Ile-Thr and 197Asn-Phe-Thr). Thus, they get dynamic and flexible properties and the glycan covers prevent intermolecular and intramolecular interactions.

tion sites with metal ions (Cu+2, Zn+2, Mn+2, Ni+2). A disulfide bond between Cys179 and

encoded by the *prion protein gene (Prpn)* which is located on chromosome 20. PrPc

) and scrapie isoform of prion protein (PrPSc). Properly folded form is denoted as PrPc

is an α-helix-rich glycoprotein that is approximately 250 amino acids in length. It is

**Aggregates**

**Inclusion**

http://dx.doi.org/10.5772/54487

59

Senile Plaques

is com‐

β-sheet Spongiosis

Role of Protein Aggregation in Neurodegenerative Diseases

β-sheet Lewy Bodies

consist of a signal peptide (1-22), five octapep‐

[9, 18-20, 24, 27].

and they compose coordina‐

β-sheet Neurofibrillary Tangles

β-sheet Huntington Inclusion

**Diseases**

#### *2.1.6. pH*

Environmental pH is to be critical for protein aggregation due to changes in net charge on protein. Protonation state of ionizable sites of protein and positive net charge are increased in acidic conditions. Especially, organization of salt bridges is changed in parallel with com‐ posed new secondary structures [2,20]. In prion diseases, acidic pH facilitates generation of PrPSc. At low pH, PrPc gains β-sheet structures and shows aggregation tendency [24]. Ac‐ cording to the Finl (2006), α-Syn incubated at different temperature and pH values, and the best formation conditions were determined as pH 7.4 and 37˚C [25]. α-Syn can be lost its na‐ tive structures and PD is accelerated in these conditions.

### **3. Neurodegenerative diseases**

In the world, millions of elder people suffer from neurodegenerative diseases and diagnosis and treatment of these diseases costs millions of dollars per year. Unfortunately, the mecha‐ nisms of these diseases are still unclear and we don't have effective treatment methods. Neurodegenerative diseases are identified as protein misfolding diseases, proteopathies and protein conformational disorders. All diseases (prion, AD, PD, and HD) show typical symp‐ toms: loss and deterioration of neurons and synaptic alterations.

Protein misfolding leads to protein aggregation and accumulation of these aggregates is im‐ plicated as the main reason of neurodegenerative diseases. In brain, some native proteins (prion, tau, β-amyloid, α-synuclein, and huntington) undergo conformational changes via genetic and environmental factors. Therefore, secondary structures of protein convert from α-helix/random coil to β-sheet (Table 1.). Consequently, neurotoxic misfolded protein aggre‐ gates are deposited in central nervous systems and brain damage and neurodegenerative diseases are formed. In this section, we will analyse the most important four neurodegenera‐ tive diseases; prion diseases, AD, PD, and HD, on the basis of protein aggregation and its molecular and cellular mechanisms [6, 8-10].


**Table 1.** Properties of neurodegenerative diseases.

#### **3.1. Prion diseases**

*2.1.5. Protein concentration*

58 Neurodegenerative Diseases

cepted as 10-50 µg/ml. [2, 23].

PrPSc. At low pH, PrPc

tive structures and PD is accelerated in these conditions.

toms: loss and deterioration of neurons and synaptic alterations.

**3. Neurodegenerative diseases**

molecular and cellular mechanisms [6, 8-10].

*2.1.6. pH*

Protein concentration is an important parameter in protein aggregation. High protein con‐ centration can increase the likelihood of aggregation. Protein-protein interactions and inter‐ molecular interactions (especially interactions among hydrophobic amino acids) may generate abnormal protein structures. Some misfolded protein aggregates can be constituted neurodegenerative diseases above a certain concentration. Moreover, proteins are refolded at low concentrations spontaneously. For example, lysozyme and immunoglobulin G refold itself at low protein concentration however, refolding yield decreases with increasing pro‐ tein concentration. Therefore, the optimum spontaneous protein concentration range is ac‐

Environmental pH is to be critical for protein aggregation due to changes in net charge on protein. Protonation state of ionizable sites of protein and positive net charge are increased in acidic conditions. Especially, organization of salt bridges is changed in parallel with com‐ posed new secondary structures [2,20]. In prion diseases, acidic pH facilitates generation of

cording to the Finl (2006), α-Syn incubated at different temperature and pH values, and the best formation conditions were determined as pH 7.4 and 37˚C [25]. α-Syn can be lost its na‐

In the world, millions of elder people suffer from neurodegenerative diseases and diagnosis and treatment of these diseases costs millions of dollars per year. Unfortunately, the mecha‐ nisms of these diseases are still unclear and we don't have effective treatment methods. Neurodegenerative diseases are identified as protein misfolding diseases, proteopathies and protein conformational disorders. All diseases (prion, AD, PD, and HD) show typical symp‐

Protein misfolding leads to protein aggregation and accumulation of these aggregates is im‐ plicated as the main reason of neurodegenerative diseases. In brain, some native proteins (prion, tau, β-amyloid, α-synuclein, and huntington) undergo conformational changes via genetic and environmental factors. Therefore, secondary structures of protein convert from α-helix/random coil to β-sheet (Table 1.). Consequently, neurotoxic misfolded protein aggre‐ gates are deposited in central nervous systems and brain damage and neurodegenerative diseases are formed. In this section, we will analyse the most important four neurodegenera‐ tive diseases; prion diseases, AD, PD, and HD, on the basis of protein aggregation and its

gains β-sheet structures and shows aggregation tendency [24]. Ac‐

The group of prion diseases, including Creutzfeldt-Jakob (CJD), fatal familial insomnia (FFI), Kuru, Gerstmann-Sträussler-Scheinker syndrome (GSS) are seen in humans, and in similar fashion scrapie, bovine spongiform encephalopathy (mad cow disease), chronic wasting diseases (CWD), transmissible mink encephalopathy (TME), feline spongiform en‐ cephalopathy (FSE) diseases are observed in animals. All of these diseases give similar neu‐ rological symptoms such as dysmnesia, depression, sense disturbances, and psychosis [26].

#### *3.1.1. Structure of PrPc and PrPSc*

In 1982, prion term coined by Stanley Prusiner and co-workers from "proteinaceous infec‐ tious particle". Prion protein is found in two different forms: a cellular form of prion protein (PrPc ) and scrapie isoform of prion protein (PrPSc). Properly folded form is denoted as PrPc while misfolded form is denoted as PrPSc [27].

The PrPc is an α-helix-rich glycoprotein that is approximately 250 amino acids in length. It is encoded by the *prion protein gene (Prpn)* which is located on chromosome 20. PrPc is com‐ monly found on neuronal cell membrane by a glycosyl phosphatidylinositol (GPI), however it is also expressed on other cells such as leukocytes and dendritic cells. PrPc has been as‐ sumed a variety of functions including cell adhesion, intracellular signaling, copper metabo‐ lism, and protective antioxidant activity [8, 12, 28].

PrPc is highly conserved protein among mammals during evolution (Fig.1.). When we exam‐ ine the primary structure of the protein, PrPc consist of a signal peptide (1-22), five octapep‐ tide repeats (PHGGGWGQ) (51-91), a highly conserved hydrophobic domain (106-126), and a GPI (glyco-sylphosphatidylinositol) anchor. Furthermore, PrPc contains two N-linked gly‐ cosylation sites (181Asn-Ile-Thr and 197Asn-Phe-Thr). Thus, they get dynamic and flexible properties and the glycan covers prevent intermolecular and intramolecular interactions. His96 and His111 are found in metal binding domains of PrPc and they compose coordina‐ tion sites with metal ions (Cu+2, Zn+2, Mn+2, Ni+2). A disulfide bond between Cys179 and Cys214 play a significant role for proper folding of PrPc [9, 18-20, 24, 27].


**<sup>6</sup>**3 , and, , and **6** 5 , and, , and **<sup>6</sup>**19 , and, , and **Figure 1.** Multiple protein sequence alignment of prion proteins. Full length amino acid sequences of rabbit (NP\_001075490.1), bovine (GenBank: CAA39368.1), human (UniProtKB/Swiss-Prot: P04156.1), and mouse (NP\_035300.1) were aligned using the program Clustal W. PHGGGWGQ repeats are shaded in red. Metal binding sites, His96, and His111 are shaded in blue. Glycosylation sites, 181NIT and 197NFT, are shaded in pink. Finally, Cys179 and Cys214 (disulfide bond sites) are shaded in black.

**7** 10 formation.Cu+2 formation. Cu+2 **7** 15 ofCu+2 of Cu+2 **7** 19 is stability-enhancing is a stability-enhancing **7** 24 layers is play layers play **7** 31 Alzheimer disease Alzheimer's disease PrPSc can be defined as an infectious isoform of PrPc and causes fatal prion diseases. PrPSc is formed by misfolding of PrPc with a lost in α-helical content. PrPSc has same amino acid se‐ quence with PrPc , but their secondary, tertiary, and quarternary structures are different. Ap‐ proximately, PrPc includes 3% β-structure and 47% α-helix structure, but nonetheless PrPSc is composed of 43% β-structure and 30% α-helix structure [24, 29]. It becomes non-soluble and resists to proteolytic degradation with conformational changes, whereas PrPc is soluble and protease sensitive. This insoluble protein accumulates in brain and causes a variety of prion diseases in human and animals.

**7** 34 then disease then the disease

#### **8** 4 in brains in brain *3.1.2. Molecular pathology of Prion diseases*

**8** 5 hearth and hearth, and **8** 8 exon 2, 3 and 10 exon 2, 3, and 10 **8** 13 R1, R2, R3, and, R4 R1, R2, R3, and R4 **8** 21 three or four three to four **9** 3 structure, and, structure, and Previous studies suggested a variety of mechanisms for explaining prion pathology. Oxida‐ tive stress and lipid peroxidation are the major factors in prion diseases [20, 30]. In central nervous systems, a variety of oxidative stresses including high level of oxygen and lipid, metal ions, and inadequate antioxidants produce free radicals. In PrPSc infected mice, super‐ oxide anion (O2 - ) is extremely increased in brain. Therefore, high levels of heme oxygenase-1 and malondialdehyde are observed as oxidative stress markers in brain. Cytochrome c oxi‐ dase is a large transmembrane protein in mitochondria and it shows antioxidant activity in mitochondria [12, 31]. The level of lipid peroxidation is increased while Cytochrome c oxi‐

2

dase activities are reduced in scrapie infected animal models. Phospholipase D catalyzes the hydrolysis of phosphatidylcholine to generate choline and phosphatidic acid, and its expres‐ sion level is induced by H2O2 [12]. According to the studies, activity of phospholipase D is increased in the brains of scrapie infected animals. As a result, PrPc transforms into PrPSc

As we discussed the effect of metal ions on protein misfolding in section 2.1.4, the metal ions also play key roles in prion formation. Cu2+ions play a critical role in prion diseases. PrPc

five conserved octapeptide repeats (PHGGGWGQ) which have an affinity for Cu2+ ions. In contrast, affinity of other metal ions (Mg2+, Mn2+, Ni2+ etc.) is weak or nonexistent. The bind‐ ing of Cu2+ provides formation of protease resistant form: PrPSc. It is also suggested that,

 protects cells from harmful redox activities. Especially, in copper-rich environment, PrPc acts as a "copper buffer" that means it inhibits toxic effects of Cu2+ ions for central nerv‐ ous system and helps maintaining neurons in high level of cupper ions. Thus, redox damage

has been involved in prion diseases. In brief, copper can convert the cellular prion

with membrane lipid layers play a significant role in conversion of PrPc

is bound to lipid membranes through its GPI anchor. While leaving PrPc

to the lipid membranes, PrPc

Role of Protein Aggregation in Neurodegenerative Diseases

http://dx.doi.org/10.5772/54487

is localized in cholesterol and sphingomyelin rich area on cell surface (known

Glycosylation is a stability-enhancing post translational modification in proteins. PrPc con‐ tains two potential glycosylation sites which are Asn181 and Asn197 in human PrPc [36]. Generally, binding of carbohydrates can protect protein surface from proteases and unde‐ sired protein-protein interactions. Moreover, N-glycosylated prion protein is anchored to

from the membranes by catalysis of phosphatidylinositol phospholipase C (PIPLC), PrPSc

Alzheimer's disease (AD) is the most frequent type of neurodegenerative disorder in the world. AD is composed of accumulation of aberrant folded tau protein and beta-amyloid protein (Aβ protein) in brain. In 1906, AD was first described by psychiatrist and pathologist

To date, there are no effective treatment methods for AD, but some nuclear medicine appli‐ cations (MRI and PET) are applied in diagnosis of AD. Dramatically, symptoms of AD rise with increasing age and the first sign is memory lapse. Cellular and molecular mechanisms of AD are not well understood yet. Researchers have been reported that AD is associated

A microtubule associated protein, Tau, is a biggest component of AD. In 1975, tau proteins were first discovered by Marc Kirschner in Princeton University. Tau was derived from

Dr. Alois Alzheimer and then the disease was named with his surname [8-10].

protein into a protease-resistant species with conformational changes [31-35].

has

61

is degraded or

because of the formation of these free radicals.

the lipid membranes via GPI [18, 19].

converted into PrPSc form [37-40].

**3.2. Alzheimer's diseases**

show resistance to PIPLC. When binding of PrPc

with genetic and environmental factors and life-style.

*3.2.1. Structure of Tau gene and proteins*

Interaction of PrPc

as lipid raft). PrPc

to PrPSc. PrPc

PrPc

of PrPc

dase activities are reduced in scrapie infected animal models. Phospholipase D catalyzes the hydrolysis of phosphatidylcholine to generate choline and phosphatidic acid, and its expres‐ sion level is induced by H2O2 [12]. According to the studies, activity of phospholipase D is increased in the brains of scrapie infected animals. As a result, PrPc transforms into PrPSc because of the formation of these free radicals.

As we discussed the effect of metal ions on protein misfolding in section 2.1.4, the metal ions also play key roles in prion formation. Cu2+ions play a critical role in prion diseases. PrPc has five conserved octapeptide repeats (PHGGGWGQ) which have an affinity for Cu2+ ions. In contrast, affinity of other metal ions (Mg2+, Mn2+, Ni2+ etc.) is weak or nonexistent. The bind‐ ing of Cu2+ provides formation of protease resistant form: PrPSc. It is also suggested that, PrPc protects cells from harmful redox activities. Especially, in copper-rich environment, PrPc acts as a "copper buffer" that means it inhibits toxic effects of Cu2+ ions for central nerv‐ ous system and helps maintaining neurons in high level of cupper ions. Thus, redox damage of PrPc has been involved in prion diseases. In brief, copper can convert the cellular prion protein into a protease-resistant species with conformational changes [31-35].

Glycosylation is a stability-enhancing post translational modification in proteins. PrPc con‐ tains two potential glycosylation sites which are Asn181 and Asn197 in human PrPc [36]. Generally, binding of carbohydrates can protect protein surface from proteases and unde‐ sired protein-protein interactions. Moreover, N-glycosylated prion protein is anchored to the lipid membranes via GPI [18, 19].

Interaction of PrPc with membrane lipid layers play a significant role in conversion of PrPc to PrPSc. PrPc is localized in cholesterol and sphingomyelin rich area on cell surface (known as lipid raft). PrPc is bound to lipid membranes through its GPI anchor. While leaving PrPc from the membranes by catalysis of phosphatidylinositol phospholipase C (PIPLC), PrPSc show resistance to PIPLC. When binding of PrPc to the lipid membranes, PrPc is degraded or converted into PrPSc form [37-40].

#### **3.2. Alzheimer's diseases**

Alzheimer's disease (AD) is the most frequent type of neurodegenerative disorder in the world. AD is composed of accumulation of aberrant folded tau protein and beta-amyloid protein (Aβ protein) in brain. In 1906, AD was first described by psychiatrist and pathologist Dr. Alois Alzheimer and then the disease was named with his surname [8-10].

To date, there are no effective treatment methods for AD, but some nuclear medicine appli‐ cations (MRI and PET) are applied in diagnosis of AD. Dramatically, symptoms of AD rise with increasing age and the first sign is memory lapse. Cellular and molecular mechanisms of AD are not well understood yet. Researchers have been reported that AD is associated with genetic and environmental factors and life-style.

#### *3.2.1. Structure of Tau gene and proteins*

2

is soluble

**Rabbit** --MAHLGYWMLLLFVATWSDVGLCKKRPKPGGGWNTGGSRYPGQSSPGGN **Bovine** MVKSHIGSWILVLFVAMWSDVGLCKKRPKPGGGWNTGGSRYPGQGSPGGN **Human** --MANLGCWMLVLFVATWSDLGLCKKRPKPGG-WNTGGSRYPGQGSPGGN **Mouse** --MANLGYWLLALFVTMWTDVGLCKKRPKPGG-WNTGGSRYPGQGSPGGN **Rabbit** RYP**PQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQ**------ **Bovine** RYP**PQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQ**P**H**GGGG **Human** RYP**PQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQ**------ **Mouse** RYP**PQGG**-T**WGQPHGGGWGQPHGGSWGQPHGGSWGQPHGGGWGQ**------ **Rabbit** --GGGT**H**NQWGKPSKPKTSMKHVAGAAAAGAVVGGLGGYMLGSAMSRPLI **Bovine** WGQGGT**H**GQWNKPSKPKTNMKHVAGAAAAGAVVGGLGGYMLGSAMSRPLI **Human** --GGGT**H**SQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPII **Mouse** --GGGT**H**NQWNKPSKPKTNLKHVAGAAAAGAVVGGLGGYMLGSAMSRPMI **Rabbit** HFGNDYEDRYYRENMYRYPNQVYYRPVDQYSNQNSFVHDCV**NIT**VKQHTV **Bovine** HFGSDYEDRYYRENMHRYPNQVYYRPVDQYSNQNNFVHDCV**NIT**VKEHTV **Human** HFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCV**NIT**IKQHTV **Mouse** HFGNDWEDRYYRENMYRYPNQVYYRPVDQYSNQNNFVHD**C**V**NIT**IKQHTV **Rabbit** TTTTKGE**NFT**ETDIKIMERVVEQMCITQYQQESQAAYQ--RAAGVLLFSS **Bovine** TTTTKGE**NFT**ETDIKMMERVVEQMCITQYQRESQAYYQ--RGASVILFSS **Human** TTTTKGE**NFT**ETDVKMMERVVEQMCITQYERESQAYYQ--RGSSMVLFSS **Mouse** TTTTKGE**NFT**ETDVKMMERVVEQM**C**VTQYQKESQAYYDGRRSSSTVLFSS

**Figure 1.** Multiple protein sequence alignment of prion proteins. Full length amino acid sequences of rabbit (NP\_001075490.1), bovine (GenBank: CAA39368.1), human (UniProtKB/Swiss-Prot: P04156.1), and mouse (NP\_035300.1) were aligned using the program Clustal W. PHGGGWGQ repeats are shaded in red. Metal binding sites, His96, and His111 are shaded in blue. Glycosylation sites, 181NIT and 197NFT, are shaded in pink. Finally, Cys179

is composed of 43% β-structure and 30% α-helix structure [24, 29]. It becomes non-soluble

and protease sensitive. This insoluble protein accumulates in brain and causes a variety of

Previous studies suggested a variety of mechanisms for explaining prion pathology. Oxida‐ tive stress and lipid peroxidation are the major factors in prion diseases [20, 30]. In central nervous systems, a variety of oxidative stresses including high level of oxygen and lipid, metal ions, and inadequate antioxidants produce free radicals. In PrPSc infected mice, super‐

and malondialdehyde are observed as oxidative stress markers in brain. Cytochrome c oxi‐ dase is a large transmembrane protein in mitochondria and it shows antioxidant activity in mitochondria [12, 31]. The level of lipid peroxidation is increased while Cytochrome c oxi‐

formation. Cu+2

, but their secondary, tertiary, and quarternary structures are different. Ap‐

includes 3% β-structure and 47% α-helix structure, but nonetheless PrPSc

) is extremely increased in brain. Therefore, high levels of heme oxygenase-1

with a lost in α-helical content. PrPSc has same amino acid se‐

and causes fatal prion diseases. PrPSc is

**Rabbit** PPVILLISFLIFLIVG **Bovine** PPVILLISFLIFLIVG **Human** PPVILLISFLIFLIVG **Mouse** PPVILLISFLIFLIVG

**<sup>6</sup>**3 , and, , and

**6** 5 , and, , and **<sup>6</sup>**19 , and, , and

**7** 15 ofCu+2 of Cu+2

PrPSc can be defined as an infectious isoform of PrPc

**7** 24 layers is play layers play

**8** 4 in brains in brain

**8** 5 hearth and hearth, and

**8** 21 three or four three to four **9** 3 structure, and, structure, and

**8** 8 exon 2, 3 and 10 exon 2, 3, and 10 **8** 13 R1, R2, R3, and, R4 R1, R2, R3, and R4

**7** 19 is stability-enhancing is a stability-enhancing

and resists to proteolytic degradation with conformational changes, whereas PrPc

**7** 31 Alzheimer disease Alzheimer's disease

**7** 34 then disease then the disease

**7** 10 formation.Cu+2

formed by misfolding of PrPc

quence with PrPc

60 Neurodegenerative Diseases

proximately, PrPc

oxide anion (O2

and Cys214 (disulfide bond sites) are shaded in black.

prion diseases in human and animals.

*3.1.2. Molecular pathology of Prion diseases*


A microtubule associated protein, Tau, is a biggest component of AD. In 1975, tau proteins were first discovered by Marc Kirschner in Princeton University. Tau was derived from "**t**ubulin **a**ssociated **u**nit" as a term. It is highly expressed in brain, but other organs such as lung, hearth, and kidney have trace amounts. Many animals (bovine, goat, monkey, goldfish etc.) also have tau proteins [41].

*3.2.2. Aggregation of Tau protein*

phosphorylation [17, 45, 46].

bule cannot function correctly [16, 50].

*3.2.3. Structure of Aβ protein and gene*

tive diseases including AD.

Microtubules are major proteins of the cytoskeleton. They have hollow and cylindrical struc‐ ture and participate in intracellular transport, protection cell structure, and continuity of cell viability. The main function of the tau protein is to stabilize microtubules with binding to microtubules and to other proteins [16, 41]. To perform these functions, tau proteins must be phosphorylated at normal level. However, if tau protein hyperphosphorylate, its biological activity can be lost. Moreover, hyperphosphorylation causes conformational changes and aggregation of tau proteins. Other post-translational modifications such as glycosylation,

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The longest isoform of tau protein (441 amino acids) has seventy nine Ser or Thr phosphory‐ lation sites which are mainly found on prolin-rich regions. Also, Ser262, Ser293, Ser324, and Ser356 are located in KXGS motif of R1, R2, R3, and R4 domains. Many of kinases and phos‐ phatases (glycogen synthase kinase-3β (GSK3β), mitogen activated protein kinase (MAPK), tau tubulin kinase 1-2 (TTBK1/2), cyclin dependent kinase 5 (CDK5), microtubule affinity regulating kinase (MARK), and stress activated protein kinase (SAP) are affected in tau

Frontotemporal dementia with parkinsonism-17 (FTDP-17) is a progressive neurodegenera‐ tive disease which is caused by mutations in the tau gene. The tau gene is mutated in fami‐ lial FTDP-17 and this mutation accelerates formation of neurofibrillary tangles (NFTs) in the

Excess of NFTs and senile plaques (SPs) are important markers in AD. NFTs are aggregates of hyperphosphorylated tau protein that are most commonly known as a primary marker of AD [50]. NFTs are originated from abnormally hyperphosphorylated tau protein. Normally, tau is a microtubule binding protein that stabilizes and assembles microtubules. However, in AD, tau protein undergoes biochemical changes because it twists into pairs of helical fila‐ ments and they twist into tangles. Also, tau is generally located in axons, but in tauopathy, it is located in dendrites. Thus, neuron's transport system may be disintegrated and microtu‐

Aβ is a relatively small peptide of 4 to 4.4 kDa that is the major component of amyloid de‐ posits. Intracellular Aβ protein is widely found in neurons and it is associated with inflam‐ matory and antioxidant activity, regulation of cholesterol transport, and activation of kinase enzyme. However, Aβ is one of the best known components in formation of neurodegenera‐

Aβ is approximately composed of 36-43 amino acids and it originates from amyloid precur‐ sor protein (APP). In human, *APP gene* is encoded on chromosome 21 and contains at least 18 exons. APP is a glycoprotein of 695-770 amino acids which has three main regions: an ex‐ tracellular N-terminal region, a hydrophobic transmembrane region, and cytoplasmic C-ter‐ minal region. Mutations in APP gene cause familial susceptibility to AD. Furthermore, mutations in other three genes, including apoE, PS1, and PS2 are associated with AD and

brain. Furthermore, hyperphosphorylation is promoted by this mutation [47,49].

glycation, polyamination, and nitration may play essential roles in AD [17].

The human *tau gene* is located on chromosome 17q21 and contains 16 exons. Among these exons of the tau gene, exon 2, 3, and 10 are alternatively spliced and these exons allow six combinations (2-3-10-; 2+3-10-; 2+3+10-; 2-3-10+; 2+3-10+; 2+3+10+). Thus, human brain con‐ tains six isoforms of tau proteins which range from 352 to 441 amino acids length and ap‐ proximate molecular weights are between 60 and 70 kDa (Fig. 2.) [16, 17].

**Figure 2.** Schematic representation of six human tau isoforms. R1, R2, R3, and R4 (blue boxes) indicate repeat regions. E2 and E3 (orange and yellow box) indicate Exon 2 and Exon 3 respectively (Redrawn from Buee et al., 2000).

Tau protein has four main regions in its primary structures. Acidic region is located in the N-terminal part and it is encoded by exon 2 and exon 3. Prolin-rich region located in the middle of the protein, is encoded by exon 7 and exon 9 and contains several PXXP motifs which can interact with tyrosine kinase. Prolin-rich region works together with acidic re‐ gion, therefore these two regions are called projection domain which interacts with neural plasma membrane and cytoskeletal elements [16, 17, 41-43].

Tau protein has three to four highly conserved repeats in the C-terminal part for binding to microtubules. Therefore, these repetitive regions are called microtubule binding domains (MBDs) which is encoded by exons 9-12. 275VQJINK280 and 306VQJVYK311are conserved hexa‐ peptides which are located at the beginning of the second and third MBDs. These peptides involve in the generation of β-sheet structure during tauopathy [44].

#### *3.2.2. Aggregation of Tau protein*

"**t**ubulin **a**ssociated **u**nit" as a term. It is highly expressed in brain, but other organs such as lung, hearth, and kidney have trace amounts. Many animals (bovine, goat, monkey, goldfish

The human *tau gene* is located on chromosome 17q21 and contains 16 exons. Among these exons of the tau gene, exon 2, 3, and 10 are alternatively spliced and these exons allow six combinations (2-3-10-; 2+3-10-; 2+3+10-; 2-3-10+; 2+3-10+; 2+3+10+). Thus, human brain con‐ tains six isoforms of tau proteins which range from 352 to 441 amino acids length and ap‐

N R1 R3 R4 C **2-3-10-**

proximate molecular weights are between 60 and 70 kDa (Fig. 2.) [16, 17].

N E2 E3 R1 R2 R3 R4 C

plasma membrane and cytoskeletal elements [16, 17, 41-43].

involve in the generation of β-sheet structure during tauopathy [44].

**Figure 2.** Schematic representation of six human tau isoforms. R1, R2, R3, and R4 (blue boxes) indicate repeat regions. E2 and E3 (orange and yellow box) indicate Exon 2 and Exon 3 respectively (Redrawn from Buee et al., 2000).

Tau protein has four main regions in its primary structures. Acidic region is located in the N-terminal part and it is encoded by exon 2 and exon 3. Prolin-rich region located in the middle of the protein, is encoded by exon 7 and exon 9 and contains several PXXP motifs which can interact with tyrosine kinase. Prolin-rich region works together with acidic re‐ gion, therefore these two regions are called projection domain which interacts with neural

Tau protein has three to four highly conserved repeats in the C-terminal part for binding to microtubules. Therefore, these repetitive regions are called microtubule binding domains (MBDs) which is encoded by exons 9-12. 275VQJINK280 and 306VQJVYK311are conserved hexa‐ peptides which are located at the beginning of the second and third MBDs. These peptides

N E2 C

N R1 R3 R4 C

N E2 E3 R1 R3 R4 C

N E2 R1 R3 R4 C

R2

R1 R2 R3 R4

**2+3-10-**

**2+3+10-**

**2-3-10+**

**2+3-10+**

**2+3+10+**

etc.) also have tau proteins [41].

62 Neurodegenerative Diseases

Microtubules are major proteins of the cytoskeleton. They have hollow and cylindrical struc‐ ture and participate in intracellular transport, protection cell structure, and continuity of cell viability. The main function of the tau protein is to stabilize microtubules with binding to microtubules and to other proteins [16, 41]. To perform these functions, tau proteins must be phosphorylated at normal level. However, if tau protein hyperphosphorylate, its biological activity can be lost. Moreover, hyperphosphorylation causes conformational changes and aggregation of tau proteins. Other post-translational modifications such as glycosylation, glycation, polyamination, and nitration may play essential roles in AD [17].

The longest isoform of tau protein (441 amino acids) has seventy nine Ser or Thr phosphory‐ lation sites which are mainly found on prolin-rich regions. Also, Ser262, Ser293, Ser324, and Ser356 are located in KXGS motif of R1, R2, R3, and R4 domains. Many of kinases and phos‐ phatases (glycogen synthase kinase-3β (GSK3β), mitogen activated protein kinase (MAPK), tau tubulin kinase 1-2 (TTBK1/2), cyclin dependent kinase 5 (CDK5), microtubule affinity regulating kinase (MARK), and stress activated protein kinase (SAP) are affected in tau phosphorylation [17, 45, 46].

Frontotemporal dementia with parkinsonism-17 (FTDP-17) is a progressive neurodegenera‐ tive disease which is caused by mutations in the tau gene. The tau gene is mutated in fami‐ lial FTDP-17 and this mutation accelerates formation of neurofibrillary tangles (NFTs) in the brain. Furthermore, hyperphosphorylation is promoted by this mutation [47,49].

Excess of NFTs and senile plaques (SPs) are important markers in AD. NFTs are aggregates of hyperphosphorylated tau protein that are most commonly known as a primary marker of AD [50]. NFTs are originated from abnormally hyperphosphorylated tau protein. Normally, tau is a microtubule binding protein that stabilizes and assembles microtubules. However, in AD, tau protein undergoes biochemical changes because it twists into pairs of helical fila‐ ments and they twist into tangles. Also, tau is generally located in axons, but in tauopathy, it is located in dendrites. Thus, neuron's transport system may be disintegrated and microtu‐ bule cannot function correctly [16, 50].

#### *3.2.3. Structure of Aβ protein and gene*

Aβ is a relatively small peptide of 4 to 4.4 kDa that is the major component of amyloid de‐ posits. Intracellular Aβ protein is widely found in neurons and it is associated with inflam‐ matory and antioxidant activity, regulation of cholesterol transport, and activation of kinase enzyme. However, Aβ is one of the best known components in formation of neurodegenera‐ tive diseases including AD.

Aβ is approximately composed of 36-43 amino acids and it originates from amyloid precur‐ sor protein (APP). In human, *APP gene* is encoded on chromosome 21 and contains at least 18 exons. APP is a glycoprotein of 695-770 amino acids which has three main regions: an ex‐ tracellular N-terminal region, a hydrophobic transmembrane region, and cytoplasmic C-ter‐ minal region. Mutations in APP gene cause familial susceptibility to AD. Furthermore, mutations in other three genes, including apoE, PS1, and PS2 are associated with AD and increased production of Aβ protein and amyloidogenicity. In contrast, a coding mutation (A673T) in APP gene shows protective effects for AD [51-53].

α-Syn is encoded with *SNCA gene* which is located on chromosome 4q21. Human α-Syn has 140 amino acids and three main domains: **(i)** an amphipathic N-terminal region (residues 1-60), **(ii)** a central hydrophobic region (residues 61-95) and **(iii)** a highly acidic C-terminal region (residues 96-140) (Fig. 3.). Amphipathic N-terminal region contains imperfect six hex‐

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amer motif repeats (KTKEGV) which involve in binding of micelles and liposomes.

Acidic residues are colored in red. Phosphorylation sites (Ser87 and Ser129) are colored in yellow.

2 1 MDVFMKGLS**K**A**KEGV**VAAAE**KTK**Q**GV**AEAAG**KTKEGV**LYVGS**KTKEGV**VHGVATVAE**KTK** 3 61 **E**Q**V**TNVGGAVVTGVTAVAQ**KT**V**EG**AG**S**IAAATGFVKK**D**QLGKN**EE**GAPQ**E**GIL**ED**MPV**D**P

**Figure 3.** Primary structure of human α-Syn (Gen Bank: AAL15443.1). Imperfect KTKEGV repeats are colored in blue.

Central hydrophobic region contains non-amyloid beta component (NAC) sequences from residue 61 to 95. This region is highly hydrophobic, and it can promote formation of β-sheet structure. The less conserved C-terminal region consists of a large number of acidic amino

At present, biological role and pathogenic processes of Lewy bodies are still unclear. As far as we know, Lewy bodies are aberrant protein aggregates and their deposits cause PD. Lewy bodies are localized not only in PD brains, but also in other neurodegenerative disor‐ ders such as AD brains. According to the electron microscopy images, Lewy bodies are 8-30 µm which are consisted of approximately 10 nm amyloidogenic fibrils such as fibrillary α-Syn and neurofilaments. Lewy bodies contain a variety of proteins including α-Syn, neurofi‐ laments, ubiquitinated proteins, and heat shock proteins (Hsp70 and Hsp90). Oxidative stress, mitochondrial dysfunction, inflammation, ubiquitin proteasome system, pH, protein concentration, and high temperature may be affected negatively by Lewy bodies. Therefore, these factors induce misfolding and aggregation of proteins in the Lewy bodies [57, 61].

α-Syn plays crucial role in PD because α-Syn is a major fibrillary component for Lewy bod‐ ies. There are many adjuvant and disincentive factors available in α-Synfibrillogenesis. Two mutations, A53T and A30P, in the α-Syn gene and overexpression of wild type α-Syn are increased misfolding processes and aggregation. Also, accumulation of abnormal form of α-

It is known that, α-Syn is natively unfolded as well as predominantly non-phosphorylated *in vivo*. In PD brains, α-Syn is found to be phosphorylated at Ser87 and Ser129 in aggregates. These serine residues are phosphorylated with casein kinase 1 (CK1) and casein kinase 2 (CK2). Several studies reported that accumulation of phosphorylated α-Syn was observed in animal models of synucleinopathies. Therefore, this post translational modification has a

1

5

4 121 **D**N**E**AY**E**MP**SEE**GYQ**D**Y**E**P**E**A

*3.3.3. Mechanisms of α-Syn misfolding and aggregation*

Syn can inhibit proteasomal functions [9, 61, 62].

pathological role in fibrillation of α-Syn [63, 64].

acids and several prolines [56-60].

*3.3.2. Structures of Lewy bodies*

APP can be cleaved into fragments by α, β, and γ secretases and Aβ protein is formed by the action of the β and γ secretases. Aβ protein contains two important regions which play a major role in the formation insoluble amyloid fibrils. C-terminal regions (residues 32 to 42) and internal hydrophobic regions (residues 16 to 23) may enhance increasing β-sheet confor‐ mation and Aβ protein misfolding [53].

#### *3.2.4. Aggregation mechanisms of Aβ protein*

In normal brain, Aβ proteins contain mixture of β-sheet and random coil structures. Howev‐ er, a number of β-sheet structures are increased at high protein concentration. Aβ protein constitutes SPs which are important markers in AD. The formation of SPs is a problem of protein folding because of misfolded and aggregated form of Aβ accumulates and shows toxicity in brain [54].

Genetically, three genes: *APP*, *PS1,* and, *PS2* are associated with AD. More than 50 different mutations in the APP gene can cause AD. The most frequent APP mutation is a single muta‐ tion in the APP at position 717. As a result of this single mutation, a valine residue is re‐ placed by an isoleucine, or phenylalanine, or glycine. *APP* mutations lead to an increased amount of the Aβ proteins which are deposited in neuritic plaques [50, 54].

#### **3.3. Parkinson's diseases**

Parkinson's disease (PD) is neurodegenerative movement disorder of the central nervous sys‐ tems. In 1817, Dr. James Parkinson published "*An Essay on the Shaking Palsy*" in which he first described paralysis agitans and then this disorder was entitled as a Parkinson's disease by Dr. Jean Martin Charcot. The occurrence of an illness is characterized by accumulation of misfold‐ ed α-synuclein protein in brain. Generally; anxiety, tremor, rigidity, depression, bradykinesia, and postural abnormalities are the most common symptoms in Parkinson's disease. Also, α-Syn related neurodegenerative diseases are known as "Synucleinopathies" [55].

#### *3.3.1. Structure of α-synuclein*

Natively unfolded α-syncline (α-Syn) is a 14 kDa and highly conserved protein that localize different regions of the brain. The name of protein was preferred as "α-synuclein" because of it shows **syn**aptic and **nuc**lear localization. α-Syn regulates dopamine neurotransmission by modulation of vesicular dopamine storage. It interacts with tubulin and can function like tau protein. Also, α-Syn shows a molecular chaperon activity in folding of SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins [56].

Natively, α-Syn is an unfolded protein, but obtains its conformation with biological interac‐ tions. In cytoplasm, α-Syn is a soluble and in an unfolded state, but it can be found in αhelical conformation for binding to lipid membranes. Also, α-Syn can be in the form of βsheet for composing Lewy bodies as explained below [56-58].

α-Syn is encoded with *SNCA gene* which is located on chromosome 4q21. Human α-Syn has 140 amino acids and three main domains: **(i)** an amphipathic N-terminal region (residues 1-60), **(ii)** a central hydrophobic region (residues 61-95) and **(iii)** a highly acidic C-terminal region (residues 96-140) (Fig. 3.). Amphipathic N-terminal region contains imperfect six hex‐ amer motif repeats (KTKEGV) which involve in binding of micelles and liposomes.

**Figure 3.** Primary structure of human α-Syn (Gen Bank: AAL15443.1). Imperfect KTKEGV repeats are colored in blue. Acidic residues are colored in red. Phosphorylation sites (Ser87 and Ser129) are colored in yellow.

Central hydrophobic region contains non-amyloid beta component (NAC) sequences from residue 61 to 95. This region is highly hydrophobic, and it can promote formation of β-sheet structure. The less conserved C-terminal region consists of a large number of acidic amino acids and several prolines [56-60].

#### *3.3.2. Structures of Lewy bodies*

1

5

increased production of Aβ protein and amyloidogenicity. In contrast, a coding mutation

APP can be cleaved into fragments by α, β, and γ secretases and Aβ protein is formed by the action of the β and γ secretases. Aβ protein contains two important regions which play a major role in the formation insoluble amyloid fibrils. C-terminal regions (residues 32 to 42) and internal hydrophobic regions (residues 16 to 23) may enhance increasing β-sheet confor‐

In normal brain, Aβ proteins contain mixture of β-sheet and random coil structures. Howev‐ er, a number of β-sheet structures are increased at high protein concentration. Aβ protein constitutes SPs which are important markers in AD. The formation of SPs is a problem of protein folding because of misfolded and aggregated form of Aβ accumulates and shows

Genetically, three genes: *APP*, *PS1,* and, *PS2* are associated with AD. More than 50 different mutations in the APP gene can cause AD. The most frequent APP mutation is a single muta‐ tion in the APP at position 717. As a result of this single mutation, a valine residue is re‐ placed by an isoleucine, or phenylalanine, or glycine. *APP* mutations lead to an increased

Parkinson's disease (PD) is neurodegenerative movement disorder of the central nervous sys‐ tems. In 1817, Dr. James Parkinson published "*An Essay on the Shaking Palsy*" in which he first described paralysis agitans and then this disorder was entitled as a Parkinson's disease by Dr. Jean Martin Charcot. The occurrence of an illness is characterized by accumulation of misfold‐ ed α-synuclein protein in brain. Generally; anxiety, tremor, rigidity, depression, bradykinesia, and postural abnormalities are the most common symptoms in Parkinson's disease. Also, α-

Natively unfolded α-syncline (α-Syn) is a 14 kDa and highly conserved protein that localize different regions of the brain. The name of protein was preferred as "α-synuclein" because of it shows **syn**aptic and **nuc**lear localization. α-Syn regulates dopamine neurotransmission by modulation of vesicular dopamine storage. It interacts with tubulin and can function like tau protein. Also, α-Syn shows a molecular chaperon activity in folding of SNARE (soluble

Natively, α-Syn is an unfolded protein, but obtains its conformation with biological interac‐ tions. In cytoplasm, α-Syn is a soluble and in an unfolded state, but it can be found in αhelical conformation for binding to lipid membranes. Also, α-Syn can be in the form of β-

amount of the Aβ proteins which are deposited in neuritic plaques [50, 54].

Syn related neurodegenerative diseases are known as "Synucleinopathies" [55].

N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins [56].

sheet for composing Lewy bodies as explained below [56-58].

(A673T) in APP gene shows protective effects for AD [51-53].

mation and Aβ protein misfolding [53].

*3.2.4. Aggregation mechanisms of Aβ protein*

toxicity in brain [54].

64 Neurodegenerative Diseases

**3.3. Parkinson's diseases**

*3.3.1. Structure of α-synuclein*

At present, biological role and pathogenic processes of Lewy bodies are still unclear. As far as we know, Lewy bodies are aberrant protein aggregates and their deposits cause PD. Lewy bodies are localized not only in PD brains, but also in other neurodegenerative disor‐ ders such as AD brains. According to the electron microscopy images, Lewy bodies are 8-30 µm which are consisted of approximately 10 nm amyloidogenic fibrils such as fibrillary α-Syn and neurofilaments. Lewy bodies contain a variety of proteins including α-Syn, neurofi‐ laments, ubiquitinated proteins, and heat shock proteins (Hsp70 and Hsp90). Oxidative stress, mitochondrial dysfunction, inflammation, ubiquitin proteasome system, pH, protein concentration, and high temperature may be affected negatively by Lewy bodies. Therefore, these factors induce misfolding and aggregation of proteins in the Lewy bodies [57, 61].

#### *3.3.3. Mechanisms of α-Syn misfolding and aggregation*

α-Syn plays crucial role in PD because α-Syn is a major fibrillary component for Lewy bod‐ ies. There are many adjuvant and disincentive factors available in α-Synfibrillogenesis. Two mutations, A53T and A30P, in the α-Syn gene and overexpression of wild type α-Syn are increased misfolding processes and aggregation. Also, accumulation of abnormal form of α-Syn can inhibit proteasomal functions [9, 61, 62].

It is known that, α-Syn is natively unfolded as well as predominantly non-phosphorylated *in vivo*. In PD brains, α-Syn is found to be phosphorylated at Ser87 and Ser129 in aggregates. These serine residues are phosphorylated with casein kinase 1 (CK1) and casein kinase 2 (CK2). Several studies reported that accumulation of phosphorylated α-Syn was observed in animal models of synucleinopathies. Therefore, this post translational modification has a pathological role in fibrillation of α-Syn [63, 64].

As known, oxidative stress is one of the major factors in many diseases as well as the formation of PD [9, 20]. As a result of oxidation, formed free radicals react rapidly with proteins thus, misfolding and aggregation are generated inside the cells. Among twenty amino acids, methionine and cysteine are capable of being easily oxidized. α-Syn doesn't have cysteine residues, but high content of methionine residues oxidize to methionine sulfoxide. Thus, due to methionine content of α-Syn, the protein is readily aggregates at oxidation conditions. On the other hand, α-Syn phosphorylation can be increased with oxidative stresses as well [65].

er CAG repeats are never generated neuropathy. However, in childhood, CAG repeats

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In 1994, Max Perutz put forward a "**polar zipper**" hypothesis about HD pathology. In this model, many polar glutamine residues can generate anti parallel β-sheet structures with hy‐ drogen bonds. Therefore, aggregation tendency of Htt protein is increased and this state lead to cell death [73-75]. Furthermore, aggregation of Htt protein induces oxidative stress,

**4. Prevention of neurodegenerative diseases and molecular chaperones**

Until this section, neurodegenerative diseases have been discussed on the basis of protein aggregation and misfolding. In healthy organisms, a variety of mechanisms work efficiently for prevention of preteopathies. Molecular chaperones are known to be critical for protein folding processes and neurodegenerative diseases. Heat shock proteins (Hsps) are well-

Hsps are highly conserved proteins among living organisms. Hsps are an important class of molecular chaperons and they are located in different parts of the cells such as endo‐ plasmic reticulum, cytosol, and mitochondria. Mainly, Hsps are related with formation of proper protein conformation, and also prevention protein aggregation, misfolding, and oligomerization. Proteins can be exposed a number of cellular and environmental factors including high temperature, inflammation, growth factors, oxidative stress etc. which can cause misfolding and protein aggregation. Overexpression of Hsps has been observed under these stress conditions. Several studies have focused on the neuroprotective role of Hsps. Therefore, the expression levels of Hsps are decreased and misfolded protein accu‐

Generally, Hsps are divided into six groups on the basis of molecular mass. In this section; Hsp40, Hsp60, Hsp70, Hsp90, Hsp100, and small Hsp (sHsp) have been examined and char‐ acterized for association with neurodegenerative diseases and protein folding processes [1].

Hsp70 is a highly conserved protein in all living organisms. It makes complex with unfolded or partially denatured proteins. Hsp70 has two functional domains: ATPase domain and substrate binding domain (SBD). The operation of these domains is controlled with hydroly‐ sis of ATP. ATPase domain binds to ATP and hydrolyzes it to ADP. This energy drives the protein folding function of the Hsp70. Similarly, Hsp70 binding to misfolded peptides, in‐ creases the ATP hydrolysis. Also, Hsp70 interacts with Hsp40 and Hsp90 to perform protein folding process. Hsp70 serves a neuroprotective role in all of neurodegenerative diseases [79-81]. Auluck and co-workers indicated that, expression of Hsp70 reduced α-Syn aggrega‐ tion, accumulation, and toxicity in PD animal models [82]. In HD models, Hsp70 shows pro‐ tective assignment in polyglutamine-induced toxicity [83]. Also, Hsp70 is involved in the

from 27 to 35 can develop neuropathy [66, 70-72].

mitochondria dysfunction, lipid peroxidation etc.

known molecular chaperons in living organisms [1, 76-78].

mulation can be occurred in brain [1, 76-78].

**4.1. The Hsp chaperons**

#### **3.4. Huntington's diseases**

Huntington's disease (HD) is a genetic neurodegenerative disorder and the disease is caused by autosomal dominant inheritance. In 1872, HD was first described as a genetic disease by Dr. George Huntington. Involuntary muscle contractions, movement, and mental disorders are progressed in HD. The disease is inherited as an autosomal dominant and effects brain and nervous systems. Huntington protein undergoes conformational changes with mutation and it shows aggregation tendency [66].

#### *3.4.1. Structure of Huntington protein and genes*

Huntington (Htt) is a large size protein of 350 kDa that is generally composed of 3144 amino acids. Normal protein is highly expressed in peripheral tissues and brain, and it is involved in endocytosis, cytosketal functions, vesicle trafficking, cellular signal transduction, and membrane recycling. In brains, Htt protein leads to cell damage and toxicity through depo‐ sition of misfolded aggregate form of Htt protein [8, 9].

The gene for HD is located on the tip of chromosome 4, and is called the *IT-15 gene*. This part of DNA contains cytosine-adenine-guanine (CAG) repeats which are called tri‐ nucleotide repeats. Number of trinucleotide repeats is determined as risk of HD develop‐ ment. The CAG repeats are translated into polyglutamine (polyQ) residues which are located in the N-terminal region [66-68].

Htt proteins interact with a variety of peptides including huntington associated protein 1 (HAP1), huntington interacting protein 1 and 2 (HIP1 and HIP2) and huntington yeast part‐ ners A, B, and C (HYPA, HYPB, and HYPC). These peptides are functioned in cell signaling, transport, and transcription processes [67, 69].

#### *3.4.2. Mechanisms of Huntington misfolding and aggregation*

In HD, the neuropathology is characterized with accumulation of Htt protein aggregates. HD is caused by a number of CAG repeats in the gene. To date, many theories have been suggested in HD, but functions of CAG repeats and mechanisms of HD cannot be understood yet. However, common opinion is long CAG repeats (polyQ) are the most important promoter for toxicity of Htt protein aggregates. The polyQ region starts at res‐ idue 18 and the number of glutamine residues are the most important marker in HD. Surprisingly, 40 or more CAG repeats are always generated neuropathy, while 35 or few‐ er CAG repeats are never generated neuropathy. However, in childhood, CAG repeats from 27 to 35 can develop neuropathy [66, 70-72].

In 1994, Max Perutz put forward a "**polar zipper**" hypothesis about HD pathology. In this model, many polar glutamine residues can generate anti parallel β-sheet structures with hy‐ drogen bonds. Therefore, aggregation tendency of Htt protein is increased and this state lead to cell death [73-75]. Furthermore, aggregation of Htt protein induces oxidative stress, mitochondria dysfunction, lipid peroxidation etc.

### **4. Prevention of neurodegenerative diseases and molecular chaperones**

Until this section, neurodegenerative diseases have been discussed on the basis of protein aggregation and misfolding. In healthy organisms, a variety of mechanisms work efficiently for prevention of preteopathies. Molecular chaperones are known to be critical for protein folding processes and neurodegenerative diseases. Heat shock proteins (Hsps) are wellknown molecular chaperons in living organisms [1, 76-78].

#### **4.1. The Hsp chaperons**

As known, oxidative stress is one of the major factors in many diseases as well as the formation of PD [9, 20]. As a result of oxidation, formed free radicals react rapidly with proteins thus, misfolding and aggregation are generated inside the cells. Among twenty amino acids, methionine and cysteine are capable of being easily oxidized. α-Syn doesn't have cysteine residues, but high content of methionine residues oxidize to methionine sulfoxide. Thus, due to methionine content of α-Syn, the protein is readily aggregates at oxidation conditions. On the other hand, α-Syn phosphorylation can be increased with

Huntington's disease (HD) is a genetic neurodegenerative disorder and the disease is caused by autosomal dominant inheritance. In 1872, HD was first described as a genetic disease by Dr. George Huntington. Involuntary muscle contractions, movement, and mental disorders are progressed in HD. The disease is inherited as an autosomal dominant and effects brain and nervous systems. Huntington protein undergoes conformational changes with mutation

Huntington (Htt) is a large size protein of 350 kDa that is generally composed of 3144 amino acids. Normal protein is highly expressed in peripheral tissues and brain, and it is involved in endocytosis, cytosketal functions, vesicle trafficking, cellular signal transduction, and membrane recycling. In brains, Htt protein leads to cell damage and toxicity through depo‐

The gene for HD is located on the tip of chromosome 4, and is called the *IT-15 gene*. This part of DNA contains cytosine-adenine-guanine (CAG) repeats which are called tri‐ nucleotide repeats. Number of trinucleotide repeats is determined as risk of HD develop‐ ment. The CAG repeats are translated into polyglutamine (polyQ) residues which are

Htt proteins interact with a variety of peptides including huntington associated protein 1 (HAP1), huntington interacting protein 1 and 2 (HIP1 and HIP2) and huntington yeast part‐ ners A, B, and C (HYPA, HYPB, and HYPC). These peptides are functioned in cell signaling,

In HD, the neuropathology is characterized with accumulation of Htt protein aggregates. HD is caused by a number of CAG repeats in the gene. To date, many theories have been suggested in HD, but functions of CAG repeats and mechanisms of HD cannot be understood yet. However, common opinion is long CAG repeats (polyQ) are the most important promoter for toxicity of Htt protein aggregates. The polyQ region starts at res‐ idue 18 and the number of glutamine residues are the most important marker in HD. Surprisingly, 40 or more CAG repeats are always generated neuropathy, while 35 or few‐

oxidative stresses as well [65].

and it shows aggregation tendency [66].

*3.4.1. Structure of Huntington protein and genes*

located in the N-terminal region [66-68].

transport, and transcription processes [67, 69].

*3.4.2. Mechanisms of Huntington misfolding and aggregation*

sition of misfolded aggregate form of Htt protein [8, 9].

**3.4. Huntington's diseases**

66 Neurodegenerative Diseases

Hsps are highly conserved proteins among living organisms. Hsps are an important class of molecular chaperons and they are located in different parts of the cells such as endo‐ plasmic reticulum, cytosol, and mitochondria. Mainly, Hsps are related with formation of proper protein conformation, and also prevention protein aggregation, misfolding, and oligomerization. Proteins can be exposed a number of cellular and environmental factors including high temperature, inflammation, growth factors, oxidative stress etc. which can cause misfolding and protein aggregation. Overexpression of Hsps has been observed under these stress conditions. Several studies have focused on the neuroprotective role of Hsps. Therefore, the expression levels of Hsps are decreased and misfolded protein accu‐ mulation can be occurred in brain [1, 76-78].

Generally, Hsps are divided into six groups on the basis of molecular mass. In this section; Hsp40, Hsp60, Hsp70, Hsp90, Hsp100, and small Hsp (sHsp) have been examined and char‐ acterized for association with neurodegenerative diseases and protein folding processes [1].

Hsp70 is a highly conserved protein in all living organisms. It makes complex with unfolded or partially denatured proteins. Hsp70 has two functional domains: ATPase domain and substrate binding domain (SBD). The operation of these domains is controlled with hydroly‐ sis of ATP. ATPase domain binds to ATP and hydrolyzes it to ADP. This energy drives the protein folding function of the Hsp70. Similarly, Hsp70 binding to misfolded peptides, in‐ creases the ATP hydrolysis. Also, Hsp70 interacts with Hsp40 and Hsp90 to perform protein folding process. Hsp70 serves a neuroprotective role in all of neurodegenerative diseases [79-81]. Auluck and co-workers indicated that, expression of Hsp70 reduced α-Syn aggrega‐ tion, accumulation, and toxicity in PD animal models [82]. In HD models, Hsp70 shows pro‐ tective assignment in polyglutamine-induced toxicity [83]. Also, Hsp70 is involved in the folding and functional maintenance of tau protein. In prion diseases, Hsp70 binds aggregate form of prion proteins and it mediates their degradation by the proteasome [81].

are characterized by progressive nervous system dysfunction. In the world, millions of peo‐ ple are affected from these diseases. For example, five million people suffer from Alzheim‐ er's disease, one million people from Parkinson's disease and 30.000 people from Huntington's diseases in USA. US government spends approximately 50-100 billions of dol‐ lars for diagnosis and therapies [91]. Today, we are using effective methods (PET, MRI, SPECT etc.) for diagnosis of neurodegenerative diseases, but we cannot treat the diseases

Role of Protein Aggregation in Neurodegenerative Diseases

http://dx.doi.org/10.5772/54487

69

All of neurodegenerative diseases are related with protein misfolding and aggregation. Dif‐ ferent native proteins lead to formation of diseases, but practically same factors involve in these diseases. Structurally, β-sheet conformation plays a key role in neuropathies. Further‐ more, oxidative stress, pH, mutations, post translational modifications etc. lead to protein

New innovations in biochemical and medicinal fields lead to development of a number of mechanisms for protein aggregation and neurodegenerative diseases. Moreover, a variety of analytical techniques such as IR (infrared spectroscopy), NMR (nuclear magnetic reso‐ nance), CD (circular dichroism), calorimeters, and electron microscopy have been used for detection of aggregation process. Nevertheless, we cannot identified cellular mechanisms of protein misfolding and related diseases clearly yet. In the future, scientists will concentrate

This work was funded through a seed grant from the Turkish National Academy of Sciences

1 Cumhuriyet University, Faculty of Pharmacy, Department of Biochemistry, Turkey

3 Cumhuriyet University, Faculty of Medicine, CUTFAM Research Center, Sivas, Turkey

4 Gaziosmanpasa University, Faculty of Natural Sciences and Engineering, Department of

5 Kahramanmaraş Sütçü İmam University, Faculty of Science and Letters, Department of Bi‐

2 Cumhuriyet University, Faculty of Medicine, Department of Biophysics, Turkey

on design of practical and fast methods for detection of protein aggregation in cells.

easily as we diagnose them.

folding diseases.

**Acknowledgements**

**Author details**

(TUBA GEBIP 2008-29 for YT).

Bioengineering, Tokat, Turkey

ology, Kahramanmaraş, Turkey

Yusuf Tutar1,2,3, Aykut Özgür4\* and Lütfi Tutar5

Hsp40 is expressed in variety of organisms in different isoforms. It associates with unfolded polypeptides and prevents protein aggregation. Hsp40 can be found in a cell in three differ‐ ent types. All types of Hsp40 contain highly conserved J domain which interacts with Hsp70 ATPase domain. Thus, ATPase activity of Hsp70 is regulated by this interaction. Hsp40 transmits substrate towards Hsp70 SBD domain with an appropriate conformation. Thus, Hsp70 help substrate peptide in its hydrophobic SBD region and assists the peptide to come to a state of proper three-dimensional structure [1, 84]. Hsp40 is extensively found in neuro‐ degenerated brains due to it is association with Hsp70 as a co-chaperon [83].

Hsp100 participates in counteraction of protein aggregation with Hsp70 and Hsp40. This hexameric 100 kDa protein has substrate and ATP binding regions. Large protein aggregates are broken by Hsp100 and formed small aggregates are carried forward by Hsp70-Hsp40 complex. In yeast, overexpression of Hsp100 leads to disassemble of large prion aggregates and generate the small prion seeds for new rounds of prion propagation [1, 83].

Hsp90 is a highly expressed cellular molecular chaperon and also stabilizes certain proteins and aids protein degradation. Hsp90 is a dimeric protein which has a highly conserved Nterminal domain and a C-terminal domain. Hsp90 is one of the main cytosolic molecular chaperons which is activated with Hsp40 and Hsp70 [1, 85]. Uryu and co-workers demon‐ strated that expression of Hsp90 is increased in transgenic mouse model of PD. Inhibition of Hsp90 lead to generation of tauopathies because of protein hyperphosphorylation and ab‐ normal neuronal activity can be increased in AD [86].

Hsp60 is a heptameric 60 kDa protein which is located particularly in mitochondria. Hsp60 works with Hsp70 coordinately for protein folding. Furthermore, it plays key roles in mito‐ chondrial protein transport, replication and transmission of mitochondrial DNA, and apop‐ tosis. For actin and tubulin, Hsp60 is a specific chaperon which is decreased in AD. In AD effected neurons, aggregated and misfolded tau protein is increased in contrast with expres‐ sion of Hsp60 is decreased [1, 83].

sHsp has a molecular mass between 12 and 30 kDa [87]. As a molecular chaperone, sHsp are located at different compartments in the cell and they can protect protein struc‐ tures and activities. Also, overexpression of sHsp have reported in many studies [87-90]. In HD, the expression level of Hsp27 is increased and it prevents polyglutamine induced toxicity in neurons. Furthermore, Hsp27 reduces α-syn-induced toxicity in PD patient brain [82, 88]. The other sHsps including Hsp10, Hsp12, Hsp20, Hsp26 are associated with protein folding diseases [89].

### **5. Conclusion**

More than 600 diseases such as Alzheimer's disease, Huntington's disease, prion diseases, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and others are characterized by progressive nervous system dysfunction. In the world, millions of peo‐ ple are affected from these diseases. For example, five million people suffer from Alzheim‐ er's disease, one million people from Parkinson's disease and 30.000 people from Huntington's diseases in USA. US government spends approximately 50-100 billions of dol‐ lars for diagnosis and therapies [91]. Today, we are using effective methods (PET, MRI, SPECT etc.) for diagnosis of neurodegenerative diseases, but we cannot treat the diseases easily as we diagnose them.

All of neurodegenerative diseases are related with protein misfolding and aggregation. Dif‐ ferent native proteins lead to formation of diseases, but practically same factors involve in these diseases. Structurally, β-sheet conformation plays a key role in neuropathies. Further‐ more, oxidative stress, pH, mutations, post translational modifications etc. lead to protein folding diseases.

New innovations in biochemical and medicinal fields lead to development of a number of mechanisms for protein aggregation and neurodegenerative diseases. Moreover, a variety of analytical techniques such as IR (infrared spectroscopy), NMR (nuclear magnetic reso‐ nance), CD (circular dichroism), calorimeters, and electron microscopy have been used for detection of aggregation process. Nevertheless, we cannot identified cellular mechanisms of protein misfolding and related diseases clearly yet. In the future, scientists will concentrate on design of practical and fast methods for detection of protein aggregation in cells.

### **Acknowledgements**

folding and functional maintenance of tau protein. In prion diseases, Hsp70 binds aggregate

Hsp40 is expressed in variety of organisms in different isoforms. It associates with unfolded polypeptides and prevents protein aggregation. Hsp40 can be found in a cell in three differ‐ ent types. All types of Hsp40 contain highly conserved J domain which interacts with Hsp70 ATPase domain. Thus, ATPase activity of Hsp70 is regulated by this interaction. Hsp40 transmits substrate towards Hsp70 SBD domain with an appropriate conformation. Thus, Hsp70 help substrate peptide in its hydrophobic SBD region and assists the peptide to come to a state of proper three-dimensional structure [1, 84]. Hsp40 is extensively found in neuro‐

Hsp100 participates in counteraction of protein aggregation with Hsp70 and Hsp40. This hexameric 100 kDa protein has substrate and ATP binding regions. Large protein aggregates are broken by Hsp100 and formed small aggregates are carried forward by Hsp70-Hsp40 complex. In yeast, overexpression of Hsp100 leads to disassemble of large prion aggregates

Hsp90 is a highly expressed cellular molecular chaperon and also stabilizes certain proteins and aids protein degradation. Hsp90 is a dimeric protein which has a highly conserved Nterminal domain and a C-terminal domain. Hsp90 is one of the main cytosolic molecular chaperons which is activated with Hsp40 and Hsp70 [1, 85]. Uryu and co-workers demon‐ strated that expression of Hsp90 is increased in transgenic mouse model of PD. Inhibition of Hsp90 lead to generation of tauopathies because of protein hyperphosphorylation and ab‐

Hsp60 is a heptameric 60 kDa protein which is located particularly in mitochondria. Hsp60 works with Hsp70 coordinately for protein folding. Furthermore, it plays key roles in mito‐ chondrial protein transport, replication and transmission of mitochondrial DNA, and apop‐ tosis. For actin and tubulin, Hsp60 is a specific chaperon which is decreased in AD. In AD effected neurons, aggregated and misfolded tau protein is increased in contrast with expres‐

sHsp has a molecular mass between 12 and 30 kDa [87]. As a molecular chaperone, sHsp are located at different compartments in the cell and they can protect protein struc‐ tures and activities. Also, overexpression of sHsp have reported in many studies [87-90]. In HD, the expression level of Hsp27 is increased and it prevents polyglutamine induced toxicity in neurons. Furthermore, Hsp27 reduces α-syn-induced toxicity in PD patient brain [82, 88]. The other sHsps including Hsp10, Hsp12, Hsp20, Hsp26 are associated

More than 600 diseases such as Alzheimer's disease, Huntington's disease, prion diseases, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and others

form of prion proteins and it mediates their degradation by the proteasome [81].

degenerated brains due to it is association with Hsp70 as a co-chaperon [83].

and generate the small prion seeds for new rounds of prion propagation [1, 83].

normal neuronal activity can be increased in AD [86].

sion of Hsp60 is decreased [1, 83].

with protein folding diseases [89].

**5. Conclusion**

68 Neurodegenerative Diseases

This work was funded through a seed grant from the Turkish National Academy of Sciences (TUBA GEBIP 2008-29 for YT).

### **Author details**

Yusuf Tutar1,2,3, Aykut Özgür4\* and Lütfi Tutar5

1 Cumhuriyet University, Faculty of Pharmacy, Department of Biochemistry, Turkey


4 Gaziosmanpasa University, Faculty of Natural Sciences and Engineering, Department of Bioengineering, Tokat, Turkey

5 Kahramanmaraş Sütçü İmam University, Faculty of Science and Letters, Department of Bi‐ ology, Kahramanmaraş, Turkey

#### **References**

[1] Tutar L, Tutar Y. Heat shock proteins: An overview. Current Pharmaceutical Biotech‐ nology 2010; 11(2):216-222.

[16] Buee L, Bussiere T, Buee-Scherrer V, Delacourte A, Hof PR. Tau protein isoforms, phosphorylation and role in neurodegenerative disorders. Brain Research Reviews

Role of Protein Aggregation in Neurodegenerative Diseases

http://dx.doi.org/10.5772/54487

71

[17] Martin L, Latypova X, Terro F. Post-translational modifications of tau protein: Impli‐ cations for Alzheimer's disease. Neurochemistry International 2011; 58: 458-471. [18] Rudd PM, Merry AH, Wormald MR, Dwek RA. Glycosylation and prion protein.

[19] Rudd PM, Wormald MR, Wing DR, Prusiner SB, Dwek RA. Prion glycoprotein: Structure, dynamics, and roles for the sugar. Biochemistry 2001; 40(13): 3759-3766. [20] Guest WC, Plotkin SS, Cashman NR. Toward a mechanisms of prion misfolding and structural models of PrPSc: Current knowledge and future directions. Journal of Toxi‐

[21] Pogocki D. Alzheimer's β-amyloid peptide as a source of neurotoxic free radicals: the

[22] Moulton PV, Yang W. Air pollution, oxidative stress, and Alzheimer's Disease. Jour‐

[23] Treuheit MJ, Kosky AA, Brems DN. Inverse relationship of protein concentration and

[24] DeMArco ML, Daggett V. Local environmental effects on the structure of the prion

[25] Finl AL. The aggregation and fibrillation of α-synuclein. AccChem Res 2006; 39:

[26] Wadsworth JDF, Collinge J. Update on human prion diseases. BiochimiaetBiophysi‐

[27] Mehrpour M, Codogno P. Prion protein: From physiology to cancer biology. Cancer

[28] Hu W, Kieseier B, Frohman E, Eagar TN, Rosenberg RN, Hartung HP, Stüve O. Prion protein: Physiological functions and role in neurological disorders. Journal of Neuro‐

[29] Cohen FE. Protein misfolding and prion diseases. Journal of Molecular Biology 1999;

[30] Milhavet O, McMahon HEM, Rachidi W, Nishida N, Katamine S, Mange A, Arlotto M, Casanova D, Riondel J, Favier A, Lehmann S. Prion infection impairs the cellular

response to oxidative stress. PNAS 2000; 97(25): 13937-13942.

nal of Environmental and Public Health 2012; DOI:10.1155/2012/472751.

Current Opinion in Structural Biology 2002; 12: 578-586.

cology and Environmental Health, Part A 2011; 74: 154-160.

role of structural effects. ActaNeurobiolExp 2003; 63: 131-145.

aggregation. Pharmaceutical Research 2002; 19(4): 511-516.

protein. C R Biologies 2005; 328: 847-862.

caActa 2007; 1772: 598-609.

logical Sciences 2008; 264: 1-8.

Letters 2010; 290: 1-23.

293: 131-320.

628-634.

2000; 33: 95-130.


[16] Buee L, Bussiere T, Buee-Scherrer V, Delacourte A, Hof PR. Tau protein isoforms, phosphorylation and role in neurodegenerative disorders. Brain Research Reviews 2000; 33: 95-130.

**References**

70 Neurodegenerative Diseases

nology 2010; 11(2):216-222.

65(2): 184-189.

ternational 2003; 43: 1-7.

Journal of Pharmaceutics 2005; 289: 1-30.

approaches. FEBS Journal 2006; 1331-1349.

gation. ActaNeurobiolExp 2004; 64: 41-52.

Medicine 2004; DOI: 10.1038/nm1066.

otechnology 2009; 10: 348-351.

Biology 2005; 38(3): 275-280.

2011; DOI:10.4061/2011/207691

[1] Tutar L, Tutar Y. Heat shock proteins: An overview. Current Pharmaceutical Biotech‐

[2] Wang W. Protein aggregation and its inhibition biopharmaceutics. International

[3] Stefani M. Protein misfolding and aggregation: new examples in medicine and biolo‐ gy of the dark side of the protein world. BiochimiaetBiophysicaActa 2004; 1739: 5-25.

[4] Chaudhuri TK, Paul S. Protein-misfoldingdiseases and chaperone-based therapeutic

[5] Adachi H, Katsuno M, Waza M, Minamiyama M, Tanaka F, Sobue G. Heat shock protins in neurodegenerative diseases: Pathogenic roles and therapeutic implica‐

[6] Soto C, Estrada, LD. Protein misfolding and neurodegeneration. Arch Neurol2008;

[7] Lee SJ, Lim HS, Masliah E, Lee HJ. Protein aggregate spreading in neurodegenerative diseases: Problems and perspectives. Neuroscience Research 2011; 70: 339-348.

[8] Shastry BS. Neurodegenerative disorders of protein aggregation. Neurochemistry In‐

[9] Trzesniewska K, Brzyska M, Elbaum D. Neurodegenerative aspects of protein aggre‐

[10] Ross CA, Poirier MA. Protein aggregation and neurodegenerative diseases. Nature

[11] Philo JS, Arakawa T. Mechanisms of protein aggregation. Current Pharmaceutical Bi‐

[12] Hur K, Kim JI, Choi SI, Choi EK, Carp RI, Kim YS. The pathogenic mechanisms of prion protein. Mechanisms of Ageing and Development 2002; 123: 1637-1647.

[13] Lee C, Y MH. Protein folding and diseases. Journal of Biochemistry and Molecular

[14] Wilson AC, Dugger BN, Dickson DW, Wang DS. TDP-43 in aging and Alzheimer's

[15] Karve TM, Cheema AK. Small changes huge impact: The role of protein posttransla‐ tional modifications in cellular homeostasis and disease. Journal of Amino Acids

disease – a review. Int J ClinExpPathol 2011;4(2):147-155.

tions. International Journal of Hyperthermia 2009; 25(8): 647-654.


[31] Westergard L, Christensen HM, Harris DA. The cellular prion protein (PrPC): Its physiological function and role in disease. BiochimicaetBiophysicaActa 2007; 1772: 629-644.

[45] Avila J. Tau phosphorylation and aggregation in Alzheimer's diseases pathology.

Role of Protein Aggregation in Neurodegenerative Diseases

http://dx.doi.org/10.5772/54487

73

[46] Du JT, Li YM, Ma QF, Qiang W, Zhao YF, Abe H, Kanazawa K, Qin XR, Aoyagi R, Ishizuka Y, Nemoto T, Nakanishi H. Synthesis and conformational properties of phosphopeptides related to the human tau protein. Regulatory Peptides 2005; 130:

[47] Connell JW, Gibb GM, Betts JC, Blacstock WP, Gallo JM, Lovestone S, Hutton M, An‐ derton BH. E¡ects of FTDP-17 mutations on the in vitro phosphorylation of tau by‐ glycogen synthase kinase 3*β* identified by mass spectrometry demonstrate certain mutations exert long-range conformational changes. FEBS Letters 2001; 493: 40-44. [48] Bunker JM, Kamath K, Wilson L, Jordan MA, Feinstein SC. FTDP-17 Mutations com‐ promise the abilityoftautoregulatemicrotubuledynamicsincells. JBC Papers 2006;

[49] Goedert M. Tau protein and neurodegeneration. Seminars in Cell&Developmental

[50] Dimakopoulos AC. Protein aggregation in Alzheimer's diseases and other neuropa‐

[51] Baumketner A, Bernstein SL, Wyttenbach T, Lazo ND, Teplow DB, Bowers MT, Shea JE. Structure of the 21-30 fragment of amyloid β-protein. Protein Science 2006; 15:

[52] Crescenzi O, Tomaselli S, Guerrini R, Salvadori S, D'Ursi AM, Temussi PA, Picone D. Solution structure of the Alzheimer amyloid β-peptide (1-42) in an apolar microen‐

[53] Estrada LD, Soto C. Disrupting β-amyloid aggregation for Alzheimer diseases treat‐

[54] Li M, Chen L, Lee DHS, Yu LC, Zhang Y. The role of intracellular amyloid β in Alz‐

[55] Dauer W, Przedborski S. Parkinson's diseases: Mechanisms and Models. Neuron

[56] Ma QL, Chan P, Yoshii M, Ueda K. α-Synuclein aggregation and neurodegenerative

[57] McNaught KSP, Olanow CW. Protein aggregation in the pathogenesis of familial and

[58] Valtierra S. α-Synuclein phosphorylation and nitration in Parkinson's disease. Eu‐

sporadic Parkinson's diseases. Neurobiology of Aging 2006; 27: 530-545.

thological disorders. Current Alzheimer Research 2005; 2: 19-28.

ment. Current Topics in Medicinal Chemistry 2007; 7: 115-126.

heimer's diseases. Progress in Neurobiology 2007; 83: 131-139.

diseases. Journal of Alzheimer's Diseases 2003; 5: 139-148.

vironment. Eur J Biochem 2002; 269: 5642-5648.

FEBS Letters 2006; 580: 2922-2927.

DOI: 10.1074/jbc.M509420200.

Biology 2004; 15: 45-49.

1239-1247.

2003; 39: 889-909.

karyon 2008; 4: 90-94.

48-56.


[45] Avila J. Tau phosphorylation and aggregation in Alzheimer's diseases pathology. FEBS Letters 2006; 580: 2922-2927.

[31] Westergard L, Christensen HM, Harris DA. The cellular prion protein (PrPC): Its physiological function and role in disease. BiochimicaetBiophysicaActa 2007; 1772:

[32] Garnett AP, Viles JH. Copper Binding to the octarepeats of the prion protein. Journal

[33] MilhauserGL. Copper binding in the prion protein. AccChem Res 2004; 37(2): 79-85.

[34] Viles JH, Cohen FD, Prusiner SB, Goodin DB, Wright PE, Dyson HJ. Copper binding to the prion protein: Structural implications of four identical cooperative binding

[35] Calabrese MF, Miranker AD. Metal binding sheds light on mechanisms of amyloid

[36] Daggett V. Structure-function aspects of prion proteins. Current Opinion in Biotech‐

[37] Taylor DR, Hooper NM. The prion protein and lipid rafts. Molecular Membrane Biol‐

[38] Wang F, Yang F, Hu Y, Wang X, Wang X, Jin C, Ma J. Lipid interaction converts prion protein to a PrPSc-like proteinase K-resistant conformation under physiological

[39] Critchley P, Kazlauskaite J, Eason R, Pinheiro TJT. Binding of prion proteins to lipid membranes. Biochemical and Biophysical Research Communications 2004; 313:

[40] Rushworth JV, Hooper NM. Lipid Rafts: Linking Alzheimer's amyloid-β production, aggregation, and toxicity at neuronal membranes. International Journal of Alzheim‐

[41] Kolarova M, Garcia-Sierra F, Bartos A, Ricny J, Ripova D. Structure and pathology of tau protein in Alzheimer Disease. International Journal of Alzheimer's Disease 2012;

[42] Bulic B, Pickharth M, Mandelkow EM, Mandelkow E. Tau protein and tau aggrega‐

[43] Li L, Bergen MV, Mandelkow EM, Mandelkow E. Structure, Stability, and Aggrega‐ tion of paired helical filaments from tau protein and FTDP-17 mutants probed by tryptophan scanning mutagenesis. Journal of Biological Chemistry 2002; 277(44):

[44] Bergen MV, Friedhoff P, Biernat J, Heberle J, Maldelkox EM, Maldelkow E. Assembly of t protein into Alzheimer paired helicalfilaments depends on a local sequence motif

(306VQIVYK311) forming β structure. Biochemistry 2000; 97(10): 5129-5134.

of Biological Chemistry 2003; 278(9): 6795-6802.

sites. ProcNatlAcadSci 1999; 96: 2042-2047.

conditions. Biochemistry 2007; 46: 7045-7053.

er's Disease 2011; DOI:10.4061/2011/603052.

tion inhibitors. Neuropharmacy 2010; 59: 276-289.

assembly. Prion 2009; 3(1): 1-4.

nology 1998; 9: 359-365.

ogy 2006; 23(1) 89-99.

DOI:10.1155/2012/731526.

41390-41400.

559-567.

629-644.

72 Neurodegenerative Diseases


[59] Khandelwal PJ, Dumanis SB, Feng LR, Maguire-Zeiss K, Rebeck GW, Lashuel HA, Moussa CEH. Parkinson-related parkin reduces α-Synuclein phosphorylation in a gene transfer model. Molecular Neurodegeneration 2010; 5(47): 1-13.

[74] Hoffner G, Djan P. Transglutamine and diseases of the central nervous system. Fron‐

Role of Protein Aggregation in Neurodegenerative Diseases

http://dx.doi.org/10.5772/54487

75

[76] Bukau B, Weissman J, Horwich A. Molecular chaperons and protein quality control.

[77] Mayer MP. Gymnastics of molecular chaperones. Cell 2010; DOI:10.1016/j.molcel.

[78] Dou F, Netzer WJ, Takashima A, Xu H. Heat shock proteins reduce aggregation and facilitate degradation of tau protein. International Congress Series 2003; 1252:

[79] Fan CY, Lee S, Douglas MC. Mechanisms for regulation of Hsp70 function by Hsp40.

[80] Mapa K, Sikor M, Kudryavtsev V, Waegemann K, Kalinin S, Seidel CAM, Neupert W, Lamb DC, Mokranjac D. The conformational dynamics of the mitochondrial

[81] Turturici G, Sconzo G, Geraci F. Hsp70 and its molecular role in nervous system dis‐ eases. Biochemistry Research International 2011; DOI: 10.1155/2011/618127.

[82] Luo GR, Chen S, Le WD. Are heat shock proteins therapeutic target for Parkinson's

[83] Söti C, Csermely P. Chaperons and aging: Role in neurodegeneration and in other

[84] Li J, Bingdon SHA. Structure-based mutagenesis studies of the peptide substrate binding fragment of type I heat-shock protein 40. Biochem J 2005; 386: 453-460. [85] Faou P, Hoogenraad NJ, Tom34: A cytosolic cochaperone of the Hsp90/Hsp70 pro‐ tein complex involved in mitochondrial protein import. BiochimiaetBiophysicaActa

[86] Luo W, Sun W, Taldone T, Rodina A, Chiosis G. Heat shock protein 90 in neurodege‐

[87] Sun Y, MacRae TH. The small heat shock proteins and their role in human diseases.

[88] Outeiro TF, Klucken J, Strathearn KE, Liu F, Nguyen P, Rochet JC, Hyman BT, McLean PJ. Small heat shock proteins protect against α-synuclein-induced toxicity and aggregation. Biochemical and Biophysical Research Communications 2006; 351:

diseases? International Journal of Biological Sciences 2007; 3(1): 20-26.

civilization diseases. Neurochemistry International 2002; 41: 383-389.

nerative diseases. Molecular Neurodegeneratin 2010; 5(24): 1-8.

tiers in Bioscience 2005; 10: 3078-3092.

Cell Stress & Chaperones 2003; 8(4): 309-316.

Hsp70 chaperone. Cell 2010; 38: 89-100.

Cell 2006; 125: 443-451.

2012; 1823: 348-357.

631-638.

FEBS Journal 2005; 272: 2613-2627.

2010.07.012

383-393.

[75] Newcomer ME. Trading places. Nature 2001; 8(4): 282-284.


[59] Khandelwal PJ, Dumanis SB, Feng LR, Maguire-Zeiss K, Rebeck GW, Lashuel HA, Moussa CEH. Parkinson-related parkin reduces α-Synuclein phosphorylation in a

[60] Mizuna Y, Hattori N, Mori H. Genetics of Parkinson's diseases. Biomed &Pharmac‐

[61] Licker V, Kövari E, Hochstrasser DF, Burkhard PR. Proteomics in human Parkinson's

[62] Gitler AD, Chesi A, Geddie ML, Strathearn KE, Hamamichi S, Hill KJ, Caldwell GA, Cooper AA, Rochet JC, Lindquist S. α-Synuclein is part of a diverse and highly con‐ served interaction network that includes PARK9 and manganese toxicity. Nature Ge‐

[63] Carla M, Tome L, Tyson T, Rey NL, Grathwohl S, Britscgi M, Brundin P. Inflammati on and α-Synuclein 's prion-like behavior in Parkinson's disease—Is there a link?

[64] Kim EJ, Sung JY, Lee HJ, Rhim Y, Hasegawa M, Iwatsubo T, Min DS, Kim J, Paik SR, Chung, KC. Dyrk1A phosphorylates α-synuclein and enhances intracellular inclu‐ sion formation. The Journal of Biological Chemistry 2006; 281(44): 33250-33257.

[65] Uverski VN, Yamin G, Souillac PO, Goers J, Glaser CB, Fink AL. Methionine oxida‐ tion inhibits fibrillation of human alphasynuclein in vitro. FEBS Letters 2002; 517:

[66] Ross RAC. Huntington 's disease: A clinical review. Journal of Rare Diseases 2010;

[67] Gusella JF, MacDonald ME. Huntington: A single bait hooks many species. Current

[68] Ramaswamy S, Shannon KM, Kordower JH. Huntington 's disease: Pathological mechanisms and therapeutic strategies. Cell Transplantation 2007; 16: 1-100.

[69] Margolis RL, Rudnicki DD, Holmes SE. Huntington's disease like-2: Review and up‐

[71] Perez De-La Cruz V, Santamaria A. Integrative hypothesis for Huntington's disease:

[72] Gudesblatt M, Tarsy D. Huntington's diseases: A clinical review. Neurobiology Re‐

[73] Perutz M. Polar zippers: Their role in human disease. Protein Science 1994; 3:

A brief review of experimental evidence. Physiol Res 2007; 56: 513-526.

gene transfer model. Molecular Neurodegeneration 2010; 5(47): 1-13.

diseases research. Journal of Proteomics 2009; 73: 10-29.

MolNeurobiol 2012; DOI 10.1007/s12035-012-8267-8.

Opinion in Neurobiology 1998; 8: 425-430.

date. ActaNeurol Taiwan 2005; 14: 1-8.

[70] Walker FO. Huntington's diseases. Lancet 2007; 369: 218-228.

other 1999; 53: 109-116.

74 Neurodegenerative Diseases

netics 2009; 41(3): 308-315.

239-244.

5(40): 1-8.

views 2011.

1629-1637.


[89] Wilhelmus MMM, Boelens WC, Otte-Höller I, Kamps B, De Wall RMW, VErbeek MM. Small heat shock proteins inhibit amyloid-β protein aggregation and cerebro‐ vascular amyloid-β protein toxicity. Brain Research 2006; 1089: 67-78.

**Chapter 4**

**Role of Oxidative Stress in Aβ**

**Vicious Circle of Apoptosis,**

Additional information is available at the end of the chapter

The aging process is believed to be closely related to increased oxidative stress. Reactive intermediates of oxidative stress affect the cellular redox status and induce apoptosis [1]. Oxidative stress due to the loss of balance between ROS production and antioxidant defenses affects all the vital organs, resulting in aging [1,2]. Oxidative damage, mitochondrial dysfunc‐ tion and inflammation underlies many common aging-related neurodegenerative diseases, including AD [2,3]. The major pathological hallmark of AD is the accumulation of Aβ peptides in the brain [4]. Oxidative insults that induce neuronal apoptosis, including agents that induce membrane lipid peroxidation, also have been shown to activate caspases [5]. Increased lipid peroxidation was consistently observed in some animal models of Alzheimer amyloidosis [4, 6]. It has been shown that a single Aβ administration into the rat hippocampus could induce increase of NOS activity and NO level [6]. Nitric oxide is a multifunctional molecule that acts as messenger/modulator in synaptogenesis and potential neurotoxin and is synthesized by three isozymes of Nitric oxide synthase (NOS) [7]. Oxidative stress reflects a situation in which ROS is continuously produced and exceeds the capacity of endogenous antioxidant defense systems. Several studies have suggested that oxidative stress plays a key role in Aβ-mediated neuronal cytotoxicity by triggering or facilitating neurodegeneration through a wide range of molecular events that eventually lead to neuronal cell loss. Aβ significantly increases produc‐ tion and enhances membrane lipid peroxidation, leading to neuronal apoptosis [8,9]. Because multiple factors are involved in the pathogenesis of the AD, it is difficult to find an ideal in vivo model. It is important to determine Aβ 1-42 injection effects especially in hippocampus,

> © 2013 Cetin; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**Nitric Oxide and Age**

http://dx.doi.org/10.5772/54718

Ferihan Cetin

**1. Introduction**

**Animal Model of Alzheimer's Disease:**


**Chapter 4**

**Role of Oxidative Stress in Aβ Animal Model of Alzheimer's Disease: Vicious Circle of Apoptosis, Nitric Oxide and Age**

Ferihan Cetin

[89] Wilhelmus MMM, Boelens WC, Otte-Höller I, Kamps B, De Wall RMW, VErbeek MM. Small heat shock proteins inhibit amyloid-β protein aggregation and cerebro‐

[90] Jiao W, Li P, Zhang J, Zhang H, Chang Z. Small heat-shock proteins function in the insoluble protein complex. Biochemical and Biophysical Research Communications

vascular amyloid-β protein toxicity. Brain Research 2006; 1089: 67-78.

[91] http://www.neurodiscovery.harvard.edu/challenge/challenge\_2.html

2005; 335: 227-231.

76 Neurodegenerative Diseases

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54718

### **1. Introduction**

The aging process is believed to be closely related to increased oxidative stress. Reactive intermediates of oxidative stress affect the cellular redox status and induce apoptosis [1]. Oxidative stress due to the loss of balance between ROS production and antioxidant defenses affects all the vital organs, resulting in aging [1,2]. Oxidative damage, mitochondrial dysfunc‐ tion and inflammation underlies many common aging-related neurodegenerative diseases, including AD [2,3]. The major pathological hallmark of AD is the accumulation of Aβ peptides in the brain [4]. Oxidative insults that induce neuronal apoptosis, including agents that induce membrane lipid peroxidation, also have been shown to activate caspases [5]. Increased lipid peroxidation was consistently observed in some animal models of Alzheimer amyloidosis [4, 6]. It has been shown that a single Aβ administration into the rat hippocampus could induce increase of NOS activity and NO level [6]. Nitric oxide is a multifunctional molecule that acts as messenger/modulator in synaptogenesis and potential neurotoxin and is synthesized by three isozymes of Nitric oxide synthase (NOS) [7]. Oxidative stress reflects a situation in which ROS is continuously produced and exceeds the capacity of endogenous antioxidant defense systems. Several studies have suggested that oxidative stress plays a key role in Aβ-mediated neuronal cytotoxicity by triggering or facilitating neurodegeneration through a wide range of molecular events that eventually lead to neuronal cell loss. Aβ significantly increases produc‐ tion and enhances membrane lipid peroxidation, leading to neuronal apoptosis [8,9]. Because multiple factors are involved in the pathogenesis of the AD, it is difficult to find an ideal in vivo model. It is important to determine Aβ 1-42 injection effects especially in hippocampus,

© 2013 Cetin; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

temporal and parietal cortex, because a definite atrophy reveals in these regions in the late stage of sporadic AD. The fact that revealing changes with age are the accumulation of ROS and the onset of apoptosis cascade, these interfering subjects have been investigated intensely nearly for two decades. β-amyloid itself is a source of free radicals. One possible mechanism for initiating apoptosis could be the generation of free radicals by the peptide leading to lipid peroxidation and oxidative stress [9,10]. Many recent studies have confirmed the toxicity of peroxynitrite to neurons and the involvement of nitric oxide in neurologic pathologies [10,11]. Other suspects include the superoxide radical hydrogen peroxide, which is implicated in amyloid neurotoxicity, and peroxynitrite, which can be formed by combining superoxide and nitric oxide [9].

Nitric oxide is a multifunctional molecule that acts as messenger/modulator in synaptogenesis and potential neurotoxin and is synthesized by three isozymes of NOS. Entorhinal cortex neurons are highly vulnerable to neurodegeneration in AD express low levels of NOS and the inducible form of NOS is upregulated in AD [14]. They found a significant decrease in the number of cells expressing detectable levels of nNOS mRNA in the white matter underlying the frontal cortex and in the dentate gyrus and CA subfields of the hippocampus in AD. Furthermore, there was also a significant decrease in the number of NADPHd-positive cells in the dentate gyrus and CA subfields of the hippocampus in AD [15].

It has been suggested that the upregulation of nNOS and subsequent NO release in certain classes of cortical interneurons may be one of the earliest events signaling the apoptosis of cortical projection neurons in lesion models [16]. Conflicts may arise from varying emphasis on different cortical areas among these investigators and the complex anatomy of cortical nitrinergic neurons that comprise at least three distinct cell populations, that is, large subcort‐ ical whiter matter neurons, large nNOS (+) interneurons, and smaller nNOS (+) interneurons that are often missed because of low levels of nNOS/NADPHd expression [16]. Not only neurons but also activated microglia are capable of releasing neurotoxic molecules, such as proinflammatory cytokines and toxic oxygen and nitrogen species [17]. The relationship between mitochondrial damage, glutathione status/GSH dependent enzymes, oxidative stress, and neuronal dysfunction has been demonstrated by the effects of excessive production of H2O2 within mitochondria, which leads to depletion of mitochondrial GSH. Besides, meas‐ urements of the activity time course of antioxidant enzymes are important when comparing the alterations induced by Aβ with those found in AD patients [18]. Glutathione is known to protect cells against apoptosis, which is consistent with the suggestion of involvement of ONOO- mediated cellular events in neuronal apoptosis [2,17].The current literature contains contradictory results about the dual role (neuroprotector/neurotoxic) that NO may play in the aging CNS [19]. It is mostly believed that NO has neurotoxic effects on neurons [20,21]. It is now known that, in aging and Alzheimer brain, nNOS-expressing hippocampal neurons are more vulnerable to oxidative stress [14]. Aβ (25–35) activated nNOS in the cerebral cortex and hippocampus without effect on iNOS activity [6]. Since Aβ neurotoxicity is believed to be mediated by NO and the potential toxic effects of NO depend on the intracellular source of the molecule (i.e. isoform-specific) [7].

**Figure 1.** Nitric oxide (NO) neurotoxicity and neuroprotectivity in relation to Alzheimer's disease (AD). Mutations of presenilins (PSs) and amyloid precursor protein (APP) are associated with increased production of Aβ. Neurofibrillary tangles (NFT) formation is the result of tau hyperphosphorylation, which leads to cell death secondary to cellular traf‐ ficking disruption. PSs have also been implicated in the process of tau hyperphosphorylation. Apolipoprotein E e4 (ApoE e4) genotype is considered a risk factor for AD. It appears to affect Aβ production and a correlation between ApoE e4 and cholinergic deficit has been established. Cholinergic deficit is one of the most significant findings in AD, and is implicated in memory impairments observed in this disease. Increased production of Aβ induces NO production either by disrupting Ca homeostasis and subsequent increased in intracellular Ca (nNOS and eNOS-mediated NO re‐ lease) or by interactions with glial cells (iNOS-mediated NO release). NO is a free radical and can produce peroxynitrite. These reactive oxygen species induce a variety of neurotoxic mechanisms, including DNA/ protein alterations, mito‐

Role of Oxidative Stress in Aβ Animal Model of Alzheimer's Disease: Vicious Circle of Apoptosis, Nitric Oxide and Age

http://dx.doi.org/10.5772/54718

79

Role of Oxidative Stress in Aβ Animal Model of Alzheimer's Disease: Vicious Circle of Apoptosis, Nitric Oxide and Age http://dx.doi.org/10.5772/54718 79

temporal and parietal cortex, because a definite atrophy reveals in these regions in the late stage of sporadic AD. The fact that revealing changes with age are the accumulation of ROS and the onset of apoptosis cascade, these interfering subjects have been investigated intensely nearly for two decades. β-amyloid itself is a source of free radicals. One possible mechanism for initiating apoptosis could be the generation of free radicals by the peptide leading to lipid peroxidation and oxidative stress [9,10]. Many recent studies have confirmed the toxicity of peroxynitrite to neurons and the involvement of nitric oxide in neurologic pathologies [10,11]. Other suspects include the superoxide radical hydrogen peroxide, which is implicated in amyloid neurotoxicity, and peroxynitrite, which can be formed by combining superoxide and

Nitric oxide is a multifunctional molecule that acts as messenger/modulator in synaptogenesis and potential neurotoxin and is synthesized by three isozymes of NOS. Entorhinal cortex neurons are highly vulnerable to neurodegeneration in AD express low levels of NOS and the inducible form of NOS is upregulated in AD [14]. They found a significant decrease in the number of cells expressing detectable levels of nNOS mRNA in the white matter underlying the frontal cortex and in the dentate gyrus and CA subfields of the hippocampus in AD. Furthermore, there was also a significant decrease in the number of NADPHd-positive cells

It has been suggested that the upregulation of nNOS and subsequent NO release in certain classes of cortical interneurons may be one of the earliest events signaling the apoptosis of cortical projection neurons in lesion models [16]. Conflicts may arise from varying emphasis on different cortical areas among these investigators and the complex anatomy of cortical nitrinergic neurons that comprise at least three distinct cell populations, that is, large subcort‐ ical whiter matter neurons, large nNOS (+) interneurons, and smaller nNOS (+) interneurons that are often missed because of low levels of nNOS/NADPHd expression [16]. Not only neurons but also activated microglia are capable of releasing neurotoxic molecules, such as proinflammatory cytokines and toxic oxygen and nitrogen species [17]. The relationship between mitochondrial damage, glutathione status/GSH dependent enzymes, oxidative stress, and neuronal dysfunction has been demonstrated by the effects of excessive production of H2O2 within mitochondria, which leads to depletion of mitochondrial GSH. Besides, meas‐ urements of the activity time course of antioxidant enzymes are important when comparing the alterations induced by Aβ with those found in AD patients [18]. Glutathione is known to protect cells against apoptosis, which is consistent with the suggestion of involvement of ONOO- mediated cellular events in neuronal apoptosis [2,17].The current literature contains contradictory results about the dual role (neuroprotector/neurotoxic) that NO may play in the aging CNS [19]. It is mostly believed that NO has neurotoxic effects on neurons [20,21]. It is now known that, in aging and Alzheimer brain, nNOS-expressing hippocampal neurons are more vulnerable to oxidative stress [14]. Aβ (25–35) activated nNOS in the cerebral cortex and hippocampus without effect on iNOS activity [6]. Since Aβ neurotoxicity is believed to be mediated by NO and the potential toxic effects of NO depend on the intracellular source of

in the dentate gyrus and CA subfields of the hippocampus in AD [15].

the molecule (i.e. isoform-specific) [7].

nitric oxide [9].

78 Neurodegenerative Diseases

**Figure 1.** Nitric oxide (NO) neurotoxicity and neuroprotectivity in relation to Alzheimer's disease (AD). Mutations of presenilins (PSs) and amyloid precursor protein (APP) are associated with increased production of Aβ. Neurofibrillary tangles (NFT) formation is the result of tau hyperphosphorylation, which leads to cell death secondary to cellular traf‐ ficking disruption. PSs have also been implicated in the process of tau hyperphosphorylation. Apolipoprotein E e4 (ApoE e4) genotype is considered a risk factor for AD. It appears to affect Aβ production and a correlation between ApoE e4 and cholinergic deficit has been established. Cholinergic deficit is one of the most significant findings in AD, and is implicated in memory impairments observed in this disease. Increased production of Aβ induces NO production either by disrupting Ca homeostasis and subsequent increased in intracellular Ca (nNOS and eNOS-mediated NO re‐ lease) or by interactions with glial cells (iNOS-mediated NO release). NO is a free radical and can produce peroxynitrite. These reactive oxygen species induce a variety of neurotoxic mechanisms, including DNA/ protein alterations, mito‐

chondrial dysfunction, poly ADP-ribose polymerase (PARP) overactivation, apoptosis, neuro-inflammation, and lipid peroxidation (which jeopardize cellular membrane integrity, which leads to further Ca influx and NO release). These mechanisms are likely to be involved in cell death and memory impairments observed in AD. Several potential rela‐ tionships may exist between various AD markers (dashed arrows). ApoE e4 may induce iNOS-mediated NO produc‐ tion. NFT formation may influence Aβ accumulation and vice versa, and that APP metabolism may play a role in tau phosphorylation and subsequent NFT formation. NO, by activating a variety of signalling molecules, may induce Aβ and NFT formation. In ischemia, eNOS-mediated NO production appears to be neuroprotective by decreasing Ca in‐ flux and neuro-inflammation (thickened arrows). This NOS isoform may have a similar role in neurodegenerative dis‐ eases such as AD. [Law A., Gauthier S., Quiron R., Say NO to Alzheimer's disease: putative links between nitric oxide and dementia of the Alzheimer's type. 2001, ref 7]

Thus, the results strongly suggest that injection of fibrillar Aβ activates both microglia and astrocytes. Although both microglia and astrocytes show a dramatic upregulation of iNOS expression in response to injection of fAβ, there are marked spatial and morphological differences in the microglia and astrocyte responses to fAβ. Whereas microglia surround and phagocytize fAβ, astrocytes show no evidence of Aβ phagocytosis but rather form a virtual wall between microglia containing fAβ and the surrounding neurons.[23] The fAβ induces neuronal loss and a significant increase in iNOS expression by microglia and astrocytes *in vivo*, suggesting that it is the release of bioactive molecules like nitric oxide by microglia and astrocytes, rather than direct contact between Aβ fibrils and neurons, that mediates Aβ

Role of Oxidative Stress in Aβ Animal Model of Alzheimer's Disease: Vicious Circle of Apoptosis, Nitric Oxide and Age

In recent years, considerable data have accrued indicating that the brain in AD is under increased oxidative stress and this may have a role in the pathogenesis of neuron degeneration and death in this disorder. The direct evidence supporting increased oxidative stress in AD is: (1) increased brain Fe, Al, and Hg in AD, capable of stimulating free radical generation; (2) increased lipid peroxidation and decreased polyunsaturated fatty acids in the AD brain, and increased 4-hydroxynonenal, an aldehyde product of lipid peroxidation in AD ventricular fluid; (3) increased protein and DNA oxidation in the AD brain; (4) diminished energy metabolism and decreased cytochrome c oxidase in the brain in AD; (5) advanced glycation end products (AGE), malondialdehyde, carbonyls, peroxynitrite, heme oxygenase-1 and SOD-1 in neurofibrillary tangles and AGE, heme oxygenase-1, SOD-1 in senile plaques; and

Oxidative insults emanating from within the cell can threaten homeostasis if they are not appropriately resolved. Mitochondria actively and continuously generate ROS during respiration, favoring a situation of mitochondrial oxidative stress. The electron transport chain is an essential mechanism for generation of cellular energy and is localized to the mitochondrial inner membrane. Autooxidation of reduced respiratory chain components cause the produc‐ tion of free radical intermediates, O2 and H2O2, which in the presence of iron can produce hydroxyl radical (•OH). There are two specific sites where electrons may leak out of the chain to partially reduce oxygen. One is the NADH dehydrogenase and the other is at the ubiquinone cytochrome b intersection. These oxygen species are dealt with by superoxide dismutases (SOD), enzymes that are considered to be the first line of defense against oxygen toxicity, and exist in two forms in mammalian tissues: copper, zinc (Cu, Zn SOD), and manganese (Mn SOD) metalloproteins. Although mitochondria are notorious for ROS production, they are not the only sites of intracellular oxidative stress. In the cytosol, the arachadonic acid cascade, yielding prostaglandins, and leukotrienes may generate ROS when the released lipid is metabolized,

nitrosyl complexes with critical FeScontaining enzymes (e.g., aconitase), may cause an

impairment of mitochondrial function and energy depletion. Second, NO-


http://dx.doi.org/10.5772/54718

81

may directly


(6) amyloid beta peptide is capable of generating free radicals. [25]

and some cytochrome P-450 isozymes are notorious O2

been proposed for the mechanisms of NO-

neurotoxicity in AD.[23]

**4. Aβ and oxidative stress**

### **2. β-amyloid peptide in Alzheimer's disease**

In 1911, Alois Alzheimer described a neuropsychiatric disorder affecting the elderly, which is widely known today as Alzheimer's disease (AD). Early studies of patients afflicted with this disease demonstrated, via silver staining, the presence of lesions in the brain cortex. Those lesions corresponded to neurofibrillary tangles (NFTs), which are histopathologic structures localized within the neuronal cells. The majority of cases of AD correspond to the sporadic form of this disorder. Approximately 5–10% of patients present an autosomal mode of transmission and account for cases called familial Alzheimer's disease (FAD). The role of amyloid deposits in brain as a triggering factor for Alzheimer's disease has obtained increasing scientific support since Glenner's discovery in 1984. The amyloid precursor protein (APP) belongs to a type 1 transmembrane family of glycoproteins that is ubiquitously expressed in several types of cells. The N-terminal moiety of APP is projected toward the extracellular domain or can be localized in the lumen of intracellular vesicles, such as those of the endo‐ plasmic reticulum, Golgi apparatus, and intracellular endosomes. On the other hand, the APP C-terminal region lies in the cytoplasmic domain. APP is sensitive to proteolysis by a set of proteases called α, β, γ secretases. Secretases are responsible for the production of Aβ(1–40) peptide or the Aβ(1–42) variant with a significantly higher capacity to self-aggregate.[22]

#### **3. Aβ animal models**

A critical question that must be addressed in examining any animal model of a human disease is how well the animal model mimics the mechanisms and ultimate pathology observed in the human disease. [23] Clearly, there are differences between the present rat model and human AD. First, whereas in a study gliosis and pathology are observed over a time course of 30 d, in human AD the time course is on the order of decades. Second, the amyloid load per unit of brain area is generally higher and the amyloid distribution more extensive in the human AD brain than in their rat model. However, a key difficulty in addressing the mechanisms of pathology in human AD is that one rarely looks at human brain tissue at the initiation of the disease but rather examines the AD brain at the end stage of the disease, long after the initial mechanisms of pathology occur. In behalf of these discrepancies intracerebral or intracere‐ broventricular Aβ injections or infusions are used to mimic AD.[23,24]

Thus, the results strongly suggest that injection of fibrillar Aβ activates both microglia and astrocytes. Although both microglia and astrocytes show a dramatic upregulation of iNOS expression in response to injection of fAβ, there are marked spatial and morphological differences in the microglia and astrocyte responses to fAβ. Whereas microglia surround and phagocytize fAβ, astrocytes show no evidence of Aβ phagocytosis but rather form a virtual wall between microglia containing fAβ and the surrounding neurons.[23] The fAβ induces neuronal loss and a significant increase in iNOS expression by microglia and astrocytes *in vivo*, suggesting that it is the release of bioactive molecules like nitric oxide by microglia and astrocytes, rather than direct contact between Aβ fibrils and neurons, that mediates Aβ neurotoxicity in AD.[23]

### **4. Aβ and oxidative stress**

chondrial dysfunction, poly ADP-ribose polymerase (PARP) overactivation, apoptosis, neuro-inflammation, and lipid peroxidation (which jeopardize cellular membrane integrity, which leads to further Ca influx and NO release). These mechanisms are likely to be involved in cell death and memory impairments observed in AD. Several potential rela‐ tionships may exist between various AD markers (dashed arrows). ApoE e4 may induce iNOS-mediated NO produc‐ tion. NFT formation may influence Aβ accumulation and vice versa, and that APP metabolism may play a role in tau phosphorylation and subsequent NFT formation. NO, by activating a variety of signalling molecules, may induce Aβ and NFT formation. In ischemia, eNOS-mediated NO production appears to be neuroprotective by decreasing Ca in‐ flux and neuro-inflammation (thickened arrows). This NOS isoform may have a similar role in neurodegenerative dis‐ eases such as AD. [Law A., Gauthier S., Quiron R., Say NO to Alzheimer's disease: putative links between nitric oxide

In 1911, Alois Alzheimer described a neuropsychiatric disorder affecting the elderly, which is widely known today as Alzheimer's disease (AD). Early studies of patients afflicted with this disease demonstrated, via silver staining, the presence of lesions in the brain cortex. Those lesions corresponded to neurofibrillary tangles (NFTs), which are histopathologic structures localized within the neuronal cells. The majority of cases of AD correspond to the sporadic form of this disorder. Approximately 5–10% of patients present an autosomal mode of transmission and account for cases called familial Alzheimer's disease (FAD). The role of amyloid deposits in brain as a triggering factor for Alzheimer's disease has obtained increasing scientific support since Glenner's discovery in 1984. The amyloid precursor protein (APP) belongs to a type 1 transmembrane family of glycoproteins that is ubiquitously expressed in several types of cells. The N-terminal moiety of APP is projected toward the extracellular domain or can be localized in the lumen of intracellular vesicles, such as those of the endo‐ plasmic reticulum, Golgi apparatus, and intracellular endosomes. On the other hand, the APP C-terminal region lies in the cytoplasmic domain. APP is sensitive to proteolysis by a set of proteases called α, β, γ secretases. Secretases are responsible for the production of Aβ(1–40) peptide or the Aβ(1–42) variant with a significantly higher capacity to self-aggregate.[22]

A critical question that must be addressed in examining any animal model of a human disease is how well the animal model mimics the mechanisms and ultimate pathology observed in the human disease. [23] Clearly, there are differences between the present rat model and human AD. First, whereas in a study gliosis and pathology are observed over a time course of 30 d, in human AD the time course is on the order of decades. Second, the amyloid load per unit of brain area is generally higher and the amyloid distribution more extensive in the human AD brain than in their rat model. However, a key difficulty in addressing the mechanisms of pathology in human AD is that one rarely looks at human brain tissue at the initiation of the disease but rather examines the AD brain at the end stage of the disease, long after the initial mechanisms of pathology occur. In behalf of these discrepancies intracerebral or intracere‐

broventricular Aβ injections or infusions are used to mimic AD.[23,24]

and dementia of the Alzheimer's type. 2001, ref 7]

80 Neurodegenerative Diseases

**3. Aβ animal models**

**2. β-amyloid peptide in Alzheimer's disease**

In recent years, considerable data have accrued indicating that the brain in AD is under increased oxidative stress and this may have a role in the pathogenesis of neuron degeneration and death in this disorder. The direct evidence supporting increased oxidative stress in AD is: (1) increased brain Fe, Al, and Hg in AD, capable of stimulating free radical generation; (2) increased lipid peroxidation and decreased polyunsaturated fatty acids in the AD brain, and increased 4-hydroxynonenal, an aldehyde product of lipid peroxidation in AD ventricular fluid; (3) increased protein and DNA oxidation in the AD brain; (4) diminished energy metabolism and decreased cytochrome c oxidase in the brain in AD; (5) advanced glycation end products (AGE), malondialdehyde, carbonyls, peroxynitrite, heme oxygenase-1 and SOD-1 in neurofibrillary tangles and AGE, heme oxygenase-1, SOD-1 in senile plaques; and (6) amyloid beta peptide is capable of generating free radicals. [25]

Oxidative insults emanating from within the cell can threaten homeostasis if they are not appropriately resolved. Mitochondria actively and continuously generate ROS during respiration, favoring a situation of mitochondrial oxidative stress. The electron transport chain is an essential mechanism for generation of cellular energy and is localized to the mitochondrial inner membrane. Autooxidation of reduced respiratory chain components cause the produc‐ tion of free radical intermediates, O2 and H2O2, which in the presence of iron can produce hydroxyl radical (•OH). There are two specific sites where electrons may leak out of the chain to partially reduce oxygen. One is the NADH dehydrogenase and the other is at the ubiquinone cytochrome b intersection. These oxygen species are dealt with by superoxide dismutases (SOD), enzymes that are considered to be the first line of defense against oxygen toxicity, and exist in two forms in mammalian tissues: copper, zinc (Cu, Zn SOD), and manganese (Mn SOD) metalloproteins. Although mitochondria are notorious for ROS production, they are not the only sites of intracellular oxidative stress. In the cytosol, the arachadonic acid cascade, yielding prostaglandins, and leukotrienes may generate ROS when the released lipid is metabolized, and some cytochrome P-450 isozymes are notorious O2 -2 producers. Several possibilities have been proposed for the mechanisms of NO- -mediated cytotoxicity. [26] First, formation of ironnitrosyl complexes with critical FeScontaining enzymes (e.g., aconitase), may cause an impairment of mitochondrial function and energy depletion. Second, NO may directly damage chromatin by deamination and cross-linking of DNA, which increases mutagenesis. Third, generation of peroxynitrite by a reaction between NO and superoxide (O2 -2) may play a significant role in the cytotoxic process. Fourth, NO may inactivate several antioxidant enzymes, including catalase, glutathione peroxidase, and superoxide dismutases. Also, NOhas been reported to induce apoptosis by increasing ceramide generation through caspase-3 activation, induction of mitochondrial permeabilittransition, and activation of the Fas system. The mechanism of action of many exogenous agents involves redox cycling whereby an electron is accepted to form a free radical and it is then transferred to oxygen. [26]

in line with the recently proposed hypothesis of an intracellular amyloid-beta toxicity cascade which suggests that the toxic amyloid-beta species intervening in molecular and biochemical abnormalities may be intracellular soluble aggregates instead of extracellular, insoluble plaques. There are many studies proposing that megalin- and/or RAGE-dependent signalling are involved in the regulation of amyloid-beta clearance and probably may contribute to amyloid pathology and cognitive dysfunction observed in the AD patients and AD mouse

Role of Oxidative Stress in Aβ Animal Model of Alzheimer's Disease: Vicious Circle of Apoptosis, Nitric Oxide and Age

http://dx.doi.org/10.5772/54718

83

**Figure 2.** Mechanism of potential neuron death in AD. Anumber of conditions may cause free radical generation that can lead to peroxidation of membrane polyunsaturated fatty acids. This leads to formation of other oxygen radicals and generates aldehydes such as 4-hydroxynonenal (HNE). 4-HNE is capable of altering membrane ATPases leading to increased intracellular calcium, which initiates a cascade of events leading to further free radical generation and neu‐

One of the most interesting events in AD is that mitochondrial oxidative stress occurs early in AD progression, before the onset of Aβ pathology. Mitochondria are the major source of ROS, and, in fact, mitochondrial dysfunction as well as hypometabolism has long been implicated in the onset of the familial and sporadic forms of AD. mtDNA defects have also been linked to an increased incidence of AD. Energy deficiency and mitochondrial dysfunction have been recognized as a prominent, early event in AD. Mitochondrial abnormalities have been found

**5. Interrelation between amyloid-beta and mitochondria**

model. [28]

ron death. [Markesbery W.R, 1997, ref 25]

Although NO has many important and beneficial physiologic functions, it also can play a role in neurodegenerative disease pathology. In these diseases, NO is produced in excess by iNOS induction owing to the proinflammatory response, which is a common feature of neurodege‐ nerative disorders. Moreover, NO is much more harmful under pathologic conditions that involve the production of reactive oxygen species (ROS), such as superoxide anion and the formation of peroxynitrite. Two important properties of NO that may contribute to its pathologic functions are its ability to modify proteins through nitrosylation and nitrotyrosi‐ nation and its ability to react with oxygen to form RNS. [27]

In mature neurons, when cytosolic nNOS is the primary producer of NO, Ca2+ entry through overactive NMDA channels stimulates nNOS; thus, NO can then enter the mitochondria, directly inhibiting complex IV (cytochrome c oxidase) of the respiratory chain, which leads to a block of ATP production and eventual cell death due to energetic failure. [27]

Furthermore, NO has been shown to activate both the constitutive and inducible isoforms of cy‐ clooxygenase, which are upregulated in brain cells under proinflammatory conditions. During the catalytic cycle of cyclooxygenase, the release of free radicals and the formation of prosta‐ glandins occur, two events closely related to the development of neuroinflammation. [27]

Hsp90 is another protein associated with AD that undergoes nitrosylation. S-Nitrosylation of Hsp90 abolishes ATPase activity that is necessary for its chaperone function; thus, inactivation of Hsp90 may allow accumulation of tau and Aβ aggregates in the AD brain. It is generally recognized that mitochondria continuously undergo two opposing processes, fission and fusion. The disruption of this dynamic equilibrium may herald cell injury or death and may contribute to developmental and neurodegenerative disorders. Nitric oxide produced in response to β-amyloid protein has been shown to trigger mitochondrial fission, synaptic loss, and neuronal damage, in part through S-nitrosylation of dynamin-related protein. [27]

Many studies showed amyloid-beta interaction with different receptors in the cellular membrane of the vasculature, neurons, oligodendrocytes, and glial cells where it is transported from cell surface into endosomal and lysosomal compartments. The aberrant signalling of these receptors in AD triggered an abnormal accumulation of amyloid-beta into cytosolinducing cellular stress underlies to neuronal dysfunction and dementia. It is described that these receptors can be megalin, also known as low-density lipoprotein related protein-2 (LRP2), LRP-1, or RAGE (receptors for advanced glycation end products). The interaction of these receptors with amyloid-beta in neurons, microglia, and vascular cells accelerates and amplifies deleterious effects on neuronal and synaptic functions. [28] These findings are further in line with the recently proposed hypothesis of an intracellular amyloid-beta toxicity cascade which suggests that the toxic amyloid-beta species intervening in molecular and biochemical abnormalities may be intracellular soluble aggregates instead of extracellular, insoluble plaques. There are many studies proposing that megalin- and/or RAGE-dependent signalling are involved in the regulation of amyloid-beta clearance and probably may contribute to amyloid pathology and cognitive dysfunction observed in the AD patients and AD mouse model. [28]

damage chromatin by deamination and cross-linking of DNA, which increases mutagenesis.

enzymes, including catalase, glutathione peroxidase, and superoxide dismutases. Also, NOhas been reported to induce apoptosis by increasing ceramide generation through caspase-3 activation, induction of mitochondrial permeabilittransition, and activation of the Fas system. The mechanism of action of many exogenous agents involves redox cycling whereby an

Although NO has many important and beneficial physiologic functions, it also can play a role in neurodegenerative disease pathology. In these diseases, NO is produced in excess by iNOS induction owing to the proinflammatory response, which is a common feature of neurodege‐ nerative disorders. Moreover, NO is much more harmful under pathologic conditions that involve the production of reactive oxygen species (ROS), such as superoxide anion and the formation of peroxynitrite. Two important properties of NO that may contribute to its pathologic functions are its ability to modify proteins through nitrosylation and nitrotyrosi‐

In mature neurons, when cytosolic nNOS is the primary producer of NO, Ca2+ entry through overactive NMDA channels stimulates nNOS; thus, NO can then enter the mitochondria, directly inhibiting complex IV (cytochrome c oxidase) of the respiratory chain, which leads to

Furthermore, NO has been shown to activate both the constitutive and inducible isoforms of cy‐ clooxygenase, which are upregulated in brain cells under proinflammatory conditions. During the catalytic cycle of cyclooxygenase, the release of free radicals and the formation of prosta‐ glandins occur, two events closely related to the development of neuroinflammation. [27]

Hsp90 is another protein associated with AD that undergoes nitrosylation. S-Nitrosylation of Hsp90 abolishes ATPase activity that is necessary for its chaperone function; thus, inactivation of Hsp90 may allow accumulation of tau and Aβ aggregates in the AD brain. It is generally recognized that mitochondria continuously undergo two opposing processes, fission and fusion. The disruption of this dynamic equilibrium may herald cell injury or death and may contribute to developmental and neurodegenerative disorders. Nitric oxide produced in response to β-amyloid protein has been shown to trigger mitochondrial fission, synaptic loss, and neuronal damage, in part through S-nitrosylation of dynamin-related protein. [27]

Many studies showed amyloid-beta interaction with different receptors in the cellular membrane of the vasculature, neurons, oligodendrocytes, and glial cells where it is transported from cell surface into endosomal and lysosomal compartments. The aberrant signalling of these receptors in AD triggered an abnormal accumulation of amyloid-beta into cytosolinducing cellular stress underlies to neuronal dysfunction and dementia. It is described that these receptors can be megalin, also known as low-density lipoprotein related protein-2 (LRP2), LRP-1, or RAGE (receptors for advanced glycation end products). The interaction of these receptors with amyloid-beta in neurons, microglia, and vascular cells accelerates and amplifies deleterious effects on neuronal and synaptic functions. [28] These findings are further

a block of ATP production and eventual cell death due to energetic failure. [27]

electron is accepted to form a free radical and it is then transferred to oxygen. [26]

and superoxide (O2

may inactivate several antioxidant


Third, generation of peroxynitrite by a reaction between NO-

nation and its ability to react with oxygen to form RNS. [27]

a significant role in the cytotoxic process. Fourth, NO-

82 Neurodegenerative Diseases

**Figure 2.** Mechanism of potential neuron death in AD. Anumber of conditions may cause free radical generation that can lead to peroxidation of membrane polyunsaturated fatty acids. This leads to formation of other oxygen radicals and generates aldehydes such as 4-hydroxynonenal (HNE). 4-HNE is capable of altering membrane ATPases leading to increased intracellular calcium, which initiates a cascade of events leading to further free radical generation and neu‐ ron death. [Markesbery W.R, 1997, ref 25]

### **5. Interrelation between amyloid-beta and mitochondria**

One of the most interesting events in AD is that mitochondrial oxidative stress occurs early in AD progression, before the onset of Aβ pathology. Mitochondria are the major source of ROS, and, in fact, mitochondrial dysfunction as well as hypometabolism has long been implicated in the onset of the familial and sporadic forms of AD. mtDNA defects have also been linked to an increased incidence of AD. Energy deficiency and mitochondrial dysfunction have been recognized as a prominent, early event in AD. Mitochondrial abnormalities have been found both in neurons and astrocytes, suggesting that both neurons and astrocytes might be damaged by free radicals in the AD brain. Superoxide radicals might be produced in mitochondrial electron transport chain complexes I and III and in components of the Krebs cycle, including αketoglutarate dehydrogenas. In addition, superoxide radicals might be generated in the outer mitochondrial membrane. H2O2 and superoxide radicals, released from the mitochondrial matrix and from the inner and outer mitochondrial membranes, might be carried to the cytoplasm and, ultimately, might lead to the oxidation of cytoplasmic proteins. [28]

Microglial activation is a key feature in Alzheimer's disease and is considered to contribute to progressive neuronal injury by release of neurotoxic products. Mononuclear phagocytes, such as microglial cells, are crucial components of the innate immune system. Cellular activation in response to pathogen-associated molecular patterns on microorganisms is mediated through interaction with innate immune receptors on the surfaces of mononuclear phagocytes, e.g., Toll-like receptors (TLRs) and the lipopolysaccharide receptor CD14. Lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria, was first identified as the TLR ligand. The innate immune receptor Toll-like-receptor 4 (TLR4), localized on the surface of microglia, is a first-line host defense receptor against invading microorganisms. It has been shown that a spontaneous loss-of-function mutation in the *Tlr4* gene strongly inhibits micro‐ glial and monocytic activation by aggregated Alzheimer amyloid peptide resulting in a significantly lower release of the inflammatory products IL-6, TNFα and nitric oxide. Treat‐ ment of primary murine neuronal cells with supernatant of amyloid peptide-stimulated microglia demonstrates that Tlr4 contributes to amyloid peptide-induced microglial neuro‐ toxicity. The reason how TLR4 becomes activated in the pathophysiology of AD is unclear. These findings further support a role of TLR4 in neuroinflammation in AD. Microglial

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85

activation in AD pathophysiology is discussed as a two-edged sword. [29]

iNOS promoter activity. [29]

It has been demonstarted that Aβ1– 40 administration induced an increase in TNF-α expression and oxidative alterations in prefrontal cortex and hippocampus. Likewise, Aβ1– 40 led to activation of both JNK (c-Jun-NH2-terminal kinase)/c-Jun and nuclear factor-κB, resulting in iNOS upregulation in both brain structures. [29] It has been shown that the anti-TNF-α antibody reduced all of the molecular and biochemical alterations promoted by Aβ1– 40. These results provide new insights in Mouse models of AD, revealing TNF-α and iNOS as central mediators of Aβ action. These pathways might be targeted for AD drug development. [29]

Tumor necrosis factor-α (TNF-α) is a cytokine thought to play a central role in the selfpropagation of neuroinflammation. TNF-α regulates many cellular processes, including inflammation, differentiation, and cell death through activation of TNF receptor 1 (TNFR1) or TNFR2. The transduction pathways activated by TNF-α include mitogen-activated protein kinases (MAPKs) and IκB kinase, which control gene expression through transcriptional factors such as activator protein-1 (AP-1) and nuclear factor-κB (NF-κB). Regarding the CNS, microglia and astrocytes are believed to be the primary sources of TNF-α. Evidence indicates the presence of increased levels of TNF-α in the brain and plasma of AD patients and an upregulation of TNFR1 have been detected in the AD brain. Aβ has been shown to interact in a synergistic manner with cytokines to induce neuronal damage via reactive oxygen species (ROS)- and NO-dependent pathways. [30] Numerous animal models have been used to evaluate the role of inflammation in the course of AD. An experimental model that mimics the progression of AD was developed using an intracerebroventricular injection of Aβ in mice. The cross talk between TNF-α and iNOS is probably mediated by activation of two major intracellular pathways: c-Jun-NH2-terminal kinase (JNK)/c-Jun and NF- κB. According to the results, they suggested that TNF-α and iNOS as important mediators of Aβ-induced cognitive impairment. TNF-α signaling effectors include JNK and c-Jun that might also contribute to

Both APP and Aβ are present in mitochondrial membrane and interact with mitochondrial proteins, block mitochondrial import channels, impair mitochondrial transport, disrupt the electron transfer chain, increase ROS levels, and cause mitochondrial damage. [28]

APP and amyloid-beta may block mitochondrial translocation of nuclear-encoded proteins, such as components of the electron transport chain, impairing mitochondrial function. Intra‐ mitochondrial amyloid-beta is able to perturb mitochondrial function in several ways by di‐ rectly influencing extracellular transport chain complex activities, impairing mitochondrial dynamics, or disturbing calcium storage, thus increasing apoptotic pathways. Moreover, amyloid-beta interacts with mitochondrial matrix components inducing an improper mito‐ chondrial complex function leads to a decreased mitochondrial membrane potential of the organelle and impairing ATP formation. [28]

It is well documented that mtDNA changes are responsible for aging phenotypes.

It has been hypothesized that ongoing oxidative damage to mtDNA may be the underlying mechanism for cellular senescence. Since mtDNA repair mechanisms are limited and be‐ cause mtDNA is situated in close proximity to the site of ROS production, mtDNA is more vulnerable to oxidative damage than nuclear DNA. With age, oxidation of mtDNA increases compared to nuclear DNA leading to an age dependent accumulation of mtDNA mutations. The effects of these mutations may lead to opening of the mitochondrial permeability transi‐ tion pore and subsequent neuronal death. Increased ROS levels act at multiple levels to im‐ pair mitochondrial function: they induce mtDNA mutations that consequently negatively influence mitochondrial function, enhance amyloid-beta production by guiding APP cleav‐ age pathway toward the amyloidogenesis, increase lipid peroxidation, activate mitophagy, leading to a reduced mitochondrial number, and augment tau hyperphosphorylation and NFT formation impairing organelle trafficking and neuronal function which leads to apop‐ tosis. [28] In addition to its well-known role in glycolysis, GAPDH contributes to nuclear signaling in apoptosis. SNitrosylation of GAPDH terminates its enzymatic activity and al‐ lows binding of GAPDH to Siah1, an E3 ubiquitin ligase. Siah1 has a nuclear localization signal and carries GAPDH to the nucleus. GAPDH stabilizes Siah1 in the nucleus and allows degradation of nuclear proteins through ubiquitination. [27]

Protein-disulfide isomerase (PDI) is an endoplasmic reticulum (ER)-associated chaperone protein that prevents neurotoxicity caused by ER stress and protein misfolding and can also function as an NO receptor or donor, depending on the cellular context. PDI is nitrosylated both in AD and PD. Both PD and AD patient postmortem brains exhibit increased levels of nitrosylated PDI as compared with those of healthy controls. PDI nitrosylation prevents PDI-mediated ER stress reduction and allows protein misfolding. [27]

both in neurons and astrocytes, suggesting that both neurons and astrocytes might be damaged by free radicals in the AD brain. Superoxide radicals might be produced in mitochondrial electron transport chain complexes I and III and in components of the Krebs cycle, including αketoglutarate dehydrogenas. In addition, superoxide radicals might be generated in the outer mitochondrial membrane. H2O2 and superoxide radicals, released from the mitochondrial matrix and from the inner and outer mitochondrial membranes, might be carried to the

Both APP and Aβ are present in mitochondrial membrane and interact with mitochondrial proteins, block mitochondrial import channels, impair mitochondrial transport, disrupt the

APP and amyloid-beta may block mitochondrial translocation of nuclear-encoded proteins, such as components of the electron transport chain, impairing mitochondrial function. Intra‐ mitochondrial amyloid-beta is able to perturb mitochondrial function in several ways by di‐ rectly influencing extracellular transport chain complex activities, impairing mitochondrial dynamics, or disturbing calcium storage, thus increasing apoptotic pathways. Moreover, amyloid-beta interacts with mitochondrial matrix components inducing an improper mito‐ chondrial complex function leads to a decreased mitochondrial membrane potential of the

It has been hypothesized that ongoing oxidative damage to mtDNA may be the underlying mechanism for cellular senescence. Since mtDNA repair mechanisms are limited and be‐ cause mtDNA is situated in close proximity to the site of ROS production, mtDNA is more vulnerable to oxidative damage than nuclear DNA. With age, oxidation of mtDNA increases compared to nuclear DNA leading to an age dependent accumulation of mtDNA mutations. The effects of these mutations may lead to opening of the mitochondrial permeability transi‐ tion pore and subsequent neuronal death. Increased ROS levels act at multiple levels to im‐ pair mitochondrial function: they induce mtDNA mutations that consequently negatively influence mitochondrial function, enhance amyloid-beta production by guiding APP cleav‐ age pathway toward the amyloidogenesis, increase lipid peroxidation, activate mitophagy, leading to a reduced mitochondrial number, and augment tau hyperphosphorylation and NFT formation impairing organelle trafficking and neuronal function which leads to apop‐ tosis. [28] In addition to its well-known role in glycolysis, GAPDH contributes to nuclear signaling in apoptosis. SNitrosylation of GAPDH terminates its enzymatic activity and al‐ lows binding of GAPDH to Siah1, an E3 ubiquitin ligase. Siah1 has a nuclear localization signal and carries GAPDH to the nucleus. GAPDH stabilizes Siah1 in the nucleus and allows

Protein-disulfide isomerase (PDI) is an endoplasmic reticulum (ER)-associated chaperone protein that prevents neurotoxicity caused by ER stress and protein misfolding and can also function as an NO receptor or donor, depending on the cellular context. PDI is nitrosylated both in AD and PD. Both PD and AD patient postmortem brains exhibit increased levels of nitrosylated PDI as compared with those of healthy controls. PDI nitrosylation prevents

cytoplasm and, ultimately, might lead to the oxidation of cytoplasmic proteins. [28]

electron transfer chain, increase ROS levels, and cause mitochondrial damage. [28]

It is well documented that mtDNA changes are responsible for aging phenotypes.

organelle and impairing ATP formation. [28]

84 Neurodegenerative Diseases

degradation of nuclear proteins through ubiquitination. [27]

PDI-mediated ER stress reduction and allows protein misfolding. [27]

Microglial activation is a key feature in Alzheimer's disease and is considered to contribute to progressive neuronal injury by release of neurotoxic products. Mononuclear phagocytes, such as microglial cells, are crucial components of the innate immune system. Cellular activation in response to pathogen-associated molecular patterns on microorganisms is mediated through interaction with innate immune receptors on the surfaces of mononuclear phagocytes, e.g., Toll-like receptors (TLRs) and the lipopolysaccharide receptor CD14. Lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria, was first identified as the TLR ligand. The innate immune receptor Toll-like-receptor 4 (TLR4), localized on the surface of microglia, is a first-line host defense receptor against invading microorganisms. It has been shown that a spontaneous loss-of-function mutation in the *Tlr4* gene strongly inhibits micro‐ glial and monocytic activation by aggregated Alzheimer amyloid peptide resulting in a significantly lower release of the inflammatory products IL-6, TNFα and nitric oxide. Treat‐ ment of primary murine neuronal cells with supernatant of amyloid peptide-stimulated microglia demonstrates that Tlr4 contributes to amyloid peptide-induced microglial neuro‐ toxicity. The reason how TLR4 becomes activated in the pathophysiology of AD is unclear. These findings further support a role of TLR4 in neuroinflammation in AD. Microglial activation in AD pathophysiology is discussed as a two-edged sword. [29]

It has been demonstarted that Aβ1– 40 administration induced an increase in TNF-α expression and oxidative alterations in prefrontal cortex and hippocampus. Likewise, Aβ1– 40 led to activation of both JNK (c-Jun-NH2-terminal kinase)/c-Jun and nuclear factor-κB, resulting in iNOS upregulation in both brain structures. [29] It has been shown that the anti-TNF-α antibody reduced all of the molecular and biochemical alterations promoted by Aβ1– 40. These results provide new insights in Mouse models of AD, revealing TNF-α and iNOS as central mediators of Aβ action. These pathways might be targeted for AD drug development. [29]

Tumor necrosis factor-α (TNF-α) is a cytokine thought to play a central role in the selfpropagation of neuroinflammation. TNF-α regulates many cellular processes, including inflammation, differentiation, and cell death through activation of TNF receptor 1 (TNFR1) or TNFR2. The transduction pathways activated by TNF-α include mitogen-activated protein kinases (MAPKs) and IκB kinase, which control gene expression through transcriptional factors such as activator protein-1 (AP-1) and nuclear factor-κB (NF-κB). Regarding the CNS, microglia and astrocytes are believed to be the primary sources of TNF-α. Evidence indicates the presence of increased levels of TNF-α in the brain and plasma of AD patients and an upregulation of TNFR1 have been detected in the AD brain. Aβ has been shown to interact in a synergistic manner with cytokines to induce neuronal damage via reactive oxygen species (ROS)- and NO-dependent pathways. [30] Numerous animal models have been used to evaluate the role of inflammation in the course of AD. An experimental model that mimics the progression of AD was developed using an intracerebroventricular injection of Aβ in mice. The cross talk between TNF-α and iNOS is probably mediated by activation of two major intracellular pathways: c-Jun-NH2-terminal kinase (JNK)/c-Jun and NF- κB. According to the results, they suggested that TNF-α and iNOS as important mediators of Aβ-induced cognitive impairment. TNF-α signaling effectors include JNK and c-Jun that might also contribute to iNOS promoter activity. [29]

### **6. Aβ and age**

The glycation hypothesis of aging suggests that modification of proteins by glucose (the ''Maillard reaction'') leads to the development of ''advanced glycation end-products''

Deregulation of distinct signaling pathways leads to aberrant phosphorylation of cellular

Role of Oxidative Stress in Aβ Animal Model of Alzheimer's Disease: Vicious Circle of Apoptosis, Nitric Oxide and Age

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They reported the identification of signal transducer and activator of transcription 3 (STAT3) as a potential key player in AD pathophysiology. STAT3 is a transcription factor that is typically associated with cytokine signaling during neuronal differentiation, inflammation, and malignancies. Interestingly, they found that tyrosine phosphorylation of STAT3, which is required for the activation of this transcription factor, is markedly elevated in neurons treated with Aβ *in vitro* or *in vivo* as well as in the brains of APP/PS1 transgenic mice. Inhibition of STAT3 activation or reduced STAT3 expression significantly attenuates Aβ-induced neuronal cell death. Moreover, activation of a tyrosine kinase Tyk2 is required for the Aβ-induced tyrosine phosphorylation of STAT3 and neuronal cell death. Notably, elevation of STAT3 tyrosine phosphorylation is evident in postmortem samples of AD brains. These observations collectively raise an intriguing possibility that STAT3 signaling is involved in neuronal

Activated microglia secrete various humoral substances (cytokines, free radicals) which influence neurons and glia. The inhibition of C3 conversion resulted in the lack of Aβ opsoni‐ sation and prevented microglia from Aβ clearance and, as a result, doubled the amyloid burden in murine brains. [33] The following may be responsible for the inconsistency in the results of

Microglia activation is followed by astrocyte activation. Astrocytes phagocyte and degrade Aβ. *In vivo* experiments suggest that the coexistence of microglia and astrocytes diminishes the ability of microglia to digest and degrade plaques and Aβ. Therefore, activated astrocytes may exert a regulative effect (negative feedback) on the phagocytic activity of microglia. Another factor influencing microglia activation is the neuronal expression of cyclooxygenase-2 (COX-2). COX-2 participates in prostaglandin production and its expression is usually elevated

In AD, initially COX-2 expression is evident in pyramidal neurons particularly involved in AD. COX-2 expression rises at the onset of the disease and then declines in the advanced stages of AD. Of note, the expression of COX-2 correlated positively with the level of prostaglandin E2 (PGE2) in cerebrospinal fluid (CSF). CSF PGE2 levels are clearly higher in people with mild

Together with astrocytes, microglia have the ability to bind and take up Aβ in vitro and in vivo, being important players for the deposition and removal of Aβ, although there are also

proteins and has a profound effect on the progression of AD. [32]

apoptosis observed in AD patients. [32]

**•** immunomodulation of astroglia, **•** immunomodulation of neurons,

**•** senescence of microglia.

in places of inflammation. [33]

**8. Interactions between Aβ and microglia**

experiments on microglia behaviour in contact with Aβ:

dementia and decrease in the late stages of AD. [33]

(AGEs). [31] In the human brain, AGEs accumulate in neuronal perikarya of the hippocampus and parahippocampus, as well as in reactive astroglia in brains after the third decade of age. In AD, this effect is twofold: AGEs accumulate extracellularly on β-amyloid plaques and intracellulary in neurons and astrocytes. The binding of AGEs to its receptor, RAGE (receptor for advanced glycation endproducts), activates NADPH-oxidase, a central participant in the production of superoxide radicals. Superoxide and its conversion product, hydrogen peroxide, were shown to activate redoxsensitive transcription factors such as NF-κB and activator protein 1 (AP-1) resulting in the upregulation of cytokines such as interleukin (IL)-1, IL-6, tumor necrosis factor (TNF-α) and inducible nitric oxide synthase (iNOS). Interestingly, RAGE is also activated by Aβ, the major pro-inflammatory peptide present in amyloid plaques in AD and HMGB1/amphoterin, a novel pro-inflammatory ligand released from dying cells. TNF-α and NO were chosen as relevant biomolecules due to their role in augmenting inflammation and/or inducing cell death. TNF-α (cachexin or cachectin) is a cytokine involved in systemic inflammation that upregulates other NF-κB-regulated cytokines and is also a member of a group of cytokines that stimulate the acute phase reaction. TNF-α is also able to induce apoptotic cell death. [31]

Although the exact target of the polyphenols along the pro-inflammatory signal cascades is not known in detail, it is most likely suggested that they interfere with the NF-κB pathway somewhere upstream of kinases phosphorylating the inhibitor of κB, IκB. There are two signaling pathways leading to the activation of NF-κB known as the canonical pathway (or classical) and the noncanonical pathway (or alternative pathway). [31] The common regulatory step in both of these cascades is activation of an IκB kinase (IκK) complex consisting of catalytic kinase subunits (IκKa and/or IκKb) and the regulatory nonenzymatic scaffold protein NEMO. Activation of NF-κB dimers is due to IκK-mediated phosphorylation-induced proteasomal degradation of the IκB inhibitor enabling the active NF-κB transcription factor subunits to translocate to the nucleus and induce target gene expression. In the canonical (or classical) activation pathway, the complex consisting of IκK-b and IκK-g/NEMO phosphorylates two critical serine residues in IκB-a. IκB-a can then be targeted for ubiquitination and degradation. Some non-canonical pathways of IκK-independent activation of NF-κB stipulate the selective activation of NF-κB subunits. [31]

### **7. Deregulation of ıntracellular signallings due to Aβ**

The studies suggest that various intracellular signaling pathways are deregulated in AD brains or during Aβ-induced neuronal apoptosis. For example, activation of stress-related kinases c-Jun N-terminal kinase (JNK) and p38 is associated with neuronal death in AD Mouse model. [32] Glycogen synthase kinase 3 (GSK-3) has also been implicated in Aβ-induced neurotoxicity. Deregulation of distinct signaling pathways leads to aberrant phosphorylation of cellular proteins and has a profound effect on the progression of AD. [32]

They reported the identification of signal transducer and activator of transcription 3 (STAT3) as a potential key player in AD pathophysiology. STAT3 is a transcription factor that is typically associated with cytokine signaling during neuronal differentiation, inflammation, and malignancies. Interestingly, they found that tyrosine phosphorylation of STAT3, which is required for the activation of this transcription factor, is markedly elevated in neurons treated with Aβ *in vitro* or *in vivo* as well as in the brains of APP/PS1 transgenic mice. Inhibition of STAT3 activation or reduced STAT3 expression significantly attenuates Aβ-induced neuronal cell death. Moreover, activation of a tyrosine kinase Tyk2 is required for the Aβ-induced tyrosine phosphorylation of STAT3 and neuronal cell death. Notably, elevation of STAT3 tyrosine phosphorylation is evident in postmortem samples of AD brains. These observations collectively raise an intriguing possibility that STAT3 signaling is involved in neuronal apoptosis observed in AD patients. [32]

### **8. Interactions between Aβ and microglia**

Activated microglia secrete various humoral substances (cytokines, free radicals) which influence neurons and glia. The inhibition of C3 conversion resulted in the lack of Aβ opsoni‐ sation and prevented microglia from Aβ clearance and, as a result, doubled the amyloid burden in murine brains. [33] The following may be responsible for the inconsistency in the results of experiments on microglia behaviour in contact with Aβ:


**6. Aβ and age**

86 Neurodegenerative Diseases

apoptotic cell death. [31]

activation of NF-κB subunits. [31]

**7. Deregulation of ıntracellular signallings due to Aβ**

The glycation hypothesis of aging suggests that modification of proteins by glucose (the ''Maillard reaction'') leads to the development of ''advanced glycation end-products''

(AGEs). [31] In the human brain, AGEs accumulate in neuronal perikarya of the hippocampus and parahippocampus, as well as in reactive astroglia in brains after the third decade of age. In AD, this effect is twofold: AGEs accumulate extracellularly on β-amyloid plaques and intracellulary in neurons and astrocytes. The binding of AGEs to its receptor, RAGE (receptor for advanced glycation endproducts), activates NADPH-oxidase, a central participant in the production of superoxide radicals. Superoxide and its conversion product, hydrogen peroxide, were shown to activate redoxsensitive transcription factors such as NF-κB and activator protein 1 (AP-1) resulting in the upregulation of cytokines such as interleukin (IL)-1, IL-6, tumor necrosis factor (TNF-α) and inducible nitric oxide synthase (iNOS). Interestingly, RAGE is also activated by Aβ, the major pro-inflammatory peptide present in amyloid plaques in AD and HMGB1/amphoterin, a novel pro-inflammatory ligand released from dying cells. TNF-α and NO were chosen as relevant biomolecules due to their role in augmenting inflammation and/or inducing cell death. TNF-α (cachexin or cachectin) is a cytokine involved in systemic inflammation that upregulates other NF-κB-regulated cytokines and is also a member of a group of cytokines that stimulate the acute phase reaction. TNF-α is also able to induce

Although the exact target of the polyphenols along the pro-inflammatory signal cascades is not known in detail, it is most likely suggested that they interfere with the NF-κB pathway somewhere upstream of kinases phosphorylating the inhibitor of κB, IκB. There are two signaling pathways leading to the activation of NF-κB known as the canonical pathway (or classical) and the noncanonical pathway (or alternative pathway). [31] The common regulatory step in both of these cascades is activation of an IκB kinase (IκK) complex consisting of catalytic kinase subunits (IκKa and/or IκKb) and the regulatory nonenzymatic scaffold protein NEMO. Activation of NF-κB dimers is due to IκK-mediated phosphorylation-induced proteasomal degradation of the IκB inhibitor enabling the active NF-κB transcription factor subunits to translocate to the nucleus and induce target gene expression. In the canonical (or classical) activation pathway, the complex consisting of IκK-b and IκK-g/NEMO phosphorylates two critical serine residues in IκB-a. IκB-a can then be targeted for ubiquitination and degradation. Some non-canonical pathways of IκK-independent activation of NF-κB stipulate the selective

The studies suggest that various intracellular signaling pathways are deregulated in AD brains or during Aβ-induced neuronal apoptosis. For example, activation of stress-related kinases c-Jun N-terminal kinase (JNK) and p38 is associated with neuronal death in AD Mouse model. [32] Glycogen synthase kinase 3 (GSK-3) has also been implicated in Aβ-induced neurotoxicity. Microglia activation is followed by astrocyte activation. Astrocytes phagocyte and degrade Aβ. *In vivo* experiments suggest that the coexistence of microglia and astrocytes diminishes the ability of microglia to digest and degrade plaques and Aβ. Therefore, activated astrocytes may exert a regulative effect (negative feedback) on the phagocytic activity of microglia. Another factor influencing microglia activation is the neuronal expression of cyclooxygenase-2 (COX-2). COX-2 participates in prostaglandin production and its expression is usually elevated in places of inflammation. [33]

In AD, initially COX-2 expression is evident in pyramidal neurons particularly involved in AD. COX-2 expression rises at the onset of the disease and then declines in the advanced stages of AD. Of note, the expression of COX-2 correlated positively with the level of prostaglandin E2 (PGE2) in cerebrospinal fluid (CSF). CSF PGE2 levels are clearly higher in people with mild dementia and decrease in the late stages of AD. [33]

Together with astrocytes, microglia have the ability to bind and take up Aβ in vitro and in vivo, being important players for the deposition and removal of Aβ, although there are also reports suggesting that Aβ formation occurs independently of microglial cells. [34] Microglial cells express several pattern-recognition receptors, which allow them to remove potentially toxic molecules such as Aβ. These receptors include scavenger receptors (SR) class A (SR-A), class B type I, low-density lipoprotein receptor-related protein (LRP), cluster of differentiation 36 (CD36), receptor for end glycation product, mannose receptor, and SR-MARCO. [34]

morphologically by a series of events that include cytoplasmic shrinkage, chromatin conden‐ sation, nuclear and cellular fragmentation, and the formation of apoptotic bodies. Although caspases are the main players involved in apoptosis, there are other molecules involved in the progression of the apoptotic cascade that are relevant to AD. The neuronal death in AD may result directly and/or indirectly from the triggering insults caused by Aβ toxicity, glutamate excitotoxicity, long-lasting oxidative stress, DNA damage, and elevation of intracellular calcium levels. Thus, the mode of cell death in AD remains a matter of controversy, and it is possible that both apoptotic and non-apoptotic cell death coexist in the brains of affected

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Previously, it was generally considered that apoptotic neuronal death in chronic neurodege‐ nerative disease, e.g., AD, Parkinson's disease, etc., is associated with classical caspase mediated cell death. However, in part, it was suggested that the caspase-independent pathway might also participate in the pathogenesis of the disease. Cross talk is extensive between different cell death pathways, which include multiple types of caspase-dependent and

Cells undergo apoptosis through two major pathways, the extrinsic pathway (death receptor pathway) and the intrinsic pathway (the mitochondrial pathway). These two pathways can be linked by caspase-8-activated truncated Bid formation. Very recently, death receptor 6 (DR6) was shown to be involved in the neurodegeneration observed in Alzheimer disease. DR6, also known as TNFRSF21, is a relatively new member of the death receptor family, and it was found that DR6 induces apoptosis when it is overexpressed. However, how the death signal mediated by DR6 is transduced intracellularly is not known. To this end, in a study they have examined the roles of caspases, apoptogenic mitochondrial factor cytochrome c, and the Bcl-2 family proteins in DR6-induced apoptosis. In their study results demonstrated that Bax translocation is absolutely required for DR6-induced apoptosis. On the other hand, they found inhibition of caspase-8 and knockdown of Bid have no effect on DR6-induced apoptosis. Their results strongly suggest that DR6-induced apoptosis occurs through a new pathway that is different

Different studies reported both necrotic and apoptotic mechanisms for Aβ-mediated neuro‐ toxicity. In particular, oxidative-mediated DNA damage, with a pattern indicative of apopto‐ sis, was found in AD brain, which is consistent with several lines of experimental evidence linking oxidative stress and neuronal apoptosis. [38] Apoptosis is induced by micromolar concentrations of Aβ in cultured CNS neurons, however, physiological nanomolar concentra‐ tions of Aβ1-40 and Aβ1-42 are insufficient to initiate significant apoptosis in cultures of human fetal neurons. In fact, both Aβ peptides downregulate "bcl-2", a key anti- apoptotic protein, while only Aβ1-42 upregulates "bax", a protein known to promote apoptotic cell death. Interestingly, Aβ treated neurons exposed to different levels of oxidative stress, unable to increase apoptosis in control neurons, show 10-20 times more apoptotic-mediated DNAdamage, suggesting that Aβ renders the neurons vulnerable to age-dependent oxidative stress and neurodegeneration. Other links between oxidative stress and apoptotic neuronal cell death in AD have been described. Apoptosis induced by 4-HNE is prevented in cells that overexpress "bcl-2" or by incubation with glutathione, which binds 4-HNE. Also, PC12 cells expressing a

from the type I and type II pathways through interacting with Bax. [37]

patients. [36]

caspase-independent programmed cell death. [36]

In fact, microglial cells secrete multiple cytokines such as interleukin-1b (IL1b), tumor necrosis factor (TNF-α), interleukin 10 (IL10), and transforming growth factor-b (TGFβ) as well as shortlived cytotoxic factors such as superoxide (O2 -2) and NO- , which contribute to neurotoxicity. [34] It has been suggested that TGFβ1 produced by astrocytes and hippocampal cells prevents induction of reactive oxygen species (ROS) and NO- production by inflammatory mediators (lipopolysaccharide [LPS] 1 interferon-γ [IFNγ]) and neurotoxicity in vitro and that the modulation of microglia is at least partially mediated by the activation of the ERK pathway. TGFβ superfamily signaling plays important roles in a diverse set of cellular responses, including cell proliferation, differentiation, extracellular matrix remodeling, and embryonic development. TGFβ1 signaling is mediated by cell surface type I and type II receptors, which phosphorylates the two R-Smad proteins Smad2 and Smad3 downstream. Phospho-Smad2/3 forms a complex with the common mediator Smad4 that binds to a Smad-binding element (SBE) in the nucleus with a large number of transcription coregulators to activate gene promoter. They showed that the TGFβ1 Smad3 pathway is involved in modulation of micro‐ glial cell activity through its effects on expression of SRs, NO secretion, and phagocytosis. Microglial cell activation can be initiated by various signal transduction pathways, including nuclear factor-jB, JAK/STAT, and p38 pathways, which are activated by several inflammatory mediators and Aβ. [34]

### **9. Aβ and apoptosis**

Apoptosis, or programmed cell death, is a highly regulated process involved in embryonic development, developmental tissue remodeling and normal cell turnover. [35] However, when dysregulation occurs in apoptotic pathways, excessive or insufficient cell death can lead to diseases such as cancers, autoimmune syndromes and/or neurodegenerative diseases. Caspases are a family of intracellular cysteine-aspartic proteases that are not only essential for triggering programmed cell death, but have also been shown to play key roles in non-apoptotic pathways, such as differentiation and proliferation of diverse cell types, axon guidance and synaptic activity and plasticity. Caspases are divided into long prodomain caspases (caspas‐ es-2, -8, -9 and -10), which are initiators of apoptosis, and short prodomain caspases (caspas‐ es-3, -6, -7 and -14), which are generally termed the effectors of apoptosis. However, some caspases, including caspase-3 (Casp3) and caspase-6 (Casp6), appear to function as both initiators and effectors. Aberrant activation of caspases has been implicated in several neurodegenerative diseases, such as AD, HD, various ataxias and amyotrophic lateral sclerosis [35].

Apoptosis is a cell death program that is central to cellular and tissue homeostasis, and is involved in many physiological and pathological processes. [36] Apoptosis is characterized morphologically by a series of events that include cytoplasmic shrinkage, chromatin conden‐ sation, nuclear and cellular fragmentation, and the formation of apoptotic bodies. Although caspases are the main players involved in apoptosis, there are other molecules involved in the progression of the apoptotic cascade that are relevant to AD. The neuronal death in AD may result directly and/or indirectly from the triggering insults caused by Aβ toxicity, glutamate excitotoxicity, long-lasting oxidative stress, DNA damage, and elevation of intracellular calcium levels. Thus, the mode of cell death in AD remains a matter of controversy, and it is possible that both apoptotic and non-apoptotic cell death coexist in the brains of affected patients. [36]

reports suggesting that Aβ formation occurs independently of microglial cells. [34] Microglial cells express several pattern-recognition receptors, which allow them to remove potentially toxic molecules such as Aβ. These receptors include scavenger receptors (SR) class A (SR-A), class B type I, low-density lipoprotein receptor-related protein (LRP), cluster of differentiation 36 (CD36), receptor for end glycation product, mannose receptor, and SR-MARCO. [34]

In fact, microglial cells secrete multiple cytokines such as interleukin-1b (IL1b), tumor necrosis factor (TNF-α), interleukin 10 (IL10), and transforming growth factor-b (TGFβ) as well as short-

[34] It has been suggested that TGFβ1 produced by astrocytes and hippocampal cells prevents induction of reactive oxygen species (ROS) and NO- production by inflammatory mediators (lipopolysaccharide [LPS] 1 interferon-γ [IFNγ]) and neurotoxicity in vitro and that the modulation of microglia is at least partially mediated by the activation of the ERK pathway. TGFβ superfamily signaling plays important roles in a diverse set of cellular responses, including cell proliferation, differentiation, extracellular matrix remodeling, and embryonic development. TGFβ1 signaling is mediated by cell surface type I and type II receptors, which phosphorylates the two R-Smad proteins Smad2 and Smad3 downstream. Phospho-Smad2/3 forms a complex with the common mediator Smad4 that binds to a Smad-binding element (SBE) in the nucleus with a large number of transcription coregulators to activate gene promoter. They showed that the TGFβ1 Smad3 pathway is involved in modulation of micro‐

Microglial cell activation can be initiated by various signal transduction pathways, including nuclear factor-jB, JAK/STAT, and p38 pathways, which are activated by several inflammatory

Apoptosis, or programmed cell death, is a highly regulated process involved in embryonic development, developmental tissue remodeling and normal cell turnover. [35] However, when dysregulation occurs in apoptotic pathways, excessive or insufficient cell death can lead to diseases such as cancers, autoimmune syndromes and/or neurodegenerative diseases. Caspases are a family of intracellular cysteine-aspartic proteases that are not only essential for triggering programmed cell death, but have also been shown to play key roles in non-apoptotic pathways, such as differentiation and proliferation of diverse cell types, axon guidance and synaptic activity and plasticity. Caspases are divided into long prodomain caspases (caspas‐ es-2, -8, -9 and -10), which are initiators of apoptosis, and short prodomain caspases (caspas‐ es-3, -6, -7 and -14), which are generally termed the effectors of apoptosis. However, some caspases, including caspase-3 (Casp3) and caspase-6 (Casp6), appear to function as both initiators and effectors. Aberrant activation of caspases has been implicated in several neurodegenerative diseases, such as AD, HD, various ataxias and amyotrophic lateral sclerosis

Apoptosis is a cell death program that is central to cellular and tissue homeostasis, and is involved in many physiological and pathological processes. [36] Apoptosis is characterized


, which contribute to neurotoxicity.

secretion, and phagocytosis.

lived cytotoxic factors such as superoxide (O2

mediators and Aβ. [34]

88 Neurodegenerative Diseases

**9. Aβ and apoptosis**

[35].

glial cell activity through its effects on expression of SRs, NO-

Previously, it was generally considered that apoptotic neuronal death in chronic neurodege‐ nerative disease, e.g., AD, Parkinson's disease, etc., is associated with classical caspase mediated cell death. However, in part, it was suggested that the caspase-independent pathway might also participate in the pathogenesis of the disease. Cross talk is extensive between different cell death pathways, which include multiple types of caspase-dependent and caspase-independent programmed cell death. [36]

Cells undergo apoptosis through two major pathways, the extrinsic pathway (death receptor pathway) and the intrinsic pathway (the mitochondrial pathway). These two pathways can be linked by caspase-8-activated truncated Bid formation. Very recently, death receptor 6 (DR6) was shown to be involved in the neurodegeneration observed in Alzheimer disease. DR6, also known as TNFRSF21, is a relatively new member of the death receptor family, and it was found that DR6 induces apoptosis when it is overexpressed. However, how the death signal mediated by DR6 is transduced intracellularly is not known. To this end, in a study they have examined the roles of caspases, apoptogenic mitochondrial factor cytochrome c, and the Bcl-2 family proteins in DR6-induced apoptosis. In their study results demonstrated that Bax translocation is absolutely required for DR6-induced apoptosis. On the other hand, they found inhibition of caspase-8 and knockdown of Bid have no effect on DR6-induced apoptosis. Their results strongly suggest that DR6-induced apoptosis occurs through a new pathway that is different from the type I and type II pathways through interacting with Bax. [37]

Different studies reported both necrotic and apoptotic mechanisms for Aβ-mediated neuro‐ toxicity. In particular, oxidative-mediated DNA damage, with a pattern indicative of apopto‐ sis, was found in AD brain, which is consistent with several lines of experimental evidence linking oxidative stress and neuronal apoptosis. [38] Apoptosis is induced by micromolar concentrations of Aβ in cultured CNS neurons, however, physiological nanomolar concentra‐ tions of Aβ1-40 and Aβ1-42 are insufficient to initiate significant apoptosis in cultures of human fetal neurons. In fact, both Aβ peptides downregulate "bcl-2", a key anti- apoptotic protein, while only Aβ1-42 upregulates "bax", a protein known to promote apoptotic cell death. Interestingly, Aβ treated neurons exposed to different levels of oxidative stress, unable to increase apoptosis in control neurons, show 10-20 times more apoptotic-mediated DNAdamage, suggesting that Aβ renders the neurons vulnerable to age-dependent oxidative stress and neurodegeneration. Other links between oxidative stress and apoptotic neuronal cell death in AD have been described. Apoptosis induced by 4-HNE is prevented in cells that overexpress "bcl-2" or by incubation with glutathione, which binds 4-HNE. Also, PC12 cells expressing a mutated presenilin-1 gene, which accounts for the majority of cases of inherited early onset forms of AD are more susceptible to apoptosis induced by micromolar concentrations of Aβ and to oxidative stress induced by nerve growth factor with- drawal. [38]

induced mitochondrial dysfunction provides an important source of ROS, which could ultimately initiate a programmed cell death pathway. In CP from APP/PS1 mice and AD patients, this pathway involves high levels of MMP-9, increased caspases expression and cell

Role of Oxidative Stress in Aβ Animal Model of Alzheimer's Disease: Vicious Circle of Apoptosis, Nitric Oxide and Age

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91

Previous studies found that expression levels of Bcl-2 family proteins, such as Bax, Bak, Bad, Bcl-2, Bim, Bcl-w and Bcl-x are altered in the vulnerable neurons in AD. [41] They demonstrated that oligomeric Aβ altered the expression levels of Bcl-2, Bim and Bax, and that the genetic or pharmacological ablation of Bax activity suppresses oligomeric Aβ-mediated neurotoxicity in both ex vivo and in vivo. These results clearly indicate that Bax has an essential role in the induction of neuronal cell death caused by oligomeric Aβ. [41] In healthy cells, Bax is located in the cytosol or loosely associated to mitochondria and endoplasmic reticulum. Bax translo‐ cation to the mitochondria, which occurs in cells with apoptotic stresses, is thought to lead to mitochondrial dysfunction and release of cytochrome c and subsequent apoptosis. Before its translocation to mitochondria, Bax changes its conformation that exposes the N-terminal residues. This conformational change is believed to be necessary for membrane insertion of Bax at mitochondria and multimerization of Bax. They demonstrated that oligomeric Aβ induced the N-terminal exposure of Bax in neurons and that the inhibition of this event by BIP rescues neurons from oligomeric Aβ's neurotoxicity, suggesting the activation of Bax by its conformational change is a key element of oligomeric Aβ-induced neurotoxicity. Although the molecular mechanism(s) of Bax activation has not been clearly defined, multiple pro-apoptotic proteins (e.g.,Bim) and anti-apoptotic proteins (e.g., Bcl-2) are known to regulate the activation of Bax thorough heterodimerization. These data strongly suggest that Bim might be an upstream regulator of Bax activation in oligomeric Aβ-induced neurotoxicity and the patho‐

As the activation of JNK by Aβ has been shown and the active form of JNK has been reported to be increased in vulnerable neurons in AD, it is plausible that the activation of JNK and subsequent activation of Bim/Bax-mediated apoptosis pathway might be the mechanism

As the chronic administration of resveratrol also protected animals from Aβ-induced neuronal loss, it is reasonably speculated that the improvement in spatial memory by resveratrol might be ascribed to its effectiveness in reducing the levels of oxidative stress and the Aβ-induced neuronal loss. [42] The activation of iNOS gene by Aβ is controlled by the transcription factor NF-κB, which is known to regulate iNOS transcription by binding to the regulatory region of iNOS gene. In addition, Aβ treatment was reported to result in the activation of NF-κB in various brain regions and cell types, and resveratrol was shown to markedly attenuate the Aβinduced NFκB nuclear translocation. Based on these, it is suggested that the regulation pathway triggered by resveratrol is to decrease the Aβ accumulation, which in turn suppresses Aβ-induced NF-κB translocation, or activation, and leads to the downregulation of iNOS. [42] Considering the strong correlation between Aβ-induced oxidative stress and cytotoxicity, ROS produced in mitochondria may leak to the cytoplasm, leading to oxidative stress and the initiation of apoptosis via the activation of apoptosis signaling. [43] It is well established that the ratio of Bcl-2/Bax is crucial in the apoptosis of the mitochondrial pathway. Bcl-2 is a potent

logical role of Bim and Bax in neuronal cell loss in AD. [41]

causing neuronal cell death in oligomeric Aβ treated neurons and AD. [41]

loss. [40]

Microglial activation can lead to microglial apoptosis, which may serve to remove highly reactive and possibly neurotoxic microglia. [39] However the loss of microglia may have consequences for future recovery, protection and repair. P53, a nuclear phosphoprotein transcription factor, is critical for activating the expression of genes involved in cell-cycle arrest and stress-induced apoptosis. In neurodegenerative diseases the expression of p53 is signifi‐ cantly increased in glial cells, and microglial numbers fall. P53 is a transcription factor, the activation of which promotes cellular apoptosis through the normal cell cycle. [39]

P53 also activates the expression of genes involved in stress-induced apoptosis and the apoptotic pathways implicated in neurodegenerative diseases which may arise from inappro‐ priate p53 activation. Changes in p53 expression occur in glial cells in neurological conditions; p53 expression increases predominantly in glial cells in Alzheimer's disease. The sustained reactivity of microglia is implicated in the pathology of many neurodegenerative diseases and microglia may secrete substances which compromise neighbouring cells. Increased microglial reactivity can lead to microglial apoptosis. This apoptosis results from microglial stress and is triggered by nitric oxide-dependent mitochondrial depolarization and caspase activation. Whilst microglial apoptosis may serve to limit the number of reactive microglia in the brain, the processes of microglial stress and apoptosis can in themselves result in the release of proapoptotic species such as soluble Fas ligand which can trigger bystander cell apoptotic cascades. Microglial numbers significantly decrease in aged human brain and in Alzheimer's brain and such loss may restrict the ability of the brain to recover from injury. Apoptotic changes in microglia precede changes in neurons or other glia in models of neurodegenerative disease. As microglia secrete neurotrophins important for neuronal survival and regeneration, the apoptosis of microglia may impact on brain health. They examined the role p53 plays in mediating microglial apoptosis following microglial activation with the Alzheimer peptides amyloid beta and chromogranin A, as well as with the activator lipopolysaccharide. [39]

Taken together, these results suggest that mitochondrial energy metabolism might be impaired by Aβ through down-regulation of mitochondrial proteins and activity. [40] In addition, the interaction between NO and cytochrome *c* oxidase controls mitochondrial production of hydrogen peroxide, which has been shown to be implicated in cellular redox signaling. In a study they used synthetic Aβ1–42 to impair the function of complexes I and IV in CP epithelial cells and to investigate whether ROS generation was also altered. [40] Their results support the hypothesis that Aβ1–42 interferes with oxidative phosphorylation, which results in oxidative stress, and demonstrate that ROS generation is secondary to mitochondrial damage. The results of the study also show that Aβ1–42-treated CP epithelial cells have an increased expression of caspases 3 and 9. [40] This result is consistent with a recent study, where APP transfected cells showed a significant increase in the expression and activity of caspases 3 and 9. The data suggest that the excessive generation of ROS would be responsible for initiating the apoptotic cell death process by up-regulating caspase signaling, as previously demon‐ strated. In view of the results from their *in vitro* and *in vivo* studies, they proposed that Aβinduced mitochondrial dysfunction provides an important source of ROS, which could ultimately initiate a programmed cell death pathway. In CP from APP/PS1 mice and AD patients, this pathway involves high levels of MMP-9, increased caspases expression and cell loss. [40]

mutated presenilin-1 gene, which accounts for the majority of cases of inherited early onset forms of AD are more susceptible to apoptosis induced by micromolar concentrations of Aβ

Microglial activation can lead to microglial apoptosis, which may serve to remove highly reactive and possibly neurotoxic microglia. [39] However the loss of microglia may have consequences for future recovery, protection and repair. P53, a nuclear phosphoprotein transcription factor, is critical for activating the expression of genes involved in cell-cycle arrest and stress-induced apoptosis. In neurodegenerative diseases the expression of p53 is signifi‐ cantly increased in glial cells, and microglial numbers fall. P53 is a transcription factor, the

P53 also activates the expression of genes involved in stress-induced apoptosis and the apoptotic pathways implicated in neurodegenerative diseases which may arise from inappro‐ priate p53 activation. Changes in p53 expression occur in glial cells in neurological conditions; p53 expression increases predominantly in glial cells in Alzheimer's disease. The sustained reactivity of microglia is implicated in the pathology of many neurodegenerative diseases and microglia may secrete substances which compromise neighbouring cells. Increased microglial reactivity can lead to microglial apoptosis. This apoptosis results from microglial stress and is triggered by nitric oxide-dependent mitochondrial depolarization and caspase activation. Whilst microglial apoptosis may serve to limit the number of reactive microglia in the brain, the processes of microglial stress and apoptosis can in themselves result in the release of proapoptotic species such as soluble Fas ligand which can trigger bystander cell apoptotic cascades. Microglial numbers significantly decrease in aged human brain and in Alzheimer's brain and such loss may restrict the ability of the brain to recover from injury. Apoptotic changes in microglia precede changes in neurons or other glia in models of neurodegenerative disease. As microglia secrete neurotrophins important for neuronal survival and regeneration, the apoptosis of microglia may impact on brain health. They examined the role p53 plays in mediating microglial apoptosis following microglial activation with the Alzheimer peptides amyloid beta and chromogranin A, as well as with the activator lipopolysaccharide. [39]

Taken together, these results suggest that mitochondrial energy metabolism might be impaired by Aβ through down-regulation of mitochondrial proteins and activity. [40] In addition, the interaction between NO and cytochrome *c* oxidase controls mitochondrial production of hydrogen peroxide, which has been shown to be implicated in cellular redox signaling. In a study they used synthetic Aβ1–42 to impair the function of complexes I and IV in CP epithelial cells and to investigate whether ROS generation was also altered. [40] Their results support the hypothesis that Aβ1–42 interferes with oxidative phosphorylation, which results in oxidative stress, and demonstrate that ROS generation is secondary to mitochondrial damage. The results of the study also show that Aβ1–42-treated CP epithelial cells have an increased expression of caspases 3 and 9. [40] This result is consistent with a recent study, where APP transfected cells showed a significant increase in the expression and activity of caspases 3 and 9. The data suggest that the excessive generation of ROS would be responsible for initiating the apoptotic cell death process by up-regulating caspase signaling, as previously demon‐ strated. In view of the results from their *in vitro* and *in vivo* studies, they proposed that Aβ-

activation of which promotes cellular apoptosis through the normal cell cycle. [39]

and to oxidative stress induced by nerve growth factor with- drawal. [38]

90 Neurodegenerative Diseases

Previous studies found that expression levels of Bcl-2 family proteins, such as Bax, Bak, Bad, Bcl-2, Bim, Bcl-w and Bcl-x are altered in the vulnerable neurons in AD. [41] They demonstrated that oligomeric Aβ altered the expression levels of Bcl-2, Bim and Bax, and that the genetic or pharmacological ablation of Bax activity suppresses oligomeric Aβ-mediated neurotoxicity in both ex vivo and in vivo. These results clearly indicate that Bax has an essential role in the induction of neuronal cell death caused by oligomeric Aβ. [41] In healthy cells, Bax is located in the cytosol or loosely associated to mitochondria and endoplasmic reticulum. Bax translo‐ cation to the mitochondria, which occurs in cells with apoptotic stresses, is thought to lead to mitochondrial dysfunction and release of cytochrome c and subsequent apoptosis. Before its translocation to mitochondria, Bax changes its conformation that exposes the N-terminal residues. This conformational change is believed to be necessary for membrane insertion of Bax at mitochondria and multimerization of Bax. They demonstrated that oligomeric Aβ induced the N-terminal exposure of Bax in neurons and that the inhibition of this event by BIP rescues neurons from oligomeric Aβ's neurotoxicity, suggesting the activation of Bax by its conformational change is a key element of oligomeric Aβ-induced neurotoxicity. Although the molecular mechanism(s) of Bax activation has not been clearly defined, multiple pro-apoptotic proteins (e.g.,Bim) and anti-apoptotic proteins (e.g., Bcl-2) are known to regulate the activation of Bax thorough heterodimerization. These data strongly suggest that Bim might be an upstream regulator of Bax activation in oligomeric Aβ-induced neurotoxicity and the patho‐ logical role of Bim and Bax in neuronal cell loss in AD. [41]

As the activation of JNK by Aβ has been shown and the active form of JNK has been reported to be increased in vulnerable neurons in AD, it is plausible that the activation of JNK and subsequent activation of Bim/Bax-mediated apoptosis pathway might be the mechanism causing neuronal cell death in oligomeric Aβ treated neurons and AD. [41]

As the chronic administration of resveratrol also protected animals from Aβ-induced neuronal loss, it is reasonably speculated that the improvement in spatial memory by resveratrol might be ascribed to its effectiveness in reducing the levels of oxidative stress and the Aβ-induced neuronal loss. [42] The activation of iNOS gene by Aβ is controlled by the transcription factor NF-κB, which is known to regulate iNOS transcription by binding to the regulatory region of iNOS gene. In addition, Aβ treatment was reported to result in the activation of NF-κB in various brain regions and cell types, and resveratrol was shown to markedly attenuate the Aβinduced NFκB nuclear translocation. Based on these, it is suggested that the regulation pathway triggered by resveratrol is to decrease the Aβ accumulation, which in turn suppresses Aβ-induced NF-κB translocation, or activation, and leads to the downregulation of iNOS. [42]

Considering the strong correlation between Aβ-induced oxidative stress and cytotoxicity, ROS produced in mitochondria may leak to the cytoplasm, leading to oxidative stress and the initiation of apoptosis via the activation of apoptosis signaling. [43] It is well established that the ratio of Bcl-2/Bax is crucial in the apoptosis of the mitochondrial pathway. Bcl-2 is a potent cell death suppressor, and its overexpression prevents cell death. However, Bax is a deathpromoting factor, and its translocation to the mitochondrial membrane may lead to the loss of mitochondrial membrane potential and an increase in mitochondrial permeability. Increased mitochondrial permeability results in the egress of cytochrome *c* from the mitochondria and the subsequent activation of procaspase-3 to caspase-3, which eventually leads to apoptosis. Aβ treatment significantly decreased the Bcl-2/Bax protein expression ratio and increased caspase-3 activity, in agreement with previous reports. [43]

downstream executioner caspases, resulting in an amplification of cascade activity. The initiator caspases consist of long N-terminal prodomains that contain caspase recruitment domains (CARDs) in caspase 2 and caspase 9, or death effector domains (DEDs) in caspase 8 and caspase 10. Another set of caspases, termed the executioner caspases, consists of caspases 3, 6, and 7 that function to directly cleave crucial cellular protein substrates that result in cell destruction. [45] The executioner caspases contain short prodomains or have no prodomains. Apoptotic injury during Alzheimer's disease may require caspase-mediated pathways. A strong body of evidence supports the premise that caspase activation is involved in the pathological process of Alzheimer's disease. The elevation of caspase genes including caspases 1, 2, 3, 5, 6, 7, 8, and 9 has been observed in human postmortem brains from Alzheimer's disease patients. In the brains of Alzheimer's patients, single neurons with DNA fragmentation have been shown to contain cytoplasmic immunoreactivity for active caspase 3, implying that apoptotic injury results during Alzheimer's disease. In addition, activation of caspase 3 was found to occur in the parahippocampal gyrus in brains from patients with mild forms of Alzheimer's disease. Caspase 3 immunoreactivity was also co-localized with paired helical filaments in neurons, suggesting that caspase 3 activation may contribute to the formation of

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93

**Figure 3.** Intracellular sources of ROS and their interaction with the apoptotic pathway. [Chandra J. 2000, ref 26]

As Aβ peptides are used to mimic AD in animal models, and because sporadic AD is known to arise mostly in older ages, we designed a study to investigate the acute effects of Aβ 1-42

neurofibrillary tangles. [45]

Classic programmed cell death is more frequently associated with a death program that requires gene transcription and protein synthesis, whereas apoptosis is generally independent of protein synthesis and represents a posttranslational response of host cells. Apoptosis is characterized by a requirement for specific proteolysis driven by caspases, although it is also reported that caspases (caspase 9 in particular) participate in nonapoptotic cell death. [35]

The name caspase is a contraction of cysteinedependent aspartate-specific protease; their enzymatic properties are governed by a dominant specificity for substrates containing Asp and by the use of a Cys side chain for catalyzing peptide bond cleavage. Mammals contain two biologically distinct caspase subfamilies: One of these participates in the processing of proinflammatory cytokines, and the other is required to elicit and execute the apoptotic response during programmed cell death. [35]

The intrinsic pathway responds primarily to cellular stress (ionizing radiation, cytotoxic drugs, etc.) as well as some neurodevelopmental cues, with the mitochondrion acting as an important integrator. Activation of the apical protease caspase 9 occurs when it is driven into a catalytic conformation by its cofactor Apaf-1, which itself requires prior binding to cytochrome c. The extrinsic apoptosis pathway is triggered through the extracellular ligation of death receptors by their cognate ligands, resulting in receptor clustering, adapter recruitment, and activation of the apical protease caspase 8. [35]

The in vivo models offer the advantage of allowing neuronal death to proceed in a more physiologic setting, where the different components of the nervous system are intact. The in vivo models, however, do not adequately allow determination of the specific caspases necessary to execute neuronal death in each disease. [35] Elucidation of the specific caspase pathways is best approached in the cell culture models, with which biochemical studies can be more easily performed. Cell culture models include both primary neuronal cultures and cell lines. Both are valid models, as long as their limitations are appreciated. Cell lines offer the advantage of providing large amounts of homogenous material in which expression levels of the different components of the death pathways can be easily manipulated. Results from cell lines must normally be validated in vivo in the cell that they are modeling. After estab‐ lishment of the potential caspase pathways in the cell culture models, the in vivo and human samples can be analyzed. [35] Caspases are a family of cysteine proteases that cleave their substrates after aspartic residues. They are usually synthesized as inactive zymogens that are proteolytically cleaved into subunits at the onset of apoptosis and function as active caspases after reconstitution to molecular heterodimers. [45] Caspases are composed of three domains including an N-terminal prodomain, a large subunit, and a small subunit. The apoptoticassociated caspases include initiator caspases, such as caspases 2, 8, 9, and 10, that activate cell death suppressor, and its overexpression prevents cell death. However, Bax is a deathpromoting factor, and its translocation to the mitochondrial membrane may lead to the loss of mitochondrial membrane potential and an increase in mitochondrial permeability. Increased mitochondrial permeability results in the egress of cytochrome *c* from the mitochondria and the subsequent activation of procaspase-3 to caspase-3, which eventually leads to apoptosis. Aβ treatment significantly decreased the Bcl-2/Bax protein expression ratio and increased

Classic programmed cell death is more frequently associated with a death program that requires gene transcription and protein synthesis, whereas apoptosis is generally independent of protein synthesis and represents a posttranslational response of host cells. Apoptosis is characterized by a requirement for specific proteolysis driven by caspases, although it is also reported that caspases (caspase 9 in particular) participate in nonapoptotic cell death. [35] The name caspase is a contraction of cysteinedependent aspartate-specific protease; their enzymatic properties are governed by a dominant specificity for substrates containing Asp and by the use of a Cys side chain for catalyzing peptide bond cleavage. Mammals contain two biologically distinct caspase subfamilies: One of these participates in the processing of proinflammatory cytokines, and the other is required to elicit and execute the apoptotic

The intrinsic pathway responds primarily to cellular stress (ionizing radiation, cytotoxic drugs, etc.) as well as some neurodevelopmental cues, with the mitochondrion acting as an important integrator. Activation of the apical protease caspase 9 occurs when it is driven into a catalytic conformation by its cofactor Apaf-1, which itself requires prior binding to cytochrome c. The extrinsic apoptosis pathway is triggered through the extracellular ligation of death receptors by their cognate ligands, resulting in receptor clustering, adapter recruitment, and activation

The in vivo models offer the advantage of allowing neuronal death to proceed in a more physiologic setting, where the different components of the nervous system are intact. The in vivo models, however, do not adequately allow determination of the specific caspases necessary to execute neuronal death in each disease. [35] Elucidation of the specific caspase pathways is best approached in the cell culture models, with which biochemical studies can be more easily performed. Cell culture models include both primary neuronal cultures and cell lines. Both are valid models, as long as their limitations are appreciated. Cell lines offer the advantage of providing large amounts of homogenous material in which expression levels of the different components of the death pathways can be easily manipulated. Results from cell lines must normally be validated in vivo in the cell that they are modeling. After estab‐ lishment of the potential caspase pathways in the cell culture models, the in vivo and human samples can be analyzed. [35] Caspases are a family of cysteine proteases that cleave their substrates after aspartic residues. They are usually synthesized as inactive zymogens that are proteolytically cleaved into subunits at the onset of apoptosis and function as active caspases after reconstitution to molecular heterodimers. [45] Caspases are composed of three domains including an N-terminal prodomain, a large subunit, and a small subunit. The apoptoticassociated caspases include initiator caspases, such as caspases 2, 8, 9, and 10, that activate

caspase-3 activity, in agreement with previous reports. [43]

92 Neurodegenerative Diseases

response during programmed cell death. [35]

of the apical protease caspase 8. [35]

downstream executioner caspases, resulting in an amplification of cascade activity. The initiator caspases consist of long N-terminal prodomains that contain caspase recruitment domains (CARDs) in caspase 2 and caspase 9, or death effector domains (DEDs) in caspase 8 and caspase 10. Another set of caspases, termed the executioner caspases, consists of caspases 3, 6, and 7 that function to directly cleave crucial cellular protein substrates that result in cell destruction. [45] The executioner caspases contain short prodomains or have no prodomains. Apoptotic injury during Alzheimer's disease may require caspase-mediated pathways. A strong body of evidence supports the premise that caspase activation is involved in the pathological process of Alzheimer's disease. The elevation of caspase genes including caspases 1, 2, 3, 5, 6, 7, 8, and 9 has been observed in human postmortem brains from Alzheimer's disease patients. In the brains of Alzheimer's patients, single neurons with DNA fragmentation have been shown to contain cytoplasmic immunoreactivity for active caspase 3, implying that apoptotic injury results during Alzheimer's disease. In addition, activation of caspase 3 was found to occur in the parahippocampal gyrus in brains from patients with mild forms of Alzheimer's disease. Caspase 3 immunoreactivity was also co-localized with paired helical filaments in neurons, suggesting that caspase 3 activation may contribute to the formation of neurofibrillary tangles. [45]

**Figure 3.** Intracellular sources of ROS and their interaction with the apoptotic pathway. [Chandra J. 2000, ref 26]

As Aβ peptides are used to mimic AD in animal models, and because sporadic AD is known to arise mostly in older ages, we designed a study to investigate the acute effects of Aβ 1-42 injection in aged rats suggesting the onset of sporadic AD. In the light of the current literature, we investigated not only the effect of Aβ 1-42 injection on aged rat brain, but also age related changes in lipid peroxidation, nNOS and iNOS expression and caspase 3 in which involves apoptotic process as an apoptosis marker. In this study, a prominent increase of caspase 3 activity in hippocampus has been shown in Aβ 1-42 injected aged rats. [24] This result suggests that apoptotic mechanism triggers neuronal death in hippocampus, and results a significant decrement nNOS expression consequently in Aβ 1-42 injected aged rats. Another explanation of these results is that because of possible neuronal death by the way of caspase-3, there may not be enough neurons remained to cause lipid peroxidation. This suggests that acute effect of Aβ 1-42 result with increase in caspase 3 activity which is seen before the oxidative stressdependent neurotoxic effects. In this study Aβ 1-42 lead to a counter effect on nNOS expression in young adults and aged rats. The results of this study suggest that the aged rat brain does not successfully offset the oxidative stress. [24] We suggest that the relationship between aging, NOS-mediated ROS-dependent toxicity of beta amyloid toxicity and apoptotic events should be bring out into the open this vicious cycle.

IκBK: Inhibitor of κB kinase JNK: c-Jun N-terminal kinase

Tyk2: tyrosine kinase 2 SR: Scavenger receptor

GSK-3: Glycogen synthase kinase 3

TGF: Transforming growth factor

SBE: Smad binding element

SOD: Superoxide dismutase

HD: Huntington disease

PD: Parkinson disease DR6: Death receptor 6

GSH: Glutathione

**Author details**

Ferihan Cetin\*

mir, Turkey

**References**

(1998). , 78, 547-581.

(2007). , 11(4), 457-63.

STAT3: Signal transducer and activator of transcription 3

LRP: Low-density lipoprotein receptor related protein

Address all correspondence to: ferihan@yahoo.com

apoptosis. IUBMB life (2000). , 49427-435.

Izmir University Faculty of Medicine, Department of Physiology, Medical Park Hospital, Iz‐

Role of Oxidative Stress in Aβ Animal Model of Alzheimer's Disease: Vicious Circle of Apoptosis, Nitric Oxide and Age

http://dx.doi.org/10.5772/54718

95

[1] Beckman, K. B, & Ames, B. N. The free radical theory of aging matures. Physiol Rev.

[2] Sastre, J, & Pallardo, F. V. Mitochondrial oxidative stress plays a key role in aging and

[3] Fiala, M, Cribbs, D. H, Rosenthal, M, & Bernard, G. Phagocytosis of amyloid-beta and inflammation: two faces of innate immunity in Alzheimer's disease. J Alzheimers Dis.

### **Abbreviations**

ROS: Reactive oxygen species AD: Alzheimer's disease Aβ: Beta amyloid peptide NO: Nitric oxide NOS: Nitric oxide synthase CA: Cornu ammonis H2O2: Hydrogen peroxide ONOO- : Peroxynitrite APP: Amyloid precursor protein RNS: Reactive nitrogen species GADPH: Glucose 6-phosphate dehydrogenase MAPK: Mitogen activated protein kinase AP-1: Activated protein NFκB: Nuclear factor kapa B CNS: Central nervous system AGE: Advanced glycation end product IκB: Inhibitor of κB

IκBK: Inhibitor of κB kinase JNK: c-Jun N-terminal kinase GSK-3: Glycogen synthase kinase 3 STAT3: Signal transducer and activator of transcription 3 Tyk2: tyrosine kinase 2 SR: Scavenger receptor LRP: Low-density lipoprotein receptor related protein TGF: Transforming growth factor SBE: Smad binding element HD: Huntington disease PD: Parkinson disease DR6: Death receptor 6 SOD: Superoxide dismutase GSH: Glutathione

### **Author details**

#### Ferihan Cetin\*

injection in aged rats suggesting the onset of sporadic AD. In the light of the current literature, we investigated not only the effect of Aβ 1-42 injection on aged rat brain, but also age related changes in lipid peroxidation, nNOS and iNOS expression and caspase 3 in which involves apoptotic process as an apoptosis marker. In this study, a prominent increase of caspase 3 activity in hippocampus has been shown in Aβ 1-42 injected aged rats. [24] This result suggests that apoptotic mechanism triggers neuronal death in hippocampus, and results a significant decrement nNOS expression consequently in Aβ 1-42 injected aged rats. Another explanation of these results is that because of possible neuronal death by the way of caspase-3, there may not be enough neurons remained to cause lipid peroxidation. This suggests that acute effect of Aβ 1-42 result with increase in caspase 3 activity which is seen before the oxidative stressdependent neurotoxic effects. In this study Aβ 1-42 lead to a counter effect on nNOS expression in young adults and aged rats. The results of this study suggest that the aged rat brain does not successfully offset the oxidative stress. [24] We suggest that the relationship between aging, NOS-mediated ROS-dependent toxicity of beta amyloid toxicity and apoptotic events should

be bring out into the open this vicious cycle.

**Abbreviations**

94 Neurodegenerative Diseases

NO: Nitric oxide

ONOO-

CA: Cornu ammonis

ROS: Reactive oxygen species

AD: Alzheimer's disease Aβ: Beta amyloid peptide

NOS: Nitric oxide synthase

H2O2: Hydrogen peroxide

AP-1: Activated protein

IκB: Inhibitor of κB

NFκB: Nuclear factor kapa B CNS: Central nervous system

: Peroxynitrite

APP: Amyloid precursor protein RNS: Reactive nitrogen species

GADPH: Glucose 6-phosphate dehydrogenase

MAPK: Mitogen activated protein kinase

AGE: Advanced glycation end product

Address all correspondence to: ferihan@yahoo.com

Izmir University Faculty of Medicine, Department of Physiology, Medical Park Hospital, Iz‐ mir, Turkey

#### **References**


[4] Yamada, K, & Nabeshima, T. Animal models of Alzheimer's disease and evaluation of anti-dementia drugs. Pharmacol. Therap. (2000). , 88-93.

Peptide-associated free radical oxidative stress. Free Rad Biol Med. (2002). , 32(11),

http://dx.doi.org/10.5772/54718

97

[18] Jhoo, J. H, Kim, H-C, Nabeshima, T, Yamada, K, Shin, E-J, Jhoo, W-K, & Kim, W. Amyloid (1-42)-induced learning and memory deficits in mice: involvement of oxidative burdens in the hippocampus and cerebral cortex. Behav Brain Res (2004). ,

Role of Oxidative Stress in Aβ Animal Model of Alzheimer's Disease: Vicious Circle of Apoptosis, Nitric Oxide and Age

[19] Siles, E, Martinez-lara, E, Canuelo, A, Sánchez, M, Hernández, R, & López-ramos, J. C. M, Age related changes of the nitric oxide system in the brain. Brain Res. November

[20] Koppal, T, Drake, J, Yatin, S, Jordan, B, Varadarajan, S, Bettenhausen, L, & Butterfield, D. A. Peroxynitrite-induced alterations in synaptosomal membrane proteins: insight into oxidative stress in Alzheimer's Disease. J. Neurochem. (1999). , 72, 310-317. [21] Malinski, T. Nitric oxide and nitro-oxidative stress in Alzheimer's Disease. J.Alz.

[22] ] Maccioni, R. B, Muñoz, J. P, & Barbeito, L. The Molecular Bases of Alzheimer's Disease and Other Neurodegenerative Disorders. Archives of Medical Research (2001). , 32,

[23] Weldon, D. T, Rogers, S. D, Ghilardi, J. R, Finke, M. P, Cleary, J. P, Hare, O, Esler, E, Maggio, W. P, Mantyh, J. E, & Fibrillar, P. W. Amyloid Induces Microglial Phagocytosis, Expression of Inducible Nitric Oxide Synthase, and Loss of a Select Population of Neurons in the Rat CNS In Vivo The Journal of Neuroscience, March 15, (1998). , 18(6),

[24] Cetin, F, Yazihan, N, Dincer, S, & Akbulut, K. G. The The Effect of Intracerebroven‐ tricular Injection of Beta Amyloid Peptide (1-42) on Caspase 3 Activity, Lipid Peroxi‐ dation and NOS expression in Young Adult and Aged Rat Brain. Turkish Neurosurgery

[25] Markesbery William ROxidative stress hypothesis in Alzheimer's Disease. Free Radical

[26] Chandra, J, Samali, A, & Orrenius, S. Triggering and modulation of apoptosis by oxidative stres. Free Radical Biology & Medicine, (2000). Nos. 3/4, , 29, 323-333. [27] ] Calabrese, V, Cornelius, C, Rizzarelli, E, Owen, J. B, Dinkova-kostova, A. T, & Butterfield, D. A. Nitric Oxide in Cell Survival: A Janus Molecule. Antioxidants &

[28] Spuch, C, Ortolano, S, & Navarro, C. New Insights in the Amyloid-Beta Interaction withMitochondria. Journal of Aging Research Article ID 324968, 9 pages doi:

[29] Walter, S, Letiembre, M, Liu, Y, Heine, H, Penke, B, Hao, W, Bode, B, & Manietta, N. Jessica Walter J., Schulz-Schäffer W. and Faßbender K., Role of the Toll-Like Receptor

(2012). date accepted May 17.DOİ. :10.5137/JTN.5855-12.1, 1019-5149.

1050-1060.

155, 185-196.

367-381.

2161-2173.

(2002). , 956(2), 385-92.

Disease. May (2007). , 11(2), 207-18.

Biology & Medicine, (1997). , 23(1), 134-147.

Redox Signaling (2009). , 11(11)

10.1155/2012/324968, 2012


Peptide-associated free radical oxidative stress. Free Rad Biol Med. (2002). , 32(11), 1050-1060.

[18] Jhoo, J. H, Kim, H-C, Nabeshima, T, Yamada, K, Shin, E-J, Jhoo, W-K, & Kim, W. Amyloid (1-42)-induced learning and memory deficits in mice: involvement of oxidative burdens in the hippocampus and cerebral cortex. Behav Brain Res (2004). , 155, 185-196.

[4] Yamada, K, & Nabeshima, T. Animal models of Alzheimer's disease and evaluation of

[5] Camandola, S, Poli, G, & Mattson, M. P. Lipid Peroxidation Product 4-hydroxynonenal increases AP-1 binding activity through caspase activation in neurons. J Neurochem.

[6] Stepanichev, M. Y, Onufriev, M. V, Yakovlev, A. A, Khrenov, A. I, & Peregud, D. I. Amyloid-β (25-35) increases activity of neuronal NO-synthase in rat brain. Neuro‐

[7] Law, A, Gauthier, S, & Quiron, R. Say NO to Alzheimer's disease: putative links between nitric oxide and dementia of the Alzheimer's type. Brain Res. Rev. (2001). ,

[8] Xie, X, Wang, H-T, Li, H-L, Gao, X-H, Ding, J, & Zhao, H-H. Ginsenoside Rb1 protects PC12 cells against β-amyloid-induced cell injury Mol Med Reports (2010). , 3, 635-639.

[10] Lovell, M. A, Ehmann, W. D, Butler, S. M, & Markesberry, W. R. Elevated thiobarbituric acid-reactive substances and antioxidant enzyme activity in the brain in Alzheimer's

[11] Machiavelli, L. I, Poliandri, A. H, Quinteros, F. A, Cabilla, J. P, & Duvilanski, B. H. Reactive oxygen species are key mediators of the nitric oxide apoptotic pathway in

[12] Vina, J, Lloret, A, Vallés, S. L, Borrás, C, Badía, M. C, Pallardó, F. V, & Sastre, J. Mitochondrial oxidant signalling in Alzheimer's disease. J Alzheimers Dis. (2007). ,

[13] Feng, Z, Qin, C, Chang, Y, & Zhang, J. Early melatonin supplementation alleviates oxidative stress in a transgenicmouse model of Alzheimer's disease. Free Rad Biol &

[14] Thorns, V, Lawrence, H. L, & Masliah, E. nNOS Expressing Neurons in the Entorhinal Cortex and Hippocampus Are Affected in Patients with Alzheimer's Disease. Exper

[15] Norris, P. J, Faull, R. L, & Emson, P. C. Neuronal nitric oxide synthase (nNOS) mRNA expression and NADPH-diaphorase staining in the frontal cortex, visual cortex and hippocampus of control and Alzheimer's disease brains. Brain Res Mol Brain Res.

[16] Koliatsos, V. E, Kecojevic, A, Troncoso, J. C, Gastard, M. C, Bennett, D. A, & Schneider, J. A. Early involvement of small inhibitory cortical interneurons in Alzheimer's disease.

[17] Butterfield, D. A, & Lauderback, C. M. Lipid peroxidation and protein oxidation in Alzheimer's Disease brain: potential causes and consequences involving Amyloid β-

[9] Christen, Y. Oxidative stress and Alzheimer disease. Am J Clin Nutr (2000).

anterior pituitary cells. Nitric Oxide Mar (2007). , 16(2), 237-46.

anti-dementia drugs. Pharmacol. Therap. (2000). , 88-93.

(2000). , 74(1), 159-68.

35-73.

96 Neurodegenerative Diseases

11(2), 175-81.

(1996).

Med (2006). , 40-101.

Neurol (1998). , 150-14.

Acta Neuropathol (2006). , 112-147.

chemistry International (2008). , 52-1114.

disease. Neurology (1995). , 45-1594.


4 in Neuroinflammation in Alzheimer's Disease. Cell Physiol Biochem (2007). , 20, 947-956.

[39] Davenport, C. M, Sevastou, I. G, Hooper, C, Pocock, J. M, & Inhibiting, P. Pathways in microglia attenuates microglial evoked neurotoxicity following exposure to Alzheimer

Role of Oxidative Stress in Aβ Animal Model of Alzheimer's Disease: Vicious Circle of Apoptosis, Nitric Oxide and Age

http://dx.doi.org/10.5772/54718

99

[40] Vargas, T, Ugalde, C, Spuch, C, Antequera, D, Moránc, M. J, Martín, M. A, Ferrer, I, Bermejo-pareja, F, & Carro, E. Aβ accumulation in choroid plexus is associated with mitochondrial-induced apoptosis. Neurobiology of Aging (2010). , 31, 1569-1581.

[41] Kudo, W, Lee, H-P, Smith, M. A, Zhu, X, Matsuyama, S, & Lee, H-g. Inhibition of Bax protects neuronal cells from oligomeric Aβ neurotoxicity. Cell Death and Disease

[42] Huang, T-C, & Lu, K-T. Peter Wo Y-Y., Wu Y-J., Yang Y-L., Resveratrol Protects Rats from Aβ-induced Neurotoxicity by the Reduction of iNOS Expression and Lipid

[43] XiaxieHai-Taowang, Chun-Lili, Xu-Hong Gao, Jin-Landing, Hai-Huazhao and Yong-Lil, Ginsenoside Rb1 protects PC12 cells against β-amyloid-induced cell injury.

[44] Troy, C. M, & Salvesen, G. S. Caspases On The Brain. Journal of Neuroscience Research

[45] Chonga, Z. Z, Lia, F, & Maiese, K. Stress in the brain: novel cellular mechanisms of injury linked to Alzheimer's disease. Brain Research Reviews (2005). , 49, 1-21.

peptides. Journal of Neurochemistry (2010). , 112, 552-563.

Peroxidation. PLoS ONE December (2011). e 29102, 6(12)

Molecular Medicine REPORTS (2010). , 3, 635-639.

(2012). e309; doi:10.1038/cddis.2012.43

(2002). , 69, 145-150.


[39] Davenport, C. M, Sevastou, I. G, Hooper, C, Pocock, J. M, & Inhibiting, P. Pathways in microglia attenuates microglial evoked neurotoxicity following exposure to Alzheimer peptides. Journal of Neurochemistry (2010). , 112, 552-563.

4 in Neuroinflammation in Alzheimer's Disease. Cell Physiol Biochem (2007). , 20,

[30] Medeiros, R, Prediger, R. D. S, Passos, G. F, Pandolfo, P, Duarte, F. S, Franco, J. L, Dafre, A. L, Giunta, G. D, Figueiredo, C. P, Takahashi, R. N, Campos, M. M, & Calixto, J. B. Connecting TNF-β Signaling Pathways to iNOS Expression in a Mouse Model of Alzheimer's Disease: Relevance for the Behavioral and Synaptic Deficits Induced by Amyloid β Protein. Neurobiology of Disease, The Journal of Neuroscience, May 16,

[31] Chandler, D, Woldu, A, Rahmadi, A, Shanmugam, K, Steiner, N, Wright, E, Benaventegarci, a O, Schulz, O, Castillo, J, & Munch, G. Effects of plant-derived polyphenols on TNF-α and nitric oxide production induced by advanced glycation endproducts. Mol.

[32] Wan, J, Fu, A. K. Y, & Ip, F. C. F. Ho-Keung Ng, Hugon J., Page G., Wang J.H., Kwok-On Lai, Wu Z., and Ip N.Y., Tyk2/STAT3 Signaling Mediates β-Amyloid-Induced Neuronal Cell Death: Implications in Alzheimer's Disease. Neurobiology of Disease

[33] Wojtera, M, Sobów, T, Kloszewska, I, Liberski, P. P, Brown, D. R, & Sikorska, B. Expression of immunohistochemical markers on microglia in Creutzfeldt-Jakob disease and Alzheimer's disease: morphometric study and review of the literature. Folia

[34] Juan, E, Tichauer, J. E, & Bernhardi, R. Transforming Growth Factor-β Stimulates β-Amyloid Uptake by Microglia Through Smad3-Dependent Mechanisms. Journal of

[35] Uribe, V, Wong, B.K.Y, Graham, R.K, Cusack, C.L, Skotte, N.H, Pouladi, M.A, Xie, Y, Feinberg, K, Ou, Y, Ouyang, Y, & Deng, . ., Rescue from excitotoxicity and axonal degeneration accompanied by age-dependent behavioral and neuroanatomical alterations in caspase-6-deficient mice. Human Molecular Genetics, 2012; Vol. 21, No.

[36] Lee, J-H, & Cheon, . ., Evidence of early involvement of apoptosis inducing factorinduced neuronal death in Alzheimer brain. Anatomy and Cell Biolog.2012; pISSN

[37] Zeng, L, Li, T, Xu, D. C, Liu, J, Mao, G, Cui, M. Z, Fu, X, & Xu, X. Death Receptor 6 Induces Apoptosis Not through Type I or Type II Pathways, but via a Unique Mito‐ chondria-dependent Pathway by Interacting with Bax Protein. J Biol Chem. 2012 Aug

[38] Miranda, S, Opazo, C, & Larrondo, L. F. MunÄ oz F.J., Ruiz F., Leighton F., Inestrosa N.C., The role of oxidative stress in the toxicity induced by amyloid b- peptide in

2093-3665 eISSN 2093-3673. http://dx.doi.org/10.5115/acb.2012.45.1.26

Alzheimer's disease. Progress in Neurobiology (2000). , 62, 633-648.

The Journal of Neuroscience, May 19, (2010). , 30(20), 6873-6881.

947-956.

98 Neurodegenerative Diseases

(2007). , 27(20), 5394-5404.

Nutr. Food Res. (2010). SS150, 141.

Neuropathol (2012). , 50(1), 74-84.

9 1954-1967

Neuroscience Research (2012). , 90, 1970-1980.

17; Epub (2012). Jul 3., 287(34), 29125-33.


**Chapter 5**

**Alterations of Mitochondria and Golgi Apparatus Are**

**Related to Synaptic Pathology in Alzheimer's Disease**

Alzheimer's disease (AD) is an insidiously progressive severe presenile and senile dementia, involving a number of cellular and biochemical mechanisms. AD affects millions of humans as the most common cause of cognitive decline worldwide, in addition to being a main medical challenge for aging population. From the clinical point of view, AD is mostly characterized by age-dependent inexorably progressing cognitive decline, affecting memory primarily associ‐ ated with behavioral and mood disorders, which increasingly appear as the disease advances [1]. From the neuropathological point of view, AD is mostly characterized by selective neuronal loss [2, 3], marked synaptic alterations [4–6], morphological mitochondrial abnor‐ malities [7, 8], tau pathology [9] resulting in neurofibrillary tangles (NFT) composed of hyperphosphorylated tau [10], inflammatory responses and by extracellular extensive deposits of polymers of Aβ peptide, in the form of neuritic plaques, which are a main hallmark of AD [11, 12]. These are dispersed in the neocortex, the hippocampus, and many subcortical structures, which play an important role in cognition. In addition, AD is characterized ultrastructurally by organelle pathology involving mostly the microtubules, the mitochondria,

Most studies have revealed that the main pathological criteria for AD, namely the neuritic plaques and neurofibrillary tangles, can account for 40%–70% of the cognitive impairment seen in advanced age, though additional cerebrovascular changes [14, 15] contribute greatly in

The two main hypotheses of AD - the amyloid cascade hypothesis, introduced in 1991 [16, 17] and the Tau protein hypothesis, are still subjects of extensive research and debate*.*The production and accumulation of Aβ peptide, a pathogenic factor leading to AD development,

and reproduction in any medium, provided the original work is properly cited.

© 2013 Baloyannis; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

Stavros J. Baloyannis

**1. Introduction**

and Golgi apparatus [13].

plotting the dramatic profile of AD.

http://dx.doi.org/10.5772/56079

Additional information is available at the end of the chapter

### **Alterations of Mitochondria and Golgi Apparatus Are Related to Synaptic Pathology in Alzheimer's Disease**

Stavros J. Baloyannis

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56079

### **1. Introduction**

Alzheimer's disease (AD) is an insidiously progressive severe presenile and senile dementia, involving a number of cellular and biochemical mechanisms. AD affects millions of humans as the most common cause of cognitive decline worldwide, in addition to being a main medical challenge for aging population. From the clinical point of view, AD is mostly characterized by age-dependent inexorably progressing cognitive decline, affecting memory primarily associ‐ ated with behavioral and mood disorders, which increasingly appear as the disease advances [1]. From the neuropathological point of view, AD is mostly characterized by selective neuronal loss [2, 3], marked synaptic alterations [4–6], morphological mitochondrial abnor‐ malities [7, 8], tau pathology [9] resulting in neurofibrillary tangles (NFT) composed of hyperphosphorylated tau [10], inflammatory responses and by extracellular extensive deposits of polymers of Aβ peptide, in the form of neuritic plaques, which are a main hallmark of AD [11, 12]. These are dispersed in the neocortex, the hippocampus, and many subcortical structures, which play an important role in cognition. In addition, AD is characterized ultrastructurally by organelle pathology involving mostly the microtubules, the mitochondria, and Golgi apparatus [13].

Most studies have revealed that the main pathological criteria for AD, namely the neuritic plaques and neurofibrillary tangles, can account for 40%–70% of the cognitive impairment seen in advanced age, though additional cerebrovascular changes [14, 15] contribute greatly in plotting the dramatic profile of AD.

The two main hypotheses of AD - the amyloid cascade hypothesis, introduced in 1991 [16, 17] and the Tau protein hypothesis, are still subjects of extensive research and debate*.*The production and accumulation of Aβ peptide, a pathogenic factor leading to AD development,

are the result of the post-translational proteolysis of the APP [18], by concerted actions of βand γ-secretases [19].

Mitochondrial dysfunction might contribute to Aβ neurotoxicity and is also associated with oxidative stress, which may play an important role in the early stages of pathogenetic mech‐ anisms in AD [35-37], presumably prior to the onset of the cognitive dysfunction, since a substantial body of evidence suggests that mitochondria play a crucial role in ageing-related

Alterations of Mitochondria and Golgi Apparatus Are Related to Synaptic Pathology in Alzheimer's Disease

http://dx.doi.org/10.5772/56079

103

Mitochondria may be the target for amyloid precursor protein (APP) and Aβ peptide, which might play an important role in impairing mitochondrial dynamics [39]. During AD processes accumulation of APP occurs mostly in the mitochondrial import channels, inducing mito‐ chondrial functional impairment [40]. APP could not be processed to generate Aβ peptide locally [41] although a fraction of active γ-secretase is associated with mitochondria [42].

In the mitochondria, Aβ peptide uptake is mediated by the translocase, which is located in the outer mitochondrial membrane (TOM) [43]. Then Aβ peptide is accumulated mostly in the outer mitochondrial membrane, the inter membrane and the matrix and interacts with large number of proteins inside mitochondria, leading eventually to mitochondrial dysfunction, whenever substantial amount of molecules of Aβ peptide would be pro‐

It is important to emphasize that mitochondrial alterations are associated with synaptic loss in AD patients, even before amyloid plaques are detected [32, 45]. Morphological and mor‐ phometric studies revealed that at early stages of AD the number of mitochondria in synaptic terminals is dramatically decrease and their structural pattern changes [45]. That modification might be attributed to enhanced nitrosative stress, generated by Aβ peptide, leading to mitochondrial fission, which is followed by mitochondrial depletion, resulting in synaptic

Golgi apparatus plays an important role in the pathogenesis of AD [13, 47], since it is associated with protein trafficking. All newly synthesized proteins, which are used for fast axoplasmic transport are processed practically through the vesicles and the cisternae of Golgi complex [48]. From the first its visualization by Camillo Golgi, in 1898, Golgi apparatus has been subject of

The electron microscopy study of the mammalian Golgi apparatus has revealed that it consists of stapled cisternae, which serve for the modification of newly synthesized proteins and lipids. At the entrance site of the Golgi apparatus, namely the cis-Golgi, numerous clusters of vesicles and tubular structures form an intermediate chain between the smooth endoplasmic reticulum and the Golgi stack. The exit site, the so called trans-Golgi network (TGN) is the main site of sorting proteins to distinct cellular destinations. Bi-directional traffic between the Golgi apparatus and the endosomal system sustains the functions of the trans-Golgi network (TGN)

The function of the Golgi complex consists mainly on vesicular transport, involving constant membrane fission and fusion, mediated by GTPases, coat proteins, Rabs, tethers and SNARE proteins, respectively, as it was documented from studies on glycosylation enzymes [50]. The

neurodegenerative diseases [38].

duced near mitochondria [44].

degeneration eventually [46].

**1.2. Golgi apparatus in Alzheimer's disease**

in secretion and organelle biogenesis [49].

intense morphological and neurochemical research.

The amyloidogenic pathway for APP is initiated by β- site amyloid precursor proteincleavage enzyme 1 (BACE- 1), resulting in the generation of the intermediate product sAPPβ [20]. γ -Secretase activity is substantial for the cleavage of the transmembrane domain, releasing the Aβ peptide and the APP intracellular domain. Generation of Aβ peptide may occur in the endoplasmic reticulum (ER), trans-Golgi network [21], in lysosomes and on the surface of the cell, whereas its intracellular accumulation has been mostly detected in the majority of neurons in the endoplasmic reticulum, the mitochon‐ dria [22], the lysosomes, the multivesicular bodies, associated with synaptic pathology [23]. Many risk factors may affect APP protein metabolism and Aβ peptide production, mainly in late-onset AD. In addition changes in cellular protein homeostasis and aspects of protein folding or misfolding [24] are also linked to AD pathogenesis [25].

All the biochemical phenomena, which may occur within the spectrum of pathogenetic mechanisms in Alzheimer's disease affect the brain metabolic activity reasonably, since increasing evidence from functional neuroimaging plead in favor of the global and regional disruptions in brain metabolism in advanced cases of AD [26].

From the etiological point of view, it would be hypothesized that the multiple genetic loci [27, 28], associated with familial Alzheimer's disease would plead in favor of the heterogeneity of the disease and support the idea that the phenomenological profile of Alzheimer's disease may be the final consequence of various metabolic, neurochemical, and morphological alterations, based on a broad genetic background [29]. Although the majority of familial or inherited AD, which manifests at an early age, are often associated with mutations in AβPP [30], the numerous sporadic ones, which manifests usually at later stages of the life, are proved to be multifactorial, including induced expression of AβPP [31] by pathological stimuli, environ‐ mental factors, as well as deprivation of trophic factors. The eventual accumulation of Aβ peptide at synaptic terminals may be associated with synaptic damage, resulting in cognitive decline in patients with AD [32]. Moreover the increased risk of AD in sporadic cases, whenever a maternal relative is afflicted with the disease, pleads reasonably in favor of a maternally derived predisposition, which might be related to mutations of mitochondrial DNA (mtDNA) [33, 34].

#### **1.1. Mitochondria in Alzheimer's disease**

Mitochondria are highly dynamic ATP-generating organelles, which play an essential role in many cellular functions, such as alteration of reduction-oxidation potential of cells, free radical scavenging, intracellular calcium regulation and activation of apoptotic process. Mitochondria are unique amongst cellular organelles, since they dispose their own, spiral, double-stranded DNA (mtDNA), which is mostly inherited from the maternal line. The number of mitochondria is very high in neurons and especially in synaptic terminals, since they are the major energy generators for the cell biological processes and the synaptic activity, through tricarboxylic cycle and oxidative phosphorylation. The shape and size of mitochondria are not stable, since they undergo continual fission and fusion leading to their fragmentation or elongation accordingly. Mitochondrial dysfunction might contribute to Aβ neurotoxicity and is also associated with oxidative stress, which may play an important role in the early stages of pathogenetic mech‐ anisms in AD [35-37], presumably prior to the onset of the cognitive dysfunction, since a substantial body of evidence suggests that mitochondria play a crucial role in ageing-related neurodegenerative diseases [38].

Mitochondria may be the target for amyloid precursor protein (APP) and Aβ peptide, which might play an important role in impairing mitochondrial dynamics [39]. During AD processes accumulation of APP occurs mostly in the mitochondrial import channels, inducing mito‐ chondrial functional impairment [40]. APP could not be processed to generate Aβ peptide locally [41] although a fraction of active γ-secretase is associated with mitochondria [42].

In the mitochondria, Aβ peptide uptake is mediated by the translocase, which is located in the outer mitochondrial membrane (TOM) [43]. Then Aβ peptide is accumulated mostly in the outer mitochondrial membrane, the inter membrane and the matrix and interacts with large number of proteins inside mitochondria, leading eventually to mitochondrial dysfunction, whenever substantial amount of molecules of Aβ peptide would be pro‐ duced near mitochondria [44].

It is important to emphasize that mitochondrial alterations are associated with synaptic loss in AD patients, even before amyloid plaques are detected [32, 45]. Morphological and mor‐ phometric studies revealed that at early stages of AD the number of mitochondria in synaptic terminals is dramatically decrease and their structural pattern changes [45]. That modification might be attributed to enhanced nitrosative stress, generated by Aβ peptide, leading to mitochondrial fission, which is followed by mitochondrial depletion, resulting in synaptic degeneration eventually [46].

#### **1.2. Golgi apparatus in Alzheimer's disease**

are the result of the post-translational proteolysis of the APP [18], by concerted actions of β-

The amyloidogenic pathway for APP is initiated by β- site amyloid precursor proteincleavage enzyme 1 (BACE- 1), resulting in the generation of the intermediate product sAPPβ [20]. γ -Secretase activity is substantial for the cleavage of the transmembrane domain, releasing the Aβ peptide and the APP intracellular domain. Generation of Aβ peptide may occur in the endoplasmic reticulum (ER), trans-Golgi network [21], in lysosomes and on the surface of the cell, whereas its intracellular accumulation has been mostly detected in the majority of neurons in the endoplasmic reticulum, the mitochon‐ dria [22], the lysosomes, the multivesicular bodies, associated with synaptic pathology [23]. Many risk factors may affect APP protein metabolism and Aβ peptide production, mainly in late-onset AD. In addition changes in cellular protein homeostasis and aspects of protein

All the biochemical phenomena, which may occur within the spectrum of pathogenetic mechanisms in Alzheimer's disease affect the brain metabolic activity reasonably, since increasing evidence from functional neuroimaging plead in favor of the global and regional

From the etiological point of view, it would be hypothesized that the multiple genetic loci [27, 28], associated with familial Alzheimer's disease would plead in favor of the heterogeneity of the disease and support the idea that the phenomenological profile of Alzheimer's disease may be the final consequence of various metabolic, neurochemical, and morphological alterations, based on a broad genetic background [29]. Although the majority of familial or inherited AD, which manifests at an early age, are often associated with mutations in AβPP [30], the numerous sporadic ones, which manifests usually at later stages of the life, are proved to be multifactorial, including induced expression of AβPP [31] by pathological stimuli, environ‐ mental factors, as well as deprivation of trophic factors. The eventual accumulation of Aβ peptide at synaptic terminals may be associated with synaptic damage, resulting in cognitive decline in patients with AD [32]. Moreover the increased risk of AD in sporadic cases, whenever a maternal relative is afflicted with the disease, pleads reasonably in favor of a maternally derived predisposition, which might be related to mutations of mitochondrial DNA

Mitochondria are highly dynamic ATP-generating organelles, which play an essential role in many cellular functions, such as alteration of reduction-oxidation potential of cells, free radical scavenging, intracellular calcium regulation and activation of apoptotic process. Mitochondria are unique amongst cellular organelles, since they dispose their own, spiral, double-stranded DNA (mtDNA), which is mostly inherited from the maternal line. The number of mitochondria is very high in neurons and especially in synaptic terminals, since they are the major energy generators for the cell biological processes and the synaptic activity, through tricarboxylic cycle and oxidative phosphorylation. The shape and size of mitochondria are not stable, since they undergo continual fission and fusion leading to their fragmentation or elongation accordingly.

folding or misfolding [24] are also linked to AD pathogenesis [25].

disruptions in brain metabolism in advanced cases of AD [26].

and γ-secretases [19].

102 Neurodegenerative Diseases

(mtDNA) [33, 34].

**1.1. Mitochondria in Alzheimer's disease**

Golgi apparatus plays an important role in the pathogenesis of AD [13, 47], since it is associated with protein trafficking. All newly synthesized proteins, which are used for fast axoplasmic transport are processed practically through the vesicles and the cisternae of Golgi complex [48]. From the first its visualization by Camillo Golgi, in 1898, Golgi apparatus has been subject of intense morphological and neurochemical research.

The electron microscopy study of the mammalian Golgi apparatus has revealed that it consists of stapled cisternae, which serve for the modification of newly synthesized proteins and lipids. At the entrance site of the Golgi apparatus, namely the cis-Golgi, numerous clusters of vesicles and tubular structures form an intermediate chain between the smooth endoplasmic reticulum and the Golgi stack. The exit site, the so called trans-Golgi network (TGN) is the main site of sorting proteins to distinct cellular destinations. Bi-directional traffic between the Golgi apparatus and the endosomal system sustains the functions of the trans-Golgi network (TGN) in secretion and organelle biogenesis [49].

The function of the Golgi complex consists mainly on vesicular transport, involving constant membrane fission and fusion, mediated by GTPases, coat proteins, Rabs, tethers and SNARE proteins, respectively, as it was documented from studies on glycosylation enzymes [50]. The activity of γ-secretase, which consists of presenilins (PSs) [51], nicastrin [52,53], pen-2 [54] and the aph proteins [54,55], is closely related with the function of Golgi complex, since it requires the presenilin-dependent trafficking of nicastrin, through the Golgi apparatus [56].

2.5% glutaraldehyde in cacodylate buffer 0.1 M, adjusted at pH 7.35. Then they were postfixed by immersion in 1% osmium tetroxide for 30 min at room temperature and dehydrated in

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Thin sections were cut in a Reichert ultratome, contrasted with uranyl acetate and lead citrate,

We studied the morphology of the mitochondria, the Golgi apparatus, and the synapses and we proceeded to morphometric estimations at electron microscope on micrographs of a

The remaining parts of the above-mentioned areas of the brain and the cerebellum were

Thus, after a four-week fixation in formalin they were immersed in potassium dichromate (7 g potassium dichromate in 300 mL water) for 10 days. Then they were immersed in 1% silver nitrate for 10 days. Following a rapid dehydration in graded alcohol solutions, the specimens were embedded in paraffin and cut, some of them at 100 *μ* and some at 25 *μ*, alternatively. Many sections of 25 *μ* were stained also with methylene blue, according to Golgi-Nissl method. All the sections were mounted in permount, between two cover slips and were studied in a

We estimated the dendritic arborization, the morphology and the number of the dendritic branches, and the morphology of the dendritic spines in light microscope on sections stained

Statistical analysis was based on the *t*-test on the basis of 5000 mitochondria and 600 Golgi apparatus from 30 specimens of AD brains and 30 specimens of normal control brains.

The silver impregnation technique or black reaction (*reazione near*) according to Golgi [61] is a simple and easy histological procedure that enables the visualization of the three-dimensional morphology of neurons and glial cells. Santiago Ramón y Cajal has been applying Golgi technique extensively for the histological analysis of the CNS, defending successfully the "neuron doctrine" and sharing with Camillo Golgi the Nobel Prize in 1906. After 140 years from its first application, the Golgi technique continues to remain a very useful and valuable method in neuropathology for the morphological and morphometric estimation of neuronal

circuits at the early stages of the degenerative processes of the brain [62].

processed for silver impregnation techniques, according to rapid Golgi method.

graded alcohol solutions and propylene oxide.

and studied in a Zeiss 9aS electron microscope.

**2.3. Light microscope, Golgi staining, Golgi-Nissl method**

standard magnification of 56. 000 X.

Zeiss Axiolab Photomicroscope.

**3.1. Silver impregnation techniques**

**2.4. Statistical analysis**

**3. Results**

according to rapid Golgi and Golgi-Nissl methods

It was hypothesized that the passage of nicastrin and other components of the γ-secretase complex through Golgi apparatus is essential for the molecular stabilization and the protease activity [56]. In addition APP, which is normally synthesized in the endoplasmic reticulum (ER), is transported to trans-Golgi network (TGN) for trafficking to the cell surface [57, 58], or to synaptic terminals. APP is transported by fast axonal transport, been recycled back for further trafficking or final storing within the lysosomal system [59], in view that TGN generates transport vesicles bound for distinct domains of the plasma membrane and early endosomes. Whereas a small proportion of APP molecules are delivered to the plasma membrane and then cleaved by α-secretase into non-amyloidogenic fragments, the majority of APP molecules undergo degradation, following amyloidogenic pathway in the trans-Golgi network [59].

### **2. Material and methods**

#### **2.1. Patients**

We studied the hippocampus, the acoustic and the visual cortices, the thalamus, the globus pallidus, the locus coeruleus, the red nucleus, the hypothalamus and many regions of the cerebellar cortex in ten brains of patients who suffered from AD, four men and six women, aged 62–87 years, who fulfilled the clinical, neuropsychological, and laboratory diagnostic criteria of AD.

The mean education of the patients was 15.2 years, and all of them spoke their native language fluently. Screening procedures were applied, which included medical history, medical examination, cardiological investigation, physical neurologic assessment, psychiatric and neuropsychological examinations. All the patients underwent EEG, carotid duplex Doppler, computerized tomography (CT) scanning and magnetic resonance imaging (MRI) of the brain, and single-photon emission computed tomography (SPECT).

The mental status of the patients was assessed by Mini mental State Examination (MMSE) and dementia rating scale (DRS) [60] and ADAS-COX test.

The cause of death of the patients was heart arrest, following to cardiac infarct one to seven months after the final neurological assessment. The postmortem examination of each one of the cases was performed within 6 h after death.

#### **2.2. Electron microscopy**

Small samples (2×2×2 mm) from the hippocampus, the acoustic and the visual cortices, the thalamus, the globus pallidus, the locus coeruleus, the red nucleus, the hypothalamus and from many regions of the cortex of the cerebellar hemispheres and the vermis were excised and immediately immersed in Sotelo's fixing solution, composed of 1% paraformaldehyde, 2.5% glutaraldehyde in cacodylate buffer 0.1 M, adjusted at pH 7.35. Then they were postfixed by immersion in 1% osmium tetroxide for 30 min at room temperature and dehydrated in graded alcohol solutions and propylene oxide.

Thin sections were cut in a Reichert ultratome, contrasted with uranyl acetate and lead citrate, and studied in a Zeiss 9aS electron microscope.

We studied the morphology of the mitochondria, the Golgi apparatus, and the synapses and we proceeded to morphometric estimations at electron microscope on micrographs of a standard magnification of 56. 000 X.

#### **2.3. Light microscope, Golgi staining, Golgi-Nissl method**

The remaining parts of the above-mentioned areas of the brain and the cerebellum were processed for silver impregnation techniques, according to rapid Golgi method.

Thus, after a four-week fixation in formalin they were immersed in potassium dichromate (7 g potassium dichromate in 300 mL water) for 10 days. Then they were immersed in 1% silver nitrate for 10 days. Following a rapid dehydration in graded alcohol solutions, the specimens were embedded in paraffin and cut, some of them at 100 *μ* and some at 25 *μ*, alternatively. Many sections of 25 *μ* were stained also with methylene blue, according to Golgi-Nissl method. All the sections were mounted in permount, between two cover slips and were studied in a Zeiss Axiolab Photomicroscope.

We estimated the dendritic arborization, the morphology and the number of the dendritic branches, and the morphology of the dendritic spines in light microscope on sections stained according to rapid Golgi and Golgi-Nissl methods

#### **2.4. Statistical analysis**

Statistical analysis was based on the *t*-test on the basis of 5000 mitochondria and 600 Golgi apparatus from 30 specimens of AD brains and 30 specimens of normal control brains.

### **3. Results**

activity of γ-secretase, which consists of presenilins (PSs) [51], nicastrin [52,53], pen-2 [54] and the aph proteins [54,55], is closely related with the function of Golgi complex, since it requires

It was hypothesized that the passage of nicastrin and other components of the γ-secretase complex through Golgi apparatus is essential for the molecular stabilization and the protease activity [56]. In addition APP, which is normally synthesized in the endoplasmic reticulum (ER), is transported to trans-Golgi network (TGN) for trafficking to the cell surface [57, 58], or to synaptic terminals. APP is transported by fast axonal transport, been recycled back for further trafficking or final storing within the lysosomal system [59], in view that TGN generates transport vesicles bound for distinct domains of the plasma membrane and early endosomes. Whereas a small proportion of APP molecules are delivered to the plasma membrane and then cleaved by α-secretase into non-amyloidogenic fragments, the majority of APP molecules undergo degradation, following amyloidogenic pathway in the trans-Golgi network [59].

We studied the hippocampus, the acoustic and the visual cortices, the thalamus, the globus pallidus, the locus coeruleus, the red nucleus, the hypothalamus and many regions of the cerebellar cortex in ten brains of patients who suffered from AD, four men and six women, aged 62–87 years, who fulfilled the clinical, neuropsychological, and laboratory diagnostic

The mean education of the patients was 15.2 years, and all of them spoke their native language fluently. Screening procedures were applied, which included medical history, medical examination, cardiological investigation, physical neurologic assessment, psychiatric and neuropsychological examinations. All the patients underwent EEG, carotid duplex Doppler, computerized tomography (CT) scanning and magnetic resonance imaging (MRI) of the brain,

The mental status of the patients was assessed by Mini mental State Examination (MMSE) and

The cause of death of the patients was heart arrest, following to cardiac infarct one to seven months after the final neurological assessment. The postmortem examination of each one of

Small samples (2×2×2 mm) from the hippocampus, the acoustic and the visual cortices, the thalamus, the globus pallidus, the locus coeruleus, the red nucleus, the hypothalamus and from many regions of the cortex of the cerebellar hemispheres and the vermis were excised and immediately immersed in Sotelo's fixing solution, composed of 1% paraformaldehyde,

and single-photon emission computed tomography (SPECT).

dementia rating scale (DRS) [60] and ADAS-COX test.

the cases was performed within 6 h after death.

**2.2. Electron microscopy**

the presenilin-dependent trafficking of nicastrin, through the Golgi apparatus [56].

**2. Material and methods**

**2.1. Patients**

104 Neurodegenerative Diseases

criteria of AD.

#### **3.1. Silver impregnation techniques**

The silver impregnation technique or black reaction (*reazione near*) according to Golgi [61] is a simple and easy histological procedure that enables the visualization of the three-dimensional morphology of neurons and glial cells. Santiago Ramón y Cajal has been applying Golgi technique extensively for the histological analysis of the CNS, defending successfully the "neuron doctrine" and sharing with Camillo Golgi the Nobel Prize in 1906. After 140 years from its first application, the Golgi technique continues to remain a very useful and valuable method in neuropathology for the morphological and morphometric estimation of neuronal circuits at the early stages of the degenerative processes of the brain [62].

The application of silver impregnation technique in our specimens revealed neuronal loss and marked abbreviation of the dendritic arborization in all the layers of the acoustic and the visual cortices, the hippocampus, the thalamus, the globus pallidus, the locus coeruleus, the red nucleus, the hypothalamus (Fig.1), the cerebellar cortex (Fig.2) and the vermis of the cerebel‐ lum (Fig.3). The layer I, of the acoustic and visual cortices, which includes Cajal-Retzius cells, which normally protrude very long horizontal axonal profiles with substantial number of collaterals [63. 64], was practically empty of neurons in patients who suffered from AD, in contrast to normal controls.

**Figure 1.** Neuron from the Hypothalamus of a case of AD, showing abbreviation of dendritic arborization and marked loss of dendritic spines (Golgi staining 2,400X)

Loss of tertiary dendritic branches was also noticed in the acoustic and visual cortices in all of the specimens. Abbreviation of the dendritic arborization was mostly prominent in neurons of layers III and V of the acoustic and visual cortices, the pyramidal neurons of the hippocam‐ pus, the polyhedral neurons of the locus coeruleus and in Purkinje cells of the cerebellar cortex, which demonstrated also a marked decrease of the number of dendritic spines (Fig.3) in comparison with the normal controls.

**Figure 3.** Purkinje cells from the vermis of the cerebellum of a case of AD, showing dramatic reduction of dendritic

**Figure 2.** Purkinje cells from the cerebellar hemisphere of a case of AD, demonstrating marked abbreviation of the

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dendritic arborization and considerable loss of dendritic spines (Golgi staining 2,000X).

branches and loss of dendritic spines (Golgi staining 3,100X)

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The application of silver impregnation technique in our specimens revealed neuronal loss and marked abbreviation of the dendritic arborization in all the layers of the acoustic and the visual cortices, the hippocampus, the thalamus, the globus pallidus, the locus coeruleus, the red nucleus, the hypothalamus (Fig.1), the cerebellar cortex (Fig.2) and the vermis of the cerebel‐ lum (Fig.3). The layer I, of the acoustic and visual cortices, which includes Cajal-Retzius cells, which normally protrude very long horizontal axonal profiles with substantial number of collaterals [63. 64], was practically empty of neurons in patients who suffered from AD, in

**Figure 1.** Neuron from the Hypothalamus of a case of AD, showing abbreviation of dendritic arborization and marked

Loss of tertiary dendritic branches was also noticed in the acoustic and visual cortices in all of the specimens. Abbreviation of the dendritic arborization was mostly prominent in neurons of layers III and V of the acoustic and visual cortices, the pyramidal neurons of the hippocam‐ pus, the polyhedral neurons of the locus coeruleus and in Purkinje cells of the cerebellar cortex, which demonstrated also a marked decrease of the number of dendritic spines (Fig.3) in

contrast to normal controls.

106 Neurodegenerative Diseases

loss of dendritic spines (Golgi staining 2,400X)

comparison with the normal controls.

**Figure 2.** Purkinje cells from the cerebellar hemisphere of a case of AD, demonstrating marked abbreviation of the dendritic arborization and considerable loss of dendritic spines (Golgi staining 2,000X).

**Figure 3.** Purkinje cells from the vermis of the cerebellum of a case of AD, showing dramatic reduction of dendritic branches and loss of dendritic spines (Golgi staining 3,100X)

In addition, the axonal collaterals in layers III, IV, V, and VI of the acoustic and visual cortices were dramatically decreased in comparison with the normal controls.

globus pallidus, the polyhedral neurons of the locus coeruleus as well as the Purkinje cells of the cerebellar hemispheres (Fig.5). Giant elongated spines were mostly seen in the hippocam‐ pus and in the Purkinje cells of the cerebellum. In a large number of presynaptic terminals of the acoustic and visual cortices of patients who suffered from AD, the ultrastructural study has revealed an considerable polymorphism and pleomorphism of the synaptic vesicles, which

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**Figure 5.** Synaptic alterations in the cerebellar cortex in a case of AD. Impressive poverty of synaptic vesicles is noticed at the presynaptic terminals associated with mitochondrial alterations and disruption of the spinal apparatus (Electron

Substatial poverty of the synaptic vesicles was particularly seen in the presynaptic terminals in layers III, IV, and V of the acoustic and visual cortices, as well as in the mossy fibers of the cerebellar cortex. Decrease of the number of synaptic vesicles associated with polymorphism of the remained vesicles was noticed in the hippocampus, the thalamus, the locus coeruleus,

Mitochondrial pathology was demonstrated in the majority of the dendritic spines in all of the specimens. That consisted of substantial change of mitochondrial shape and size, fragmenta‐ tion of cristae, and accumulation of osmiophilic material in a considerable number of mitochon‐ drial profiles (Fig.6). Many dendritic branches included mitochondria, which showed an unusual polymorphic arrangement of the cristae, which either showed a concentric configuration or they were arranged in a parallel way to the long axis of the organelle. Some Purkinje cell dendrites

and the parallel and climbing fibers of the cerebellar cortex.

micrograph 128,000X).

were dramatically decreased in number in comparison with normal controls.

The decrease of the branches of the apical dendrites of the cortical neurons as well as decrease in spine density was widespread phenomena seen in the large majority of neurons of the acoustic (Fig.4) and the visual cortices, in the hippocampus, the thalamus, the globus pallidus, the red nucleus, the locus coeruleus, the hypothalamus and the cerebellar cortex (Figs 1,2).

**Figure 4.** Neurons from the acoustic cortex of a case of AD, showing marked decrease of tertiary dendritic branches and tremendous loss of spines (Golgi staining 2,400X).

#### **3.2. Electron microscopy**

Electron microscopy is the most valuable and precise method for the morphological and morphometric study of the cell organelles, the synapses, the dendritic spines as well as the neuron- glial relationships in the CNS both in health and disease.

In our study, the electron microscopy revealed pathological alterations of the dendritic spines and impressive decrease in spine density in the secondary and tertiary dendritic branches in all of the layers of the acoustic and visual cortices.

The reduction in spine size was prominent in neurons of layers II, III, and V of the acoustic cortex. A substantial number of dendritic spines demonstrated large multivesicular bodies, abnormal spine apparatus, and mitochondria, which were characterized by marked morpho‐ logical alterations. Morphological alterations of the dendritic spines were also seen in the pyramidal neurons of the hippocampus, the large polyhedral neurons of the thalamus, the globus pallidus, the polyhedral neurons of the locus coeruleus as well as the Purkinje cells of the cerebellar hemispheres (Fig.5). Giant elongated spines were mostly seen in the hippocam‐ pus and in the Purkinje cells of the cerebellum. In a large number of presynaptic terminals of the acoustic and visual cortices of patients who suffered from AD, the ultrastructural study has revealed an considerable polymorphism and pleomorphism of the synaptic vesicles, which were dramatically decreased in number in comparison with normal controls.

In addition, the axonal collaterals in layers III, IV, V, and VI of the acoustic and visual cortices

The decrease of the branches of the apical dendrites of the cortical neurons as well as decrease in spine density was widespread phenomena seen in the large majority of neurons of the acoustic (Fig.4) and the visual cortices, in the hippocampus, the thalamus, the globus pallidus, the red nucleus, the locus coeruleus, the hypothalamus and the cerebellar cortex (Figs 1,2).

**Figure 4.** Neurons from the acoustic cortex of a case of AD, showing marked decrease of tertiary dendritic branches

Electron microscopy is the most valuable and precise method for the morphological and morphometric study of the cell organelles, the synapses, the dendritic spines as well as the

In our study, the electron microscopy revealed pathological alterations of the dendritic spines and impressive decrease in spine density in the secondary and tertiary dendritic branches in

The reduction in spine size was prominent in neurons of layers II, III, and V of the acoustic cortex. A substantial number of dendritic spines demonstrated large multivesicular bodies, abnormal spine apparatus, and mitochondria, which were characterized by marked morpho‐ logical alterations. Morphological alterations of the dendritic spines were also seen in the pyramidal neurons of the hippocampus, the large polyhedral neurons of the thalamus, the

neuron- glial relationships in the CNS both in health and disease.

and tremendous loss of spines (Golgi staining 2,400X).

all of the layers of the acoustic and visual cortices.

**3.2. Electron microscopy**

108 Neurodegenerative Diseases

were dramatically decreased in comparison with the normal controls.

**Figure 5.** Synaptic alterations in the cerebellar cortex in a case of AD. Impressive poverty of synaptic vesicles is noticed at the presynaptic terminals associated with mitochondrial alterations and disruption of the spinal apparatus (Electron micrograph 128,000X).

Substatial poverty of the synaptic vesicles was particularly seen in the presynaptic terminals in layers III, IV, and V of the acoustic and visual cortices, as well as in the mossy fibers of the cerebellar cortex. Decrease of the number of synaptic vesicles associated with polymorphism of the remained vesicles was noticed in the hippocampus, the thalamus, the locus coeruleus, and the parallel and climbing fibers of the cerebellar cortex.

Mitochondrial pathology was demonstrated in the majority of the dendritic spines in all of the specimens. That consisted of substantial change of mitochondrial shape and size, fragmenta‐ tion of cristae, and accumulation of osmiophilic material in a considerable number of mitochon‐ drial profiles (Fig.6). Many dendritic branches included mitochondria, which showed an unusual polymorphic arrangement of the cristae, which either showed a concentric configuration or they were arranged in a parallel way to the long axis of the organelle. Some Purkinje cell dendrites (Fig.6) and a substantial number of climbing fibers included very large elongated mitochon‐ dria. Small round mitochondria intermixed with dense bodies were seen in association with fragmentation or dilatation of the cisternae of the Golgi apparatus in the soma of a considera‐ ble number of neurons of the visual cortex (Fig.7), the hippocampus, the locus coeruleus, the red nucleus, the polyhedral neurons of globus pallidus, and the Purkinje cells in AD brains.

It is worth to emphasize that morphological alterations of the mitochondria were also seen in the soma of astrocytes, the perivascular processes, and the astrocytic sheaths in AD brains, in

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From the morphometric point of view the ellipsoid mitochondria of the dendritic spines in normal control brains appear to have an average diameter of 650 ± 250 nm and a mean axial ratio of 1.9 ± 0.2. The round or global mitochondria appeared to have in normal controls a mean radius of 350 nm. In AD brains, the ellipsoid mitochondria of the neurons in acoustic and visual cortices appear to have an average diameter of 480 ± 250 nm and a mean axial ratio of 1.7 ± 0.2.

**Chart 1.** Decrease of the diameter of mitochondria in the visual cortex in Alzheimer's cases in comparison with normal

In the majority of Purkinje cells of the cerebellum, in the hippocampal neurons and in neurons of the parietal, frontal, acoustic and visual cortices the Golgi apparatus was mostly fragmented

**Figure 9.** Fragmentation of the cisternae of the Golgi apparatus in a Purkinje cell of the cerebellar cortex of a case of

AD (left) (Electron micrograph 128,000). Normal control (right) (Electron micrograph 65,000X)

and atrophic (Fig.8) in comparison with normal controls.

The round mitochondria appear to have a mean radius of 280 nm (Chart.1).

contrast to normal controls.

controls.

**Figure 6.** Abnormal mitochondria in a dendritic profile of Purkinje cell in a case of AD. The disruption of the cristae is a prominent phenomenon (Electron micrograph 128,000X).

**Figure 7.** Elongated mitocondria and mitochondria in fusión, intermixed with dense bodies and dilated cisternae of Golgi apparatus in a case of AD (Electron micrograph 25,000X).

It is worth to emphasize that morphological alterations of the mitochondria were also seen in the soma of astrocytes, the perivascular processes, and the astrocytic sheaths in AD brains, in contrast to normal controls.

(Fig.6) and a substantial number of climbing fibers included very large elongated mitochon‐ dria. Small round mitochondria intermixed with dense bodies were seen in association with fragmentation or dilatation of the cisternae of the Golgi apparatus in the soma of a considera‐ ble number of neurons of the visual cortex (Fig.7), the hippocampus, the locus coeruleus, the red

**Figure 6.** Abnormal mitochondria in a dendritic profile of Purkinje cell in a case of AD. The disruption of the cristae is a

**Figure 7.** Elongated mitocondria and mitochondria in fusión, intermixed with dense bodies and dilated cisternae of

prominent phenomenon (Electron micrograph 128,000X).

110 Neurodegenerative Diseases

Golgi apparatus in a case of AD (Electron micrograph 25,000X).

nucleus, the polyhedral neurons of globus pallidus, and the Purkinje cells in AD brains.

From the morphometric point of view the ellipsoid mitochondria of the dendritic spines in normal control brains appear to have an average diameter of 650 ± 250 nm and a mean axial ratio of 1.9 ± 0.2. The round or global mitochondria appeared to have in normal controls a mean radius of 350 nm. In AD brains, the ellipsoid mitochondria of the neurons in acoustic and visual cortices appear to have an average diameter of 480 ± 250 nm and a mean axial ratio of 1.7 ± 0.2. The round mitochondria appear to have a mean radius of 280 nm (Chart.1).

**Chart 1.** Decrease of the diameter of mitochondria in the visual cortex in Alzheimer's cases in comparison with normal controls.

In the majority of Purkinje cells of the cerebellum, in the hippocampal neurons and in neurons of the parietal, frontal, acoustic and visual cortices the Golgi apparatus was mostly fragmented and atrophic (Fig.8) in comparison with normal controls.

**Figure 9.** Fragmentation of the cisternae of the Golgi apparatus in a Purkinje cell of the cerebellar cortex of a case of AD (left) (Electron micrograph 128,000). Normal control (right) (Electron micrograph 65,000X)

It is important to underline that the fragmentation of the Golgi apparatus was seen in neurons which did not showed any tau pathology, such as accumulation of intracellular NFTs, and were located in areas with minimal deposits of Aβ peptide. However the atrophy and the fragmentation of Golgi apparatus coexisted with mitochondrial alterations and dendritic and spinal pathology in the large majority of neurons (Fig.9).

Mitochondrial alterations and dysfunction have been reported in several neurodegenerative diseases [75] mostly associated with oxidative damage [76-78] and vascular lesions [79]. Oxidative stress is mostly related with the accumulation of Aβ peptide in the neocortex [80, 81], playing an important role in the pathogenesis of AD [82], since it is not only involved in the formation of senile plaques and in damage to the proteins of NFT [83], but also induces

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It is also well documented that Aβ peptide may increase mitochondrial reactive oxygen species (ROS) production [85, 86], causing further impairment of mitochondrial function [87], since the lack of histones in mitochondrial DNA renders them a vulnerable target to oxidative stress. In major examples of neurodegenerative diseases, there is strong evidence that mitochondrial dysfunction occurs early and acts causally in disease pathogenesis. Mutations in mitochondrial DNA and oxidative stress, on the other hand, may contribute to ageing [88, 89], which is the substantial biological background for the majority of the neurodegenerative diseases [65].

Mitochondrial dysfunction has been associated with energy crisis of the cell and excitotoxic cell death and is considered to be of substantial importance in the cascade of phenomena, which eventually lead to apoptosis. Some observations in early cases of AD [90] indicate that morphological alterations of the mitochondria and oxidative damage may be one of the earliest

The morphological alteration of the mitochondria seen in subcortical centers, such as in the thalamus, the globus pallidus, the red nucleus, the hypothalamus and the locus coeruleus, pleads in favor of a generalized mitochondrial dysfunction in AD, which may be associated with wide neuronal loss and synaptic alterations, seriously affecting the mental faculties, which are basically related to extensive neural networks [91] and synaptic activity. Moreover, an impressive number of disease-specific proteins interact with mitochondria. Well-docu‐ mented studies [92] demonstrate that a significant amount of the N-terminal domain of APP targeted the mitochondria of cortical neurons and select regions of the brain of a transgenic mouse model for AD. The accumulation of trans membrane-arrested APP blocked protein translocation, disrupted mitochondrial function, and impaired brain energy metabolism.

In AD it may be considered that mitochondria-associated Aβ peptide may directly cause neurotoxicity [93, 94]. Mitochondrial dysfunction, therefore, might be a hallmark of amyloidbeta-induced neuronal toxicity in Alzheimer's disease [95]. The binding site for amyloid beta peptide has been identified as alcohol dehydrogenase in the matrix space of the organelle. Recent evidence also suggest that PS1, PS2, APP, and γ-secretase activity are not homogene‐ ously distributed in the ER, but rather are enriched in mitochondria-associated ER membranes (ER-MAMs or MAMs), which is a dynamic sub-compartment of the ER, which is connected with mitochondria [96]. Mitochondria and ER are closely connected in several functions such

as transfer of Calium, lipid metabolism, the control of apoptosis and autophagy [96]

Many morphological alterations of AD could be well linked to mitochondria changes, since blockage of mitochondrial energy production shifts APP metabolism to the production of more amyloidogenic forms of amyloid [97]. In addition amyloid beta peptide promotes permeability transition pore in brain mitochondria [85, 98]. It is important to mention that many protein

events.

extensive damage to the cytoplasm of neuronal populations vulnerable to death [84].

**Figure 9.** Degeneration of dendritic spine in the visual cortex of a case of AD (Electron micrograph 130,000X)

### **4. Discussion**

The mitochondria, which are the only nonnuclear constituents of the cell with their own DNA (mtDNA), having machinery for synthesizing RNA and proteins, are critical to homeostasis of the cell, by virtue of providing most of the energy for cellular processes, since energy, realized by oxidative phosphorylation, comes through the mitochondria, which generate most of the cell's supply of ATP. Mitochondria are also critical regulators of cell apoptosis, as being involved in a considerable number of neurodegenerative diseases [65, 66],

From the morphological point of view the shape and size of the mitochondria are highly variable [67], depending on fission and fusion [68]. Their morphology is occasionally controlled by cytoskeletal elements, namely the neurofilaments and the microtubules [69]. The change of the mitochondrial shape occurs mostly through their move to axons, dendrites, and synaptic terminals via anterograde transport [70]. During the various neuronal processes approximately one-third of the mitochondria are in motion along microtubules and actin filaments [71–73]. Mitochondrial motility and accumulation are coordinated, since mitochondria are transported to regions where ATP consumption and necessity for energy are particularly high, as it occurs in the synapses, which have high energy demand reasonably, for serving neuronal communication [74].

Mitochondrial alterations and dysfunction have been reported in several neurodegenerative diseases [75] mostly associated with oxidative damage [76-78] and vascular lesions [79]. Oxidative stress is mostly related with the accumulation of Aβ peptide in the neocortex [80, 81], playing an important role in the pathogenesis of AD [82], since it is not only involved in the formation of senile plaques and in damage to the proteins of NFT [83], but also induces extensive damage to the cytoplasm of neuronal populations vulnerable to death [84].

It is important to underline that the fragmentation of the Golgi apparatus was seen in neurons which did not showed any tau pathology, such as accumulation of intracellular NFTs, and were located in areas with minimal deposits of Aβ peptide. However the atrophy and the fragmentation of Golgi apparatus coexisted with mitochondrial alterations and dendritic and

**Figure 9.** Degeneration of dendritic spine in the visual cortex of a case of AD (Electron micrograph 130,000X)

involved in a considerable number of neurodegenerative diseases [65, 66],

energy demand reasonably, for serving neuronal communication [74].

The mitochondria, which are the only nonnuclear constituents of the cell with their own DNA (mtDNA), having machinery for synthesizing RNA and proteins, are critical to homeostasis of the cell, by virtue of providing most of the energy for cellular processes, since energy, realized by oxidative phosphorylation, comes through the mitochondria, which generate most of the cell's supply of ATP. Mitochondria are also critical regulators of cell apoptosis, as being

From the morphological point of view the shape and size of the mitochondria are highly variable [67], depending on fission and fusion [68]. Their morphology is occasionally controlled by cytoskeletal elements, namely the neurofilaments and the microtubules [69]. The change of the mitochondrial shape occurs mostly through their move to axons, dendrites, and synaptic terminals via anterograde transport [70]. During the various neuronal processes approximately one-third of the mitochondria are in motion along microtubules and actin filaments [71–73]. Mitochondrial motility and accumulation are coordinated, since mitochondria are transported to regions where ATP consumption and necessity for energy are particularly high, as it occurs in the synapses, which have high

spinal pathology in the large majority of neurons (Fig.9).

**4. Discussion**

112 Neurodegenerative Diseases

It is also well documented that Aβ peptide may increase mitochondrial reactive oxygen species (ROS) production [85, 86], causing further impairment of mitochondrial function [87], since the lack of histones in mitochondrial DNA renders them a vulnerable target to oxidative stress. In major examples of neurodegenerative diseases, there is strong evidence that mitochondrial dysfunction occurs early and acts causally in disease pathogenesis. Mutations in mitochondrial DNA and oxidative stress, on the other hand, may contribute to ageing [88, 89], which is the substantial biological background for the majority of the neurodegenerative diseases [65].

Mitochondrial dysfunction has been associated with energy crisis of the cell and excitotoxic cell death and is considered to be of substantial importance in the cascade of phenomena, which eventually lead to apoptosis. Some observations in early cases of AD [90] indicate that morphological alterations of the mitochondria and oxidative damage may be one of the earliest events.

The morphological alteration of the mitochondria seen in subcortical centers, such as in the thalamus, the globus pallidus, the red nucleus, the hypothalamus and the locus coeruleus, pleads in favor of a generalized mitochondrial dysfunction in AD, which may be associated with wide neuronal loss and synaptic alterations, seriously affecting the mental faculties, which are basically related to extensive neural networks [91] and synaptic activity. Moreover, an impressive number of disease-specific proteins interact with mitochondria. Well-docu‐ mented studies [92] demonstrate that a significant amount of the N-terminal domain of APP targeted the mitochondria of cortical neurons and select regions of the brain of a transgenic mouse model for AD. The accumulation of trans membrane-arrested APP blocked protein translocation, disrupted mitochondrial function, and impaired brain energy metabolism.

In AD it may be considered that mitochondria-associated Aβ peptide may directly cause neurotoxicity [93, 94]. Mitochondrial dysfunction, therefore, might be a hallmark of amyloidbeta-induced neuronal toxicity in Alzheimer's disease [95]. The binding site for amyloid beta peptide has been identified as alcohol dehydrogenase in the matrix space of the organelle. Recent evidence also suggest that PS1, PS2, APP, and γ-secretase activity are not homogene‐ ously distributed in the ER, but rather are enriched in mitochondria-associated ER membranes (ER-MAMs or MAMs), which is a dynamic sub-compartment of the ER, which is connected with mitochondria [96]. Mitochondria and ER are closely connected in several functions such as transfer of Calium, lipid metabolism, the control of apoptosis and autophagy [96]

Many morphological alterations of AD could be well linked to mitochondria changes, since blockage of mitochondrial energy production shifts APP metabolism to the production of more amyloidogenic forms of amyloid [97]. In addition amyloid beta peptide promotes permeability transition pore in brain mitochondria [85, 98]. It is important to mention that many protein systems are also essential in mitochondrial function and morphological integrity as well as in binding to the cytoskeleton [99]. Mitochondrial porin is an outer-membrane protein that forms regulated channels (Voltage-Dependent Anionic Channels) between the mitochondrial intermembrane space and the cytosol [100]. Porin may play an important role in binding to neurofilaments and microtubules, since porin-rich domains contain most of the binding sites for MAP2 [101, 102]. In addition, preselinin-2 modulates endoplasmic reticulum-mitochon‐ drial interactions [103], a fact that would plead in favor of the crucial role that mitochondria play in the pathogenetic cascade of AD.

system [48, 49]. Current data clearly suggest that perturbations to the endosomal retrograde sorting pathway promote the production of Aβ. The recent discovery of an integral membrane protein, Gamma-secretase activating protein (GSAP) [114,115],which associates with γsecretase, APP and promotes the production Aβ peptide may provide an important link for understanding how γ-secretase is directed to APP and help to clarify the site(s) of Aβ pro‐ duction. GSAP might be also an ideal target for designing γ-secretase modulators in an attempt

Alterations of Mitochondria and Golgi Apparatus Are Related to Synaptic Pathology in Alzheimer's Disease

http://dx.doi.org/10.5772/56079

115

1 Department of Neurology, School of Medicine, Aristotle University of Thessaloniki,

[1] Alzheimer's Association. Alzheimer's disease facts and figures. Alzheimer's and De‐

[2] Wisniewski H M, Wegiel J, Kotula L. Some neuropathological aspects of Alzheimer's disease and its relevance to other disciplines. Neuropathology and Applied Neurobi‐

[3] Duyckaerts C, Delatour B,. Potier M C. Classification and basic pathology of Alz‐

[4] Baloyannis S, Costa V, Arnaoutoglou A, Arnaoutoglou H. Synaptic alterations in the molecular layer of the cerebellum in Alzheimer's disease. Neuropathology and Ap‐

[5] Baloyannis S J, Manolidis S L, Manolidis L S. The acoustic cortex in Alzheimer's dis‐

[6] Baloyannis S J, Manolidis S L, Manolidis L S. Synaptic alterations in the vestibule-cer‐ ebellar system in Alzheimer's disease- a Golgi and electron microscope study. Acta

[7] Baloyannis S J, Costa V. Michmizos D. Mitochondrial alterations in Alzheimer's dis‐ ease. American Journal of Alzheimer's Disease and other Dementias. 2004; 19: 89–93.

2 Institute for research on Alzheimer's disease, Thessaloniki, Greece

heimer disease. Acta Neuropathologica, 2009; 118: 5–36.

for treating AD [116,117].

**Author details**

Greece

**References**

mentia. 2010; 6: 158–194.

ology, 1996; 22: 3–11.

plied Neurobiology 1996; 22: 78–79.

Oto-Laryngolo-gica 2000; 120: 247–250.

ease. Acta Oto-Laryngologica. 1992; 494: 1–13.

Stavros J. Baloyannis1,2

Recent studies reported increased mitochondrial fission and decreased fusion, due to increased amyloid beta (Aβ) interaction with the mitochondrial fission protein Drp 1, inducing increased mitochondrial fragmentation, impaired axonal transport of mitochondria and synaptic degeneration in AD [104]. In addition, the interaction of the voltage-dependent anion channel 1 protein (VDAC1) with Aβ peptide and phosphorylated tau block mitochondrial pores, leading to mitochondrial dysfunction in AD [105].

The number of the mitochondria varies, according to energy state of the cell. Some evidence suggests that the mitochondria redistribute towards the dendritic profiles, in response to stimulation as a manifestation of synaptic plasticity [106]. Normally, a limited number of dendritic spines contain mitochondria, which are mostly small and round, which are increased in number inside the dendritic branches during the synaptogenesis. Decrease in energy metabolism and altered cytochrome c oxidase (CytOX) activity are among the earliest detect‐ able defects in AD [107], affecting presumably neuronal plasticity and synaptogenesis. Some observations suggest that mitochondrial cytochrome c oxidase may be inhibited by a dimeric conformer of A*β*35, a phenomenon which further emphasizes the role of the A*β* peptide on the mitochondrial dysfunction in AD [108].

Among the ongoing therapeutic efforts [109], those targeting basic mitochondrial processes, such as energy metabolism, free-radical generation, or specific interactions of disease-related proteins with mitochondria, hold great promise. On the basis of the mitochondrial pathology, in the pathogenetic spectrum in AD, new strategies inducing protection to mitochondria by the administration of efficient antioxidant factors could be introduced in the treatment of early cases of AD.

Golgi apparatus, on the other hand plays a very important role in posttranslational modifica‐ tions, transport, and targeting of large number of proteins, which participate in axoplasmic transport or are transported to plasma membrane, to lysosomes, to synaptic terminals and dendritic spines. A substantial body of evidence suggest that Golgi complex is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) [110-112] and AD, as it is well documented by highly specific immunocytochemistry techniques [47, 113].

The size of the Golgi apparatus may be an index of neuronal activity. Thus the fragmentation of the cisternae of Golgi apparatus may be associated with impaired trafficking of proteins to synapses and dendritic spines resulting in synaptic degeneration and cognitive impairment eventually. It is well documented that the trans-Golgi network (TGN) is the major sorting structure of the secretory pathway and the main site of intersection with the endo-lysosomal system [48, 49]. Current data clearly suggest that perturbations to the endosomal retrograde sorting pathway promote the production of Aβ. The recent discovery of an integral membrane protein, Gamma-secretase activating protein (GSAP) [114,115],which associates with γsecretase, APP and promotes the production Aβ peptide may provide an important link for understanding how γ-secretase is directed to APP and help to clarify the site(s) of Aβ pro‐ duction. GSAP might be also an ideal target for designing γ-secretase modulators in an attempt for treating AD [116,117].

### **Author details**

systems are also essential in mitochondrial function and morphological integrity as well as in binding to the cytoskeleton [99]. Mitochondrial porin is an outer-membrane protein that forms regulated channels (Voltage-Dependent Anionic Channels) between the mitochondrial intermembrane space and the cytosol [100]. Porin may play an important role in binding to neurofilaments and microtubules, since porin-rich domains contain most of the binding sites for MAP2 [101, 102]. In addition, preselinin-2 modulates endoplasmic reticulum-mitochon‐ drial interactions [103], a fact that would plead in favor of the crucial role that mitochondria

Recent studies reported increased mitochondrial fission and decreased fusion, due to increased amyloid beta (Aβ) interaction with the mitochondrial fission protein Drp 1, inducing increased mitochondrial fragmentation, impaired axonal transport of mitochondria and synaptic degeneration in AD [104]. In addition, the interaction of the voltage-dependent anion channel 1 protein (VDAC1) with Aβ peptide and phosphorylated tau block mitochondrial pores,

The number of the mitochondria varies, according to energy state of the cell. Some evidence suggests that the mitochondria redistribute towards the dendritic profiles, in response to stimulation as a manifestation of synaptic plasticity [106]. Normally, a limited number of dendritic spines contain mitochondria, which are mostly small and round, which are increased in number inside the dendritic branches during the synaptogenesis. Decrease in energy metabolism and altered cytochrome c oxidase (CytOX) activity are among the earliest detect‐ able defects in AD [107], affecting presumably neuronal plasticity and synaptogenesis. Some observations suggest that mitochondrial cytochrome c oxidase may be inhibited by a dimeric conformer of A*β*35, a phenomenon which further emphasizes the role of the A*β* peptide on the

Among the ongoing therapeutic efforts [109], those targeting basic mitochondrial processes, such as energy metabolism, free-radical generation, or specific interactions of disease-related proteins with mitochondria, hold great promise. On the basis of the mitochondrial pathology, in the pathogenetic spectrum in AD, new strategies inducing protection to mitochondria by the administration of efficient antioxidant factors could be introduced in the treatment of early

Golgi apparatus, on the other hand plays a very important role in posttranslational modifica‐ tions, transport, and targeting of large number of proteins, which participate in axoplasmic transport or are transported to plasma membrane, to lysosomes, to synaptic terminals and dendritic spines. A substantial body of evidence suggest that Golgi complex is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) [110-112] and AD, as it is well documented

The size of the Golgi apparatus may be an index of neuronal activity. Thus the fragmentation of the cisternae of Golgi apparatus may be associated with impaired trafficking of proteins to synapses and dendritic spines resulting in synaptic degeneration and cognitive impairment eventually. It is well documented that the trans-Golgi network (TGN) is the major sorting structure of the secretory pathway and the main site of intersection with the endo-lysosomal

by highly specific immunocytochemistry techniques [47, 113].

play in the pathogenetic cascade of AD.

114 Neurodegenerative Diseases

mitochondrial dysfunction in AD [108].

cases of AD.

leading to mitochondrial dysfunction in AD [105].

Stavros J. Baloyannis1,2

1 Department of Neurology, School of Medicine, Aristotle University of Thessaloniki, Greece

2 Institute for research on Alzheimer's disease, Thessaloniki, Greece

### **References**


[8] Baloyannis S J, Baloyannis J S. Mitochondrial alterations in Alzheimer's disease. Neu‐ robiology of Aging. 2004; 25: 405–406.

[23] Selkoe DJ. Soluble oligomers of the amyloid beta-protein impair synaptic plasticity

Alterations of Mitochondria and Golgi Apparatus Are Related to Synaptic Pathology in Alzheimer's Disease

http://dx.doi.org/10.5772/56079

117

[25] Morawe T, Hiebel C, Kern A, Behl C Protein Homeostasis, Aging and Alzheimer's

[26] Ishii K, Sasaki M, Kitagaki H, Yamaji S, Sakamoto S, Matsuda K, *et al.* Reduction of cerebellar glucose metabolism in advanced Alzheimer's disease. J Nucl Med 1997:38:

[27] Price D L, Sisodia S S. Mutant genes in familial Alzheimer's disease and transgenic

[28] Tanzi R E. A genetic dichotomy model for the inheritance of Alzheimer's disease and common age-related disorders. Journal of Clinical Investigation. 1999; 104: 1175–

[29] Caruso C, Franceschi C, Licastro F. Genetics of neurodegenerative disorders. The

[30] Hashimoto Y, Niikura T, Ito Y, Nishimoto I. Multiple mechanisms underlie neurotox‐ icity by different types of Alzheimer's disease mutations of amyloid precursor pro‐

[31] Selkoe D J. Alzheimer's disease results from the cerebral accumulation and cytotoxic‐ ity of amyloid beta-protein. Journal of Alzheimer's Disease.2001; 3: 75–80.

[32] Reddy P H, Beal M F.Amyloid beta, mitochondrial dysfunction and synaptic dam‐ age: implications for cognitive decline in aging and Alzheimer's disease. Trends in

[33] Shoffner J M, Brown MD, Torroni A, et al. Mitochondrial DNA variants observed in Alzheimer disease and Parkinson disease patients. Genomics, 1993; 17: 171–184.

[34] Hirai K, Aliev G, Nunomura A, et al. Mitochondrial abnormalities in Alzheimer's

[35] Lin MT, Beal MF. Mitochondrial dysfunction and oxidative stress in neurodegenera‐

[36] Manczak M, Anekonda T S, Henson E, Park B S, Quinn J, Reddy P H. Mitochondria are a direct site of A beta accumulation in Alzheimer's disease neurons: implications for free radical generation and oxidative damage in disease progression. Human Mo‐

[37] Sheng B, Wang X, Su B, Lee HG, Casadesus G, Perry G, *et al.* Impaired mitochondrial biogenesis contributes to mitochondrial dysfunction in Alzheimer's disease. J Neuro‐

and behavior. Behav. Brain Res. 2008; 192:106–113.

Disease. Molecular Neurobiology. 2012; 46: 41-54.

925–928.

1179.

[24] Dobson CM Protein folding and misfolding. Nature 2003; 426:884–890.

models. Annual Review of Neuroscience. 1998; 21: 479–505.

New England Journal of Medicine.2003; 349: 193–194.

disease. Journal of Neuroscience. 2001; 21: 3017–3023.

Molecular Medicine. 2008; 14: 45–53.

tive diseases. Nature 2006; 443: 787–795.

lecular Genetics 2006; 15: 1437–1449.

chem 2012; 120: 419–429.

tein. Journal of Biological Chemistry. 2000; 275: 34541–34551.


[8] Baloyannis S J, Baloyannis J S. Mitochondrial alterations in Alzheimer's disease. Neu‐

[9] Iqbal K, Liu F, Gong C X, Grundke-Iqbal I. Tau in Alzheimer disease and related

[10] Mattson M P. Pathways towards and away from Alzheimer's disease. Nature. 2004;

[11] Dickson D M. The pathogenesis of senile plaques. Journal of Neuropathology and

[12] Gandy S. The role of cerebral amyloid beta accumulation in common forms of Alz‐

[13] Baloyannis S. The Golgi apparatus of Purkinje cells in Alzheimer's disease. in Neuro‐ pathology Back to the Roots, J. Bohl, (Ed)., pp. 1–10, Shaker Vertag, Aachen, Germa‐

[14] Dolan D, Troncoso J, Crain B, Resnick S, Zonderman A, O'Brien R. Age, dementia

[15] Baloyannis SJ, Baloyannis IS The vascular factor in Alzheimer's disease: A study in Golgi technique and electron microscopy. J Neurol Sci. 2012; 322:117-121.

[16] Hardy J, Allsop D. Amyloid deposition as the central event in the aetiology of Alz‐

[17] Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer's disease: progress and

[18] Tanzi RE, Bertram L. Twenty years of the Alzheimer's disease amyloid hypothesis: A

[19] Selkoe D J. Alzheimer's disease: genes, proteins, and therapy. Physiol. Rev. 2001; 81:

[20] Greenfield J, Tsai J, Gouras G, Hai B, Thinakaran G, Checler F, Sisodia S, Greengard P, Xu H. Endoplasmic reticulum and trans-Golgi network generate distinct popula‐ tions of Alzheimer β-amyloid peptides. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 742–

[21] Takahashi R, Milner T, Li F, Nam E, Edgar M, Yamaguchi H, Beal M, Xu H, Green‐ gard P, Gouras G. Intraneuronal Alzheimer A42 accumulates in multivesicular bod‐ ies and is associated with synaptic pathology. Am. J. Pathol. 2002; 161: 1869–1879.

[22] Caspersen C, Wang N, Yao Y, Sosunov A, Chen X, Lustbader J W, Xu H W, Stern D, McKhann G, Yan S D. Mitochondrial A: a potential focal point for neuronal metabol‐

ic dysfunction in Alzheimer's disease. FASEB J. 2005;19: 2040–2041.

heimer disease. Journal of Clinical Investigation. 2005; 115: 1121–1129.

and Alzheimer's disease in the BLSA. Brain. 2010; 133: 2225–2231.

heimer's disease. Trends Pharmacol. Sci. 1991; 12: 383–388.

genetic perspective. Cell. 2005; 120:545–555.

problems on the road to therapeutics. Science 2002; 297:353–356

robiology of Aging. 2004; 25: 405–406.

Experimental Neurology1997; 56: 321–339.

430: 631–639.

116 Neurodegenerative Diseases

ny, 2002.

741–766.

747.

tauopathies. Current Alzheimer Research.2010; 7: 656–664.


[38] Beal MF, Hyman B T, Koroshetz W. Do defects in mitochondrial energy metabolism underlie the pathology of neurodegenerative diseases? Trends in Neurosciences 1993; 16:125–131.

[51] Zhang Z, Nadeau P, Song W, Donoviel D, Yuan M, Bernstein A, Yankner BA. Prese‐ nilins are required for gamma-secretase cleavage of beta-APP and trans-membrane

Alterations of Mitochondria and Golgi Apparatus Are Related to Synaptic Pathology in Alzheimer's Disease

http://dx.doi.org/10.5772/56079

119

[52] Chung H M, Struhl G. Nicastrin is required for Presenilin mediated trans-membrane

[53] Hu Y, Ye Y, Fortini M E. Nicastrin is required for gamma secretase cleavage of the

[54] Francis R, McGrath G, Zhang J, Ruddy D A, Sym M, Apfeld J, Nicoll M, Maxwell M, Hai B, Ellis M C. et al. aph-1 and pen-2 are required for Notch pathway signaling, gamma-secretase cleavage of beta APP, and presenilin protein accumulation. Dev

[55] Goutte C, Tsunozaki M, Hale V A, Priess J R. APH-1 is a multipass membrane pro‐ tein essential for the Notch signaling pathway in Caenorhabditis elegans embryos.

[56] Herreman An, Van Gassen G, Bentahir M, Nyabi O, Craessaerts K. Mueller U, An‐ naert W, De Strooper B. g-Secretase activity requires the presenilin-dependent traf‐ ficking of nicastrin through the Golgi apparatus but not its complex glycosylation.

[57] Sisodia SS. Beta-amyloid precursor protein cleavage by a membrane bound protease.

[58] Vetrivel KS, Thinakaran G. Amyloidogenic processing of beta-amyloid precursor

[59] Zheng L, Cedazo-Minguez A, Hallbeck M, Jerhammar F, Marcusson J, Terman A. In‐ tracellular distribution of amyloid beta Peptide and its relationship to the lysosomal

[60] Mattis S., Dementia Rating Scale Professional Manual, Psychological Assessment Re‐

[61] Pannese E. The Golgi stain: invention, diffusion and impact on neurosciences. J Hist

[62] Baloyannis SJ. Morphological and morphometric alterations of Cajal-Retzius cells in early cases of Alzheimer's disease: a Golgi and electron microscope study. Int J Neu‐

[63] Marín-Padilla M. Cajal-Retzius cells and the development of the neocortex. Trends in

[64] Marín-Padilla M. Three-dimensional structural organization of layer I of the human cerebral cortex: a Golgi study. Journal of Comparative Neurology.1990; 299: 89–105.

protein in intracellular compartments, Neurology 2006;66: 69-73.

system. Translational neurodegeneration 2012, 19:1-7.

cleavage of Notch-1. Nat Cell Biol. 2000; 2: 463-465.

Drosophila notch receptor. Dev Cell 2002; 2: 69-78.

Proc. Natl. Acad. Sci. US A 2002;99: 775-779.

Journal of Cell Science. 2003; 116:1127-1136.

Proc Natl Acad Sci USA 1992; 89:6075–6079.

sources, Odessa, Fla, USA, 1988.

Neurosci. 1999; 8:132- 140.

rosci. 2005; 115:965-80.

Neurosciences. 1998; 21: 64–71.

Cell. 2002; 3: 85-97.

cleavage in Drosophila. Nat Cell Biol. 2001; 3: 1129-1132.


[51] Zhang Z, Nadeau P, Song W, Donoviel D, Yuan M, Bernstein A, Yankner BA. Prese‐ nilins are required for gamma-secretase cleavage of beta-APP and trans-membrane cleavage of Notch-1. Nat Cell Biol. 2000; 2: 463-465.

[38] Beal MF, Hyman B T, Koroshetz W. Do defects in mitochondrial energy metabolism underlie the pathology of neurodegenerative diseases? Trends in Neurosciences

[39] Lin M T, Beal M F. Alzheimer's APP mangles mitochondria. Nat. Med. 2006; 12:

[40] Anandatheerthavarada H K, Biswas G, Robin M A, Avadhani N G. Mitochondrial targeting and a novel transmembrane arrest of Alzheimer's amyloid precursor pro‐ tein impairs mitochondrial function in neuronal cells. Journal of Cell Biology. 2003;

[41] Pagani L, Eckert A. Amyloid-Beta Interaction with Mitochondria. International Jour‐ nal of Alzheimer's Disease Volume 2011, Article ID 925050, 12 pages doi:

[42] Hansson C A, Fryckman S, Farmery M. R, Tjernberg L O, Nilsberth C, Pursglove S E, Ito A, Winblad B, Cowburn R F, Thyberg J, Ankarcrona, M. Nicastrin, preseni‐ lin,APH-1, and PEN-2 form active γ-secretase complexes in mitochondria J. Biol.

[43] Petersen H, Alikhani CA, Behbahani N, Wiehager H, Pavlov B, Alafuzoff I, Leinonen V, Ito A, Winblad B, Glaser E, Ankarcrona M. The amyloid beta peptide is imported into mitochondria via the TOM import machinery and localized to mitochondrial

[44] Caspersen C, Wang N, Yao Y, Sosunov A, Chen X, Lustbader J W, Xu H W, Stern D, McKhann G, Yan S D. Mitochondrial Aβ: a potential focal point for neuronal meta‐

[45] Baloyannis SJ. Mitochondria are related to synaptic pathology in Alzheimer's dis‐

[46] Cho D H, Nakamura T, Fang J. et al., S-nitrosylation of Drp1 mediates beta-amyloidrelated mitochondrial fission and neuronal injury. Science. 2009; 324:102–105.

[47] Stieber A, Mourelatos Z, Gonatas N K. In Alzheimer's Disease the Golgi Apparatus of a Population of Neurons without neurofibrillary Tangles Is Fragmented and Atro‐

[48] Hammerschlag R, Stone GC, Bolen FA, Lindsey JD, Ellisman MH. Evidence that all newly synthesized proteins destined for fast axonal transport pass through the Golgi

[49] Burd C G. Physiology and Pathology of Endosome-to-Golgi Retrograde Sorting Traf‐

[50] Duncan JR, Kornfeld S. Intra cellular movement of two mannose 6-phosphate recep‐

tors: return to the Golgi apparatus. J Cell Biol. 1988; 106: 617–628.

bolic dysfunction in Alzheimer's disease. FASEB J. 2005;19: 2040–2041.

ease. Int J Alzheimers Dis. 2011; 2011:305395. Epub 2011 Sep 12

phic. American Journal of Pathology. 1996; 148: 415-426.

apparatus. J Cell Biol. 1982; 93:568-575

fic 2011; 12: 948–955.

cristae. Proc. Natl. Acad. Sci. U.S.A. 2008; 105:13145–13150.

1993; 16:125–131.

1241–1243.

118 Neurodegenerative Diseases

161: 41–54

10.4061/2011/925050.

Chem. 2004; 279: 51654–51660.


[65] Beal MF. Mitochondrial dysfunction in neurodegenerative diseases. Biochimica et Bi‐ ophysica Acta.1998; 1366: 211–223.

[79] Aliev G, Smith M A, Seyidova D. et al. The role of oxidative stress in the pathophysi‐ ology of cerebrovascular lesions in Alzheimer's disease. Brain Pathology. 2002; 12:21–

Alterations of Mitochondria and Golgi Apparatus Are Related to Synaptic Pathology in Alzheimer's Disease

http://dx.doi.org/10.5772/56079

121

[80] Arias C, Montiel T, Quiroz-Baez R, Massieu L. Beta-Amyloid neurotoxicity is exacer‐ bated during glycolysis inhibition and mitochondrial impairment in the rat hippo‐ campus in vivo and in isolated nerve terminals: implications for Alzheimer's disease.

[81] Nunomura A, Perry G, Aliev G. et al. Oxidative damage is the earliest event in Alz‐ heimer's disease. Journal of Neuropathology and Experimental Neurology 2001; 60:

[82] Aliev G. Oxidative stress induced-metabolic imbalance, mitochondrial failure, and cellular hypoperfusion as primary pathogenetic factors for the development of Alz‐ heimer disease which can be used as a alternate and successful drug treatment strat‐ egy: past, present and future.CNS and Neurological Disorders—Drug Targets. 2011;

[83] Hugon J, Esclaire F, Lesort M, Kisby G, Spencer P. Toxic neuronal apoptosis and modifications of tau and APP gene and protein expressions. Drug Metabolism Re‐

[84] Moreira P I, Cardoso S M, Santos M S, Oliveira C R. The key role of mitochondria in

[85] Moreira PI, Santos MS, Moreno A, Oliveira C. Amyloid beta-peptide promotes per‐ meability transition pore in brain mitochondria. Bioscience Reports. 2001; 21: 789–

[86] Maurer I, Zierz S, Moller HJ. A selective defect of cytochrome c oxidase is present in brain of Alzheimer disease patients. Neurobiology of Aging. 2000; 21: 455–462. [87] Mecocci P, Cherubini A, Beal M F. et al. Altered mitochondrial membrane fluidity in

[88] Luo Y, Bond J D, Ingram VM. Compromised mitochondrial function leads to in‐ creased cytosolic calcium and to activation of MAP kinases. Proceedings of the Na‐

[89] Hsin-Chen Lee, Yau-Huei Wei. Mitochondria and Aging. Advances in Experimental

[90] Baloyannis SJ. Mitochondrial alterations in Alzheimer's disease. Journal of Alzheim‐

[91] Mesulam M M. Large-scale neurocognitive networks and distributed processing for attention, language, and memory. Annals of Neurology. 1990; 28: 597–613.

[92] Choia K, Kima M, Jea A, Ryua K, Chaea H, Kweona H, Leea C. A Three-Dimensional Study of Ultrastructural Variation of Abnormal Mitochondria in APP/PSEN1 Trans‐

Alzheimer's disease. Journal of Alzheimer's Disease 2006; 9: 101–110.

AD brain. Neuroscience Letters.1996; 207: 129–132.

tional Academy of Sciences USA 1997; 94: 9705–9710.

Medicine and Biology. 2012; 942: 311-327.

er's Disease, 2006; 9: 119–126.

Experimental Neurology 2002; 176:163–174.

35.

759–767.

10: 147–148.

800.

views. 1999; 31: 635–647.


[79] Aliev G, Smith M A, Seyidova D. et al. The role of oxidative stress in the pathophysi‐ ology of cerebrovascular lesions in Alzheimer's disease. Brain Pathology. 2002; 12:21– 35.

[65] Beal MF. Mitochondrial dysfunction in neurodegenerative diseases. Biochimica et Bi‐

[66] Mizuno Y, Ikebe S I, Hattori N.et al. Role of mitochondria in the etiology and patho‐ genesis of Parkinson's disease. Biochimica et Biophysica Acta. 1995; 1271: 265–274.

[67] Bereiter-Hahn J, Vöth M. Dynamics of mitochondria in living cells: shape changes, dislocations, fusion, and fission of mitochondria. Microscopy Research and Techni‐

[68] Chen H, McCaffery JM, Chan DC. Mitochondrial fusion protects against neurode‐

[69] Knowles M K, Guenza M G, Capaldi R A, Marcus A H. Cytoskeletal-assisted dynam‐ ics of the mitochondrial reticulum in living cells. Proceedings of the National Acade‐

[70] Hollenbeck PJ, Saxton WM. The axonal transport of mitochondria. Journal of Cell Sci‐

[71] Krendel M, Sgourdas G, Bonder EM. Disassembly of actin filaments leads to in‐ creased rate and frequency of mitochondrial movement along microtubules. Cell Mo‐

[72] Leterrier J F, Rusakov D A, Nelson B D, Linden M. Interactions between brain mito‐ chondria and cytoskeleton: evidence for specialized outer membrane domains in‐ volved in the association of cytoskeleton-associated proteins to mitochondria in situ

[73] Lifshitz J, Janmey PA, Linden M, McIntosh TK, Leterrier J F. Mechanisms of mito‐ chondria-neurofilament interactions. The Journal of Neuroscience. 2003; 23: 9046–

[74] Brown M R, Sullivan P G, Geddes J W. Synaptic mitochondria are more susceptible to Ca2+ overload than nonsynaptic mitochondria. Journal of Biological Chemistry.

[75] Mattson M P, Gleichmann M, Cheng A. Mitochondria in neuroplasticity and neuro‐

[76] Baloyannis S. Oxidative stress and mitochondria alterations in Alzheimer's disease.

[77] Pereira C, Santos M S, Oliveira C. Involvement of oxidative stress on the impairment of energy metabolism induced by Abeta peptides on PC12 cells: protection by antiox‐

[78] Zhu X, Perry G, Moreira P I. et al. Mitochondrial abnormalities and oxidative imbal‐ ance in Alzheimer disease. Journal of Alzheimer's Disease. 2006; 9: 147–153.

and in vitro. Microscopy Research and Technique. 1994; 27: 233–261.

generation in the cerebellum. Cell. 2007; 130: 548-562.

my of Sciences USA. 2002; 99: 14772–14777.

tility and the Cytoskeleton. 1998; 40: 368–378.

logical disorders. Neuron. 2008; 60: 748–766.

idants. Neurobiology of Disease. 1999; 6: 209–219.

Neurobiology of Aging. 21: 264. 2000.

ophysica Acta.1998; 1366: 211–223.

que.1994; 27:198–219.

120 Neurodegenerative Diseases

ence. 2005; 118: 5411–5419.

9058.

2006; 281:11658–11668.


genic Mouse. Brain Microscopy and Microanalysis. 2012; 18 (Supplement S2): 212-213.

[105] Manczak M, Reddy PH. Abnormal interaction of VDAC1 with amyloid beta and phosphorylated tau causes mitochondrial dysfunction in Alzheimer's disease. Hum

Alterations of Mitochondria and Golgi Apparatus Are Related to Synaptic Pathology in Alzheimer's Disease

http://dx.doi.org/10.5772/56079

123

[106] Li Z, Okamoto K.-I, Hayashi Y, Sheng M. The importance of dendritic mitochondria in the morphogenesis and plasticity of spines and synapses. Cell 2004; 119: 873–887.

[107] Cardoso S, Proenca M, Santos S, Santana I, Oliveira C. Cytochrome c oxidase is de‐ creased in Alzheimer's disease platelets. Neurobiology of Aging 2004; 25:105–110. [108] Lynch T, Cherny RA, Bush A I. Oxidative processes in Alzheimer's disease: the role

[109] He G, Luo W, Li P, Remmers C, Netzer WJ, Hendrick J, Bettayeb K, Flajolet M, Gore‐ lick F, Wennogle LP, Greengard P. Gamma-secretase activating protein is a therapeu‐

[110] Gonatas NK, Stieber A, Mourelatos Z, Chen Y, Gonatas JO, Appel SH, Hays AP, Hickey WF, Hauw JJ. Fragmentation of the Golgi apparatus of motor neurons in

[111] Mourelatos Z, Hirano A, Rosenquist A, Gonatas NK. Fragmentation of the Golgi ap‐ paratus of motor neurons in amyotrophic lateral sclerosis (ALS): clinical studies in ALS of Guam and experimental studies in deafferented neurons and in f3, f-iminodi‐

[112] Mourelatos Z, Yachnis A, Rorke L, Mikol J, Gonatas NK. The Golgi apparatus of mo‐ tor neurons in amyotrophic lateral sclerosis. Ann Neurol. 1993, 33:608-615.

[113] Mourelatos Z, Adler H, Hirano A, Donnenfeld H, Gonatas JO, Gonatas NK. Fragmenta‐ tion of the Golgi apparatus of motor neurons in amyotrophic lateral sclerosis revealed

[114] Deatherage CL, Hadziselimovic A, Sanders C R. Purification and Characterization of the Human γ-Secretase Activating Protein Biochemistry. 2012; 51: 5153–5159. [115] Satoh J, Tabunoki H, Ishida T, Saito Y, Arima K. Immunohistochemical characteriza‐ tion of γ-secretase activating protein expression in Alzheimer's disease brains Neuro‐

[116] Borgegard T, Jureus A, Olsson F, Rosqvist S, Sabirsh A, Rotticci D, Paulsen K, Klinten‐ berg R, Yan H, Waldman M, Stromberg K, Nord J, Johansson J, Regner A, Parpal S, Mali‐ nowsky D, Radesater A-C, Li T, Singh R, Eriksson H, Lundkvist J. First and second generation gamma-secretase modulators (GSMs) modulate Abeta production through different mechanisms. The Journal of Biological Chemistry. 2012; 287: 11810-11819. [117] Borgegård T, Gustavsson S, Nilsson C, Parpal S, Klintenberg R, Berg A-L, Rosqvist S, Serneels L, Svensson S, Olsson F, Jin S, Yan H, Wanngren J, Jureus A, Ridderstad-Wollberg A, Wollberg P, Stockling K, Karlström H, Malmberg Å, Lund J, Arvidsson PI, De Strooper B, Lendah U, Lundkvist J. Alzheimer's Disease: Presenilin 2-Sparing γ-Secretase Inhibition Is a Tolerable Aβ Peptide-Lowering Strategy. The Journal of

by organelle-specific antibodies. Proc Natl Acad Sci USA. 1990, 87:4393-4395.

of Ab-metal interactions. Experimental Gerontology 2000; 35: 445–451.

tic target for Alzheimer's disease. Nature 2010; 467:95–98.

amyotrophic lateral sclerosis. Am J Pathol. 1992, 140:731-737.

proprionitrile axonopathy. Am J Pathol 1994, 144:1288-1300.

pathology and Applied Neurobiology. 2012; 38: 132–141.

Neuroscience. 2012; 32: 17297-17305.

Mol Genet. 2012; 21: 5131-5146.


[105] Manczak M, Reddy PH. Abnormal interaction of VDAC1 with amyloid beta and phosphorylated tau causes mitochondrial dysfunction in Alzheimer's disease. Hum Mol Genet. 2012; 21: 5131-5146.

genic Mouse. Brain Microscopy and Microanalysis. 2012; 18 (Supplement S2):

[93] Eckert A, Schmitt K, Gotz J. Mitochondrial dysfunction, the beginning of the end in Alzheimer's disease? Separate and synergistic modes of tau and amyloid beta toxici‐

[94] Spuch C, Ortolano S, Navarro C. New Insights in the Amyloid-Beta Interaction with Mitochondria. Journal of Aging Research Volume 2012, Article ID 324968. 9 pages

[95] de Brito OM, Scorrano L. An intimate liaison: spatial organization of the endoplasmic

[96] De Strooper Bart, Scorrano L. Close encounter: mitochondria, endoplasmic reticulum

[97] Crouch P J, Blake R,. Duce J A. et al. Copper-dependent inhibition of human cyto‐ chrome c oxidase by a dimeric conformer of amyloid-β1-42. Journal of Neuroscience

[98] Moreira P I, Santos M S, Moreno A, Rego A C, Oliveira C. Effect of amyloid beta-pep‐ tide on permeability transition pore: a comparative study. Journal of Neuroscience

[99] Truscott K, Pfanner N, Voos W. Transport of proteins into mitochondria. Reviews of

[100] Summersa WAT, Wilkinsb JA, Dwivedib RC, Ezzatib P, Courta DA. Mitochondrial dysfunction resulting from the absence of mitochondrial porin in Neurospora crassa.

[101] Reddy PH. Is the mitochondrial outer membrane protein VDAC1 therapeutic target for Alzheimer's disease? Biochimica et Biophysica Acta (BBA) - Molecular Basis of

[102] Calkins M J, Reddy PH. Assessment of newly synthesized mitochondrial DNA using BrdU labeling in primary neurons from Alzheimer's disease mice: Implications for impaired mitochondrial biogenesis and synaptic damage. Biochimica et Biophysica

[103] Zampese E, Fasolato C, Kipanyula J, Bortolozzi M, Pozzan T, Pizzo P. Presenilin 2 modulates endoplasmic reticulum (ER)—mitochondria interactions and Ca2+ crosstalk. Proceedings of the National Academy of Sciences USA. 2011; 7: 2777–2782.

[104] Manczak M, Reddy P H. Abnormal interaction between the mitochondrial fission protein Drp1 and hyperphosphorylated tau in Alzheimer's disease neurons: implica‐ tions for mitochondrial dysfunction and neuronal damage. Hum. Mol. Genet. 2012;

reticulum-mitochondria relationship. EMBO J 2010; 29: 2715–2723.

andAlzheimer's disease The EMBO Journal.2012; 31: 4095–4097.

Physiology, Biochemistry and Pharmacology. 2001; 143: 81–136.

Acta (BBA) - Molecular Basis of Disease. 2011; 1812:1182–1189.

ty. Alzheimer's Research and Therapy, 2011; 3:15.

212-213.

122 Neurodegenerative Diseases

doi: 10. 1155/ 2012/324968

2005; 25: 672–679.

Research. 2002; 69: 257–267.

Mitochondrion. 2012;12: 220–229.

Disease2013;1832: 67–75.

21: 2538-2547.


**Chapter 6**

**Caspases in Alzheimer's Disease**

Additional information is available at the end of the chapter

tin, neuropeptide Y and substance P (Terry, 2000).

In AD, a significant synaptic loss ranging from 20% to 50% is reported. Biochemistry, elec‐ tron microscopy and immunocytochemistry have shown a decrease in synaptic density, presynaptic terminals, synaptic vesicle and synaptic protein markers in AD brains com‐ pared with the normal aged controls (Terry et al., 1991; Geula, 1998; Larson et al., 1999; Yao et al., 1999; Ashe, 2000; Baloyannis et al., 2000; Terry, 2000; Masliah, 2001; Masliah et al., 2001; Price et al., 2001; Scheff and Price, 2001; Scheff et al., 2001; Stephan et al., 2001; Callahan et al., 2002; Chan et al., 2002; Dodd, 2002). Although synaptic loss is remarkable in AD, it is not specific to AD. Reduction in synaptic density is also found in Pick's dis‐ ease, Huntington's disease, Parkinson's disease as well as in vascular dementia (Geula, 1998; Larson et al., 1999; Yao et al., 1999; Ashe, 2000; Baloyannis et al., 2000; Terry, 2000; Masliah, 2001; Masliah et al., 2001; Price et al., 2001; Scheff and Price, 2001; Scheff et al.,

2001; Stephan et al., 2001; Callahan et al., 2002; Chan et al., 2002; Dodd, 2002).

Since one of the most important physiological functions of synapses is to release and ac‐ cept neurotransmitters, the changes of activity of these neurotransmitters in neurodege‐ nerative diseases have also been intensively studied (Terry, 2000). In AD, most significant lesions happen in the cholinergic, adrenergic and serotoninergic systems (Davies and Ma‐ loney, 1976; Geula, 1998; Larson et al., 1999; Yao et al., 1999; Ashe, 2000; Baloyannis et al., 2000; Terry, 2000; Masliah, 2001; Masliah et al., 2001; Price et al., 2001; Scheff and Price, 2001; Scheff et al., 2001; Stephan et al., 2001; Callahan et al., 2002; Chan et al., 2002; Dodd, 2002). Some other peptidergic neurotransmitters also decrease in AD, such as somatosta‐

Synaptic loss might be one of the first events in AD development (Terry et al., 1991; Ter‐ ry, 2000; Selkoe, 2002). Decrease in presynaptic terminals, synaptic vesicle and synaptic protein markers occur in very early stage of AD (Ashe, 2000; Terry, 2000; Masliah et al.,

and reproduction in any medium, provided the original work is properly cited.

© 2013 Zhang; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

Yan Zhang

**1. Introduction**

http://dx.doi.org/10.5772/54627

### **Chapter 6**

### **Caspases in Alzheimer's Disease**

Yan Zhang

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54627

### **1. Introduction**

In AD, a significant synaptic loss ranging from 20% to 50% is reported. Biochemistry, elec‐ tron microscopy and immunocytochemistry have shown a decrease in synaptic density, presynaptic terminals, synaptic vesicle and synaptic protein markers in AD brains com‐ pared with the normal aged controls (Terry et al., 1991; Geula, 1998; Larson et al., 1999; Yao et al., 1999; Ashe, 2000; Baloyannis et al., 2000; Terry, 2000; Masliah, 2001; Masliah et al., 2001; Price et al., 2001; Scheff and Price, 2001; Scheff et al., 2001; Stephan et al., 2001; Callahan et al., 2002; Chan et al., 2002; Dodd, 2002). Although synaptic loss is remarkable in AD, it is not specific to AD. Reduction in synaptic density is also found in Pick's dis‐ ease, Huntington's disease, Parkinson's disease as well as in vascular dementia (Geula, 1998; Larson et al., 1999; Yao et al., 1999; Ashe, 2000; Baloyannis et al., 2000; Terry, 2000; Masliah, 2001; Masliah et al., 2001; Price et al., 2001; Scheff and Price, 2001; Scheff et al., 2001; Stephan et al., 2001; Callahan et al., 2002; Chan et al., 2002; Dodd, 2002).

Since one of the most important physiological functions of synapses is to release and ac‐ cept neurotransmitters, the changes of activity of these neurotransmitters in neurodege‐ nerative diseases have also been intensively studied (Terry, 2000). In AD, most significant lesions happen in the cholinergic, adrenergic and serotoninergic systems (Davies and Ma‐ loney, 1976; Geula, 1998; Larson et al., 1999; Yao et al., 1999; Ashe, 2000; Baloyannis et al., 2000; Terry, 2000; Masliah, 2001; Masliah et al., 2001; Price et al., 2001; Scheff and Price, 2001; Scheff et al., 2001; Stephan et al., 2001; Callahan et al., 2002; Chan et al., 2002; Dodd, 2002). Some other peptidergic neurotransmitters also decrease in AD, such as somatosta‐ tin, neuropeptide Y and substance P (Terry, 2000).

Synaptic loss might be one of the first events in AD development (Terry et al., 1991; Ter‐ ry, 2000; Selkoe, 2002). Decrease in presynaptic terminals, synaptic vesicle and synaptic protein markers occur in very early stage of AD (Ashe, 2000; Terry, 2000; Masliah et al.,

© 2013 Zhang; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

2001; Price et al., 2001; Scheff et al., 2001; Callahan et al., 2002; Chan et al., 2002; Dodd, 2002). In the transgenic mice with FAD mutations, synaptophysin, marker for presynaptic protein, decreases before the appearance of Aβ deposits and formation of plaques (Hamos et al., 1989; Masliah et al., 1989; Selkoe, 2002). Most important, the decline of function of synaptic transmission occurs even before synaptic structural changes (Masliah, 2001; Scheff and Price, 2001; Chan et al., 2002; Selkoe, 2002). Long-term potentiation (LTP) is commonly accepted as a measurement for capacity of synaptic plasticity, which is the ba‐ sis of learning, memory and complex information processing. The incidence and duration of LTP formation are used as an indication for formation and maintenance of working memory. Several lines of FAD mutant transgenic mice show a decline in the formation of LTP and synaptic excitation before the appearance of synaptic loss, plaques and other AD pathology (Geula, 1998; Ashe, 2000; Masliah, 2001; Masliah et al., 2001; Scheff and Price, 2001; Callahan et al., 2002; Chan et al., 2002; Selkoe, 2002). In summary, synaptic loss seems to appear earlier than all other pathological markers and the functional loss of syn‐ apses may be responsible for the initiation of cognitive decline in AD patients.

of the early events in AD. Besides the main pathology discussed above, some other pathologies of AD include granulovacuolar degeneration, cerebral amyloid angiopathy, blood-brain barrier disorder, white matter lesions, neuropil thread and gliosis (Jellinger, 2002a; Jellinger,

Caspases in Alzheimer's Disease http://dx.doi.org/10.5772/54627 127

As discussed above, stereological cell counting shows that densities of neurons in the AD cerebral cortex, the entorhinal cortex, the association cortex, the basal nucleus of Meynert, the locus coeruleus and the dorsal raphe decrease significantly compared to the age-matched non-AD controls (Bondareff et al., 1982; Lippa et al., 1992; Gomez-Isla et al., 1996; Gomez-Isla et al., 1997; Gomez-Isla et al., 1999; Colle et al., 2000). Neuronal cell loss is one of the first events during AD development. In the mild AD patient brains, remarkable neuronal cell loss of more than 40% is seen in the entorhinal cortex (Gomez-Isla et al., 1996; Gomez-Isla et al., 1997). Even in the mild cognitive impairment patient brains, significant neuronal loss is also observed in the entorhinal cortex (Gomez-Isla et al., 1996). Furthermore, the degree of neuronal loss

It was thought until recently that neuronal loss is mainly due to passive neurotrophic factor withdrawal. In 1988, Martin et al. (1988) showed that sympathetic neuronal death could be prevented by inhibiting RNA and protein synthesis, indicating that some of the neuronal death

Apoptosis, or programmed cell death (PCD), is a term proposed by Kerr, Wyllie and Currie in 1972 (Kerr et al., 1972) to describe a common type of cell death characterized by membrane blebbing, cell shrinkage, protein fragmentation, chromatin condensation and DNA degrada‐ tion followed by rapid engulfment of corpses by surrounding cells (Kerr et al., 1972). It rapidly cleans out dysfunctional cells, limits toxic effects, saves energy and recycles molecules for

Two major apoptotic pathways in mammalian cells are mediated through either death re‐ ceptors or mitochondria (Figure 1). The so-called extrinsic pathway is initiated by death re‐ ceptor CD95 (Apo-1/Fas), or other death receptors, such as tumor necrosis factor (TNF) receptor and tumor necrosis factor related apoptosis inducing ligand (TRAIL) receptor. CD95 is linked to pro-caspase-8 by an adapter factor FADD. Pro-caspase-8 has a death ef‐ fecter domain (DED) which binds to the death domain (DD) on death receptor adapter FADD, and a caspase activation and recruitment domain (CARD), which binds to other downstream caspases and further processes the downstream caspases. Ligand binding sig‐ nals the activation of pro-caspase-8 to form a tetramer of active caspase-8. Caspase-8 then mediates the cleavage of pro-caspase-3 and initiates the caspase activation cascade, which leads to protein and DNA cleavage and final termination of the cells. In this pathway, cas‐ pase-8 activation can be prevented by its natural inhibitors, such as cellular FADD-like in‐ terlukin-1-β-converting enzyme (FLICE/caspase-8)-inhibitory proteins (c-FLIPs) (Irmler et

correlates better with the clinical dementia level in AD than other pathology.

might be actively programmed (apoptotic) instead of passive (necrotic).

2002b, c; Jellinger and Attems, 2003).

**3. Apoptosis**

future *de novo* synthesis.

### **2. Neuronal loss in AD**

Synaptic loss and degeneration induce neuronal dysfunction and cell body loss. Neuronal loss in the cerebral cortex and the hippocampus is a hallmark feature of AD. Some of AD patients at late stage of the disease can have a severe decrease in brain volume and weight due to either neuronal loss or atrophy (Smale et al., 1995; Cotman and Su, 1996; Gomez-Isla et al., 1996; Gomez-Isla et al., 1997; Li et al., 1997; Su et al., 1997; Gomez-Isla et al., 1999). Assumption-based and design-based unbiased stereological cell counting show decreased density of neurons in the cerebral cortex, the entorhinal cortex, the association cortex, the basal nucleus of Meynert, the locus coeruleus and the dorsal raphe of AD brains (Bondareff et al., 1982; Lippa et al., 1992; Gomez-Isla et al., 1996; Gomez-Isla et al., 1997; Gomez-Isla et al., 1999; Colle et al., 2000). Profound neuronal loss is especially observed in the entorhinal cortex in the mild AD brains (Gomez-Isla et al., 1996; Gomez-Isla et al., 1997; Gomez-Isla et al., 1999). Besides AD, significant neuronal loss is also observed in the entorhinal cortex in very mild cognitive impairment patient brains (Gomez-Isla et al., 1996; Gomez-Isla et al., 1997; Gomez-Isla et al., 1999). These data suggest that neuronal loss may be one of the early events before formation of SPs and NFTs in AD development.

The loss of cholinergic neurons in AD is widely studied. The hippocampus and cortex receive major cholinergic input from the basal forebrain nuclei (Hohmann et al., 1987). Decrease of choline acetyltransferase activity and acetylcholine synthesis correlate well with the degree of cognitive impairment in AD patients (Mesulam, 1986; Hohmann et al., 1987; Pearson and Powell, 1987). Cholinergic neuronal lesion can be detected in the patients that have showed clinical memory loss symptoms for less than 1 year (Whitehouse et al., 1981; Whitehouse et al., 1982; Francis et al., 1993; Weinstock, 1997). However, markers for dopamine, γ-aminobutyric acid (GABA), or somatostatin are not altered (Whitehouse et al., 1981; Whitehouse et al., 1982; Francis et al., 1993). These results suggest that cholinergic neuronal loss is probably one of the early events in AD. Besides the main pathology discussed above, some other pathologies of AD include granulovacuolar degeneration, cerebral amyloid angiopathy, blood-brain barrier disorder, white matter lesions, neuropil thread and gliosis (Jellinger, 2002a; Jellinger, 2002b, c; Jellinger and Attems, 2003).

As discussed above, stereological cell counting shows that densities of neurons in the AD cerebral cortex, the entorhinal cortex, the association cortex, the basal nucleus of Meynert, the locus coeruleus and the dorsal raphe decrease significantly compared to the age-matched non-AD controls (Bondareff et al., 1982; Lippa et al., 1992; Gomez-Isla et al., 1996; Gomez-Isla et al., 1997; Gomez-Isla et al., 1999; Colle et al., 2000). Neuronal cell loss is one of the first events during AD development. In the mild AD patient brains, remarkable neuronal cell loss of more than 40% is seen in the entorhinal cortex (Gomez-Isla et al., 1996; Gomez-Isla et al., 1997). Even in the mild cognitive impairment patient brains, significant neuronal loss is also observed in the entorhinal cortex (Gomez-Isla et al., 1996). Furthermore, the degree of neuronal loss correlates better with the clinical dementia level in AD than other pathology.

It was thought until recently that neuronal loss is mainly due to passive neurotrophic factor withdrawal. In 1988, Martin et al. (1988) showed that sympathetic neuronal death could be prevented by inhibiting RNA and protein synthesis, indicating that some of the neuronal death might be actively programmed (apoptotic) instead of passive (necrotic).

### **3. Apoptosis**

2001; Price et al., 2001; Scheff et al., 2001; Callahan et al., 2002; Chan et al., 2002; Dodd, 2002). In the transgenic mice with FAD mutations, synaptophysin, marker for presynaptic protein, decreases before the appearance of Aβ deposits and formation of plaques (Hamos et al., 1989; Masliah et al., 1989; Selkoe, 2002). Most important, the decline of function of synaptic transmission occurs even before synaptic structural changes (Masliah, 2001; Scheff and Price, 2001; Chan et al., 2002; Selkoe, 2002). Long-term potentiation (LTP) is commonly accepted as a measurement for capacity of synaptic plasticity, which is the ba‐ sis of learning, memory and complex information processing. The incidence and duration of LTP formation are used as an indication for formation and maintenance of working memory. Several lines of FAD mutant transgenic mice show a decline in the formation of LTP and synaptic excitation before the appearance of synaptic loss, plaques and other AD pathology (Geula, 1998; Ashe, 2000; Masliah, 2001; Masliah et al., 2001; Scheff and Price, 2001; Callahan et al., 2002; Chan et al., 2002; Selkoe, 2002). In summary, synaptic loss seems to appear earlier than all other pathological markers and the functional loss of syn‐

apses may be responsible for the initiation of cognitive decline in AD patients.

Synaptic loss and degeneration induce neuronal dysfunction and cell body loss. Neuronal loss in the cerebral cortex and the hippocampus is a hallmark feature of AD. Some of AD patients at late stage of the disease can have a severe decrease in brain volume and weight due to either neuronal loss or atrophy (Smale et al., 1995; Cotman and Su, 1996; Gomez-Isla et al., 1996; Gomez-Isla et al., 1997; Li et al., 1997; Su et al., 1997; Gomez-Isla et al., 1999). Assumption-based and design-based unbiased stereological cell counting show decreased density of neurons in the cerebral cortex, the entorhinal cortex, the association cortex, the basal nucleus of Meynert, the locus coeruleus and the dorsal raphe of AD brains (Bondareff et al., 1982; Lippa et al., 1992; Gomez-Isla et al., 1996; Gomez-Isla et al., 1997; Gomez-Isla et al., 1999; Colle et al., 2000). Profound neuronal loss is especially observed in the entorhinal cortex in the mild AD brains (Gomez-Isla et al., 1996; Gomez-Isla et al., 1997; Gomez-Isla et al., 1999). Besides AD, significant neuronal loss is also observed in the entorhinal cortex in very mild cognitive impairment patient brains (Gomez-Isla et al., 1996; Gomez-Isla et al., 1997; Gomez-Isla et al., 1999). These data suggest that neuronal loss may be one of the early events before formation of SPs and

The loss of cholinergic neurons in AD is widely studied. The hippocampus and cortex receive major cholinergic input from the basal forebrain nuclei (Hohmann et al., 1987). Decrease of choline acetyltransferase activity and acetylcholine synthesis correlate well with the degree of cognitive impairment in AD patients (Mesulam, 1986; Hohmann et al., 1987; Pearson and Powell, 1987). Cholinergic neuronal lesion can be detected in the patients that have showed clinical memory loss symptoms for less than 1 year (Whitehouse et al., 1981; Whitehouse et al., 1982; Francis et al., 1993; Weinstock, 1997). However, markers for dopamine, γ-aminobutyric acid (GABA), or somatostatin are not altered (Whitehouse et al., 1981; Whitehouse et al., 1982; Francis et al., 1993). These results suggest that cholinergic neuronal loss is probably one

**2. Neuronal loss in AD**

126 Neurodegenerative Diseases

NFTs in AD development.

Apoptosis, or programmed cell death (PCD), is a term proposed by Kerr, Wyllie and Currie in 1972 (Kerr et al., 1972) to describe a common type of cell death characterized by membrane blebbing, cell shrinkage, protein fragmentation, chromatin condensation and DNA degrada‐ tion followed by rapid engulfment of corpses by surrounding cells (Kerr et al., 1972). It rapidly cleans out dysfunctional cells, limits toxic effects, saves energy and recycles molecules for future *de novo* synthesis.

Two major apoptotic pathways in mammalian cells are mediated through either death re‐ ceptors or mitochondria (Figure 1). The so-called extrinsic pathway is initiated by death re‐ ceptor CD95 (Apo-1/Fas), or other death receptors, such as tumor necrosis factor (TNF) receptor and tumor necrosis factor related apoptosis inducing ligand (TRAIL) receptor. CD95 is linked to pro-caspase-8 by an adapter factor FADD. Pro-caspase-8 has a death ef‐ fecter domain (DED) which binds to the death domain (DD) on death receptor adapter FADD, and a caspase activation and recruitment domain (CARD), which binds to other downstream caspases and further processes the downstream caspases. Ligand binding sig‐ nals the activation of pro-caspase-8 to form a tetramer of active caspase-8. Caspase-8 then mediates the cleavage of pro-caspase-3 and initiates the caspase activation cascade, which leads to protein and DNA cleavage and final termination of the cells. In this pathway, cas‐ pase-8 activation can be prevented by its natural inhibitors, such as cellular FADD-like in‐ terlukin-1-β-converting enzyme (FLICE/caspase-8)-inhibitory proteins (c-FLIPs) (Irmler et al., 1997; Thome et al., 1997) and apoptosis repressor with caspase recruitment domain (ARC) (Koseki et al., 1998) (Figure 1).

chondrial membrane, increases mitochondrial outer membrane permeability, facilitates pore formation and potentiates cytochrome c release on the mitochondrial outer mem‐

This apoptosis machinery is self-amplifying. For example, second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI (Smac/DIABLO) and apoptosisinducing factor (AIF) proteins (Hengartner, 2000; Cregan et al., 2002) are released from the mitochondria with cytochrome c to facilitate apoptosis. In addition, recent studies showed that caspase-3, -6, -7 and –8 can initiate cytochrome c release by activating cytosolic factors (Figure 1). Since cytochrome c acts as an initiator for the caspase activation cascade, this self-amplified

**Extrinsic pathway Intrinsic pathway**

Bid

DNA damage

p53

Bax

mitochondria

Bcl-xL

Procaspase-9

Apaf-1

IAPs Smac/DIABLO

AIF

Bcl-2

Caspases in Alzheimer's Disease http://dx.doi.org/10.5772/54627 129

Cytochrome c

loop facilitates cellular apoptosis (Figure 1) (Bossy-Wetzel and Green, 1999).

brane (Figure 1) (Hengartner, 2000).

CD95 FADD

Procaspase-8

Active caspase-8

c-FLIP

Active caspase-3, -6, -7

Apoptotic substrates

apoptosis inducing factor.

Truncated bid Procaspase-3, -6, -7

Apoptosome

**Figure 1.** Schematic drawing of two major pathways involved in apoptosis. Schematic digram showing the two major pathways mediating apoptosis. The extrinsic pathway is mediated by death receptors, such as FADD, and caspase-8 activation. The intrinsic pathway is induced by DNA damage and mediated by caspase-9. IAP: inhibitor of apoptosis protein, Smac/DIABLO: second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI, AIF:

The intrinsic pathway is triggered mainly by internal insults such as DNA damage. Dam‐ aging insults such as UV irradiation activate tumor suppression gene product p53, a tran‐ scriptional factor. There are several hypotheses for p53 activation mechanism. First, stressactivated protein kinases, such as DNA-dependent protein kinase (DNA-PK), phosphorylates p53. This phosphorylation prevents p53 from degradation. For example, DNA-PK can be activated by DNA damage, and then phosphorylate p53 at Ser-15 in the N terminal, which prevents the interaction between p53 and its inhibitory protein MDM-2. In addition to phosphorylation, dephosphorylation, such as the one caused by 14-3-3 at Ser-376, can also enable DNA binding of p53 and activate its function. The second model states that p53 is constitutively active and is regulated by the negative regulator MDM-2. MDM-2 protein can bind to p53 and send it out of the nucleus for degradation. Interesting‐ ly, the MDM-2 gene can be activated by p53, therefore, p53 is negatively self-regulated. Af‐ ter activation, p53 acts as a transcriptional factor controlling the expression of certain genes. These genes are involved in cell cycle control (eg. p21, GADD45, 14-3-3, CyclinD1, Cy‐ clinG), DNA repair (eg. GADD45, p21), apoptosis (eg. Bax, Bcl-2, FASL, DR5), angiogenesis (eg. TSP-1, BAI1) and cellular stress response (eg. TP53TG1, CSR, PIG3).

During apoptosis, p53 activates transcription of the pro-apoptotic Bax and, at the same time, transcriptionally represses the anti-apoptotic Bcl-2. A family of Bcl-2 proteins is impli‐ cated in apoptosis. This family contains three subgroups of proteins, some of them are proapoptotic while some are anti-apoptotic. The Group I proteins, such as Bcl-2, have a transmembrane domain and conserved Bcl-2 homology (BH) 1-4 domains. The Group II lacks the BH4 domain, such as Bax, while in the Group III, only the BH3 domain is in com‐ mon, such as Bid and Bik (Hengartner, 2000). Bax is a pro-apoptotic protein causing the de‐ polarization of mitochondrial membrane and release of cytochrome c from the mitochondria to the cytosol. The detailed mechanism of Bax leading to cytochrome c re‐ lease is unknown. Bax is located in the cytosol as monomers and upon the apoptotic stimu‐ lation, Bax oligomerizes and translocates to the mitochondria. There are several models suggested to explain how Bax oligomers cause intermembrane protein cytochrome c re‐ lease (Degli Esposti and Dive, 2003). First, Bax oligomers may form a pore structure on the mitochondria outer membrane leading to cytochrome c release. Second, interaction be‐ tween Bax and other mitochondrial proteins, such as voltage dependent anion selective channel (VDAC) and adenosine nucleotide transporter (ANT) may induce pore formation by VDAC and ANT, through which cytochrome c is released. Third, the pore may form on the membrane by low-selective ion channels and induce cytochrome c release through these channels (Degli Esposti and Dive, 2003). Cytochrome c, together with the adapter molecule Apaf-1 and pro-caspase-9, forms an apoptosome that cleaves pro-caspase-3 into its active form and initiates apoptosis. Bcl-2, the other member of Bcl-2 family in the Group I, is an anti-apoptotic protein preventing Bax-mediated cytochrome c release efficiently.

The two apoptotic pathways cross at Bid, a pro-apoptotic protein from Bcl-2 family Group III. Bid can be cleaved by active caspase-8 and the truncated Bid translocates to the mito‐ chondrial membrane, increases mitochondrial outer membrane permeability, facilitates pore formation and potentiates cytochrome c release on the mitochondrial outer mem‐ brane (Figure 1) (Hengartner, 2000).

al., 1997; Thome et al., 1997) and apoptosis repressor with caspase recruitment domain

The intrinsic pathway is triggered mainly by internal insults such as DNA damage. Dam‐ aging insults such as UV irradiation activate tumor suppression gene product p53, a tran‐ scriptional factor. There are several hypotheses for p53 activation mechanism. First, stressactivated protein kinases, such as DNA-dependent protein kinase (DNA-PK), phosphorylates p53. This phosphorylation prevents p53 from degradation. For example, DNA-PK can be activated by DNA damage, and then phosphorylate p53 at Ser-15 in the N terminal, which prevents the interaction between p53 and its inhibitory protein MDM-2. In addition to phosphorylation, dephosphorylation, such as the one caused by 14-3-3 at Ser-376, can also enable DNA binding of p53 and activate its function. The second model states that p53 is constitutively active and is regulated by the negative regulator MDM-2. MDM-2 protein can bind to p53 and send it out of the nucleus for degradation. Interesting‐ ly, the MDM-2 gene can be activated by p53, therefore, p53 is negatively self-regulated. Af‐ ter activation, p53 acts as a transcriptional factor controlling the expression of certain genes. These genes are involved in cell cycle control (eg. p21, GADD45, 14-3-3, CyclinD1, Cy‐ clinG), DNA repair (eg. GADD45, p21), apoptosis (eg. Bax, Bcl-2, FASL, DR5), angiogenesis

During apoptosis, p53 activates transcription of the pro-apoptotic Bax and, at the same time, transcriptionally represses the anti-apoptotic Bcl-2. A family of Bcl-2 proteins is impli‐ cated in apoptosis. This family contains three subgroups of proteins, some of them are proapoptotic while some are anti-apoptotic. The Group I proteins, such as Bcl-2, have a transmembrane domain and conserved Bcl-2 homology (BH) 1-4 domains. The Group II lacks the BH4 domain, such as Bax, while in the Group III, only the BH3 domain is in com‐ mon, such as Bid and Bik (Hengartner, 2000). Bax is a pro-apoptotic protein causing the de‐ polarization of mitochondrial membrane and release of cytochrome c from the mitochondria to the cytosol. The detailed mechanism of Bax leading to cytochrome c re‐ lease is unknown. Bax is located in the cytosol as monomers and upon the apoptotic stimu‐ lation, Bax oligomerizes and translocates to the mitochondria. There are several models suggested to explain how Bax oligomers cause intermembrane protein cytochrome c re‐ lease (Degli Esposti and Dive, 2003). First, Bax oligomers may form a pore structure on the mitochondria outer membrane leading to cytochrome c release. Second, interaction be‐ tween Bax and other mitochondrial proteins, such as voltage dependent anion selective channel (VDAC) and adenosine nucleotide transporter (ANT) may induce pore formation by VDAC and ANT, through which cytochrome c is released. Third, the pore may form on the membrane by low-selective ion channels and induce cytochrome c release through these channels (Degli Esposti and Dive, 2003). Cytochrome c, together with the adapter molecule Apaf-1 and pro-caspase-9, forms an apoptosome that cleaves pro-caspase-3 into its active form and initiates apoptosis. Bcl-2, the other member of Bcl-2 family in the Group I, is an anti-apoptotic protein preventing Bax-mediated cytochrome c release efficiently.

The two apoptotic pathways cross at Bid, a pro-apoptotic protein from Bcl-2 family Group III. Bid can be cleaved by active caspase-8 and the truncated Bid translocates to the mito‐

(eg. TSP-1, BAI1) and cellular stress response (eg. TP53TG1, CSR, PIG3).

(ARC) (Koseki et al., 1998) (Figure 1).

128 Neurodegenerative Diseases

This apoptosis machinery is self-amplifying. For example, second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI (Smac/DIABLO) and apoptosisinducing factor (AIF) proteins (Hengartner, 2000; Cregan et al., 2002) are released from the mitochondria with cytochrome c to facilitate apoptosis. In addition, recent studies showed that caspase-3, -6, -7 and –8 can initiate cytochrome c release by activating cytosolic factors (Figure 1). Since cytochrome c acts as an initiator for the caspase activation cascade, this self-amplified loop facilitates cellular apoptosis (Figure 1) (Bossy-Wetzel and Green, 1999).

**Figure 1.** Schematic drawing of two major pathways involved in apoptosis. Schematic digram showing the two major pathways mediating apoptosis. The extrinsic pathway is mediated by death receptors, such as FADD, and caspase-8 activation. The intrinsic pathway is induced by DNA damage and mediated by caspase-9. IAP: inhibitor of apoptosis protein, Smac/DIABLO: second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI, AIF: apoptosis inducing factor.

### **4. Apoptosis involvement in neuronal cell loss of AD**

There is evidence for apoptosis involvement in neuronal loss in AD, but the evidence so far is not sufficient to support a significant role for apoptosis in the neuronal cell loss in AD. Evidence of apoptosis in AD is as follows. First, overexpression of FAD-related mutations causes apoptosis in the transfected cell lines, cultured neurons and transgenic mice. For ex‐ ample, overexpression of FAD mutations of APPV642I, APPV642F and APPV642G in COS or F11 cells increases number of apoptotic cells as determined by DNA fragmentation and termi‐ nal dUTP nick end labeling (TUNEL) staining assay, which can be inhibited by anti-apop‐ totic protein Bcl-2 overexpression (Yamatsuji et al., 1996). These results support the role of FAD mutations in inducing apoptosis. Similarly, the data from transgenic mice confirm the above observations. Transgenic mice overexpressing FAD mutant APPV717F develop neuritic dystrophy similar to some pathological features in AD patients (Games et al., 1995; Hol‐ comb et al., 1998). The degenerating neurons in these mice also show typical apoptotic fea‐ tures, such as chromatin segmentation and condensation, and positive TUNEL staining (Nijhawan et al., 2000). However, in these studies, the FAD mutant proteins are normally overexpressed far beyond the physiological levels. However, FAD neurons do not necessa‐ rily undergo apoptosis. In the mutant PS1 expressing neurons, increased apoptosis is not reported (Bursztajn et al., 1998). Also, there is no neuronal loss found in mutant PS1 trans‐ genic mice (Takeuchi et al., 2000).

or anti-apoptotic proteins could be explained by either the neurons undergoing apoptosis or

Caspases in Alzheimer's Disease http://dx.doi.org/10.5772/54627 131

Therefore, in summary, to date, there is no strong evidence showing significant involvement of apoptosis in AD neuronal loss. Since caspases, a family of cysteinyl proteases, play an important role in cell death, especially in apoptosis as discussed in the previous section (Thompson, 1995; Strasser et al., 2000; Yuan and Yankner, 2000), it is of interest to identify which caspase is responsible for human neuronal cell loss, how it is regulated, and whether it can be inhibited. In addition, caspase activation may be easier to use for identification of apoptosis since it is an upstream event of DNA fragmentation. Therefore, the apoptotic cells

neurons responding against apoptotic insults to prevent initiation of apoptosis.

**5. Caspase involvement in apoptosis and APP metabolism in AD**

Caspases (cysteinyl aspartate-specific proteases) belong to a cysteinyl protease family that cleaves specifically after an aspartic acid. To date, 14 caspases (11 of them are found in human) have been identified in mammals. The connection between apoptosis and caspases was first reported by Yuan et al. (1993) that caspase-1 is a homolog to CED-3, a gene regulating cell death in *Caenorhaditis elegans* (Yuan et al., 1993). The important role of caspases in apoptosis is also supported by the strong correlation between caspase activity and apoptosis in various cell types. Furthermore, caspase inhibition prevents apoptosis both *in vitro* and *in vivo* (Yuan et al., 1993; Kuida et al., 1995; Schwartz and Milligan, 1996; Alnemri, 1997; Thornberry, 1997; Li and

Inactive caspases contain a pro-domain, a large subunit and a small subunit. According to their pro-domain structure and function, caspases are divided into "inflammatory", "initiator" and "effecter" caspases (Figure 2A) (Nicholson, 1999; Hengartner, 2000). Cas‐ pases are normally present as inactive precursors in cells. After receiving apoptotic sig‐ nals, caspase pro-enzymes undergo proteolytic processing to remove the N terminal prodomain and cleave between large and small subunits to produce the active form of a tetrameric enzyme formed by 2 copies of the large subunit and 2 copies of small subu‐ nit. To date, three caspase activation pathways are known in mammalian cells: recruit‐ ment-activation, trans-activation and autoactivation (Nicholson, 1999). Recruitmentactivation is triggered by death receptors of the tumor necrosis factor receptor family. The so-called initiator caspases, namely caspase-2, -8, -9 and –10 (Figure 3A), are thought to be directly activated through the signals from death receptor (Boldin et al., 1996; Mu‐ zio et al., 1996). In addition, more recent data show that the initiator caspase-8, -9 and – 10 can be activated by homodimerization of their monomeric zymogens, a so called "proximity-induced activation" (Boatright et al., 2003). Trans-activation occurs through downstream or executioner caspases, caspase-3, -6 and –7 that can be activated by direct proteolysis by their upstream initiator caspases. By such trans-activation cascade, apopto‐ sis is well controlled and regulated (Darmon et al., 1995; Martin et al., 1996; Andrade et

have not been cleared yet.

Yuan, 1999; Yuan and Yankner, 2000).

**5.1. Caspases**

Second, some studies indicate apoptosis in AD patient brains using *in situ* detection of DNA fragmentation by TUNEL staining (Su et al., 1994; Dragunow et al., 1995; Lassmann et al., 1995; Smale et al., 1995; Anderson et al., 1996; Su et al., 1996; Troncoso et al., 1996; Gervais et al., 1999; Anderson et al., 2000). However, in the AD brain tissues, some TUNEL-positive neurons show typical apoptotic morphology whereas some do not, suggesting degenerating neurons in AD may undergo both apoptosis and passive cell death, necrosis (Su et al., 1994; Troncoso et al., 1996; Lucassen et al., 1997; Yuan and Yankner, 2000). It is now commonly accepted that TUNEL staining sometimes is not specific to apoptosis (Stadelmann et al., 1998). The staining of TUNEL may indicate increased vulnerability of cells to a secondary insult, not necessarily undergoing apoptosis. On the other hand, the number of apoptotic neurons is difficult to measure precisely due to the chronic nature, relatively long progress of the disease and rapid clearance mechanism of dead cells.

Third, there are reports of changes of expression of apoptosis related proteins, such as p53, Bcl-2 and Bcl-xL, in AD brains (Paradis et al., 1996; Kitamura et al., 1997; MacGibbon et al., 1997; Su et al., 1997; Cotman, 1998; Torp et al., 1998; Tortosa et al., 1998). For example, proapoptotic protein p53 is increased in AD brains (Kitamura et al., 1997), while anti-apoptotic proteins Bcl-2 and Bcl-xL are decreased in AD brains (Kitamura et al., 1998; Tortosa et al., 1998). Also, another pro-apoptotic protein Bax is increased in AD brains (Paradis et al., 1996; Kitamura et al., 1997; MacGibbon et al., 1997; Su et al., 1997; Cotman, 1998; Torp et al., 1998; Tortosa et al., 1998). However, the regulation of these pro- or anti-apoptotic proteins could be primary or secondary response to insults. On the other hand, the upregulation of either proor anti-apoptotic proteins could be explained by either the neurons undergoing apoptosis or neurons responding against apoptotic insults to prevent initiation of apoptosis.

Therefore, in summary, to date, there is no strong evidence showing significant involvement of apoptosis in AD neuronal loss. Since caspases, a family of cysteinyl proteases, play an important role in cell death, especially in apoptosis as discussed in the previous section (Thompson, 1995; Strasser et al., 2000; Yuan and Yankner, 2000), it is of interest to identify which caspase is responsible for human neuronal cell loss, how it is regulated, and whether it can be inhibited. In addition, caspase activation may be easier to use for identification of apoptosis since it is an upstream event of DNA fragmentation. Therefore, the apoptotic cells have not been cleared yet.

### **5. Caspase involvement in apoptosis and APP metabolism in AD**

#### **5.1. Caspases**

**4. Apoptosis involvement in neuronal cell loss of AD**

genic mice (Takeuchi et al., 2000).

130 Neurodegenerative Diseases

the disease and rapid clearance mechanism of dead cells.

There is evidence for apoptosis involvement in neuronal loss in AD, but the evidence so far is not sufficient to support a significant role for apoptosis in the neuronal cell loss in AD. Evidence of apoptosis in AD is as follows. First, overexpression of FAD-related mutations causes apoptosis in the transfected cell lines, cultured neurons and transgenic mice. For ex‐ ample, overexpression of FAD mutations of APPV642I, APPV642F and APPV642G in COS or F11 cells increases number of apoptotic cells as determined by DNA fragmentation and termi‐ nal dUTP nick end labeling (TUNEL) staining assay, which can be inhibited by anti-apop‐ totic protein Bcl-2 overexpression (Yamatsuji et al., 1996). These results support the role of FAD mutations in inducing apoptosis. Similarly, the data from transgenic mice confirm the above observations. Transgenic mice overexpressing FAD mutant APPV717F develop neuritic dystrophy similar to some pathological features in AD patients (Games et al., 1995; Hol‐ comb et al., 1998). The degenerating neurons in these mice also show typical apoptotic fea‐ tures, such as chromatin segmentation and condensation, and positive TUNEL staining (Nijhawan et al., 2000). However, in these studies, the FAD mutant proteins are normally overexpressed far beyond the physiological levels. However, FAD neurons do not necessa‐ rily undergo apoptosis. In the mutant PS1 expressing neurons, increased apoptosis is not reported (Bursztajn et al., 1998). Also, there is no neuronal loss found in mutant PS1 trans‐

Second, some studies indicate apoptosis in AD patient brains using *in situ* detection of DNA fragmentation by TUNEL staining (Su et al., 1994; Dragunow et al., 1995; Lassmann et al., 1995; Smale et al., 1995; Anderson et al., 1996; Su et al., 1996; Troncoso et al., 1996; Gervais et al., 1999; Anderson et al., 2000). However, in the AD brain tissues, some TUNEL-positive neurons show typical apoptotic morphology whereas some do not, suggesting degenerating neurons in AD may undergo both apoptosis and passive cell death, necrosis (Su et al., 1994; Troncoso et al., 1996; Lucassen et al., 1997; Yuan and Yankner, 2000). It is now commonly accepted that TUNEL staining sometimes is not specific to apoptosis (Stadelmann et al., 1998). The staining of TUNEL may indicate increased vulnerability of cells to a secondary insult, not necessarily undergoing apoptosis. On the other hand, the number of apoptotic neurons is difficult to measure precisely due to the chronic nature, relatively long progress of

Third, there are reports of changes of expression of apoptosis related proteins, such as p53, Bcl-2 and Bcl-xL, in AD brains (Paradis et al., 1996; Kitamura et al., 1997; MacGibbon et al., 1997; Su et al., 1997; Cotman, 1998; Torp et al., 1998; Tortosa et al., 1998). For example, proapoptotic protein p53 is increased in AD brains (Kitamura et al., 1997), while anti-apoptotic proteins Bcl-2 and Bcl-xL are decreased in AD brains (Kitamura et al., 1998; Tortosa et al., 1998). Also, another pro-apoptotic protein Bax is increased in AD brains (Paradis et al., 1996; Kitamura et al., 1997; MacGibbon et al., 1997; Su et al., 1997; Cotman, 1998; Torp et al., 1998; Tortosa et al., 1998). However, the regulation of these pro- or anti-apoptotic proteins could be primary or secondary response to insults. On the other hand, the upregulation of either proCaspases (cysteinyl aspartate-specific proteases) belong to a cysteinyl protease family that cleaves specifically after an aspartic acid. To date, 14 caspases (11 of them are found in human) have been identified in mammals. The connection between apoptosis and caspases was first reported by Yuan et al. (1993) that caspase-1 is a homolog to CED-3, a gene regulating cell death in *Caenorhaditis elegans* (Yuan et al., 1993). The important role of caspases in apoptosis is also supported by the strong correlation between caspase activity and apoptosis in various cell types. Furthermore, caspase inhibition prevents apoptosis both *in vitro* and *in vivo* (Yuan et al., 1993; Kuida et al., 1995; Schwartz and Milligan, 1996; Alnemri, 1997; Thornberry, 1997; Li and Yuan, 1999; Yuan and Yankner, 2000).

Inactive caspases contain a pro-domain, a large subunit and a small subunit. According to their pro-domain structure and function, caspases are divided into "inflammatory", "initiator" and "effecter" caspases (Figure 2A) (Nicholson, 1999; Hengartner, 2000). Cas‐ pases are normally present as inactive precursors in cells. After receiving apoptotic sig‐ nals, caspase pro-enzymes undergo proteolytic processing to remove the N terminal prodomain and cleave between large and small subunits to produce the active form of a tetrameric enzyme formed by 2 copies of the large subunit and 2 copies of small subu‐ nit. To date, three caspase activation pathways are known in mammalian cells: recruit‐ ment-activation, trans-activation and autoactivation (Nicholson, 1999). Recruitmentactivation is triggered by death receptors of the tumor necrosis factor receptor family. The so-called initiator caspases, namely caspase-2, -8, -9 and –10 (Figure 3A), are thought to be directly activated through the signals from death receptor (Boldin et al., 1996; Mu‐ zio et al., 1996). In addition, more recent data show that the initiator caspase-8, -9 and – 10 can be activated by homodimerization of their monomeric zymogens, a so called "proximity-induced activation" (Boatright et al., 2003). Trans-activation occurs through downstream or executioner caspases, caspase-3, -6 and –7 that can be activated by direct proteolysis by their upstream initiator caspases. By such trans-activation cascade, apopto‐ sis is well controlled and regulated (Darmon et al., 1995; Martin et al., 1996; Andrade et al., 1998). Caspase activation can also occur by activation of the dormant enzyme mole‐ cule by the already active one (autoactivation). The supporting evidence of this mecha‐ nism comes from the observation that arginine-glycine-aspartate (RGD) motif-containing peptides can induce caspase-3 autoactivation by triggering conformational changes of pro-caspase-3 (Buckley et al., 1999). A similar mechanism has been suggested for pro-cas‐ pase-8 and –2 activation (Hengartner, 2000).

After activation, caspases recognize four amino acid substrate sites as their targets and cleave the C terminal to an obligatory aspartic acid (XXXD). According to their specific substrates, caspases are divided into 3 groups: caspase-1, -4, -5 and –13 (substrates: WEHD and YVAD); caspase-2, -3 and -7 (substrate: DEXD) and caspase-6, -8, –9 and -10 (substrates: (I, V, L)EXD) (reviewed by (Thornberry, 1997)). Once activated, caspases cleave downstream substrates in a highly specific and rapid manner. More than 250 substrates are found including downstream caspases or apoptosis-related proteins (e.g. Bid, Bcl-2), structural proteins (e.g. lamins, actin, fodrin, gelsolin), DNA repair proteins (e.g. PARP, p21) and some other proteins involved in neurodegenerative diseases (eg. APP, tau, PSs, Huntingtin) (reviewed by (Bounhar et al., 2002).

Caspases in Alzheimer's Disease http://dx.doi.org/10.5772/54627 133

Most of the morphological changes in apoptosis described by Kerr et al. (1972) are caused by caspases that are activated specifically in PCD. Caspase cleavage of nuclear lamins results in nuclear shrinking and blebbing (McCarthy et al., 1997; Sakahira et al., 1998). Loss of cell structure may be due to caspase cleavage of cytoskeletal proteins, such as fodrin (Vanags et al., 1996; Janicke et al., 1998) and gelsolin (Kothakota et al., 1997). DNA fragmentation is also caused by caspase-activated DNase (CAD), which is activated by caspases through removing the inhibitory subunit (ICAD) from the inactive CAD enzyme complex (Hengartner, 2000).

In general, the evidence supporting the involvement of caspases in neuronal loss in AD is still not conclusive. The involvement of caspases in AD is first suggested by the finding that caspases, acting as proteases, are involved in PSs and APP metabolism and Aβ peptide generation in AD. Caspase-3 directly cleaves PSs during apoptosis (Kim et al., 1997). Caspase-3, -6, -7, -8 and –9 can cleave APP and generate Aβ or Aβ-containing fragments, therefore, giving a possibility of Aβ accumulation in AD (Barnes et al., 1998; Gervais et al., 1999; LeBlanc et al., 1999; Pellegrini et al., 1999; Weidemann et al., 1999). In chick motor neurons, caspase-3 cleaves APP and generates Aβ products (Barnes et al., 1998). In human 293 cells, caspase-9 cleaves APP at the C terminal and produces a "C31" peptide, which is cytotoxic to cells (Lu et al., 2000). Caspase-3 cleaves APP in NT-2 cells and is involved in Aβ formation (Gervais et al., 1999). APP is a caspase substrate in staurosporine-treated NT-2 cells and Fas-treated human Jurkat cells (Pellegrini et al., 1999). In addition, caspase inhibitors can prevent the formation

Second, more direct proof of caspase involvement in AD comes from immuno-detection of active caspases in AD brain tissues, although the evidence is not sufficient enough to be conclusive. Some studies have shown the activation of caspase-8 in AD brains (Rohn et al., 2001; Rohn et al., 2002), while others found decreased level of caspase-8 in AD brains (Engidawork et al., 2001a). In yet another study, both inactive and active caspase-8 immunor‐ eactivity are not changed in AD compared to control brains (Engidawork et al., 2001b). The cleavage fragment of caspase-9 is also detected in AD but not in the control brains (Lu et al., 2000; Goyal, 2001; Rohn et al., 2002), but the alteration of two caspase-9 activation co-factors, Apaf-1 and cytochrome c, is not detected (Engidawork et al., 2001b). Furthermore, there is recent evidence showing that pro-caspase-9 is activated through dimerization, but not cleavage (Boatright et al., 2003). Among all caspases, caspase-3 is the most intensively studied

**5.2. Involvement of caspases in neuronal loss in AD**

of Aβ (Barnes et al., 1998; Gervais et al., 1999; LeBlanc et al., 1999).

**Figure 2.** Caspases are cysteinyl proteases responsible for protein degradation during apoptosis. A. Schematic dia‐ gram of caspase category according to function. There are three groups of caspases according to their basic functions: inflammation, initiation and execution. B. Schematic drawing of pro-caspase-6 activation into active caspase-6. Cas‐ pase-6 activation by cleavage at the sites between pro-domain and large subunit (p20) as well as large subunit and small subunit (p10). Arrow head: cleavage. C. Schematic drawing of caspase-6-like cleavage sites on APP. There are three caspase-6-like cleavage sites on APP. Arrow: cleavage. TM: transmembrane domain.

After activation, caspases recognize four amino acid substrate sites as their targets and cleave the C terminal to an obligatory aspartic acid (XXXD). According to their specific substrates, caspases are divided into 3 groups: caspase-1, -4, -5 and –13 (substrates: WEHD and YVAD); caspase-2, -3 and -7 (substrate: DEXD) and caspase-6, -8, –9 and -10 (substrates: (I, V, L)EXD) (reviewed by (Thornberry, 1997)). Once activated, caspases cleave downstream substrates in a highly specific and rapid manner. More than 250 substrates are found including downstream caspases or apoptosis-related proteins (e.g. Bid, Bcl-2), structural proteins (e.g. lamins, actin, fodrin, gelsolin), DNA repair proteins (e.g. PARP, p21) and some other proteins involved in neurodegenerative diseases (eg. APP, tau, PSs, Huntingtin) (reviewed by (Bounhar et al., 2002).

Most of the morphological changes in apoptosis described by Kerr et al. (1972) are caused by caspases that are activated specifically in PCD. Caspase cleavage of nuclear lamins results in nuclear shrinking and blebbing (McCarthy et al., 1997; Sakahira et al., 1998). Loss of cell structure may be due to caspase cleavage of cytoskeletal proteins, such as fodrin (Vanags et al., 1996; Janicke et al., 1998) and gelsolin (Kothakota et al., 1997). DNA fragmentation is also caused by caspase-activated DNase (CAD), which is activated by caspases through removing the inhibitory subunit (ICAD) from the inactive CAD enzyme complex (Hengartner, 2000).

#### **5.2. Involvement of caspases in neuronal loss in AD**

al., 1998). Caspase activation can also occur by activation of the dormant enzyme mole‐ cule by the already active one (autoactivation). The supporting evidence of this mecha‐ nism comes from the observation that arginine-glycine-aspartate (RGD) motif-containing peptides can induce caspase-3 autoactivation by triggering conformational changes of pro-caspase-3 (Buckley et al., 1999). A similar mechanism has been suggested for pro-cas‐

> Group III Execution

Caspase-1 Caspase-4

Caspase-5

Caspase-13

Active Caspase-6

Caspase-3

Caspase-6

Caspase-7

Caspase-14

pase-8 and –2 activation (Hengartner, 2000).

Group II Initiation

A

132 Neurodegenerative Diseases

B

C

Prodomain Large domain Small domain

three caspase-6-like cleavage sites on APP. Arrow: cleavage. TM: transmembrane domain.

Inactive Proenzyme Caspase-6

Group I Inflammation

Caspase-2

Caspase-8

Caspase-9

Caspase-10

Csp-6 like Csp-6 like Csp-6 like

**Figure 2.** Caspases are cysteinyl proteases responsible for protein degradation during apoptosis. A. Schematic dia‐ gram of caspase category according to function. There are three groups of caspases according to their basic functions: inflammation, initiation and execution. B. Schematic drawing of pro-caspase-6 activation into active caspase-6. Cas‐ pase-6 activation by cleavage at the sites between pro-domain and large subunit (p20) as well as large subunit and small subunit (p10). Arrow head: cleavage. C. Schematic drawing of caspase-6-like cleavage sites on APP. There are

<sup>A</sup> TM APP

In general, the evidence supporting the involvement of caspases in neuronal loss in AD is still not conclusive. The involvement of caspases in AD is first suggested by the finding that caspases, acting as proteases, are involved in PSs and APP metabolism and Aβ peptide generation in AD. Caspase-3 directly cleaves PSs during apoptosis (Kim et al., 1997). Caspase-3, -6, -7, -8 and –9 can cleave APP and generate Aβ or Aβ-containing fragments, therefore, giving a possibility of Aβ accumulation in AD (Barnes et al., 1998; Gervais et al., 1999; LeBlanc et al., 1999; Pellegrini et al., 1999; Weidemann et al., 1999). In chick motor neurons, caspase-3 cleaves APP and generates Aβ products (Barnes et al., 1998). In human 293 cells, caspase-9 cleaves APP at the C terminal and produces a "C31" peptide, which is cytotoxic to cells (Lu et al., 2000). Caspase-3 cleaves APP in NT-2 cells and is involved in Aβ formation (Gervais et al., 1999). APP is a caspase substrate in staurosporine-treated NT-2 cells and Fas-treated human Jurkat cells (Pellegrini et al., 1999). In addition, caspase inhibitors can prevent the formation of Aβ (Barnes et al., 1998; Gervais et al., 1999; LeBlanc et al., 1999).

Second, more direct proof of caspase involvement in AD comes from immuno-detection of active caspases in AD brain tissues, although the evidence is not sufficient enough to be conclusive. Some studies have shown the activation of caspase-8 in AD brains (Rohn et al., 2001; Rohn et al., 2002), while others found decreased level of caspase-8 in AD brains (Engidawork et al., 2001a). In yet another study, both inactive and active caspase-8 immunor‐ eactivity are not changed in AD compared to control brains (Engidawork et al., 2001b). The cleavage fragment of caspase-9 is also detected in AD but not in the control brains (Lu et al., 2000; Goyal, 2001; Rohn et al., 2002), but the alteration of two caspase-9 activation co-factors, Apaf-1 and cytochrome c, is not detected (Engidawork et al., 2001b). Furthermore, there is recent evidence showing that pro-caspase-9 is activated through dimerization, but not cleavage (Boatright et al., 2003). Among all caspases, caspase-3 is the most intensively studied since the caspase-3 knockouts develop abnormal brains with significantly excessive numbers of neurons (Cregan et al., 1999; Keramaris et al., 2000; Simpson et al., 2001; Fernando et al., 2002). Some studies report increased caspase-3-like immunoreactivity in AD brains (Masliah et al., 1998; Gervais et al., 1999). However, the protein and mRNA levels of caspase-3 do not appear altered in AD compared to control brains (Desjardins and Ledoux, 1998; LeBlanc et al., 1999). Active caspase-3 is detected in granulovacuolar degeneration, an aging associated pathology that is not necessarily specific to AD (Stadelmann et al., 1999; Roth, 2001). Given that apoptosis results in a rapid clearance of dysfunctional cells, only small amount of caspase activation can be detected at a certain time window. Therefore, the extensive detection of active caspase-3 in some studies may be due to lack of immunospecificity of the caspase-3 antibody. Furthermore, although caspase-3 is critical for apoptosis in many cell types, it does not have significant role in the human neuronal loss in AD (Desjardins and Ledoux, 1998; LeBlanc et al., 1999; Selznick et al., 1999; Stadelmann et al., 1999).

although thus increase is not dramatic. Given that AD is a long progressive disease, at a certain postmortem time window, there is only limited amount of cell death where caspase activation can be detected. Meanwhile, in contrast to caspase-6, changes in the levels of pro- and active caspase-3 levels are not detectable in AD brains, suggesting that caspase-6, but not caspase-3, may be involved in human neuronal cell death (LeBlanc et al., 1999). Caspase-6 can also alter APP metabolism to generate Aβ-containing fragments. There are several caspase-6-like sites on APP695 (Figure 2C). However, incubating recombinant caspase-6 and APP695-containing neuronal extract does not show APP cleavage. One of these cleavages at 591VKMD594 generates a 6.5 kDa fragment denoted "Capp6.5" containing the Aβ sequence (Figure 2C) (LeBlanc et al., 1999). Although caspase-6 does not directly induce 4 kDa Aβ, pulse-chase experiments showed that this Capp6.5 fragment is able to generate 4 kDa Aβ in human neurons (Figure 2C) (LeBlanc et al., 1999). Caspase-6 can cleave APP close to β-secretase site at D653 to further generate Aβ2-40 or Aβ2-42 (Gervais et al., 1999). In the Swedish mutation of APP, which changes the VKMD653 sequence at the β-secretase site to VNLD653, the caspase-6 cleavage of VNLD-AMC is 6 fold higher than the VKMD-AMC fluorogenic peptide *in vitro* (Gervais et al., 1999). Also, caspase-6 can cleave APP after the γ-secretase site at VEVD720/A (Gervais et al., 1999). Therefore, caspase-6 may process APP similar to β- and γ-secretase activity. Direct microinjecting caspase-6 into human neurons induces dramatic cell death (Zhang et al., 2000). Taken together, the above evidence suggests that caspase-6 plays important role in human neuronal cell death,

Caspases in Alzheimer's Disease http://dx.doi.org/10.5772/54627 135

Aβ formation, and may be responsible for neuronal loss in AD (LeBlanc et al., 1999).

The natural inhibitors to caspases include Cowpox virus product cytokine response modifier A (crmA), FLIPs, protease inhibitor 9 (PI-9), p35, ARC and inhibitor of apoptosis proteins (IAPs). CrmA is a member of serpin family, a group of serine protease inhibitors. CrmA inhibits caspases by acting as a pseudosubstrate that binds to active caspases, such as caspase-1, -4, -5, -8 and –9 (Ray et al., 1992; Komiyana et al., 1994). Besides crmA, FLIPs inhibits caspase-8 by acting as the dominant negative form to suppress caspase-8 mRNA expression (Thornberry, 1997). The mammalian homolog of crmA is PI-9. PI-9 mRNA expression can be rapidly induced by estrogen in human liver (Kanamori et al., 2000). PI-9 is a granzyme B inhibitor (GBI). Granzyme B is a 30 to 32 kDa serine protease, which cleaves peptides at aspartyl residue in the killer T thymocytes and natural killer cells. Granzyme B is involved in the perforation of target cells and then initiation of proteolysis that leads to apoptosis. Although PI-9 can inhibit granzyme B and granzyme B-mediated apoptosis, *In vitro* experiments do not show that PI-9

p35 is a baculoviral protein that can block the defensive apoptotic response of insect cells to viral infection (Clem et al., 1991; Ekert et al., 1999). p35 inhibits CED-3 in *C. elegans* and mammalian caspase-1, -3, -6, -7, -8 and -10 (Ekert et al., 1999). P35 is cleaved at its P1 resi‐ due by caspases, and the cleaved fragment forms an inhibitory complex to block caspase activation (Zhou et al., 1998). ARC interacts with caspase-2, -8 and CED-3, but not cas‐

**5.4. Inhibition of active caspases**

*5.4.1. Natural inhibitors to caspases*

inhibits active caspases (Bird et al., 1998; Bird, 1999).

Interestingly, LeBlanc et al. have shown that caspase-6 is activated in serum deprivation induced cell death of human neurons in primary cultured (LeBlanc et al., 1999). Therefore, could caspase-6 be the responsible caspase in human neuronal cell death in AD?

#### **5.3. Caspase-6 is activated during human neuronal cell death**

#### *5.3.1. Introduction to caspase-6*

Caspase-6 (Mch-2), located on human chromosome 4q25, is an "effecter" caspase with a short pro-domain. It recognizes VEID substrates and cleaves after the amino acid D. Its common substrates include APP (LeBlanc et al., 1999), cytoskeleton proteins, such as keratin 18 (Caulin et al., 1997), focal adhesion kinase (Gervais et al., 1998), tau (LeBlanc et al., 1999), β-catenin (Van de Craen et al., 1999), vimentin (Byun et al., 2001) and desmin (Chen et al., 2003), nuclear proteins, such as lamin A (Ruchaud et al., 2002), lamin B (Slee et al., 2001), PARP (Fernandes-Alnemri et al., 1995), DNA topoisomerase I (Samejima et al., 1999) and emerin (Columbaro et al., 2001), several transcriptional factors, such as SATB1, and AP-2α (Galande et al., 2001; Nyormoi et al., 2001). It is highly expressed in the heart, lung, liver, kidney and testis in murine tissues. There is immunodetectable caspase-6 in human brain and neurons (LeBlanc et al., 1999; Zheng et al., 1999; Harrison et al., 2001). Caspase-6 can be activated by caspase-1, -3, -7, -8 and –11 (Fernandes-Alnemri et al., 1995; Chinnaiyan et al., 1996; Orth et al., 1996). Procaspase-6 is a ~ 34 kDa protein, that can be cleaved into ~20 kDa (p20) and ~10 kDa (p10) fragments. The p20 and p10 fragments form a tetramer, which is the active form of the enzyme (Figure 3C) (Fernandes-Alnemri et al., 1995). Caspase-6 knockout mice do not show abnormal phenotype during development, which does not rule out the role of caspase-6 in cell death in later stage of life or under stress conditions (Zheng et al., 1999).

#### *5.3.2. Caspase-6 involvement in human neuronal cell death and APP processing*

Pro-caspase-6 decreases during apoptosis induced by serum deprivation in human neurons in primary cultures (LeBlanc et al., 1999). Moreover, caspase-6 active 10 kDa fragments is detected only in AD brains, and not in the normal aging control brains (LeBlanc et al., 1999), although thus increase is not dramatic. Given that AD is a long progressive disease, at a certain postmortem time window, there is only limited amount of cell death where caspase activation can be detected. Meanwhile, in contrast to caspase-6, changes in the levels of pro- and active caspase-3 levels are not detectable in AD brains, suggesting that caspase-6, but not caspase-3, may be involved in human neuronal cell death (LeBlanc et al., 1999). Caspase-6 can also alter APP metabolism to generate Aβ-containing fragments. There are several caspase-6-like sites on APP695 (Figure 2C). However, incubating recombinant caspase-6 and APP695-containing neuronal extract does not show APP cleavage. One of these cleavages at 591VKMD594 generates a 6.5 kDa fragment denoted "Capp6.5" containing the Aβ sequence (Figure 2C) (LeBlanc et al., 1999). Although caspase-6 does not directly induce 4 kDa Aβ, pulse-chase experiments showed that this Capp6.5 fragment is able to generate 4 kDa Aβ in human neurons (Figure 2C) (LeBlanc et al., 1999). Caspase-6 can cleave APP close to β-secretase site at D653 to further generate Aβ2-40 or Aβ2-42 (Gervais et al., 1999). In the Swedish mutation of APP, which changes the VKMD653 sequence at the β-secretase site to VNLD653, the caspase-6 cleavage of VNLD-AMC is 6 fold higher than the VKMD-AMC fluorogenic peptide *in vitro* (Gervais et al., 1999). Also, caspase-6 can cleave APP after the γ-secretase site at VEVD720/A (Gervais et al., 1999). Therefore, caspase-6 may process APP similar to β- and γ-secretase activity. Direct microinjecting caspase-6 into human neurons induces dramatic cell death (Zhang et al., 2000). Taken together, the above evidence suggests that caspase-6 plays important role in human neuronal cell death, Aβ formation, and may be responsible for neuronal loss in AD (LeBlanc et al., 1999).

#### **5.4. Inhibition of active caspases**

since the caspase-3 knockouts develop abnormal brains with significantly excessive numbers of neurons (Cregan et al., 1999; Keramaris et al., 2000; Simpson et al., 2001; Fernando et al., 2002). Some studies report increased caspase-3-like immunoreactivity in AD brains (Masliah et al., 1998; Gervais et al., 1999). However, the protein and mRNA levels of caspase-3 do not appear altered in AD compared to control brains (Desjardins and Ledoux, 1998; LeBlanc et al., 1999). Active caspase-3 is detected in granulovacuolar degeneration, an aging associated pathology that is not necessarily specific to AD (Stadelmann et al., 1999; Roth, 2001). Given that apoptosis results in a rapid clearance of dysfunctional cells, only small amount of caspase activation can be detected at a certain time window. Therefore, the extensive detection of active caspase-3 in some studies may be due to lack of immunospecificity of the caspase-3 antibody. Furthermore, although caspase-3 is critical for apoptosis in many cell types, it does not have significant role in the human neuronal loss in AD (Desjardins and Ledoux, 1998; LeBlanc et

Interestingly, LeBlanc et al. have shown that caspase-6 is activated in serum deprivation induced cell death of human neurons in primary cultured (LeBlanc et al., 1999). Therefore,

Caspase-6 (Mch-2), located on human chromosome 4q25, is an "effecter" caspase with a short pro-domain. It recognizes VEID substrates and cleaves after the amino acid D. Its common substrates include APP (LeBlanc et al., 1999), cytoskeleton proteins, such as keratin 18 (Caulin et al., 1997), focal adhesion kinase (Gervais et al., 1998), tau (LeBlanc et al., 1999), β-catenin (Van de Craen et al., 1999), vimentin (Byun et al., 2001) and desmin (Chen et al., 2003), nuclear proteins, such as lamin A (Ruchaud et al., 2002), lamin B (Slee et al., 2001), PARP (Fernandes-Alnemri et al., 1995), DNA topoisomerase I (Samejima et al., 1999) and emerin (Columbaro et al., 2001), several transcriptional factors, such as SATB1, and AP-2α (Galande et al., 2001; Nyormoi et al., 2001). It is highly expressed in the heart, lung, liver, kidney and testis in murine tissues. There is immunodetectable caspase-6 in human brain and neurons (LeBlanc et al., 1999; Zheng et al., 1999; Harrison et al., 2001). Caspase-6 can be activated by caspase-1, -3, -7, -8 and –11 (Fernandes-Alnemri et al., 1995; Chinnaiyan et al., 1996; Orth et al., 1996). Procaspase-6 is a ~ 34 kDa protein, that can be cleaved into ~20 kDa (p20) and ~10 kDa (p10) fragments. The p20 and p10 fragments form a tetramer, which is the active form of the enzyme (Figure 3C) (Fernandes-Alnemri et al., 1995). Caspase-6 knockout mice do not show abnormal phenotype during development, which does not rule out the role of caspase-6 in cell death in

could caspase-6 be the responsible caspase in human neuronal cell death in AD?

al., 1999; Selznick et al., 1999; Stadelmann et al., 1999).

*5.3.1. Introduction to caspase-6*

134 Neurodegenerative Diseases

**5.3. Caspase-6 is activated during human neuronal cell death**

later stage of life or under stress conditions (Zheng et al., 1999).

*5.3.2. Caspase-6 involvement in human neuronal cell death and APP processing*

Pro-caspase-6 decreases during apoptosis induced by serum deprivation in human neurons in primary cultures (LeBlanc et al., 1999). Moreover, caspase-6 active 10 kDa fragments is detected only in AD brains, and not in the normal aging control brains (LeBlanc et al., 1999),

#### *5.4.1. Natural inhibitors to caspases*

The natural inhibitors to caspases include Cowpox virus product cytokine response modifier A (crmA), FLIPs, protease inhibitor 9 (PI-9), p35, ARC and inhibitor of apoptosis proteins (IAPs). CrmA is a member of serpin family, a group of serine protease inhibitors. CrmA inhibits caspases by acting as a pseudosubstrate that binds to active caspases, such as caspase-1, -4, -5, -8 and –9 (Ray et al., 1992; Komiyana et al., 1994). Besides crmA, FLIPs inhibits caspase-8 by acting as the dominant negative form to suppress caspase-8 mRNA expression (Thornberry, 1997). The mammalian homolog of crmA is PI-9. PI-9 mRNA expression can be rapidly induced by estrogen in human liver (Kanamori et al., 2000). PI-9 is a granzyme B inhibitor (GBI). Granzyme B is a 30 to 32 kDa serine protease, which cleaves peptides at aspartyl residue in the killer T thymocytes and natural killer cells. Granzyme B is involved in the perforation of target cells and then initiation of proteolysis that leads to apoptosis. Although PI-9 can inhibit granzyme B and granzyme B-mediated apoptosis, *In vitro* experiments do not show that PI-9 inhibits active caspases (Bird et al., 1998; Bird, 1999).

p35 is a baculoviral protein that can block the defensive apoptotic response of insect cells to viral infection (Clem et al., 1991; Ekert et al., 1999). p35 inhibits CED-3 in *C. elegans* and mammalian caspase-1, -3, -6, -7, -8 and -10 (Ekert et al., 1999). P35 is cleaved at its P1 resi‐ due by caspases, and the cleaved fragment forms an inhibitory complex to block caspase activation (Zhou et al., 1998). ARC interacts with caspase-2, -8 and CED-3, but not cas‐ pase-1, -3, or –9. ARC inhibits caspase-8 enzyme activity in 293 cells, and further attenu‐ ates apoptosis induced by FADD through stimulation of death receptors coupled with pro-caspase-8 (Koseki et al., 1998).

neuronal loss. If the synthetic caspase-6 inhibitor is applied and it would inhibit active caspase-6 activity in all types of cells, which has a large potential to develop cancer in cells that proliferate. If a natural inhibitor to active caspase-6 can be activated somehow specifically in

Caspases in Alzheimer's Disease http://dx.doi.org/10.5772/54627 137

Since neuronal loss is a striking feature of AD, decreasing or suppressing neuronal cell loss may benefit early AD patients in retaining cognitive capacities and prevent or delay disease progression. Caspases seem to play a significant role in human neuronal cell loss in AD, thus it is intriguing to determine if caspase activity can be inhibited after activa‐

State Key Laboratory of Biomembrane and Membrane Biotechnology, College of Life Sciences,

[1] Alnemri, E. S. (1997). Mammalian cell death proteases: a family of highly conserved

[3] Anderson, A. J, Stoltzner, S, Lai, F, Su, J, & Nixon, R. A. (2000). Morphological and biochemical assessment of DNA damage and apoptosis in Down syndrome and Alzheimer disease, and effect of postmortem issue archival on TUNEL. Neurobiol

[4] Anderson, A. J, Su, J. H, & Cotman, C. W. (1996). DNA damage and apoptosis in Alzheimer's disease: colocalization with c-Jun immunoreactivity, relationship to brain

[5] Andrade, F, Roy, S, Nicholson, D, Thornberry, N, Rosen, A, & Casciola-rosen, L. (1998). Granzyme B directly and efficiently cleaves several downstream caspase substrates:

[6] Arnold, S. E, Hyman, B. T, Flory, J, Damasio, A. R, & Van Hoesen, G. W. (1991). The topographical and neuroanatomical distribution of neurofibrillary tangles and neuritic plaques in the cerebral cortex of patients with Alzheimer's disease. Cereb Cortex , 1,

aspartate specific cysteine proteases. J Cell Biochem , 64, 33-42.

area, and effect of postmortem delay. J Neurosci , 16, 1710-1719.

implications for CTL-induced apoptosis. Immunity , 8, 451-460.

[2] Alorainy, I. (2000). Senile scleral plaques: CT. Neuroradiology , 42, 145-148.

human neurons, the risk of oncogenesis in the brain would be greatly reduced.

tion by neuroprotective agents.

Peking University, Beijing, China

Aging , 21, 511-524.

103-116.

Address all correspondence to: yanzhang@pku.edu.cn

**Author details**

Yan Zhang\*

**References**

IAPs were found in a search for viral genes with a similar function to p35. A group of cellular IAP homologs are found in yeast, *C. elegans*, *Drosophila* and vertebrates (Ekert et al., 1999). To date, the IAP family contains about a dozen proteins from viruses, *Drosophila*, mice and humans. All known IAPs share a baculovirus inhibitory repeat (BIR) domain, which contains a number of conserved residues including a zinc-binding region. Most of the IAPs also contain a RING zinc-binding finger motif at the C terminal. Both BIR motifs and RING finger are important for IAP function (Ekert et al., 1999). The neuronal apoptosis inhibitory protein (NAIP) in human was found by searching for mutations in spinal muscular atrophy (SMA), which is characterized by degeneration of the anterior horn cells in the spinal cord. The NAIP gene is deleted in the SMA patients. During development, excessive neurons are ultimately needed to send out axons, the neurons do not target properly undergo apoptosis. It is suggested that NAIP is involved by preventing apoptosis in the "successful" cells (Miller, 1997).

The most studied IAP is the X-linked IAP (XIAP). XIAP binds to active caspase-9 small subunit through its BIR3 domain and cleaves the small subunit to inactivate caspase-9 (Srinivasula et al., 2001). On the other hand, XIAP binds to active caspase-3 or –7 through BIR2 domain and mask the active site of these caspases (Huang et al., 2001; Riedl et al., 2001; Stennicke et al., 2002). In addition, evidence shows that in insects, some IAPs interact with apoptosis-related proteins, such as Grim, Reaper and Hid (Hay et al., 1995; Miller, 1997; Vucic et al., 1997; Vucic et al., 1998). IAPs can also inhibit caspase-3 directly and cytochrome c-induced caspase-9 (Deveraux et al., 1998). However, up to now, although IAPs may inhibit caspase-6 activation by blocking upstream caspase-9 or –3 activation (Deveraux et al., 1998), there is no evidence showing that mammalian IAPs directly inhibit active caspase-6 enzyme activity.

#### *5.4.2. Synthetic inhibitors to active caspases*

Synthetic caspase inhibitors function as pseudosubstrates for active caspases and therefore, they are competitive inhibitors of active caspases (Ekert et al., 1999). The N terminal blocking groups of the pseudosubstrate peptides are usually acetyl- (Ac-) or benzocarbonyl (Z-). The biochemical property of these synthetic inhibitors depends on the chemical group linked to pseudosubstrate peptides. Aldehyde (CHO-) group inhibitors are reversible since there is no covalent bond formed between these inhibitors and caspases. These CHO inhibitors have poor cell membrane permeability, which limits largely the use of these inhibitors on live cells and animals (Schotte et al., 1999; Bounhar et al., 2002). The inhibitors linked to methylketone (chloromethylketone, cmk or fluoromethylketone, fmk) irreversibly inhibit caspases due to the formation of thyomethylketone bonds with the cysteine in the active site of caspases (Bounhar et al., 2002). These inhibitors are membrane permeable, but have less specificity of inhibitory action to caspases (Schotte et al., 1999; Bounhar et al., 2002).

Although synthetic caspase inhibitors are widely used in research, they may not be the ideal candidates for disease treatment since these inhibitors cannot inhibit caspase activation in specific certain cell types. For example, in AD, caspase-6 is the key caspase responsible for neuronal loss. If the synthetic caspase-6 inhibitor is applied and it would inhibit active caspase-6 activity in all types of cells, which has a large potential to develop cancer in cells that proliferate. If a natural inhibitor to active caspase-6 can be activated somehow specifically in human neurons, the risk of oncogenesis in the brain would be greatly reduced.

Since neuronal loss is a striking feature of AD, decreasing or suppressing neuronal cell loss may benefit early AD patients in retaining cognitive capacities and prevent or delay disease progression. Caspases seem to play a significant role in human neuronal cell loss in AD, thus it is intriguing to determine if caspase activity can be inhibited after activa‐ tion by neuroprotective agents.

### **Author details**

Yan Zhang\*

pase-1, -3, or –9. ARC inhibits caspase-8 enzyme activity in 293 cells, and further attenu‐ ates apoptosis induced by FADD through stimulation of death receptors coupled with

IAPs were found in a search for viral genes with a similar function to p35. A group of cellular IAP homologs are found in yeast, *C. elegans*, *Drosophila* and vertebrates (Ekert et al., 1999). To date, the IAP family contains about a dozen proteins from viruses, *Drosophila*, mice and humans. All known IAPs share a baculovirus inhibitory repeat (BIR) domain, which contains a number of conserved residues including a zinc-binding region. Most of the IAPs also contain a RING zinc-binding finger motif at the C terminal. Both BIR motifs and RING finger are important for IAP function (Ekert et al., 1999). The neuronal apoptosis inhibitory protein (NAIP) in human was found by searching for mutations in spinal muscular atrophy (SMA), which is characterized by degeneration of the anterior horn cells in the spinal cord. The NAIP gene is deleted in the SMA patients. During development, excessive neurons are ultimately needed to send out axons, the neurons do not target properly undergo apoptosis. It is suggested

that NAIP is involved by preventing apoptosis in the "successful" cells (Miller, 1997).

showing that mammalian IAPs directly inhibit active caspase-6 enzyme activity.

The most studied IAP is the X-linked IAP (XIAP). XIAP binds to active caspase-9 small subunit through its BIR3 domain and cleaves the small subunit to inactivate caspase-9 (Srinivasula et al., 2001). On the other hand, XIAP binds to active caspase-3 or –7 through BIR2 domain and mask the active site of these caspases (Huang et al., 2001; Riedl et al., 2001; Stennicke et al., 2002). In addition, evidence shows that in insects, some IAPs interact with apoptosis-related proteins, such as Grim, Reaper and Hid (Hay et al., 1995; Miller, 1997; Vucic et al., 1997; Vucic et al., 1998). IAPs can also inhibit caspase-3 directly and cytochrome c-induced caspase-9 (Deveraux et al., 1998). However, up to now, although IAPs may inhibit caspase-6 activation by blocking upstream caspase-9 or –3 activation (Deveraux et al., 1998), there is no evidence

Synthetic caspase inhibitors function as pseudosubstrates for active caspases and therefore, they are competitive inhibitors of active caspases (Ekert et al., 1999). The N terminal blocking groups of the pseudosubstrate peptides are usually acetyl- (Ac-) or benzocarbonyl (Z-). The biochemical property of these synthetic inhibitors depends on the chemical group linked to pseudosubstrate peptides. Aldehyde (CHO-) group inhibitors are reversible since there is no covalent bond formed between these inhibitors and caspases. These CHO inhibitors have poor cell membrane permeability, which limits largely the use of these inhibitors on live cells and animals (Schotte et al., 1999; Bounhar et al., 2002). The inhibitors linked to methylketone (chloromethylketone, cmk or fluoromethylketone, fmk) irreversibly inhibit caspases due to the formation of thyomethylketone bonds with the cysteine in the active site of caspases (Bounhar et al., 2002). These inhibitors are membrane permeable, but have less specificity of inhibitory

Although synthetic caspase inhibitors are widely used in research, they may not be the ideal candidates for disease treatment since these inhibitors cannot inhibit caspase activation in specific certain cell types. For example, in AD, caspase-6 is the key caspase responsible for

pro-caspase-8 (Koseki et al., 1998).

136 Neurodegenerative Diseases

*5.4.2. Synthetic inhibitors to active caspases*

action to caspases (Schotte et al., 1999; Bounhar et al., 2002).

Address all correspondence to: yanzhang@pku.edu.cn

State Key Laboratory of Biomembrane and Membrane Biotechnology, College of Life Sciences, Peking University, Beijing, China

#### **References**


[7] Ashe, K. H. (2000). Synaptic structure and function in transgenic APP mice. Ann N Y Acad Sci , 924, 39-41.

[21] Callahan, L. M, Vaules, W. A, & Coleman, P. D. (2002). Progressive reduction of synaptophysin message in single neurons in Alzheimer disease. J Neuropathol Exp

Caspases in Alzheimer's Disease http://dx.doi.org/10.5772/54627 139

[22] Caulin, C, Salvesen, G. S, & Oshima, R. G. (1997). Caspase cleavage of keratin 18 and reorganization of intermediate filaments during epithelial cell apoptosis. J Cell Biol ,

[23] Chan, S. L, Furukawa, K, & Mattson, M. P. (2002). Presenilins and APP in neuritic and synaptic plasticity: implications for the pathogenesis of Alzheimer's disease. Neuro‐

[24] Chen, F, Chang, R, Trivedi, M, Capetanaki, Y, & Cryns, V. L. (2003). Caspase proteolysis of desmin produces a dominant-negative inhibitor of intermediate filaments and

[25] Chinnaiyan, A. M, Orth, K, Rourke, O, Duan, K, Poirier, H, & Dixit, G. G. VM ((1996). Molecular ordering of the cell death pathway. Bcl-2 and Bcl-xL function upstream of

[26] Clem, R. J, Fechheimer, M, & Miller, L. K. (1991). Prevention of apoptosis by a baculo‐

[27] Colle, M. A, Duyckaerts, C, Laquerriere, A, Pradier, L, Czech, C, Checler, F, & Hauw, J. J. (2000). Laminar specific loss of isocortical presenilin 1 immunoreactivity in Alzheimer's disease. Correlations with the amyloid load and the density of tau-positive

[28] Columbaro, M, Mattioli, E, Lattanzi, G, Rutigliano, C, Ognibene, A, Maraldi, N. M, & Squarzoni, S. (2001). Staurosporine treatment and serum starvation promote the cleavage of emerin in cultured mouse myoblasts: involvement of a caspase-dependent

[29] Cotman, C. W. (1998). Apoptosis decision cascades and neuronal degeneration in

[30] Cotman, C. W, & Su, J. H. (1996). Mechanism of neuronal death in Alzheimer's disease.

[31] Cregan, S. P, & Fortin, A. MacLaurin JG, Callaghan SM, Cecconi F, Yu SW, Dawson TM, Dawson VL, Park DS, Kroemer G, Slack RS ((2002). Apoptosis-inducing factor is involved in the regulation of caspase-independent neuronal cell death. J Cell Biol , 158,

[32] Cregan, S. P. MacLaurin JG, Craig CG, Robertson GS, Nicholson DW, Park DS, Slack RS ((1999). Bax-dependent caspase-3 activation is a key determinant in apoptosis in

[33] Darmon, A. J, Nicholson, D. W, & Bleackley, R. C. (1995). Activation of the apoptotic protease CPP32 by cytotoxic T-cell-derived granzyme B. Nature , 377, 446-448.

the CED-3-like apoptotic proteases. J Biol Chem , 271, 4573-4576.

virus gene during infection of insect cells. Science , 254, 1388-1390.

neurofibrillary tangles. Neuropathol Appl Neurobiol , 26, 117-123.

Neurol , 61, 384-395.

molecular Med , 2, 167-196.

promotes apoptosis. J Biol Chem , 278, 6848-6853.

mechanism. FEBS Lett , 509, 423-429.

neurons. J Neurosci 19:7860-7869., 53.

Brain Pathol , 6, 493-506.

507-517.

Alzheimer's disease. Neurobiol Aging 19:S, 29-32.

138, 1379-1394.


[21] Callahan, L. M, Vaules, W. A, & Coleman, P. D. (2002). Progressive reduction of synaptophysin message in single neurons in Alzheimer disease. J Neuropathol Exp Neurol , 61, 384-395.

[7] Ashe, K. H. (2000). Synaptic structure and function in transgenic APP mice. Ann N Y

[8] Baloyannis, S. J, Manolidis, S. L, & Manolidis, L. S. (2000). Synaptic alterations in the vestibulocerebellar system in Alzheimer's disease--a Golgi and electron microscope

[9] Barnes, N. Y, Li, L, Yoshikawa, K, Schwartz, L. M, Oppenhein, R. W, & Milligan, C. E. (1998). Increased production of amyloid precursor protein provides a substrate for

[10] Bird, C. H, Sutton, V. R, Sun, J. R, Hirst, C. E, Novak, A, Kumar, S, Trapani, J. A, & Bird, P. I. (1998). Selective regulation of apoptosis-the cytotoxic lymphocyte serpin protease inhibitor 9 protects against granzyme B-mediated apoptosis without perturbing the Fas

[11] Bird, P. I. (1999). Regulation of pro-apoptotic leukocyte granule serine proteinases by

[12] Boatright, K. M, Renatus, M, Scott, F. L, Sperandio, S, Shin, H, Pedersen, I. M, Ricci, J. E, Edris, W. A, Sutherlin, D. P, Green, D. R, & Salvesen, G. S. (2003). A unified model

[13] Boldin, M. P, Goncharov, T. V, Goltsev, Y. V, & Wallach, D. (1996). Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1 and TNF receptor-

[14] Bondareff, W, Mountjoy, C. Q, & Roth, M. (1982). Loss of neurons of origin of the adrenergic projection to cerebral cortex (nucleus locus ceruleus) in senile dementia.

[15] Bossy-wetzel, E, & Green, D. R. (1999). Caspases induce cytochrome c release from mitochondria by activating cytosolic factors. J Biol Chem , 274, 17484-17490.

[16] Bounhar, Y, & Tounekti, O. LeBlanc A ((2002). Monitoring Caspases in Neuronal Cell

[17] Braak, H, & Braak, E. (1994). Pathology of Alzheimer's disease. In: Neurodegenerative

[18] Buckley, S, Driscoll, B, Barsky, L, Weinberg, K, Anderson, K, & Warburton, D. (1999). ERK activation protects against DNA damage and apoptosis in byperoxic rat AEC2.

[19] Bursztajn, S, De Souza, R, & Mcphie, D. L. (1998). Overexpression in neurons of human presenilin-1 familial Alzheimer disease mutant does not enhance apoptosis. J Neurosci ,

[20] Byun, Y, Chen, F, Chang, R, Trivedi, M, Green, K. J, & Cryns, V. L. (2001). Caspase cleavage of vimentin disrupts intermediate filaments and promotes apoptosis. Cell

caspase-3 in dying motoneurons. J Neurosci , 18, 5869-5880.

cell death pathway. Mol Cell Biol , 18, 6387-6398.

intracellular serpins. Immunol Cell Biol , 77, 47-57.

for apical caspase activation. Mol Cell , 11, 529-541.

Death, 2nd Edition. Totowa: Humana Press Inc.

Disease., Philadelphia: Saunders., 585-613.

Am J Physiol 277:LL166., 159.

Death Differ , 8, 443-450.

18, 9790-9799.

induced cell death. Cell , 85, 803-815.

Neurology , 32, 164-168.

Acad Sci , 924, 39-41.

138 Neurodegenerative Diseases

study. Acta Otolaryngol , 120, 247-250.


[34] Davies, P, & Maloney, A. J. (1976). Selective loss of central cholinergic neurons in Alzheimer's disease. Lancet 2:1403.

[48] Games, D, Adams, D, Alesandrini, R, Barbour, R, & Berthelette, P. (1995). Alzheimertype neuropathology in transgenic mice overexpression beta-amyloid precursor

Caspases in Alzheimer's Disease http://dx.doi.org/10.5772/54627 141

[49] Garver, T. D, Harris, K. A, Lehman, R. A, Lee, V. M, Trojanowski, J. Q, & Billingsley, M. L. (1994). Tau phosphorylation in human, primate, and rat brain: evidence that a pool of tau is highly phosphorylated in vivo and is rapidly dephosphorylated in vitro.

[50] Gervais, F. G, Thornberry, N. A, Ruffolo, S. C, Nicholson, D. W, & Roy, S. (1998). Caspases cleave focal adhesion kinase during apoptosis to generate a FRNK-like

[51] Gervais, FG, Xu, D, Robertson, GS, Vaillancourt, JP, Zhu, Y, & Le, J. H. , Zheng H, van Der Ploeg LHT, Ruffolo SC, Thornberry NS, Xanthoudakis S, Zamboni RJ, Roy S, Nicholson DW (1999) Involvement of caspases in proteolytic cleavage of Alzheimer's amyloid-b precursor protein and amyloidogenic Ab peptide formation. Cell 97:395-406.

[52] Geula, C. (1998). Abnormalities of neural circuitry in Alzheimer's disease: hippocam‐ pus and cortical cholinergic innervation. Neurology 51:Sdiscussion S65-17., 18-29. [53] Glenner, G. G, & Wong, C. W. (1984). Alzheimer's disease: initial report of the purifi‐ cation and characterization of a novel cerebrovascular amyloid protein. biochem

[54] Gold, M. (2002). Tau therapeutics for Alzheimer's disease: the promise and the

[55] Gomez-isla, T, Growdon, W. B, Mcnamara, M, Newell, K, Gomez-tortosa, E, Hedleywhyte, E. T, & Hyman, B. T. (1999). Clinicopathologic correlates in temporal cortex in

[56] Gomez-isla, T, Hollister, R, West, H, Mui, S, Growdon, J. H, Petersen, R. C, Parisi, J. E, & Hyman, B. T. (1997). Neuronal loss correlates with but exceeds neurofibrillary tangles

[57] Gomez-isla, T, Price, J. L, & Mckeel, D. W. Jr., Morris JC, Growdon JH, Hyman BT ((1996). Profound loss of layer II entorhinal cortex neurons occurs in very mild

[59] Hamos, J. E, Degennaro, L. J, & Drachman, D. A. (1989). Synaptic loss in Alzheimer's

[60] Hardy, J. (2003). The Relationship between Amyloid and Tau. J Mol Neurosci , 20,

[61] Harrison, D. C, Davis, R. P, Bond, B. C, Campbell, C. A, James, M. F, Parsons, A. A, & Philpott, K. L. (2001). Caspase mRNA expression in a rat model of focal cerebral

[58] Goyal, L. (2001). Cell death inhibition: Keeping caspases in check. Cell 104:805.

protein. Nature 373:523-527., 717F

J Neurochem , 63, 2279-2287.

Biophys Res Comm , 120, 885-890.

challenges. J Mol Neurosci , 19, 331-334.

dementia with Lewy bodies. Neurology , 53, 2003-2009.

in Alzheimer's disease. Ann Neurol , 41, 17-24.

Alzheimer's disease. J Neurosci , 16, 4491-4500.

ischemia. Brain Res Mol Brain Res , 89, 133-146.

203-206.

disease and other dementias. Neurology , 39, 355-361.

polypeptide. J Biol Chem , 273, 17102-17108.


[48] Games, D, Adams, D, Alesandrini, R, Barbour, R, & Berthelette, P. (1995). Alzheimertype neuropathology in transgenic mice overexpression beta-amyloid precursor protein. Nature 373:523-527., 717F

[34] Davies, P, & Maloney, A. J. (1976). Selective loss of central cholinergic neurons in

[35] Defigueiredo, R. J, Cummings, B. J, Mundkur, P. Y, & Cotman, C. W. (1995). Color image analysis in neuroanatomical research: application to senile plaque subtype quantifica‐

[36] Degli Esposti MDive C ((2003). Mitochondrial membrane permeabilisation by Bax/Bak.

[37] Desjardins, P, & Ledoux, S. (1998). Expression of ced-3 and ced-9 homologs in Alz‐

[38] Deveraux, Q. L, Roy, N, Stennick, H. R, Van Arsdale, T, Zhou, Q, Srinivasula, S. M, Alnemri, E. S, Salvesen, G. S, & Reed, J. C. (1998). IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. EMBO J , 17,

[39] Dodd, P. R. (2002). Excited to death: different ways to lose your neurones. Biogeron‐

[40] Dragunow, M, Faull, R. L, Lawlor, P, Beilharz, E. J, Singleton, K, Walker, E. B, & Mee, E. (1995). In situ evidence for DNA fragmentation in Huntington's disease striatum and

[41] Ekert, P. G, Silke, J, & Vaux, D. L. (1999). Caspase inhibitors. Cell Death Differe , 6,

[42] Engidawork, E, Gulesserian, T, Yoo, B. C, Cairns, N, & Lubec, G. (2001a). Alteration of caspases and apoptosis-related proteins in brains of patients with Alzheimer's disease.

[43] Engidawork, E, Gulesserian, T, Yoo, B. C, Cairns, N, & Lubec, G. (2001b). Alternation of caspase- and apoptosis-related proteins in brains of patients with Alzheimer's

[44] Fernandes-alnemri, T, Litwack, G, & Alnemri, E. S. (1995). Mch2, a new member of the apoptotic Ced-3/Ice cysteine protease gene family. Cancer Res , 55, 2737-2742.

[45] Fernando, P, Kelly, J. F, Balazsi, K, Slack, R. S, & Megeney, L. A. (2002). Caspase 3 activity is required for skeletal muscle differentiation. Proc Natl Acad Sci U S A , 99,

[46] Francis, P. T, Webster, M. T, Chessell, I. P, Holmes, C, Stratmann, G. C, Procter, A. W, Cross, A. J, Green, A. R, & Bowen, D. M. (1993). Neurotransmitters and second

[47] Galande, S, Dickinson, L. A, Mian, I. S, Sikorska, M, & Kohwi-shigematsu, T. (2001). SATB1 cleavage by caspase 6 disrupts PDZ domain-mediated dimerization, causing detachment from chromatin early in T-cell apoptosis. Mol Cell Biol , 21, 5591-5604.

messengers in aging and Alzheimer's disease. Ann N Y Acad Sci , 695, 19-26.

tion in Alzheimer's disease. Neurobiol Aging , 16, 211-223.

heimer's disease cerebral cortex. Neurosci Lett , 244, 69-72.

Alzheimer's disease temporal lobes. Neuroreport , 6, 1053-1057.

Biochem Biophys Res Commun , 304, 455-461.

Biochem Biophys Res Commun , 281, 84-93.

disease. Biochem Biophys Res Comm , 281, 84-93.

Alzheimer's disease. Lancet 2:1403.

2215-2223.

140 Neurodegenerative Diseases

1081-1086.

11025-11030.

tology , 3, 51-56.


[62] Hartig, W, Klein, C, Brauer, K, Schuppel, K. F, Arendt, T, Bruckner, G, & Bigl, V. (2000). Abnormally phosphorylated protein tau in the cortex of aged individuals of various mammalian orders. Acta Neuropathol (Berl) , 100, 305-312.

[77] Kim, T. W, Pettingell, W. H, Jung, Y. K, Kovacs, D. M, & Tanzi, R. E. (1997). Alternative cleavage of Alzheimer-associated presenilins during apoptosis by a caspase-3 family

Caspases in Alzheimer's Disease http://dx.doi.org/10.5772/54627 143

[78] Kitamura, Y, Shimohama, S, Kamoshima, W, Matsuoka, Y, Nomura, Y, & Taniguchi, T. (1997). Changes of in the brains of patients with Alzheimer's disease. Biochem

[79] Kitamura, Y, Shimohama, S, Kamoshima, W, Ota, T, Matsuoka, Y, Nomura, Y, Smith, M. A, Perry, G, Whitehouse, P. J, & Taniguchi, T. (1998). Alteration of proteins regu‐ lating apoptosis, Bcl-2, Bcl-x, Bax, Bak, Bad, ICH-1 and CPP32, in Alzheimer's disease.

[80] Komiyana, T, Ray, C. A, Pickup, D. J, Howard, A. D, Thornberry, N. A, Peterson, E. P, & Salvesen, G. (1994). Inhibitorion of interleukin-1-Beta converting enzyme by the cowpox virus serpin CrmA-an example of cross-class inhibition. J Biol Chem , 269,

[81] Koseki, T, Inohara, H, Chen, S, Ez, N, & Are, G. an inhibitor of apoptosis expressed in skeletal muscle and heart that interacts selectively with caspases. Proc Natl Acad Sci

[82] Kothakota, S, Azuma, T, Reinhard, C, Klippel, A, Tang, J, Chu, K, Mcgarry, T. J, Kirschner, M. W, Koths, K, Kwiatkowski, D. J, & Williams, L. T. (1997). Caspase-3 generated fragment of gelsolin: effector of morphological change in apoptosis. Science ,

[83] Kuida, K, Lippke, J. A, Ku, G, Harding, M. W, Livingston, D. J, Su, M. S, & Flavell, R. A. (1995). Altered cytokine export and apoptosis in mice deficient in interleukin-1 beta

[84] Larson, J, Lynch, G, Games, D, & Seubert, P. (1999). Alterations in synaptic transmission and long-term potentiation in hippocampal slices from young and aged PDAPP mice.

[85] Lassmann, H, Bancher, C, Breitschopf, H, Wegiel, J, & Bobinski, M. (1995). Cell death in Alzheimer's disease evaluated by DNA fragmentation in situ. Acta Neuropathol ,

[86] LeBlanc ALiu H, Goodyer C, Bergeron C, Hammond J ((1999). Caspase-6 role in apoptosis of human neurons, amyloidogenesis, and Alzheimer's disease. J Biol Chem ,

[87] Lee VMYBalin BJ, Otvos LJ, Trojanowski JQ ((1991). A68: a major subunit of paired helical filaments and derivatized forms of normal tau. Science , 251, 675-678.

[88] Li, H, & Yuan, J. (1999). Deciphering the pathways of life and death. Curr Opin Cell

protease. Science , 277, 373-376.

Brain Res , 780, 260-269.

19331-19337.

278, 294-298.

89, 35-41.

274, 23426-23436.

Biol , 11, 261-266.

USA , 95, 5156-5160.

Brain Res , 840, 23-35.

Biophys Res Commun 232:418-421., 53.

converting enzyme. Science , 267, 2000-2003.


[77] Kim, T. W, Pettingell, W. H, Jung, Y. K, Kovacs, D. M, & Tanzi, R. E. (1997). Alternative cleavage of Alzheimer-associated presenilins during apoptosis by a caspase-3 family protease. Science , 277, 373-376.

[62] Hartig, W, Klein, C, Brauer, K, Schuppel, K. F, Arendt, T, Bruckner, G, & Bigl, V. (2000). Abnormally phosphorylated protein tau in the cortex of aged individuals of various

[63] Hay, B. A, Wassaman, D. A, & Rubin, G. M. (1995). Drosophila homologs of baculoirus inhibitor of apoptosis proteins function to block cell death. Cell , 83, 1253-1262.

[65] Hohmann, G. F, Wenk, G. L, Lowenstein, P, Brown, M. E, & Coyle, J. T. (1987). Agerelated recurrence of basal forebrain lesion-induced cholinergic deficits. Neurosci Lett ,

[66] Holcomb, L, Gordon, M. N, Mcgowan, E, Yu, X, & Benkovic, S. (1998). Accelerate Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor

[67] Huang, Y, Park, Y. C, Rich, R. L, Segal, D, Myszka, D. G, & Wu, H. (2001). Structural basis of caspase inhibition by XIAP: differential roles of the linker versus the BIR

[68] Irmler, M, Thome, M, Hahne, M, Schneider, P, Hofmann, K, Steiner, V, Bodmer, J. L, Schroter, M, Burns, K, Mattmann, C, Rimoldi, D, French, L. E, & Tschopp, J. (1997).

[69] Janicke, R. U, Ng, P, Sprengart, M. L, & Porter, A. G. (1998). Caspase-3 is required for alpha-fodrin cleavage but dispensable for cleavage of other death substrates in

[70] Jellinger, K. (2002a). Prevalence of Alzheimer's disease in very elderly people: a prospective neuropathological study. Neurology author reply 671-672., 58, 671-672.

[72] Jellinger, K. A. (2002c). Alzheimer disease and cerebrovascular pathology: an update.

[73] Jellinger, K. A, & Attems, J. (2003). Incidence of cerebrovascular lesions in Alzheimer's

[74] Kanamori, H, Krieg, S, & Mao, C. Di Pippo VA, Wang S, Zajchowski DA, Shapiro DJ ((2000). Proteinase inhibitor 9, an inhibitor of granzyme B-mediated apoptosis, is a primary estrogen-inducible gene in human liver cells. J Biol Chem , 275, 5867-5873. [75] Keramaris, E, & Stefanis, L. MacLaurin J, Harada N, Takaku K, Ishikawa T, Taketo MM, Robertson GS, Nicholson DW, Slack RS, Park DS ((2000). Involvement of caspase 3 in apoptotic death of cortical neurons evoked by DNA damage. Mol Cell Neurosci , 15,

[76] Kerr, J. E, Wyllie, A. H, & Currie, A. R. (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer , 26, 239-257.

[71] Jellinger, K. A. dementia: an update. J Neural Transm Suppl:, 1-23.

disease: a postmortem study. Acta Neuropathol (Berl) , 105, 14-17.

Inhibition of death receptor signals by cellular FLIP. Nature , 388, 190-195.

[64] Hengartner, M. O. (2000). The biochemistry of apoptosis. Nature , 407, 770-776.

mammalian orders. Acta Neuropathol (Berl) , 100, 305-312.

protein and presenilin 1 transgenes. Nat Med , 4, 97-100.

82, 253-259.

142 Neurodegenerative Diseases

domain. Cell , 104, 781-790.

apoptosis. J Biol Chem , 273, 15540-15545.

J Neural Transm , 109, 813-836.

368-379.


[89] Li, W. P, Chan, W. Y, Lai, H. W, & Yew, D. T. (1997). Terminal dUTP nick end labeling (TUNEL) positive cells in the different regions of the brain in normal aging and Alzheimer patients. J Mol Neurosci , 8, 75-82.

[101] Mckhann, G, Drachman, D, Folstein, M, Katzman, R, Price, D. L, & Stadlan, E. M. (1984). Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on

Caspases in Alzheimer's Disease http://dx.doi.org/10.5772/54627 145

[102] Mesulam, M. M. (1986). Alzheimer plaques and cortical cholinergic innervation.

[103] Michaelis, M. L, Dobrowsky, R. T, & Li, G. (2002). Tau neurofibrillary pathology and

[104] Miller, L. K. (1997). Baculovirus interaction with host apoptotic pathways. J Cell

[105] Mori, H, Takio, k, Ogawara, M, & Selkie, D. (1992). Mass spectrometry of purified amyloid b protein in Alzheimer's disease. J Biol Chem , 267, 17082-17086.

[106] Morris, J. C. McKeel DWJ, Storandt M, Rubin EH, Price JL, Grant EA, Ball MJ, Berg L ((1991). Very mild Alzheimer's disease: informant-based clinical, psychometric, and

[107] Muzio, M, Chinnaiyan, A. M, Kischkel, F. C, Rourke, O, Shevchenko, K, Ni, A, Scaffidi, J, Bretz, C, Zhang, J. D, Gentz, M, Mann, R, Krammer, M, Peter, P. H, Dixit, M. E, & Flice, V. M. a novel FADD-homologous ICE/CED-3-like protease, is recruited to the

[108] Nicholson, D. W. (1999). Caspase structure, proteilytic substrates, and function during

[109] Nijhawan, D, Honarpour, N, & Wang, X. (2000). Apoptosis in neural development and

[110] Nyormoi, O, Wang, Z, Doan, D, Ruiz, M, Mcconkey, D, & Bar-eli, M. (2001). Transcrip‐ tion factor AP-2alpha is preferentially cleaved by caspase 6 and degraded by protea‐ some during tumor necrosis factor alpha-induced apoptosis in breast cancer cells. Mol

[111] Orth, K, Rourke, O, Salvesen, K, & Dixit, G. S. VM ((1996). Molecular ordering of apoptotic mammalian CED-3/ICE-like proteases. J Biol Chem , 271, 20977-20980.

[112] Paradis, E, Douillard, H, Koutroumanis, M, & Goodyer, C. LeBlanc A ((1996). Amyloid beta peptide of Alzheimer's disease downregulates Bcl-2 and upregulates bax expres‐

[113] Pearson, R. C, & Powell, T. P. (1987). Anterograde vs. retrograde degeneration of the nucleus basalis medialis in Alzheimer's disease. J Neural Transm Suppl , 24, 139-146.

[114] Pellegrini, L, Passer, B. J, Tabaton, M, Ganjei, J. K, Adamio, D, & Alternative, L. nonsecretase processing of Alzheimer's b-amyloid precursor protein during apoptosis by

pathologic distinction from normal aging. Neurology , 41, 469-478.

CD95 (Fas/APO-1) death-inducing signaling complex. Cell , 85, 817-827.

apoptotic cell death. Cell Death Differe , 6, 1028-1042.

sion in human neurons. J Neurosci , 16, 7533-7539.

caspase-6 and-8. J Biol Chem , 274, 21011-21016.

disease. Annu Rev Neurosci , 23, 73-87.

Cell Biol , 21, 4856-4867.

Alzheimer's disease. Neurology , 34, 939-944.

microtubule stability. J Mol Neurosci , 19, 289-293.

Neuroscience , 17, 275-276.

Physiol , 173, 178-182.


[101] Mckhann, G, Drachman, D, Folstein, M, Katzman, R, Price, D. L, & Stadlan, E. M. (1984). Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's disease. Neurology , 34, 939-944.

[89] Li, W. P, Chan, W. Y, Lai, H. W, & Yew, D. T. (1997). Terminal dUTP nick end labeling (TUNEL) positive cells in the different regions of the brain in normal aging and

[90] Lippa, C. F, Hamos, J. E, Pulaski-salo, D, Degennaro, L. J, & Drachman, D. A. (1992). Alzheimer's disease and aging: effects on perforant pathway perikarya and synapses.

[91] Lu, D. C, Rabizadeh, S, Chandra, S, Shayya, R. F, Ellerby, L. M, Ye, X, Salvesen, G. S, Koo, E. H, & Bredesen, D. E. (2000). A second cytotoxic proteolytic peptide derived

[92] Lucassen, P. J, Chung, W. C, Kamphorst, W, & Swaab, D. F. (1997). DNA damage distribution in the human brain as shown by in situ end labeling; area-specific differ‐ ences in aging and Alzheimer disease in the absence of apoptotic morphology. J

[93] MacGibbon GALawlor PA, Sirimanne ES, Walton MR, Connor B, Young D, Williams C, Gluckman P, Faull RL, Hughes P, Dragunow M ((1997). Bax expression in mamma‐ lian neurons undergoing apoptosis, and in Alzheimer's disease hippocampus. Brain

[94] Martin, S. J, Finucane, D. M, Amarante-mendes, G. P, Brien, O, & Green, G. A. DR ((1996). Phosphatidylserine externalization during CD95-induced apoptosis of cells and cytoplasts requires ICE/CED-3 protease activity. J Biol Chem , 271, 28753-28756.

[95] Masliah, E. (2001). Recent advances in the understanding of the role of synaptic proteins in Alzheimer's Disease and other neurodegenerative disorders. J Alzheimers Dis , 3,

[96] Masliah, E, Mallory, M, Alford, M, Deteresa, R, Hansen, L. A, & Mckeel, D. W. Jr., Morris JC ((2001). Altered expression of synaptic proteins occurs early during progression of

[97] Masliah, E, Mallory, M, Alford, M, Tanaka, S, & Hansen, L. A. (1998). Caspase depend‐ ent DNA fragmentation might be associated with excitotoxicity in Alzheimer disease.

[98] Masliah, E, Terry, R. D, Deteresa, R. M, & Hansen, L. A. (1989). Immunohistochemical quantification of the synapse-related protein synaptophysin in Alzheimer disease.

[99] Masters, C. L, Simms, G, Weinman, N. A, Multhaup, G, Mcdonald, B. L, & Beyreuther, K. (1985). Amyloid plaque core protein in Alzheimer disease and Down syndrome.

[100] Mccarthy, N. J, Whyte, M. K, Gilbert, C. S, & Evan, G. I. (1997). Inhibition of Ced-3/ICErelated proteases does not prevent cell death induced by oncogenes, DNA damage, or

Alzheimer patients. J Mol Neurosci , 8, 75-82.

from amyloid b-protein precursor. Nature Med , 6, 397-404.

Neurobiol Aging , 13, 405-411.

144 Neurodegenerative Diseases

Neurophathol Exp Neuorl , 56, 887-900.

Alzheimer's disease. Neurology , 56, 127-129.

J Neuropathol Exp Neurol , 57, 1041-1052.

Proc Natl Acad Sci USA , 82, 4245-4249.

the Bcl-2 homologue Bak. J Cell Biol , 136, 215-227.

Neurosci Lett , 103, 234-239.

Res , 750, 223-234.

121-129.


[115] Price, D. L, & Sisodia, S. S. (1998). Mutant genes in familial Alzheimer's disease and transgenic models. Annu Reve Neurosci , 21, 479-505.

[129] Schotte, P, Declercq, W, Van Huffel, S, Vandenabeele, P, & Beyaert, R. (1999). Nonspecific effects of methyl ketone peptide inhibitors of caspases. FEBS Lett , 442, 117-121.

Caspases in Alzheimer's Disease http://dx.doi.org/10.5772/54627 147

[130] Schwartz, L. M, & Milligan, C. E. (1996). Cold thoughts of death : the role of ICE

[131] Selkoe, D. J. (2002). Alzheimer's disease is a synaptic failure. Science , 298, 789-791.

[132] Selznick, L. A, Holtzman, D. M, Han, B. H, Gokden, M, & Srinivassan, A. N. Johnson EMJ, Roth KA ((1999). In situ immunodetection of neuronal caspase-3 activation in

[133] Simpson, M. T. MacLaurin JG, Xu D, Ferguson KL, Vanderluit JL, Davoli MA, Roy S, Nicholson DW, Robertson GS, Park DS, Slack RS ((2001). Caspase 3 deficiency rescues peripheral nervous system defect in retinoblastoma nullizygous mice. J Neurosci , 21,

[134] Slee, E. A, Adrain, C, & Martin, S. J. (2001). Executioner caspase-3,-6, and-7 perform distinct, non-redundant roles during the demolition phase of apoptosis. J Biol Chem ,

[135] Smale, G, Nichols, N. R, Brady, D. R, & Finch, C. E. Horton WEJ ((1995). Evidence for

[136] Srinivasula, S. M, Hegde, R, Saleh, A, Datta, P, Shiozaki, E, Chai, J, Lee, R. A, Robbins, P. D, Fernandes-alnemri, T, Shi, Y, & Alnemri, E. S. (2001). A conserved XIAP-interac‐ tion motif in caspase-9 and Smac/DIABLO regulates caspase activity and apoptosis.

[137] Stadelmann, C, Br, k W, Bancher, C, Jellinger, K, & Lassmann, H. (1998). Alzheimer disease: DNA fragmentation indicates increased neuronal vulnerability, but not

[138] Stadelmann, C, Deckwerth, T, Srinivasan, A, Bancher, C, Br, k W, Jellinger, K, & Lassmann, H. (1999). Activation of caspase-3 in single neurons and autophagic granules of granulovacuolar degeneration in Alzheimer's disease. Am J Path , 155, 1459-1466.

[139] Stennicke, H. R, Ryan, C. A, & Salvesen, G. S. (2002). Reprieval from execution: the

[140] Stephan, A, Laroche, S, & Davis, S. (2001). Generation of aggregated beta-amyloid in the rat hippocampus impairs synaptic transmission and plasticity and causes memory

[141] Strasser, A, Connor, O, & Dixit, L. Apoptosis signaling. Annu Rev Biochem 69:217-245.,

[142] Su, J. H, Anderson, A. J, Cummings, B. J, & Cotman, C. W. (1994). Immunohistochemical evidence for apoptosis in Alzheimer's disease. Neuroreport , 5, 2529-2533.

molecular basis of caspase inhibition. Trends Biochem Sci , 27, 94-101.

apoptotic cell death in Alzheimer's disease. Exp Neurol , 133, 225-230.

apoptosis. J Neuropathol Exp Neurol , 57, 456-464.

deficits. J Neurosci , 21, 5703-5714.

2000

proteases in neuronal cell death. Trends Neurosci , 19, 555-562.

Alzheimer disease. J Neurophathol Exp Neurol , 58, 1020-1026.

7089-7098.

276, 7320-7326.

Nature , 410, 112-116.


[129] Schotte, P, Declercq, W, Van Huffel, S, Vandenabeele, P, & Beyaert, R. (1999). Nonspecific effects of methyl ketone peptide inhibitors of caspases. FEBS Lett , 442, 117-121.

[115] Price, D. L, & Sisodia, S. S. (1998). Mutant genes in familial Alzheimer's disease and

[116] Price, J. L, & Mckeel, D. W. Jr., Morris JC ((2001). Synaptic loss and pathological change

[117] Ray, C. A, Black, R. A, Kronheim, S. R, Greensheet, T. A, Sleath, P. R, Salvesen, G. S, & Pickup, D. J. (1992). Viral inhibition of inflammation: cowpox virus encodes an inhibitor

[118] Riedl, S. J, Renatus, M, Schwarzenbacher, R, Zhou, Q, Sun, C, Fesik, S. W, Liddington, R. C, & Salvesen, G. S. (2001). Structural basis for the inhibition of caspase-3 by XIAP.

[119] Roder, H. (2003). Prospect of therapeutic approaches to tauopathies. J Mol Neurosci ,

[120] Roher, A, Lowenson, J, Clarke, S, Woods, S, Cotter, R, Gowing, E, & Ball, M. J. (1993). beta-Amyloid-(1-42) is a major component of cerebrovascular amyloid deposits: implications for the pathology of Alzheimer's disease. Proc Natl Acad Sci USA , 90,

[121] Rohn, T. T, Head, E, Nesse, W. H, Cotman, C. W, & Cribbs, D. H. (2001). Activation of caspase-8 in the Alzheimer's disease brain. Neurobiol Dis , 8, 1006-1016.

[122] Rohn, T. T, Rissman, R. A, Davis, M. C, Kim, Y. E, Cotman, C. W, & Head, E. (2002). Caspase-9 activation and caspase cleavage of tau in the Alzheimer's disease brain.

[123] Roth, K. A. (2001). Caspases, apoptosis, and Alzheimer disease: Causation, correlation,

[124] Ruchaud, S, Korfali, N, Villa, P, Kottke, T. J, Dingwall, C, Kaufmann, S. H, & Earnshaw, W. C. (2002). Caspase-6 gene disruption reveals a requirement for lamin A cleavage in

[125] Sakahira, H, Enari, M, & Nagata, S. (1998). Cleavage of CAD inhibitor in CAD activation

[126] Samejima, K, Svingen, P. A, Basi, G. S, Kottke, T, & Mesner, P. W. Jr., Stewart L, Durrieu F, Poirier GG, Alnemri ES, Champoux JJ, Kaufmann SH, Earnshaw WC ((1999). Caspase-mediated cleavage of DNA topoisomerase I at unconventional sites during

[127] Scheff, S. W, & Price, D. A. (2001). Alzheimer's disease-related synapse loss in the

[128] Scheff, S. W, Price, D. A, & Sparks, D. L. (2001). Quantitative assessment of possible age-related change in synaptic numbers in the human frontal cortex. Neurobiol Aging ,

and confusion. J Neuropathol Exp Neurol , 60, 829-838.

apoptotic chromatin condensation. Embo J , 21, 1967-1977.

and DNA degradation during apoptosis. Nature , 391, 96-99.

apoptosis. J Biol Chem , 274, 4335-4340.

cingulate cortex. J Alzheimers Dis , 3, 495-505.

in older adults--aging versus disease? Neurobiol Aging , 22, 351-352.

of the interleukin-1 beta converting enzyme. Cell , 69, 597-604.

transgenic models. Annu Reve Neurosci , 21, 479-505.

Cell , 104, 791-800.

20, 195-202.

146 Neurodegenerative Diseases

10836-10840.

22, 355-365.

Neurobiol Dis , 11, 341-354.


[143] Su, J. H, Deng, G, & Cotman, C. W. (1997). Bax protein expression is increased in Alzheimer's brain: correlations with DNA damage, Bcl-2 expression, and brain pathology. J NeuropatholExp Neurol , 56, 86-93.

[156] Van de Craen MBerx G, Van den Brande I, Fiers W, Declercq W, Vandenabeele Proteolytic cleavage of beta-catenin by caspases: an in vitro analysis. FEBS Lett

Caspases in Alzheimer's Disease http://dx.doi.org/10.5772/54627 149

[157] Vanags, D. M, Porn-ares, M. I, Coppola, S, Burgess, D. H, & Orrenius, S. (1996). Protease involvement in fodrin cleavage and phosphatidylserine exposure in apoptosis. J Biol

[158] Vucic, D, Kaiser, W. J, Harvey, A. J, & Miller, L. K. (1997). Inhibition of reaper-induced apoptosis by interaction with inhibitor of apoptosis proteins (IAPs). Proc Natl Acd Sci

[159] Vucic, D, Kaiser, W. J, & Miller, L. K. (1998). Inhibitor of apoptosis proteins physically interact with and block apoptosis induced by Drosophila proteins hid and grim. Mol

[160] Weidemann, A, Paliga, K, Drwang, U, Reinhard, F. B, Schuckert, O, Evin, G, & Masters, C. L. (1999). Proteolytic processing of the Alzheimer/s disease amyloid precursor protein within its cytoplasmic domain by caspase-like proteases. J Biol Chem , 274,

[161] Weinstock, M. (1997). Possible role of the cholinergic system and disease models. J

[162] Whitehouse, P. J, Price, D. L, Clark, A. W, Coyle, J. T, & Delong, M. R. (1981). Alzheimer disease: evidence for selective loss of cholinergic neurons in the nucleus basalis. Ann

[163] Whitehouse, P. J, Price, D. L, Struble, R. G, Clark, A. W, Coyle, J. T, & Delon, M. R. (1982). Alzheimer's disease and senile dementia: loss of neurons in the basal forebrain.

[164] Yamatsuji, T, Okamoto, T, Takeda, S, Murayama, Y, Tanaka, N, & Nishinoto, I. (1996). Expression of APP mutant causes cellular apoptosis as Alzheimer trait-linked pheno‐

[165] Yao, P. J, Morsch, R, Callahan, L. M, & Coleman, P. D. (1999). Changes in synaptic expression of clathrin assembly protein AP180 in Alzheimer's disease analysed by

[166] Yuan, J, Shaham, S, Ledoux, S, Ellis, H. M, & Horvitz, H. R. Elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1beta-converting enzyme.

[167] Yuan, J, & Yankner, B. A. (2000). Apoptosis in the nervous system. Nature , 407, 802-809.

[168] Zhang, Y, & Goodyer, C. LeBlanc A ((2000). Selective and protracted apoptosis in human primary neurons microinjected with active caspase-3,-6,-7, and-8. J Neurosci ,

458:167-170., 1999.

Chem , 271, 31075-31085.

USA , 94, 10183-10188.

Cell Biol , 18, 3300-3309.

Neurol , 10, 122-126.

Science , 215, 1237-1239.

Cell , 75, 641-652.

20, 8384-8389.

type. EMBO J 15:498-509., 642

immunohistochemistry. Neuroscience , 94, 389-394.

Neural Transm Suppl , 49, 93-102.

5823-5829.


[156] Van de Craen MBerx G, Van den Brande I, Fiers W, Declercq W, Vandenabeele Proteolytic cleavage of beta-catenin by caspases: an in vitro analysis. FEBS Lett 458:167-170., 1999.

[143] Su, J. H, Deng, G, & Cotman, C. W. (1997). Bax protein expression is increased in Alzheimer's brain: correlations with DNA damage, Bcl-2 expression, and brain

[144] Su, J. H, Satou, T, Anderson, A. J, & Cotman, C. W. (1996). Up-regulation of Bcl-2 is associated with neuronal DNA damage in Alzheimer's disease. Neuroreport , 7,

[145] Takeuchi, A, Irizarry, M. C, & Duff, K. (2000). Age-related amyloid beta deposition in transgenic mice overexpressing both Alzheimer mutant presenilin 1 and amyloid beta precursor protein Swedish mutant is not associated with global neuronal loss. Am J

[146] Terry, R. D. (2000). Cell death or synaptic loss in Alzheimer disease. J Neuropathol Exp

[147] Terry, R. D, Masliah, E, & Salmon, D. P. (1991). Physical basis of cognitive alterations in Alzheimer's disease: Synaptic loss is a major correlate of cognitive impairment. Ann

[148] Thome, M, Schneider, P, Hofmann, K, Fickenscher, H, Meinl, E, Neipel, F, Mattmann, C, Burns, K, Bodmer, J. L, Schroter, M, Scaffidi, C, Karammer, P. H, Peter, M. E, & Tachopp, J. (1997). Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced

[149] Thompson, C. S. (1995). Apoptosis in the pathogenesis and treatment of disease.

[150] Thornberry, N. A. (1997). The caspase family of cysteine proteases. Br Med Bull , 53,

[151] Torp, R, Su, J. H, Deng, G, & Cotman, C. W. (1998). GADD45 is induced in Alzheimer's disease, and protects against apoptosis in vitro. Neurobiol Dis , 5, 245-252.

[152] Tortosa, A, Lopez, E, & Ferrer, I. (1998). Bcl-2 and Bax protein expression in Alzheimer's

[153] Troncoso, J. C, Sukhov, R. R, Kawas, C. H, & Koliatsos, V. E. (1996). In situ labeling of dying cortical neurons in normal aging and in Alzheimer's disease: correlations with senile plaques and disease progression. J Nuropathol Exp Neurol , 55, 1134-1142.

[154] Tseng, B. P, Esler, W. P, Clish, C. B, Stimson, E. R, Ghilardi, J. R, Vinters, H. V, Mantyh, P. W, Lee, J. P, & Maggio, J. E. (1999). Deposition of monomeric, not oligomeric, Abeta mediates growth of Alzheimer's disease amyloid plaques in human brain preparations.

[155] Urbanc, B, Cruz, L, Buldyrev, S. V, Havlin, S, Irizarry, M. C, Stanley, H. E, & Hyman, B. T. (1999). Dynamics of plaque formation in Alzheimer's disease. Biophys J , 76,

pathology. J NeuropatholExp Neurol , 56, 86-93.

437-440.

148 Neurodegenerative Diseases

Path , 157, 331-339.

Neurol , 59, 1118-1119.

Neurol , 30, 572-580.

Science , 2667, 1456-1462.

478-490.

1330-1334.

by death receptors. Nature , 386, 517-521.

disease. Acta Neuropathol (Berl) , 95, 407-412.

Biochemistry , 38, 10424-10431.


[169] Zheng, T. S, Hunot, S, Kuida, K, & Flavell, R. A. (1999). Caspase knockouts: matters of life and death. Cell Death Differe , 6, 1043-1053.

**Chapter 7**

**Brain Reserve Regulators in Alzheimer's Disease**

Brain reserve refers to the ability of the brain to tolerate pathological changes such as those seen in AD before manifesting clinical signs and symptoms [1-3]. Neurotrophic factors (NTFs), most notably Brain Derived Neurotrophic Factor (BDNF) and its receptor Tyrosine kinase B (TrkB), regulate synaptic plasticity and functional efficiency in adulthood [4-6] and thus may influence brain reserve. BDNF/TrkB signaling affects memory formation and retention [7,8], determines neurite length [9,10], and governs regeneration upon neuronal injury [11,12] by modifying neuronal cytoskeleton. Abnormalities in the neuronal cytoskeleton are well documented in AD. However, how these abnormalities affect AD progression remains unclear. In *Drosophila*, neurodegeneration stems directly from mutations in alpha and beta subunits of the actin capping protein (CP), demonstrating that a mutation in a gene encoding an actin cytoskeleton regulator can lead to demise of neurons [13]. Further, a causative role for actin cytoskeleton abnormalities in neurotoxicity has been documented in a *Drosophila* tauopathy

Important evidence that cytoskeletal abnormalities are critically involved in the pathogenesis of neurodegeneration stems from the studies demonstrating the effect of apolipoprotein E isoform ε4 (ApoE ε4), the well-documented genetic risk factor for the most common form of AD, late-onset AD [15], on neuronal cytoskeleton. In the United States, the ApoE ε4 allele occurs in 60% of AD patients. ApoE ε4 inhibits neurite outgrowth in cultured neuronal cells [16] and correlates with the simplification of dendritic branching patterns in the brains of AD patients [17]. ApoE ε4 dose inversely correlates with dendritic spine density in dentate gyrus neurons of both AD and aged normal controls [18]. Overexpression and neuron-specific proteolytic cleavage of ApoE ε4 result in tau hyperphosphorylation in neurons of transgenic mice, suggesting a role of ApoE ε4 in cytoskeletal destabilization and the development of ADrelated neuronal deficits [19,20]. Humanized ApoE ε4 knock-in homozygous transgenic mice

> © 2013 Delalle; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

Ivana Delalle

**1. Introduction**

model [14].

http://dx.doi.org/10.5772/53658

[170] Zhou, Q, Krebs, J. F, Snipas, S. J, Price, A, Alnemri, E. S, Tomaselli, K. J, & Salvesen, G. S. (1998). Interaction of the baculovirus anti-apoptotic protein with caspase-specificity, kinetics, and characterization of the caspase/p35 complex. Biochemistry 37:10757-10765., 35.

### **Brain Reserve Regulators in Alzheimer's Disease**

### Ivana Delalle

[169] Zheng, T. S, Hunot, S, Kuida, K, & Flavell, R. A. (1999). Caspase knockouts: matters of

[170] Zhou, Q, Krebs, J. F, Snipas, S. J, Price, A, Alnemri, E. S, Tomaselli, K. J, & Salvesen, G. S. (1998). Interaction of the baculovirus anti-apoptotic protein with caspase-specificity, kinetics, and characterization of the caspase/p35 complex. Biochemistry

life and death. Cell Death Differe , 6, 1043-1053.

37:10757-10765., 35.

150 Neurodegenerative Diseases

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53658

### **1. Introduction**

Brain reserve refers to the ability of the brain to tolerate pathological changes such as those seen in AD before manifesting clinical signs and symptoms [1-3]. Neurotrophic factors (NTFs), most notably Brain Derived Neurotrophic Factor (BDNF) and its receptor Tyrosine kinase B (TrkB), regulate synaptic plasticity and functional efficiency in adulthood [4-6] and thus may influence brain reserve. BDNF/TrkB signaling affects memory formation and retention [7,8], determines neurite length [9,10], and governs regeneration upon neuronal injury [11,12] by modifying neuronal cytoskeleton. Abnormalities in the neuronal cytoskeleton are well documented in AD. However, how these abnormalities affect AD progression remains unclear. In *Drosophila*, neurodegeneration stems directly from mutations in alpha and beta subunits of the actin capping protein (CP), demonstrating that a mutation in a gene encoding an actin cytoskeleton regulator can lead to demise of neurons [13]. Further, a causative role for actin cytoskeleton abnormalities in neurotoxicity has been documented in a *Drosophila* tauopathy model [14].

Important evidence that cytoskeletal abnormalities are critically involved in the pathogenesis of neurodegeneration stems from the studies demonstrating the effect of apolipoprotein E isoform ε4 (ApoE ε4), the well-documented genetic risk factor for the most common form of AD, late-onset AD [15], on neuronal cytoskeleton. In the United States, the ApoE ε4 allele occurs in 60% of AD patients. ApoE ε4 inhibits neurite outgrowth in cultured neuronal cells [16] and correlates with the simplification of dendritic branching patterns in the brains of AD patients [17]. ApoE ε4 dose inversely correlates with dendritic spine density in dentate gyrus neurons of both AD and aged normal controls [18]. Overexpression and neuron-specific proteolytic cleavage of ApoE ε4 result in tau hyperphosphorylation in neurons of transgenic mice, suggesting a role of ApoE ε4 in cytoskeletal destabilization and the development of ADrelated neuronal deficits [19,20]. Humanized ApoE ε4 knock-in homozygous transgenic mice

© 2013 Delalle; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

exhibit cognitive deficits before the onset of age-dependent neuropathology including ADassociated neurofibrillary tangles and neuritic plaques [21,22].

Capzb2 - βIII-tubulin interaction is indispensable for normal growth cone morphology and

Brain Reserve Regulators in Alzheimer's Disease

http://dx.doi.org/10.5772/53658

153

**Figure 1.** [37]: Capzb2-EGFP (green) expression in mouse hippocampal neurons. In addition to soma and processes,

Interestingly, the interaction between actin capping protein and ß-tubulin has been uncovered in a mass spectrometry screen for the alterations in protein-target binding *in vivo* in response

In line with the previously documented increased cytoskeletal reorganization including dendritic proliferation and sprouting in neurons of AD patients [40,41,31], we recently demonstrated increased expression of Capzb2 (Figure 2) and TrkB in mid-stages (Braak and

**Figure 2.** [42]: Hippocampal pyramidal neurons from a control case contain less Capzb2 mRNA (higher ΔCT) than the

BDNF binding to the TrkB receptor initiates intracellular cascades involving cell survival, growth, and differentiation via mitogen-activated protein kinase (MAPK), phosphatidylino‐ sitol 3-kinase (PI3K), and phospholipase C-g (PLCγ) signaling pathways, as recently reviewed [43]. PI3K and MAPK simultaneous triggering alters both actin and microtubule dynamics needed for dendrite branching [43]. BDNF has been shown to promote growth of undifferen‐

neurite length (Figure 1) [37].

Capzb2 is expressed in growth cones (red- ß-tubulin, blue- nuclei).

Braak III-IV, BBIII-IV) AD pathology[42].

to spatial learning [38], a process that requires BDNF [39].

neurons from Braak and Braak III-IV AD cases (\*\*p<0.01, Student's t test).

While the relationships between BDNF gene polymorphisms and AD are not yet fully understood [23-25], there is compelling evidence that epigenetic regulation connects BDNF/ TrkB signaling with learning and memory. Exercise restored TrkB in ApoE ε4 mice to the level observed in ε3 mice and increased synaptophysin (a marker of synaptic function) in ε4 mice; hippocampal BDNF levels were similarly increased in both ε3 and ε4 mice after exercise [26]. Exposure to an enriched environment for three to four weeks also caused dramatic increase in BDNF mRNA in mouse hippocampus [27]. Understanding the regulation of BDNF/TrkB signaling in AD pathogenesis, particularly in individuals carrying ApoE ε4, could be of great clinical and public health importance because BDNF is inducible and may be one of the key molecules mediating the beneficial effect of certain lifestyle measures (environmental enrich‐ ment, increased aerobic physical activity, lower caloric intake) [28-30] on the risk of developing dementia.

### **2. Neuronal cytoskeleton regulator actin capping protein ß2 (Capzb2) and BDNF/TrkB signaling**

As hyperphosphorylated tau gives rise to neurofibrillary tangles in AD, dystrophic neurites, marked by reduced length and poor branching, become apparent. In parallel, perisomatic proliferation of dendrites and sprouting of distal dystrophic neurites take place[31]. These morphological changes in neurons during AD progression indicate major cytoskeletal reorganization raising the possibility that microtubules and microfilaments may represent a target for pathobiological mechanisms underlying AD. The presence of growth cone-like structures on distal ends of dystrophic neurites suggests that regenerative response accom‐ panies cytoskeleton degeneration in AD [31].

Changes in growth cone morphology, motility, and direction of growth are controlled by interactions between F-actin and microtubules and their associated proteins [32]. The growth cone morphology is characterized by lamellipodia, which are the veil-like extensions at the periphery, and filopodia, which are narrow, spiky extensions coming from the periphery of the growth cone. Interestingly, APP concentrates in lamellipodia where it is proposed to play a role in growth cone motility and neurite outgrowth [33]. Upon acute neuronal injury, the first critical steps that initiate regenerative response are microtubule polymerization and F-actin cytoskeleton rearrangement leading to the formation of a motile growth cone [34]. Actin cytoskeleton regulator CP (F-actin capping protein, CapZ) is an α/β heterodimer that binds the barbed end of F-actin thus blocking the access of actin monomers to the fast growing end. Both mammalian and *Drosophila* CP subunits play a critical role in the organization and dynamics of lamellipodia and filopodia in non-neuronal cells [35]. One of the mammalian β-subunit isoforms, Capzb2, is predominantly expressed in the brain [36]. Capzb2 not only caps F-actin barbed end but also binds βIII-tubulin directly, affecting the rate and the extent of microtubule polymerization in the presence of tau [37]. Moreover, Capzb2 - βIII-tubulin interaction is indispensable for normal growth cone morphology and neurite length (Figure 1) [37].

exhibit cognitive deficits before the onset of age-dependent neuropathology including AD-

While the relationships between BDNF gene polymorphisms and AD are not yet fully understood [23-25], there is compelling evidence that epigenetic regulation connects BDNF/ TrkB signaling with learning and memory. Exercise restored TrkB in ApoE ε4 mice to the level observed in ε3 mice and increased synaptophysin (a marker of synaptic function) in ε4 mice; hippocampal BDNF levels were similarly increased in both ε3 and ε4 mice after exercise [26]. Exposure to an enriched environment for three to four weeks also caused dramatic increase in BDNF mRNA in mouse hippocampus [27]. Understanding the regulation of BDNF/TrkB signaling in AD pathogenesis, particularly in individuals carrying ApoE ε4, could be of great clinical and public health importance because BDNF is inducible and may be one of the key molecules mediating the beneficial effect of certain lifestyle measures (environmental enrich‐ ment, increased aerobic physical activity, lower caloric intake) [28-30] on the risk of developing

**2. Neuronal cytoskeleton regulator actin capping protein ß2 (Capzb2) and**

As hyperphosphorylated tau gives rise to neurofibrillary tangles in AD, dystrophic neurites, marked by reduced length and poor branching, become apparent. In parallel, perisomatic proliferation of dendrites and sprouting of distal dystrophic neurites take place[31]. These morphological changes in neurons during AD progression indicate major cytoskeletal reorganization raising the possibility that microtubules and microfilaments may represent a target for pathobiological mechanisms underlying AD. The presence of growth cone-like structures on distal ends of dystrophic neurites suggests that regenerative response accom‐

Changes in growth cone morphology, motility, and direction of growth are controlled by interactions between F-actin and microtubules and their associated proteins [32]. The growth cone morphology is characterized by lamellipodia, which are the veil-like extensions at the periphery, and filopodia, which are narrow, spiky extensions coming from the periphery of the growth cone. Interestingly, APP concentrates in lamellipodia where it is proposed to play a role in growth cone motility and neurite outgrowth [33]. Upon acute neuronal injury, the first critical steps that initiate regenerative response are microtubule polymerization and F-actin cytoskeleton rearrangement leading to the formation of a motile growth cone [34]. Actin cytoskeleton regulator CP (F-actin capping protein, CapZ) is an α/β heterodimer that binds the barbed end of F-actin thus blocking the access of actin monomers to the fast growing end. Both mammalian and *Drosophila* CP subunits play a critical role in the organization and dynamics of lamellipodia and filopodia in non-neuronal cells [35]. One of the mammalian β-subunit isoforms, Capzb2, is predominantly expressed in the brain [36]. Capzb2 not only caps F-actin barbed end but also binds βIII-tubulin directly, affecting the rate and the extent of microtubule polymerization in the presence of tau [37]. Moreover,

associated neurofibrillary tangles and neuritic plaques [21,22].

dementia.

152 Neurodegenerative Diseases

**BDNF/TrkB signaling**

panies cytoskeleton degeneration in AD [31].

**Figure 1.** [37]: Capzb2-EGFP (green) expression in mouse hippocampal neurons. In addition to soma and processes, Capzb2 is expressed in growth cones (red- ß-tubulin, blue- nuclei).

Interestingly, the interaction between actin capping protein and ß-tubulin has been uncovered in a mass spectrometry screen for the alterations in protein-target binding *in vivo* in response to spatial learning [38], a process that requires BDNF [39].

In line with the previously documented increased cytoskeletal reorganization including dendritic proliferation and sprouting in neurons of AD patients [40,41,31], we recently demonstrated increased expression of Capzb2 (Figure 2) and TrkB in mid-stages (Braak and Braak III-IV, BBIII-IV) AD pathology[42].

**Figure 2.** [42]: Hippocampal pyramidal neurons from a control case contain less Capzb2 mRNA (higher ΔCT) than the neurons from Braak and Braak III-IV AD cases (\*\*p<0.01, Student's t test).

BDNF binding to the TrkB receptor initiates intracellular cascades involving cell survival, growth, and differentiation via mitogen-activated protein kinase (MAPK), phosphatidylino‐ sitol 3-kinase (PI3K), and phospholipase C-g (PLCγ) signaling pathways, as recently reviewed [43]. PI3K and MAPK simultaneous triggering alters both actin and microtubule dynamics needed for dendrite branching [43]. BDNF has been shown to promote growth of undifferen‐ tiated dendrites and axons in cultured hippocampal pyramidal neurons [44], a process that requires Capzb2 [37]. Thus, the expression of Capzb2 may represent one of the likely downstream read-outs for BDNF-TrkB neuronal signaling. In a rat model of dementia there is activity-dependent, synapse-specific regulation of CapZ redistribution possibly important in both maintenance and remodeling of synaptic connections receiving spatial and temporal patterns of inputs [45].

of Alzheimer Disease (AD) and whether that response depends on an up-regulation of the BDNF pathway. The expression of TrkB and Capzb2 in CA1 hippocampal neurons of indi‐ viduals with preserved cognitive status (CDR 0) and initial neurofibrillary tangle formation was increased in comparison to cognitively intact individuals without any neurofibrillary tangles (Figure 3) [46]. In contrast, BDNF expression remained unchanged, raising the possibility that the up-regulated TrkB expression in CDR0 individuals is responsible for the increase in BDNF/TrkB signaling tapping the brain reserve (Figure 3) [46]. In the group of individuals with more advanced tangle formation and early to mild dementia (CDR 0.5-1), the increase in TrkB expression and the unchanged expression of BDNF might have been insuffi‐

Brain Reserve Regulators in Alzheimer's Disease

http://dx.doi.org/10.5772/53658

155

**Figure 3.** [46]: TrkB, BDNF, and Capzb2 mRNAs expression in control, CDR 0 (no dementia) and CDR 0.5-1 (mild de‐ mentia) subjects. TrkB mRNA expression is significantly increased (lower ΔCT) in subjects with early AD pathology (BBI-II) but no dementia (A). BDNF mRNA expression is similar in all groups examined (B). Capzb2 mRNA is significantly increased (lower ΔCT) in cases with AD pathology (C). Fold-increases of mean mRNA expression of TrkB and Capzb2 in

cient to provide adequate brain reserve (Figure 3) [46].

cases with AD pathology in comparison to controls (D).

### **3. Increased expression of TrkB and Capzb2 accompanies preserved cognitive status in early AD pathology**

Recent study compared mRNA (Figure 3) and protein (Figure 4) expression of BDNF, TrkB and Capzb2 in samples of neuropathologically normal and cognitively intact subjects (con‐ trols), with samples of persons with AD-related pathological changes who were cognitively intact prior to death (Clinical Dementia Rating zero, CDR0), and samples of persons with ADrelated pathological changes as well as early clinical dementia (CDR0.5 – 1) [46]. This approach was possible due to the existence of a unique sample of Framingham Heart Study (FHS) participants who have undergone repeated antemortem cognitive testing and brain imaging [47,48]. All FHS participants in the FHS have undergone screening cognitive tests (an MMSE) once in two years and have also had a more detailed cognitive assessment examining multiple cognitive domains once in 1974-75, once in 1999-2004 and at least twice thereafter. The presence or absence of dementia in all FHS participants is defined using DSM-IV criteria that require impairment in memory and in at least one other area of cognitive function, as well as docu‐ mented functional disability. AD is defined using NINCDS-ADRDA criteria for definite, probable or possible AD[49]. All FHS participants are invited to become brain donors and the nearly 700 persons who have accepted this invitation undergo a detailed neuropsychological testing [50,51], at least once every 2 years beyond age 75 years. Persons who screen positive or are otherwise referred (by self, family or treating physicians) undergo detailed neurological and neuropsychological assessment, informant interview (with a physician administered CDR) and a review of hospital records, nursing home notes, brain imaging and laboratory tests. A structured family interview (including Blessed Dementia and Hachinski scales) [52,53] is conducted with the next-of-kin based on which a retrospective CDR score is assigned after the participant dies. The retrospective CDR is very similar to the retrospective collateral dementia interview validated by Davis and colleagues (1991)[54]. A final clinical decision regarding the presence or absence of dementia, diagnosis of dementia type and date of onset/ diagnosis is made by a clinical consensus panel including behavioral neurologists and neuropsychologists who review all available records including records at the time of death. All deaths are reviewed to assign a cause of death and to determine if dementia was present or absent at the time of death. The neuropathological report is generated prior to a final clinicopathological conference during which the clinical diagnoses and pathological findings are discussed.

Hippocampi from selected FHS cases were used to determine whether specifically vulnerable population of CA1 neurons shows a compensatory response to the neuropathological changes of Alzheimer Disease (AD) and whether that response depends on an up-regulation of the BDNF pathway. The expression of TrkB and Capzb2 in CA1 hippocampal neurons of indi‐ viduals with preserved cognitive status (CDR 0) and initial neurofibrillary tangle formation was increased in comparison to cognitively intact individuals without any neurofibrillary tangles (Figure 3) [46]. In contrast, BDNF expression remained unchanged, raising the possibility that the up-regulated TrkB expression in CDR0 individuals is responsible for the increase in BDNF/TrkB signaling tapping the brain reserve (Figure 3) [46]. In the group of individuals with more advanced tangle formation and early to mild dementia (CDR 0.5-1), the increase in TrkB expression and the unchanged expression of BDNF might have been insuffi‐ cient to provide adequate brain reserve (Figure 3) [46].

tiated dendrites and axons in cultured hippocampal pyramidal neurons [44], a process that requires Capzb2 [37]. Thus, the expression of Capzb2 may represent one of the likely downstream read-outs for BDNF-TrkB neuronal signaling. In a rat model of dementia there is activity-dependent, synapse-specific regulation of CapZ redistribution possibly important in both maintenance and remodeling of synaptic connections receiving spatial and temporal

**3. Increased expression of TrkB and Capzb2 accompanies preserved**

Recent study compared mRNA (Figure 3) and protein (Figure 4) expression of BDNF, TrkB and Capzb2 in samples of neuropathologically normal and cognitively intact subjects (con‐ trols), with samples of persons with AD-related pathological changes who were cognitively intact prior to death (Clinical Dementia Rating zero, CDR0), and samples of persons with ADrelated pathological changes as well as early clinical dementia (CDR0.5 – 1) [46]. This approach was possible due to the existence of a unique sample of Framingham Heart Study (FHS) participants who have undergone repeated antemortem cognitive testing and brain imaging [47,48]. All FHS participants in the FHS have undergone screening cognitive tests (an MMSE) once in two years and have also had a more detailed cognitive assessment examining multiple cognitive domains once in 1974-75, once in 1999-2004 and at least twice thereafter. The presence or absence of dementia in all FHS participants is defined using DSM-IV criteria that require impairment in memory and in at least one other area of cognitive function, as well as docu‐ mented functional disability. AD is defined using NINCDS-ADRDA criteria for definite, probable or possible AD[49]. All FHS participants are invited to become brain donors and the nearly 700 persons who have accepted this invitation undergo a detailed neuropsychological testing [50,51], at least once every 2 years beyond age 75 years. Persons who screen positive or are otherwise referred (by self, family or treating physicians) undergo detailed neurological and neuropsychological assessment, informant interview (with a physician administered CDR) and a review of hospital records, nursing home notes, brain imaging and laboratory tests. A structured family interview (including Blessed Dementia and Hachinski scales) [52,53] is conducted with the next-of-kin based on which a retrospective CDR score is assigned after the participant dies. The retrospective CDR is very similar to the retrospective collateral dementia interview validated by Davis and colleagues (1991)[54]. A final clinical decision regarding the presence or absence of dementia, diagnosis of dementia type and date of onset/ diagnosis is made by a clinical consensus panel including behavioral neurologists and neuropsychologists who review all available records including records at the time of death. All deaths are reviewed to assign a cause of death and to determine if dementia was present or absent at the time of death. The neuropathological report is generated prior to a final clinicopathological conference during which the clinical diagnoses and pathological findings are

Hippocampi from selected FHS cases were used to determine whether specifically vulnerable population of CA1 neurons shows a compensatory response to the neuropathological changes

patterns of inputs [45].

154 Neurodegenerative Diseases

discussed.

**cognitive status in early AD pathology**

**Figure 3.** [46]: TrkB, BDNF, and Capzb2 mRNAs expression in control, CDR 0 (no dementia) and CDR 0.5-1 (mild de‐ mentia) subjects. TrkB mRNA expression is significantly increased (lower ΔCT) in subjects with early AD pathology (BBI-II) but no dementia (A). BDNF mRNA expression is similar in all groups examined (B). Capzb2 mRNA is significantly increased (lower ΔCT) in cases with AD pathology (C). Fold-increases of mean mRNA expression of TrkB and Capzb2 in cases with AD pathology in comparison to controls (D).

In light of the reported restoration of learning and memory functions in AD animal models upon BDNF gene delivery [55], exogenous intervention to boost BDNF/TrkB signaling might appear a compelling therapy in early AD. However, the experiments by Frank et al. (1996) suggest that the exposure of developing and adult rodent hippocampal neurons to BDNF *in vitro* and *in vivo* results in long-term functional desensitization to BDNF and down regulation of TrkB mRNA [56]. It is possible that BDNF/TrkB signaling is differentially regulated in healthy vs. diseased hippocampal neurons. Nevertheless, the reported increase in TrkB mRNA expression in astrocytes occasionally associated with senile plaques in hippocampi of AD brains raises concerns that the administration of neurotrophic factors could promote gliosis and plaque formation [57]. Importantly, if the observed increase in TrkB expression in cognitively intact FHS subjects with initial formation of neurofibrillary tangles constitutes brain reserve, down regulation of TrkB might represent a potentially harmful side-effect of

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157

One in five persons currently 65 years old will develop clinical Alzheimer's dementia in their lifetime. However, postponing the onset of clinical disease by as little as five years could halve individual risk and population burden of disease [58,59]. Since the timing of clinical dementia onset is determined not only by the pace of pathological changes but also by brain reserve, that postponement might be possible. The study of CA1 hippocampal neurons in FHS participant brain donors[46] adds to the emerging evidence that the BDNF/TrkB pathway may be involved in the compensatory response to early AD pathology, i.e. it may underlie the biology of cognitive reserve. Consequently, an epigenetic enhancement of BDNF/TrkB signaling in persons with early cognitive changes associated with AD pathology (mild cognitive impairment, MCI, due to AD pathology)[60] and in persons with no clinical symp‐ toms but with biomarker evidence of AD pathology (so-called preMCI due to AD pathology) [61] may be beneficial in delaying the onset of clinical dementia. The lifestyle modifications that are thought to reduce the risk of developing clinical AD, such as intake of docosahexaenoic acid (DHA) and increased exercise, appear to interact with BDNF-related synaptic plastici‐ ty[62]. As reviewed by Sananbenesi and Fischer (2009)[63], deregulation of "plasticity genes", in particular synaptic plasticity genes, accompanies aging, a major risk for AD. Histone deacetylase inhibitors (HDACs) and environmental enrichment have been shown to reinstate learning behavior and improve memory in a CK-p25 mouse model of neurodegeneration[64], whereas altered histone acetylation is associated with age-dependent memory impairment in mice[65]. These findings make deciphering of epigenetic signatures in preserved vs. failing human cognitive functions urgent and necessary for the development of rational interventions in the progression of AD [66,67]. The prevention of clinical AD will likely require a multidimensional approach and the modulation of the BDNF/TrkB pathway, calibrated to each

individual's needs, might be one facet of this multi-dimensional approach.

exogenous BDNF delivery.

**4. Conclusion**

**Figure 4.** [46]: Immunohistochemistry for TrkB (A-C), BDNF (D-F), and Capzb2 (G-I) in representative individuals from control, CDR0, and CDR1 groups reflects established trends in mRNA expression. Immunohistochemistry for tau high‐ lights intensity of neurofibrillary changes in a CDR0 subject (K) and in a CDR1 subject (L), while the control is free of neuropathology (J).

In light of the reported restoration of learning and memory functions in AD animal models upon BDNF gene delivery [55], exogenous intervention to boost BDNF/TrkB signaling might appear a compelling therapy in early AD. However, the experiments by Frank et al. (1996) suggest that the exposure of developing and adult rodent hippocampal neurons to BDNF *in vitro* and *in vivo* results in long-term functional desensitization to BDNF and down regulation of TrkB mRNA [56]. It is possible that BDNF/TrkB signaling is differentially regulated in healthy vs. diseased hippocampal neurons. Nevertheless, the reported increase in TrkB mRNA expression in astrocytes occasionally associated with senile plaques in hippocampi of AD brains raises concerns that the administration of neurotrophic factors could promote gliosis and plaque formation [57]. Importantly, if the observed increase in TrkB expression in cognitively intact FHS subjects with initial formation of neurofibrillary tangles constitutes brain reserve, down regulation of TrkB might represent a potentially harmful side-effect of exogenous BDNF delivery.

#### **4. Conclusion**

**Figure 4.** [46]: Immunohistochemistry for TrkB (A-C), BDNF (D-F), and Capzb2 (G-I) in representative individuals from control, CDR0, and CDR1 groups reflects established trends in mRNA expression. Immunohistochemistry for tau high‐ lights intensity of neurofibrillary changes in a CDR0 subject (K) and in a CDR1 subject (L), while the control is free of

neuropathology (J).

156 Neurodegenerative Diseases

One in five persons currently 65 years old will develop clinical Alzheimer's dementia in their lifetime. However, postponing the onset of clinical disease by as little as five years could halve individual risk and population burden of disease [58,59]. Since the timing of clinical dementia onset is determined not only by the pace of pathological changes but also by brain reserve, that postponement might be possible. The study of CA1 hippocampal neurons in FHS participant brain donors[46] adds to the emerging evidence that the BDNF/TrkB pathway may be involved in the compensatory response to early AD pathology, i.e. it may underlie the biology of cognitive reserve. Consequently, an epigenetic enhancement of BDNF/TrkB signaling in persons with early cognitive changes associated with AD pathology (mild cognitive impairment, MCI, due to AD pathology)[60] and in persons with no clinical symp‐ toms but with biomarker evidence of AD pathology (so-called preMCI due to AD pathology) [61] may be beneficial in delaying the onset of clinical dementia. The lifestyle modifications that are thought to reduce the risk of developing clinical AD, such as intake of docosahexaenoic acid (DHA) and increased exercise, appear to interact with BDNF-related synaptic plastici‐ ty[62]. As reviewed by Sananbenesi and Fischer (2009)[63], deregulation of "plasticity genes", in particular synaptic plasticity genes, accompanies aging, a major risk for AD. Histone deacetylase inhibitors (HDACs) and environmental enrichment have been shown to reinstate learning behavior and improve memory in a CK-p25 mouse model of neurodegeneration[64], whereas altered histone acetylation is associated with age-dependent memory impairment in mice[65]. These findings make deciphering of epigenetic signatures in preserved vs. failing human cognitive functions urgent and necessary for the development of rational interventions in the progression of AD [66,67]. The prevention of clinical AD will likely require a multidimensional approach and the modulation of the BDNF/TrkB pathway, calibrated to each individual's needs, might be one facet of this multi-dimensional approach.

#### **Summary**

During the progression of Alzheimer's disease (AD), hippocampal neurons show degenerative as well as regenerative changes, possibly influenced by genes that may modify brain reserve, the ability of the brain to tolerate pathological changes in AD before manifesting clinical signs and symptoms. Recent data suggest that the expression of these genes in the hippocampal neurons correlates with the cognitive function. Identifying molecules that may promote regenerative potential and/or increase brain reserve provides novel targets for interventions in late-onset AD.

[8] Lu, Y, Christian, K, & Lu, B. (2008). BDNF: a key regulator for protein synthesis-de‐ pendent LTP and long-term memory? Neurobiol Learn Mem , 89, 312-323.

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159

[9] Miyamoto, Y, Yamauchi, J, Tanoue, A, Wu, C, & Mobley, W. C. (2006). TrkB binds and tyrosine-phosphorylates Tiam1, leading to activation of Rac1 and induction of

changes in cellular morphology. Proc Natl Acad Sci U S A , 103, 10444-10449.

173-180.

Res , 59, 154-164.

[10] Luo, L. (2000). Rho GTPases in neuronal morphogenesis. Nat Rev Neurosci , 1,

[11] Goutan, E, Marti, E, & Ferrer, I. (1998). BDNF, and full length and truncated TrkB ex‐ pression in the hippocampus of the rat following kainic acid excitotoxic damage. Evi‐ dence of complex time-dependent and cell-specific responses. Brain Res Mol Brain

[12] Avwenagha, O, Campbell, G, & Bird, M. M. (2003). Distribution of GAP-43, beta-III tubulin and F-actin in developing and regenerating axons and their growth cones in

[13] Delalle, I, Pfleger, C. M, Buff, E, Lueras, P, & Hariharan, I. K. (2005). Mutations in the Drosophila orthologs of the F-actin capping protein alpha- and beta-subunits cause actin accumulation and subsequent retinal degeneration. Genetics , 171, 1757-1765.

[14] Fulga, T. A, Elson-schwab, I, Khurana, V, Steinhilb, M. L, Spires, T. L, et al. (2007). Abnormal bundling and accumulation of F-actin mediates tau-induced neuronal de‐

[15] Li, Y, & Grupe, A. (2007). Genetics of late-onset Alzheimer's disease: progress and

[16] Nathan, B. P, Chang, K. C, Bellosta, S, Brisch, E, Ge, N, et al. (1995). The inhibitory effect of apolipoprotein E4 on neurite outgrowth is associated with microtubule de‐

[17] Arendt, T, Schindler, C, Bruckner, M. K, Eschrich, K, Bigl, V, et al. (1997). Plastic neu‐ ronal remodeling is impaired in patients with Alzheimer's disease carrying apolipo‐

[18] Ji, Y, Gong, Y, Gan, W, Beach, T, Holtzman, D. M, et al. (2003). Apolipoprotein E iso‐ form-specific regulation of dendritic spine morphology in apolipoprotein E transgen‐

[19] Tesseur, I, Van Dorpe, J, & Spittaels, K. Van den Haute C, Moechars D, et al. ((2000). Expression of human apolipoprotein E4 in neurons causes hyperphosphorylation of

[20] Brecht, W. J, Harris, F. M, Chang, S, Tesseur, I, Yu, G. Q, et al. (2004). Neuron-specific apolipoprotein e4 proteolysis is associated with increased tau phosphorylation in

ic mice and Alzheimer's disease patients. Neuroscience , 122, 305-315.

protein tau in the brains of transgenic mice. Am J Pathol , 156, 951-964.

vitro, following neurotrophin treatment. J Neurocytol , 32, 1077-1089.

generation in vivo. Nat Cell Biol , 9, 139-148.

prospect. Pharmacogenomics , 8, 1747-1755.

polymerization. J Biol Chem , 270, 19791-19799.

protein epsilon 4 allele. J Neurosci , 17, 516-529.

brains of transgenic mice. J Neurosci , 24, 2527-2534.

### **Author details**

#### Ivana Delalle

Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, USA

#### **References**


[8] Lu, Y, Christian, K, & Lu, B. (2008). BDNF: a key regulator for protein synthesis-de‐ pendent LTP and long-term memory? Neurobiol Learn Mem , 89, 312-323.

**Summary**

158 Neurodegenerative Diseases

in late-onset AD.

**Author details**

Ivana Delalle

Boston, USA

**References**

During the progression of Alzheimer's disease (AD), hippocampal neurons show degenerative as well as regenerative changes, possibly influenced by genes that may modify brain reserve, the ability of the brain to tolerate pathological changes in AD before manifesting clinical signs and symptoms. Recent data suggest that the expression of these genes in the hippocampal neurons correlates with the cognitive function. Identifying molecules that may promote regenerative potential and/or increase brain reserve provides novel targets for interventions

Department of Pathology and Laboratory Medicine, Boston University School of Medicine,

[1] Valenzuela, M. J, & Sachdev, P. (2006). Brain reserve and cognitive decline: a non-

[2] Riley, K. P, Snowdon, D. A, Desrosiers, M. F, & Markesbery, W. R. (2005). Early life linguistic ability, late life cognitive function, and neuropathology: findings from the

[3] Snowdon, D. A, Kemper, S. J, Mortimer, J. A, Greiner, L. H, Wekstein, D. R, et al. (1996). Linguistic ability in early life and cognitive function and Alzheimer's disease

[4] Waterhouse, E. G, & Xu, B. (2009). New insights into the role of brain-derived neuro‐

[5] Minichiello, L. (2009). TrkB signalling pathways in LTP and learning. Nat Rev Neu‐

[6] Cowansage, K. K, Ledoux, J. E, & Monfils, M. H. (2009). Brain-Derived Neurotrophic

[7] Rex, C. S, Lin, C. Y, Kramar, E. A, Chen, L. Y, Gall, C. M, et al. (2007). Brain-derived neurotrophic factor promotes long-term potentiation-related cytoskeletal changes in

Factor: A Dynamic Gatekeeper of Neural Plasticity. Curr Mol Pharmacol.

parametric systematic review. Psychol Med , 36, 1065-1073.

in late life. Findings from the Nun Study. JAMA , 275, 528-532.

trophic factor in synaptic plasticity. Mol Cell Neurosci , 42, 81-89.

Nun Study. Neurobiol Aging , 26, 341-347.

adult hippocampus. J Neurosci , 27, 3017-3029.

rosci , 10, 850-860.


[21] Wang, C, Wilson, W. A, Moore, S. D, Mace, B. E, Maeda, N, et al. (2005). Human apoE4-targeted replacement mice display synaptic deficits in the absence of neuropa‐ thology. Neurobiol Dis , 18, 390-398.

[34] Spira, M. E, Oren, R, Dormann, A, & Gitler, D. (2003). Critical calpain-dependent ul‐ trastructural alterations underlie the transformation of an axonal segment into a growth cone after axotomy of cultured Aplysia neurons. J Comp Neurol , 457,

Brain Reserve Regulators in Alzheimer's Disease

http://dx.doi.org/10.5772/53658

161

[35] Mejillano, M. R, Kojima, S, Applewhite, D. A, Gertler, F. B, Svitkina, T. M, et al. (2004). Lamellipodial versus filopodial mode of the actin nanomachinery: pivotal role

[36] Schafer, D. A, Korshunova, Y. O, Schroer, T. A, & Cooper, J. A. (1994). Differential localization and sequence analysis of capping protein beta-subunit isoforms of verte‐

[37] Davis, D. A, Wilson, M. H, Giraud, J, Xie, Z, Tseng, H. C, et al. (2009). Capzb2 inter‐ acts with beta-tubulin to regulate growth cone morphology and neurite outgrowth.

[38] Nelson, T. J, & Backlund, P. S. Jr., Alkon DL ((2004). Hippocampal protein-protein in‐

[39] Linnarsson, S, Bjorklund, A, & Ernfors, P. (1997). Learning deficit in BDNF mutant

[40] Scheibel, A. B, & Tomiyasu, U. (1978). Dendritic sprouting in Alzheimer's presenile

[41] Scheibel, A. B. (1979). Dendritic changes in senile and presenile dementias. Res Publ

[42] Kao, P. F, Davis, D. A, Banigan, M. G, Vanderburg, C. R, Seshadri, S, et al. Modula‐ tors of cytoskeletal reorganization in CA1 hippocampal neurons show increased ex‐

[43] Grande, I, Fries, G. R, Kunz, M, & Kapczinski, F. (2010). The role of BDNF as a medi‐ ator of neuroplasticity in bipolar disorder. Psychiatry Investig , 7, 243-250.

[44] Labelle, C, & Leclerc, N. (2000). Exogenous BDNF, NT-3 and NT-4 differentially reg‐ ulate neurite outgrowth in cultured hippocampal neurons. Brain Res Dev Brain Res ,

[45] Kitanishi, T, Sakai, J, Kojima, S, Saitoh, Y, Inokuchi, K, et al. Activity-dependent lo‐ calization in spines of the F-actin capping protein CapZ screened in a rat model of

[46] Kao, P. F, Banigan, M. G, Vanderburg, C. R, Mckee, A. C, Polgar, P. R, et al. (2012). Increased Expression of TrkB and Capzb2 Accompanies Preserved Cognitive Status in Early Alzheimer Disease Pathology. J Neuropathol Exp Neurol , 71, 654-664.

pression in patients at mid-stage Alzheimer's disease. PLoS One 5: e13337.

of the filament barbed end. Cell , 118, 363-373.

teractions in spatial memory. Hippocampus , 14, 46-57.

brates. J Cell Biol , 127, 453-465.

mice. Eur J Neurosci , 9, 2581-2587.

Assoc Res Nerv Ment Dis , 57, 107-124.

dementia. Exp Neurol , 60, 1-8.

123, 1-11.

dementia. Genes Cells.

PLoS Biol 7: e1000208.

293-312.


[34] Spira, M. E, Oren, R, Dormann, A, & Gitler, D. (2003). Critical calpain-dependent ul‐ trastructural alterations underlie the transformation of an axonal segment into a growth cone after axotomy of cultured Aplysia neurons. J Comp Neurol , 457, 293-312.

[21] Wang, C, Wilson, W. A, Moore, S. D, Mace, B. E, Maeda, N, et al. (2005). Human apoE4-targeted replacement mice display synaptic deficits in the absence of neuropa‐

[22] Klein, R. C, Mace, B. E, & Moore, S. D. Sullivan PM Progressive loss of synaptic in‐ tegrity in human apolipoprotein E4 targeted replacement mice and attenuation by

[23] Riemenschneider, M, Schwarz, S, Wagenpfeil, S, Diehl, J, Muller, U, et al. (2002). A polymorphism of the brain-derived neurotrophic factor (BDNF) is associated with Alzheimer's disease in patients lacking the Apolipoprotein E epsilon4 allele. Mol

[24] Lee, J, Fukumoto, H, Orne, J, Klucken, J, Raju, S, et al. (2005). Decreased levels of BDNF protein in Alzheimer temporal cortex are independent of BDNF polymor‐

[25] Forlenza, O. V, Diniz, B. S, Teixeira, A. L, Ojopi, E. B, Talib, L. L, et al. Effect of brainderived neurotrophic factor Val66Met polymorphism and serum levels on the pro‐

[26] Nichol, K, Deeny, S. P, Seif, J, Camaclang, K, & Cotman, C. W. (2009). Exercise im‐ proves cognition and hippocampal plasticity in APOE epsilon4 mice. Alzheimers De‐

[27] Kuzumaki, N, Ikegami, D, Tamura, R, Hareyama, N, Imai, S, et al. Hippocampal epi‐ genetic modification at the brain-derived neurotrophic factor gene induced by an en‐

[28] Verghese, J, Lipton, R. B, Katz, M. J, Hall, C. B, Derby, C. A, et al. (2003). Leisure ac‐ tivities and the risk of dementia in the elderly. N Engl J Med , 348, 2508-2516.

[29] Larson, E. B, Wang, L, Bowen, J. D, Mccormick, W. C, Teri, L, et al. (2006). Exercise is associated with reduced risk for incident dementia among persons 65 years of age

[30] Pasinetti, G. M, Zhao, Z, Qin, W, Ho, L, Shrishailam, Y, et al. (2007). Caloric intake and Alzheimer's disease. Experimental approaches and therapeutic implications. In‐

[31] Mckee, A. C, Kowall, N. W, & Kosik, K. S. (1989). Microtubular reorganization and dendritic growth response in Alzheimer's disease. Ann Neurol , 26, 652-659.

[32] Dent, E. W, & Gertler, F. B. (2003). Cytoskeletal dynamics and transport in growth

[33] Sabo, S. L, Ikin, A. F, Buxbaum, J. D, & Greengard, P. (2003). The amyloid precursor protein and its regulatory protein, FE65, in growth cones and synapses in vitro and

gression of mild cognitive impairment. World J Biol Psychiatry , 11, 774-780.

thology. Neurobiol Dis , 18, 390-398.

Psychiatry , 7, 782-785.

160 Neurodegenerative Diseases

ment , 5, 287-294.

phisms. Exp Neurol , 194, 91-96.

apolipoprotein E2. Neuroscience , 171, 1265-1272.

riched environment. Hippocampus , 21, 127-132.

cone motility and axon guidance. Neuron , 40, 209-227.

and older. Ann Intern Med , 144, 73-81.

terdiscip Top Gerontol , 35, 159-175.

in vivo. J Neurosci , 23, 5407-5415.


[47] Farmer, M. E, White, L. R, Kittner, S. J, Kaplan, E, Moes, E, et al. (1987). Neuropsy‐ chological test performance in Framingham: a descriptive study. Psychol Rep , 60, 1023-1040.

[60] Albert, M. S, Dekosky, S. T, Dickson, D, Dubois, B, Feldman, H. H, et al. (2011). The diagnosis of mild cognitive impairment due to Alzheimer's disease: recommenda‐ tions from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease. Alzheimers Dement , 7, 270-279. [61] Sperling, R. A, Aisen, P. S, Beckett, L. A, Bennett, D. A, Craft, S, et al. (2011). Toward defining the preclinical stages of Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic

Brain Reserve Regulators in Alzheimer's Disease

http://dx.doi.org/10.5772/53658

163

[62] Wu, A, Ying, Z, & Gomez-pinilla, F. (2008). Docosahexaenoic acid dietary supple‐ mentation enhances the effects of exercise on synaptic plasticity and cognition. Neu‐

[63] Sananbenesi, F, & Fischer, A. (2009). The epigenetic bottleneck of neurodegenerative

[64] Fischer, A, Sananbenesi, F, Wang, X, Dobbin, M, & Tsai, L. H. (2007). Recovery of learning and memory is associated with chromatin remodelling. Nature , 447,

[65] Been, E, Barash, A, Pessah, H, & Peleg, S. (2010). A new look at the geometry of the

[66] Fischer, A, Sananbenesi, F, Mungenast, A, & Tsai, L. H. (2010). Targeting the correct HDAC(s) to treat cognitive disorders. Trends Pharmacol Sci , 31, 605-617.

[67] Stilling, R. M, & Fischer, A. (2011). The role of histone acetylation in age-associated memory impairment and Alzheimer's disease. Neurobiol Learn Mem , 96, 19-26.

guidelines for Alzheimer's disease. Alzheimers Dement , 7, 280-292.

and psychiatric diseases. Biol Chem , 390, 1145-1153.

lumbar spine. Spine (Phila Pa 1976) 35: E , 1014-1017.

roscience , 155, 751-759.

178-182.


[60] Albert, M. S, Dekosky, S. T, Dickson, D, Dubois, B, Feldman, H. H, et al. (2011). The diagnosis of mild cognitive impairment due to Alzheimer's disease: recommenda‐ tions from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease. Alzheimers Dement , 7, 270-279.

[47] Farmer, M. E, White, L. R, Kittner, S. J, Kaplan, E, Moes, E, et al. (1987). Neuropsy‐ chological test performance in Framingham: a descriptive study. Psychol Rep , 60,

[48] Au, R, Seshadri, S, Wolf, P. A, Elias, M, Elias, P, et al. (2004). New norms for a new generation: cognitive performance in the framingham offspring cohort. Exp Aging

[49] Mckhann, G, Drachman, D, Folstein, M, Katzman, R, Price, D, et al. (1984). Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheim‐

[50] Morris, J. C, Weintraub, S, Chui, H. C, Cummings, J, Decarli, C, et al. (2006). The Uni‐ form Data Set (UDS): clinical and cognitive variables and descriptive data from Alz‐

[51] Weintraub, S, Salmon, D, Mercaldo, N, Ferris, S, Graff-radford, N. R, et al. (2009). The Alzheimer's Disease Centers' Uniform Data Set (UDS): the neuropsychologic test bat‐

[52] Blessed, G, Tomlinson, B. E, & Roth, M. (1968). The association between quantitative measures of dementia and of senile change in the cerebral grey matter of elderly sub‐

[53] Hachinski, V. C, Iliff, L. D, & Zilhka, E. Du Boulay GH, McAllister VL, et al. ((1975).

[54] Davis, P. B, White, H, Price, J. L, Mckeel, D, & Robins, L. N. (1991). Retrospective postmortem dementia assessment. Validation of a new clinical interview to assist

[55] Nagahara, A. H, Merrill, D. A, Coppola, G, Tsukada, S, Schroeder, B. E, et al. (2009). Neuroprotective effects of brain-derived neurotrophic factor in rodent and primate

[56] Frank, L, Ventimiglia, R, Anderson, K, Lindsay, R. M, & Rudge, J. S. (1996). BDNF down-regulates neurotrophin responsiveness, TrkB protein and TrkB mRNA levels

[57] Connor, B, Young, D, Lawlor, P, Gai, W, Waldvogel, H, et al. (1996). Trk receptor al‐

[58] Seshadri, S, Wolf, P. A, Beiser, A, Au, R, Mcnulty, K, et al. (1997). Lifetime risk of de‐ mentia and Alzheimer's disease. The impact of mortality on risk estimates in the Fra‐

[59] Seshadri, S, & Wolf, P. A. (2007). Lifetime risk of stroke and dementia: current con‐ cepts, and estimates from the Framingham Study. Lancet Neurol , 6, 1106-1114.

in cultured rat hippocampal neurons. Eur J Neurosci , 8, 1220-1230.

terations in Alzheimer's disease. Brain Res Mol Brain Res , 42, 1-17.

heimer Disease Centers. Alzheimer Dis Assoc Disord , 20, 210-216.

Cerebral blood flow in dementia. Arch Neurol , 32, 632-637.

neuropathologic study. Arch Neurol , 48, 613-617.

models of Alzheimer's disease. Nat Med , 15, 331-337.

mingham Study. Neurology , 49, 1498-1504.

1023-1040.

162 Neurodegenerative Diseases

Res , 30, 333-358.

er's Disease. Neurology , 34, 939-944.

tery. Alzheimer Dis Assoc Disord , 23, 91-101.

jects. Br J Psychiatry , 114, 797-811.


**Chapter 8**

**Cholesterol and Alzheimer's Disease**

Additional information is available at the end of the chapter

Cholesterol plays a central role in the brain's metabolism: the fact that the brain accounts for only 2% of the body mass and brain cholesterol represents 25% of the total body cholesterol speaks for itself. Overall, the brain is the organ with the highest content of

Direct analysis of brain cholesterol metabolism is further complicated by the separation of

It is commonly assumed that all brain cholesterol originates from in situ neo-synthesis. This conclusion is based mainly on studies tracking the incorporation of tritiated water into the

Most brain cholesterol is unesterified (free) and is found within the specialized membranes of myelin. Since myelin has a very slow turnover rate, myelin-associated cholesterol is virtually

The remaining brain cholesterol is found in neurons, glial cells and extracellular lipoproteins, and these pools of cholesterol participate in cholesterol homeostatis of the CNS. Considering that the large mass of cholesterol sequestered into myelin membranes, makes the analysis of

Direct quantification of plasma cholesterol in the brain, which is very difficult, has thus far

The membrane concentration of cholesterol is maintained within extremely fine variation limits owing to numerous homeostasis mechanisms. These mechanisms can be altered

and reproduction in any medium, provided the original work is properly cited.

© 2013 Nicola-Antoniu; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

cholesterol distribution in the brain (brain cholesterol) technically challenging.

genetically, by environment factors, through aging or dietary induced.

brain cholesterol from plasma cholesterol owing to the blood-brain barrier.

Iuliana Nicola-Antoniu

http://dx.doi.org/10.5772/55502

**1. Introduction**

cholesterol in the body.

immobilized.

yielded negative results.

pool of sterols contained in the brain.

**Chapter 8**

### **Cholesterol and Alzheimer's Disease**

Iuliana Nicola-Antoniu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55502

### **1. Introduction**

Cholesterol plays a central role in the brain's metabolism: the fact that the brain accounts for only 2% of the body mass and brain cholesterol represents 25% of the total body cholesterol speaks for itself. Overall, the brain is the organ with the highest content of cholesterol in the body.

Direct analysis of brain cholesterol metabolism is further complicated by the separation of brain cholesterol from plasma cholesterol owing to the blood-brain barrier.

It is commonly assumed that all brain cholesterol originates from in situ neo-synthesis. This conclusion is based mainly on studies tracking the incorporation of tritiated water into the pool of sterols contained in the brain.

Most brain cholesterol is unesterified (free) and is found within the specialized membranes of myelin. Since myelin has a very slow turnover rate, myelin-associated cholesterol is virtually immobilized.

The remaining brain cholesterol is found in neurons, glial cells and extracellular lipoproteins, and these pools of cholesterol participate in cholesterol homeostatis of the CNS. Considering that the large mass of cholesterol sequestered into myelin membranes, makes the analysis of cholesterol distribution in the brain (brain cholesterol) technically challenging.

Direct quantification of plasma cholesterol in the brain, which is very difficult, has thus far yielded negative results.

The membrane concentration of cholesterol is maintained within extremely fine variation limits owing to numerous homeostasis mechanisms. These mechanisms can be altered genetically, by environment factors, through aging or dietary induced.

© 2013 Nicola-Antoniu; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The studies carried out throughout the years have shown cholesterol's vital importance for the brain:

Membrane rafts and isoprenylation play an important role in transforming the amyloid

Cholesterol and Alzheimer's Disease http://dx.doi.org/10.5772/55502 167

Lipid rafts are heterogenous, cholesterol and sphingolipid-rich membrane microdomains that mediate compartmentalized cellular processes by clustering receptors and signalling mole‐ cules. These dynamic lipid–protein assemblies are enriched in saturated glycerophospholipids

Raft lipids are believed to be held together by relatively weak covalent bonds, establishing a dynamic equilibrium of raft and non-raft regions within the plasma membrane. The density, size, duration and exact composition of the rafts varies based on the cell type. In average they have a 50 nm diameter (between 10-100 nm), each comprising approximately 20 protein molecules. Theoretically, a cell may have approximately 1,000,00 membrane rafts, representing

When activated, membrane rafts have been shown to function as a concentrating platform for a variety of signal transduction molecules. During activation, several rafts cluster forming large platforms in order to allow the functional proteins to concentrate and interact. An especially important aspect is that this concentration is cholesterol dependant. Thus, mem‐ brane rafts have a central role in regulating a few cellular processes, such as membrane sorting,

Even clearer evidence shows that amyloid processing of APP takes place at raft level, especially due to the presence of the active functional pool of BACE 1 and gamma-secretase at the level of these micro-domains. An even more supported idea is that the decreased association of these proteins at raft level would be beneficial for AD. When supporting this theory, the possibility

Although, APP, BACE1 and presenile domains are present both in raft areas, as well as in membrane areas without rafts, APP processing at raft level seems to be predominantly amyloidogenic, while outside the raft level, the APP processing is predominantly non

Cholesterol decrease does not favour the association of BACE1 with the lipid rafts, which correlates with the decrease of amyloidogenesis. In contrast, the acute exposure of cells to cholesterol stimulates the co clustering of APP and BACE1 at raft level and their fast

Besides gamma-secretase, the beta-secretase component seems to be located at membrane raft

Thus, we can draw the conclusion that the production of cholesterol-influenced amyloid is determined, at least partially, at BACE1 level present in lipid rafts. Moreover, APP's associa‐

Genetic, metabolic and biochemical studies have shown that intracellular cholesterol distribution, rather than total cholesterol levels, regulates APP processing and β-amyloid

and protein molecules with a high inherent affinity for ordered lipid domains.

precursor protein at gamma-secretase level.

more than half of the total membrane surface.

of interfering with bodily functions must be taken into account.

amyloidogenic, on the alpha-secretase pathway.

level. Cholesterol depletion abrogates this localization.

tion with rafts stimulates the formation of amyloid.

trafficking and signal transduction.

endocytosis.

generation.

Here are just a few examples:


The interplay between Alzheimer's disease and cholesterol is very controversial, being supported by some authors and denied by others.

### **2. The involvement of cholesterol in the generation and deposit of amyloid beta**

Although it is not completely elucidated, today it is generally acknowledged that the debut and evolution of AD's molecular mechanism consists in the overproduction and accumulation of toxic amyloid beta proteins.

Numerous studies show the direct relation between the cholesterol levels and the amyloid volume at brain level. High cholesterol level (more than 5.8 mmol/L) is significantly related to the brain plaques associated with Alzheimer's disease in autopsied people; there wasn't found any link between high cholesterol and the tangles that develop in the brain (plaques and tangles both known to be trademark signs of Alzheimer's disease)

Of the other part, a series of prospective studies did not support the hypothesis according to which high levels of cholesterol represent a risk factor for Alzheimer's disease.

**•** Cholesterol and the formation of the β-amyloid precursor protein

The amyloid processing of the amyloid precursor protein takes place only in certain areas of the cell membrane and is favored by its high cholesterol content.

Membrane rafts and isoprenylation play an important role in transforming the amyloid precursor protein at gamma-secretase level.

The studies carried out throughout the years have shown cholesterol's vital importance for

**•** It is a limiting factor in the ability to form synapses. The "factor" searched for since 1997, that acts on the glial cells, stimulating them to increase synapse formation, was identified

**•** Synapses formed in the presence of cholesterol rich glial cells proved to be more effective and functional. When neurons deprived of this glial secretion were exposed to a solution of

**•** THUS, cholesterol helps us think, learn and memorize by regulating the plasticity of

**•** Cholesterol has also been discovered to play an important role in forming what are called *lipid rafts,* areas in the plasma membrane of cells that anchor certain proteins important to cell signaling. Several of the proteins anchored in these *rafts* are responsible for stimulating

The interplay between Alzheimer's disease and cholesterol is very controversial, being

**2. The involvement of cholesterol in the generation and deposit of amyloid**

Although it is not completely elucidated, today it is generally acknowledged that the debut and evolution of AD's molecular mechanism consists in the overproduction and accumulation

Numerous studies show the direct relation between the cholesterol levels and the amyloid volume at brain level. High cholesterol level (more than 5.8 mmol/L) is significantly related to the brain plaques associated with Alzheimer's disease in autopsied people; there wasn't found any link between high cholesterol and the tangles that develop in the brain (plaques and tangles

Of the other part, a series of prospective studies did not support the hypothesis according to

The amyloid processing of the amyloid precursor protein takes place only in certain areas of

which high levels of cholesterol represent a risk factor for Alzheimer's disease.

**•** Cholesterol and the formation of the β-amyloid precursor protein

the cell membrane and is favored by its high cholesterol content.

**•** It is the main regulator of the structure of lipids and neuronal membrane fluidity.

the brain:

166 Neurodegenerative Diseases

synapses.

**beta**

of toxic amyloid beta proteins.

Here are just a few examples:

in 2001. It proved to be cholesterol.

and guiding the growth of nerve axons.

supported by some authors and denied by others.

both known to be trademark signs of Alzheimer's disease)

cholesterol, synapse formation increased by twelve times.

Lipid rafts are heterogenous, cholesterol and sphingolipid-rich membrane microdomains that mediate compartmentalized cellular processes by clustering receptors and signalling mole‐ cules. These dynamic lipid–protein assemblies are enriched in saturated glycerophospholipids and protein molecules with a high inherent affinity for ordered lipid domains.

Raft lipids are believed to be held together by relatively weak covalent bonds, establishing a dynamic equilibrium of raft and non-raft regions within the plasma membrane. The density, size, duration and exact composition of the rafts varies based on the cell type. In average they have a 50 nm diameter (between 10-100 nm), each comprising approximately 20 protein molecules. Theoretically, a cell may have approximately 1,000,00 membrane rafts, representing more than half of the total membrane surface.

When activated, membrane rafts have been shown to function as a concentrating platform for a variety of signal transduction molecules. During activation, several rafts cluster forming large platforms in order to allow the functional proteins to concentrate and interact. An especially important aspect is that this concentration is cholesterol dependant. Thus, mem‐ brane rafts have a central role in regulating a few cellular processes, such as membrane sorting, trafficking and signal transduction.

Even clearer evidence shows that amyloid processing of APP takes place at raft level, especially due to the presence of the active functional pool of BACE 1 and gamma-secretase at the level of these micro-domains. An even more supported idea is that the decreased association of these proteins at raft level would be beneficial for AD. When supporting this theory, the possibility of interfering with bodily functions must be taken into account.

Although, APP, BACE1 and presenile domains are present both in raft areas, as well as in membrane areas without rafts, APP processing at raft level seems to be predominantly amyloidogenic, while outside the raft level, the APP processing is predominantly non amyloidogenic, on the alpha-secretase pathway.

Cholesterol decrease does not favour the association of BACE1 with the lipid rafts, which correlates with the decrease of amyloidogenesis. In contrast, the acute exposure of cells to cholesterol stimulates the co clustering of APP and BACE1 at raft level and their fast endocytosis.

Besides gamma-secretase, the beta-secretase component seems to be located at membrane raft level. Cholesterol depletion abrogates this localization.

Thus, we can draw the conclusion that the production of cholesterol-influenced amyloid is determined, at least partially, at BACE1 level present in lipid rafts. Moreover, APP's associa‐ tion with rafts stimulates the formation of amyloid.

Genetic, metabolic and biochemical studies have shown that intracellular cholesterol distribution, rather than total cholesterol levels, regulates APP processing and β-amyloid generation.

At cellular level, the highest concentration of cholesterol is at plasmatic membrane level, just like in the endocytic recycling compartment. Because the blood-brain barrier prevents any exchange of lipoproteins between the serum and the brain, most of the brain cholesterol is synthesized de novo de novo, at glial level.

equilibrium (report) between free cholesterol and cholesteryl-esters is the key element for

Cholesterol and Alzheimer's Disease http://dx.doi.org/10.5772/55502 169

Abeta in turn regulates cholesterol metabolism. Abeta 40 supresses the mevalonate pathway

The HMG-CoA reductase pathway, also called the mevalonate pathway, leads to cholesterol synthesis, as well as supplies indispensable lipids, such as isoprenoids, to eukaryotic cells. (The statins that block the HMG-CoA reductase pathway can thus manifest their effects

Long-chain isoprenoids participate in membrane organization and proteic glycosylation, as

The short-chain isoprenoids (farnesylpyrophosphate-FPP) and metabolite or geranylgeranyl‐ pyrophosphate (GGPP) are used for the isoprenylation of complex proteins, including nuclear

Isoprenylation of proteins, respectively farnsylation and geranylgeranylation influence the

Recent studies indicate that AD has a metabolism interference at FPP and GGPP level, consequently affecting signalling through GTPases. This is relevant for AD because signalling through GTPases controls multiple aspects of amyloidogenesis, including trafficking of APP,

Intracellular amyloid beta induces the increase of oxidative stress through mitochondrial

Recent studies evince that the specific mitochondrial cholesterol pool sensitizes neurons to cell death (induced by oxidative stress) as well as at caspase-independent apoptosis. This is due to due to selective mitochondrial GSH (mGSH) depletion induced by cholesterol-mediated

"MGSH replenishment by permeable precursors such as glutatione ethyl ester protected

Thus, mitochondrial cholesterol determines amyloid beta neurotoxicity by regulating mito‐

In the relation between CHOLESTEROL-AMYLOID it is essential to analyze the hypercho‐

The increased levels of serum cholesterol are the result of different factors that should be taken into account. For instance, familial hypercholesterolemia involves a dysfunction of LDL

by inhibiting HMG CoA reductase, thus decreasing the cholesterol level.

through a cholesterol independent mechanism).

well as in mitochondrial oxygen consumption.

signalling capacity of the respective proteins.

**•** Cholesterol and intracellular accumulation of amyloid beta

perturbation of mitochondrial membrane dynamics.

against Ab-mediated neurotoxicity and inflammation"

BACE1 and the secretase complex.

controlling amyloidogenesis.

**3. Isoprenylation**

lamins and GTPases.

perturbation.

chondrial GSH.

lesterolemia *subtypes.*

The excess free cholesterol is transformed by acyl-conezyme A: cholesterol acyltransferase (ACAT) resulting with the intracellular accumulation of lipid droplets or trans-membrane efflux in the extra-cellular environment.

ACAT is essential for the regulation of intracellular cholesterol homeostasis and distribution of cholesterol throughout the body. In the small intestine and liver it also regulates the secretion of chylomicrons and very large-density lipoproteins (VLDL).

Mammals, including humans, express two different isoforms of ACAT, called ACAT-1 and ACAT-2. Whereas ACAT-1 is almost uniformly distributed among several tissues, including the brain, ACAT-2 is selectively expressed in the intestine and liver. Both forms of ACAT are ER resident enzymes, allosterically regulated by cholesterol available in the ER membrane.

Acyl-coenzyme A cholesterol acyltrasferase (ACAT) catalyzes the formation of cholesterylesters from cholesterol and long-chain fatty acids. ACAT controls the dynamic equilibrium between these two forms of cellular cholesterol, ultimately affecting cholesterol homeostasis. This dynamic equilibrium ultimately regulates the generation of β-amyloid (Aβ). A selective increase in cholesteryl-esters is sufficient to up-regulate the generation of Aβ and increase the steady-state levels of β-APP CTFs (C-terminal excerpt). It has been shown that at neuron level, the ACAT competitive inhibitors reduce both cholesteryl-ester and Aβ biosynthesis in a dosedependant manner, while increasing free cholesterol. Similar results were obtained with agents that block delivery of free cholesterol to ACAT.

How free cholesterol and cholesteryl-ester distribution affects the Aβ synthesis is not yet clear. HOWEVER, the participation of free cholesterol in cellular membranes must be also taken into account because altered cholesteryl-ester levels modulate the free cholesterol pool. Even undetectable changes in free cholesterol levels may affect APP processing.

Second, studies have shown that ACAT regulates ALL three cleavages of APP. Thus, altered cholesterol distribution affects either the activity of all three secretases, APP itself, or an-yetunidentified protetin that controls APP processing.

The increase of cholesteryl-ester level in the cell cultures increases the release of Abeta, while the pharmacological inhibition of ACAT reduces the formation of cholesteryl-esters, as well as Aβ. The genetic ablation of ACAT1 in the mouse model with AD reduces the formation of AD as well as cognitive decline.

The ACAT1 ablation also increases the oxy-cholesterol levels and 24S-hydrocholesterol, thus suggesting a potential reduction role of amyloidogenesis for these cholesterol metabolites. THUS, a possible mechanism is that the excess brain cholesterol resulted from ACAT1 ablation can be transformed in 24S-hydroxycholesterol and in this form cross the blood-brain barrier to the periphery, reducing the brain cholesterol level. The corroborated data suggests that the equilibrium (report) between free cholesterol and cholesteryl-esters is the key element for controlling amyloidogenesis.

### **3. Isoprenylation**

At cellular level, the highest concentration of cholesterol is at plasmatic membrane level, just like in the endocytic recycling compartment. Because the blood-brain barrier prevents any exchange of lipoproteins between the serum and the brain, most of the brain cholesterol is

The excess free cholesterol is transformed by acyl-conezyme A: cholesterol acyltransferase (ACAT) resulting with the intracellular accumulation of lipid droplets or trans-membrane

ACAT is essential for the regulation of intracellular cholesterol homeostasis and distribution of cholesterol throughout the body. In the small intestine and liver it also regulates the secretion

Mammals, including humans, express two different isoforms of ACAT, called ACAT-1 and ACAT-2. Whereas ACAT-1 is almost uniformly distributed among several tissues, including the brain, ACAT-2 is selectively expressed in the intestine and liver. Both forms of ACAT are ER resident enzymes, allosterically regulated by cholesterol available in the ER membrane.

Acyl-coenzyme A cholesterol acyltrasferase (ACAT) catalyzes the formation of cholesterylesters from cholesterol and long-chain fatty acids. ACAT controls the dynamic equilibrium between these two forms of cellular cholesterol, ultimately affecting cholesterol homeostasis. This dynamic equilibrium ultimately regulates the generation of β-amyloid (Aβ). A selective increase in cholesteryl-esters is sufficient to up-regulate the generation of Aβ and increase the steady-state levels of β-APP CTFs (C-terminal excerpt). It has been shown that at neuron level, the ACAT competitive inhibitors reduce both cholesteryl-ester and Aβ biosynthesis in a dosedependant manner, while increasing free cholesterol. Similar results were obtained with agents

How free cholesterol and cholesteryl-ester distribution affects the Aβ synthesis is not yet clear. HOWEVER, the participation of free cholesterol in cellular membranes must be also taken into account because altered cholesteryl-ester levels modulate the free cholesterol pool. Even

Second, studies have shown that ACAT regulates ALL three cleavages of APP. Thus, altered cholesterol distribution affects either the activity of all three secretases, APP itself, or an-yet-

The increase of cholesteryl-ester level in the cell cultures increases the release of Abeta, while the pharmacological inhibition of ACAT reduces the formation of cholesteryl-esters, as well as Aβ. The genetic ablation of ACAT1 in the mouse model with AD reduces the formation of

The ACAT1 ablation also increases the oxy-cholesterol levels and 24S-hydrocholesterol, thus suggesting a potential reduction role of amyloidogenesis for these cholesterol metabolites. THUS, a possible mechanism is that the excess brain cholesterol resulted from ACAT1 ablation can be transformed in 24S-hydroxycholesterol and in this form cross the blood-brain barrier to the periphery, reducing the brain cholesterol level. The corroborated data suggests that the

undetectable changes in free cholesterol levels may affect APP processing.

synthesized de novo de novo, at glial level.

168 Neurodegenerative Diseases

efflux in the extra-cellular environment.

that block delivery of free cholesterol to ACAT.

unidentified protetin that controls APP processing.

AD as well as cognitive decline.

of chylomicrons and very large-density lipoproteins (VLDL).

Abeta in turn regulates cholesterol metabolism. Abeta 40 supresses the mevalonate pathway by inhibiting HMG CoA reductase, thus decreasing the cholesterol level.

The HMG-CoA reductase pathway, also called the mevalonate pathway, leads to cholesterol synthesis, as well as supplies indispensable lipids, such as isoprenoids, to eukaryotic cells.

(The statins that block the HMG-CoA reductase pathway can thus manifest their effects through a cholesterol independent mechanism).

Long-chain isoprenoids participate in membrane organization and proteic glycosylation, as well as in mitochondrial oxygen consumption.

The short-chain isoprenoids (farnesylpyrophosphate-FPP) and metabolite or geranylgeranyl‐ pyrophosphate (GGPP) are used for the isoprenylation of complex proteins, including nuclear lamins and GTPases.

Isoprenylation of proteins, respectively farnsylation and geranylgeranylation influence the signalling capacity of the respective proteins.

Recent studies indicate that AD has a metabolism interference at FPP and GGPP level, consequently affecting signalling through GTPases. This is relevant for AD because signalling through GTPases controls multiple aspects of amyloidogenesis, including trafficking of APP, BACE1 and the secretase complex.

**•** Cholesterol and intracellular accumulation of amyloid beta

Intracellular amyloid beta induces the increase of oxidative stress through mitochondrial perturbation.

Recent studies evince that the specific mitochondrial cholesterol pool sensitizes neurons to cell death (induced by oxidative stress) as well as at caspase-independent apoptosis. This is due to due to selective mitochondrial GSH (mGSH) depletion induced by cholesterol-mediated perturbation of mitochondrial membrane dynamics.

"MGSH replenishment by permeable precursors such as glutatione ethyl ester protected against Ab-mediated neurotoxicity and inflammation"

Thus, mitochondrial cholesterol determines amyloid beta neurotoxicity by regulating mito‐ chondrial GSH.

In the relation between CHOLESTEROL-AMYLOID it is essential to analyze the hypercho‐ lesterolemia *subtypes.*

The increased levels of serum cholesterol are the result of different factors that should be taken into account. For instance, familial hypercholesterolemia involves a dysfunction of LDL receptors. On the one hand, cholesterol levels in the blood increase because they cannot be received into cells, and on the other, the absence of properly functioning LDL receptors could be causing other intracellular problems. Thus this more likely results from a specific *defect of receptors*, rather than from high cholesterol, or even a *deficiency of intracellular cholesterol*.

**5. HDL colesterol and AD**

Launer on "Japanese-American men".)

the HDL levels to try to prevent AD.

and under 50 mg/dL for women.

brain.

of sporadic AD.

**6. APOE- cholesterol and AD**

heart disease, you also reduce the risk of AD.

via associated receptor for LDL (LRP) and VLDL.

is synthesized in situ, especially by astrocytes.

present in at least one copy in less than 25% of the population.

relevant with stroke, and stroke is associated with AD.

HDL is one of the main protein carriers in the brain. It can cross the brain-blood barrier. It is difficult to estimate to what extent its serum value reflects its brain concentration. Studies carried out throughout the years regarding the correlation between cholesterol HDL – AD have not allowed to reach a decisive conclusion. Some authors say that the HDL association is more

Cholesterol and Alzheimer's Disease http://dx.doi.org/10.5772/55502 171

Other authors have found an interplay between the high levels of HDL cholesterol and the presence of plaques and intraneuronal neurofibrillary tangles (study carried out in 2001 by

Until now, the data offered by the studies do not allow to make a recommendation regarding the HDL cholesterol levels for preventing AD. We have no reasonable argument for boosting

It should be considered to keep cholesterol under control (within the recommended values) in order to reduce the risk of heart diseases. This has sense because by reducing the risk of

Guidelines recommend an optimal HDL level of 60 mg/dL or higher, under 40 mg/dL for men

ApoE is one of the major plasma apolipoproteins and the principal cholesterol carrier in the

ApoE encodes a 34kDa protein that is an essential regulator of the brain cholesterol metabolism and triglycerides in the body. It mediates the capturing of lipoprotein particles from the brain

In humans, there are three common alleles of the ApoE gene: ε2, ε3 and ε4. The protein isoforms produced by these alleles differ in the amino acids at positions 112 and/or 158: E2 (Cys112, Cys158), E3 (Cys112, Arg158), which is the most common, and E4 (Arg112,Arg158), which is

Peripheral apolipoprotein E is synthesized in the liver, while apolipoprotein E from the brain

It has been confirmed that APOE ε4 allele is the most prevalent risk factor for the development

The risk for Alzheimer's disease conferred by APOE ε4 increases in a dose-dependant manner; individuals that are homozygous for APOE ε4 alleles are 8 times more likely to develop AD than are homozygotes for APOE ε3. Less than 2% of the population is homozygous for APOE ε4. This is neither necessary nor sufficient to cause AD; it only increases risk for the disease.

The role of the LDL receptor-related protein is so much more important and disputed now that we know that its associated proteins are responsible in both increased free brain choles‐ terol and increased beta-amyloid in Alzheimer's disease. They are also responsible for bringing apolipoprotein-E-associated cholesterol into cells. Apparently, the deficiency or dysfunction of LRP could be a factor that results in both increased free brain cholesterol and increased betaamyloid, which could be accidental.

### **4. Tau pathology and cholesterol**

Together with amyloid deposits, the second defining element of AD from a histopathological point of view are the intraneuronal neurofibrillary tangles, formed from hyperphosphoryla‐ tion of Tau protein.

In contrast with APP and secretases, which are membrane bound, tau is a protein that stabilizes microtubules, which are found in cytoplasm.

However there is an interaction between cholesterol and its pathology.


### **5. HDL colesterol and AD**

receptors. On the one hand, cholesterol levels in the blood increase because they cannot be received into cells, and on the other, the absence of properly functioning LDL receptors could be causing other intracellular problems. Thus this more likely results from a specific *defect of receptors*, rather than from high cholesterol, or even a *deficiency of intracellular cholesterol*.

The role of the LDL receptor-related protein is so much more important and disputed now that we know that its associated proteins are responsible in both increased free brain choles‐ terol and increased beta-amyloid in Alzheimer's disease. They are also responsible for bringing apolipoprotein-E-associated cholesterol into cells. Apparently, the deficiency or dysfunction of LRP could be a factor that results in both increased free brain cholesterol and increased beta-

Together with amyloid deposits, the second defining element of AD from a histopathological point of view are the intraneuronal neurofibrillary tangles, formed from hyperphosphoryla‐

In contrast with APP and secretases, which are membrane bound, tau is a protein that stabilizes

**•** The Aβ signalling pathway perturbs pathways involving lipid metabolizing enzymes

**•** The cholesterol levels are a key parameter controlling Aβ-induced tau proteolysis by calpain and proteolytic cleavage of tau by this protease, but also by caspases. These seem to be the

**•** A pool of hyperphosphorylated tau is present in lipid rafts, along with APP metabolites, BACE1, the γ-secretase complex and ApoE. Thus, evincing that significant crosstalk may

**•** Kinases implicated in tau phosphorylation, such as Cdk5 and GSK-3β, are activated on cellular membranes and thus dysregulation of lipid metabolism may affect the activity of

**•** The aggregate treatment pathway is also influenced by the lipidic metabolism. Cdk5 phosphorylates the lipid kinase Vps34, whose product PtdIns3P, may regulate the clearance

**•** More and more theories support that the propagation of the tau pathology is similar to prionlike infections, by crossing cell membranes, case in which the importance of the cell

However there is an interaction between cholesterol and its pathology.

amyloid, which could be accidental.

tion of Tau protein.

170 Neurodegenerative Diseases

these kinases.

**4. Tau pathology and cholesterol**

microtubules, which are found in cytoplasm.

ultimately affecting tau phosphorylation

early steps leading to tau pathology.

exist between Aβ and tau at the raft interface.

MEMBRANE's functionality comes forefront.

of tau aggregates by stimulating the autophagy pathway

HDL is one of the main protein carriers in the brain. It can cross the brain-blood barrier. It is difficult to estimate to what extent its serum value reflects its brain concentration. Studies carried out throughout the years regarding the correlation between cholesterol HDL – AD have not allowed to reach a decisive conclusion. Some authors say that the HDL association is more relevant with stroke, and stroke is associated with AD.

Other authors have found an interplay between the high levels of HDL cholesterol and the presence of plaques and intraneuronal neurofibrillary tangles (study carried out in 2001 by Launer on "Japanese-American men".)

Until now, the data offered by the studies do not allow to make a recommendation regarding the HDL cholesterol levels for preventing AD. We have no reasonable argument for boosting the HDL levels to try to prevent AD.

It should be considered to keep cholesterol under control (within the recommended values) in order to reduce the risk of heart diseases. This has sense because by reducing the risk of heart disease, you also reduce the risk of AD.

Guidelines recommend an optimal HDL level of 60 mg/dL or higher, under 40 mg/dL for men and under 50 mg/dL for women.

### **6. APOE- cholesterol and AD**

ApoE is one of the major plasma apolipoproteins and the principal cholesterol carrier in the brain.

ApoE encodes a 34kDa protein that is an essential regulator of the brain cholesterol metabolism and triglycerides in the body. It mediates the capturing of lipoprotein particles from the brain via associated receptor for LDL (LRP) and VLDL.

In humans, there are three common alleles of the ApoE gene: ε2, ε3 and ε4. The protein isoforms produced by these alleles differ in the amino acids at positions 112 and/or 158: E2 (Cys112, Cys158), E3 (Cys112, Arg158), which is the most common, and E4 (Arg112,Arg158), which is present in at least one copy in less than 25% of the population.

Peripheral apolipoprotein E is synthesized in the liver, while apolipoprotein E from the brain is synthesized in situ, especially by astrocytes.

It has been confirmed that APOE ε4 allele is the most prevalent risk factor for the development of sporadic AD.

The risk for Alzheimer's disease conferred by APOE ε4 increases in a dose-dependant manner; individuals that are homozygous for APOE ε4 alleles are 8 times more likely to develop AD than are homozygotes for APOE ε3. Less than 2% of the population is homozygous for APOE ε4. This is neither necessary nor sufficient to cause AD; it only increases risk for the disease.

The mechanisms through which APOE leads to the development of the disease are partially unknown.

Soluble Aβ interacts with APOE associated with lipoparticles and under-goes receptormediated endocytosis. The lipoproteins are enzymatically digested in the lysosomal compart‐ ment, releasing cholesterol to the cell. A fraction of APOE and Aβ undergoes degradation at lysosomal level. The rest of APOE remains associated with Aβ and promotes its aggregation

Cholesterol and Alzheimer's Disease http://dx.doi.org/10.5772/55502 173

The internalization of Aβ is not necessarily followed by its degradation. Amyloid aggregates from the endocytic compartment may be secreted in a more toxic fibrillar form. Finally, APOE

Moreover, the non-lypidic form of APOE seems to have a high-affinity binding activity to

Epidemiologic studies indicate that people with risk factors such as high blood pressure, diabetes, cerebrovascular disease and high cholesterol are two times more likely to develop

The causal chain between the high plasma cholesterol level and atherosclerosis is well defined. We can discuss a risk factor profile for sporadic cases of AD, with late-onset, which account for 90-95% of all cases. In elderly patients that suffer from AD there is an obvious link between this disease and vascular risk factors and atherosclerosis. However, the nature of this link remains partially speculative. Some authors have suggested that AD occurs as a secondary event related to atherosclerosis of extracranial or intracranial vessels with secondary cerebral hypoperfusion or small cerebral strokes. The toxic effects of vascular risk factors on susceptible

Another approach is that both AD and atherosclerosis are common among elderly persons. They are similar with regard to the long prodromal period when specific, clinically "silent" lesions accumulate that manifest according to the symptomatology plan when the disease is already after many years of evolution. However, this fact does not impede us from considering them as two independent diseases with convergent evolution. This idea is supported by epidemiological observations, pathological elements and the answer to common therapy for

The APOE risk factor is also associated with atherosclerosis. In cell cultures, APOE is linked with the decreased cholesterol efflux from macrophages, as well as from neurons and astro‐ cytes. At macrophage level, APOE promotes foam cell formation, at neuronal level it promotes

Is has also been evinced that APOE 4 has a more reduced antioxidant capacity than its isoform APOE3. The pathogenic strain of both atherosclerosis and AD is the increase of the oxidative stress associated with the production of reactive oxygen and nitrogen species that oxidize

into amyloid fibrils and is then secreted back in the extracellular milieu.

Aβ and favour the formation of Aβ fibrils rather than APOE3 isoforms.

areas of the brain, such as the temporal lobe, have also been discussed.

Alzheimer disease than those without vascular risk factor.

both diseases.

the increased APP processing for forming Aβ.

may contribute to the aggregation of amyloid-beta through its internalization.

**7. Cholesterol — Common risk factor for AD and atherosclerosis**

ApoE may alter brain cholesterol homeostasis by modifying lipoprotein-particle formation. In the plasma, ApoE4 is associated with VLDL particles, which contain more cholesterol, whereas ApoE3 is associated with HDL. Subjects homozygous for the ApoE ε4 allele have higher levels of cholesterol in the plasma and 25-hydroxycholesterol in the cerebrospinal fluid. 25-hydrox‐ ycholesterol is a catabolic derivative of cholesterol and represents the major metabolic route for cholesterol clearance from the brain. The lipoproteins produced in the brain are very different from the particles found in the plasma, in what concerns composition as well density or other properties.

As ApoE4 shows differential preference for VLDL in the plasma, it is possible that different ApoE isoforms modify brain cholesterol homeostasis by preferentially associating with specific lipoprotein particles. The predominant nature of Apoe in brain lipoproteins would accentuate small differences in lipoprotein affinity for ApoE isoforms. The role of apoE in maintaining cholesterol homeostasis in the brain may contribute to the increased risk for AD associated with APOE ε4.

ApoE may up-regulate the rate of Aβ generation by increasing cellular cholesterol. After receptor-mediated internalization and enzymatic digestion of the lipoproteins, cholesterol is released to cellular membranes. APOEA tend to contain more cholesterol. The increased cholesterol content of cellular membranes promotes the increased production of amyloid-beta from its precursor, APP.

Epidemiologic studies suggest that high levels of cholesterol may contribute to the pathogen‐ esis of AD. Individuals with elevated levels of plasma cholesterol have an increased suscept‐ ibility to AD, apparently influenced by the APOE ε4 genotype. Moreover, AD patients have increased levels of total serum and low density lipoprotein (LDL) cholesterol along with reduced levels of apoA/high-density lipoprotein (HDL) in their plasma, as compared to agematched controls. This is strengthened by the significantly decreased lecithin cholesterol acyltransferase activity (LCAT) in AD patients. LCAT is an enzyme found in plasma that catalyzes an acyltransferase reaction on lipoprotein-associated cholesterol and is a key step in reverse cholesterol transport in humans (the process that eliminates cholesterol from periph‐ eral cells).

APOE may mediate the internalization of amyloid-beta by binding to the proteins associated to the LDL receptor.

LRP is a multi-ligand receptor, a member of the LDL receptor family, with many common structural motifs and functional particularities, such as the high-affinity binding activity for LDL particles. LRP's main protein ligand is APOE, but it also binds other molecules such as α2-macroglobulin and APP751/750 containing the KPI domain.

LRP may be linked to the degradation of secreted amyloid-beta by facilitating the internali‐ zation of Aβ bound to APOE. The internalization of Aβ mediated by LRP is more likely followed by its intracellular aggregation, rather than degradation, suggesting that LRP might be involved in the process of Aβ deposition.

Soluble Aβ interacts with APOE associated with lipoparticles and under-goes receptormediated endocytosis. The lipoproteins are enzymatically digested in the lysosomal compart‐ ment, releasing cholesterol to the cell. A fraction of APOE and Aβ undergoes degradation at lysosomal level. The rest of APOE remains associated with Aβ and promotes its aggregation into amyloid fibrils and is then secreted back in the extracellular milieu.

The mechanisms through which APOE leads to the development of the disease are partially

ApoE may alter brain cholesterol homeostasis by modifying lipoprotein-particle formation. In the plasma, ApoE4 is associated with VLDL particles, which contain more cholesterol, whereas ApoE3 is associated with HDL. Subjects homozygous for the ApoE ε4 allele have higher levels of cholesterol in the plasma and 25-hydroxycholesterol in the cerebrospinal fluid. 25-hydrox‐ ycholesterol is a catabolic derivative of cholesterol and represents the major metabolic route for cholesterol clearance from the brain. The lipoproteins produced in the brain are very different from the particles found in the plasma, in what concerns composition as well density

As ApoE4 shows differential preference for VLDL in the plasma, it is possible that different ApoE isoforms modify brain cholesterol homeostasis by preferentially associating with specific lipoprotein particles. The predominant nature of Apoe in brain lipoproteins would accentuate small differences in lipoprotein affinity for ApoE isoforms. The role of apoE in maintaining cholesterol homeostasis in the brain may contribute to the increased risk for AD

ApoE may up-regulate the rate of Aβ generation by increasing cellular cholesterol. After receptor-mediated internalization and enzymatic digestion of the lipoproteins, cholesterol is released to cellular membranes. APOEA tend to contain more cholesterol. The increased cholesterol content of cellular membranes promotes the increased production of amyloid-beta

Epidemiologic studies suggest that high levels of cholesterol may contribute to the pathogen‐ esis of AD. Individuals with elevated levels of plasma cholesterol have an increased suscept‐ ibility to AD, apparently influenced by the APOE ε4 genotype. Moreover, AD patients have increased levels of total serum and low density lipoprotein (LDL) cholesterol along with reduced levels of apoA/high-density lipoprotein (HDL) in their plasma, as compared to agematched controls. This is strengthened by the significantly decreased lecithin cholesterol acyltransferase activity (LCAT) in AD patients. LCAT is an enzyme found in plasma that catalyzes an acyltransferase reaction on lipoprotein-associated cholesterol and is a key step in reverse cholesterol transport in humans (the process that eliminates cholesterol from periph‐

APOE may mediate the internalization of amyloid-beta by binding to the proteins associated

LRP is a multi-ligand receptor, a member of the LDL receptor family, with many common structural motifs and functional particularities, such as the high-affinity binding activity for LDL particles. LRP's main protein ligand is APOE, but it also binds other molecules such as

LRP may be linked to the degradation of secreted amyloid-beta by facilitating the internali‐ zation of Aβ bound to APOE. The internalization of Aβ mediated by LRP is more likely followed by its intracellular aggregation, rather than degradation, suggesting that LRP might

α2-macroglobulin and APP751/750 containing the KPI domain.

be involved in the process of Aβ deposition.

unknown.

172 Neurodegenerative Diseases

or other properties.

associated with APOE ε4.

from its precursor, APP.

eral cells).

to the LDL receptor.

The internalization of Aβ is not necessarily followed by its degradation. Amyloid aggregates from the endocytic compartment may be secreted in a more toxic fibrillar form. Finally, APOE may contribute to the aggregation of amyloid-beta through its internalization.

Moreover, the non-lypidic form of APOE seems to have a high-affinity binding activity to Aβ and favour the formation of Aβ fibrils rather than APOE3 isoforms.

### **7. Cholesterol — Common risk factor for AD and atherosclerosis**

Epidemiologic studies indicate that people with risk factors such as high blood pressure, diabetes, cerebrovascular disease and high cholesterol are two times more likely to develop Alzheimer disease than those without vascular risk factor.

The causal chain between the high plasma cholesterol level and atherosclerosis is well defined.

We can discuss a risk factor profile for sporadic cases of AD, with late-onset, which account for 90-95% of all cases. In elderly patients that suffer from AD there is an obvious link between this disease and vascular risk factors and atherosclerosis. However, the nature of this link remains partially speculative. Some authors have suggested that AD occurs as a secondary event related to atherosclerosis of extracranial or intracranial vessels with secondary cerebral hypoperfusion or small cerebral strokes. The toxic effects of vascular risk factors on susceptible areas of the brain, such as the temporal lobe, have also been discussed.

Another approach is that both AD and atherosclerosis are common among elderly persons. They are similar with regard to the long prodromal period when specific, clinically "silent" lesions accumulate that manifest according to the symptomatology plan when the disease is already after many years of evolution. However, this fact does not impede us from considering them as two independent diseases with convergent evolution. This idea is supported by epidemiological observations, pathological elements and the answer to common therapy for both diseases.

The APOE risk factor is also associated with atherosclerosis. In cell cultures, APOE is linked with the decreased cholesterol efflux from macrophages, as well as from neurons and astro‐ cytes. At macrophage level, APOE promotes foam cell formation, at neuronal level it promotes the increased APP processing for forming Aβ.

Is has also been evinced that APOE 4 has a more reduced antioxidant capacity than its isoform APOE3. The pathogenic strain of both atherosclerosis and AD is the increase of the oxidative stress associated with the production of reactive oxygen and nitrogen species that oxidize


People with high cholesterol in their early 40s are more likely to develop Alzheimer's disease than those with low cholesterol. High levels of high-density lipoprotein-"good cholesterol"

Cholesterol and Alzheimer's Disease http://dx.doi.org/10.5772/55502 175

Statins have a pleiotropic effect – they act on cholesterol, as well as on isoprenylation. They block the HGM-Co A reductase pathway and thus manifest their effects through a cholesterol

The usefulness of hyper or hypo-cholesterolemic diets in preventing AD is also another controversial issue. Most of the studies are carried out on rodents: 1998 studies found that the hypercholesterolemic diet lowers beta-amyloid at cerebral level, while 2000 studies found the

Although the link between cholesterol and AD remains an open issue, that still has many unresolved aspects, the studies have found strong evidence that cholesterol is a factor in the

Cholesterol is involved in the development of AD owing to its intervention, at different levels, on the formation and deposit of amyloid-beta and the genesis of intraneuronal neurofibrillary

Based on these data and knowing cholesterol's essential role both at cerebral and body level, as well as the relative difficulty to estimate the exact link between the plasma cholesterol and the brain cholesterol level, it is important to maintain throughout your entire life, in correlation with age and sex, the cholesterol concentration (and its fractions) within the recommended

It is also important to avoid the drastic "correction" of cholesterol, sometimes even under normal levels, especially in the case of elderly persons, in order to prevent Alzheimer's disease,

considering that cholesterol is an essential substance for the entire body.

appear to be associated with a reduced risk for Alzheimer disease in older adults.

**9. Statins and the evolution of AD**

**10. Diet and the evolution of Alzheimer's disease**

Is statin treatment protective?

independent mechanism.

opposite.

**11. Conclusions**

pathogenesis of AD.

tangles, also favoring the atherogenesis process.

limits in order to prevent cardio-vascular diseases.

**Table 1.** Common risk factors for AD and atherosclerosis

amino acids and lipids. The loss of the antioxidant protection conferred by APOE may lead to increased alterations produced by oxidative stress.

Epidemiologic studies suggest that AD patients have increased levels of total serum and low density lipoprotein (LDL) cholesterol along with reduced levels of apoA/high-density lipoprotein (HDL) in their plasma, as compared to age-matched controls. LCAT is an enzyme found in plasma that catalyzes an acyltransferase reaction on lipoprotein-associated choles‐ terol and is a key step in reverse cholesterol transport in humans (the process that eliminates cholesterol from peripheral cells).

The same metabolic profile with high plasma cholesterol level, high LDL cholesterol and low HDL cholesterol is found with atherosclerosis patients.

### **8. Predictive value of cholesterol based on AGE**

The study carried out in 2010 on Swedish women has not evinced any increase in AD risk associated with high plasma cholesterol values between the ages of 40 and 60. This finding contradicts several previous studies which did suggest a role for elevated plasma cholesterol at middle age in the later development of AD.

However, this study has evinced another association of cholesterol level to DA based on age. Namely, women whose cholesterol decreased the most from middle age to old age were more than twice (x2!) as likely to develop AD as women whose cholesterol levels increased or stayed the same between the same age intervals.

Rapidly declining cholesterol late in life (over 60) appears to be associated with increased frailty and may be an early sign of dementia.

It seems that this is due to the fact that 10 years earlier people develop symptoms of dementia, they tend to become more frail. They forget to eat and start to lose weight, which can impact cholesterol levels.

People with high cholesterol in their early 40s are more likely to develop Alzheimer's disease than those with low cholesterol. High levels of high-density lipoprotein-"good cholesterol" appear to be associated with a reduced risk for Alzheimer disease in older adults.

### **9. Statins and the evolution of AD**

Is statin treatment protective?

Statins have a pleiotropic effect – they act on cholesterol, as well as on isoprenylation. They block the HGM-Co A reductase pathway and thus manifest their effects through a cholesterol independent mechanism.

### **10. Diet and the evolution of Alzheimer's disease**

The usefulness of hyper or hypo-cholesterolemic diets in preventing AD is also another controversial issue. Most of the studies are carried out on rodents: 1998 studies found that the hypercholesterolemic diet lowers beta-amyloid at cerebral level, while 2000 studies found the opposite.

### **11. Conclusions**

amino acids and lipids. The loss of the antioxidant protection conferred by APOE may lead to

Epidemiologic studies suggest that AD patients have increased levels of total serum and low density lipoprotein (LDL) cholesterol along with reduced levels of apoA/high-density lipoprotein (HDL) in their plasma, as compared to age-matched controls. LCAT is an enzyme found in plasma that catalyzes an acyltransferase reaction on lipoprotein-associated choles‐ terol and is a key step in reverse cholesterol transport in humans (the process that eliminates

The same metabolic profile with high plasma cholesterol level, high LDL cholesterol and low

The study carried out in 2010 on Swedish women has not evinced any increase in AD risk associated with high plasma cholesterol values between the ages of 40 and 60. This finding contradicts several previous studies which did suggest a role for elevated plasma cholesterol

However, this study has evinced another association of cholesterol level to DA based on age. Namely, women whose cholesterol decreased the most from middle age to old age were more than twice (x2!) as likely to develop AD as women whose cholesterol levels increased or stayed

Rapidly declining cholesterol late in life (over 60) appears to be associated with increased frailty

It seems that this is due to the fact that 10 years earlier people develop symptoms of dementia, they tend to become more frail. They forget to eat and start to lose weight, which can impact

increased alterations produced by oxidative stress.

**Table 1.** Common risk factors for AD and atherosclerosis

HDL cholesterol is found with atherosclerosis patients.

at middle age in the later development of AD.

the same between the same age intervals.

and may be an early sign of dementia.

cholesterol levels.

**8. Predictive value of cholesterol based on AGE**

cholesterol from peripheral cells).

• ApoEe 4 polmorphism • Hypercholesterolemia • High blood pressure • Hyperhomocysteinemia • Diabetes mellitus • Metabolic syndrome

174 Neurodegenerative Diseases

• Systemic inflammatory response • High level of fats and obesity

• Smoking

Although the link between cholesterol and AD remains an open issue, that still has many unresolved aspects, the studies have found strong evidence that cholesterol is a factor in the pathogenesis of AD.

Cholesterol is involved in the development of AD owing to its intervention, at different levels, on the formation and deposit of amyloid-beta and the genesis of intraneuronal neurofibrillary tangles, also favoring the atherogenesis process.

Based on these data and knowing cholesterol's essential role both at cerebral and body level, as well as the relative difficulty to estimate the exact link between the plasma cholesterol and the brain cholesterol level, it is important to maintain throughout your entire life, in correlation with age and sex, the cholesterol concentration (and its fractions) within the recommended limits in order to prevent cardio-vascular diseases.

It is also important to avoid the drastic "correction" of cholesterol, sometimes even under normal levels, especially in the case of elderly persons, in order to prevent Alzheimer's disease, considering that cholesterol is an essential substance for the entire body.

#### **Author details**

Iuliana Nicola-Antoniu

Departement of Neurology,"Colentina" Clinical Hospital, Bucharest, Romania

#### **References**

[1] Bu. Apolipoprotein E and its receptors in Alzheimer,s disease:patways,pathogenesis and therapy. Nat Rev Neurosci. 2008;9:333 – 344

[14] Kim J,Bassak JM,Holtzman DM. The role af apolipoprotein E in Alzheimer's dis‐

Cholesterol and Alzheimer's Disease http://dx.doi.org/10.5772/55502 177

[15] Kivipelto M,Helkala EL,Laakso MP,et al.Midlife vascular risk factors and Alzheim‐ er's disease in latter life:longitudinal,populational base3d study.BMJ

[16] Kojoro E,GimplG,Lammich S, Marz W,Fahrenholz F. Low cholesterol stimulates the nonamyloidogenic pathway by its effct on the alpha-secretase Alzheimer's disease

[17] Lingwood D,Simons K.Lipid rafts as a membrane-orhganisind principle. Science,

[18] Nicholson AM,Ferreira A. Increased membrane cholesterol might render mature hi‐ pocampal neurons more susceptible to the beta-amyloid-induced calpain activation

[19] Puglielli L et al. Acyl-coenzyme A:cholesterol acyltranferase modulate tehe genera‐

[20] Puglielli L, Tanzi R, Kovacs D. Alzheimer's diasease: the closterol connection. Nature

[21] Refolo LM,Malestre B,La Francois J et al. Hypercolesterolemia accelerates the Alz‐ heimer's amyloid pathology in a trangenic mouse model. Neurobiol Dis 2000;7:321-

[22] Runz H,et al.Inhibition of intracellular cholesterol transport alters presenilin localiza‐ tion and amyloid precursor protein processing in neuronal in neuronal cells. J Neu‐

[23] Salynn Boyls,Mielke,Cholesterol level doesn't predict Alzheiemr's in old age,new

[24] Simons M,Keller P,Dichgans J Schulz JB. Cholesterol and Alzheimer's disease:is there

[25] Sparks DL,Scheff SW,Hunsanker JC, Liu H,Landers T, Gross DR. Induction of Alz‐ heimer -like beta-amyloid immnureactivity in the brain of rabbits with dietary cho‐

[26] Tall AR.Cholesterol efflux pathways and other potential mechanisms involved in the athero-protective effect of high density lipoproteins. J Intern Med 2008;263:256 -273

[27] de la Torre JC. Alzheimer disease as a vascular disorder:nosological evidence. Stroke

[28] Vance JE,Hayashi H,Karten B.Cholesterol homeostasis in neurons and glial cells.

tion of the amyloid beta-peptide. Nat.Cell Biol,2001;3:905 – 912

study finds,MM Neurology, Nov. 23,2010;vol 75 pp1888-1895

ease.Nuron.2009;63:287 -303

AM 10 Proc Natl Acad Sci USA 2001;98:5815 -5820

and tau toxicity. J Neurosci 2009;29:4640 -4651

2001;322:1447-51

2010;327: 46-50

Publishing Group,2003

rosci,2001;22:1679- 1689

2002;33:1152-62

a link? Neurology,2001;57:1089 -93

lesterol. Exp Neurol.1994;126;88-94

Semin Cell Dev Biol.2005;16:193-212

31


[14] Kim J,Bassak JM,Holtzman DM. The role af apolipoprotein E in Alzheimer's dis‐ ease.Nuron.2009;63:287 -303

**Author details**

176 Neurodegenerative Diseases

**References**

Iuliana Nicola-Antoniu

ro Dis 2006; 22:209 -22

ease. Mol Mem Bio.2006; 23:111-22

Nat Rev Neurosci,2011;12(5):284-96

topsy,MA, Neurology,sept 2011

Neurochem,2007;103(Suppl 1):159-170

417-23

2011

2001;12:105- 112

Departement of Neurology,"Colentina" Clinical Hospital, Bucharest, Romania

mation,cholesterol,and misfolded proteins.Lancet 2004;363:1139-46

and therapy. Nat Rev Neurosci. 2008;9:333 – 344

[1] Bu. Apolipoprotein E and its receptors in Alzheimer,s disease:patways,pathogenesis

[2] Casserly I, Topol E. Convergence of atherosclerosis and Alzheiemr's disease:inflam‐

[3] Chang TY,Chang CC,Lu X&Lin S Catalysis of ACAT may be completed with the plane of the membrane. A working ipothesis. J Lipig Res.2001;42:1933-1938

[4] Cole S, Vassar R. Isoprenoids and Alzheiemr's disease: a complex realtionship. Neu‐

[5] Colell A,Fernandez A,Fernandez-Checa JC. Mitochondria,cholesterol and amyloid beta peptide; a dangerous tri in Alzheimer,s disease. J Bioenerg Biomembr.2009;41(5):

[6] Cordy JM, Hooper NM, Turner AJ. The involvement of lipid rafts in Alzheimer's dis‐

[7] Di Paolo G,Kim TW,Linking lipids to Alzheiemr,s disease:cholesterol and beyond,

[8] Dietschy JM & Turley SD. Cholesterol metabolism in the brain. Curr. Opin. Lipiol

[9] Frears ER, Stephens DJ,Walters CE, Davies H&Austen BM. The role of cholesterol in

[10] Goodman Brenda, High cholesterol predicts brain-clagging protein deposits on au‐

[11] Hartmann T,Kuchenbecker J, Grimm MO. Alzheiemr's disese: the lipid connection. J

[12] Hoffman A,Ott A, Breteler MM et alAtherosclerosis,apolipoprotein E and prevalence of dementia and Alzheimer's disease in the Rotterdam study. Lancet 1997;349:151 54

[13] Ivanhoe Newswere,Study links high cholesterol and Alzheimer's-Neurology,sept

the biosyntesis of beta-amyloid. Neuroreport 1995;10:1699-1705


[29] Vetrivel KS,Thinakaran G. Membrane rafts in Alzheimer's disease beta-amyloid pro‐ duction. Biochim Biophy Acta. 2010

**Section 2**

**Therapeutic Aspects of Alzheimer**

**Therapeutic Aspects of Alzheimer**

[29] Vetrivel KS,Thinakaran G. Membrane rafts in Alzheimer's disease beta-amyloid pro‐

duction. Biochim Biophy Acta. 2010

178 Neurodegenerative Diseases

**Chapter 9**

**Emerging Therapeutic Strategies in Alzheimer's Disease**

Alzheimer's disease (AD) is the most common cause of dementia in mid- to late-life. It currently affects about 10% of individuals older than 65 years old, counting for more than 25 million people in the world (Chiba et al., 2007; Huang and Mucke, 2012; Mattson, 2004). A century has passed since the famous neurologist Alois Alzheimer first described about a 51-year-old female patient with severe progressive memory deficit, brain atrophy, senile plaques (SPs, or neuritic plaques), and neurofibrillary tangles (NFTs) in 1906 (1987). SPs and NFTs are abnormal protein aggregates consisting of extracellular amyloid β protein (Aβ) and intracellular hyperphos‐ phorylated microtubule associated protein tau (Braak and Braak, 1991; Perrin et al., 2009). The worst affected areas in AD brains are the olfactory bulb, the cerebral neocortex, and the hippocampus. SPs and NFTs are spatially and temporally disjointed: SPs occur in the cerebral neocortex, preceding the occurrence of NFTs, which is predominant in the entorhinal cortex, by about 10 years (Fig. 1). The onset of mild dementia, or currently described as mild cognitive impairment (MCI), is better correlated with significant synaptic and neuronal loss, which is

Knowledge of AD has considerably expanded in the last two decades with the progress in molecular biology and genetics although curative therapy for AD is not yet available. Most AD cases occur sporadically (sporadic AD or SAD), while about 1% of AD cases are inherited in an autosomal dominant manner (familial AD, FAD). In 1984, Glenner and Wong first purified and sequenced Aβ (Glenner and Wong, 1984), which was followed by the identifica‐ tion of amyloid precursor protein (APP) as a source of Aβ (Kang et al., 1987). Genetic analysis of FAD then revealed that mutations in APP co-segregate with FAD (Chartier-Harlin et al., 1991; Goate et al., 1991). Consequently, drug candidates have been developed for AD based on a putative pathogenic hypothesis that increase in Aβ production and aggregation, which can be accelerated by the mutations in genes related to FAD, results in tau hyperphosphory‐ lation and neuronal death. This is termed the "amyloid cascade" hypothesis (Hardy and

> © 2013 Chiba; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Tomohiro Chiba

**1. Introduction**

http://dx.doi.org/10.5772/55293

Additional information is available at the end of the chapter

about 10-15 years behind the occurrence of NFTs.

### **Emerging Therapeutic Strategies in Alzheimer's Disease**

### Tomohiro Chiba

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55293

### **1. Introduction**

Alzheimer's disease (AD) is the most common cause of dementia in mid- to late-life. It currently affects about 10% of individuals older than 65 years old, counting for more than 25 million people in the world (Chiba et al., 2007; Huang and Mucke, 2012; Mattson, 2004). A century has passed since the famous neurologist Alois Alzheimer first described about a 51-year-old female patient with severe progressive memory deficit, brain atrophy, senile plaques (SPs, or neuritic plaques), and neurofibrillary tangles (NFTs) in 1906 (1987). SPs and NFTs are abnormal protein aggregates consisting of extracellular amyloid β protein (Aβ) and intracellular hyperphos‐ phorylated microtubule associated protein tau (Braak and Braak, 1991; Perrin et al., 2009). The worst affected areas in AD brains are the olfactory bulb, the cerebral neocortex, and the hippocampus. SPs and NFTs are spatially and temporally disjointed: SPs occur in the cerebral neocortex, preceding the occurrence of NFTs, which is predominant in the entorhinal cortex, by about 10 years (Fig. 1). The onset of mild dementia, or currently described as mild cognitive impairment (MCI), is better correlated with significant synaptic and neuronal loss, which is about 10-15 years behind the occurrence of NFTs.

Knowledge of AD has considerably expanded in the last two decades with the progress in molecular biology and genetics although curative therapy for AD is not yet available. Most AD cases occur sporadically (sporadic AD or SAD), while about 1% of AD cases are inherited in an autosomal dominant manner (familial AD, FAD). In 1984, Glenner and Wong first purified and sequenced Aβ (Glenner and Wong, 1984), which was followed by the identifica‐ tion of amyloid precursor protein (APP) as a source of Aβ (Kang et al., 1987). Genetic analysis of FAD then revealed that mutations in APP co-segregate with FAD (Chartier-Harlin et al., 1991; Goate et al., 1991). Consequently, drug candidates have been developed for AD based on a putative pathogenic hypothesis that increase in Aβ production and aggregation, which can be accelerated by the mutations in genes related to FAD, results in tau hyperphosphory‐ lation and neuronal death. This is termed the "amyloid cascade" hypothesis (Hardy and

© 2013 Chiba; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Higgins, 1992; Reitz, 2012). In this review, therapeutic strategies for AD will be discussed from a viewpoint of neuronal death and neuroprotection.

APP is ubiquitously expressed type-I single transmembrane glycoprotein (Kang et al., 1987). From its primary structure, it is likely to be a cell surface receptor (Fig. 2A). APP undergoes sequential processing by three proteases: α-, β- and γ-secretase. β- and γ-cleavage generates Aβ, secretory APPβ (sAPPβ), and APP intracellular domain (AICD), whereas α- and γcleavage generates secretory APPα (sAPPα), AICD, and p3 fragment (De Strooper and Annaert, 2000; Kawasumi et al., 2002). Since β- and γ-cleavage generates Aβ, it is believed that initial cleavage by β-secretase is pathogenic and one by α-secreatese is non-pathogenic on the other hands. Val 642 Ile (V642I, numbering by a neuron-specific APP695 form) mutation within the transmembrane domain of APP was identified as the first FAD-linked mutation and is called as London-type mutation (Fig. 2B) (Chartier-Harlin et al., 1991; Goate et al., 1991). Then, several other types of mutations in APP, including K595N/M596L (Swedish type mutant or NL mutant), were identified (pathological impact of these mutations will be discussed later, see 2.1.3). It is notable that E618Q (Dutch type, numbering by APP695) mutation within Aβ sequence does not cause FAD but causes cerebral amyloid angiopathy called as hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) (Fernandez-Madrid et al., 1991), while E618G (Arctic type, numbering by APP695) mutation at the same position causes FAD without severe cerebral amyloid angiopathy (Nilsberth et al., 2001). Recently, a rare recessive (A598V, numbering by APP695; A673V, numbering by a ubiquitous APP770 form) and a disease-resistant mutation (A598T, by APP695; A672T, by APP770) have been identified in APP at Ala 598, the second amino acid in Aβ (Di Fede et al., 2009; Jonsson et al., 2012).

Emerging Therapeutic Strategies in Alzheimer's Disease

http://dx.doi.org/10.5772/55293

183

PSEN1 and PSEN2 are highly homologous proteins consisting of 467 and 448 amino acid residues respectively with 8 or 9 transmembrane domains (Laudon et al., 2005; Marjaux et al., 2004) (Fig. 2C). They are ubiquitously expressed and localized in the endoplasmic reticulum (ER) and golgi apparatus. Mutations in the *PSEN1* gene are the most frequently involved in FAD accounting for about 10-20% of all FAD cases. More than one hundred mutations throughout entire *PSEN1* gene have been reported while about ten mutations have been reported in *PSEN2* (Bertram et al., 2010; Schellenberg and Montine, 2012). *PSEN1* and *PSEN2* are now regarded as the active core of the γ-secretase which cleaves the hydrophobic integral membrane domain of APP to generate Aβ. It is now widely recognized that γ-secretase, at least, involves four different proteins: PSEN, nicastrin, Aph-1, and Pen-2 (De Strooper, 2003; Takasugi et al., 2003). It should be noted that FAD-linked mutations in *PSEN1* and *PSEN2* induce neuronal death or enhance neuronal vulnerability to several toxic insults independent of the γ-secretase activity (Guo et al., 1996; Hashimoto et al., 2002a; Hashimoto et al., 2002b;

NFTs are comprised of paired helical filaments (PHFs), which is resulted from hyperphos‐ phorylation of tau at Ser/Thr residues. NFT formation is one of the hallmarks of AD although tau dysfunction is not limited to AD but is widely observed in neurological disorders (usually termed as tauopathy) (Goedert et al., 1998; Johnson and Stoothoff, 2004). It is reported that the number of NFT correlates well with neuronal loss in AD brain and severity of dementia than that of SPs (Braak and Braak, 1991; Perrin et al., 2009). It is also notable that putative tau kinases

Zhang et al., 1998).

*2.1.2. Tau-hyperphosphorylation in AD*

### **2. Molecular pathogenesis of Alzheimer's Disease (AD)**

In this section, the overview of molecular pathogenesis of AD will be discussed. This is important to elaborate therapeutic strategies for AD. The current knowledge of pathogenic mechanisms for AD can be classified into two major categories, namely the "amyloid cascade" hypothesis and the alternative hypotheses. Recently, pathogenic roles of signal transducer and activator of transcription 3 (Stat3) and related intracellular signaling pathways in neurons has been described, which is attracting attention of AD researchers (Chiba et al., 2009a; Chiba et al., 2009b; Nicolas et al., 2012). In addition, genome-wide association studies (GWAS) have been carried out rigorously to seek for novel genes and loci related AD. These unbiased global genetic studies are now providing novel insights into both canonical and alternative patho‐ genic mechanisms for AD.

**Figure 1. Pathological and clinical features of Alzheimer's disease (AD).** (left) Senile plaques (SPs) in the cerebral cortex of an AD model mouse at the age of 12 monthes were stained with Thioflavin T. (right) Clinical process of AD and its relationship with pathological hallmarks of AD (mild cognitive impairment, MCI; cognitive function [blue]; SPs [red]; neurofibrillary tangles, NFTs [green]; neuronal loss [purple]). SP formation precedes NFT formation, which is fol‐ lowed by neuronal loss. Cognitive decline occurs as a result of neuronal loss.

#### **2.1. The "amyloid cascade" hypothesis**

#### *2.1.1. Causative genes for FAD and their roles*

FAD, which accounts for up to 1% of total AD cases, usually occurs between the age of 40-65 years (early-onset AD, EOAD) and is inherited in an autosomal dominant manner (Bertram et al., 2010; Schellenberg and Montine, 2012). Genetic analysis has identified so far three causative genes for FAD: *amyloid precursor protein (APP)* on chromosome 21, *presenilin 1 (PSEN1)* on chromosome 14 (Sherrington et al., 1995), and *presenilin 2 (PSEN2)* on chromosome 1 (Rogaev et al., 1995).

APP is ubiquitously expressed type-I single transmembrane glycoprotein (Kang et al., 1987). From its primary structure, it is likely to be a cell surface receptor (Fig. 2A). APP undergoes sequential processing by three proteases: α-, β- and γ-secretase. β- and γ-cleavage generates Aβ, secretory APPβ (sAPPβ), and APP intracellular domain (AICD), whereas α- and γcleavage generates secretory APPα (sAPPα), AICD, and p3 fragment (De Strooper and Annaert, 2000; Kawasumi et al., 2002). Since β- and γ-cleavage generates Aβ, it is believed that initial cleavage by β-secretase is pathogenic and one by α-secreatese is non-pathogenic on the other hands. Val 642 Ile (V642I, numbering by a neuron-specific APP695 form) mutation within the transmembrane domain of APP was identified as the first FAD-linked mutation and is called as London-type mutation (Fig. 2B) (Chartier-Harlin et al., 1991; Goate et al., 1991). Then, several other types of mutations in APP, including K595N/M596L (Swedish type mutant or NL mutant), were identified (pathological impact of these mutations will be discussed later, see 2.1.3). It is notable that E618Q (Dutch type, numbering by APP695) mutation within Aβ sequence does not cause FAD but causes cerebral amyloid angiopathy called as hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) (Fernandez-Madrid et al., 1991), while E618G (Arctic type, numbering by APP695) mutation at the same position causes FAD without severe cerebral amyloid angiopathy (Nilsberth et al., 2001). Recently, a rare recessive (A598V, numbering by APP695; A673V, numbering by a ubiquitous APP770 form) and a disease-resistant mutation (A598T, by APP695; A672T, by APP770) have been identified in APP at Ala 598, the second amino acid in Aβ (Di Fede et al., 2009; Jonsson et al., 2012).

PSEN1 and PSEN2 are highly homologous proteins consisting of 467 and 448 amino acid residues respectively with 8 or 9 transmembrane domains (Laudon et al., 2005; Marjaux et al., 2004) (Fig. 2C). They are ubiquitously expressed and localized in the endoplasmic reticulum (ER) and golgi apparatus. Mutations in the *PSEN1* gene are the most frequently involved in FAD accounting for about 10-20% of all FAD cases. More than one hundred mutations throughout entire *PSEN1* gene have been reported while about ten mutations have been reported in *PSEN2* (Bertram et al., 2010; Schellenberg and Montine, 2012). *PSEN1* and *PSEN2* are now regarded as the active core of the γ-secretase which cleaves the hydrophobic integral membrane domain of APP to generate Aβ. It is now widely recognized that γ-secretase, at least, involves four different proteins: PSEN, nicastrin, Aph-1, and Pen-2 (De Strooper, 2003; Takasugi et al., 2003). It should be noted that FAD-linked mutations in *PSEN1* and *PSEN2* induce neuronal death or enhance neuronal vulnerability to several toxic insults independent of the γ-secretase activity (Guo et al., 1996; Hashimoto et al., 2002a; Hashimoto et al., 2002b; Zhang et al., 1998).

#### *2.1.2. Tau-hyperphosphorylation in AD*

Higgins, 1992; Reitz, 2012). In this review, therapeutic strategies for AD will be discussed from

In this section, the overview of molecular pathogenesis of AD will be discussed. This is important to elaborate therapeutic strategies for AD. The current knowledge of pathogenic mechanisms for AD can be classified into two major categories, namely the "amyloid cascade" hypothesis and the alternative hypotheses. Recently, pathogenic roles of signal transducer and activator of transcription 3 (Stat3) and related intracellular signaling pathways in neurons has been described, which is attracting attention of AD researchers (Chiba et al., 2009a; Chiba et al., 2009b; Nicolas et al., 2012). In addition, genome-wide association studies (GWAS) have been carried out rigorously to seek for novel genes and loci related AD. These unbiased global genetic studies are now providing novel insights into both canonical and alternative patho‐

**Figure 1. Pathological and clinical features of Alzheimer's disease (AD).** (left) Senile plaques (SPs) in the cerebral cortex of an AD model mouse at the age of 12 monthes were stained with Thioflavin T. (right) Clinical process of AD and its relationship with pathological hallmarks of AD (mild cognitive impairment, MCI; cognitive function [blue]; SPs [red]; neurofibrillary tangles, NFTs [green]; neuronal loss [purple]). SP formation precedes NFT formation, which is fol‐

FAD, which accounts for up to 1% of total AD cases, usually occurs between the age of 40-65 years (early-onset AD, EOAD) and is inherited in an autosomal dominant manner (Bertram et al., 2010; Schellenberg and Montine, 2012). Genetic analysis has identified so far three causative genes for FAD: *amyloid precursor protein (APP)* on chromosome 21, *presenilin 1 (PSEN1)* on chromosome 14 (Sherrington et al., 1995), and *presenilin 2 (PSEN2)*

lowed by neuronal loss. Cognitive decline occurs as a result of neuronal loss.

**2.1. The "amyloid cascade" hypothesis**

*2.1.1. Causative genes for FAD and their roles*

on chromosome 1 (Rogaev et al., 1995).

a viewpoint of neuronal death and neuroprotection.

genic mechanisms for AD.

182 Neurodegenerative Diseases

**2. Molecular pathogenesis of Alzheimer's Disease (AD)**

NFTs are comprised of paired helical filaments (PHFs), which is resulted from hyperphos‐ phorylation of tau at Ser/Thr residues. NFT formation is one of the hallmarks of AD although tau dysfunction is not limited to AD but is widely observed in neurological disorders (usually termed as tauopathy) (Goedert et al., 1998; Johnson and Stoothoff, 2004). It is reported that the number of NFT correlates well with neuronal loss in AD brain and severity of dementia than that of SPs (Braak and Braak, 1991; Perrin et al., 2009). It is also notable that putative tau kinases

chromosome 17 (FTDP-17) rather than AD (Hutton et al., 1998; Lewis et al., 2000). Recently, however, several observations supporting the relation between tau and AD have been

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The "amyloid cascade hypothesis" postulates that excessive formation of insoluble fibrillar Aβ, with consequent formation of SPs, is the initial event in AD pathogenesis (Hardy and Higgins, 1992; Reitz, 2012). Then, a neurotoxic cascade, including NFT formation, secondarily occurs, leading to synaptic and neuronal loss. The hypothesis is originally based on the two key observations: the detection of Aβ as a main constituent of SPs, and identification of the FAD-causative mutations in the Aβ precursor (*APP*) and γ-secretase genes (*PSEN1* and *PSEN2*) (Bertram et al., 2010; Chiba et al., 2007; Schellenberg and Montine, 2012). Aβ can vary in length at the c-terminus; Aβ1-40 (Aβ40, 40 amino acids) is the most prevalent, followed by Aβ1-42 (Aβ42). The latter has hydrophobic properties and aggregates more readily than Aβ40, which leads to the notion that Aβ42 is the toxic Aβ property. Mutations of all three FAD genes generally increase the ratio of Aβ42 to Aβ40 (Aβ42/Aβ40) and promote Aβ oligomerization

As already mentioned, SPs and NFTs are distributed independently of each other. Researchers, however, have postulated that NFT formation lies downstream from SP formation to integrate NFTs into the "amyloid cascade", based on the experimental observations showing the relationship between Aβ and NFT formation: (i) APP or PSEN1 transgene enhanced NFT formation in tau transgenic mice (Gotz et al., 2001; Lewis et al., 2001; Oddo et al., 2003), (ii) fetal rat hippocampal neurons and human cortical neurons treated with fibrillar Aβ display an increased degree of tau phosphorylation (Busciglio et al., 1995; Rapoport et al., 2002), (iii) reduction of endogenous levels of tau can ameliorate some of the behavioral and other dificits mediated by Aβ (Roberson et al., 2007), and (iv) mutations in the tau gene cause FTDP-17 with a tau pathology similar to that in AD

There are several objections to the "amyloid cascade" hypothesis. There is only a weak correlation between cerebral SPs and the severity of dementia. SPs and NFTs may be reactive products resulting from neurodegeneration in AD rather than being its cause. It remains unclear whether and how the deposition of Aβ leads to the formation of NFTs. These should

Signal transducer and activator of transcription 3 (Stat3) is an important mediator of cellular physiological functions such as cell proliferation, differentiation, and survival, mainly upon cytokine receptor stimulation (Chiba et al., 2009a; Stephanou and Latchman, 2005). Immuno‐ histochemical analysis using a specific antibody against phosphorylated (p-) or activated form of Stat3 revealed that p-Stat3 levels were significantly reduced in hippocampal neurons of clinically and pathologically diagnosed AD patients and several lines of AD model mice as

reported, which led to the "amyloid cascade" hypothesis for AD (Fig. 3).

and aggregation, followed by the synaptic and neuronal loss.

without SP formation (Hutton et al., 1998; Lewis et al., 2000).

be addressed in the future investigations.

**2.2. Stat3 inactivation in the "amyloid casdade"**

*2.1.3. Basis of the "amyloid cascade" hypothesis*

**Figure 2.** Familial AD (FAD) causative genes. (A) Proteolytic processing of APP. APP is consisting of extracellular, transmem‐ brane and intracellular domains. There are several isoforms of APP with or without a Kunitz protease inhibitor (KPI) do‐ main. Intracellular domain has signal transducing domains such as a G protein-binding motif (Go-binding domain) and an NPXY motif (GYENPTY) recognized by phosphotyrosine-binding domains. Sequential cleavage by α-/γ- or β-/γ-secretases produces soluble APP (sAPPα or sAPPβ, respectively), APP intracellular domain (AICD) and small peptides (Aβ or p3 frag‐ ment, respectively). (B) APP mutations were indicated in the Aβ coding region (amino acids). FAD mutations are indicated in red characters (numbering by a neuronal isoform of APP [APP695]); Swedish (K595N/M596N, NL mutation), London (V642I), Flemish (A617G, AD with strokes), Arctic (A618G), Iowa (A619N), German (V640A), French (V640M), Florida (I641V) and Indiana (V642F). Dutch type mutation (E618Q) is related to hereditary cerebral hemorrhage with amyloido‐ sis, Dutch type (HCHWA-D, [orange]). E618K is also related to cerebral hemorrhage with amyloidosis. A598V [blue] is a rare recessive mutation and A598T [green] has been identified as a disease-resistant mutation. (C) Presenilin 1 (PSEN1) and 2 (PSEN2) were also schematically indicated with mutations (red dots). Autocatalytic cleavage of PSENs (arrow) into N- and C-terminal fragments is required for the activation of the γ-secretase.

including glycogen synthase kinase 3β (GSK3β), cyclin dependent kinase 5 (cdk5), c-Jun Nterminal kinase (JNK), p38 mitogen-activated protein kinase (p38-MAPK) and Calcium/ calmodulin-dependent kinase II (CaMKII) are reported to be upregulated in AD (Ferrer et al., 2002; Ferrer et al., 2001; Sato et al., 2002; Takashima et al., 2001). These data suggest that the mechanism of NFT formation plays an important role in neuronal loss in AD brain. Genetic mutations in tau, however, cause frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) rather than AD (Hutton et al., 1998; Lewis et al., 2000). Recently, however, several observations supporting the relation between tau and AD have been reported, which led to the "amyloid cascade" hypothesis for AD (Fig. 3).

#### *2.1.3. Basis of the "amyloid cascade" hypothesis*

The "amyloid cascade hypothesis" postulates that excessive formation of insoluble fibrillar Aβ, with consequent formation of SPs, is the initial event in AD pathogenesis (Hardy and Higgins, 1992; Reitz, 2012). Then, a neurotoxic cascade, including NFT formation, secondarily occurs, leading to synaptic and neuronal loss. The hypothesis is originally based on the two key observations: the detection of Aβ as a main constituent of SPs, and identification of the FAD-causative mutations in the Aβ precursor (*APP*) and γ-secretase genes (*PSEN1* and *PSEN2*) (Bertram et al., 2010; Chiba et al., 2007; Schellenberg and Montine, 2012). Aβ can vary in length at the c-terminus; Aβ1-40 (Aβ40, 40 amino acids) is the most prevalent, followed by Aβ1-42 (Aβ42). The latter has hydrophobic properties and aggregates more readily than Aβ40, which leads to the notion that Aβ42 is the toxic Aβ property. Mutations of all three FAD genes generally increase the ratio of Aβ42 to Aβ40 (Aβ42/Aβ40) and promote Aβ oligomerization and aggregation, followed by the synaptic and neuronal loss.

As already mentioned, SPs and NFTs are distributed independently of each other. Researchers, however, have postulated that NFT formation lies downstream from SP formation to integrate NFTs into the "amyloid cascade", based on the experimental observations showing the relationship between Aβ and NFT formation: (i) APP or PSEN1 transgene enhanced NFT formation in tau transgenic mice (Gotz et al., 2001; Lewis et al., 2001; Oddo et al., 2003), (ii) fetal rat hippocampal neurons and human cortical neurons treated with fibrillar Aβ display an increased degree of tau phosphorylation (Busciglio et al., 1995; Rapoport et al., 2002), (iii) reduction of endogenous levels of tau can ameliorate some of the behavioral and other dificits mediated by Aβ (Roberson et al., 2007), and (iv) mutations in the tau gene cause FTDP-17 with a tau pathology similar to that in AD without SP formation (Hutton et al., 1998; Lewis et al., 2000).

There are several objections to the "amyloid cascade" hypothesis. There is only a weak correlation between cerebral SPs and the severity of dementia. SPs and NFTs may be reactive products resulting from neurodegeneration in AD rather than being its cause. It remains unclear whether and how the deposition of Aβ leads to the formation of NFTs. These should be addressed in the future investigations.

#### **2.2. Stat3 inactivation in the "amyloid casdade"**

including glycogen synthase kinase 3β (GSK3β), cyclin dependent kinase 5 (cdk5), c-Jun Nterminal kinase (JNK), p38 mitogen-activated protein kinase (p38-MAPK) and Calcium/ calmodulin-dependent kinase II (CaMKII) are reported to be upregulated in AD (Ferrer et al., 2002; Ferrer et al., 2001; Sato et al., 2002; Takashima et al., 2001). These data suggest that the mechanism of NFT formation plays an important role in neuronal loss in AD brain. Genetic mutations in tau, however, cause frontotemporal dementia with parkinsonism linked to

C-terminal fragments is required for the activation of the γ-secretase.

184 Neurodegenerative Diseases

**Figure 2.** Familial AD (FAD) causative genes. (A) Proteolytic processing of APP. APP is consisting of extracellular, transmem‐ brane and intracellular domains. There are several isoforms of APP with or without a Kunitz protease inhibitor (KPI) do‐ main. Intracellular domain has signal transducing domains such as a G protein-binding motif (Go-binding domain) and an NPXY motif (GYENPTY) recognized by phosphotyrosine-binding domains. Sequential cleavage by α-/γ- or β-/γ-secretases produces soluble APP (sAPPα or sAPPβ, respectively), APP intracellular domain (AICD) and small peptides (Aβ or p3 frag‐ ment, respectively). (B) APP mutations were indicated in the Aβ coding region (amino acids). FAD mutations are indicated in red characters (numbering by a neuronal isoform of APP [APP695]); Swedish (K595N/M596N, NL mutation), London (V642I), Flemish (A617G, AD with strokes), Arctic (A618G), Iowa (A619N), German (V640A), French (V640M), Florida (I641V) and Indiana (V642F). Dutch type mutation (E618Q) is related to hereditary cerebral hemorrhage with amyloido‐ sis, Dutch type (HCHWA-D, [orange]). E618K is also related to cerebral hemorrhage with amyloidosis. A598V [blue] is a rare recessive mutation and A598T [green] has been identified as a disease-resistant mutation. (C) Presenilin 1 (PSEN1) and 2 (PSEN2) were also schematically indicated with mutations (red dots). Autocatalytic cleavage of PSENs (arrow) into N- and

> Signal transducer and activator of transcription 3 (Stat3) is an important mediator of cellular physiological functions such as cell proliferation, differentiation, and survival, mainly upon cytokine receptor stimulation (Chiba et al., 2009a; Stephanou and Latchman, 2005). Immuno‐ histochemical analysis using a specific antibody against phosphorylated (p-) or activated form of Stat3 revealed that p-Stat3 levels were significantly reduced in hippocampal neurons of clinically and pathologically diagnosed AD patients and several lines of AD model mice as

compared with age-matched controls (Chiba et al., 2009b). Animal experiments further showed that (i) i.c.v. injection of toxic Aβ peptide reduces p-Stat3 in hippocampal neurons to induce memory impairment (a toxic Aβ gain of function effect) and that (ii) Aβ passive immunotherapy reduces cerebral Aβ burden and recovers cognitive function of Tg2576 mice in parallel with Stat3 activation in hippocampal neurons (a toxic Aβ loss of function effect). These data suggest that cerebral Aβ levels are inversely correlated with p-STAT3 levels in hippocampal neurons *in vivo* (Chiba et al., 2009b).

Aging, one of the most common risk factor for AD, seems to be one of the other factors responsible for the AD-related neuronal inactivation of Stat3 because p-Stat3 immunoreactiv‐ ity in hippocampal neurons of young subjects was substantially higher than that of elder cognitively normal subjects in both humans and rodents (Chiba et al., 2009b). Aging-depend‐ ent reduction of p-Stat3 levels may be due to age-dependent decrease in endogenous neuro‐ trophic factors, which play a role in sustaining neuronal p-Stat3 levels. In support of this idea, it is reported that endogenous levels of insulin-like growth factor I (IGF-I), which activates Stat3, decrease with aging and that this decrease may be linked to the pathogenesis of AD (Rollero et al., 1998).

Relationship between Stat3 and tau is yet to be elucidated. Stat3 binds to and inhibits stathmin, which depolymerizes microtubules, while tau binds to and stabilizes microtubules (Ng et al., 2006). Involvement of STAT3-mediated stathmin regulation in tau phosphorylation should be addressed in the future research. There are some reports describing Stat3 inactivation and cell death; i.e. Stat3 deletion sensitizes cells to oxidative stress (Barry et al., 2009; Sarafian et al.). Combined with the fact that Aβ induces neuronal death by inducing oxidative stress *in vitro* and *in vivo* (Butterfield et al., 2002; De Felice et al., 2007; Guglielmotto et al., 2009), Stat3 may sensitize neurons to Aβ neurotoxicity through oxidative stress.

In addition to the role of Stat3 in neuronal death, roles of Jak2/Stat3 pathway in synaptic plasticity have been reported. Activation of the Jak2/Stat3 pathway induces presynaptic transcriptional upregulation of cholinergic genes including choline acetyletransferase (ChAT) and vesicular acetylcholine transporter (VAChT) and postsynaptic sensitization of m1-type muscarinic acetylcholine receptor (m1-mAChR) to support cholinergic neurotransmission (Chiba et al., 2009b). The Jak2/Stat3 pathway also plays an essential role in the induction of NMDA-receptor dependent long-term depression (NMDAR-LTD) in the hippocampus (Nicolas et al., 2012).

Poirier, 2005). One copy of *APOE* ε4 increases the risk for AD fourfold and two copies further raise the risk tenfold. *APOE* is not a causative gene, i.e. *APOE* ε4 is neither necessary nor sufficient for LOAD. In addition to *APOE* itself, a variable-length poly-T (deoxythymidine homopolymer) polymorphism in the *TOMM40* gene, located in a region of strong linkage disequilibrium next to *APOE*, was reported to be associated with the age of onset of LOAD

**Figure 3. A schematic overview of the "amyloid cascade" hypothesis and therapeutic strategies.** (Red arrows) The "amyloid cascade" begins with the proteolytic cleavage of APP by the γ-secretase. Aβ forms toxic oligomers or aggregates, inducing neuronal Stat3 inactivation, NFT formation, caspase activation and neuronal death. Therapies targeting the canonical "amyloid cascade" such as secretase inhibitors and immunotherapy are indicated in blue char‐ acters. Therapies targeting on the downstream pathways of the "amyloid cascade" are indicated in green characters. Alternative pathological mechanisms, which can be caused by intracellular Aβ, extracellular Aβ, or FAD mutants them‐ selves, are also targeted (purple). (NEP, neprilysin; IDE, insulin degrading enzyme; VPA, valproic acid; HN, humanin).

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Genetic association studies on LOAD susceptibility loci and small nucleotide polymor‐ phisms (SNPs) revealed two genes: *ubiquilin 1 (UBQLN1)* on chromosome 9 (Bertram et al., 2005) and *sortilin-related receptor (SORL1)* (Rogaeva et al., 2007). Identification of *UBQLN1* is intriguing since it not only interacts with PSEN1, PSEN2 and APP but also may play a role in the proteasome degradation of them (Mah et al., 2000; Massey et al., 2004). SORL1 is involved in both trafficking of APP from the cell surface into recycling pathways and processing of APP by γ-secretase. A recent meta-analysis of genetic data and GWAS supported that mutations in *SORL1* may play a role in LOAD although the effect on the AD risk is moderate (Schellenberg and Montine, 2012). On the other hand, no evidence for

(Lutz et al., 2010; Roses et al.).

*UBQLN1* has been provided from GWAS.

#### **2.3. Emerging genetic risk factors for AD**

AD can be classified into early onset (EOAD, <65 years) and late onset (LOAD, >65 years) form. According to family history, AD can be also classified into FAD and sporadic (SAD) (Alves et al., 2010). Although aforementioned three FAD genes generally result in EOAD, there is a substantial genetic component in LOAD as well (an estimated heritability of 58-79%). Accord‐ ingly, *apolipoprotein E (APOE)* gene on chromosome 19 has also been shown to be a genetic risk factor for LOAD in the early 1990s (Bertram et al., 2010; Schellenberg and Montine, 2012). *APOE* contains three major alleles: ε2, ε3, and ε4. Inherited ε4 allele is reported to worsen the loss of neuronal function in AD patients and decrease the age of onset (Huang and Mucke, 2012;

compared with age-matched controls (Chiba et al., 2009b). Animal experiments further showed that (i) i.c.v. injection of toxic Aβ peptide reduces p-Stat3 in hippocampal neurons to induce memory impairment (a toxic Aβ gain of function effect) and that (ii) Aβ passive immunotherapy reduces cerebral Aβ burden and recovers cognitive function of Tg2576 mice in parallel with Stat3 activation in hippocampal neurons (a toxic Aβ loss of function effect). These data suggest that cerebral Aβ levels are inversely correlated with p-STAT3 levels in

Aging, one of the most common risk factor for AD, seems to be one of the other factors responsible for the AD-related neuronal inactivation of Stat3 because p-Stat3 immunoreactiv‐ ity in hippocampal neurons of young subjects was substantially higher than that of elder cognitively normal subjects in both humans and rodents (Chiba et al., 2009b). Aging-depend‐ ent reduction of p-Stat3 levels may be due to age-dependent decrease in endogenous neuro‐ trophic factors, which play a role in sustaining neuronal p-Stat3 levels. In support of this idea, it is reported that endogenous levels of insulin-like growth factor I (IGF-I), which activates Stat3, decrease with aging and that this decrease may be linked to the pathogenesis of AD

Relationship between Stat3 and tau is yet to be elucidated. Stat3 binds to and inhibits stathmin, which depolymerizes microtubules, while tau binds to and stabilizes microtubules (Ng et al., 2006). Involvement of STAT3-mediated stathmin regulation in tau phosphorylation should be addressed in the future research. There are some reports describing Stat3 inactivation and cell death; i.e. Stat3 deletion sensitizes cells to oxidative stress (Barry et al., 2009; Sarafian et al.). Combined with the fact that Aβ induces neuronal death by inducing oxidative stress *in vitro* and *in vivo* (Butterfield et al., 2002; De Felice et al., 2007; Guglielmotto et al., 2009), Stat3 may

In addition to the role of Stat3 in neuronal death, roles of Jak2/Stat3 pathway in synaptic plasticity have been reported. Activation of the Jak2/Stat3 pathway induces presynaptic transcriptional upregulation of cholinergic genes including choline acetyletransferase (ChAT) and vesicular acetylcholine transporter (VAChT) and postsynaptic sensitization of m1-type muscarinic acetylcholine receptor (m1-mAChR) to support cholinergic neurotransmission (Chiba et al., 2009b). The Jak2/Stat3 pathway also plays an essential role in the induction of NMDA-receptor dependent long-term depression (NMDAR-LTD) in the hippocampus

AD can be classified into early onset (EOAD, <65 years) and late onset (LOAD, >65 years) form. According to family history, AD can be also classified into FAD and sporadic (SAD) (Alves et al., 2010). Although aforementioned three FAD genes generally result in EOAD, there is a substantial genetic component in LOAD as well (an estimated heritability of 58-79%). Accord‐ ingly, *apolipoprotein E (APOE)* gene on chromosome 19 has also been shown to be a genetic risk factor for LOAD in the early 1990s (Bertram et al., 2010; Schellenberg and Montine, 2012). *APOE* contains three major alleles: ε2, ε3, and ε4. Inherited ε4 allele is reported to worsen the loss of neuronal function in AD patients and decrease the age of onset (Huang and Mucke, 2012;

hippocampal neurons *in vivo* (Chiba et al., 2009b).

sensitize neurons to Aβ neurotoxicity through oxidative stress.

(Rollero et al., 1998).

186 Neurodegenerative Diseases

(Nicolas et al., 2012).

**2.3. Emerging genetic risk factors for AD**

**Figure 3. A schematic overview of the "amyloid cascade" hypothesis and therapeutic strategies.** (Red arrows) The "amyloid cascade" begins with the proteolytic cleavage of APP by the γ-secretase. Aβ forms toxic oligomers or aggregates, inducing neuronal Stat3 inactivation, NFT formation, caspase activation and neuronal death. Therapies targeting the canonical "amyloid cascade" such as secretase inhibitors and immunotherapy are indicated in blue char‐ acters. Therapies targeting on the downstream pathways of the "amyloid cascade" are indicated in green characters. Alternative pathological mechanisms, which can be caused by intracellular Aβ, extracellular Aβ, or FAD mutants them‐ selves, are also targeted (purple). (NEP, neprilysin; IDE, insulin degrading enzyme; VPA, valproic acid; HN, humanin).

Poirier, 2005). One copy of *APOE* ε4 increases the risk for AD fourfold and two copies further raise the risk tenfold. *APOE* is not a causative gene, i.e. *APOE* ε4 is neither necessary nor sufficient for LOAD. In addition to *APOE* itself, a variable-length poly-T (deoxythymidine homopolymer) polymorphism in the *TOMM40* gene, located in a region of strong linkage disequilibrium next to *APOE*, was reported to be associated with the age of onset of LOAD (Lutz et al., 2010; Roses et al.).

Genetic association studies on LOAD susceptibility loci and small nucleotide polymor‐ phisms (SNPs) revealed two genes: *ubiquilin 1 (UBQLN1)* on chromosome 9 (Bertram et al., 2005) and *sortilin-related receptor (SORL1)* (Rogaeva et al., 2007). Identification of *UBQLN1* is intriguing since it not only interacts with PSEN1, PSEN2 and APP but also may play a role in the proteasome degradation of them (Mah et al., 2000; Massey et al., 2004). SORL1 is involved in both trafficking of APP from the cell surface into recycling pathways and processing of APP by γ-secretase. A recent meta-analysis of genetic data and GWAS supported that mutations in *SORL1* may play a role in LOAD although the effect on the AD risk is moderate (Schellenberg and Montine, 2012). On the other hand, no evidence for *UBQLN1* has been provided from GWAS.

Since 2009, several GWAS results have been published (Alves et al., 2010; Bertram, 2011; Eisenstein, 2011; Reitz, 2012; Schellenberg and Montine, 2012). In GWAS, researchers analyze millions of SNPs in affected and healthy individuals. There is a criticism that two many comparisons at the same time will just lead to a number of false positive associations, which cannot be reproduced by other studies. This is partially true. GWAS, however, have identified nine novel loci reproducibly associated with LOAD: (i) *clusterin (CLU)*, (ii) *phosphatidylinositolbinding clathrin assembly protein (PICALM)*, (iii) *complement receptor 1 (CR1)*, (iv) *bridging integrator 1 (BIN1)*, (v) *ATP-binding cassette, subfamily A, member 7 (ABCA7)*, (vi) *membranespanning 4-domains, subfamily A, members 4 and 6E (MS4A4/MS4A6E, MS4A cluster)*, (vii) *CD2 associated protein (CD2AP)*, (viii) *CD33 molecule (CD33)* and (ix) *EPH receptor A1 (EPHA1)* (Bertram, 2011). These genes significantly increase risk for LOAD although they rather have small effects on susceptibility to AD (within 1.5 times increase or decrease in odds ratio).

by an enzyme ChAT in certain neurons. There are two major classes of ACh receptors (AChRs); i.e. ionotropic nicotinic AChR (nAChR) and metabotropic muscarinic AChR (mAChR). ACh in the synaptic cleft is rapidly degraded into two inactive metabolites choline and acetate by an enzyme, ChE. Thus, cholinergic neurotransmission can be improved with ChEIs via

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ChEIs, such as donepezil (Aricept®), galantamine (Razadyne®) and rivastigmine (Exelon®) are approved by the Food and Drug Administration (FDA) for the treatment of AD (Farrimond et al., 2012; Pettenati et al., 2003). Donepezil is a reversible ChEI with high specificity with few side effects and can be used for all stages of AD. Galantamine, which is approved for mild-tomoderate AD, has multiple functions such as inhibition of ChE, induction of ACh release and allosteric stimulation of nAChR. Rivastigmine, which is also for mild-to-moderate AD, suppresses both acetyl- and butyryl-ChE. Some placebo-controlled, double-blind trials have demonstrated that ChEI therapy results in significant improvement in cognitive performance of AD patients. Unfortunately, however, a long-term rondamized trial named 'AD2000' failed to prove therapeutic benefits of Donepezil (Courtney et al., 2004; Tariot et al., 2001; Winblad et al., 2006). Although ChEIs may slow worsening of AD symptoms to some extent, it should be noted that the effectiveness of ChEIs and how long they work, would vary from person to person. In addition, direct activation of acetylcholine receptors, such as m1-mAChR and α7

Excitotoxicity by excess amount of glutamatergic neurotransmission is also implicated in the pathogenesis of AD. Memantine (Namenda®) is a non-competitive, voltage-dependent and of moderate affinity N-methyl-D-aspartate (NMDA) receptor antagonist, preventing neuronal cells from excitotoxic death caused by a glutamate-induced excessive income of calcium ions. Memantine was approved by the FDA in 2003 for the treatment of moderate-to-severe AD since it improved cognitive function of AD patients who were receiving stable doses of a ChEI (Tariot et al., 2004). Serious side effects have not been reported to occur at high frequency. In addition to the NMDA antagonism, memantine is reported to decrease Aβ production (Alley et al., 2009; Ray et al., 2009), suppress synaptic dysfunction (Klyubin et al., 2009) and induce protein phosphatase-2A (PP2A)-mediated reduction of tau hyperphosphorylation (Chohan et

AD patients also suffer from neuropsychiatric symptoms such as apathy, depression, anxiety, psychosis, aggression and sleep disturbances, which are generally called as behavioral and psychological symptoms in dementia (BPSD) (Alves et al., 2010). When BPSD are severe and persist despite the use of ChEIs and memantine, pharmacological treatment will be started. For severe depression, antidepressive drugs such as sertraline, which is one of the selective serotonin reuptake inhibitors (SSRIs) used to treat major depression. Neuroleptic drugs such as risperidone, which is a dopamine antagonist and mainly used to treat schizophrenia, are used to treat aggression, agitation and psychosis. Anxiolytics such as loracepam and oxacepam

prevention of ACh degradation.

nAChR, is currently under development.

al., 2006; Martinez-Coria et al., 2010).

**3.3. Other supporting drugs**

**3.2. N-methyl-D-aspartate (NMDA) receptor antagonists**

One of the important aims of GWAS is to identify novel pathogenic pathways for AD. Although Morgan, for example, classified risk genes identified from GWAS into three new pathogenic pathways (Morgan): immune system function (*CLU, CR1, ABCA7, MS4A cluster, CD33 and EPHA1*), cholesterol metabolism (*APOE, CLU and ABCA7*) and synaptic dysfunction (*PICALM, BIN1, CD33, CD2AP and EPHA1*). Others assume that most of risk genes are related to the "amyloid cascade", providing a solid support for the "amyloid cascade" hypothesis. This discrepancy is due to multiple functions of each risk gene. E.g. *CLU*, which is also known as *apolipoprotein-J (APOJ)*, helps *APOE* in cholesterol trafficking in the central nervous system and is also involved in Aβ aggregation and clearance. *CR1* is an important component of the innate immune response against infection and is also involved in the clearance of circulating Aβ. In summary, *APOE* is still the biggest risk gene for LOAD and detailed functional analyses are necessary for other novel risk genes to discuss novel or relevant pathogenic mechanisms for AD.

### **3. Clinically available drugs for AD**

In this section, currently available drugs in AD clinics, such as cholinesterase inhibitors (ChEIs) and N-methyl-D-aspartate (NMDA) receptor antagonists will be discussed (Lleo et al., 2006; Mangialasche et al., 2010). More than 15 years have passed since the first ChEI, donepezil (Aricept®), was approved as a clinical drug for AD in the United States. Many clinical trials and follow-up surveys have revealed marginal to moderate effects of ChEIs and NMDA antagonists in AD patients (Riordan et al., 2011). They show only symptomatic effects although potential neuroprotective effects have been postulated.

#### **3.1. Cholinesterase inhibitors (ChEIs)**

Deteriorated cholinergic function such as loss of basal forebrain cholinergic neurons is one of the pathological features of AD (Bartus et al., 1982; Farrimond et al., 2012; Van der Zee et al.). Acetylcholine (ACh), which is one of neurotransmitters functioning in both central and peripheral nervous system, is synthesized from choline and acetyl coenzyme A (acetyl-CoA) by an enzyme ChAT in certain neurons. There are two major classes of ACh receptors (AChRs); i.e. ionotropic nicotinic AChR (nAChR) and metabotropic muscarinic AChR (mAChR). ACh in the synaptic cleft is rapidly degraded into two inactive metabolites choline and acetate by an enzyme, ChE. Thus, cholinergic neurotransmission can be improved with ChEIs via prevention of ACh degradation.

ChEIs, such as donepezil (Aricept®), galantamine (Razadyne®) and rivastigmine (Exelon®) are approved by the Food and Drug Administration (FDA) for the treatment of AD (Farrimond et al., 2012; Pettenati et al., 2003). Donepezil is a reversible ChEI with high specificity with few side effects and can be used for all stages of AD. Galantamine, which is approved for mild-tomoderate AD, has multiple functions such as inhibition of ChE, induction of ACh release and allosteric stimulation of nAChR. Rivastigmine, which is also for mild-to-moderate AD, suppresses both acetyl- and butyryl-ChE. Some placebo-controlled, double-blind trials have demonstrated that ChEI therapy results in significant improvement in cognitive performance of AD patients. Unfortunately, however, a long-term rondamized trial named 'AD2000' failed to prove therapeutic benefits of Donepezil (Courtney et al., 2004; Tariot et al., 2001; Winblad et al., 2006). Although ChEIs may slow worsening of AD symptoms to some extent, it should be noted that the effectiveness of ChEIs and how long they work, would vary from person to person. In addition, direct activation of acetylcholine receptors, such as m1-mAChR and α7 nAChR, is currently under development.

#### **3.2. N-methyl-D-aspartate (NMDA) receptor antagonists**

Excitotoxicity by excess amount of glutamatergic neurotransmission is also implicated in the pathogenesis of AD. Memantine (Namenda®) is a non-competitive, voltage-dependent and of moderate affinity N-methyl-D-aspartate (NMDA) receptor antagonist, preventing neuronal cells from excitotoxic death caused by a glutamate-induced excessive income of calcium ions. Memantine was approved by the FDA in 2003 for the treatment of moderate-to-severe AD since it improved cognitive function of AD patients who were receiving stable doses of a ChEI (Tariot et al., 2004). Serious side effects have not been reported to occur at high frequency. In addition to the NMDA antagonism, memantine is reported to decrease Aβ production (Alley et al., 2009; Ray et al., 2009), suppress synaptic dysfunction (Klyubin et al., 2009) and induce protein phosphatase-2A (PP2A)-mediated reduction of tau hyperphosphorylation (Chohan et al., 2006; Martinez-Coria et al., 2010).

#### **3.3. Other supporting drugs**

Since 2009, several GWAS results have been published (Alves et al., 2010; Bertram, 2011; Eisenstein, 2011; Reitz, 2012; Schellenberg and Montine, 2012). In GWAS, researchers analyze millions of SNPs in affected and healthy individuals. There is a criticism that two many comparisons at the same time will just lead to a number of false positive associations, which cannot be reproduced by other studies. This is partially true. GWAS, however, have identified nine novel loci reproducibly associated with LOAD: (i) *clusterin (CLU)*, (ii) *phosphatidylinositolbinding clathrin assembly protein (PICALM)*, (iii) *complement receptor 1 (CR1)*, (iv) *bridging integrator 1 (BIN1)*, (v) *ATP-binding cassette, subfamily A, member 7 (ABCA7)*, (vi) *membranespanning 4-domains, subfamily A, members 4 and 6E (MS4A4/MS4A6E, MS4A cluster)*, (vii) *CD2 associated protein (CD2AP)*, (viii) *CD33 molecule (CD33)* and (ix) *EPH receptor A1 (EPHA1)* (Bertram, 2011). These genes significantly increase risk for LOAD although they rather have small effects on susceptibility to AD (within 1.5 times increase or decrease in odds ratio).

One of the important aims of GWAS is to identify novel pathogenic pathways for AD. Although Morgan, for example, classified risk genes identified from GWAS into three new pathogenic pathways (Morgan): immune system function (*CLU, CR1, ABCA7, MS4A cluster, CD33 and EPHA1*), cholesterol metabolism (*APOE, CLU and ABCA7*) and synaptic dysfunction (*PICALM, BIN1, CD33, CD2AP and EPHA1*). Others assume that most of risk genes are related to the "amyloid cascade", providing a solid support for the "amyloid cascade" hypothesis. This discrepancy is due to multiple functions of each risk gene. E.g. *CLU*, which is also known as *apolipoprotein-J (APOJ)*, helps *APOE* in cholesterol trafficking in the central nervous system and is also involved in Aβ aggregation and clearance. *CR1* is an important component of the innate immune response against infection and is also involved in the clearance of circulating Aβ. In summary, *APOE* is still the biggest risk gene for LOAD and detailed functional analyses are necessary for other novel risk genes to discuss novel or relevant pathogenic mechanisms

In this section, currently available drugs in AD clinics, such as cholinesterase inhibitors (ChEIs) and N-methyl-D-aspartate (NMDA) receptor antagonists will be discussed (Lleo et al., 2006; Mangialasche et al., 2010). More than 15 years have passed since the first ChEI, donepezil (Aricept®), was approved as a clinical drug for AD in the United States. Many clinical trials and follow-up surveys have revealed marginal to moderate effects of ChEIs and NMDA antagonists in AD patients (Riordan et al., 2011). They show only symptomatic effects although

Deteriorated cholinergic function such as loss of basal forebrain cholinergic neurons is one of the pathological features of AD (Bartus et al., 1982; Farrimond et al., 2012; Van der Zee et al.). Acetylcholine (ACh), which is one of neurotransmitters functioning in both central and peripheral nervous system, is synthesized from choline and acetyl coenzyme A (acetyl-CoA)

for AD.

188 Neurodegenerative Diseases

**3. Clinically available drugs for AD**

potential neuroprotective effects have been postulated.

**3.1. Cholinesterase inhibitors (ChEIs)**

AD patients also suffer from neuropsychiatric symptoms such as apathy, depression, anxiety, psychosis, aggression and sleep disturbances, which are generally called as behavioral and psychological symptoms in dementia (BPSD) (Alves et al., 2010). When BPSD are severe and persist despite the use of ChEIs and memantine, pharmacological treatment will be started. For severe depression, antidepressive drugs such as sertraline, which is one of the selective serotonin reuptake inhibitors (SSRIs) used to treat major depression. Neuroleptic drugs such as risperidone, which is a dopamine antagonist and mainly used to treat schizophrenia, are used to treat aggression, agitation and psychosis. Anxiolytics such as loracepam and oxacepam (benzodiazepines) are also used to ameliorate anxiety and verbal problems. For sleeping disturbances, non-pharmacological interventions are common because sleeping drugs or sedative-hypnotic medications have only a limited efficacy and significant adverse effects (David et al., 2010; Shub et al., 2009). In addition, melatonin, which is an endogenous regulator of circadian rhythms and is used as a chronobiotic or soporific, showed no benefits in a multicenter, placebo-controlled trial for sleep disturbance in AD (Singer et al., 2003). These drugs are used to reduce BPSD and to improve patients' activities of daily living and quality of life.

Europe and USA, however, were suspended since 6% of the subjects gave a sign of menin‐ goencephalitis such as fever, vomiting, headache, loss of consciousness, and so on after the second vaccination (Orgogozo et al., 2003). Patients who received AN-1792 have been reported to show elevated levels of antibodies against Aβ (Hock et al., 2002) and some immunized patients resulted in reduction of amyloid deposits without significant decrease in both NFT

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191

As reported, there is a potential risk in the Aβ immunothrerapy that antibodies against APP induce neuronal cell death, which may lead to meningoencephalitis (Rohn et al., 2000; Sudo et al., 2000). Although it is reported that antibodies elevated in patients mainly recognize SPs, there is still a certain possibility that small fraction of elevated antibodies binds to APP and induces neuronal death. Another potential risk is that cytotoxic T cells, which might cause cellular toxicity on neurons, are also activated by active immuniza‐ tion. In agreement, there were a number of lymphocytic infiltration in brains of immu‐

Recently, however, several alternatives of Aβ immunotherapy have been proposed. Passive immunization with anti-Aβ monoclonal antibodies is first one (Dodart et al., 2002; Kotilinek et al., 2002). In this approach, it seems easier to control side-effects by using monoclonal antibody than non-specific activation of immune system by active immunization. Active immunization can be safer by specific targeting of immunogen to T-helper cells. Other types of immunization have been also developed. Hara *et al.* developed an oral vaccine using recombinant adeno-associated virus (AAV) vector encoding Aβ cDNA (Hara et al., 2004) and Okura *et al.* developed nonviral Aβ DNA vaccine (Okura et al., 2006). With these vaccines, serum antibodies against Aβ were elevated by ectopic Aβ expression without T cell prolifer‐

New vaccines that selectively target B-cell epitopes (N-terminus of Aβ peptide) have been developed. CAD-106, consisting of Aβ1-6 peptide coupled to virus-like carrier particle Qβ (a T-helper cell epitope) could efficiently induce Aβ antibodies without induction of Aβ-specific T-cells (Wiessner et al., 2011). Similar strategy is employed in other vaccines: ACC-01 (Aβ1-6 donjugated to the mutated diphtheria toxin protein CRM19), V-950 (Aβ N-terminus with an aluminium-containing adjuvant with or without ISCOMATRIX), ACI-24 (Aβ1-15 closely apposed to the surface of the liposome), UB-311 (Aβ1-14 associated with the UBITh peptide, a T-helper cell epitope) (Mangialasche et al., 2010; Reitz, 2012). Another active immunization strategy is based on Affitopes, short peptides mimicking parts of native Aβ42 without its sequence identity. AD-01 and AD-02 mimick the N-terminal Aβ fragments. These vaccines are generally reported to be safe and well-tolerated in phase I studies. Results of phase II and III

Passive immunotherapy is based on the administration of antibodies against Aβ. This can be achieved by both monoclonal antibodies and polyclonal immunoglobulins. In animal models,

ative response. Safety of these vaccines, however, should be carefully tested.

formation and neuronal cell death (Nicoll et al., 2003).

nized AD patients (Nicoll et al., 2003). **Improvement of Aβ immunotherapy**

**Improved active Aβ immunotherapy**

studies are awaited.

**Passive Aβ immunotherapy**

### **4. Emerging therapeutic strategies**

Finally, emerging therapeutic strategies will be discussed. For better understanding, strategies are first classified into two major categories: AD-specific and AD-non-specific disease modi‐ fying treatments (Table 1). As to AD-specific disease-modifying treatments, the amyloidogenic processes and their regulatory mechanisms are defined as the canonical "amyloid cascade". Strategies based on other findings related to the downstream pathways of the "amyloid cascade" are summarized in the other drugs based on alternative pathways. Drugs related to the downstream mechanisms from the amyloidogenesis including tau phosphorylation and Stat3 inactivation are classified into the alternative pathways here because the connection between the canonical "amyloid cascade" and the downstream mechanisms is still elusive. As to AD-non-specific treatments, regenerative therapy may be realized in the near future with an enormous progress in the field of stem cell biology.

#### **4.1. AD-specific drugs targeting the canonical "amyloid cascade"**

In the last 10 years, most pharmaceutical companies were trying hard to develop secretase inhibitors such as α-, β- and γ-secretase inhibitors to suppress amyloid beta production, the initial step of the "amyloid cascade" (Ghosh et al., 2012; Wolfe, 2012). A number of clinical trials of β-secretase inhibitors and γ-secretase inhibitors have been carried out or are ongoing.

Another major attempt has been the development of Aβ immunotherapy to remove amyloid deposits, which is also targeting the initial step of the cascade (Delrieu et al., 2012). Both active and passive immunization have been developed by utilizing Aβ peptide and its antigenic epitopes, or anti-Aβ antibodies.

#### *4.1.1. Aβ immunotherapy to remove brain Aβ*

#### **Initial findings from a clinical trial of active Aβ immunotherapy**

Aβ immunotherapy is an approach to remove accumulated Aβ in AD brains immunologically: Aβ vaccination therapy and passive immunization with anti-Aβ antibody. Schenk *et al.* reported that the Aβ vaccine named AN-1792, which contains Aβ42 peptide and adjuvants, significantly reduced amyloid plaques in brain of PD-APP mice (Schenk et al., 1999). With the evidence in transgenic mice, clinical trial was started in 2001. Phase II studies performed in Europe and USA, however, were suspended since 6% of the subjects gave a sign of menin‐ goencephalitis such as fever, vomiting, headache, loss of consciousness, and so on after the second vaccination (Orgogozo et al., 2003). Patients who received AN-1792 have been reported to show elevated levels of antibodies against Aβ (Hock et al., 2002) and some immunized patients resulted in reduction of amyloid deposits without significant decrease in both NFT formation and neuronal cell death (Nicoll et al., 2003).

As reported, there is a potential risk in the Aβ immunothrerapy that antibodies against APP induce neuronal cell death, which may lead to meningoencephalitis (Rohn et al., 2000; Sudo et al., 2000). Although it is reported that antibodies elevated in patients mainly recognize SPs, there is still a certain possibility that small fraction of elevated antibodies binds to APP and induces neuronal death. Another potential risk is that cytotoxic T cells, which might cause cellular toxicity on neurons, are also activated by active immuniza‐ tion. In agreement, there were a number of lymphocytic infiltration in brains of immu‐ nized AD patients (Nicoll et al., 2003).

#### **Improvement of Aβ immunotherapy**

(benzodiazepines) are also used to ameliorate anxiety and verbal problems. For sleeping disturbances, non-pharmacological interventions are common because sleeping drugs or sedative-hypnotic medications have only a limited efficacy and significant adverse effects (David et al., 2010; Shub et al., 2009). In addition, melatonin, which is an endogenous regulator of circadian rhythms and is used as a chronobiotic or soporific, showed no benefits in a multicenter, placebo-controlled trial for sleep disturbance in AD (Singer et al., 2003). These drugs are used to reduce BPSD and to improve patients' activities of daily living and quality

Finally, emerging therapeutic strategies will be discussed. For better understanding, strategies are first classified into two major categories: AD-specific and AD-non-specific disease modi‐ fying treatments (Table 1). As to AD-specific disease-modifying treatments, the amyloidogenic processes and their regulatory mechanisms are defined as the canonical "amyloid cascade". Strategies based on other findings related to the downstream pathways of the "amyloid cascade" are summarized in the other drugs based on alternative pathways. Drugs related to the downstream mechanisms from the amyloidogenesis including tau phosphorylation and Stat3 inactivation are classified into the alternative pathways here because the connection between the canonical "amyloid cascade" and the downstream mechanisms is still elusive. As to AD-non-specific treatments, regenerative therapy may be realized in the near future with

In the last 10 years, most pharmaceutical companies were trying hard to develop secretase inhibitors such as α-, β- and γ-secretase inhibitors to suppress amyloid beta production, the initial step of the "amyloid cascade" (Ghosh et al., 2012; Wolfe, 2012). A number of clinical trials of β-secretase inhibitors and γ-secretase inhibitors have been carried out or are ongoing.

Another major attempt has been the development of Aβ immunotherapy to remove amyloid deposits, which is also targeting the initial step of the cascade (Delrieu et al., 2012). Both active and passive immunization have been developed by utilizing Aβ peptide and its antigenic

Aβ immunotherapy is an approach to remove accumulated Aβ in AD brains immunologically: Aβ vaccination therapy and passive immunization with anti-Aβ antibody. Schenk *et al.* reported that the Aβ vaccine named AN-1792, which contains Aβ42 peptide and adjuvants, significantly reduced amyloid plaques in brain of PD-APP mice (Schenk et al., 1999). With the evidence in transgenic mice, clinical trial was started in 2001. Phase II studies performed in

of life.

190 Neurodegenerative Diseases

**4. Emerging therapeutic strategies**

an enormous progress in the field of stem cell biology.

epitopes, or anti-Aβ antibodies.

*4.1.1. Aβ immunotherapy to remove brain Aβ*

**4.1. AD-specific drugs targeting the canonical "amyloid cascade"**

**Initial findings from a clinical trial of active Aβ immunotherapy**

Recently, however, several alternatives of Aβ immunotherapy have been proposed. Passive immunization with anti-Aβ monoclonal antibodies is first one (Dodart et al., 2002; Kotilinek et al., 2002). In this approach, it seems easier to control side-effects by using monoclonal antibody than non-specific activation of immune system by active immunization. Active immunization can be safer by specific targeting of immunogen to T-helper cells. Other types of immunization have been also developed. Hara *et al.* developed an oral vaccine using recombinant adeno-associated virus (AAV) vector encoding Aβ cDNA (Hara et al., 2004) and Okura *et al.* developed nonviral Aβ DNA vaccine (Okura et al., 2006). With these vaccines, serum antibodies against Aβ were elevated by ectopic Aβ expression without T cell prolifer‐ ative response. Safety of these vaccines, however, should be carefully tested.

#### **Improved active Aβ immunotherapy**

New vaccines that selectively target B-cell epitopes (N-terminus of Aβ peptide) have been developed. CAD-106, consisting of Aβ1-6 peptide coupled to virus-like carrier particle Qβ (a T-helper cell epitope) could efficiently induce Aβ antibodies without induction of Aβ-specific T-cells (Wiessner et al., 2011). Similar strategy is employed in other vaccines: ACC-01 (Aβ1-6 donjugated to the mutated diphtheria toxin protein CRM19), V-950 (Aβ N-terminus with an aluminium-containing adjuvant with or without ISCOMATRIX), ACI-24 (Aβ1-15 closely apposed to the surface of the liposome), UB-311 (Aβ1-14 associated with the UBITh peptide, a T-helper cell epitope) (Mangialasche et al., 2010; Reitz, 2012). Another active immunization strategy is based on Affitopes, short peptides mimicking parts of native Aβ42 without its sequence identity. AD-01 and AD-02 mimick the N-terminal Aβ fragments. These vaccines are generally reported to be safe and well-tolerated in phase I studies. Results of phase II and III studies are awaited.

#### **Passive Aβ immunotherapy**

Passive immunotherapy is based on the administration of antibodies against Aβ. This can be achieved by both monoclonal antibodies and polyclonal immunoglobulins. In animal models,

anti-Aβ antibodies are reported to prevent oligomer formation with reduced brain Aβ load. Several monoclonal antibodies are currently tested in clinical trials: bapineuzumab (AAB-001), solanezumab(LY-2062430),ponezumab(PF-04360365),GSK-933776,R-1450 (RO-4909832), and MABT-5102A (Mangialasche et al., 2010). A phase II study of bapineuzumab in patients with mild to moderate AD did not attain statistical significance on the primary efficacy endpoints in the overall study population (Salloway et al., 2009). They only found some statistically signifi‐ cant benefits in limited subpopulations without the APOE e4 allele. A phase III study of bapineuzumab finally again failed recently (Aug. 6, 2012: www.pfizer.com). Solanezumab, which mobilized brain Aβ in a phase II study (Farlow et al., 2012), also failed to slow cognitive decline inphase III clinicaltrials includingmore than2,050ADpatients (Aug. 24, 2012: www.lil‐ ly.com). Ponezumab is a humanized monoclonal IgG2 antibody binding to the C-terminus of Aβ40 and a phase I trial showed that the antibody is well tolerated in AD patients (Freeman et al., 2012). Other antibodies are also currently tested in phase I and II studies.

2001). Consequently, it is assumed that β-secretase inhibitors are safer than γ-secretase inhibi‐ tors. Recently, however, BACE1/2 double knockout mice was generated and resulted in lethal phenotype,presumablybecauseBACE1/2havemanysubstrates(includingneuregulin-1,which

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http://dx.doi.org/10.5772/55293

193

Pioglitazone and rosiglitazone are thiazolidinediones, which control blood sugar by stimulat‐ ing the nuclear peroxisome proliferator-activated receptor γ (PPARγ), clinically used for type II diabetes mellitus (DM). They turned out to be good BACE1 inhibitors: i.e. PPARγ activation by the thiazolidinediones suppresses BACE1 and APP expression (Mangialasche et al., 2010). In addition, pioglitazone and rosiglitazone promote Aβ degradation, which is competitively inhibited by insulin, by reducing insulin concentrations. Results from clinical studies are, however, disappointing (Miller et al., 2011). Neither pioglitazone nor rosiglitazone showed

Development of γ-secretase inhibitors, which suppress the final step in amyloidgenesis, is one of the major issues in AD research. Transition state analogs of γ-secretase, such as L-685458 (Merk), inhibit the γ-secretase activity and decreases production of Aβ40 and Aβ42 (Shearman et al., 2000). Chemical compunds including pepstatin A, sulfonamide derivatives and benzo‐ diazepines are also shown to inhibit the γ-secretase activity in mechanisms other than competitive inhibition (Tian et al., 2002). Since γ-secretase is also involved in processing of other membrane proteins including Notch, γ-secretase inhibitors may induce severe side effects. In agreement, the chronic administration of a γ-secretase inhibitor triggered abnormal blood cell differentiation and damage on digestive tracts (Searfoss et al., 2003). APP-specific

Several γ-secretase inhibitors are currently tested in clinical studies: semagacestat (LY450139), begacestat (GSI-953), avagacestat (BMS-708163), PF-3084014, MK-0752, E-2012 and NIC5-15 (Mangialasche et al., 2010). Phase III trials for semagacestat, which inhibits Notch cleavage as well as APP cleavage, not only failed but also worsened clinical measures of cognition and activity of daily living with increased incidence of skin cancer (Cummings, 2010). Other Notchsparing γ-secretase inhibitors (second-generation inhibitors) such as begacestat and avagace‐ stat are now in phase I or II studies and they show reduction of plasma and/or cerebrospinal fluid (CSF) Aβ levels. PF-3084014, a γ-secretase inhibitor with high selectivity for APP, and NIC5-15, a Notch-sparing γ-secretase inhibitor with insulin sensitizing activity, showed some positive effects with good tolerance and now proceeded into phase II studies (Mangialasche et al., 2010). Effects of a γ-secretase modulator, tarenflurbil, will be discussed later (see 4.3.2.).

Upregulation of α-cleavage, which results in non-amyloidogenic cleavage of APP, can competitively inhibit β-cleavage, leading to downregulation of Aβ production and reciprocal upregulation of neuroprotective soluble APPα (sAPPα) secretion (Vingtdeux and Maram‐ baud, 2012). α-Secretase is a member of the ADAM (a disintegrin and metalloprotease) family of proteases including ADAM10, ADAM17/TACE (TNFα converting enzyme), or ADAM9. There is a number of ways to activate α-secretase (Mangialasche et al., 2010): (i) activation of

γ-secretase inhibitors or γ-secretase modulators may resolve this problem.

is involved in myelination) required in the development (Dominguez et al., 2005).

any efficacy on cognition in AD patients.

**γ-secretase inhibitors**

**α-secretase activators**

Passive immunization can also be achieved by intravenous infusion of immunoglobins (IVIg), from healthy donors, which are assumed to include naturally occurring polyclonal antibodies againstpathogenicAβ(Britschgietal.,2009;Dodeletal.,2002;Dodeletal.,2004;Fillitetal.,2009). IVIg is already approved for immune deficiency, meaning that it is safe and well tolerated. Preliminarydata fromaphase II study showedapositive effect oncognition(Relkinet al., 2009).

#### **Active versus passive Aβ immunotherapy**

Active immunotherapy will keep high antibody titers for a long period, enabling few followup observations and reduced costs. The control of the antibody concentrations and adverse effectsarelimited,however.Passiveimmunotherapyoffersagoodandrapidcontrolofantibody properties and concentrations. In addition, passive immunotherapy could be more effective in elderly population with reduced immune responses. However, administration of antibodies is time- and money-consuming. The effects of active and passive Aβ immunotherapy are not so much different and unfortunately both immunotherapy are not successful so far.

#### *4.1.2. γ-secretase and β-secretase inhibitors for reduction of Aβ production*

Secretases have become the targets to control Aβ production and prevent the progress of AD since Aβ is generated from APP via sequential cleavage by β- and γ-secretases (Chiba et al., 2007; De Strooper and Annaert, 2000). This is the initial step in the canonical "amyloid cascade". Considering that most of the mutant *APP* and *PSEN1* result in increased Aβ42/Aβ40 ratio, abnormal γ-secretase function seems to be closely related to the pathogenesis of AD. In addition, the choice of the first cleavage by α- or β-secretase decides the generation of Aβ, which gives us the notion that β-cleavage inhibition and α-cleavage activation greatly reduce Aβ production.

#### **β-secretase inhibitors**

In 1999, β-site APP cleaving enzyme (BACE1), a membrane-anchored aspartyl protease, was reported to have β-secretase activity (John et al., 2003; Vassar et al., 1999). The BACE1 knock out mice studies showed that Aβ levels in brain was drastically reduced, and they remain healthy without any anomaly in clear contrast to that of PSEN knockout mice (Cai et al., 2001; Luo et al.,

2001). Consequently, it is assumed that β-secretase inhibitors are safer than γ-secretase inhibi‐ tors. Recently, however, BACE1/2 double knockout mice was generated and resulted in lethal phenotype,presumablybecauseBACE1/2havemanysubstrates(includingneuregulin-1,which is involved in myelination) required in the development (Dominguez et al., 2005).

Pioglitazone and rosiglitazone are thiazolidinediones, which control blood sugar by stimulat‐ ing the nuclear peroxisome proliferator-activated receptor γ (PPARγ), clinically used for type II diabetes mellitus (DM). They turned out to be good BACE1 inhibitors: i.e. PPARγ activation by the thiazolidinediones suppresses BACE1 and APP expression (Mangialasche et al., 2010). In addition, pioglitazone and rosiglitazone promote Aβ degradation, which is competitively inhibited by insulin, by reducing insulin concentrations. Results from clinical studies are, however, disappointing (Miller et al., 2011). Neither pioglitazone nor rosiglitazone showed any efficacy on cognition in AD patients.

#### **γ-secretase inhibitors**

anti-Aβ antibodies are reported to prevent oligomer formation with reduced brain Aβ load. Several monoclonal antibodies are currently tested in clinical trials: bapineuzumab (AAB-001), solanezumab(LY-2062430),ponezumab(PF-04360365),GSK-933776,R-1450 (RO-4909832), and MABT-5102A (Mangialasche et al., 2010). A phase II study of bapineuzumab in patients with mild to moderate AD did not attain statistical significance on the primary efficacy endpoints in the overall study population (Salloway et al., 2009). They only found some statistically signifi‐ cant benefits in limited subpopulations without the APOE e4 allele. A phase III study of bapineuzumab finally again failed recently (Aug. 6, 2012: www.pfizer.com). Solanezumab, which mobilized brain Aβ in a phase II study (Farlow et al., 2012), also failed to slow cognitive decline inphase III clinicaltrials includingmore than2,050ADpatients (Aug. 24, 2012: www.lil‐ ly.com). Ponezumab is a humanized monoclonal IgG2 antibody binding to the C-terminus of Aβ40 and a phase I trial showed that the antibody is well tolerated in AD patients (Freeman et

Passive immunization can also be achieved by intravenous infusion of immunoglobins (IVIg), from healthy donors, which are assumed to include naturally occurring polyclonal antibodies againstpathogenicAβ(Britschgietal.,2009;Dodeletal.,2002;Dodeletal.,2004;Fillitetal.,2009). IVIg is already approved for immune deficiency, meaning that it is safe and well tolerated. Preliminarydata fromaphase II study showedapositive effect oncognition(Relkinet al., 2009).

Active immunotherapy will keep high antibody titers for a long period, enabling few followup observations and reduced costs. The control of the antibody concentrations and adverse effectsarelimited,however.Passiveimmunotherapyoffersagoodandrapidcontrolofantibody properties and concentrations. In addition, passive immunotherapy could be more effective in elderly population with reduced immune responses. However, administration of antibodies is time- and money-consuming. The effects of active and passive Aβ immunotherapy are not so

Secretases have become the targets to control Aβ production and prevent the progress of AD since Aβ is generated from APP via sequential cleavage by β- and γ-secretases (Chiba et al., 2007; De Strooper and Annaert, 2000). This is the initial step in the canonical "amyloid cascade". Considering that most of the mutant *APP* and *PSEN1* result in increased Aβ42/Aβ40 ratio, abnormal γ-secretase function seems to be closely related to the pathogenesis of AD. In addition, the choice of the first cleavage by α- or β-secretase decides the generation of Aβ, which gives us the notion that β-cleavage inhibition and α-cleavage activation greatly reduce

In 1999, β-site APP cleaving enzyme (BACE1), a membrane-anchored aspartyl protease, was reported to have β-secretase activity (John et al., 2003; Vassar et al., 1999). The BACE1 knock out mice studies showed that Aβ levels in brain was drastically reduced, and they remain healthy without any anomaly in clear contrast to that of PSEN knockout mice (Cai et al., 2001; Luo et al.,

much different and unfortunately both immunotherapy are not successful so far.

*4.1.2. γ-secretase and β-secretase inhibitors for reduction of Aβ production*

al., 2012). Other antibodies are also currently tested in phase I and II studies.

**Active versus passive Aβ immunotherapy**

Aβ production.

192 Neurodegenerative Diseases

**β-secretase inhibitors**

Development of γ-secretase inhibitors, which suppress the final step in amyloidgenesis, is one of the major issues in AD research. Transition state analogs of γ-secretase, such as L-685458 (Merk), inhibit the γ-secretase activity and decreases production of Aβ40 and Aβ42 (Shearman et al., 2000). Chemical compunds including pepstatin A, sulfonamide derivatives and benzo‐ diazepines are also shown to inhibit the γ-secretase activity in mechanisms other than competitive inhibition (Tian et al., 2002). Since γ-secretase is also involved in processing of other membrane proteins including Notch, γ-secretase inhibitors may induce severe side effects. In agreement, the chronic administration of a γ-secretase inhibitor triggered abnormal blood cell differentiation and damage on digestive tracts (Searfoss et al., 2003). APP-specific γ-secretase inhibitors or γ-secretase modulators may resolve this problem.

Several γ-secretase inhibitors are currently tested in clinical studies: semagacestat (LY450139), begacestat (GSI-953), avagacestat (BMS-708163), PF-3084014, MK-0752, E-2012 and NIC5-15 (Mangialasche et al., 2010). Phase III trials for semagacestat, which inhibits Notch cleavage as well as APP cleavage, not only failed but also worsened clinical measures of cognition and activity of daily living with increased incidence of skin cancer (Cummings, 2010). Other Notchsparing γ-secretase inhibitors (second-generation inhibitors) such as begacestat and avagace‐ stat are now in phase I or II studies and they show reduction of plasma and/or cerebrospinal fluid (CSF) Aβ levels. PF-3084014, a γ-secretase inhibitor with high selectivity for APP, and NIC5-15, a Notch-sparing γ-secretase inhibitor with insulin sensitizing activity, showed some positive effects with good tolerance and now proceeded into phase II studies (Mangialasche et al., 2010). Effects of a γ-secretase modulator, tarenflurbil, will be discussed later (see 4.3.2.).

#### **α-secretase activators**

Upregulation of α-cleavage, which results in non-amyloidogenic cleavage of APP, can competitively inhibit β-cleavage, leading to downregulation of Aβ production and reciprocal upregulation of neuroprotective soluble APPα (sAPPα) secretion (Vingtdeux and Maram‐ baud, 2012). α-Secretase is a member of the ADAM (a disintegrin and metalloprotease) family of proteases including ADAM10, ADAM17/TACE (TNFα converting enzyme), or ADAM9. There is a number of ways to activate α-secretase (Mangialasche et al., 2010): (i) activation of neurotransmitter receptors such as muscarinic, glutamate, γ-aminobutyric acid (GABA) (e.g. etazolate [SQ-20009, EHT-0202]) and serotonin receptors (e.g. SB-742457, PRX-03140, AVN-322 and RQ-00000009), (ii) steroid hormones such as estrogens and testosterones, and (iii) protein kinase C (PKC) activation (e.g. bryostatin-1). No conclusive results are published yet from clinical trials.

*4.1.4. Drugs inducing Aβ degradation*

An endopeptidase named neprilysin (NEP) was identified to degrade Aβ aggregates and regulate Aβ metabolism *in vivo* (Iwata et al., 2001; Iwata et al., 2000). It is a type II transmembra‐ nous ectoenzyme, and cleaves peptide bond of N-terminal hydrophobic amino acid residue in lower than 5kDa peptide. In knock out mice study, Aβ levels in brain elevated to twice as that in the control mice (Iwata et al., 2001), and NEP gets lower in old wild type mice brain (Iwata et al., 2002). Furthermore, the expression levels of NEP were decreased to the half levels in sporadic AD patients' brain as compared with healthy controls' (Yasojima et al., 2001a; Yasojima et al., 2001b). The cleavege of Aβ by NEP, however, is not so potent that NEP itself may be difficult to be clinically effective.Recently, Saito *et al.*have reportedthat somatostatin regulates brainAβ42 through modulating proteolytic degradation catalyzed by NEP (Saito et al., 2005). Somatosta‐

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Insulin degrading enzyme (IDE) is another enzyme degrading Aβ (Qiu et al., 1998). Overex‐ pression of IDE in AD mouse models retarded or even completely prevented SP formation, while IDE knockout mice showed increased levels of Aβ and insulin (Farris et al., 2003; Leissring et al., 2003). Cabrol *et al.* performed high-throughput compound screening for smallmolecule activator of IDE and found promising compound activating IDE by binding to its putative ATP-binding domain (Cabrol et al., 2009). Current investigations have further suggested that Aβ degradation is also mediated by multiple types of proteases including presequence peptidase, endothelin converting enzyme (ECE), angiotensin-converting enzyme (ACE), the uPA/tPA-plasmin system, transthyretin (TTR, gelsolin, α2-macroglobulin and

Drugs targeting the downstream pathways of the "amyloid cascade" have been also devel‐ oped. These include tau inhibitors and neuronal death inhibitors. Tau inhibitors are either based on suppression of tau phosphorylation or inhibition of tau aggregation. Glycogensynthase-kinase-3β (GSK3β) inhibitors are shown to reduce tau phosphorylation and methyl‐ ene blue inhibits tau aggretation. Neuronal death can be suppressed by endogenous soluble factors such as leptin, AL-108 (NAP) and humanin (HN) as well as artificially modified small peptides such as colivelin (CLN) (Chiba et al., 2009a). Some of them activate Stat3, which is specifically inactivated in AD patients. AL-108 shows anti-Aβ activity and anti-tau activity at

Alternative therapeutic targets are also emerging. One of the major targets is related to the metabolic syndrome. Several lines of evidence supported that the metabolic syndrome increases the relative risk of AD (Misiak et al., 2012). Especially, there seems to be a link between type 2 diabetes mellitus (DM) and AD. Thiazolidinediones, commonly used for type 2 DM such as piaglitazone and rosiglitazone, are already mentioned focusing on their BACE1 inhibiting activity. In addition, there is epidemiological evidence that drugs used for high cholesterol (hypercholesterolemia) and high blood pressure also lower the risk of AD (Burgos

tin and its receptor could also be utilized in the Aβ degradation therapy for AD.

matrix metalloproteinases (MMP-2 and 9) (Nalivaeva et al., 2012).

**4.2. AD-specific drugs based on the alternative pathways**

the same time.

et al., 2012; Davies et al., 2011).

#### *4.1.3. Drugs preventing Aβ aggregation, destabilizing Aβ oligomers and inducing Aβ clearance*

Neurotoxic activity of Aβ is likely to be mediated by certain types of soluble oligomers (Di Carlo, 2010; Lesne et al., 2006; Schilling et al., 2008). Oligomerization and aggregation of Aβ further promote SP formation or accumulation of brain Aβ load. Therefore, compounds preventing Aβ aggregation or destabilizing Aβ oligomers seem to be promising drug candi‐ dates for AD. Compounds binding to Aβ monomers may prevent oligomerization and promote Aβ degradation, while compounds recognizing Aβ oligomers may neurtralize the neurotoxicity and facilitate Aβ clearance.

Tramiprosate (homotaurine, 3APS), which is one of the non-peptidic anti-aggregants binding to Aβ monomers, failed to show clinical efficacy in a phase III study (the Alphase study) (Aisen et al., 2011). An antifungal and antiprotozoal drug clioquinol (PBT1) (Tabira, 2001), which disrupts interactions between Aβ, copper and zinc, showed positive results in phase II stud‐ ies, but further studies were halted due to manufacturing toxicity issues (Ritchie et al., 2003; Sampson et al., 2008). PBT2, a second-generation inhibitor without toxicity, also showed promising results in aphase II study (Lannfelt et al., 2008). Scyllo-inositol(ELND-005), an orally administered stereoisomer of inositol, binds to Aβ, inhibits Aβ aggregation and promotes dissociation of aggregates. Phase II studies, however, revealed serious adverse events among patients with high-dose treatments (Salloway et al., 2011). Epigallocatechin-3-gallate (EGCg), a polyphenol from green tea, preventing Aβ aggregation by binding to unfolded Aβ, is current‐ ly tested in phase II/III studies (Mandel et al., 2008; Rezai-Zadeh et al., 2005).

Aβ clearance itself is also a target of novel therapeutics, which is based on the fact that Aβ clearance from brains is impaired in AD patients, especially in those with *APOE* ε4. Aβ clearance can be achieved by local Aβ uptake and degradation in the CNS (Aβ degradation will be discussed in 4.1.4), or pumping out of Aβ through the blood-brain-barrier (BBB) from the brain to the plasma. Aβ clearance is facilitated by APOE, the transcription or expression levels of which are regulated by PPARγ and heterodimeric receptors consisting of liver X receptors (LXRs) and retinoid X receptors (RXRs) (Cramer et al., 2012; Huang and Mucke, 2012). Agonists of these receptors could be used to reduce Aβ load in AD brains. Aβ translo‐ cation through BBB is regulated by lipoprotein receptor related protein-1 (LRP-1), which is a receptor for APOE and α2-macroglobulin, and the receptor for advanced glycation endprod‐ ucts (RAGE) (Deane et al., 2003; Deane et al., 2008). LRP-1 pumps Aβ out of the CNS across the BBB, while RAGE supports Aβ influx into the CNS. The soluble form of RAGE (sRAGE) competes with the membrane-linked RAGE, promoting removal of circulating Aβ. A phase II study on a RAGE inhibitor (TTP-488) is ongoing (Mangialasche et al., 2010). Lactoferrin, a globular glycoprotein found in various secretory fluids such as milk, saliva and tears, is known to stimulate LRP-1 (Ito et al., 2007). LRP-1 agonists are also candidates for AD therapy.

#### *4.1.4. Drugs inducing Aβ degradation*

neurotransmitter receptors such as muscarinic, glutamate, γ-aminobutyric acid (GABA) (e.g. etazolate [SQ-20009, EHT-0202]) and serotonin receptors (e.g. SB-742457, PRX-03140, AVN-322 and RQ-00000009), (ii) steroid hormones such as estrogens and testosterones, and (iii) protein kinase C (PKC) activation (e.g. bryostatin-1). No conclusive results are published yet from

*4.1.3. Drugs preventing Aβ aggregation, destabilizing Aβ oligomers and inducing Aβ clearance*

Neurotoxic activity of Aβ is likely to be mediated by certain types of soluble oligomers (Di Carlo, 2010; Lesne et al., 2006; Schilling et al., 2008). Oligomerization and aggregation of Aβ further promote SP formation or accumulation of brain Aβ load. Therefore, compounds preventing Aβ aggregation or destabilizing Aβ oligomers seem to be promising drug candi‐ dates for AD. Compounds binding to Aβ monomers may prevent oligomerization and promote Aβ degradation, while compounds recognizing Aβ oligomers may neurtralize the

Tramiprosate (homotaurine, 3APS), which is one of the non-peptidic anti-aggregants binding to Aβ monomers, failed to show clinical efficacy in a phase III study (the Alphase study) (Aisen et al., 2011). An antifungal and antiprotozoal drug clioquinol (PBT1) (Tabira, 2001), which disrupts interactions between Aβ, copper and zinc, showed positive results in phase II stud‐ ies, but further studies were halted due to manufacturing toxicity issues (Ritchie et al., 2003; Sampson et al., 2008). PBT2, a second-generation inhibitor without toxicity, also showed promising results in aphase II study (Lannfelt et al., 2008). Scyllo-inositol(ELND-005), an orally administered stereoisomer of inositol, binds to Aβ, inhibits Aβ aggregation and promotes dissociation of aggregates. Phase II studies, however, revealed serious adverse events among patients with high-dose treatments (Salloway et al., 2011). Epigallocatechin-3-gallate (EGCg), a polyphenol from green tea, preventing Aβ aggregation by binding to unfolded Aβ, is current‐

Aβ clearance itself is also a target of novel therapeutics, which is based on the fact that Aβ clearance from brains is impaired in AD patients, especially in those with *APOE* ε4. Aβ clearance can be achieved by local Aβ uptake and degradation in the CNS (Aβ degradation will be discussed in 4.1.4), or pumping out of Aβ through the blood-brain-barrier (BBB) from the brain to the plasma. Aβ clearance is facilitated by APOE, the transcription or expression levels of which are regulated by PPARγ and heterodimeric receptors consisting of liver X receptors (LXRs) and retinoid X receptors (RXRs) (Cramer et al., 2012; Huang and Mucke, 2012). Agonists of these receptors could be used to reduce Aβ load in AD brains. Aβ translo‐ cation through BBB is regulated by lipoprotein receptor related protein-1 (LRP-1), which is a receptor for APOE and α2-macroglobulin, and the receptor for advanced glycation endprod‐ ucts (RAGE) (Deane et al., 2003; Deane et al., 2008). LRP-1 pumps Aβ out of the CNS across the BBB, while RAGE supports Aβ influx into the CNS. The soluble form of RAGE (sRAGE) competes with the membrane-linked RAGE, promoting removal of circulating Aβ. A phase II study on a RAGE inhibitor (TTP-488) is ongoing (Mangialasche et al., 2010). Lactoferrin, a globular glycoprotein found in various secretory fluids such as milk, saliva and tears, is known to stimulate LRP-1 (Ito et al., 2007). LRP-1 agonists are also candidates for AD therapy.

ly tested in phase II/III studies (Mandel et al., 2008; Rezai-Zadeh et al., 2005).

clinical trials.

194 Neurodegenerative Diseases

neurotoxicity and facilitate Aβ clearance.

An endopeptidase named neprilysin (NEP) was identified to degrade Aβ aggregates and regulate Aβ metabolism *in vivo* (Iwata et al., 2001; Iwata et al., 2000). It is a type II transmembra‐ nous ectoenzyme, and cleaves peptide bond of N-terminal hydrophobic amino acid residue in lower than 5kDa peptide. In knock out mice study, Aβ levels in brain elevated to twice as that in the control mice (Iwata et al., 2001), and NEP gets lower in old wild type mice brain (Iwata et al., 2002). Furthermore, the expression levels of NEP were decreased to the half levels in sporadic AD patients' brain as compared with healthy controls' (Yasojima et al., 2001a; Yasojima et al., 2001b). The cleavege of Aβ by NEP, however, is not so potent that NEP itself may be difficult to be clinically effective.Recently, Saito *et al.*have reportedthat somatostatin regulates brainAβ42 through modulating proteolytic degradation catalyzed by NEP (Saito et al., 2005). Somatosta‐ tin and its receptor could also be utilized in the Aβ degradation therapy for AD.

Insulin degrading enzyme (IDE) is another enzyme degrading Aβ (Qiu et al., 1998). Overex‐ pression of IDE in AD mouse models retarded or even completely prevented SP formation, while IDE knockout mice showed increased levels of Aβ and insulin (Farris et al., 2003; Leissring et al., 2003). Cabrol *et al.* performed high-throughput compound screening for smallmolecule activator of IDE and found promising compound activating IDE by binding to its putative ATP-binding domain (Cabrol et al., 2009). Current investigations have further suggested that Aβ degradation is also mediated by multiple types of proteases including presequence peptidase, endothelin converting enzyme (ECE), angiotensin-converting enzyme (ACE), the uPA/tPA-plasmin system, transthyretin (TTR, gelsolin, α2-macroglobulin and matrix metalloproteinases (MMP-2 and 9) (Nalivaeva et al., 2012).

#### **4.2. AD-specific drugs based on the alternative pathways**

Drugs targeting the downstream pathways of the "amyloid cascade" have been also devel‐ oped. These include tau inhibitors and neuronal death inhibitors. Tau inhibitors are either based on suppression of tau phosphorylation or inhibition of tau aggregation. Glycogensynthase-kinase-3β (GSK3β) inhibitors are shown to reduce tau phosphorylation and methyl‐ ene blue inhibits tau aggretation. Neuronal death can be suppressed by endogenous soluble factors such as leptin, AL-108 (NAP) and humanin (HN) as well as artificially modified small peptides such as colivelin (CLN) (Chiba et al., 2009a). Some of them activate Stat3, which is specifically inactivated in AD patients. AL-108 shows anti-Aβ activity and anti-tau activity at the same time.

Alternative therapeutic targets are also emerging. One of the major targets is related to the metabolic syndrome. Several lines of evidence supported that the metabolic syndrome increases the relative risk of AD (Misiak et al., 2012). Especially, there seems to be a link between type 2 diabetes mellitus (DM) and AD. Thiazolidinediones, commonly used for type 2 DM such as piaglitazone and rosiglitazone, are already mentioned focusing on their BACE1 inhibiting activity. In addition, there is epidemiological evidence that drugs used for high cholesterol (hypercholesterolemia) and high blood pressure also lower the risk of AD (Burgos et al., 2012; Davies et al., 2011).


tau (Schirmer et al., 2011). Rember also has antioxidant activity and supports mitochondrial function. A phase II study showed slower disease progression in patients receiving a middledose (60 mg) although the highest-dose (100 mg) failed to show its efficacy presumably due to drug formulation defects (Mangialasche et al., 2010). A phase III study is now on-going.

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Neuronal death is directly implicated in the pathogenesis of AD (Rohn and Head, 2009). Neuronal loss, but not SPs, correlates with the cognitive impairment in AD. Consequently, it is supposed that neuronal death suppression will result in potent therapeutic effect or even a curative one. Neuronal death could be caused by not only toxic Aβ oligomers but also death signals activated by mutations in the FAD genes. Notably, the death signals can differ depending on the types of FAD genes and the types of mutations (Chiba et al., 2007; Kawasumi et al., 2002). Apoptosis is implicated in the neuronal loss related to AD: terminal deoxyuridine triphosphate nick endlabeling (TUNEL)-staining and caspase activation are observed in

It was a milestone in AD research that Yamatsuji *et al.* first showed that expression of FADassociated mutants of *APP* (V642I/F/G) induce apoptosis via a mechanism independent of Aβ because it suggested not only that there might be neurotoxic insults other than Aβ underlying AD pathogenesis but also that APP might have physiological function besides its role as a precursor of Aβ (Yamatsuji et al., 1996). Actually, multiple groups have confirmed that overexpression of FAD mutants of *APP* induces neuronal cell death by triggering intracellular death signaling cascades (Hashimoto et al., 2000; Zhao et al., 1997). In addition, a number of observations support the idea that APP might function as a cell-surface receptor inducing cell death signals. Given that APP-dimerization activates the intracellular death signals identical to that induced by FAD mutants of APP without ligand stimulation, it is likely that there is a natural ligand for APP to trigger intracellular death signals. In accordance with the idea, transforming growth factor β2 (TGFβ2) and death receptor 6 (DR6) are reported to be physiological ligands for APP triggering the fore-mentioned death signals (Hashimoto et

FAD-linked mutants of *PSEN1* and *PSEN2* also enhance cell death in several cell lines and primary neurons. Mechanisms of death induced by mutants of *PSEN1* is likely to be distinct from that by *PSEN2*. *PSEN1* mutants induce calcium-dependent oxidative stress (Guo et al., 1996), destabilization of β-catenin (Zhang et al., 1998), down-regulation of Akt (Weihl et al., 1999), ER stress (Mattson et al., 1998), and activation of nitric oxide synthase (NOS)-mediated caspase-independent death signals (Hashimoto et al., 2002a), while PSEN2 mutants induce downregulation of Bcl-X(L) (Passer et al., 1999) and Bcl-2 (Araki et al., 2001), activation of

Activated caspases may serve as positive feedback regulators of death. APP is reported to be a substrate for caspase-3, which may contribute to Aβ formation and synaptic loss (Gervais et

NADPH oxidase and xanthine oxidase (XO) (Hashimoto et al., 2002b).

*4.2.2. Neuronal death inhibitors*

neurons of patients' brains.

al., 2005; Nikolaev et al., 2009).

**Complicated mechanisms of neuronal loss**

**Table 1. Therapeutic strategy for AD.** Drugs which failed in clinical trials are indicated with "(f)".

#### *4.2.1. Drugs based on tau pathology*

In AD, abnormally hyperphosphorylated tau forms aggregates called NFT. This pathway can be inhibited by preventing either hyperphosphorylation or aggregation. Tau phosphorylation is regulated by the equilibrium between tau kinases (e.g. cdk5, JNK and GSK3β) and tau phosphatases (e.g. protein phosphatase 2A [PP2A]). Aggregated tau shows β-sheet structure similar to Aβ aggregates although tau locates only in the cytoplasm.

Valproate (valproic acid, VPA) is reported to suppress tau phosphorylation via inhibiting cdk5 and GSK3β (Hu et al., 2011). This is so far the only compound, which reached phase III studies, inthis category.Unfortunately,VPAshowednoefficacyoncognition(Mangialasche et al., 2010; Tariot and Aisen, 2009). Lithium (Li), as well as VPA, is a well-known drug for psychiatric disorder, inhibiting GSK3β. A small clinical study with Li, however, did not show any cogni‐ tivebenefitoranychangeinbiomarkers(Hampeletal.,2009).Regardlessofthesefailures,several GSK3 inhibitors areunderdevelopment.NP-031112 (NP-12)isoneofthoseGSK3 inhibitors and is currently tested in a phase II study, the result of which have not been reported.

Methylene blue (Rember®), which is used as a redox indicator in analytical chemistry and as a dye for nuclear staining in histology, is recently attracting attention as an anti-aggregant for tau (Schirmer et al., 2011). Rember also has antioxidant activity and supports mitochondrial function. A phase II study showed slower disease progression in patients receiving a middledose (60 mg) although the highest-dose (100 mg) failed to show its efficacy presumably due to drug formulation defects (Mangialasche et al., 2010). A phase III study is now on-going.

#### *4.2.2. Neuronal death inhibitors*

*4.2.1. Drugs based on tau pathology*

196 Neurodegenerative Diseases

In AD, abnormally hyperphosphorylated tau forms aggregates called NFT. This pathway can be inhibited by preventing either hyperphosphorylation or aggregation. Tau phosphorylation is regulated by the equilibrium between tau kinases (e.g. cdk5, JNK and GSK3β) and tau phosphatases (e.g. protein phosphatase 2A [PP2A]). Aggregated tau shows β-sheet structure

Valproate (valproic acid, VPA) is reported to suppress tau phosphorylation via inhibiting cdk5 and GSK3β (Hu et al., 2011). This is so far the only compound, which reached phase III studies, inthis category.Unfortunately,VPAshowednoefficacyoncognition(Mangialasche et al., 2010; Tariot and Aisen, 2009). Lithium (Li), as well as VPA, is a well-known drug for psychiatric disorder, inhibiting GSK3β. A small clinical study with Li, however, did not show any cogni‐ tivebenefitoranychangeinbiomarkers(Hampeletal.,2009).Regardlessofthesefailures,several GSK3 inhibitors areunderdevelopment.NP-031112 (NP-12)isoneofthoseGSK3 inhibitors and

Methylene blue (Rember®), which is used as a redox indicator in analytical chemistry and as a dye for nuclear staining in histology, is recently attracting attention as an anti-aggregant for

similar to Aβ aggregates although tau locates only in the cytoplasm.

**Table 1. Therapeutic strategy for AD.** Drugs which failed in clinical trials are indicated with "(f)".

is currently tested in a phase II study, the result of which have not been reported.

Neuronal death is directly implicated in the pathogenesis of AD (Rohn and Head, 2009). Neuronal loss, but not SPs, correlates with the cognitive impairment in AD. Consequently, it is supposed that neuronal death suppression will result in potent therapeutic effect or even a curative one. Neuronal death could be caused by not only toxic Aβ oligomers but also death signals activated by mutations in the FAD genes. Notably, the death signals can differ depending on the types of FAD genes and the types of mutations (Chiba et al., 2007; Kawasumi et al., 2002). Apoptosis is implicated in the neuronal loss related to AD: terminal deoxyuridine triphosphate nick endlabeling (TUNEL)-staining and caspase activation are observed in neurons of patients' brains.

#### **Complicated mechanisms of neuronal loss**

It was a milestone in AD research that Yamatsuji *et al.* first showed that expression of FADassociated mutants of *APP* (V642I/F/G) induce apoptosis via a mechanism independent of Aβ because it suggested not only that there might be neurotoxic insults other than Aβ underlying AD pathogenesis but also that APP might have physiological function besides its role as a precursor of Aβ (Yamatsuji et al., 1996). Actually, multiple groups have confirmed that overexpression of FAD mutants of *APP* induces neuronal cell death by triggering intracellular death signaling cascades (Hashimoto et al., 2000; Zhao et al., 1997). In addition, a number of observations support the idea that APP might function as a cell-surface receptor inducing cell death signals. Given that APP-dimerization activates the intracellular death signals identical to that induced by FAD mutants of APP without ligand stimulation, it is likely that there is a natural ligand for APP to trigger intracellular death signals. In accordance with the idea, transforming growth factor β2 (TGFβ2) and death receptor 6 (DR6) are reported to be physiological ligands for APP triggering the fore-mentioned death signals (Hashimoto et al., 2005; Nikolaev et al., 2009).

FAD-linked mutants of *PSEN1* and *PSEN2* also enhance cell death in several cell lines and primary neurons. Mechanisms of death induced by mutants of *PSEN1* is likely to be distinct from that by *PSEN2*. *PSEN1* mutants induce calcium-dependent oxidative stress (Guo et al., 1996), destabilization of β-catenin (Zhang et al., 1998), down-regulation of Akt (Weihl et al., 1999), ER stress (Mattson et al., 1998), and activation of nitric oxide synthase (NOS)-mediated caspase-independent death signals (Hashimoto et al., 2002a), while PSEN2 mutants induce downregulation of Bcl-X(L) (Passer et al., 1999) and Bcl-2 (Araki et al., 2001), activation of NADPH oxidase and xanthine oxidase (XO) (Hashimoto et al., 2002b).

Activated caspases may serve as positive feedback regulators of death. APP is reported to be a substrate for caspase-3, which may contribute to Aβ formation and synaptic loss (Gervais et al., 1999). Tau is also a substrate for caspases and truncation of tau by caspases may lead to the formation of paired helical filaments (PHFs) and further NFTs (Gamblin et al., 2003).

HN derivatives ameliorate cognitive deficits observed in several types of AD model mice, presumably through activating its receptor consisting of the cytokine receptor gp130 and the downstream intracellular signaling pathways including the Jak2/Stat3 pathway (Chiba et al.,

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Effects of HN on the metabolic syndrome including DM and atherosclerosis are also reported: (i) HN suppresses pancreatic β-cell death (Hoang et al., 2010), (ii) HN increases peripheral insulin sensitivity (Muzumdar et al., 2009), and (iii) HN protects endothelial cells from LDLinduced oxidative stress (Bachar et al., 2010; Oh et al., 2011). These findings suggest that HN

ADNF-9 or SAL (SALLRSIPA, a 9 amino-acid peptide) is an active core domain of ADNF, which antagonizes various types of neurotoxicity, such as tetrodotoxin (TTX), oxidative stress, NMDA, Aβ, ALS and gp120 of human immunodeficiency virus (HIV) (Brenneman and Gozes, 1996; Chiba et al., 2004; Chiba et al., 2007; Chiba et al., 2006; Dibbern et al., 1997). ADNF-9 exerts a neuroprotective effect at extremely low concentrations, such as hundred femtomolar concentrations, while it loses its neuroprotective effect at 1 nM or greater concentrations. Although ADNF receptors have not been identified, ADNF-mediated prosurvival mecha‐ nisms have been reported to involve activation of (i) CREB, (ii) NFkB, (iii) CaMKIV, (iv) hsp60, (v) transcriptional up-regulation of IGF-I, and (vi) poly ADP-ribosylation (Chiba et al., 2007). Through expression screening of proteins recognized by antiserum against ADNF, a geneencoding, activity-dependent neurotrophic protein (ADNP) was identified (Bassan et al., 1999). An eight-amino-acid sequence termed NAP (NAPVSIPQ, an 8 amino-acid peptide) in ADNP shows homology with ADNF and is recognized by antiserum against ADNF. NAP exhibits ADNF-like neuroprotective activity against various insults. NAP and its relative peptides, such as ADNF-9, D-SAL, and D-NAP have been reported to bind to and stabilize tubulin (Gozes and Divinski, 2004). NAP suppressed zinc-mediated microtubule depolyme‐ rization in astrocytes by promoting microtubule assembly and reorganization (Gozes and Divinski, 2007; Vulih-Shultzman et al., 2007). Accordingly, NAP is reported to protect neurons

AL-108 (Davunetide), an intranasal formulation of NAP, and AL-208, an intravenous formu‐ lation of NAP, have been developed for clinical use (Shiryaev et al., 2011). A phase II study was safe and well tolerated and had positive effects on cognition. Currently, a phase III study

Stat3 activation is another option for AD therapy, which can be achieved by soluble factors activating Stat3 signals; i.e. activation of upstream kinases and suppression of endogenous Stat3 inhibitors or regulators (Chiba et al., 2009a). As mentioned above, HN and its derivatives activate the Jak2/Stat3 signaling pathway. There are also many types of cytokines activating Stat3, such as interleukin-6 (IL-6) family cytokines (IL-6, ciliary neurotrophic factor [CNTF], leukemia inhibitory factor [LIF] and cardiotrophin-1 [CT-1]), erythropoietin (EPO), IL-27,

may reduce risks for LOAD through improving glucose and lipid metabolisms.

**Activity-dependent neurotrophic factor (ADNF) and NAP**

2009a; Yamada et al., 2008).

from tau-related neurotoxicity.

*4.2.3. Stat3 activation therapy for AD*

is on-going.

#### **Caspase inhibitors for AD**

Evidence supports a role for executioner caspases including caspases-3, -6 and -7 in the pathogenesis of AD. Caspase activation can be prevented by peptide-based inhibitors such as Z-VAD-fmk and Z-DEVD-fmk (more specific to caspase-3). Although these inhibitors are potent and efficient *in vitro*, establishment of a proper drug delivery system and formulation is necessary for *in vivo* treatment of AD patients (Rohn and Head, 2009). An alternative small molecule, the quinolyl-valyl-O-methylaspartyl-[-2, 6-difluorophenoxy]-methyl ketone (Q-VD-OPh), has been developed to substitute Z-VAD-fmk (Caserta et al., 2003). Q-VD-OPh is not toxic to cells at high concentrations and is systemically active, demonstrating efficacy in animal models of Parkinson's disease (PD), Huntington's disease (HD) and stroke (Renolleau et al., 2007; Yang et al., 2004).

Anotherpotentialtherapeuticstrategyliesonupregulationofanti-apoptoticBcl-2protein.Small compounds inhibiting Bcl-2 have been synthesized for the treatment of cancer, while com‐ pounds activating Bcl-2 have not been obtained so far. In addition, Bcl-2 should not be activat‐ ed systemically because of expected severe side effects such as tumorigenesis. Consequently, the delivery of *Bcl-2* gene by viral vectors, such as adenoviruses, AAV and lentiviruses, is considered. Local delivery of *Bcl-2* and its family genes by viral vectors are shown to be efficient in several animal models of neurodegeneration including cerebral ischemia, axotomy and amyotrophic lateral sclerosis (ALS) (Caleo et al., 2002; Kilic et al., 2002; Yamashita et al., 2001).

Another potential drug candidate is minocycline, which is reported to prevent mitochondrial release of cytochrome c and the following caspase-3 activation (Hashimoto, 2011). Minocy‐ cline is anorallyavailable second-generationtetracycline,whichcancross theBBB.Itis reported thatminocycline elicitedtherapeutic effects inanimalmodels ofischemic braininjury,ALS,PD, HDandmultiple sclerosis (Berger, 2000;Duet al., 2001;Zhuet al., 2002).Recent studies revealed that minocycline protects neurons and reduces Aβ deposition in AD mice although efficacy of minocycline in AD is yet to be carefully addressed (Choi et al., 2007; Seabrook et al., 2006).

#### **Multi-spectrum neuroprotective factor, humanin and its derivatives**

A functional screening for a death-suppressing factor, which antagonizes death induced by overexpression of V642I-APP, was carried out with a cDNA library established from occipital lobes of AD patients, which are relatively preserved regions in AD brains (Hashimoto et al., 2001b). As a result, cDNA encoding a novel 24 amino-acid peptide, termed humanin (HN), was identified (MAPRGFSCLLLLTSEIDLPVKRRA). Notably, HN specifically abolished death induced by AD-related neurotoxicity such as soluble oligomeric Aβ and *PSEN1/PSEN2* mutants as well as overexpressed *APP* mutants, but did not suppress neurotoxicity related to PD, HD and prion diseases (Hashimoto et al., 2001a; Hashimoto et al., 2001b). As a result of detailed structural characterization of HN, several types of HN derivatives with higher efficacy, such as S14G-HN (HNG), have been developed (Chiba et al., 2007; Hashimoto et al., 2001a). One of HN derivatives termed colivelin (SALLRSIPA-PAGASRLLLLTGEIDLP, a 26 amino-acid peptide) elicits 108 -fold more potent effects than authentic HN (Chiba et al., 2005). HN derivatives ameliorate cognitive deficits observed in several types of AD model mice, presumably through activating its receptor consisting of the cytokine receptor gp130 and the downstream intracellular signaling pathways including the Jak2/Stat3 pathway (Chiba et al., 2009a; Yamada et al., 2008).

Effects of HN on the metabolic syndrome including DM and atherosclerosis are also reported: (i) HN suppresses pancreatic β-cell death (Hoang et al., 2010), (ii) HN increases peripheral insulin sensitivity (Muzumdar et al., 2009), and (iii) HN protects endothelial cells from LDLinduced oxidative stress (Bachar et al., 2010; Oh et al., 2011). These findings suggest that HN may reduce risks for LOAD through improving glucose and lipid metabolisms.

#### **Activity-dependent neurotrophic factor (ADNF) and NAP**

al., 1999). Tau is also a substrate for caspases and truncation of tau by caspases may lead to the

Evidence supports a role for executioner caspases including caspases-3, -6 and -7 in the pathogenesis of AD. Caspase activation can be prevented by peptide-based inhibitors such as Z-VAD-fmk and Z-DEVD-fmk (more specific to caspase-3). Although these inhibitors are potent and efficient *in vitro*, establishment of a proper drug delivery system and formulation is necessary for *in vivo* treatment of AD patients (Rohn and Head, 2009). An alternative small molecule, the quinolyl-valyl-O-methylaspartyl-[-2, 6-difluorophenoxy]-methyl ketone (Q-VD-OPh), has been developed to substitute Z-VAD-fmk (Caserta et al., 2003). Q-VD-OPh is not toxic to cells at high concentrations and is systemically active, demonstrating efficacy in animal models of Parkinson's disease (PD), Huntington's disease (HD) and stroke (Renolleau et al.,

Anotherpotentialtherapeuticstrategyliesonupregulationofanti-apoptoticBcl-2protein.Small compounds inhibiting Bcl-2 have been synthesized for the treatment of cancer, while com‐ pounds activating Bcl-2 have not been obtained so far. In addition, Bcl-2 should not be activat‐ ed systemically because of expected severe side effects such as tumorigenesis. Consequently, the delivery of *Bcl-2* gene by viral vectors, such as adenoviruses, AAV and lentiviruses, is considered. Local delivery of *Bcl-2* and its family genes by viral vectors are shown to be efficient in several animal models of neurodegeneration including cerebral ischemia, axotomy and amyotrophic lateral sclerosis (ALS) (Caleo et al., 2002; Kilic et al., 2002; Yamashita et al., 2001). Another potential drug candidate is minocycline, which is reported to prevent mitochondrial release of cytochrome c and the following caspase-3 activation (Hashimoto, 2011). Minocy‐ cline is anorallyavailable second-generationtetracycline,whichcancross theBBB.Itis reported thatminocycline elicitedtherapeutic effects inanimalmodels ofischemic braininjury,ALS,PD, HDandmultiple sclerosis (Berger, 2000;Duet al., 2001;Zhuet al., 2002).Recent studies revealed that minocycline protects neurons and reduces Aβ deposition in AD mice although efficacy of minocycline in AD is yet to be carefully addressed (Choi et al., 2007; Seabrook et al., 2006).

A functional screening for a death-suppressing factor, which antagonizes death induced by overexpression of V642I-APP, was carried out with a cDNA library established from occipital lobes of AD patients, which are relatively preserved regions in AD brains (Hashimoto et al., 2001b). As a result, cDNA encoding a novel 24 amino-acid peptide, termed humanin (HN), was identified (MAPRGFSCLLLLTSEIDLPVKRRA). Notably, HN specifically abolished death induced by AD-related neurotoxicity such as soluble oligomeric Aβ and *PSEN1/PSEN2* mutants as well as overexpressed *APP* mutants, but did not suppress neurotoxicity related to PD, HD and prion diseases (Hashimoto et al., 2001a; Hashimoto et al., 2001b). As a result of detailed structural characterization of HN, several types of HN derivatives with higher efficacy, such as S14G-HN (HNG), have been developed (Chiba et al., 2007; Hashimoto et al., 2001a). One of HN derivatives termed colivelin (SALLRSIPA-PAGASRLLLLTGEIDLP, a 26


**Multi-spectrum neuroprotective factor, humanin and its derivatives**

formation of paired helical filaments (PHFs) and further NFTs (Gamblin et al., 2003).

**Caspase inhibitors for AD**

198 Neurodegenerative Diseases

2007; Yang et al., 2004).

amino-acid peptide) elicits 108

ADNF-9 or SAL (SALLRSIPA, a 9 amino-acid peptide) is an active core domain of ADNF, which antagonizes various types of neurotoxicity, such as tetrodotoxin (TTX), oxidative stress, NMDA, Aβ, ALS and gp120 of human immunodeficiency virus (HIV) (Brenneman and Gozes, 1996; Chiba et al., 2004; Chiba et al., 2007; Chiba et al., 2006; Dibbern et al., 1997). ADNF-9 exerts a neuroprotective effect at extremely low concentrations, such as hundred femtomolar concentrations, while it loses its neuroprotective effect at 1 nM or greater concentrations. Although ADNF receptors have not been identified, ADNF-mediated prosurvival mecha‐ nisms have been reported to involve activation of (i) CREB, (ii) NFkB, (iii) CaMKIV, (iv) hsp60, (v) transcriptional up-regulation of IGF-I, and (vi) poly ADP-ribosylation (Chiba et al., 2007).

Through expression screening of proteins recognized by antiserum against ADNF, a geneencoding, activity-dependent neurotrophic protein (ADNP) was identified (Bassan et al., 1999). An eight-amino-acid sequence termed NAP (NAPVSIPQ, an 8 amino-acid peptide) in ADNP shows homology with ADNF and is recognized by antiserum against ADNF. NAP exhibits ADNF-like neuroprotective activity against various insults. NAP and its relative peptides, such as ADNF-9, D-SAL, and D-NAP have been reported to bind to and stabilize tubulin (Gozes and Divinski, 2004). NAP suppressed zinc-mediated microtubule depolyme‐ rization in astrocytes by promoting microtubule assembly and reorganization (Gozes and Divinski, 2007; Vulih-Shultzman et al., 2007). Accordingly, NAP is reported to protect neurons from tau-related neurotoxicity.

AL-108 (Davunetide), an intranasal formulation of NAP, and AL-208, an intravenous formu‐ lation of NAP, have been developed for clinical use (Shiryaev et al., 2011). A phase II study was safe and well tolerated and had positive effects on cognition. Currently, a phase III study is on-going.

#### *4.2.3. Stat3 activation therapy for AD*

Stat3 activation is another option for AD therapy, which can be achieved by soluble factors activating Stat3 signals; i.e. activation of upstream kinases and suppression of endogenous Stat3 inhibitors or regulators (Chiba et al., 2009a). As mentioned above, HN and its derivatives activate the Jak2/Stat3 signaling pathway. There are also many types of cytokines activating Stat3, such as interleukin-6 (IL-6) family cytokines (IL-6, ciliary neurotrophic factor [CNTF], leukemia inhibitory factor [LIF] and cardiotrophin-1 [CT-1]), erythropoietin (EPO), IL-27, granulocyte-colony stimulating factor (G-CSF), and leptin. Administration of these soluble factors could be considered although the bioavailability and delivery of these cytokines into the CNS should be addressed. Stat3 is phosphorylated by cellular Tyr kinases such as Jaks and Src family kinases. Synthetic activators such as Src family activator (EPQYEEIPIYL, Src family activator from Santa Cruz Biothechnology, sc-3052) may activate Stat3 to suppress the pathogenesis of AD although it may bring a large risk of carcinogenesis.

results in severe side effect (Lim et al., 2000; Lleo et al., 2004; Weggen et al., 2001). Flurizan (Tarenflurbil or R-flurbiprofen) is originally developed as an NSAID and is shown to reduce toxic longer Aβ42 levels as a γ-secretase modifier. In 2008, Myriad Genetics, Inc. announced that an 18-month phase III study of Flurizan in patients with mild AD (the Act-Earil-AD trial), unfortunately failed to achieve significant disease-modifying effect and that they decided to discontinue development of Flurizan (www.myriad.com) (Green et al., 2009). It should be noted that several COX-2 inhibitors may increase the production of the toxic Aβ42 peptide (Kukar et al., 2005). Further basic study about how and which NSAIDs work on AD should be

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http://dx.doi.org/10.5772/55293

201

High cholesterol level is now recognized as a risk factor for LOAD (Shobab et al., 2005). As already mentioned, *APOE*, which is involved in lipid metabolisms, is reported to be a risk factor for LOAD (Bertram, 2011; Schellenberg and Montine, 2012). In this line, it was reported that long-term taking of HMG-CoA reductase inhibitors (statins) lowered the risk of AD significantly (Jick et al., 2000; Wolozin et al., 2000). In APP transgenic mice, statins improved brain pathology (Refolo et al., 2001). The mechanism in detail is unclear, but one possible mechanism is that statins modulate secretases: activation of α-secretase and inhibition of βsecretase to exert its anti-AD effect (Kojro et al., 2001; Parsons et al., 2006). These facts and clinical safety of chronic use of statins led this therapy to clinical trials. Recently, no significant clinical benefit on cognition or global functioning was, unfortunately, reported for atorvastatin

Dimebon (latrepirdine) received a huge attention as a potential therapy for AD after a publication in the *Lancet* of a positive phase II study carried out in Russia (Doody et al., 2008; Jones, 2010). Dimebon is an orally available and well-tolerated drug, which used to be approved for clinical use in Russia as a non-selective anti-histamine drug. Dimebon is reported to elicit multiple anti-AD effects such as ChE inhibition, prevention of NMDA-mediated excitotoxicity and inhibition of mitochondrial permeability transition pore opening. The phase III study of dimebon, called the CONNECTION study, unfortunately, showed no clinical benefit on co-primary (cognition and global function) and secondary endpoints. Moreover, an additional phase III study called the CONCERT study again failed to show efficacy of dimebon (announcement from Pfizer and Medivation on Jan 17, 2012, www.medivation.com). Complete discrepancy between the results of phase II and phase III studies has brought researchers a huge confusion. Negative comments about the underlying rationale for the use of dimebon in AD are currently increasing because of the complete failure of dimebon in the phase III studies.

AD-non-specific drugs are also possible. The therapeutic bases of these drugs lie on protection of neurons from aging-related toxicity such as oxidative stress, promotion of synaptic plasticity

and simvastatin in phase III studies (Feldman et al., 2010; Sano et al., 2011).

piled up and elaborate clinical trials are essential.

*4.3.2. Inhibitors for cholesterol synthesis: Statins*

**4.4. AD-non-specific drugs for dementia**

and regeneration of neural tissues using stem cell technologies.

*4.3.3. Dimebon*

There are several endogenous regulator of Stat3: protein Tyr phosphatases (PTPs) like Src homology region 2 domain-containing phosphatase-1 (SHP-1) and SHP-2, suppressor of cytokine signaling3 (SOCS3) andproteininhibitorof activatedStat3 (PIAS3)(Chiba et al., 2009a; Hendriks et al., 2012; Stephanou and Latchman, 2005). There are a number of PTPs and some of them are involved in the dephosphorylation or inactivation of the Jak2/Stat3 signaling path‐ way. SOCS3 is transcriptionally induced by activated Stat3 as a negative feedback regulator. SOCS3 binds to and inhibits Jak2 to prevent Stat3 phosphorylation. PIAS3 is a nuclear protein inhibiting activated Stat3 via multiple mechanisms. Expression of PIAS3 seems to be epigenet‐ ically regulated. Regulation of PIAS3 may contribute to recover Stat3 phosphorylation in AD.

#### *4.2.4. Drugs based on prion protein*

Prion diseases such as Creutzfeldt-Jakob disease are transmitted from patients to others by infectious agents consisting of misfolded proteins. The prion hypothesis for AD, suspected during 1970s and 80s, is re-emerging based on the reports that AD pathology can be transmit‐ ted to mice as prions do (Eisele et al., 2009; Eisele et al., 2010). Recent findings support the notion that there is a considerable similarity between Aβ and prion protein: (i) Aβ aggregation has similar structure to prion protein aggregation (Nussbaum et al., 2012), (ii) Aβ oligomer binds to postsynaptic prion protein to impair neuronal function (Um et al., 2012), and (iii) Aβ aggregates infect like prions (Stohr et al., 2012). Accordingly, AD therapy targeting prion proteins or prophylaxis for AD based on the prion hypothesis may be possible.

#### **4.3. AD-specific drugs based on epidemiological findings**

Epidemiology, as well as genetics, has pointed out several important aspects of the pathogen‐ esis of AD. Based on the findings, a number of clinical trials have been carried out.

#### *4.3.1. Nonsteroidal Anti-Inflammatory Drugs (NSAIDs)*

Anti-inflammatory agents have been attracting attentions based on the fact that there is severe astrogliosis in AD brains and that epidemiological study showed that long-term users of nonsteroidal anti-inflammatory drugs (NSAIDs) have the lower risk of AD (Etminan et al., 2003). Considering that there are also several NSAIDs without benefits for AD patients including rofecoxib (a selective cyclooxygenase-2 [COX-2] inhibitor), and naproxen (a mixed COX-1 and 2 inhibitor) (Aisen et al., 2003), there seems to be additional anti-AD mechanisms other than attenuation of inflammatory response in effective NSAIDs including ibuprofen and indomethacin. For example, some NSAIDs may directly modify the γ-secretase activity and reduce Aβ42 levels without any evidence of inhibition of Notch processing, which might results in severe side effect (Lim et al., 2000; Lleo et al., 2004; Weggen et al., 2001). Flurizan (Tarenflurbil or R-flurbiprofen) is originally developed as an NSAID and is shown to reduce toxic longer Aβ42 levels as a γ-secretase modifier. In 2008, Myriad Genetics, Inc. announced that an 18-month phase III study of Flurizan in patients with mild AD (the Act-Earil-AD trial), unfortunately failed to achieve significant disease-modifying effect and that they decided to discontinue development of Flurizan (www.myriad.com) (Green et al., 2009). It should be noted that several COX-2 inhibitors may increase the production of the toxic Aβ42 peptide (Kukar et al., 2005). Further basic study about how and which NSAIDs work on AD should be piled up and elaborate clinical trials are essential.

#### *4.3.2. Inhibitors for cholesterol synthesis: Statins*

High cholesterol level is now recognized as a risk factor for LOAD (Shobab et al., 2005). As already mentioned, *APOE*, which is involved in lipid metabolisms, is reported to be a risk factor for LOAD (Bertram, 2011; Schellenberg and Montine, 2012). In this line, it was reported that long-term taking of HMG-CoA reductase inhibitors (statins) lowered the risk of AD significantly (Jick et al., 2000; Wolozin et al., 2000). In APP transgenic mice, statins improved brain pathology (Refolo et al., 2001). The mechanism in detail is unclear, but one possible mechanism is that statins modulate secretases: activation of α-secretase and inhibition of βsecretase to exert its anti-AD effect (Kojro et al., 2001; Parsons et al., 2006). These facts and clinical safety of chronic use of statins led this therapy to clinical trials. Recently, no significant clinical benefit on cognition or global functioning was, unfortunately, reported for atorvastatin and simvastatin in phase III studies (Feldman et al., 2010; Sano et al., 2011).

#### *4.3.3. Dimebon*

granulocyte-colony stimulating factor (G-CSF), and leptin. Administration of these soluble factors could be considered although the bioavailability and delivery of these cytokines into the CNS should be addressed. Stat3 is phosphorylated by cellular Tyr kinases such as Jaks and Src family kinases. Synthetic activators such as Src family activator (EPQYEEIPIYL, Src family activator from Santa Cruz Biothechnology, sc-3052) may activate Stat3 to suppress the

There are several endogenous regulator of Stat3: protein Tyr phosphatases (PTPs) like Src homology region 2 domain-containing phosphatase-1 (SHP-1) and SHP-2, suppressor of cytokine signaling3 (SOCS3) andproteininhibitorof activatedStat3 (PIAS3)(Chiba et al., 2009a; Hendriks et al., 2012; Stephanou and Latchman, 2005). There are a number of PTPs and some of them are involved in the dephosphorylation or inactivation of the Jak2/Stat3 signaling path‐ way. SOCS3 is transcriptionally induced by activated Stat3 as a negative feedback regulator. SOCS3 binds to and inhibits Jak2 to prevent Stat3 phosphorylation. PIAS3 is a nuclear protein inhibiting activated Stat3 via multiple mechanisms. Expression of PIAS3 seems to be epigenet‐ ically regulated. Regulation of PIAS3 may contribute to recover Stat3 phosphorylation in AD.

Prion diseases such as Creutzfeldt-Jakob disease are transmitted from patients to others by infectious agents consisting of misfolded proteins. The prion hypothesis for AD, suspected during 1970s and 80s, is re-emerging based on the reports that AD pathology can be transmit‐ ted to mice as prions do (Eisele et al., 2009; Eisele et al., 2010). Recent findings support the notion that there is a considerable similarity between Aβ and prion protein: (i) Aβ aggregation has similar structure to prion protein aggregation (Nussbaum et al., 2012), (ii) Aβ oligomer binds to postsynaptic prion protein to impair neuronal function (Um et al., 2012), and (iii) Aβ aggregates infect like prions (Stohr et al., 2012). Accordingly, AD therapy targeting prion

Epidemiology, as well as genetics, has pointed out several important aspects of the pathogen‐

Anti-inflammatory agents have been attracting attentions based on the fact that there is severe astrogliosis in AD brains and that epidemiological study showed that long-term users of nonsteroidal anti-inflammatory drugs (NSAIDs) have the lower risk of AD (Etminan et al., 2003). Considering that there are also several NSAIDs without benefits for AD patients including rofecoxib (a selective cyclooxygenase-2 [COX-2] inhibitor), and naproxen (a mixed COX-1 and 2 inhibitor) (Aisen et al., 2003), there seems to be additional anti-AD mechanisms other than attenuation of inflammatory response in effective NSAIDs including ibuprofen and indomethacin. For example, some NSAIDs may directly modify the γ-secretase activity and reduce Aβ42 levels without any evidence of inhibition of Notch processing, which might

proteins or prophylaxis for AD based on the prion hypothesis may be possible.

esis of AD. Based on the findings, a number of clinical trials have been carried out.

**4.3. AD-specific drugs based on epidemiological findings**

*4.3.1. Nonsteroidal Anti-Inflammatory Drugs (NSAIDs)*

pathogenesis of AD although it may bring a large risk of carcinogenesis.

*4.2.4. Drugs based on prion protein*

200 Neurodegenerative Diseases

Dimebon (latrepirdine) received a huge attention as a potential therapy for AD after a publication in the *Lancet* of a positive phase II study carried out in Russia (Doody et al., 2008; Jones, 2010). Dimebon is an orally available and well-tolerated drug, which used to be approved for clinical use in Russia as a non-selective anti-histamine drug. Dimebon is reported to elicit multiple anti-AD effects such as ChE inhibition, prevention of NMDA-mediated excitotoxicity and inhibition of mitochondrial permeability transition pore opening. The phase III study of dimebon, called the CONNECTION study, unfortunately, showed no clinical benefit on co-primary (cognition and global function) and secondary endpoints. Moreover, an additional phase III study called the CONCERT study again failed to show efficacy of dimebon (announcement from Pfizer and Medivation on Jan 17, 2012, www.medivation.com). Complete discrepancy between the results of phase II and phase III studies has brought researchers a huge confusion. Negative comments about the underlying rationale for the use of dimebon in AD are currently increasing because of the complete failure of dimebon in the phase III studies.

#### **4.4. AD-non-specific drugs for dementia**

AD-non-specific drugs are also possible. The therapeutic bases of these drugs lie on protection of neurons from aging-related toxicity such as oxidative stress, promotion of synaptic plasticity and regeneration of neural tissues using stem cell technologies.

#### *4.4.1. Antioxidants*

Antioxidants are assumed to trap toxic reactive oxygen species (ROS), which increase as aging. ROS is also present in the damaged neurons containing NFTs or close to SPs. The principal antioxidant strategy involves treatments with vitamin E (α-tocopherol), which resulted in benefit for AD patients in a randomized, placebo-controlled trial (Sano et al., 1997). Vitamin E, however, does not seem to be so effective because it failed to show reproducible efficacy in a double-blind study performed recently (Petersen et al., 2005). Natural polyphenolic com‐ pounds such as ginkgo biloba extracts, curcumin, green tea catechins and grape-seed oil extract (resveratrol) are also attracting attention as antioxidants (Aranda-Abreu et al., 2011).

(cGMP) positively regulates synaptic plasticity in this scheme, suggesting that PDE should be suppressed to increase cGMP. Consequently, PDE9A inhibitors are reported to promote synaptic plasticity via activation of cGMP signaling pathways (Andreeva et al., 2001). PF-04447943 is a selective PDE9A inhibitor, increasing cGMP concentrations in the CSF of healthy volunteers, and is being tested in a phase II study among mild-to-moderate AD

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203

Omega-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), are involved in neurite outgrowth, remodeling of membrane lipid rafts and neurogenesis (Aranda-Abreu et al., 2011). They are also reported to suppress tau hyperphosphorylation and Aβ aggregation. Omega-3 fatty acids may repair damages in neuronal membranes and restore lipid rafts for appropriate trafficking of membrane proteins, leading to promotion of synaptic activity. Some clinical trials have reported beneficial effects of DHA in elderly people with cognitive impair‐

Adult neurogenesis is now generally recognized. In adult brains, neurogenesis occurs mainly in two regions of the CNS including the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of hippocampal dentate gyri (Rodriguez and Verkhratsky, 2011). Multipotent neural stem cells (NSCs), localized in these regions, undergo self-renewal and differentiation into neuronal progenitor cells (NPCs) or glial progenitor cells (GPCs) with a faster cell cycle, which ultimately differentiate into neurons or glia. Impaired neurogenesis is expected for AD patients with massive neuronal loss. There is, however, a prevalent contro‐ versy among neurogenesis in AD brains. In a number of animal models for AD, toxic Aβ oligomers seem to enhance neurogenesis (Jin et al., 2004a; Sotthibundhu et al., 2009) although there are still several inconsistent observations that neurogenesis is disturbed in AD models (Feng et al., 2001; Haughey et al., 2002; Rodriguez et al., 2008). In post mortem AD brains, reduction of NPCs in the SVZ (Ziabreva et al., 2006) and an increase in NPCs in the DG (Jin et al., 2004b) are reported inconsistently as well. Accordingly, further clarification is mandatory. Regardless of the discussion on the neurogenesis in AD brains, neuroregeneration, which can be achieved by either enhancement of endogenous neurogenesis or implantation of neurons or their progenitors (cell therapy), is getting to be considered as a valid therapeutic strategy for AD. This is mainly due to the progress in stem cell biology and a number of successful observations in preclinical studies. NSCs can be cultured and expanded *in vitro*. Moreover, NSCs can be differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSs) established from AD patients themselves. Stem cell transplantation experiments have been carried out in animal models with some positive observations (Moghadam et al., 2009; Park et al., 2012; Wu et al., 2008; Yamasaki et al., 2007). These experiments, however, did not show significant regeneration or replacement of neural circuits by transplanted stem cells. Some of them used gene-modified stem cells with NGF or ChAT expression, supporting function of endogenous neurons. Delivery methods for NSCs/NPCs, cell viability and efficiency of engraftment should be considerably improved as well as resolving the safety issues. Endogenous neurogenesis is shown to be upregulated by soluble factors such as

ment but other studies resulted in no effects on AD patients (Quinn et al., 2010).

patients (Mangialasche et al., 2010).

*4.4.4. Neuroregenerative therapy*

**Omega-3 polyunsaturated fatty acids (docosahexaenoic acid etc.)**

#### *4.4.2. Neurotrophic factors to promote synaptic plasticity*

Neurotrophins are well-characterized "trophic" factors, which generally promote neuronal survival and plasticity (Chiba et al., 2007). Each factor has its own relevance in AD. Nerve growth factor (NGF), which is discovered in the 1950s to be the first neurotrophic factor, mainly expressed in the peripheral nervous system, but also plays a key role in stimulation, mainte‐ nance, and survival of basal forebrain cholinergic neurons, which are destroyed in AD. Brainderived neurotrophic factor (BDNF), purified from porcine brain homogenates in 1982, is highly expressed in cortical and hippocampal structures and plays roles in neuronal survival, neurite outgrowth and synaptic plasticity. A SNP in the *BDNF* gene that results in Met substitution of Val 66 in the pro-domain (V66M-BDNF or BDNFMet) causes dysregulation in BDNF secretion, which is linked to memory impairment as well as to altered susceptibility to neuropsychiatric disorders, such as AD, PD, depression, eating disorder, and bipolar disorder (Hong et al., 2011; Nagata et al., 2012).

Basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) are also considered to be neurotrophic or neuroprotective factors whose receptors belong to the tyrosine kinase receptor family. bFGF and HGF exhibit a neuroprotective effect against Aβ neurotoxicity (Hashimoto et al., 2004; Takeuchi et al., 2008). Genetic variations in the VEGF gene modifies risks of AD (Del Bo et al., 2009).

Clinical application of these factors seem to be relatively difficult because proteins are not stable and are not easily delivered through BBB. Clinical studies on NGF, however, are promising. NGF treatments were initially based on intracerebroventricular infusion, which counterbalanced the positive effects with adverse effects. Accordingly, other drug delivery systems are currently tested in clinical trials: gene therapy with AAV or genetically modified fibroblasts producing NGF, encapsulated-cell biodelivery, intranasal delivery and topical application on the ocular surface (Mangialasche et al., 2010; Tuszynski et al., 2005).

#### *4.4.3. Other therapies promoting synaptic plasticity*

#### **Phosphodiesterase inhibitors**

Phosphodiesterases (PDEs) such as PDE-2, -4, -5 and -9 are expressed in the brain and play a key role in synaptic plasticity (Domek-Lopacinska and Strosznajder, 2010). Cyclic GMP (cGMP) positively regulates synaptic plasticity in this scheme, suggesting that PDE should be suppressed to increase cGMP. Consequently, PDE9A inhibitors are reported to promote synaptic plasticity via activation of cGMP signaling pathways (Andreeva et al., 2001). PF-04447943 is a selective PDE9A inhibitor, increasing cGMP concentrations in the CSF of healthy volunteers, and is being tested in a phase II study among mild-to-moderate AD patients (Mangialasche et al., 2010).

#### **Omega-3 polyunsaturated fatty acids (docosahexaenoic acid etc.)**

Omega-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), are involved in neurite outgrowth, remodeling of membrane lipid rafts and neurogenesis (Aranda-Abreu et al., 2011). They are also reported to suppress tau hyperphosphorylation and Aβ aggregation. Omega-3 fatty acids may repair damages in neuronal membranes and restore lipid rafts for appropriate trafficking of membrane proteins, leading to promotion of synaptic activity. Some clinical trials have reported beneficial effects of DHA in elderly people with cognitive impair‐ ment but other studies resulted in no effects on AD patients (Quinn et al., 2010).

#### *4.4.4. Neuroregenerative therapy*

*4.4.1. Antioxidants*

202 Neurodegenerative Diseases

Antioxidants are assumed to trap toxic reactive oxygen species (ROS), which increase as aging. ROS is also present in the damaged neurons containing NFTs or close to SPs. The principal antioxidant strategy involves treatments with vitamin E (α-tocopherol), which resulted in benefit for AD patients in a randomized, placebo-controlled trial (Sano et al., 1997). Vitamin E, however, does not seem to be so effective because it failed to show reproducible efficacy in a double-blind study performed recently (Petersen et al., 2005). Natural polyphenolic com‐ pounds such as ginkgo biloba extracts, curcumin, green tea catechins and grape-seed oil extract

(resveratrol) are also attracting attention as antioxidants (Aranda-Abreu et al., 2011).

Neurotrophins are well-characterized "trophic" factors, which generally promote neuronal survival and plasticity (Chiba et al., 2007). Each factor has its own relevance in AD. Nerve growth factor (NGF), which is discovered in the 1950s to be the first neurotrophic factor, mainly expressed in the peripheral nervous system, but also plays a key role in stimulation, mainte‐ nance, and survival of basal forebrain cholinergic neurons, which are destroyed in AD. Brainderived neurotrophic factor (BDNF), purified from porcine brain homogenates in 1982, is highly expressed in cortical and hippocampal structures and plays roles in neuronal survival, neurite outgrowth and synaptic plasticity. A SNP in the *BDNF* gene that results in Met substitution of Val 66 in the pro-domain (V66M-BDNF or BDNFMet) causes dysregulation in BDNF secretion, which is linked to memory impairment as well as to altered susceptibility to neuropsychiatric disorders, such as AD, PD, depression, eating disorder, and bipolar disorder

Basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) are also considered to be neurotrophic or neuroprotective factors whose receptors belong to the tyrosine kinase receptor family. bFGF and HGF exhibit a neuroprotective effect against Aβ neurotoxicity (Hashimoto et al., 2004; Takeuchi et al.,

Clinical application of these factors seem to be relatively difficult because proteins are not stable and are not easily delivered through BBB. Clinical studies on NGF, however, are promising. NGF treatments were initially based on intracerebroventricular infusion, which counterbalanced the positive effects with adverse effects. Accordingly, other drug delivery systems are currently tested in clinical trials: gene therapy with AAV or genetically modified fibroblasts producing NGF, encapsulated-cell biodelivery, intranasal delivery and topical

Phosphodiesterases (PDEs) such as PDE-2, -4, -5 and -9 are expressed in the brain and play a key role in synaptic plasticity (Domek-Lopacinska and Strosznajder, 2010). Cyclic GMP

2008). Genetic variations in the VEGF gene modifies risks of AD (Del Bo et al., 2009).

application on the ocular surface (Mangialasche et al., 2010; Tuszynski et al., 2005).

*4.4.2. Neurotrophic factors to promote synaptic plasticity*

(Hong et al., 2011; Nagata et al., 2012).

*4.4.3. Other therapies promoting synaptic plasticity*

**Phosphodiesterase inhibitors**

Adult neurogenesis is now generally recognized. In adult brains, neurogenesis occurs mainly in two regions of the CNS including the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of hippocampal dentate gyri (Rodriguez and Verkhratsky, 2011). Multipotent neural stem cells (NSCs), localized in these regions, undergo self-renewal and differentiation into neuronal progenitor cells (NPCs) or glial progenitor cells (GPCs) with a faster cell cycle, which ultimately differentiate into neurons or glia. Impaired neurogenesis is expected for AD patients with massive neuronal loss. There is, however, a prevalent contro‐ versy among neurogenesis in AD brains. In a number of animal models for AD, toxic Aβ oligomers seem to enhance neurogenesis (Jin et al., 2004a; Sotthibundhu et al., 2009) although there are still several inconsistent observations that neurogenesis is disturbed in AD models (Feng et al., 2001; Haughey et al., 2002; Rodriguez et al., 2008). In post mortem AD brains, reduction of NPCs in the SVZ (Ziabreva et al., 2006) and an increase in NPCs in the DG (Jin et al., 2004b) are reported inconsistently as well. Accordingly, further clarification is mandatory.

Regardless of the discussion on the neurogenesis in AD brains, neuroregeneration, which can be achieved by either enhancement of endogenous neurogenesis or implantation of neurons or their progenitors (cell therapy), is getting to be considered as a valid therapeutic strategy for AD. This is mainly due to the progress in stem cell biology and a number of successful observations in preclinical studies. NSCs can be cultured and expanded *in vitro*. Moreover, NSCs can be differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSs) established from AD patients themselves. Stem cell transplantation experiments have been carried out in animal models with some positive observations (Moghadam et al., 2009; Park et al., 2012; Wu et al., 2008; Yamasaki et al., 2007). These experiments, however, did not show significant regeneration or replacement of neural circuits by transplanted stem cells. Some of them used gene-modified stem cells with NGF or ChAT expression, supporting function of endogenous neurons. Delivery methods for NSCs/NPCs, cell viability and efficiency of engraftment should be considerably improved as well as resolving the safety issues. Endogenous neurogenesis is shown to be upregulated by soluble factors such as serotonin (5-HT) agonists and BDNF in rodents (Lee et al., 2002; Santarelli et al., 2003). Effects in humans should be carefully addressed.

**References**

2819-2826.

9068-9076.

posal. Clin Interv Aging *6*, 53-59.

oxidative stress. Cardiovasc Res *88*, 360-366.

ochem Biophys Res Commun *385*, 324-329.

esis of geriatric memory dysfunction. Science *217*, 408-414.

[1] (1987). About a peculiar disease of the cerebral cortex. By Alois Alzheimer, 1907 (Translated by L. Jarvik and H. Greenson). Alzheimer Dis Assoc Disord *1*, 3-8.

Emerging Therapeutic Strategies in Alzheimer's Disease

http://dx.doi.org/10.5772/55293

205

[2] Aisen, P.S., Gauthier, S., Ferris, S.H., Saumier, D., Haine, D., Garceau, D., Duong, A., Suhy, J., Oh, J., Lau, W.C.*, et al.* (2011). Tramiprosate in mild-to-moderate Alzheim‐ er's disease - a randomized, double-blind, placebo-controlled, multi-centre study (the

[3] Aisen, P.S., Schafer, K.A., Grundman, M., Pfeiffer, E., Sano, M., Davis, K.L., Farlow, M.R., Jin, S., Thomas, R.G., and Thal, L.J. (2003). Effects of rofecoxib or naproxen vs placebo on Alzheimer disease progression: a randomized controlled trial. JAMA *289*,

[4] Alley, G.M., Bailey, J.A., Chen, D., Ray, B., Puli, L.K., Tanila, H., Banerjee, P.K., and Lahiri, D.K. (2009). Memantine lowers amyloid-beta peptide levels in neuronal cul‐

[5] Alves, L., Correia, A.S., Miguel, R., Alegria, P., and Bugalho, P. (2010). Alzheimer's

[6] Andreeva, S.G., Dikkes, P., Epstein, P.M., and Rosenberg, P.A. (2001). Expression of cGMP-specific phosphodiesterase 9A mRNA in the rat brain. J Neurosci *21*,

[7] Araki, W., Yuasa, K., Takeda, S., Takeda, K., Shirotani, K., Takahashi, K., and Tabira, T. (2001). Pro-apoptotic effect of presenilin 2 (PS2) overexpression is associated with

[8] Aranda-Abreu, G.E., Hernandez-Aguilar, M.E., Manzo Denes, J., Garcia Hernandez, L.I., and Herrera Rivero, M. (2011). Rehabilitating a brain with Alzheimer's: a pro‐

[9] Bachar, A.R., Scheffer, L., Schroeder, A.S., Nakamura, H.K., Cobb, L.J., Oh, Y.K., Ler‐ man, L.O., Pagano, R.E., Cohen, P., and Lerman, A. (2010). Humanin is expressed in human vascular walls and has a cytoprotective effect against oxidized LDL-induced

[10] Barry, S.P., Townsend, P.A., McCormick, J., Knight, R.A., Scarabelli, T.M., Latchman, D.S., and Stephanou, A. (2009). STAT3 deletion sensitizes cells to oxidative stress. Bi‐

[11] Bartus, R.T., Dean, R.L., 3rd, Beer, B., and Lippa, A.S. (1982). The cholinergic hypoth‐

[12] Bassan, M., Zamostiano, R., Davidson, A., Pinhasov, A., Giladi, E., Perl, O., Bassan, H., Blat, C., Gibney, G., Glazner, G.*, et al.* (1999). Complete sequence of a novel pro‐

down-regulation of Bcl-2 in cultured neurons. J Neurochem *79*, 1161-1168.

tures and in APP/PS1 transgenic mice. J Neurosci Res *88*, 143-154.

disease: a clinical practice-oriented review. Front Neurol *3*, 63.

Alphase Study). Arch Med Sci *7*, 102-111.

### **5. Conclusion**

Disease-modifying therapy for AD is not yet available despite vast efforts on drug develop‐ ment and plenty of candidate drugs. As shown in Table 1, failure rate of phase II and III clinical trials for AD are extremely high, meaning not only that current *in vitro* or pre-clinical models of AD can hardly predict the clinical efficacy but also that drugs, which showed only a mild effect in phase II studies, would eventually fail in phase III studies. Consequently, ChEIs and memantine are still in the center of clinical therapies for AD. The "amyloid cascade" hypothesis certainly gave us important insights into AD pathogenesis and provided a number of candi‐ dates, most of which are still under assessment in clinical trials. However, the results obtained from completed clinical trials are rather negative for the "amyloid cascade" hypothesis: e.g. Aβ immunotherapy and β-/γ-secretase inhibitors continue to fail in the trials. In addition, there are critical objections to the "amyloid cascade", which has not been properly answered: (i) about one third of the cases of a cognitively normal elder population showed AD-like brain pathology such as SPs and NFTs (Bennett et al., 2006), and (ii) post-mortem pathological analyses of AD brains with immunotherapy revealed that a certain population of patients with Aβ immunotherapy resulted in significant decrease in senile plaques without significant recovery of cognitive function (Gilman et al., 2005; Nicoll et al., 2003). Time might have come to further modify the "amyloid cascade", integrate alternative hypotheses into it, or reconsti‐ tute a novel hypothesis for AD, in order to develop clinically effective AD therapies.

### **Acknowledgements**

This work was supported in part by a grant from the Japan Research Foundation for Clinical Pharmacology. I thank Drs. Ikuo Nishimoto (1956-2003), Sadakazu Aiso and Bernd Groner.

### **Author details**

Tomohiro Chiba1,2,3

Address all correspondence to: chibatomohero@gmail.com

1 Georg-Speyer-Haus, Institute for Biomedical Research, Frankfurt am Main, Germany


#### **References**

serotonin (5-HT) agonists and BDNF in rodents (Lee et al., 2002; Santarelli et al., 2003). Effects

Disease-modifying therapy for AD is not yet available despite vast efforts on drug develop‐ ment and plenty of candidate drugs. As shown in Table 1, failure rate of phase II and III clinical trials for AD are extremely high, meaning not only that current *in vitro* or pre-clinical models of AD can hardly predict the clinical efficacy but also that drugs, which showed only a mild effect in phase II studies, would eventually fail in phase III studies. Consequently, ChEIs and memantine are still in the center of clinical therapies for AD. The "amyloid cascade" hypothesis certainly gave us important insights into AD pathogenesis and provided a number of candi‐ dates, most of which are still under assessment in clinical trials. However, the results obtained from completed clinical trials are rather negative for the "amyloid cascade" hypothesis: e.g. Aβ immunotherapy and β-/γ-secretase inhibitors continue to fail in the trials. In addition, there are critical objections to the "amyloid cascade", which has not been properly answered: (i) about one third of the cases of a cognitively normal elder population showed AD-like brain pathology such as SPs and NFTs (Bennett et al., 2006), and (ii) post-mortem pathological analyses of AD brains with immunotherapy revealed that a certain population of patients with Aβ immunotherapy resulted in significant decrease in senile plaques without significant recovery of cognitive function (Gilman et al., 2005; Nicoll et al., 2003). Time might have come to further modify the "amyloid cascade", integrate alternative hypotheses into it, or reconsti‐

tute a novel hypothesis for AD, in order to develop clinically effective AD therapies.

This work was supported in part by a grant from the Japan Research Foundation for Clinical Pharmacology. I thank Drs. Ikuo Nishimoto (1956-2003), Sadakazu Aiso and Bernd Groner.

1 Georg-Speyer-Haus, Institute for Biomedical Research, Frankfurt am Main, Germany

2 Department of Anatomy, Keio University, School of Medicine, Tokyo, Japan

3 Department of Pathology, Kyorin University School of Medicine, Tokyo, Japan

in humans should be carefully addressed.

**5. Conclusion**

204 Neurodegenerative Diseases

**Acknowledgements**

**Author details**

Tomohiro Chiba1,2,3

Address all correspondence to: chibatomohero@gmail.com


tein containing a femtomolar-activity-dependent neuroprotective peptide. J Neuro‐ chem *72*, 1283-1293.

[25] Cai, H., Wang, Y., McCarthy, D., Wen, H., Borchelt, D.R., Price, D.L., and Wong, P.C. (2001). BACE1 is the major beta-secretase for generation of Abeta peptides by neu‐

Emerging Therapeutic Strategies in Alzheimer's Disease

http://dx.doi.org/10.5772/55293

207

[26] Caleo, M., Cenni, M.C., Costa, M., Menna, E., Zentilin, L., Giadrossi, S., Giacca, M., and Maffei, L. (2002). Expression of BCL-2 via adeno-associated virus vectors rescues thalamic neurons after visual cortex lesion in the adult rat. Eur J Neurosci *15*,

[27] Caserta, T.M., Smith, A.N., Gultice, A.D., Reedy, M.A., and Brown, T.L. (2003). Q-VD-OPh, a broad spectrum caspase inhibitor with potent antiapoptotic properties.

[28] Chartier-Harlin, M.C., Crawford, F., Houlden, H., Warren, A., Hughes, D., Fidani, L., Goate, A., Rossor, M., Roques, P., Hardy, J.*, et al.* (1991). Early-onset Alzheimer's dis‐ ease caused by mutations at codon 717 of the beta-amyloid precursor protein gene.

[29] Chiba, T., Hashimoto, Y., Tajima, H., Yamada, M., Kato, R., Niikura, T., Terashita, K., Schulman, H., Aiso, S., Kita, Y.*, et al.* (2004). Neuroprotective effect of activity-de‐ pendent neurotrophic factor against toxicity from familial amyotrophic lateral sclero‐

[30] Chiba, T., Nishimoto, I., Aiso, S., and Matsuoka, M. (2007). Neuroprotection against neurodegenerative diseases: development of a novel hybrid neuroprotective peptide

[31] Chiba, T., Yamada, M., and Aiso, S. (2009a). Targeting the JAK2/STAT3 axis in Alz‐

[32] Chiba, T., Yamada, M., Hashimoto, Y., Sato, M., Sasabe, J., Kita, Y., Terashita, K., Ai‐ so, S., Nishimoto, I., and Matsuoka, M. (2005). Development of a femtomolar-acting humanin derivative named colivelin by attaching activity-dependent neurotrophic factor to its N terminus: characterization of colivelin-mediated neuroprotection against Alzheimer's disease-relevant insults in vitro and in vivo. J Neurosci *25*,

[33] Chiba, T., Yamada, M., Sasabe, J., Terashita, K., Aiso, S., Matsuoka, M., and Nishimo‐ to, I. (2006). Colivelin prolongs survival of an ALS model mouse. Biochem Biophys

[34] Chiba, T., Yamada, M., Sasabe, J., Terashita, K., Shimoda, M., Matsuoka, M., and Ai‐ so, S. (2009b). Amyloid-beta causes memory impairment by disturbing the JAK2/

[35] Chohan, M.O., Khatoon, S., Iqbal, I.G., and Iqbal, K. (2006). Involvement of I2PP2A in the abnormal hyperphosphorylation of tau and its reversal by Memantine. FEBS Lett

STAT3 axis in hippocampal neurons. Mol Psychiatry *14*, 206-222.

sis-linked mutant SOD1 in vitro and in vivo. J Neurosci Res *78*, 542-552.

heimer's disease. Expert Opin Ther Targets *13*, 1155-1167.

rons. Nat Neurosci *4*, 233-234.

1271-1277.

Apoptosis *8*, 345-352.

Nature *353*, 844-846.

10252-10261.

*580*, 3973-3979.

Res Commun *343*, 793-798.

Colivelin. Mol Neurobiol *35*, 55-84.


[25] Cai, H., Wang, Y., McCarthy, D., Wen, H., Borchelt, D.R., Price, D.L., and Wong, P.C. (2001). BACE1 is the major beta-secretase for generation of Abeta peptides by neu‐ rons. Nat Neurosci *4*, 233-234.

tein containing a femtomolar-activity-dependent neuroprotective peptide. J Neuro‐

[13] Bennett, D.A., Schneider, J.A., Arvanitakis, Z., Kelly, J.F., Aggarwal, N.T., Shah, R.C., and Wilson, R.S. (2006). Neuropathology of older persons without cognitive impair‐

[14] Berger, A. (2000). Minocycline slows progress of Huntington's disease in mice. BMJ

[15] Bertram, L. (2011). Alzheimer's genetics in the GWAS era: a continuing story of 'repli‐

[16] Bertram, L., Hiltunen, M., Parkinson, M., Ingelsson, M., Lange, C., Ramasamy, K., Mullin, K., Menon, R., Sampson, A.J., Hsiao, M.Y.*, et al.* (2005). Family-based associa‐ tion between Alzheimer's disease and variants in UBQLN1. N Engl J Med *352*,

[17] Bertram, L., Lill, C.M., and Tanzi, R.E. (2010). The genetics of Alzheimer disease:

[18] Braak, H., and Braak, E. (1991). Neuropathological stageing of Alzheimer-related

[19] Brenneman, D.E., and Gozes, I. (1996). A femtomolar-acting neuroprotective peptide.

[20] Britschgi, M., Olin, C.E., Johns, H.T., Takeda-Uchimura, Y., LeMieux, M.C., Rufibach, K., Rajadas, J., Zhang, H., Tomooka, B., Robinson, W.H.*, et al.* (2009). Neuroprotective natural antibodies to assemblies of amyloidogenic peptides decrease with normal ag‐ ing and advancing Alzheimer's disease. Proc Natl Acad Sci U S A *106*, 12145-12150.

[21] Burgos, J.S., Benavides, J., Douillet, P., Velasco, J., and Valdivieso, F. (2012). How sta‐ tins could be evaluated successfully in clinical trials for Alzheimer's disease? Am J

[22] Busciglio, J., Lorenzo, A., Yeh, J., and Yankner, B.A. (1995). beta-amyloid fibrils in‐ duce tau phosphorylation and loss of microtubule binding. Neuron *14*, 879-888. [23] Butterfield, D.A., Castegna, A., Lauderback, C.M., and Drake, J. (2002). Evidence that amyloid beta-peptide-induced lipid peroxidation and its sequelae in Alzheimer's dis‐

[24] Cabrol, C., Huzarska, M.A., Dinolfo, C., Rodriguez, M.C., Reinstatler, L., Ni, J., Yeh, L.A., Cuny, G.D., Stein, R.L., Selkoe, D.J.*, et al.* (2009). Small-molecule activators of in‐ sulin-degrading enzyme discovered through high-throughput compound screening.

ease brain contribute to neuronal death. Neurobiol Aging *23*, 655-664.

ment from two community-based studies. Neurology *66*, 1837-1844.

cations and refutations'. Curr Neurol Neurosci Rep *11*, 246-253.

back to the future. Neuron *68*, 270-281.

changes. Acta Neuropathol *82*, 239-259.

Alzheimers Dis Other Demen *27*, 151-153.

J Clin Invest *97*, 2299-2307.

PLoS One *4*, e5274.

chem *72*, 1283-1293.

*321*, 70.

206 Neurodegenerative Diseases

884-894.


[36] Choi, Y., Kim, H.S., Shin, K.Y., Kim, E.M., Kim, M., Park, C.H., Jeong, Y.H., Yoo, J., Lee, J.P., Chang, K.A.*, et al.* (2007). Minocycline attenuates neuronal cell death and improves cognitive impairment in Alzheimer's disease models. Neuropsychophar‐ macology *32*, 2393-2404.

[48] Delrieu, J., Ousset, P.J., Caillaud, C., and Vellas, B. (2012). 'Clinical trials in Alzheim‐ er's disease': immunotherapy approaches. J Neurochem *120 Suppl 1*, 186-193.

Emerging Therapeutic Strategies in Alzheimer's Disease

http://dx.doi.org/10.5772/55293

209

[49] Di Carlo, M. (2010). Beta amyloid peptide: from different aggregation forms to the ac‐

[50] Di Fede, G., Catania, M., Morbin, M., Rossi, G., Suardi, S., Mazzoleni, G., Merlin, M., Giovagnoli, A.R., Prioni, S., Erbetta, A.*, et al.* (2009). A recessive mutation in the APP gene with dominant-negative effect on amyloidogenesis. Science *323*, 1473-1477.

[51] Dibbern, D.A., Jr., Glazner, G.W., Gozes, I., Brenneman, D.E., and Hill, J.M. (1997). Inhibition of murine embryonic growth by human immunodeficiency virus envelope protein and its prevention by vasoactive intestinal peptide and activity-dependent

[52] Dodart, J.C., Bales, K.R., Gannon, K.S., Greene, S.J., DeMattos, R.B., Mathis, C., De‐ Long, C.A., Wu, S., Wu, X., Holtzman, D.M.*, et al.* (2002). Immunization reverses memory deficits without reducing brain Abeta burden in Alzheimer's disease model.

[53] Dodel, R., Hampel, H., Depboylu, C., Lin, S., Gao, F., Schock, S., Jackel, S., Wei, X., Buerger, K., Hoft, C.*, et al.* (2002). Human antibodies against amyloid beta peptide: a

[54] Dodel, R.C., Du, Y., Depboylu, C., Hampel, H., Frolich, L., Haag, A., Hemmeter, U., Paulsen, S., Teipel, S.J., Brettschneider, S.*, et al.* (2004). Intravenous immunoglobulins containing antibodies against beta-amyloid for the treatment of Alzheimer's disease.

[55] Domek-Lopacinska, K.U., and Strosznajder, J.B. (2010). Cyclic GMP and nitric oxide

[56] Dominguez, D., Tournoy, J., Hartmann, D., Huth, T., Cryns, K., Deforce, S., Serneels, L., Camacho, I.E., Marjaux, E., Craessaerts, K.*, et al.* (2005). Phenotypic and biochemi‐ cal analyses of BACE1- and BACE2-deficient mice. J Biol Chem *280*, 30797-30806.

[57] Doody, R.S., Gavrilova, S.I., Sano, M., Thomas, R.G., Aisen, P.S., Bachurin, S.O., See‐ ly, L., and Hung, D. (2008). Effect of dimebon on cognition, activities of daily living, behaviour, and global function in patients with mild-to-moderate Alzheimer's dis‐ ease: a randomised, double-blind, placebo-controlled study. Lancet *372*, 207-215.

[58] Du, Y., Ma, Z., Lin, S., Dodel, R.C., Gao, F., Bales, K.R., Triarhou, L.C., Chernet, E., Perry, K.W., Nelson, D.L.*, et al.* (2001). Minocycline prevents nigrostriatal dopaminer‐ gic neurodegeneration in the MPTP model of Parkinson's disease. Proc Natl Acad Sci

[59] Eisele, Y.S., Bolmont, T., Heikenwalder, M., Langer, F., Jacobson, L.H., Yan, Z.X., Roth, K., Aguzzi, A., Staufenbiel, M., Walker, L.C.*, et al.* (2009). Induction of cerebral

potential treatment for Alzheimer's disease. Ann Neurol *52*, 253-256.

synthase in aging and Alzheimer's disease. Mol Neurobiol *41*, 129-137.

tivation of different biochemical pathways. Eur Biophys J *39*, 877-888.

neurotrophic factor. J Clin Invest *99*, 2837-2841.

J Neurol Neurosurg Psychiatry *75*, 1472-1474.

Nat Neurosci *5*, 452-457.

U S A *98*, 14669-14674.


[48] Delrieu, J., Ousset, P.J., Caillaud, C., and Vellas, B. (2012). 'Clinical trials in Alzheim‐ er's disease': immunotherapy approaches. J Neurochem *120 Suppl 1*, 186-193.

[36] Choi, Y., Kim, H.S., Shin, K.Y., Kim, E.M., Kim, M., Park, C.H., Jeong, Y.H., Yoo, J., Lee, J.P., Chang, K.A.*, et al.* (2007). Minocycline attenuates neuronal cell death and improves cognitive impairment in Alzheimer's disease models. Neuropsychophar‐

[37] Courtney, C., Farrell, D., Gray, R., Hills, R., Lynch, L., Sellwood, E., Edwards, S., Har‐ dyman, W., Raftery, J., Crome, P.*, et al.* (2004). Long-term donepezil treatment in 565 patients with Alzheimer's disease (AD2000): randomised double-blind trial. Lancet

[38] Cramer, P.E., Cirrito, J.R., Wesson, D.W., Lee, C.Y., Karlo, J.C., Zinn, A.E., Casali, B.T., Restivo, J.L., Goebel, W.D., James, M.J.*, et al.* (2012). ApoE-directed therapeutics rapidly clear beta-amyloid and reverse deficits in AD mouse models. Science *335*,

[39] Cummings, J. (2010). What can be inferred from the interruption of the semagacestat

[40] David, R., Zeitzer, J., Friedman, L., Noda, A., O'Hara, R., Robert, P., and Yesavage, J.A. (2010). Non-pharmacologic management of sleep disturbance in Alzheimer's dis‐

[41] Davies, N.M., Kehoe, P.G., Ben-Shlomo, Y., and Martin, R.M. (2011). Associations of anti-hypertensive treatments with Alzheimer's disease, vascular dementia, and other

[42] De Felice, F.G., Velasco, P.T., Lambert, M.P., Viola, K., Fernandez, S.J., Ferreira, S.T., and Klein, W.L. (2007). Abeta oligomers induce neuronal oxidative stress through an N-methyl-D-aspartate receptor-dependent mechanism that is blocked by the Alz‐

[43] De Strooper, B. (2003). Aph-1, Pen-2, and Nicastrin with Presenilin generate an active

[44] De Strooper, B., and Annaert, W. (2000). Proteolytic processing and cell biological functions of the amyloid precursor protein. J Cell Sci *113 ( Pt 11)*, 1857-1870.

[45] Deane, R., Du Yan, S., Submamaryan, R.K., LaRue, B., Jovanovic, S., Hogg, E., Welch, D., Manness, L., Lin, C., Yu, J.*, et al.* (2003). RAGE mediates amyloid-beta peptide transport across the blood-brain barrier and accumulation in brain. Nat Med *9*,

[46] Deane, R., Sagare, A., Hamm, K., Parisi, M., Lane, S., Finn, M.B., Holtzman, D.M., and Zlokovic, B.V. (2008). apoE isoform-specific disruption of amyloid beta peptide

[47] Del Bo, R., Ghezzi, S., Scarpini, E., Bresolin, N., and Comi, G.P. (2009). VEGF genetic variability is associated with increased risk of developing Alzheimer's disease. J Neu‐

trial for treatment of Alzheimer's disease? Biol Psychiatry *68*, 876-878.

macology *32*, 2393-2404.

ease. J Nutr Health Aging *14*, 203-206.

dementias. J Alzheimers Dis *26*, 699-708.

gamma-Secretase complex. Neuron *38*, 9-12.

heimer drug memantine. J Biol Chem *282*, 11590-11601.

clearance from mouse brain. J Clin Invest *118*, 4002-4013.

*363*, 2105-2115.

208 Neurodegenerative Diseases

1503-1506.

907-913.

rol Sci *283*, 66-68.


beta-amyloidosis: intracerebral versus systemic Abeta inoculation. Proc Natl Acad Sci U S A *106*, 12926-12931.

ed protein kinase (SAPK/JNK-P), and calcium/calmodulin-dependent kinase II (CaM kinase II) are differentially expressed in tau deposits in neurons and glial cells in

Emerging Therapeutic Strategies in Alzheimer's Disease

http://dx.doi.org/10.5772/55293

211

[71] Fillit, H., Hess, G., Hill, J., Bonnet, P., and Toso, C. (2009). IV immunoglobulin is as‐ sociated with a reduced risk of Alzheimer disease and related disorders. Neurology

[72] Freeman, G.B., Lin, J.C., Pons, J., and Raha, N.M. (2012). 39-week toxicity and toxico‐ kinetic study of ponezumab (PF-04360365) in cynomolgus monkeys with 12-week re‐

[73] Gamblin, T.C., Chen, F., Zambrano, A., Abraha, A., Lagalwar, S., Guillozet, A.L., Lu, M., Fu, Y., Garcia-Sierra, F., LaPointe, N.*, et al.* (2003). Caspase cleavage of tau: link‐ ing amyloid and neurofibrillary tangles in Alzheimer's disease. Proc Natl Acad Sci U

[74] Gervais, F.G., Xu, D., Robertson, G.S., Vaillancourt, J.P., Zhu, Y., Huang, J., LeBlanc, A., Smith, D., Rigby, M., Shearman, M.S.*, et al.* (1999). Involvement of caspases in proteolytic cleavage of Alzheimer's amyloid-beta precursor protein and amyloido‐

[75] Ghosh, A.K., Brindisi, M., and Tang, J. (2012). Developing beta-secretase inhibitors

[76] Gilman, S., Koller, M., Black, R.S., Jenkins, L., Griffith, S.G., Fox, N.C., Eisner, L., Kir‐ by, L., Rovira, M.B., Forette, F.*, et al.* (2005). Clinical effects of Abeta immunization (AN1792) in patients with AD in an interrupted trial. Neurology *64*, 1553-1562. [77] Glenner, G.G., and Wong, C.W. (1984). Alzheimer's disease: initial report of the puri‐ fication and characterization of a novel cerebrovascular amyloid protein. Biochem Bi‐

[78] Goate, A., Chartier-Harlin, M.C., Mullan, M., Brown, J., Crawford, F., Fidani, L., Giuffra, L., Haynes, A., Irving, N., James, L.*, et al.* (1991). Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease.

[79] Goedert, M., Crowther, R.A., and Spillantini, M.G. (1998). Tau mutations cause fron‐

[80] Gotz, J., Chen, F., van Dorpe, J., and Nitsch, R.M. (2001). Formation of neurofibrillary tangles in P301l tau transgenic mice induced by Abeta 42 fibrils. Science *293*,

[81] Gozes, I., and Divinski, I. (2004). The femtomolar-acting NAP interacts with microtu‐

bules: Novel aspects of astrocyte protection. J Alzheimers Dis *6*, S37-41.

for treatment of Alzheimer's disease. J Neurochem *120 Suppl 1*, 71-83.

tauopathies. J Neural Transm *108*, 1397-1415.

covery period. J Alzheimers Dis *28*, 531-541.

genic A beta peptide formation. Cell *97*, 395-406.

ophys Res Commun *120*, 885-890.

totemporal dementias. Neuron *21*, 955-958.

Nature *349*, 704-706.

1491-1495.

*73*, 180-185.

S A *100*, 10032-10037.


ed protein kinase (SAPK/JNK-P), and calcium/calmodulin-dependent kinase II (CaM kinase II) are differentially expressed in tau deposits in neurons and glial cells in tauopathies. J Neural Transm *108*, 1397-1415.

[71] Fillit, H., Hess, G., Hill, J., Bonnet, P., and Toso, C. (2009). IV immunoglobulin is as‐ sociated with a reduced risk of Alzheimer disease and related disorders. Neurology *73*, 180-185.

beta-amyloidosis: intracerebral versus systemic Abeta inoculation. Proc Natl Acad

[60] Eisele, Y.S., Obermuller, U., Heilbronner, G., Baumann, F., Kaeser, S.A., Wolburg, H., Walker, L.C., Staufenbiel, M., Heikenwalder, M., and Jucker, M. (2010). Peripherally applied Abeta-containing inoculates induce cerebral beta-amyloidosis. Science *330*,

[62] Etminan, M., Gill, S., and Samii, A. (2003). Effect of non-steroidal anti-inflammatory drugs on risk of Alzheimer's disease: systematic review and meta-analysis of obser‐

[63] Farlow, M., Arnold, S.E., van Dyck, C.H., Aisen, P.S., Snider, B.J., Porsteinsson, A.P., Friedrich, S., Dean, R.A., Gonzales, C., Sethuraman, G.*, et al.* (2012). Safety and bio‐ marker effects of solanezumab in patients with Alzheimer's disease. Alzheimers De‐

[64] Farrimond, L.E., Roberts, E., and McShane, R. (2012). Memantine and cholinesterase inhibitor combination therapy for Alzheimer's disease: a systematic review. BMJ

[65] Farris, W., Mansourian, S., Chang, Y., Lindsley, L., Eckman, E.A., Frosch, M.P., Eck‐ man, C.B., Tanzi, R.E., Selkoe, D.J., and Guenette, S. (2003). Insulin-degrading en‐ zyme regulates the levels of insulin, amyloid beta-protein, and the beta-amyloid precursor protein intracellular domain in vivo. Proc Natl Acad Sci U S A *100*,

[66] Feldman, H.H., Doody, R.S., Kivipelto, M., Sparks, D.L., Waters, D.D., Jones, R.W., Schwam, E., Schindler, R., Hey-Hadavi, J., DeMicco, D.A.*, et al.* (2010). Randomized controlled trial of atorvastatin in mild to moderate Alzheimer disease: LEADe. Neu‐

[67] Feng, R., Rampon, C., Tang, Y.P., Shrom, D., Jin, J., Kyin, M., Sopher, B., Miller, M.W., Ware, C.B., Martin, G.M.*, et al.* (2001). Deficient neurogenesis in forebrain-spe‐ cific presenilin-1 knockout mice is associated with reduced clearance of hippocampal

[68] Fernandez-Madrid, I., Levy, E., Marder, K., and Frangione, B. (1991). Codon 618 var‐ iant of Alzheimer amyloid gene associated with inherited cerebral hemorrhage. Ann

[69] Ferrer, I., Barrachina, M., and Puig, B. (2002). Glycogen synthase kinase-3 is associat‐ ed with neuronal and glial hyperphosphorylated tau deposits in Alzheimer's disease, Pick's disease, progressive supranuclear palsy and corticobasal degeneration. Acta

[70] Ferrer, I., Blanco, R., Carmona, M., and Puig, B. (2001). Phosphorylated mitogen-acti‐ vated protein kinase (MAPK/ERK-P), protein kinase of 38 kDa (p38-P), stress-activat‐

[61] Eisenstein, M. (2011). Genetics: finding risk factors. Nature *475*, S20-22.

Sci U S A *106*, 12926-12931.

vational studies. BMJ *327*, 128.

980-982.

210 Neurodegenerative Diseases

ment *8*, 261-271.

Open *2*.

4162-4167.

rology *74*, 956-964.

Neurol *30*, 730-733.

Neuropathol *104*, 583-591.

memory traces. Neuron *32*, 911-926.


[82] Gozes, I., and Divinski, I. (2007). NAP, a neuroprotective drug candidate in clinical trials, stimulates microtubule assembly in the living cell. Curr Alzheimer Res *4*, 507-509.

[93] Hashimoto, Y., Niikura, T., Ito, Y., Kita, Y., Terashita, K., and Nishimoto, I. (2002b). Neurotoxic mechanisms by Alzheimer's disease-linked N141I mutant presenilin 2. J

Emerging Therapeutic Strategies in Alzheimer's Disease

http://dx.doi.org/10.5772/55293

213

[94] Hashimoto, Y., Niikura, T., Ito, Y., and Nishimoto, I. (2000). Multiple mechanisms underlie neurotoxicity by different types of Alzheimer's disease mutations of amy‐

[95] Hashimoto, Y., Niikura, T., Ito, Y., Sudo, H., Hata, M., Arakawa, E., Abe, Y., Kita, Y., and Nishimoto, I. (2001a). Detailed characterization of neuroprotection by a rescue factor humanin against various Alzheimer's disease-relevant insults. J Neurosci *21*,

[96] Hashimoto, Y., Niikura, T., Tajima, H., Yasukawa, T., Sudo, H., Ito, Y., Kita, Y., Ka‐ wasumi, M., Kouyama, K., Doyu, M.*, et al.* (2001b). A rescue factor abolishing neuro‐ nal cell death by a wide spectrum of familial Alzheimer's disease genes and Abeta.

[97] Haughey, N.J., Nath, A., Chan, S.L., Borchard, A.C., Rao, M.S., and Mattson, M.P. (2002). Disruption of neurogenesis by amyloid beta-peptide, and perturbed neural progenitor cell homeostasis, in models of Alzheimer's disease. J Neurochem *83*,

[98] Hendriks, W.J., Elson, A., Harroch, S., Pulido, R., Stoker, A., and den Hertog, J.

[99] Hoang, P.T., Park, P., Cobb, L.J., Paharkova-Vatchkova, V., Hakimi, M., Cohen, P., and Lee, K.W. (2010). The neurosurvival factor Humanin inhibits beta-cell apoptosis via signal transducer and activator of transcription 3 activation and delays and amel‐

[100] Hock, C., Konietzko, U., Papassotiropoulos, A., Wollmer, A., Streffer, J., von Rotz, R.C., Davey, G., Moritz, E., and Nitsch, R.M. (2002). Generation of antibodies specific for beta-amyloid by vaccination of patients with Alzheimer disease. Nat Med *8*,

[101] Hong, C.J., Liou, Y.J., and Tsai, S.J. (2011). Effects of BDNF polymorphisms on brain

[102] Hu, J.P., Xie, J.W., Wang, C.Y., Wang, T., Wang, X., Wang, S.L., Teng, W.P., and Wang, Z.Y. (2011). Valproate reduces tau phosphorylation via cyclin-dependent kin‐ ase 5 and glycogen synthase kinase 3 signaling pathways. Brain Res Bull *85*, 194-200.

[103] Huang, Y., and Mucke, L. (2012). Alzheimer mechanisms and therapeutic strategies.

[104] Hutton, M., Lendon, C.L., Rizzu, P., Baker, M., Froelich, S., Houlden, H., Pickering-Brown, S., Chakraverty, S., Isaacs, A., Grover, A.*, et al.* (1998). Association of mis‐

function and behavior in health and disease. Brain Res Bull *86*, 287-297.

(2012). Protein tyrosine phosphatases in health and disease. FEBS J.

iorates diabetes in nonobese diabetic mice. Metabolism *59*, 343-349.

Pharmacol Exp Ther *300*, 736-745.

Proc Natl Acad Sci U S A *98*, 6336-6341.

9235-9245.

1509-1524.

1270-1275.

Cell *148*, 1204-1222.

loid precursor protein. J Biol Chem *275*, 34541-34551.


[93] Hashimoto, Y., Niikura, T., Ito, Y., Kita, Y., Terashita, K., and Nishimoto, I. (2002b). Neurotoxic mechanisms by Alzheimer's disease-linked N141I mutant presenilin 2. J Pharmacol Exp Ther *300*, 736-745.

[82] Gozes, I., and Divinski, I. (2007). NAP, a neuroprotective drug candidate in clinical trials, stimulates microtubule assembly in the living cell. Curr Alzheimer Res *4*,

[83] Green, R.C., Schneider, L.S., Amato, D.A., Beelen, A.P., Wilcock, G., Swabb, E.A., and Zavitz, K.H. (2009). Effect of tarenflurbil on cognitive decline and activities of daily living in patients with mild Alzheimer disease: a randomized controlled trial. JAMA

[84] Guglielmotto, M., Tamagno, E., and Danni, O. (2009). Oxidative stress and hypoxia contribute to Alzheimer's disease pathogenesis: two sides of the same coin. Scientific‐

[85] Guo, Q., Furukawa, K., Sopher, B.L., Pham, D.G., Xie, J., Robinson, N., Martin, G.M., and Mattson, M.P. (1996). Alzheimer's PS-1 mutation perturbs calcium homeostasis and sensitizes PC12 cells to death induced by amyloid beta-peptide. Neuroreport *8*,

[86] Hampel, H., Ewers, M., Burger, K., Annas, P., Mortberg, A., Bogstedt, A., Frolich, L., Schroder, J., Schonknecht, P., Riepe, M.W.*, et al.* (2009). Lithium trial in Alzheimer's disease: a randomized, single-blind, placebo-controlled, multicenter 10-week study. J

[87] Hara, H., Monsonego, A., Yuasa, K., Adachi, K., Xiao, X., Takeda, S., Takahashi, K., Weiner, H.L., and Tabira, T. (2004). Development of a safe oral Abeta vaccine using recombinant adeno-associated virus vector for Alzheimer's disease. J Alzheimers Dis

[88] Hardy, J.A., and Higgins, G.A. (1992). Alzheimer's disease: the amyloid cascade hy‐

[89] Hashimoto, K. (2011). Can minocycline prevent the onset of Alzheimer's disease?

[90] Hashimoto, Y., Chiba, T., Yamada, M., Nawa, M., Kanekura, K., Suzuki, H., Terashi‐ ta, K., Aiso, S., Nishimoto, I., and Matsuoka, M. (2005). Transforming growth factor beta2 is a neuronal death-inducing ligand for amyloid-beta precursor protein. Mol

[91] Hashimoto, Y., Ito, Y., Arakawa, E., Kita, Y., Terashita, K., Niikura, T., and Nishimo‐ to, I. (2002a). Neurotoxic mechanisms triggered by Alzheimer's disease-linked mu‐ tant M146L presenilin 1: involvement of NO synthase via a novel pertussis toxin

[92] Hashimoto, Y., Kaneko, Y., Tsukamoto, E., Frankowski, H., Kouyama, K., Kita, Y., Niikura, T., Aiso, S., Bredesen, D.E., Matsuoka, M.*, et al.* (2004). Molecular characteri‐ zation of neurohybrid cell death induced by Alzheimer's amyloid-beta peptides via

507-509.

212 Neurodegenerative Diseases

379-383.

*6*, 483-488.

*302*, 2557-2564.

WorldJournal *9*, 781-791.

Clin Psychiatry *70*, 922-931.

pothesis. Science *256*, 184-185.

Cell Biol *25*, 9304-9317.

target. J Neurochem *80*, 426-437.

p75NTR/PLAIDD. J Neurochem *90*, 549-558.

Ann Neurol *69*, 739; author reply 739-740.


sense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17. Nature *393*, 702-705.

[117] Kawasumi, M., Hashimoto, Y., Chiba, T., Kanekura, K., Yamagishi, Y., Ishizaka, M., Tajima, H., Niikura, T., and Nishimoto, I. (2002). Molecular mechanisms for neuronal cell death by Alzheimer's amyloid precursor protein-relevant insults. Neurosignals

Emerging Therapeutic Strategies in Alzheimer's Disease

http://dx.doi.org/10.5772/55293

215

[118] Kilic, E., Hermann, D.M., Kugler, S., Kilic, U., Holzmuller, H., Schmeer, C., and Bahr, M. (2002). Adenovirus-mediated Bcl-X(L) expression using a neuron-specific synap‐ sin-1 promoter protects against disseminated neuronal injury and brain infarction

[119] Klyubin, I., Wang, Q., Reed, M.N., Irving, E.A., Upton, N., Hofmeister, J., Cleary, J.P., Anwyl, R., and Rowan, M.J. (2009). Protection against Abeta-mediated rapid disrup‐ tion of synaptic plasticity and memory by memantine. Neurobiol Aging *32*, 614-623.

[120] Kojro, E., Gimpl, G., Lammich, S., Marz, W., and Fahrenholz, F. (2001). Low choles‐ terol stimulates the nonamyloidogenic pathway by its effect on the alpha -secretase

[121] Kotilinek, L.A., Bacskai, B., Westerman, M., Kawarabayashi, T., Younkin, L., Hyman, B.T., Younkin, S., and Ashe, K.H. (2002). Reversible memory loss in a mouse trans‐

[122] Kukar, T., Murphy, M.P., Eriksen, J.L., Sagi, S.A., Weggen, S., Smith, T.E., Ladd, T., Khan, M.A., Kache, R., Beard, J.*, et al.* (2005). Diverse compounds mimic Alzheimer disease-causing mutations by augmenting Abeta42 production. Nat Med *11*, 545-550.

[123] Lannfelt, L., Blennow, K., Zetterberg, H., Batsman, S., Ames, D., Harrison, J., Masters, C.L., Targum, S., Bush, A.I., Murdoch, R.*, et al.* (2008). Safety, efficacy, and biomarker findings of PBT2 in targeting Abeta as a modifying therapy for Alzheimer's disease: a phase IIa, double-blind, randomised, placebo-controlled trial. Lancet Neurol *7*,

[124] Laudon, H., Hansson, E.M., Melen, K., Bergman, A., Farmery, M.R., Winblad, B., Lendahl, U., von Heijne, G., and Naslund, J. (2005). A nine-transmembrane domain

[125] Lee, J., Duan, W., and Mattson, M.P. (2002). Evidence that brain-derived neurotro‐ phic factor is required for basal neurogenesis and mediates, in part, the enhancement of neurogenesis by dietary restriction in the hippocampus of adult mice. J Neuro‐

[126] Leissring, M.A., Farris, W., Chang, A.Y., Walsh, D.M., Wu, X., Sun, X., Frosch, M.P., and Selkoe, D.J. (2003). Enhanced proteolysis of beta-amyloid in APP transgenic mice prevents plaque formation, secondary pathology, and premature death. Neuron *40*,

following focal cerebral ischemia in mice. Neurobiol Dis *11*, 275-284.

ADAM 10. Proc Natl Acad Sci U S A *98*, 5815-5820.

genic model of Alzheimer's disease. J Neurosci *22*, 6331-6335.

topology for presenilin 1. J Biol Chem *280*, 35352-35360.

*11*, 236-250.

779-786.

chem *82*, 1367-1375.

1087-1093.


[117] Kawasumi, M., Hashimoto, Y., Chiba, T., Kanekura, K., Yamagishi, Y., Ishizaka, M., Tajima, H., Niikura, T., and Nishimoto, I. (2002). Molecular mechanisms for neuronal cell death by Alzheimer's amyloid precursor protein-relevant insults. Neurosignals *11*, 236-250.

sense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17.

[105] Ito, S., Ohtsuki, S., Kamiie, J., Nezu, Y., and Terasaki, T. (2007). Cerebral clearance of human amyloid-beta peptide (1-40) across the blood-brain barrier is reduced by selfaggregation and formation of low-density lipoprotein receptor-related protein-1 li‐

[106] Iwata, N., Takaki, Y., Fukami, S., Tsubuki, S., and Saido, T.C. (2002). Region-specific reduction of A beta-degrading endopeptidase, neprilysin, in mouse hippocampus

[107] Iwata, N., Tsubuki, S., Takaki, Y., Shirotani, K., Lu, B., Gerard, N.P., Gerard, C., Ha‐ ma, E., Lee, H.J., and Saido, T.C. (2001). Metabolic regulation of brain Abeta by nepri‐

[108] Iwata, N., Tsubuki, S., Takaki, Y., Watanabe, K., Sekiguchi, M., Hosoki, E., Kawashi‐ ma-Morishima, M., Lee, H.J., Hama, E., Sekine-Aizawa, Y.*, et al.* (2000). Identification of the major Abeta1-42-degrading catabolic pathway in brain parenchyma: suppres‐

[109] Jick, H., Zornberg, G.L., Jick, S.S., Seshadri, S., and Drachman, D.A. (2000). Statins

[110] Jin, K., Galvan, V., Xie, L., Mao, X.O., Gorostiza, O.F., Bredesen, D.E., and Greenberg, D.A. (2004a). Enhanced neurogenesis in Alzheimer's disease transgenic (PDGF-

[111] Jin, K., Peel, A.L., Mao, X.O., Xie, L., Cottrell, B.A., Henshall, D.C., and Greenberg, D.A. (2004b). Increased hippocampal neurogenesis in Alzheimer's disease. Proc Natl

[112] John, V., Beck, J.P., Bienkowski, M.J., Sinha, S., and Heinrikson, R.L. (2003). Human

[113] Johnson, G.V., and Stoothoff, W.H. (2004). Tau phosphorylation in neuronal cell

[115] Jonsson, T., Atwal, J.K., Steinberg, S., Snaedal, J., Jonsson, P.V., Bjornsson, S., Stefans‐ son, H., Sulem, P., Gudbjartsson, D., Maloney, J.*, et al.* (2012). A mutation in APP pro‐ tects against Alzheimer's disease and age-related cognitive decline. Nature *488*,

[116] Kang, J., Lemaire, H.G., Unterbeck, A., Salbaum, J.M., Masters, C.L., Grzeschik, K.H., Multhaup, G., Beyreuther, K., and Muller-Hill, B. (1987). The precursor of Alzheim‐ er's disease amyloid A4 protein resembles a cell-surface receptor. Nature *325*,

beta-secretase (BACE) and BACE inhibitors. J Med Chem *46*, 4625-4630.

[114] Jones, R.W. (2010). Dimebon disappointment. Alzheimers Res Ther *2*, 25.

sion leads to biochemical and pathological deposition. Nat Med *6*, 143-150.

Nature *393*, 702-705.

214 Neurodegenerative Diseases

gand complexes. J Neurochem *103*, 2482-2490.

and the risk of dementia. Lancet *356*, 1627-1631.

function and dysfunction. J Cell Sci *117*, 5721-5729.

APPSw,Ind) mice. Proc Natl Acad Sci U S A *101*, 13363-13367.

upon aging. J Neurosci Res *70*, 493-500.

lysin. Science *292*, 1550-1552.

Acad Sci U S A *101*, 343-347.

96-99.

733-736.


[127] Lesne, S., Koh, M.T., Kotilinek, L., Kayed, R., Glabe, C.G., Yang, A., Gallagher, M., and Ashe, K.H. (2006). A specific amyloid-beta protein assembly in the brain impairs memory. Nature *440*, 352-357.

proves cognition and reduces Alzheimer's-like neuropathology in transgenic mice.

Emerging Therapeutic Strategies in Alzheimer's Disease

http://dx.doi.org/10.5772/55293

217

[140] Massey, L.K., Mah, A.L., Ford, D.L., Miller, J., Liang, J., Doong, H., and Monteiro, M.J. (2004). Overexpression of ubiquilin decreases ubiquitination and degradation of

[141] Mattson, M.P. (2004). Pathways towards and away from Alzheimer's disease. Nature

[142] Mattson, M.P., Guo, Q., Furukawa, K., and Pedersen, W.A. (1998). Presenilins, the en‐ doplasmic reticulum, and neuronal apoptosis in Alzheimer's disease. J Neurochem

[143] Miller, B.W., Willett, K.C., and Desilets, A.R. (2011). Rosiglitazone and pioglitazone for the treatment of Alzheimer's disease. Ann Pharmacother *45*, 1416-1424.

[144] Misiak, B., Leszek, J., and Kiejna, A. (2012). Metabolic syndrome, mild cognitive im‐ pairment and Alzheimer's disease-The emerging role of systemic low-grade inflam‐

[145] Moghadam, F.H., Alaie, H., Karbalaie, K., Tanhaei, S., Nasr Esfahani, M.H., and Ba‐ harvand, H. (2009). Transplantation of primed or unprimed mouse embryonic stem cell-derived neural precursor cells improves cognitive function in Alzheimerian rats.

[146] Morgan, K. The three new pathways leading to Alzheimer's disease. Neuropathol

[147] Muzumdar, R.H., Huffman, D.M., Atzmon, G., Buettner, C., Cobb, L.J., Fishman, S., Budagov, T., Cui, L., Einstein, F.H., Poduval, A.*, et al.* (2009). Humanin: a novel cen‐

[148] Nagata, T., Shinagawa, S., Nukariya, K., Yamada, H., and Nakayama, K. (2012). As‐ sociation between BDNF Polymorphism (Val66Met) and Executive Function in Pa‐ tients with Amnestic Mild Cognitive Impairment or Mild Alzheimer Disease.

[149] Nalivaeva, N.N., Beckett, C., Belyaev, N.D., and Turner, A.J. (2012). Are amyloid-de‐ grading enzymes viable therapeutic targets in Alzheimer's disease? J Neurochem *120*

[150] Ng, D.C., Lin, B.H., Lim, C.P., Huang, G., Zhang, T., Poli, V., and Cao, X. (2006). Stat3 regulates microtubules by antagonizing the depolymerization activity of stathmin. J

[151] Nicolas, C.S., Peineau, S., Amici, M., Csaba, Z., Fafouri, A., Javalet, C., Collett, V.J., Hildebrandt, L., Seaton, G., Choi, S.L.*, et al.* (2012). The Jak/STAT pathway is in‐

tral regulator of peripheral insulin action. PLoS One *4*, e6334.

Am J Pathol *176*, 870-880.

Differentiation *78*, 59-68.

*Suppl 1*, 167-185.

Cell Biol *172*, 245-257.

Appl Neurobiol *37*, 353-357.

Dement Geriatr Cogn Disord *33*, 266-272.

volved in synaptic plasticity. Neuron *73*, 374-390.

*430*, 631-639.

*70*, 1-14.

presenilin proteins. J Alzheimers Dis *6*, 79-92.

mation and adiposity. Brain Res Bull *89*, 144-149.


proves cognition and reduces Alzheimer's-like neuropathology in transgenic mice. Am J Pathol *176*, 870-880.

[140] Massey, L.K., Mah, A.L., Ford, D.L., Miller, J., Liang, J., Doong, H., and Monteiro, M.J. (2004). Overexpression of ubiquilin decreases ubiquitination and degradation of presenilin proteins. J Alzheimers Dis *6*, 79-92.

[127] Lesne, S., Koh, M.T., Kotilinek, L., Kayed, R., Glabe, C.G., Yang, A., Gallagher, M., and Ashe, K.H. (2006). A specific amyloid-beta protein assembly in the brain impairs

[128] Lewis, J., Dickson, D.W., Lin, W.L., Chisholm, L., Corral, A., Jones, G., Yen, S.H., Sa‐ hara, N., Skipper, L., Yager, D.*, et al.* (2001). Enhanced neurofibrillary degeneration in

[129] Lewis, J., McGowan, E., Rockwood, J., Melrose, H., Nacharaju, P., Van Slegtenhorst, M., Gwinn-Hardy, K., Paul Murphy, M., Baker, M., Yu, X.*, et al.* (2000). Neurofibril‐ lary tangles, amyotrophy and progressive motor disturbance in mice expressing mu‐

[130] Lim, G.P., Yang, F., Chu, T., Chen, P., Beech, W., Teter, B., Tran, T., Ubeda, O., Ashe, K.H., Frautschy, S.A.*, et al.* (2000). Ibuprofen suppresses plaque pathology and in‐ flammation in a mouse model for Alzheimer's disease. J Neurosci *20*, 5709-5714. [131] Lleo, A., Berezovska, O., Herl, L., Raju, S., Deng, A., Bacskai, B.J., Frosch, M.P., Irizar‐ ry, M., and Hyman, B.T. (2004). Nonsteroidal anti-inflammatory drugs lower Abe‐

[132] Lleo, A., Greenberg, S.M., and Growdon, J.H. (2006). Current pharmacotherapy for

[133] Luo, Y., Bolon, B., Kahn, S., Bennett, B.D., Babu-Khan, S., Denis, P., Fan, W., Kha, H., Zhang, J., Gong, Y.*, et al.* (2001). Mice deficient in BACE1, the Alzheimer's beta-secre‐ tase, have normal phenotype and abolished beta-amyloid generation. Nat Neurosci

[134] Lutz, M.W., Crenshaw, D.G., Saunders, A.M., and Roses, A.D. (2010). Genetic varia‐ tion at a single locus and age of onset for Alzheimer's disease. Alzheimers Dement *6*,

[135] Mah, A.L., Perry, G., Smith, M.A., and Monteiro, M.J. (2000). Identification of ubiqui‐ lin, a novel presenilin interactor that increases presenilin protein accumulation. J Cell

[136] Mandel, S.A., Amit, T., Kalfon, L., Reznichenko, L., Weinreb, O., and Youdim, M.B. (2008). Cell signaling pathways and iron chelation in the neurorestorative activity of green tea polyphenols: special reference to epigallocatechin gallate (EGCG). J Alz‐

[137] Mangialasche, F., Solomon, A., Winblad, B., Mecocci, P., and Kivipelto, M. (2010). Alzheimer's disease: clinical trials and drug development. Lancet Neurol *9*, 702-716.

[138] Marjaux, E., Hartmann, D., and De Strooper, B. (2004). Presenilins in memory, Alz‐

[139] Martinez-Coria, H., Green, K.N., Billings, L.M., Kitazawa, M., Albrecht, M., Rammes, G., Parsons, C.G., Gupta, S., Banerjee, P., and LaFerla, F.M. (2010). Memantine im‐

ta42 and change presenilin 1 conformation. Nat Med *10*, 1065-1066.

transgenic mice expressing mutant tau and APP. Science *293*, 1487-1491.

memory. Nature *440*, 352-357.

216 Neurodegenerative Diseases

tant (P301L) tau protein. Nat Genet *25*, 402-405.

Alzheimer's disease. Annu Rev Med *57*, 513-533.

heimer's disease, and therapy. Neuron *42*, 189-192.

*4*, 231-232.

125-131.

Biol *151*, 847-862.

heimers Dis *15*, 211-222.


[152] Nicoll, J.A., Wilkinson, D., Holmes, C., Steart, P., Markham, H., and Weller, R.O. (2003). Neuropathology of human Alzheimer disease after immunization with amy‐ loid-beta peptide: a case report. Nat Med *9*, 448-452.

[163] Perrin, R.J., Fagan, A.M., and Holtzman, D.M. (2009). Multimodal techniques for di‐

Emerging Therapeutic Strategies in Alzheimer's Disease

http://dx.doi.org/10.5772/55293

219

[164] Petersen, R.C., Thomas, R.G., Grundman, M., Bennett, D., Doody, R., Ferris, S., Gala‐ sko, D., Jin, S., Kaye, J., Levey, A.*, et al.* (2005). Vitamin E and donepezil for the treat‐

[165] Pettenati, C., Annicchiarico, R., and Caltagirone, C. (2003). Clinical pharmacology of

[166] Poirier, J. (2005). Apolipoprotein E, cholesterol transport and synthesis in sporadic

[167] Qiu, W.Q., Walsh, D.M., Ye, Z., Vekrellis, K., Zhang, J., Podlisny, M.B., Rosner, M.R., Safavi, A., Hersh, L.B., and Selkoe, D.J. (1998). Insulin-degrading enzyme regulates extracellular levels of amyloid beta-protein by degradation. J Biol Chem *273*,

[168] Quinn, J.F., Raman, R., Thomas, R.G., Yurko-Mauro, K., Nelson, E.B., Van Dyck, C., Galvin, J.E., Emond, J., Jack, C.R., Jr., Weiner, M.*, et al.* (2010). Docosahexaenoic acid supplementation and cognitive decline in Alzheimer disease: a randomized trial. JA‐

[169] Rapoport, M., Dawson, H.N., Binder, L.I., Vitek, M.P., and Ferreira, A. (2002). Tau is essential to beta -amyloid-induced neurotoxicity. Proc Natl Acad Sci U S A *99*,

[170] Ray, B., Banerjee, P.K., Greig, N.H., and Lahiri, D.K. (2009). Memantine treatment de‐ creases levels of secreted Alzheimer's amyloid precursor protein (APP) and amyloid beta (A beta) peptide in the human neuroblastoma cells. Neurosci Lett *470*, 1-5. [171] Refolo, L.M., Pappolla, M.A., LaFrancois, J., Malester, B., Schmidt, S.D., Thomas-Bry‐ ant, T., Tint, G.S., Wang, R., Mercken, M., Petanceska, S.S.*, et al.* (2001). A cholesterollowering drug reduces beta-amyloid pathology in a transgenic mouse model of

[172] Reitz, C. (2012). Alzheimer's disease and the amyloid cascade hypothesis: a critical

[173] Relkin, N.R., Szabo, P., Adamiak, B., Burgut, T., Monthe, C., Lent, R.W., Younkin, S., Younkin, L., Schiff, R., and Weksler, M.E. (2009). 18-Month study of intravenous im‐ munoglobulin for treatment of mild Alzheimer disease. Neurobiol Aging *30*,

[174] Renolleau, S., Fau, S., Goyenvalle, C., Joly, L.M., Chauvier, D., Jacotot, E., Mariani, J., and Charriaut-Marlangue, C. (2007). Specific caspase inhibitor Q-VD-OPh prevents

[175] Rezai-Zadeh, K., Shytle, D., Sun, N., Mori, T., Hou, H., Jeanniton, D., Ehrhart, J., Townsend, K., Zeng, J., Morgan, D.*, et al.* (2005). Green tea epigallocatechin-3-gallate

neonatal stroke in P7 rat: a role for gender. J Neurochem *100*, 1062-1071.

agnosis and prognosis of Alzheimer's disease. Nature *461*, 916-922.

ment of mild cognitive impairment. N Engl J Med *352*, 2379-2388.

anti-Alzheimer drugs. Fundam Clin Pharmacol *17*, 659-672.

Alzheimer's disease. Neurobiol Aging *26*, 355-361.

Alzheimer's disease. Neurobiol Dis *8*, 890-899.

review. Int J Alzheimers Dis *2012*, 369808.

32730-32738.

6364-6369.

1728-1736.

MA *304*, 1903-1911.


[163] Perrin, R.J., Fagan, A.M., and Holtzman, D.M. (2009). Multimodal techniques for di‐ agnosis and prognosis of Alzheimer's disease. Nature *461*, 916-922.

[152] Nicoll, J.A., Wilkinson, D., Holmes, C., Steart, P., Markham, H., and Weller, R.O. (2003). Neuropathology of human Alzheimer disease after immunization with amy‐

[153] Nikolaev, A., McLaughlin, T., O'Leary, D.D., and Tessier-Lavigne, M. (2009). APP binds DR6 to trigger axon pruning and neuron death via distinct caspases. Nature

[154] Nilsberth, C., Westlind-Danielsson, A., Eckman, C.B., Condron, M.M., Axelman, K., Forsell, C., Stenh, C., Luthman, J., Teplow, D.B., Younkin, S.G.*, et al.* (2001). The 'Arc‐ tic' APP mutation (E693G) causes Alzheimer's disease by enhanced Abeta protofibril

[155] Nussbaum, J.M., Schilling, S., Cynis, H., Silva, A., Swanson, E., Wangsanut, T., Tay‐ ler, K., Wiltgen, B., Hatami, A., Ronicke, R.*, et al.* (2012). Prion-like behaviour and tau-dependent cytotoxicity of pyroglutamylated amyloid-beta. Nature *485*, 651-655.

[156] Oddo, S., Caccamo, A., Shepherd, J.D., Murphy, M.P., Golde, T.E., Kayed, R., Methe‐ rate, R., Mattson, M.P., Akbari, Y., and LaFerla, F.M. (2003). Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic

[157] Oh, Y.K., Bachar, A.R., Zacharias, D.G., Kim, S.G., Wan, J., Cobb, L.J., Lerman, L.O., Cohen, P., and Lerman, A. (2011). Humanin preserves endothelial function and pre‐ vents atherosclerotic plaque progression in hypercholesterolemic ApoE deficient

[158] Okura, Y., Miyakoshi, A., Kohyama, K., Park, I.K., Staufenbiel, M., and Matsumoto, Y. (2006). Nonviral Abeta DNA vaccine therapy against Alzheimer's disease: long-

[159] Orgogozo, J.M., Gilman, S., Dartigues, J.F., Laurent, B., Puel, M., Kirby, L.C., Jouan‐ ny, P., Dubois, B., Eisner, L., Flitman, S.*, et al.* (2003). Subacute meningoencephalitis in a subset of patients with AD after Abeta42 immunization. Neurology *61*, 46-54.

[160] Park, D., Lee, H.J., Joo, S.S., Bae, D.K., Yang, G., Yang, Y.H., Lim, I., Matsuo, A., Tooyama, I., Kim, Y.B.*, et al.* (2012). Human neural stem cells over-expressing choline acetyltransferase restore cognition in rat model of cognitive dysfunction. Exp Neurol

[161] Parsons, R.B., Price, G.C., Farrant, J.K., Subramaniam, D., Adeagbo-Sheikh, J., and Austen, B.M. (2006). Statins inhibit the dimerization of beta-secretase via both isopre‐

[162] Passer, B.J., Pellegrini, L., Vito, P., Ganjei, J.K., and D'Adamio, L. (1999). Interaction of Alzheimer's presenilin-1 and presenilin-2 with Bcl-X(L). A potential role in modu‐

noid- and cholesterol-mediated mechanisms. Biochem J *399*, 205-214.

lating the threshold of cell death. J Biol Chem *274*, 24007-24013.

term effects and safety. Proc Natl Acad Sci U S A *103*, 9619-9624.

loid-beta peptide: a case report. Nat Med *9*, 448-452.

formation. Nat Neurosci *4*, 887-893.

dysfunction. Neuron *39*, 409-421.

mice. Atherosclerosis *219*, 65-73.

*234*, 521-526.

*457*, 981-989.

218 Neurodegenerative Diseases


(EGCG) modulates amyloid precursor protein cleavage and reduces cerebral amyloi‐ dosis in Alzheimer transgenic mice. J Neurosci *25*, 8807-8814.

[187] Saito, T., Iwata, N., Tsubuki, S., Takaki, Y., Takano, J., Huang, S.M., Suemoto, T., Hi‐ guchi, M., and Saido, T.C. (2005). Somatostatin regulates brain amyloid beta peptide

Emerging Therapeutic Strategies in Alzheimer's Disease

http://dx.doi.org/10.5772/55293

221

Abeta42 through modulation of proteolytic degradation. Nat Med *11*, 434-439. [188] Salloway, S., Sperling, R., Gilman, S., Fox, N.C., Blennow, K., Raskind, M., Sabbagh, M., Honig, L.S., Doody, R., van Dyck, C.H.*, et al.* (2009). A phase 2 multiple ascend‐ ing dose trial of bapineuzumab in mild to moderate Alzheimer disease. Neurology

[189] Salloway, S., Sperling, R., Keren, R., Porsteinsson, A.P., van Dyck, C.H., Tariot, P.N., Gilman, S., Arnold, D., Abushakra, S., Hernandez, C.*, et al.* (2011). A phase 2 random‐ ized trial of ELND005, scyllo-inositol, in mild to moderate Alzheimer disease. Neu‐

[190] Sampson, E., Jenagaratnam, L., and McShane, R. (2008). Metal protein attenuating compounds for the treatment of Alzheimer's disease. Cochrane Database Syst Rev,

[191] Sano, M., Bell, K.L., Galasko, D., Galvin, J.E., Thomas, R.G., van Dyck, C.H., and Ai‐ sen, P.S. (2011). A randomized, double-blind, placebo-controlled trial of simvastatin

[192] Sano, M., Ernesto, C., Thomas, R.G., Klauber, M.R., Schafer, K., Grundman, M., Woodbury, P., Growdon, J., Cotman, C.W., Pfeiffer, E.*, et al.* (1997). A controlled trial of selegiline, alpha-tocopherol, or both as treatment for Alzheimer's disease. The Alz‐

[193] Santarelli, L., Saxe, M., Gross, C., Surget, A., Battaglia, F., Dulawa, S., Weisstaub, N., Lee, J., Duman, R., Arancio, O.*, et al.* (2003). Requirement of hippocampal neurogene‐

[194] Sarafian, T.A., Montes, C., Imura, T., Qi, J., Coppola, G., Geschwind, D.H., and Sofro‐ niew, M.V. Disruption of astrocyte STAT3 signaling decreases mitochondrial func‐

[195] Sato, S., Tatebayashi, Y., Akagi, T., Chui, D.H., Murayama, M., Miyasaka, T., Planel, E., Tanemura, K., Sun, X., Hashikawa, T.*, et al.* (2002). Aberrant tau phosphorylation by glycogen synthase kinase-3beta and JNK3 induces oligomeric tau fibrils in COS-7

[196] Schellenberg, G.D., and Montine, T.J. (2012). The genetics and neuropathology of

[197] Schenk, D., Barbour, R., Dunn, W., Gordon, G., Grajeda, H., Guido, T., Hu, K., Huang, J., Johnson-Wood, K., Khan, K.*, et al.* (1999). Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature *400*,

[198] Schilling, S., Zeitschel, U., Hoffmann, T., Heiser, U., Francke, M., Kehlen, A., Holzer, M., Hutter-Paier, B., Prokesch, M., Windisch, M.*, et al.* (2008). Glutaminyl cyclase in‐

heimer's Disease Cooperative Study. N Engl J Med *336*, 1216-1222.

sis for the behavioral effects of antidepressants. Science *301*, 805-809.

tion and increases oxidative stress in vitro. PLoS One *5*, e9532.

Alzheimer's disease. Acta Neuropathol *124*, 305-323.

cells. J Biol Chem *277*, 42060-42065.

to treat Alzheimer disease. Neurology *77*, 556-563.

*73*, 2061-2070.

CD005380.

173-177.

rology *77*, 1253-1262.


[187] Saito, T., Iwata, N., Tsubuki, S., Takaki, Y., Takano, J., Huang, S.M., Suemoto, T., Hi‐ guchi, M., and Saido, T.C. (2005). Somatostatin regulates brain amyloid beta peptide Abeta42 through modulation of proteolytic degradation. Nat Med *11*, 434-439.

(EGCG) modulates amyloid precursor protein cleavage and reduces cerebral amyloi‐

[176] Riordan, K.C., Hoffman Snyder, C.R., Wellik, K.E., Caselli, R.J., Wingerchuk, D.M., and Demaerschalk, B.M. (2011). Effectiveness of adding memantine to an Alzheimer dementia treatment regimen which already includes stable donepezil therapy: a criti‐

[177] Ritchie, C.W., Bush, A.I., Mackinnon, A., Macfarlane, S., Mastwyk, M., MacGregor, L., Kiers, L., Cherny, R., Li, Q.X., Tammer, A.*, et al.* (2003). Metal-protein attenuation with iodochlorhydroxyquin (clioquinol) targeting Abeta amyloid deposition and tox‐ icity in Alzheimer disease: a pilot phase 2 clinical trial. Arch Neurol *60*, 1685-1691.

[178] Roberson, E.D., Scearce-Levie, K., Palop, J.J., Yan, F., Cheng, I.H., Wu, T., Gerstein, H., Yu, G.Q., and Mucke, L. (2007). Reducing endogenous tau ameliorates amyloid beta-induced deficits in an Alzheimer's disease mouse model. Science *316*, 750-754.

[179] Rodriguez, J.J., Jones, V.C., Tabuchi, M., Allan, S.M., Knight, E.M., LaFerla, F.M., Oddo, S., and Verkhratsky, A. (2008). Impaired adult neurogenesis in the dentate gy‐ rus of a triple transgenic mouse model of Alzheimer's disease. PLoS One *3*, e2935.

[180] Rodriguez, J.J., and Verkhratsky, A. (2011). Neurogenesis in Alzheimer's disease. J

[181] Rogaev, E.I., Sherrington, R., Rogaeva, E.A., Levesque, G., Ikeda, M., Liang, Y., Chi, H., Lin, C., Holman, K., Tsuda, T.*, et al.* (1995). Familial Alzheimer's disease in kin‐ dreds with missense mutations in a gene on chromosome 1 related to the Alzheimer's

[182] Rogaeva, E., Meng, Y., Lee, J.H., Gu, Y., Kawarai, T., Zou, F., Katayama, T., Baldwin, C.T., Cheng, R., Hasegawa, H.*, et al.* (2007). The neuronal sortilin-related receptor SORL1 is genetically associated with Alzheimer disease. Nat Genet *39*, 168-177. [183] Rohn, T.T., and Head, E. (2009). Caspases as therapeutic targets in Alzheimer's dis‐

[184] Rohn, T.T., Ivins, K.J., Bahr, B.A., Cotman, C.W., and Cribbs, D.H. (2000). A monoclo‐ nal antibody to amyloid precursor protein induces neuronal apoptosis. J Neurochem

[185] Rollero, A., Murialdo, G., Fonzi, S., Garrone, S., Gianelli, M.V., Gazzerro, E., Barreca, A., and Polleri, A. (1998). Relationship between cognitive function, growth hormone and insulin-like growth factor I plasma levels in aged subjects. Neuropsychobiology

[186] Roses, A.D., Lutz, M.W., Amrine-Madsen, H., Saunders, A.M., Crenshaw, D.G., Sundseth, S.S., Huentelman, M.J., Welsh-Bohmer, K.A., and Reiman, E.M. A TOMM40 variable-length polymorphism predicts the age of late-onset Alzheimer's

ease: is it time to "cut" to the chase? Int J Clin Exp Pathol *2*, 108-118.

dosis in Alzheimer transgenic mice. J Neurosci *25*, 8807-8814.

cally appraised topic. Neurologist *17*, 121-123.

disease type 3 gene. Nature *376*, 775-778.

disease. Pharmacogenomics J *10*, 375-384.

Anat *219*, 78-89.

220 Neurodegenerative Diseases

*74*, 2331-2342.

*38*, 73-79.


hibition attenuates pyroglutamate Abeta and Alzheimer's disease-like pathology. Nat Med *14*, 1106-1111.

toxic function of cell-surface beta-amyloid precursor protein. Mol Cell Neurosci *16*,

Emerging Therapeutic Strategies in Alzheimer's Disease

http://dx.doi.org/10.5772/55293

223

[212] Tabira, T. (2001). Clioquinol's return: cautions from Japan. Science *292*, 2251-2252.

phorylation in mouse brain. Neurosci Lett *306*, 37-40.

secretase complex. Nature *422*, 438-441.

mouse model. Gene Ther *15*, 561-571.

heimer's disease? J Clin Psychiatry *70*, 919-921.

home setting. J Am Geriatr Soc *49*, 1590-1599.

[213] Takashima, A., Murayama, M., Yasutake, K., Takahashi, H., Yokoyama, M., and Ishi‐ guro, K. (2001). Involvement of cyclin dependent kinase5 activator p25 on tau phos‐

[214] Takasugi, N., Tomita, T., Hayashi, I., Tsuruoka, M., Niimura, M., Takahashi, Y., Thi‐ nakaran, G., and Iwatsubo, T. (2003). The role of presenilin cofactors in the gamma-

[215] Takeuchi, D., Sato, N., Shimamura, M., Kurinami, H., Takeda, S., Shinohara, M., Su‐ zuki, S., Kojima, M., Ogihara, T., and Morishita, R. (2008). Alleviation of Abeta-in‐ duced cognitive impairment by ultrasound-mediated gene transfer of HGF in a

[216] Tariot, P.N., and Aisen, P.S. (2009). Can lithium or valproate untie tangles in Alz‐

[217] Tariot, P.N., Cummings, J.L., Katz, I.R., Mintzer, J., Perdomo, C.A., Schwam, E.M., and Whalen, E. (2001). A randomized, double-blind, placebo-controlled study of the efficacy and safety of donepezil in patients with Alzheimer's disease in the nursing

[218] Tariot, P.N., Farlow, M.R., Grossberg, G.T., Graham, S.M., McDonald, S., and Gergel, I. (2004). Memantine treatment in patients with moderate to severe Alzheimer dis‐ ease already receiving donepezil: a randomized controlled trial. JAMA *291*, 317-324.

[219] Tian, G., Sobotka-Briner, C.D., Zysk, J., Liu, X., Birr, C., Sylvester, M.A., Edwards, P.D., Scott, C.D., and Greenberg, B.D. (2002). Linear non-competitive inhibition of solubilized human gamma-secretase by pepstatin A methylester, L685458, sulfona‐

[220] Tuszynski, M.H., Thal, L., Pay, M., Salmon, D.P., U, H.S., Bakay, R., Patel, P., Blesch, A., Vahlsing, H.L., Ho, G.*, et al.* (2005). A phase 1 clinical trial of nerve growth factor

[221] Um, J.W., Nygaard, H.B., Heiss, J.K., Kostylev, M.A., Stagi, M., Vortmeyer, A., Wis‐ niewski, T., Gunther, E.C., and Strittmatter, S.M. (2012). Alzheimer amyloid-beta oligomer bound to postsynaptic prion protein activates Fyn to impair neurons. Nat

[222] Van der Zee, E.A., Platt, B., and Riedel, G. Acetylcholine: future research and per‐

[223] Vassar, R., Bennett, B.D., Babu-Khan, S., Kahn, S., Mendiaz, E.A., Denis, P., Teplow, D.B., Ross, S., Amarante, P., Loeloff, R.*, et al.* (1999). Beta-secretase cleavage of Alz‐

mides, and benzodiazepines. J Biol Chem *277*, 31499-31505.

gene therapy for Alzheimer disease. Nat Med *11*, 551-555.

Neurosci *15*, 1227-1235.

spectives. Behav Brain Res *221*, 583-586.

708-723.


toxic function of cell-surface beta-amyloid precursor protein. Mol Cell Neurosci *16*, 708-723.

[212] Tabira, T. (2001). Clioquinol's return: cautions from Japan. Science *292*, 2251-2252.

hibition attenuates pyroglutamate Abeta and Alzheimer's disease-like pathology.

[199] Schirmer, R.H., Adler, H., Pickhardt, M., and Mandelkow, E. (2011). "Lest we forget

[200] Seabrook, T.J., Jiang, L., Maier, M., and Lemere, C.A. (2006). Minocycline affects mi‐ croglia activation, Abeta deposition, and behavior in APP-tg mice. Glia *53*, 776-782.

[201] Searfoss, G.H., Jordan, W.H., Calligaro, D.O., Galbreath, E.J., Schirtzinger, L.M., Ber‐ ridge, B.R., Gao, H., Higgins, M.A., May, P.C., and Ryan, T.P. (2003). Adipsin, a bio‐ marker of gastrointestinal toxicity mediated by a functional gamma-secretase

[202] Shearman, M.S., Beher, D., Clarke, E.E., Lewis, H.D., Harrison, T., Hunt, P., Nadin, A., Smith, A.L., Stevenson, G., and Castro, J.L. (2000). L-685,458, an aspartyl protease transition state mimic, is a potent inhibitor of amyloid beta-protein precursor gam‐

[203] Sherrington, R., Rogaev, E.I., Liang, Y., Rogaeva, E.A., Levesque, G., Ikeda, M., Chi, H., Lin, C., Li, G., Holman, K.*, et al.* (1995). Cloning of a gene bearing missense muta‐

[204] Shiryaev, N., Pikman, R., Giladi, E., and Gozes, I. (2011). Protection against tauop‐ athy by the drug candidates NAP (davunetide) and D-SAL: biochemical, cellular and

[205] Shobab, L.A., Hsiung, G.Y., and Feldman, H.H. (2005). Cholesterol in Alzheimer's

[206] Shub, D., Darvishi, R., and Kunik, M.E. (2009). Non-pharmacologic treatment of in‐

[207] Singer, C., Tractenberg, R.E., Kaye, J., Schafer, K., Gamst, A., Grundman, M., Tho‐ mas, R., and Thal, L.J. (2003). A multicenter, placebo-controlled trial of melatonin for

[208] Sotthibundhu, A., Li, Q.X., Thangnipon, W., and Coulson, E.J. (2009). Abeta(1-42) stimulates adult SVZ neurogenesis through the p75 neurotrophin receptor. Neuro‐

[209] Stephanou, A., and Latchman, D.S. (2005). Opposing actions of STAT-1 and STAT-3.

[210] Stohr, J., Watts, J.C., Mensinger, Z.L., Oehler, A., Grillo, S.K., DeArmond, S.J., Prusin‐ er, S.B., and Giles, K. (2012). Purified and synthetic Alzheimer's amyloid beta (Abeta)

[211] Sudo, H., Jiang, H., Yasukawa, T., Hashimoto, Y., Niikura, T., Kawasumi, M., Matsu‐ da, S., Takeuchi, Y., Aiso, S., Matsuoka, M.*, et al.* (2000). Antibody-regulated neuro‐

tions in early-onset familial Alzheimer's disease. Nature *375*, 754-760.

you--methylene blue...". Neurobiol Aging *32*, 2325 e2327-2316.

Nat Med *14*, 1106-1111.

222 Neurodegenerative Diseases

inhibitor. J Biol Chem *278*, 46107-46116.

ma-secretase activity. Biochemistry *39*, 8698-8704.

behavioral aspects. Curr Pharm Des *17*, 2603-2612.

somnia in persons with dementia. Geriatrics *64*, 22-26.

prions. Proc Natl Acad Sci U S A *109*, 11025-11030.

sleep disturbance in Alzheimer's disease. Sleep *26*, 893-901.

disease. Lancet Neurol *4*, 841-852.

biol Aging *30*, 1975-1985.

Growth Factors *23*, 177-182.


heimer's amyloid precursor protein by the transmembrane aspartic protease BACE. Science *286*, 735-741.

viral vectors with Cre-loxP recombination system in motor neurons of mutant SOD1

Emerging Therapeutic Strategies in Alzheimer's Disease

http://dx.doi.org/10.5772/55293

225

[236] Yamatsuji, T., Matsui, T., Okamoto, T., Komatsuzaki, K., Takeda, S., Fukumoto, H., Iwatsubo, T., Suzuki, N., Asami-Odaka, A., Ireland, S.*, et al.* (1996). G protein-mediat‐ ed neuronal DNA fragmentation induced by familial Alzheimer's disease-associated

[237] Yang, L., Sugama, S., Mischak, R.P., Kiaei, M., Bizat, N., Brouillet, E., Joh, T.H., and Beal, M.F. (2004). A novel systemically active caspase inhibitor attenuates the toxici‐

[238] Yasojima, K., Akiyama, H., McGeer, E.G., and McGeer, P.L. (2001a). Reduced nepri‐ lysin in high plaque areas of Alzheimer brain: a possible relationship to deficient

[239] Yasojima, K., McGeer, E.G., and McGeer, P.L. (2001b). Relationship between beta amyloid peptide generating molecules and neprilysin in Alzheimer disease and nor‐

[240] Zhang, Z., Hartmann, H., Do, V.M., Abramowski, D., Sturchler-Pierrat, C., Staufen‐ biel, M., Sommer, B., van de Wetering, M., Clevers, H., Saftig, P.*, et al.* (1998). Desta‐ bilization of beta-catenin by mutations in presenilin-1 potentiates neuronal

[241] Zhao, B., Chrest, F.J., Horton, W.E., Jr., Sisodia, S.S., and Kusiak, J.W. (1997). Expres‐ sion of mutant amyloid precursor proteins induces apoptosis in PC12 cells. J Neuro‐

[242] Zhu, S., Stavrovskaya, I.G., Drozda, M., Kim, B.Y., Ona, V., Li, M., Sarang, S., Liu, A.S., Hartley, D.M., Wu, D.C.*, et al.* (2002). Minocycline inhibits cytochrome c release and delays progression of amyotrophic lateral sclerosis in mice. Nature *417*, 74-78.

[243] Ziabreva, I., Perry, E., Perry, R., Minger, S.L., Ekonomou, A., Przyborski, S., and Bal‐ lard, C. (2006). Altered neurogenesis in Alzheimer's disease. J Psychosom Res *61*,

ties of MPTP, malonate, and 3NP in vivo. Neurobiol Dis *17*, 250-259.

degradation of beta-amyloid peptide. Neurosci Lett *297*, 97-100.

transgenic mice. Gene Ther *8*, 977-986.

mutants of APP. Science *272*, 1349-1352.

mal brain. Brain Res *919*, 115-121.

apoptosis. Nature *395*, 698-702.

sci Res *47*, 253-263.

311-316.


viral vectors with Cre-loxP recombination system in motor neurons of mutant SOD1 transgenic mice. Gene Ther *8*, 977-986.

[236] Yamatsuji, T., Matsui, T., Okamoto, T., Komatsuzaki, K., Takeda, S., Fukumoto, H., Iwatsubo, T., Suzuki, N., Asami-Odaka, A., Ireland, S.*, et al.* (1996). G protein-mediat‐ ed neuronal DNA fragmentation induced by familial Alzheimer's disease-associated mutants of APP. Science *272*, 1349-1352.

heimer's amyloid precursor protein by the transmembrane aspartic protease BACE.

[224] Vingtdeux, V., and Marambaud, P. (2012). Identification and biology of alpha-secre‐

[225] Vulih-Shultzman, I., Pinhasov, A., Mandel, S., Grigoriadis, N., Touloumi, O., Pittel, Z., and Gozes, I. (2007). Activity-dependent neuroprotective protein snippet NAP re‐ duces tau hyperphosphorylation and enhances learning in a novel transgenic mouse

[226] Weggen, S., Eriksen, J.L., Das, P., Sagi, S.A., Wang, R., Pietrzik, C.U., Findlay, K.A., Smith, T.E., Murphy, M.P., Bulter, T.*, et al.* (2001). A subset of NSAIDs lower amyloi‐ dogenic Abeta42 independently of cyclooxygenase activity. Nature *414*, 212-216. [227] Weihl, C.C., Ghadge, G.D., Kennedy, S.G., Hay, N., Miller, R.J., and Roos, R.P. (1999). Mutant presenilin-1 induces apoptosis and downregulates Akt/PKB. J Neurosci *19*,

[228] Wiessner, C., Wiederhold, K.H., Tissot, A.C., Frey, P., Danner, S., Jacobson, L.H., Jen‐ nings, G.T., Luond, R., Ortmann, R., Reichwald, J.*, et al.* (2011). The second-genera‐ tion active Abeta immunotherapy CAD106 reduces amyloid accumulation in APP transgenic mice while minimizing potential side effects. J Neurosci *31*, 9323-9331. [229] Winblad, B., Kilander, L., Eriksson, S., Minthon, L., Batsman, S., Wetterholm, A.L., Jansson-Blixt, C., and Haglund, A. (2006). Donepezil in patients with severe Alz‐ heimer's disease: double-blind, parallel-group, placebo-controlled study. Lancet *367*,

[230] Wolfe, M.S. (2012). gamma-Secretase inhibitors and modulators for Alzheimer's dis‐

[231] Wolozin, B., Kellman, W., Ruosseau, P., Celesia, G.G., and Siegel, G. (2000). De‐ creased prevalence of Alzheimer disease associated with 3-hydroxy-3-methyglutaryl

[232] Wu, S., Sasaki, A., Yoshimoto, R., Kawahara, Y., Manabe, T., Kataoka, K., Asashima, M., and Yuge, L. (2008). Neural stem cells improve learning and memory in rats with

[233] Yamada, M., Chiba, T., Sasabe, J., Terashita, K., Aiso, S., and Matsuoka, M. (2008). Nasal Colivelin treatment ameliorates memory impairment related to Alzheimer's

[234] Yamasaki, T.R., Blurton-Jones, M., Morrissette, D.A., Kitazawa, M., Oddo, S., and La‐ Ferla, F.M. (2007). Neural stem cells improve memory in an inducible mouse model

[235] Yamashita, S., Mita, S., Arima, T., Maeda, Y., Kimura, E., Nishida, Y., Murakami, T., Okado, H., and Uchino, M. (2001). Bcl-2 expression by retrograde transport of adeno‐

coenzyme A reductase inhibitors. Arch Neurol *57*, 1439-1443.

Science *286*, 735-741.

224 Neurodegenerative Diseases

5360-5369.

1057-1065.

tase. J Neurochem *120 Suppl 1*, 34-45.

model. J Pharmacol Exp Ther *323*, 438-449.

ease. J Neurochem *120 Suppl 1*, 89-98.

Alzheimer's disease. Pathobiology *75*, 186-194.

of neuronal loss. J Neurosci *27*, 11925-11933.

disease. Neuropsychopharmacology *33*, 2020-2032.


**Chapter 10**

**Dropping the BACE: Beta Secretase (BACE1) as an**

The β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is an important regulator for the production of amyloid plaques, a characteristic of the Alzheimer's disease (AD) brain. The proteolytic cleavage of the amyloid precursor protein (APP), by BACE1, produces an insoluble amyloid-β (Aβ) fragment which has the ability to aggregate and migrate onto the dendrites and cell body of neuronal cells, initiating chronic immune responses of inflam‐

The cleavage of Aβ fragments trigger a feedback system that complements production num‐ bers. This increases Aβ loading to such an extent that it exceeds the defences required for natural elimination. The fragments aggregate, developing into an insoluble plaque that has the ability to effect normal functioning by causing dysfunction. Without early identification and effective inhibition of this pathogenic pathway, the disease is anticipated to become

Attempts to inhibit BACE1 have been relatively fruitless with most therapeutic trials being aborted in the early stages. A number of obstacles can be detrimental to the ability of an in‐ hibitor like solubility, bioavailability, potency and effectiveness. In addition to the complexi‐ ty, there are also a number of substrates cleaved by BACE1 which are important in other pathways like voltage gated sodium channels and axon myelination [1, 2]. This can create

Inhibitors of BACE1 have, so far, looked at active-site mimics that diverge from small and large molecules to short peptidic structures and expression modulators. Other approaches look to utilize technology with modelling software to determine the BACE1 3D structure and formulate an effective inhibitor via domain analysis. The underlying issue is translat‐

and reproduction in any medium, provided the original work is properly cited.

© 2013 Read and Suphioglu; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

more widespread with an aging population, in which AD is most prevalent.

adverse reactions beyond the reduction of plaques.

**Alzheimer's Disease Intervention Target**

Justin Read and Cenk Suphioglu

http://dx.doi.org/10.5772/53603

mation and microglia activation.

**1. Introduction**

Additional information is available at the end of the chapter

## **Dropping the BACE: Beta Secretase (BACE1) as an Alzheimer's Disease Intervention Target**

Justin Read and Cenk Suphioglu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53603

### **1. Introduction**

The β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is an important regulator for the production of amyloid plaques, a characteristic of the Alzheimer's disease (AD) brain. The proteolytic cleavage of the amyloid precursor protein (APP), by BACE1, produces an insoluble amyloid-β (Aβ) fragment which has the ability to aggregate and migrate onto the dendrites and cell body of neuronal cells, initiating chronic immune responses of inflam‐ mation and microglia activation.

The cleavage of Aβ fragments trigger a feedback system that complements production num‐ bers. This increases Aβ loading to such an extent that it exceeds the defences required for natural elimination. The fragments aggregate, developing into an insoluble plaque that has the ability to effect normal functioning by causing dysfunction. Without early identification and effective inhibition of this pathogenic pathway, the disease is anticipated to become more widespread with an aging population, in which AD is most prevalent.

Attempts to inhibit BACE1 have been relatively fruitless with most therapeutic trials being aborted in the early stages. A number of obstacles can be detrimental to the ability of an in‐ hibitor like solubility, bioavailability, potency and effectiveness. In addition to the complexi‐ ty, there are also a number of substrates cleaved by BACE1 which are important in other pathways like voltage gated sodium channels and axon myelination [1, 2]. This can create adverse reactions beyond the reduction of plaques.

Inhibitors of BACE1 have, so far, looked at active-site mimics that diverge from small and large molecules to short peptidic structures and expression modulators. Other approaches look to utilize technology with modelling software to determine the BACE1 3D structure and formulate an effective inhibitor via domain analysis. The underlying issue is translat‐

ing an analytically based, site-directed mimic into a potential inhibitor with pharmaceuti‐ cal integrity. This process is generally thwarted by the blood brain barrier (BBB), a specialized endothelium that separates systemic blood flow from the central nervous sys‐ tem (CNS). The aim is to fabricate an inhibitor that can pass through the BBB whilst main‐ taining structure and function.

lular atrophy, which includes degeneration in the temporal and parietal lobes, parts of the frontal cortex and cingulate gyrus [9, 10]. Progression of brain atrophy to well-defined brain structures results in symptoms of delusions, hallucinations, agitation, depression, anxiety, elation, apathy, disinhibition, irritability, aberrant motor behaviour and sleep disorders [11]. As the chronic nature of the disease progresses, the number of symptoms increases, becom‐ ing severely debilitating. Eventually, death results after long-term stress and reduction of

Dropping the BACE: Beta Secretase (BACE1) as an Alzheimer's Disease Intervention Target

http://dx.doi.org/10.5772/53603

229

Currently there are a variety of different intervention strategies being investigated to reme‐ dy these debilitating pathologies and focus to reduce the cause of neuronal cell death. Up‐ wards of 100 irregular protein changes occur in a brain with AD, including hyperexpression, fragmentation and phosphorylation [12]. A number of these irregular protein modifications could be a result of degenerating neurons causing further damage to associated structures of the central nervous system [12]. The overall progression of AD is however, not restricted to the dysfunction of one mechanism. The number of different symptoms result from a con‐ glomerate of sporadic cellular events that, as a consequence, stimulate the disease. The trig‐ ger or initial changes are not entirely clear and do not occur simultaneously which adds to

Histopathological review of AD patients highlighted amyloid plaques and neurofibrillary tangles (NFT) as the two significant characteristics of cerebral regression identifying them as targets for the prevention of neuronal cell death [4, 13]. Investigation into the onset of NFTs implicate the tau protein as the leading cause [14]. The tau protein becomes hyperphos‐ phorylated and releases from the intracellular microtubules decreasing structure and func‐ tion and causing axons to become dishevelled and dedifferentiated. These subsequent microtubules bind together to become an insoluble fibre decreasing the ability of neurons to transmit action potentials along the axon and neurotransmitters across the synapse [15]. Al‐

Amyloid plaque formation is characterized by an accumulation of amyloid-β (Aβ) sub-units on the cell membrane of neurons that, as a result, cause a decrease in cellular function [16]. The progressive damage of amyloid plaques cause neuronal apoptosis by activating the complement cascade and stimulating the membrane attack complex [17]. In developed cases of AD, where there is a high concentration of Aβ, significant damage to the physical struc‐ ture of the brain can be a result of the immune response. The activation of the complement cascade involves microglia and astrocytes, which have both protective and destructive at‐ tributes. The eventual outcome is apoptosis because of the overwhelming immune response, which cannot be hindered by anti-inflammatory drugs [18]. The aggregations of Aβ deposits are neurotoxic, insoluble and become vastly distributed around the brain as they increase in numbers [19]. AD needs to be proactively prevented before amyloid plaque formation gains

the ability to manipulate regular brain function and cause irreversible brain damage.

Closer investigation of the AD brain will encounter a plethora of imperative neuronal cell functions failures like synaptic failure [20], depletion of neurotransmitters [21], mitochondri‐ al dysfunction [22], decreased cholesterol metabolism [23], reduction of neurotrophin [24] and axonal transport deficiencies [25] which can be classified as associated effects of the dis‐

leviation from this facet of the disease is yet to be elucidated.

brain structure and function [9].

the ambiguity of the disease.

The importance of BACE1 and the influence it has on AD, has been investigated thoroughly since it was first identified in 1999. The enzyme is an intricate part of Aβ cleavage and pla‐ que formation instigating that inhibition could be the mechanism for relief in AD. In this chapter, we will summarize the structure and function of BACE1 and identify past attempts at its inhibition. The identification of important features regarding the protein-enzyme cleavage of APP will aid the understanding of the process and help theorize future perspec‐ tives of research.

### **2. Neurodegenerative disorders**

Neurodegenerative disorders cover a wide range of brain conditions relating to the dam‐ age or death of neuronal cells [3]. Clinical characterisation suggests a regression in struc‐ ture and function of the brain and central nervous system which is usually the final stage of a preceding period of neuronal dysfunction [4]. Specifically, dementia is of particular significance because of the devastating influence it bares on an ageing population and as the life expectancy of the general public increases, disease rates are predicted to escalate accordingly.

The burden of disease in relation to AD, the most predominant form of dementia, was calcu‐ lated to be 26.6 million cases worldwide in 2006 [5]. This figure implicated 34% of the popu‐ lation over the age of 65 and 45% over the age of 85. The World Health Organization revised this figure in 2010 to incorporate dementia as a whole because of the difficulty to diagnose the varieties of neurodegenerative disorders [6]. The worldwide incidence was tallied at ap‐ proximately 35.6 million with an estimated 7.7 million new cases annually instigating a new case arising every 4 seconds. The significance of this in regards to AD is that it covers 60-70% of dementia cases [6]. These figures could be inflated further because most cases go undiagnosed due to the requirement of post-mortem autopsy for confirmation.

The ripple effect of dementia extends far from those affected and into patient support net‐ works. The worldwide costs are estimated to be US\$604 billion annually and as the number of affected increases this number is expected to follow suit [6]. The chronic onset of the dis‐ ease indicates an eventual requirement for long term care. The financial burdens relating to carers, as the disease evolves and symptoms increase with severity, the eventual require‐ ment for long term formal care is inevitable [7]. AD patients specifically require increased supervision after diagnosis because of the increased risk of developing associated diseases like cardiovascular disease, diabetes, and bone weakening [8].

AD is largely defined by chronic symptoms of progressive neuronal and synaptic apoptosis in the cerebral cortex and subcortical regions of the brain [4]. The direct consequence is cel‐ lular atrophy, which includes degeneration in the temporal and parietal lobes, parts of the frontal cortex and cingulate gyrus [9, 10]. Progression of brain atrophy to well-defined brain structures results in symptoms of delusions, hallucinations, agitation, depression, anxiety, elation, apathy, disinhibition, irritability, aberrant motor behaviour and sleep disorders [11]. As the chronic nature of the disease progresses, the number of symptoms increases, becom‐ ing severely debilitating. Eventually, death results after long-term stress and reduction of brain structure and function [9].

ing an analytically based, site-directed mimic into a potential inhibitor with pharmaceuti‐ cal integrity. This process is generally thwarted by the blood brain barrier (BBB), a specialized endothelium that separates systemic blood flow from the central nervous sys‐ tem (CNS). The aim is to fabricate an inhibitor that can pass through the BBB whilst main‐

The importance of BACE1 and the influence it has on AD, has been investigated thoroughly since it was first identified in 1999. The enzyme is an intricate part of Aβ cleavage and pla‐ que formation instigating that inhibition could be the mechanism for relief in AD. In this chapter, we will summarize the structure and function of BACE1 and identify past attempts at its inhibition. The identification of important features regarding the protein-enzyme cleavage of APP will aid the understanding of the process and help theorize future perspec‐

Neurodegenerative disorders cover a wide range of brain conditions relating to the dam‐ age or death of neuronal cells [3]. Clinical characterisation suggests a regression in struc‐ ture and function of the brain and central nervous system which is usually the final stage of a preceding period of neuronal dysfunction [4]. Specifically, dementia is of particular significance because of the devastating influence it bares on an ageing population and as the life expectancy of the general public increases, disease rates are predicted to escalate

The burden of disease in relation to AD, the most predominant form of dementia, was calcu‐ lated to be 26.6 million cases worldwide in 2006 [5]. This figure implicated 34% of the popu‐ lation over the age of 65 and 45% over the age of 85. The World Health Organization revised this figure in 2010 to incorporate dementia as a whole because of the difficulty to diagnose the varieties of neurodegenerative disorders [6]. The worldwide incidence was tallied at ap‐ proximately 35.6 million with an estimated 7.7 million new cases annually instigating a new case arising every 4 seconds. The significance of this in regards to AD is that it covers 60-70% of dementia cases [6]. These figures could be inflated further because most cases go

The ripple effect of dementia extends far from those affected and into patient support net‐ works. The worldwide costs are estimated to be US\$604 billion annually and as the number of affected increases this number is expected to follow suit [6]. The chronic onset of the dis‐ ease indicates an eventual requirement for long term care. The financial burdens relating to carers, as the disease evolves and symptoms increase with severity, the eventual require‐ ment for long term formal care is inevitable [7]. AD patients specifically require increased supervision after diagnosis because of the increased risk of developing associated diseases

AD is largely defined by chronic symptoms of progressive neuronal and synaptic apoptosis in the cerebral cortex and subcortical regions of the brain [4]. The direct consequence is cel‐

undiagnosed due to the requirement of post-mortem autopsy for confirmation.

like cardiovascular disease, diabetes, and bone weakening [8].

taining structure and function.

**2. Neurodegenerative disorders**

tives of research.

228 Neurodegenerative Diseases

accordingly.

Currently there are a variety of different intervention strategies being investigated to reme‐ dy these debilitating pathologies and focus to reduce the cause of neuronal cell death. Up‐ wards of 100 irregular protein changes occur in a brain with AD, including hyperexpression, fragmentation and phosphorylation [12]. A number of these irregular protein modifications could be a result of degenerating neurons causing further damage to associated structures of the central nervous system [12]. The overall progression of AD is however, not restricted to the dysfunction of one mechanism. The number of different symptoms result from a con‐ glomerate of sporadic cellular events that, as a consequence, stimulate the disease. The trig‐ ger or initial changes are not entirely clear and do not occur simultaneously which adds to the ambiguity of the disease.

Histopathological review of AD patients highlighted amyloid plaques and neurofibrillary tangles (NFT) as the two significant characteristics of cerebral regression identifying them as targets for the prevention of neuronal cell death [4, 13]. Investigation into the onset of NFTs implicate the tau protein as the leading cause [14]. The tau protein becomes hyperphos‐ phorylated and releases from the intracellular microtubules decreasing structure and func‐ tion and causing axons to become dishevelled and dedifferentiated. These subsequent microtubules bind together to become an insoluble fibre decreasing the ability of neurons to transmit action potentials along the axon and neurotransmitters across the synapse [15]. Al‐ leviation from this facet of the disease is yet to be elucidated.

Amyloid plaque formation is characterized by an accumulation of amyloid-β (Aβ) sub-units on the cell membrane of neurons that, as a result, cause a decrease in cellular function [16]. The progressive damage of amyloid plaques cause neuronal apoptosis by activating the complement cascade and stimulating the membrane attack complex [17]. In developed cases of AD, where there is a high concentration of Aβ, significant damage to the physical struc‐ ture of the brain can be a result of the immune response. The activation of the complement cascade involves microglia and astrocytes, which have both protective and destructive at‐ tributes. The eventual outcome is apoptosis because of the overwhelming immune response, which cannot be hindered by anti-inflammatory drugs [18]. The aggregations of Aβ deposits are neurotoxic, insoluble and become vastly distributed around the brain as they increase in numbers [19]. AD needs to be proactively prevented before amyloid plaque formation gains the ability to manipulate regular brain function and cause irreversible brain damage.

Closer investigation of the AD brain will encounter a plethora of imperative neuronal cell functions failures like synaptic failure [20], depletion of neurotransmitters [21], mitochondri‐ al dysfunction [22], decreased cholesterol metabolism [23], reduction of neurotrophin [24] and axonal transport deficiencies [25] which can be classified as associated effects of the dis‐ ease. These associated deficiencies become apparent with the onset of plaque formation, which precedes tau deconstruction [26]. The repercussions of tau mutations are a character‐ istic of frontotemporal dementia (FTD) and Parkinson's like defects rather than AD, which makes plaque formation the focus of this chapter [27].

and neuroprotective properties as well as promoting neurite expansion, synapse production and cell adhesion, while AICD has a role in p53 expression and caspase 3 activation, both associated with cell death, and in maintaining cellular actin dynamics [36-38]. This indicates that the non-amyloidogenic cleavage of APP is imperative to the maintenance of neuronal

Dropping the BACE: Beta Secretase (BACE1) as an Alzheimer's Disease Intervention Target

http://dx.doi.org/10.5772/53603

231

**Figure 1.** The cleavage of APP, depending whether by BACE1 or α-secretase, results in the production of an amyloid plaque. The non-amyloidogenic pathway (non-pathogenic) begins with α-secretase, which releases sAPPα externally from the endosome or cell membrane. The resulting C83 fragment is cleaved by γ-secretase in the intermembrane space releasing both the AICD and p3 fragments. The release of p3 into the extracellular space is not associated with plaque formation. Alternatively, the amyloidogenic (pathogenic) pathway begins with BACE1, instead of α-secretase, and cleaves APP in a lipid raft region of the membrane. The sAPPβ released is considerably shorter than sAPPα and is still released externally. The γ-secretase cleaves APP, the same as in the non-amyloidogenic pathway, but releases the 38-42 amino acid Aβ, and AICD. The externally released fragment, Aβ, has the capability to form an amyloid plaque by

The underlying difference between the two processes is the release of the Aβ and p3 frag‐ ments. The p3 is a shorter bi-product of the overall APP cleavage that has no known func‐ tion. However, the p3 fragment does not have the same ability to form stable oligomeric intermediates like that of Aβ, which poses no threat to synaptic function instigating that it is not the cause of amyloid plaques and is the reason for being the non-amyloidogenic path‐ way [39, 40]. This suggests that the longer Aβ peptide is the main neurotoxic fragment es‐ tablished from the APP fragment. The cleaved product becomes a peptide of 40 amino acid residues but this can be varied between 38 and 42 depending on where the γ-secretase

growth and function.

aggregation.

cleaves APP.

### **3. Amyloid plaque formation**

Amyloid plaques were originally purified in the early 80s and examined to contain pepti‐ des of approximately 40 amino acids that aggregated as oligomers, later to be generically named amyloid-beta (Aβ) [28]. Gene cloning and cDNA analysis of these monomers lead to the realization that the origins of this peptide remained part of a larger precursor pro‐ tein [29]. This protein was later identified as the 695 amino acid, membrane bound cell re‐ ceptor, amyloid precursor protein (APP), which contained the Aβ sequence in the extracellular domain [30]. It is the proteolytic cleavage, by that of multiple secretases, which releases the Aβ product.

Proteolytic processing of APP occurs by one of two pathways, the amyloidogenic (pathogen‐ ic) or the non-amyloidogenic (non-pathogenic). Transport to the membrane via endosomes is chaperoned by the intracellular adaptor protein, sorting nexin 17 (SNX17), where it be‐ comes available for processing by the secretases [31]. The determinant of amyloidogenesis depends on the initial proteolytic cleavage in the extracellular space by either α- or β- secre‐ tase to create a soluble or insoluble fragment [19]. Cleavage of APP by the α-secretase, a pro‐ tein investigated as part of the disintigrin and metalloprotease (ADAM) family, cleaves APP at the α-site releasing a soluble fragment sAPPα into the extracellular space [32]. This leaves the C-terminal fragment of 83 amino acid residues (C-83), which is abruptly cleaved by γsecretase in the intramembrane space. This creates two subsequent fragments of the APP in‐ tracellular domain (AICD), released in the cytosol, and p3 which is released into the extracellular space (Figure 1).

The amyloidogenic pathway also utilises APP as a target. This pathway is much like that of the non-amyloidogenic pathway but instead α-secretase is substituted for β-secretase or beta site APP converting enzyme 1 (BACE1). This enzyme also cleaves APP in the extracellular space at the β-site, which is 18 amino acids towards the *N*-terminal, releasing a much shorter soluble APP-β (sAPPβ) fragment [33, 34]. The remaining C-terminal fragment of 99 amino acid residues (C-99) remains membrane bound until cleaved by γ-secretase. This releases two fragments of AICD and Aβ (Figure 1).

The non-amyloidogenic pathway has the ability to nullify the amyloidogenic pathway by simply having α-secretase cleave APP prior to that of BACE1. Since the α-site of APP is situ‐ ated between that of β- and γ-, the shorter p3 fragment is produced instead of Aβ. The ques‐ tion over competition for the cleavage of APP is still debated but there is evidence suggesting that α-secretase nullification does not increase BACE1 activity *in vitro* [35]. The subsequent fragments of sAPPα and AICD from the non-amyloidogenic pathway suggest a purpose from the subsequent cleavage of APP. The sAPPα has shown to have neurotrophic and neuroprotective properties as well as promoting neurite expansion, synapse production and cell adhesion, while AICD has a role in p53 expression and caspase 3 activation, both associated with cell death, and in maintaining cellular actin dynamics [36-38]. This indicates that the non-amyloidogenic cleavage of APP is imperative to the maintenance of neuronal growth and function.

ease. These associated deficiencies become apparent with the onset of plaque formation, which precedes tau deconstruction [26]. The repercussions of tau mutations are a character‐ istic of frontotemporal dementia (FTD) and Parkinson's like defects rather than AD, which

Amyloid plaques were originally purified in the early 80s and examined to contain pepti‐ des of approximately 40 amino acids that aggregated as oligomers, later to be generically named amyloid-beta (Aβ) [28]. Gene cloning and cDNA analysis of these monomers lead to the realization that the origins of this peptide remained part of a larger precursor pro‐ tein [29]. This protein was later identified as the 695 amino acid, membrane bound cell re‐ ceptor, amyloid precursor protein (APP), which contained the Aβ sequence in the extracellular domain [30]. It is the proteolytic cleavage, by that of multiple secretases,

Proteolytic processing of APP occurs by one of two pathways, the amyloidogenic (pathogen‐ ic) or the non-amyloidogenic (non-pathogenic). Transport to the membrane via endosomes is chaperoned by the intracellular adaptor protein, sorting nexin 17 (SNX17), where it be‐ comes available for processing by the secretases [31]. The determinant of amyloidogenesis depends on the initial proteolytic cleavage in the extracellular space by either α- or β- secre‐ tase to create a soluble or insoluble fragment [19]. Cleavage of APP by the α-secretase, a pro‐ tein investigated as part of the disintigrin and metalloprotease (ADAM) family, cleaves APP at the α-site releasing a soluble fragment sAPPα into the extracellular space [32]. This leaves the C-terminal fragment of 83 amino acid residues (C-83), which is abruptly cleaved by γsecretase in the intramembrane space. This creates two subsequent fragments of the APP in‐ tracellular domain (AICD), released in the cytosol, and p3 which is released into the

The amyloidogenic pathway also utilises APP as a target. This pathway is much like that of the non-amyloidogenic pathway but instead α-secretase is substituted for β-secretase or beta site APP converting enzyme 1 (BACE1). This enzyme also cleaves APP in the extracellular space at the β-site, which is 18 amino acids towards the *N*-terminal, releasing a much shorter soluble APP-β (sAPPβ) fragment [33, 34]. The remaining C-terminal fragment of 99 amino acid residues (C-99) remains membrane bound until cleaved by γ-secretase. This releases

The non-amyloidogenic pathway has the ability to nullify the amyloidogenic pathway by simply having α-secretase cleave APP prior to that of BACE1. Since the α-site of APP is situ‐ ated between that of β- and γ-, the shorter p3 fragment is produced instead of Aβ. The ques‐ tion over competition for the cleavage of APP is still debated but there is evidence suggesting that α-secretase nullification does not increase BACE1 activity *in vitro* [35]. The subsequent fragments of sAPPα and AICD from the non-amyloidogenic pathway suggest a purpose from the subsequent cleavage of APP. The sAPPα has shown to have neurotrophic

makes plaque formation the focus of this chapter [27].

**3. Amyloid plaque formation**

230 Neurodegenerative Diseases

which releases the Aβ product.

extracellular space (Figure 1).

two fragments of AICD and Aβ (Figure 1).

**Figure 1.** The cleavage of APP, depending whether by BACE1 or α-secretase, results in the production of an amyloid plaque. The non-amyloidogenic pathway (non-pathogenic) begins with α-secretase, which releases sAPPα externally from the endosome or cell membrane. The resulting C83 fragment is cleaved by γ-secretase in the intermembrane space releasing both the AICD and p3 fragments. The release of p3 into the extracellular space is not associated with plaque formation. Alternatively, the amyloidogenic (pathogenic) pathway begins with BACE1, instead of α-secretase, and cleaves APP in a lipid raft region of the membrane. The sAPPβ released is considerably shorter than sAPPα and is still released externally. The γ-secretase cleaves APP, the same as in the non-amyloidogenic pathway, but releases the 38-42 amino acid Aβ, and AICD. The externally released fragment, Aβ, has the capability to form an amyloid plaque by aggregation.

The underlying difference between the two processes is the release of the Aβ and p3 frag‐ ments. The p3 is a shorter bi-product of the overall APP cleavage that has no known func‐ tion. However, the p3 fragment does not have the same ability to form stable oligomeric intermediates like that of Aβ, which poses no threat to synaptic function instigating that it is not the cause of amyloid plaques and is the reason for being the non-amyloidogenic path‐ way [39, 40]. This suggests that the longer Aβ peptide is the main neurotoxic fragment es‐ tablished from the APP fragment. The cleaved product becomes a peptide of 40 amino acid residues but this can be varied between 38 and 42 depending on where the γ-secretase cleaves APP.

#### **3.1. Amyloid-β**

The γ-secretase complex is composed of four proteins: either one of presenilin 1 (PS1) or presenilin 2 (PS2), nicastrin (NCT), anterior pharynx-defective phenotype 1 (Aph-1) and presenilin enhancer 2 (Pen-2) [41]. The complex formation is initiated with a sub complex that forms between NCT and Aph-1 which then binds to one of the PS proteins [42]. Pen-2 is used to activate the complex by selective binding [43]. Either PS can be used in this process, with a 67% amino acid homology, including two separate aspartate residues, asp257 and asp385, considered essential to catalytic ability, both have the ability to cleave APP [44, 45]. Consequently, the decrease in availability of the γ-secretase components re‐ sults in a reduction of overall functionality, which can be targeted for use when consider‐ ing inhibitors and the vulnerabilities of this complex [46]. Mutation to either the presenilins, whether it be PS1 or PS2, can be seen as a potential threat to the production of Aβ40-42, as one can compensate for the other.

dysfunction of the Notch signalling pathway because of its involvement with cell prolifer‐ ation, apoptosis and myelin formation, not to mention it is a substrate of γ-secretase [61]. This indicates that there are a number of associated risks with nullifying γ-secretase that with any inhibition, the complex will invariably prevent the cleavage of two regulatory

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233

The increase of intra and extracellular Aβ has a direct effect on the complement system and the recruitment of microglia, instigating the activation of inflammatory mechanisms, a trademark of stress on an Alzheimer's brain [62]. The microglia, derived from the mes‐ enchyme and transferred to the CNS where proliferation occurs, are classified as the mac‐ rophages of the brain and regulate apoptotic cell abundance [63]. Stress incurred, mainly by amyloid plaque formation, triggers an immune response which leads to the activation of astrocytes and microglia to dismantle the ailing cell. The Aβ fragment has the ability to activate this process via the receptor for advanced glycation end products (RAGE), a mul‐ ti-ligand member of the immunoglobulin family that is increased in production in an AD brain, and by CD40, an inflammatory signalling receptor [64-66]. The general immune re‐ sponse is aimed at eliminating Aβ, removing disease ridden cells and restoring tissue in‐ tegrity but does more damage when it becomes of a chronic nature. The activation of reactive oxygen species (ROS), prostaglandins, and pro-inflammatory cytokines are a char‐

acteristic of the chronic inflammation and neuronal dysfunction in AD [67].

ways that can be involved in the pathogenesis of amyloid plaques.

The onset of AD encourages the migration of microglia to the plaque affected areas. Cul‐ tured microglia, from elderly human patients, showed this migration as they coupled with Aβ for the purpose of deconstruction [68]. Similar findings were discovered in sections of cortex tested in vitro for migration but growing evidence suggests that once endocytosed, the microglia struggle to breakdown the Aβ, causing stress and functional changes to the cell [68-70]. Additionally, the APP mRNA translation is upregulated in the event of trauma, nerve damage and brain ischemia, which can be beneficial for the release of more AICD, but can also result in the production of more Aβ [71-73]. BACE1 has also been found to be regu‐ lated by Aβ42 via the c-Jun N-terminal kinase (JNK) pathway, otherwise known as the stress-activated signalling pathway which is important for the mediation of pro-inflammato‐ ry cytokines [74, 75]. This reiterates the important influence that a variety of stressors have when responding to the amyloidogenic pathway and that there are many different path‐

All aspects of the amyloid plaque progression relates back to the effectiveness of the BACE1 enzyme. Taking into consideration alternative BACE1 substrates, the proteolytic cleavage of APP can be regulated with an inhibitor. The Aβ concentration increases as the disease gets worse but could be diverted with the use of an effective pharmaceutical intervention. The possibilities and implications will not be recognised until BACE1 is inhibited successfully *in*

pathways.

*vivo*.

**3.2. Neuroinflammation**

The γ-secretase, being the consistent piece of the APP cleavage puzzle, is actually respon‐ sible for the diseased state in which plaques are formed. There are upwards of 100 mis‐ sense mutations identified in the presenilins, mostly creating the difference in cleaved fragments of APP [47]. The Aβ peptide created will be influenced with a varying overall length of between 38-42 amino acids [19]. The majority of these mutations influence a higher number of Aβ42 peptides, which have been found to be more amyloidogenic and neurotoxic [48, 49]. The wild-type presenilin generates Aβ40, which is considered to be less neurotoxic even though it is present in amyloid plaques. γ-secretase cuts down‐ stream from the transmembrane domain into the ε-site of APP and therefore slightly shortens the AICD cleaving domain [50]. Aβ42 provides the basis for oligomerisation, fi‐ brillation and plaque generation, even with Aβ40 being found with a limited ability to protect neurons in mouse models [51]. The ratio of Aβ42/Aβ40 increases after the AD age of onset but is still relatively low. However, with a binding affinity to Aβ40, fibrils can, as a consequence, bind together, meaning there does not need to be a high concentration to be effective [52].

The Aβ fragments are not always cleaved to the extracellular space either. There are a num‐ ber of conformations that Aβ fragments can take including monomers, which are found free in neurons as a consequence of APP cleavage. Studies have also shown that fragments can form the shape of α-helices, random coils and even as β-sheets in a neutual pH to add to the complexity [53-56]. These individual fragments maintain the ability to block synaptic neuro‐ transmitter transfer and instigate an apoptotic response via activation of the p53 promoter leading to cell death [57]. Alternatively, soluble Aβ oligomers have been referred to as Aβderived diffusible ligands (ADDLs) that have the ability to aggregate into protofibrils, spherical structures of 7-10 nm wide, that can have the ability to interrupt nerve signal transduction leading to cell death [58]. Initial investigations into the Aβ fibrils showed there was a toxic response from the neurons in which they were attached [59].

To target the Aβ fibrils, gene knockout studies of PS1 have found adverse reactions relat‐ ing to formation of the axial skeleton, neurogenesis and neuronal survival causing effect‐ ed mice to die late in embryogenesis [60]. These reactions however, can be attributed to a dysfunction of the Notch signalling pathway because of its involvement with cell prolifer‐ ation, apoptosis and myelin formation, not to mention it is a substrate of γ-secretase [61]. This indicates that there are a number of associated risks with nullifying γ-secretase that with any inhibition, the complex will invariably prevent the cleavage of two regulatory pathways.

#### **3.2. Neuroinflammation**

**3.1. Amyloid-β**

232 Neurodegenerative Diseases

be effective [52].

of Aβ40-42, as one can compensate for the other.

The γ-secretase complex is composed of four proteins: either one of presenilin 1 (PS1) or presenilin 2 (PS2), nicastrin (NCT), anterior pharynx-defective phenotype 1 (Aph-1) and presenilin enhancer 2 (Pen-2) [41]. The complex formation is initiated with a sub complex that forms between NCT and Aph-1 which then binds to one of the PS proteins [42]. Pen-2 is used to activate the complex by selective binding [43]. Either PS can be used in this process, with a 67% amino acid homology, including two separate aspartate residues, asp257 and asp385, considered essential to catalytic ability, both have the ability to cleave APP [44, 45]. Consequently, the decrease in availability of the γ-secretase components re‐ sults in a reduction of overall functionality, which can be targeted for use when consider‐ ing inhibitors and the vulnerabilities of this complex [46]. Mutation to either the presenilins, whether it be PS1 or PS2, can be seen as a potential threat to the production

The γ-secretase, being the consistent piece of the APP cleavage puzzle, is actually respon‐ sible for the diseased state in which plaques are formed. There are upwards of 100 mis‐ sense mutations identified in the presenilins, mostly creating the difference in cleaved fragments of APP [47]. The Aβ peptide created will be influenced with a varying overall length of between 38-42 amino acids [19]. The majority of these mutations influence a higher number of Aβ42 peptides, which have been found to be more amyloidogenic and neurotoxic [48, 49]. The wild-type presenilin generates Aβ40, which is considered to be less neurotoxic even though it is present in amyloid plaques. γ-secretase cuts down‐ stream from the transmembrane domain into the ε-site of APP and therefore slightly shortens the AICD cleaving domain [50]. Aβ42 provides the basis for oligomerisation, fi‐ brillation and plaque generation, even with Aβ40 being found with a limited ability to protect neurons in mouse models [51]. The ratio of Aβ42/Aβ40 increases after the AD age of onset but is still relatively low. However, with a binding affinity to Aβ40, fibrils can, as a consequence, bind together, meaning there does not need to be a high concentration to

The Aβ fragments are not always cleaved to the extracellular space either. There are a num‐ ber of conformations that Aβ fragments can take including monomers, which are found free in neurons as a consequence of APP cleavage. Studies have also shown that fragments can form the shape of α-helices, random coils and even as β-sheets in a neutual pH to add to the complexity [53-56]. These individual fragments maintain the ability to block synaptic neuro‐ transmitter transfer and instigate an apoptotic response via activation of the p53 promoter leading to cell death [57]. Alternatively, soluble Aβ oligomers have been referred to as Aβderived diffusible ligands (ADDLs) that have the ability to aggregate into protofibrils, spherical structures of 7-10 nm wide, that can have the ability to interrupt nerve signal transduction leading to cell death [58]. Initial investigations into the Aβ fibrils showed there

To target the Aβ fibrils, gene knockout studies of PS1 have found adverse reactions relat‐ ing to formation of the axial skeleton, neurogenesis and neuronal survival causing effect‐ ed mice to die late in embryogenesis [60]. These reactions however, can be attributed to a

was a toxic response from the neurons in which they were attached [59].

The increase of intra and extracellular Aβ has a direct effect on the complement system and the recruitment of microglia, instigating the activation of inflammatory mechanisms, a trademark of stress on an Alzheimer's brain [62]. The microglia, derived from the mes‐ enchyme and transferred to the CNS where proliferation occurs, are classified as the mac‐ rophages of the brain and regulate apoptotic cell abundance [63]. Stress incurred, mainly by amyloid plaque formation, triggers an immune response which leads to the activation of astrocytes and microglia to dismantle the ailing cell. The Aβ fragment has the ability to activate this process via the receptor for advanced glycation end products (RAGE), a mul‐ ti-ligand member of the immunoglobulin family that is increased in production in an AD brain, and by CD40, an inflammatory signalling receptor [64-66]. The general immune re‐ sponse is aimed at eliminating Aβ, removing disease ridden cells and restoring tissue in‐ tegrity but does more damage when it becomes of a chronic nature. The activation of reactive oxygen species (ROS), prostaglandins, and pro-inflammatory cytokines are a char‐ acteristic of the chronic inflammation and neuronal dysfunction in AD [67].

The onset of AD encourages the migration of microglia to the plaque affected areas. Cul‐ tured microglia, from elderly human patients, showed this migration as they coupled with Aβ for the purpose of deconstruction [68]. Similar findings were discovered in sections of cortex tested in vitro for migration but growing evidence suggests that once endocytosed, the microglia struggle to breakdown the Aβ, causing stress and functional changes to the cell [68-70]. Additionally, the APP mRNA translation is upregulated in the event of trauma, nerve damage and brain ischemia, which can be beneficial for the release of more AICD, but can also result in the production of more Aβ [71-73]. BACE1 has also been found to be regu‐ lated by Aβ42 via the c-Jun N-terminal kinase (JNK) pathway, otherwise known as the stress-activated signalling pathway which is important for the mediation of pro-inflammato‐ ry cytokines [74, 75]. This reiterates the important influence that a variety of stressors have when responding to the amyloidogenic pathway and that there are many different path‐ ways that can be involved in the pathogenesis of amyloid plaques.

All aspects of the amyloid plaque progression relates back to the effectiveness of the BACE1 enzyme. Taking into consideration alternative BACE1 substrates, the proteolytic cleavage of APP can be regulated with an inhibitor. The Aβ concentration increases as the disease gets worse but could be diverted with the use of an effective pharmaceutical intervention. The possibilities and implications will not be recognised until BACE1 is inhibited successfully *in vivo*.

### **4. BACE1**

#### **4.1. BACE1 production and natural degradation**

The BACE1 enzyme (also called β-secretase, Asp2 or memapsin2) is developed in the endo‐ plasmic reticulum from a 501 amino acid, 60 kDa, immature precursor protein. The transfer from the endoplasmic reticulum to the Golgi is dependent on the prodomain, which is re‐ moved by the proprotein convertase, furin [76]. At this stage the immature BACE1 can al‐ ready cleave APP so is not a true zymogen but the removal of the prodomain can double the proteolytic effectiveness whilst increasing structural stability [77, 78]. The immature BACE1 is deacetylated and transformed in the golgi by post-translational modification into a type 1 transmembrane protein [79]. A bi-lobal structure is formed with two aspartate motifs (D93TG and D289SG in a D-T/S-G-T/S conformation), which forms the active site that stimu‐ lates water molecules to hydrolyse APP peptide bonds, a defining characteristic of aspartic proteases [80, 81]. The proteolytic ability of this motif is only limited by site-directed muta‐ genesis of the aspartate residues [81]. The active site remains an important structure of this enzyme because of its supposed vulnerability. However, although it has been the focus of inhibition studies since its highly contested discovery in 1999, no effective pharmaceutical options have been elucidated since [34, 82-85]. The distinctiveness surrounding BACE1 is ex‐ posed when comparing it to other aspartic proteases and the contrasting characteristics of a C-terminal cytosolic tail and a transmembrane domain.

BACE1 is found in a number of tissues throughout the body but majority of expression is found in the brain [91]. The levels of this expression have been of much debate due to con‐ flicting reports separating different tissues in which the samples were taken, animal or hu‐ man models and the use of controls [92]. The increase in expression could then be argued as a reason for the uncontrollable aggregation of Aβ but reports of little or no increase in ex‐

Dropping the BACE: Beta Secretase (BACE1) as an Alzheimer's Disease Intervention Target

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235

The natural degradation of BACE1 has been found to be involved the ubiquitin proteasome pathway. BACE1 can be transported from endosomes to lysosomes by ubiquitination with help from the sorting proteins, ADP ribosylation factor 6 (ARF6) and GGA3, another mem‐ ber of the GGA family [94-97]. The degradation by lysosomes is still ambiguous with an in‐ crease in BACE1 protein available in an AD brain, whilst there is little to no increase in mRNA levels. The failed or impaired lysosome could be a contributing factor to the increase in cellular Aβ or BACE1 could be getting recycled back into endosomes [93] (Figure 2D).

**Figure 2.** BACE1 Trafficking. BACE1 is constructed in the ER and processed, with the removal of the proBACE1 domain, in the Golgi. A) BACE1 is a membrane bound protease and so is either exocytosed from the Golgi or transported to an endosome with the help of the sorting protein GGA1. From here, the cleavage of APP to produce extracellular Aβ takes place in the lipid raft region of the membrane. The synthesis of Aβ occurs through the amyloidogenic (patho‐ genic) pathway. B) Processing of APP can otherwise occur whilst present in the endosome and so can release Aβ into the cytosoplasm. C) The Aβ can then either aggregate together or be endocytosed by lysosomes and degraded. D) Once APP proteolysis has occurred, BACE1 is either internalized to endosomes via GGA1 and ARF6 or labelled with ubiquitin and endocytosed. With assistance from the sorting protein, GGA3, the lysosomes will either recycle or de‐

grade BACE1.

pression of BACE1 instigates the influence of another factor is involved [93].

The transfer from the Golgi to the cell membrane is mediated by transport vesicles because BACE1 is membrane bound. The serine and di-leucine residues of BACE1 act as a signal for the Golgi-localized γ-ear-containing ARF binding protein 1 (GGA1), a sorting protein that aids the link between the transporting endosome and the cell membrane [86]. The endosome provides an optimal transport vesicle because of its internal acidic nature (approximately pH 4.5), a fundamental for the conformational shape and functionality of BACE1, but also because it is much more stable environment for transporting proteins [87, 88]. Changes in pH below 4.0 can have a negative effect on Wat1, a molecule considered to be the nucleo‐ phile with the ability to attack the carbonyl carbon of a peptide bond in the active site result‐ ing in the loss of functionality. Changes at the other end of the scale, above that of pH 7.0, render the enzyme inactive and unable to cleave a substrate [87] (Figure 2A,B,C).

The active site placement is also vulnerable as it remains exposed to the extracellular space once it makes the transition to the lipid bi-layer of the membrane where it concludes its in‐ tracellular transport. BACE1 becomes susceptible to post-translational modification, protein to protein interactions or even inhibitor attachment because of its availability. It is here, however, that the BACE1 enzyme comes into contact with the membrane bound APP [89]. In some instances, the help of increased cellular cholesterol producing lipid rafts helps im‐ prove the availability of APP for BACE1. So what is considered a negative of vulnerability of the enzyme actually serves an important purpose in that a specific environment is created for APP to become more available. The lipid rafts are formed as an essential membrane sta‐ bilizer of intermediate space but have also controversially, improved signal transduction and intracellular trafficking ability of the cell [90].

BACE1 is found in a number of tissues throughout the body but majority of expression is found in the brain [91]. The levels of this expression have been of much debate due to con‐ flicting reports separating different tissues in which the samples were taken, animal or hu‐ man models and the use of controls [92]. The increase in expression could then be argued as a reason for the uncontrollable aggregation of Aβ but reports of little or no increase in ex‐ pression of BACE1 instigates the influence of another factor is involved [93].

**4. BACE1**

234 Neurodegenerative Diseases

**4.1. BACE1 production and natural degradation**

C-terminal cytosolic tail and a transmembrane domain.

and intracellular trafficking ability of the cell [90].

The BACE1 enzyme (also called β-secretase, Asp2 or memapsin2) is developed in the endo‐ plasmic reticulum from a 501 amino acid, 60 kDa, immature precursor protein. The transfer from the endoplasmic reticulum to the Golgi is dependent on the prodomain, which is re‐ moved by the proprotein convertase, furin [76]. At this stage the immature BACE1 can al‐ ready cleave APP so is not a true zymogen but the removal of the prodomain can double the proteolytic effectiveness whilst increasing structural stability [77, 78]. The immature BACE1 is deacetylated and transformed in the golgi by post-translational modification into a type 1 transmembrane protein [79]. A bi-lobal structure is formed with two aspartate motifs (D93TG and D289SG in a D-T/S-G-T/S conformation), which forms the active site that stimu‐ lates water molecules to hydrolyse APP peptide bonds, a defining characteristic of aspartic proteases [80, 81]. The proteolytic ability of this motif is only limited by site-directed muta‐ genesis of the aspartate residues [81]. The active site remains an important structure of this enzyme because of its supposed vulnerability. However, although it has been the focus of inhibition studies since its highly contested discovery in 1999, no effective pharmaceutical options have been elucidated since [34, 82-85]. The distinctiveness surrounding BACE1 is ex‐ posed when comparing it to other aspartic proteases and the contrasting characteristics of a

The transfer from the Golgi to the cell membrane is mediated by transport vesicles because BACE1 is membrane bound. The serine and di-leucine residues of BACE1 act as a signal for the Golgi-localized γ-ear-containing ARF binding protein 1 (GGA1), a sorting protein that aids the link between the transporting endosome and the cell membrane [86]. The endosome provides an optimal transport vesicle because of its internal acidic nature (approximately pH 4.5), a fundamental for the conformational shape and functionality of BACE1, but also because it is much more stable environment for transporting proteins [87, 88]. Changes in pH below 4.0 can have a negative effect on Wat1, a molecule considered to be the nucleo‐ phile with the ability to attack the carbonyl carbon of a peptide bond in the active site result‐ ing in the loss of functionality. Changes at the other end of the scale, above that of pH 7.0,

render the enzyme inactive and unable to cleave a substrate [87] (Figure 2A,B,C).

The active site placement is also vulnerable as it remains exposed to the extracellular space once it makes the transition to the lipid bi-layer of the membrane where it concludes its in‐ tracellular transport. BACE1 becomes susceptible to post-translational modification, protein to protein interactions or even inhibitor attachment because of its availability. It is here, however, that the BACE1 enzyme comes into contact with the membrane bound APP [89]. In some instances, the help of increased cellular cholesterol producing lipid rafts helps im‐ prove the availability of APP for BACE1. So what is considered a negative of vulnerability of the enzyme actually serves an important purpose in that a specific environment is created for APP to become more available. The lipid rafts are formed as an essential membrane sta‐ bilizer of intermediate space but have also controversially, improved signal transduction

The natural degradation of BACE1 has been found to be involved the ubiquitin proteasome pathway. BACE1 can be transported from endosomes to lysosomes by ubiquitination with help from the sorting proteins, ADP ribosylation factor 6 (ARF6) and GGA3, another mem‐ ber of the GGA family [94-97]. The degradation by lysosomes is still ambiguous with an in‐ crease in BACE1 protein available in an AD brain, whilst there is little to no increase in mRNA levels. The failed or impaired lysosome could be a contributing factor to the increase in cellular Aβ or BACE1 could be getting recycled back into endosomes [93] (Figure 2D).

**Figure 2.** BACE1 Trafficking. BACE1 is constructed in the ER and processed, with the removal of the proBACE1 domain, in the Golgi. A) BACE1 is a membrane bound protease and so is either exocytosed from the Golgi or transported to an endosome with the help of the sorting protein GGA1. From here, the cleavage of APP to produce extracellular Aβ takes place in the lipid raft region of the membrane. The synthesis of Aβ occurs through the amyloidogenic (patho‐ genic) pathway. B) Processing of APP can otherwise occur whilst present in the endosome and so can release Aβ into the cytosoplasm. C) The Aβ can then either aggregate together or be endocytosed by lysosomes and degraded. D) Once APP proteolysis has occurred, BACE1 is either internalized to endosomes via GGA1 and ARF6 or labelled with ubiquitin and endocytosed. With assistance from the sorting protein, GGA3, the lysosomes will either recycle or de‐ grade BACE1.

Recently, a natural inhibition of BACE1 has been found via the sAPPa fragment produced from the non-amyloidogenic pathway [98]. The sAPPa, once produced from APP, has been found to implicate its own cleavage. The findings showed that sAPPa can bind to BACE1 and interfere with the APP cleavage in a mouse model. This resulted in an overall reduction in Aβ formation under physiological conditions. The consequence of this pathway being im‐ paired in any way, could result in a decrease in BACE1 production thus reducing the cleav‐ age of further units of APP. The sAPPa production or cellular concentration could be a therapeutic intervention strategy in future research.

yet to be characterised in vivo and subsequently cannot be verified as a substrate until this time. With the interaction between BACE1 and APP being widely of interest in the preven‐ tion of AD, the actual damage by completely inhibiting the enzyme from normal function‐ ing could prove detrimental in its own right. APP may not even be the primary substrate of the enzyme. Therefore, partial inhibition of BACE1 is all that may be required as an inter‐

Dropping the BACE: Beta Secretase (BACE1) as an Alzheimer's Disease Intervention Target

http://dx.doi.org/10.5772/53603

237

Alternatively, both of the amyloidogenic creating enzymes, BACE1 and γ-secretase, have alternative purposes relating to other substrates. Gene knockout studies in mouse models produced the reaction of hyperactivity, premature death and seizure like behaviours for BACE1 whilst presenilin -/- mice displayed early neurodegenerative behaviours and an in‐ crease of Aβ species which could possibly be due to the large number of substrates in which both are effective [108-111]. The γ-secretase complex is essential for the Notch sig‐ nalling pathway, which is important for the development of the nervous system and this would also be in deficit if inhibited completely [112]. Likewise, the γ-secretase complex is also required for the non-amyloidogenic pathway, suggesting that the complete inhibition or deletion of these proteins will be detrimental to the homeostatic processing related to

Inhibition of BACE1 has been the focus of many studies since it was discovered. The main interest in this enzyme remains with the obvious involvement in plaque formation but also to the positive intervention target for which it provides. Firstly, mouse models have shown that without BACE1, Aβ is no longer produced, which indicates that if a product can reduce the enzymatic ability of BACE1 then plaques will not be formed [109]. The enzyme has been characterised and 3-D structure produced, which will allow for inhibitor modelling [80]. BACE1 also has the ability to bind to a wide variety of peptidic structures, while specific binding is an attribute there is still access to the active site for potential targeting [113]. BACE1 is a part of the large aspartic protease family, which have had the success of at least two members (renin and HIV protease-1(HIV-1) inhibited with success [114]. This shows confidence that BACE1 is a viable option for inhibition and, if successful, could influence the

Drug intervention of AD is limited between therapeutic relief of symptoms and the preven‐ tion of the underlying etiology of the disease [115]. The treatment to minimise symptoms is the only viable option for sufferers of AD. This method slows the decline of cognition, func‐ tion and behaviour but only masks the underlying neurodegeneration taking place [116]. The drugs currently available to treat AD are divided into two categories of cholinesterase

Cholinesterase inhibitors attempt to prevent the metabolism of acetylcholine allowing the neurotransmitter to maintain effect in the neuron [117]. Cholinesterase inhibiting drugs like

vention therapy to normalize the enhanced BACE1 activity seen in AD patients.

normal development and function.

**5. Challenges**

production of Aβ.

inhibitors and receptor antagonists [115].

**5.1. Past BACE1 inhibitors**

#### **4.2. BACE2 and closely related proteins**

BACE2 is a closely associated β-secretase to BACE1. Sequence analysis show amino acid res‐ idue similarities of ~45% and structural comparisons of 75% homology [99]. BACE2 is also classified as an aspartic protease with the ability to cleave APP at the β-site [100]. In vitro studies have also implicated BACE2 as a possible competitor of BACE1 for the cleavage of the APP protein but it is not formally expressed in excess in the brain and does not compen‐ sate for the loss of BACE1 in gene knockout models in mice [89, 101]. In fact, over expression of BACE2 has actually been found to reduce Aβ production in primary neuronal cultures derived from APP transgenic mice. This could be caused by the ability of BACE2 to cleave APP before BACE1 or because BACE2 cleaves a longer 79 residue Aβ fragment from APP, which is closer to the cleaved fragment of α-secretase then that of BACE1 [89, 99].

Apart from the sequence homology, BACE2 is not primarily expressed in the brain. It is more commonly expressed in the colon, kidney and pancreas, showing that whilst theo‐ retically still having the ability to increase the pathogenesis of AD it is not considered a threat to Aβ generation [100, 102]. BACE1 is the main amyloidogenic enzyme and still re‐ mains the major contributor to amyloid plaque formation but does show some sequence homology with a number other aspartic proteases including renin and cathepsin D. These homologs are generally used for selectivity testing with *in vitro* assays to confirm no un‐ wanted binding occurs.

#### **4.3. Substrates of BACE1**

The BACE1 protein is not solely defined to the cleavage of APP and has the ability to cleave other proteins like amyloid like precursor proteins 1 and 2, APPε (which is another closely related *N*-terminal product of APP), neuregulin-1 and -3 involved in axon myelination, βsubunits of voltage gated sodium channels required for neuronal action potentials, P-selec‐ tin glycoprotein ligand-1 (PSGL-1), which regulates leukocyte adhesion in the inflammatory process, the interleukin 1 receptor type-II (IL1-R2) and the low density lipoprotein receptorrelated protein 1(LLRP1), which is a multifunctional endocytic and signalling receptor [1, 2, 103-106]. With the use of an unbiased, quantitative proteomics method, the identification of 64 new potential substrates were elucidated. The majority of these were type I transmem‐ brane proteins but an added 3 glycophosphatidylinositol anchors and one type II transmem‐ brane protein were also identified, all of which are membrane bound [107]. This suggests that BACE1 has a significant purpose in normal cellular functions but a number of these are yet to be characterised in vivo and subsequently cannot be verified as a substrate until this time. With the interaction between BACE1 and APP being widely of interest in the preven‐ tion of AD, the actual damage by completely inhibiting the enzyme from normal function‐ ing could prove detrimental in its own right. APP may not even be the primary substrate of the enzyme. Therefore, partial inhibition of BACE1 is all that may be required as an inter‐ vention therapy to normalize the enhanced BACE1 activity seen in AD patients.

Alternatively, both of the amyloidogenic creating enzymes, BACE1 and γ-secretase, have alternative purposes relating to other substrates. Gene knockout studies in mouse models produced the reaction of hyperactivity, premature death and seizure like behaviours for BACE1 whilst presenilin -/- mice displayed early neurodegenerative behaviours and an in‐ crease of Aβ species which could possibly be due to the large number of substrates in which both are effective [108-111]. The γ-secretase complex is essential for the Notch sig‐ nalling pathway, which is important for the development of the nervous system and this would also be in deficit if inhibited completely [112]. Likewise, the γ-secretase complex is also required for the non-amyloidogenic pathway, suggesting that the complete inhibition or deletion of these proteins will be detrimental to the homeostatic processing related to normal development and function.

### **5. Challenges**

Recently, a natural inhibition of BACE1 has been found via the sAPPa fragment produced from the non-amyloidogenic pathway [98]. The sAPPa, once produced from APP, has been found to implicate its own cleavage. The findings showed that sAPPa can bind to BACE1 and interfere with the APP cleavage in a mouse model. This resulted in an overall reduction in Aβ formation under physiological conditions. The consequence of this pathway being im‐ paired in any way, could result in a decrease in BACE1 production thus reducing the cleav‐ age of further units of APP. The sAPPa production or cellular concentration could be a

BACE2 is a closely associated β-secretase to BACE1. Sequence analysis show amino acid res‐ idue similarities of ~45% and structural comparisons of 75% homology [99]. BACE2 is also classified as an aspartic protease with the ability to cleave APP at the β-site [100]. In vitro studies have also implicated BACE2 as a possible competitor of BACE1 for the cleavage of the APP protein but it is not formally expressed in excess in the brain and does not compen‐ sate for the loss of BACE1 in gene knockout models in mice [89, 101]. In fact, over expression of BACE2 has actually been found to reduce Aβ production in primary neuronal cultures derived from APP transgenic mice. This could be caused by the ability of BACE2 to cleave APP before BACE1 or because BACE2 cleaves a longer 79 residue Aβ fragment from APP,

Apart from the sequence homology, BACE2 is not primarily expressed in the brain. It is more commonly expressed in the colon, kidney and pancreas, showing that whilst theo‐ retically still having the ability to increase the pathogenesis of AD it is not considered a threat to Aβ generation [100, 102]. BACE1 is the main amyloidogenic enzyme and still re‐ mains the major contributor to amyloid plaque formation but does show some sequence homology with a number other aspartic proteases including renin and cathepsin D. These homologs are generally used for selectivity testing with *in vitro* assays to confirm no un‐

The BACE1 protein is not solely defined to the cleavage of APP and has the ability to cleave other proteins like amyloid like precursor proteins 1 and 2, APPε (which is another closely related *N*-terminal product of APP), neuregulin-1 and -3 involved in axon myelination, βsubunits of voltage gated sodium channels required for neuronal action potentials, P-selec‐ tin glycoprotein ligand-1 (PSGL-1), which regulates leukocyte adhesion in the inflammatory process, the interleukin 1 receptor type-II (IL1-R2) and the low density lipoprotein receptorrelated protein 1(LLRP1), which is a multifunctional endocytic and signalling receptor [1, 2, 103-106]. With the use of an unbiased, quantitative proteomics method, the identification of 64 new potential substrates were elucidated. The majority of these were type I transmem‐ brane proteins but an added 3 glycophosphatidylinositol anchors and one type II transmem‐ brane protein were also identified, all of which are membrane bound [107]. This suggests that BACE1 has a significant purpose in normal cellular functions but a number of these are

which is closer to the cleaved fragment of α-secretase then that of BACE1 [89, 99].

therapeutic intervention strategy in future research.

**4.2. BACE2 and closely related proteins**

236 Neurodegenerative Diseases

wanted binding occurs.

**4.3. Substrates of BACE1**

#### **5.1. Past BACE1 inhibitors**

Inhibition of BACE1 has been the focus of many studies since it was discovered. The main interest in this enzyme remains with the obvious involvement in plaque formation but also to the positive intervention target for which it provides. Firstly, mouse models have shown that without BACE1, Aβ is no longer produced, which indicates that if a product can reduce the enzymatic ability of BACE1 then plaques will not be formed [109]. The enzyme has been characterised and 3-D structure produced, which will allow for inhibitor modelling [80]. BACE1 also has the ability to bind to a wide variety of peptidic structures, while specific binding is an attribute there is still access to the active site for potential targeting [113]. BACE1 is a part of the large aspartic protease family, which have had the success of at least two members (renin and HIV protease-1(HIV-1) inhibited with success [114]. This shows confidence that BACE1 is a viable option for inhibition and, if successful, could influence the production of Aβ.

Drug intervention of AD is limited between therapeutic relief of symptoms and the preven‐ tion of the underlying etiology of the disease [115]. The treatment to minimise symptoms is the only viable option for sufferers of AD. This method slows the decline of cognition, func‐ tion and behaviour but only masks the underlying neurodegeneration taking place [116]. The drugs currently available to treat AD are divided into two categories of cholinesterase inhibitors and receptor antagonists [115].

Cholinesterase inhibitors attempt to prevent the metabolism of acetylcholine allowing the neurotransmitter to maintain effect in the neuron [117]. Cholinesterase inhibiting drugs like donepezil, galantamine and revastigmine are only effective in the early stages of AD by al‐ lowing the retention of acetylcholine [115]. By inhibiting the hydrolysis of acetylcholine by cholinesterase, once the neurotransmitter has crossed the synapse, the cholinergic neuron re‐ mains active [117]. This action becomes redundant when inadvertent progression of NFTs interfere with the normal signal transmission of the axon [15]. Cholinesterase inhibitors have shown that they can actually increase the amount of phosphorylated tau which can further the progression of AD, minimising the effectiveness of the drug [118, 119].

as other peptidic structures. To their detriment, hydroxyethylamine derivatives had poor brain exposure, mainly because of p-glycoprotein (PgP) mediated efflux [128-130]. Bristol-Myers Squibb produced a HEA-derivative that contained an indole and a 7-azoindole car‐ boxamide but struggled to maintain brain Aβ levels in vivo [131]. Unfortunately, the inhibitor showed a high potency *in vitro* that had an IC50 of 10 nM and a low affinity for

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239

Another study has looked to hybridize the HEA isotere and replace the sulfonate ester present in one molecule with the sulphonamide of another [132]. The result was a com‐ pound that had high potency IC50 of 15 nm to BACE1 that was able to bind to the hydro‐ phobic space of the active site. Unfortunately, this compound was able to bind to BACE2 which minimised the selectivity to BACE1. After problems with HEA structures and brain penetration, Merck decided to use a macrocyclic inhibitor, produced by cross linking the P1 and P3 sidechains of an isophthalamide-based inhibitor. This process helped improve poten‐ cy but a bolus of 100 mg/kg fed to APP-YAC mouse model could only obtain Aβ decreases

From another perspective, BACE1 inhibitors have been manufactured from peptide sequen‐ ces by a substrate-based method. The initial peptide sequences showed promise with high potency and selectivity but did not progress as a viable pharmaceutical target because of the large, unstable products it produced. Branded OM99-2, this peptide was originally used to determine many functions and shapes of BACE1 including the active site. Unfortunately, it was too bulky to cross the BBB but maintained appropriate potency. Revision of this method looked specifically at the structure and function of BACE1 in order to manufacture a peptide sequence that would exploit its weaknesses [134]. It was designed to competitively inhibit the binding regions and shut down the enzymatic properties. The finished product was a long peptide upwards of 18 amino acid residues, which produced a high potency but was

These studies made it clear that the peptidic structure would fulfil the desired properties re‐ quired for an inhibitor, the only issue being BBB transport which has motivated the research into small molecules that can freely penetrate the brain. The potential inhibitor needs to be lipophilic enough to permeate the BBB by passive diffusion via the use of a transporter that can maintain entry to the CNS without exiting the same way. Another issue with the pep‐ tide inhibitors is the added convolution of ubiquity throughout the body. Since amino acids, peptides and proteins are the building blocks of the human form and function, there is the added complication of anonymity with other proteins and the possibility of unwanted bind‐ ing causing adverse reactions. If a severe enough reaction to the peptide is apparent, it

The BBB is a network of brain capillaries that regulate the transfer of nutrients and co-fac‐ tors that are important to the functioning and maintenance of the brain. The conformation begins with the lumen that is lined with a monolayer of capillary endothelial cells, held to‐ gether with tight gap junctions. This monolayer is complemented by pericytes for the pur‐

BACE2 and cathepsin D.

of 25% in the first hour and 10% after the third [133].

not stable enough for use in therapeutic trials.

would be more detrimental then beneficial.

**5.2. Crossing the blood brain barrier**

The other pharmaceutical option is memantine, an uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, which prevents binding of the primary excitatory neurotrans‐ mitter in the brain, glutamate [120, 121]. As a result, glutamatergic overstimulation can oc‐ cur causing neuronal cell damage by increased localized calcium stores or excitotoxicity [122]. Both drug options have important side effects and can only offer therapeutic relief of symptoms and are not useful as a long-term intervention strategy.

Therapeutic relief by drugs like donepezil, galantamine and memantine are required to opti‐ mize the productivity of the brain whilst the disease progresses and cannot translate to a definite cure. The production of Aβ is the basis of senile plaques and should be investigated as an important therapeutic target. Theories suggest that by inhibiting BACE1 before it cleaves APP, the formation of the beta amyloid residues would be reduced [123, 124]. To prevent this process, inhibition would be paramount between the translation of BACE1 and its binding to APP, before sAPPβ has been cleaved.

Modern drug discovery for a pharmaceutical intervention aims to hinder the BACE1 enzy‐ matic activity by exactly this process. Notable methods look at high-throughput screening (HTS), fragment-based drug discovery (FBDD) and substrate-based inhibitors [125]. Howev‐ er, none of these methods have been successful in therapeutic trials. The more complex ap‐ proach, generally taken by larger pharmaceutical companies, relate to either the HTS strategy or a FBDD approach. Both processes use a large library of complex compounds in order to find a suitable hit followed by a long modification process to refine it into a suitable chemical. Initial use of HTS sourced complex, high-molecular weight, compounds that were difficult to manipulate. Potential therapeutic candidates showed strong oral availability and good brain permeability but could not provide the standards of potency and selectivity for therapeutic trials [125, 126]

The HTS method has been substituted by the FBDD approach because it uses smaller and more specific compounds [127]. The screening of a fragment library is more appealing be‐ cause a higher hit ratio is produced and the options show favourable drug properties. The main problem with the hit compounds is again, the low potency and selectivity. Often, frag‐ ments that showed promise were too small to be effective and have not provided any real inhibition with the effectiveness required for therapeutic trials.

A large range of inhibitors are being researched including statins, primary, secondary and tertiary amines, hydroethylamines (HEA) of many different conformations, arylamino compounds and acyclic acylguanidines. Most of these compounds seek to act as transition state mimics with a reduced peptidic structure that preclude them from the same scrutiny as other peptidic structures. To their detriment, hydroxyethylamine derivatives had poor brain exposure, mainly because of p-glycoprotein (PgP) mediated efflux [128-130]. Bristol-Myers Squibb produced a HEA-derivative that contained an indole and a 7-azoindole car‐ boxamide but struggled to maintain brain Aβ levels in vivo [131]. Unfortunately, the inhibitor showed a high potency *in vitro* that had an IC50 of 10 nM and a low affinity for BACE2 and cathepsin D.

Another study has looked to hybridize the HEA isotere and replace the sulfonate ester present in one molecule with the sulphonamide of another [132]. The result was a com‐ pound that had high potency IC50 of 15 nm to BACE1 that was able to bind to the hydro‐ phobic space of the active site. Unfortunately, this compound was able to bind to BACE2 which minimised the selectivity to BACE1. After problems with HEA structures and brain penetration, Merck decided to use a macrocyclic inhibitor, produced by cross linking the P1 and P3 sidechains of an isophthalamide-based inhibitor. This process helped improve poten‐ cy but a bolus of 100 mg/kg fed to APP-YAC mouse model could only obtain Aβ decreases of 25% in the first hour and 10% after the third [133].

From another perspective, BACE1 inhibitors have been manufactured from peptide sequen‐ ces by a substrate-based method. The initial peptide sequences showed promise with high potency and selectivity but did not progress as a viable pharmaceutical target because of the large, unstable products it produced. Branded OM99-2, this peptide was originally used to determine many functions and shapes of BACE1 including the active site. Unfortunately, it was too bulky to cross the BBB but maintained appropriate potency. Revision of this method looked specifically at the structure and function of BACE1 in order to manufacture a peptide sequence that would exploit its weaknesses [134]. It was designed to competitively inhibit the binding regions and shut down the enzymatic properties. The finished product was a long peptide upwards of 18 amino acid residues, which produced a high potency but was not stable enough for use in therapeutic trials.

These studies made it clear that the peptidic structure would fulfil the desired properties re‐ quired for an inhibitor, the only issue being BBB transport which has motivated the research into small molecules that can freely penetrate the brain. The potential inhibitor needs to be lipophilic enough to permeate the BBB by passive diffusion via the use of a transporter that can maintain entry to the CNS without exiting the same way. Another issue with the pep‐ tide inhibitors is the added convolution of ubiquity throughout the body. Since amino acids, peptides and proteins are the building blocks of the human form and function, there is the added complication of anonymity with other proteins and the possibility of unwanted bind‐ ing causing adverse reactions. If a severe enough reaction to the peptide is apparent, it would be more detrimental then beneficial.

#### **5.2. Crossing the blood brain barrier**

donepezil, galantamine and revastigmine are only effective in the early stages of AD by al‐ lowing the retention of acetylcholine [115]. By inhibiting the hydrolysis of acetylcholine by cholinesterase, once the neurotransmitter has crossed the synapse, the cholinergic neuron re‐ mains active [117]. This action becomes redundant when inadvertent progression of NFTs interfere with the normal signal transmission of the axon [15]. Cholinesterase inhibitors have shown that they can actually increase the amount of phosphorylated tau which can further

The other pharmaceutical option is memantine, an uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, which prevents binding of the primary excitatory neurotrans‐ mitter in the brain, glutamate [120, 121]. As a result, glutamatergic overstimulation can oc‐ cur causing neuronal cell damage by increased localized calcium stores or excitotoxicity [122]. Both drug options have important side effects and can only offer therapeutic relief of

Therapeutic relief by drugs like donepezil, galantamine and memantine are required to opti‐ mize the productivity of the brain whilst the disease progresses and cannot translate to a definite cure. The production of Aβ is the basis of senile plaques and should be investigated as an important therapeutic target. Theories suggest that by inhibiting BACE1 before it cleaves APP, the formation of the beta amyloid residues would be reduced [123, 124]. To prevent this process, inhibition would be paramount between the translation of BACE1 and

Modern drug discovery for a pharmaceutical intervention aims to hinder the BACE1 enzy‐ matic activity by exactly this process. Notable methods look at high-throughput screening (HTS), fragment-based drug discovery (FBDD) and substrate-based inhibitors [125]. Howev‐ er, none of these methods have been successful in therapeutic trials. The more complex ap‐ proach, generally taken by larger pharmaceutical companies, relate to either the HTS strategy or a FBDD approach. Both processes use a large library of complex compounds in order to find a suitable hit followed by a long modification process to refine it into a suitable chemical. Initial use of HTS sourced complex, high-molecular weight, compounds that were difficult to manipulate. Potential therapeutic candidates showed strong oral availability and good brain permeability but could not provide the standards of potency and selectivity for

The HTS method has been substituted by the FBDD approach because it uses smaller and more specific compounds [127]. The screening of a fragment library is more appealing be‐ cause a higher hit ratio is produced and the options show favourable drug properties. The main problem with the hit compounds is again, the low potency and selectivity. Often, frag‐ ments that showed promise were too small to be effective and have not provided any real

A large range of inhibitors are being researched including statins, primary, secondary and tertiary amines, hydroethylamines (HEA) of many different conformations, arylamino compounds and acyclic acylguanidines. Most of these compounds seek to act as transition state mimics with a reduced peptidic structure that preclude them from the same scrutiny

the progression of AD, minimising the effectiveness of the drug [118, 119].

symptoms and are not useful as a long-term intervention strategy.

its binding to APP, before sAPPβ has been cleaved.

inhibition with the effectiveness required for therapeutic trials.

therapeutic trials [125, 126]

238 Neurodegenerative Diseases

The BBB is a network of brain capillaries that regulate the transfer of nutrients and co-fac‐ tors that are important to the functioning and maintenance of the brain. The conformation begins with the lumen that is lined with a monolayer of capillary endothelial cells, held to‐ gether with tight gap junctions. This monolayer is complemented by pericytes for the pur‐ pose of BBB-specific gene expression and inducing polarisation of the astrocyte end-feet with the surrounding basal lamina [135]. The tight junctions are known to prevent the para‐ cellular diffusion of polar molecules from systemic circulation and brain parenchyma [136]. Complementing the endothelium are a number of compounds that defend against foreign transfer like cytochrome p450 and glutathione S-transferase in conjunction with transporters and the multidrug resistance associated proteins 1 and 2 (Mrp1/2) [137-139].

can be transformed for BACE1 inhibitors, the transfer of larger, more potent molecules with

Dropping the BACE: Beta Secretase (BACE1) as an Alzheimer's Disease Intervention Target

http://dx.doi.org/10.5772/53603

241

The ability to transport potential inhibitors across the blood brain barrier can determine the success or failure of the potential therapeutic intervention. The BBB has motivated the re‐ search base to investigate small <450 Da molecules that have the potential to pass the barrier with minimal scrutiny. The issue being a lack of potency and selectivity *in vivo* which indicates that a greater effectiveness could be achieved with a larger style inhibitor. The potential for transporters could revolutionize the future of drug production by providing a medium in

The earlier transporters aimed to cross the BBB by utilising the already formed channels and receptors. The idea was to couple an inhibitor with the specific ligand of a surface receptor like that of the H1 histone, insulin, insulin-like growth factors or transferrin [148-151]. These systems can also work by binding the inhibitor to antibodies that identi‐ fy surface epitopes already present in the BBB. The coupling of an 18mer peptide to transferrin receptor specific antibodies were used to infiltrate the BBB via the transferrin receptor for the inhibition of the rev gene of HIV-1 [152]. This study demonstrated a 15 fold increase in transfer across the BBB. This system can be modified to display a num‐ ber of different biotinylated peptides specifically but could also be manipulated for other

A more advanced method of targeted liposomes progresses from the inhibitor-ligand struc‐ ture to a colloidal carrier system. The concept still utilizes BBB receptors and channels but is improved with an increase in the concentration of the inhibitor that it can carry. The liposo‐ mal system can hold >10,000 drug molecules [153]. The main question regarding this system is the avoidance of opsonins which are members of the complement system and immuno‐ globulins that cover the colloidal particle and are able to activate phagocytosis. This method has already been used to transfer monoclonal antibodies across the BBB [153]. The negative of this system however, is the vulnerability to macrophages via opsonisation and the lack of selectivity that liposomes have in regards to the brain and BBB. The targeted liposome sys‐ tem has already been used successfully to transfer sodium borocaptate in defence against malignant glioma, which suggests that further research could provide a neurodegenerative

Alternatively, fullerenes are an allotropic form of carbon that form an arrangement of 60 atoms into the shape of a hollow sphere that is 1 nanometre in diameter and can be coupled with an inhibitor [155]. These fullerene systems have shown promise in the fight against chronic multiple sclerosis [156]. Whilst still in the early stages of research, recovery has been attributed to a reduction in axon loss and demyelination in the spinal region of the CNS in a mouse model. Another study has shown a reduction of infarct size in gerbils and rats with the use of a hexasulfinonated C60 that was administered intravenously [157, 158]. Further

an increased selectivity, could be achieved.

which the larger more effective inhibitors can cross this barrier.

**5.3. Transporters**

effective compounds.

diseases template [154].

The defence mechanisms that protect the brain from systemic circulation become a chal‐ lenging interference when fabricating an inhibitor, especially when it comes to testing the administration of localised drug performance. The molecular weight threshold, which is a relevant property of all membranes, is <450 Da [140]. A molecular model can be used to determine the effect of molecular size on membrane permeability and should be a consideration when investigating an inhibitor [141]. This creates a challenge that goes beyond the production of protein specific intervention strategies. In the case of BACE1, there are a number of different challenges, as previously mentioned, influencing the in‐ hibition of APP cleavage. With the influence of the BBB, the drug will be required to maintain an ability to inhibit BACE1 but also maintain solubility across the tight junc‐ tions of vessel epithelium.

The natural functioning of the BBB is to maintain the tight junctions, for which it is idiosyn‐ cratic, but in order to transport a required drug, the concept of BBB disruption can be con‐ sidered. The relaxing of these junctions with hyperosmotic chemicals, for example, could be enough to encourage transport to the brain. The only concern with this method is due to the unfavourable uptake of plasma proteins, like albumin, which is toxic to astrocytes [142]. Consequently, it could also affect drug retention and enhance unwanted migration of other‐ wise rejected contaminants.

Another drastic, invasive technique involves drilling into the cranium and administering the treatment via intracerebroventricular injection, intracerebral implantation and convec‐ tion enhanced diffusion. While all are incredibly invasive and risky, the required re‐ sponses from the drug once applied are generally not that positive [143]. Intracerebroventricular injection specifically has been found to cause haemorrhage after the insertion of a needle into the brain as an adverse reaction but has otherwise has shown good responses [144]. This suggests these invasive techniques are questionable and should be used with extreme caution.

The interesting, non-invasive, concept of lipid carriers has the most potential. The carriers are attached to water soluble inhibitors, that cannot otherwise penetrate the BBB, and turn them lipid soluble [145]. This will allow the transfer into the BBB but once across, the re‐ quirement to either shed the carrier or be able to function with it attached in order to main‐ tain functionality, becomes apparent. The naturally occurring, 60 amino acid, galanin-like peptide (GALP) has the ability to cross the BBB where needed with the use of a saturable transport system [146]. The glucose transporter (GLUT1) is another natural carrier system that is used frequently as glucose is the main energy source of the brain [147]. If this method can be transformed for BACE1 inhibitors, the transfer of larger, more potent molecules with an increased selectivity, could be achieved.

#### **5.3. Transporters**

pose of BBB-specific gene expression and inducing polarisation of the astrocyte end-feet with the surrounding basal lamina [135]. The tight junctions are known to prevent the para‐ cellular diffusion of polar molecules from systemic circulation and brain parenchyma [136]. Complementing the endothelium are a number of compounds that defend against foreign transfer like cytochrome p450 and glutathione S-transferase in conjunction with transporters

The defence mechanisms that protect the brain from systemic circulation become a chal‐ lenging interference when fabricating an inhibitor, especially when it comes to testing the administration of localised drug performance. The molecular weight threshold, which is a relevant property of all membranes, is <450 Da [140]. A molecular model can be used to determine the effect of molecular size on membrane permeability and should be a consideration when investigating an inhibitor [141]. This creates a challenge that goes beyond the production of protein specific intervention strategies. In the case of BACE1, there are a number of different challenges, as previously mentioned, influencing the in‐ hibition of APP cleavage. With the influence of the BBB, the drug will be required to maintain an ability to inhibit BACE1 but also maintain solubility across the tight junc‐

The natural functioning of the BBB is to maintain the tight junctions, for which it is idiosyn‐ cratic, but in order to transport a required drug, the concept of BBB disruption can be con‐ sidered. The relaxing of these junctions with hyperosmotic chemicals, for example, could be enough to encourage transport to the brain. The only concern with this method is due to the unfavourable uptake of plasma proteins, like albumin, which is toxic to astrocytes [142]. Consequently, it could also affect drug retention and enhance unwanted migration of other‐

Another drastic, invasive technique involves drilling into the cranium and administering the treatment via intracerebroventricular injection, intracerebral implantation and convec‐ tion enhanced diffusion. While all are incredibly invasive and risky, the required re‐ sponses from the drug once applied are generally not that positive [143]. Intracerebroventricular injection specifically has been found to cause haemorrhage after the insertion of a needle into the brain as an adverse reaction but has otherwise has shown good responses [144]. This suggests these invasive techniques are questionable

The interesting, non-invasive, concept of lipid carriers has the most potential. The carriers are attached to water soluble inhibitors, that cannot otherwise penetrate the BBB, and turn them lipid soluble [145]. This will allow the transfer into the BBB but once across, the re‐ quirement to either shed the carrier or be able to function with it attached in order to main‐ tain functionality, becomes apparent. The naturally occurring, 60 amino acid, galanin-like peptide (GALP) has the ability to cross the BBB where needed with the use of a saturable transport system [146]. The glucose transporter (GLUT1) is another natural carrier system that is used frequently as glucose is the main energy source of the brain [147]. If this method

and the multidrug resistance associated proteins 1 and 2 (Mrp1/2) [137-139].

tions of vessel epithelium.

240 Neurodegenerative Diseases

wise rejected contaminants.

and should be used with extreme caution.

The ability to transport potential inhibitors across the blood brain barrier can determine the success or failure of the potential therapeutic intervention. The BBB has motivated the re‐ search base to investigate small <450 Da molecules that have the potential to pass the barrier with minimal scrutiny. The issue being a lack of potency and selectivity *in vivo* which indicates that a greater effectiveness could be achieved with a larger style inhibitor. The potential for transporters could revolutionize the future of drug production by providing a medium in which the larger more effective inhibitors can cross this barrier.

The earlier transporters aimed to cross the BBB by utilising the already formed channels and receptors. The idea was to couple an inhibitor with the specific ligand of a surface receptor like that of the H1 histone, insulin, insulin-like growth factors or transferrin [148-151]. These systems can also work by binding the inhibitor to antibodies that identi‐ fy surface epitopes already present in the BBB. The coupling of an 18mer peptide to transferrin receptor specific antibodies were used to infiltrate the BBB via the transferrin receptor for the inhibition of the rev gene of HIV-1 [152]. This study demonstrated a 15 fold increase in transfer across the BBB. This system can be modified to display a num‐ ber of different biotinylated peptides specifically but could also be manipulated for other effective compounds.

A more advanced method of targeted liposomes progresses from the inhibitor-ligand struc‐ ture to a colloidal carrier system. The concept still utilizes BBB receptors and channels but is improved with an increase in the concentration of the inhibitor that it can carry. The liposo‐ mal system can hold >10,000 drug molecules [153]. The main question regarding this system is the avoidance of opsonins which are members of the complement system and immuno‐ globulins that cover the colloidal particle and are able to activate phagocytosis. This method has already been used to transfer monoclonal antibodies across the BBB [153]. The negative of this system however, is the vulnerability to macrophages via opsonisation and the lack of selectivity that liposomes have in regards to the brain and BBB. The targeted liposome sys‐ tem has already been used successfully to transfer sodium borocaptate in defence against malignant glioma, which suggests that further research could provide a neurodegenerative diseases template [154].

Alternatively, fullerenes are an allotropic form of carbon that form an arrangement of 60 atoms into the shape of a hollow sphere that is 1 nanometre in diameter and can be coupled with an inhibitor [155]. These fullerene systems have shown promise in the fight against chronic multiple sclerosis [156]. Whilst still in the early stages of research, recovery has been attributed to a reduction in axon loss and demyelination in the spinal region of the CNS in a mouse model. Another study has shown a reduction of infarct size in gerbils and rats with the use of a hexasulfinonated C60 that was administered intravenously [157, 158]. Further research is required for the use in human studies but there is anticipation for its use against the aggregation of Aβ.

Once an inhibitor has been potentiated, the focus can move into research looking at plaque clearing and neurogenesis that will aid regeneration of the AD brain. One option is the utiliza‐ tion of stem cells to replace lost brain mass by utilizing multi-potent adult neural stem cells found in the subgranular and subventricular zones. The CNS has the ability to regenerate a number of neuronal cell lines with astrocytes, oligodendrocytes, and functional neurons [161]. By utilizing these cell lines the brain could be "rebuilt" to maintain the structures affected by the plaque formation. However, this would be implicated by numerous ethical challenges.

Dropping the BACE: Beta Secretase (BACE1) as an Alzheimer's Disease Intervention Target

http://dx.doi.org/10.5772/53603

243

The onset of AD is relatively unknown, but early stages of the disease are met with the accu‐ mulation of amyloid plaques. The amyloidogenic pathway, mediated by the proteolytic cleavage of APP, is the major focus for the alleviation of AD. BACE1, the defining enzyme of this process, is responsible for the cleavage of the Aβ fragment. The determinant of fragment length, 38-42 amino acids, is coordinated by γ-secretase. The focus is placed specifically on fragments Aβ40 and Aβ42 because of the influence they maintain over plaque formation and

Whilst there is an indication that γ-secretase maintains the ability to determine the severity of plaque formation, there are a number of reasons to avoid inhibition, mainly because of its involvement in the Notch pathway and the extreme consequences which brings to light the cost outweighing the means. This is a hallmark of plaque formation as the enzymes in‐ volved have a number of substrates in different pathways that do not involve AD. This mo‐ tivates the notion of regulation, as opposed to complete inhibition, to aid the process

The recent association between the cleaved product of APP by α-secretase, sAPPα, having an influence on the amyloidogenic pathway adds another perspective to AD and the mecha‐ nisms relating to brain homeostasis. It prompts the idea of maintaining normal processing as opposed to initiating inhibition. The BACE1 cleavage of APP is not a response, or trigger, to AD progression but is fundamental in Aβ production, which implies that normal conser‐

A large obstacle with potential BACE1 inhibitors is the ability to maintain the pharmaceuti‐ cal properties between *in vitro* and *in vivo* testing, namely the BBB. The transfer between the Tight junctions of brain endothelium and the neuronal cells has defined the way BACE1 in‐ hibitors are engaged. The change from basic peptidic structures to that of small <450 Da chemicals has solved one hurdle but has inadvertently created others. The original *in vitro* studies of peptidic inhibitors maintained positive selectivity for BACE1 but were too bulky for transport. The application of a BACE1 inhibitor, whatever structure it is formed from, will be rudimentary in treating and alleviating the devastating prognosis of AD in the years

vation is a natural process and regulation can be achieved with an external stimulus.

**7. Conclusions**

an increased affinity for aggregation.

alleviating majority of the proteolysis.

to come.

### **6. Future perspectives**

There are a number of exciting directions that future research will take to determine a safe and effective treatment for AD. The regulation of internal pathways for the natural manage‐ ment of BACE1 activity is of key interest as it involves the crossroad from compound based, active site mimics. The recent study identifying the sAPPα as a BACE1 modulator could mo‐ tivate the investigation of the broader pathway rather than focussing on the enzyme itself [98]. Pathway regulation could reduce side effects and associated pathologies because of the broad spectrum enzymes that envelope amyloidogenesis.

The implication of Aβ signalling in plaque formation is another concept for investigation for the possibility of a feedback system. The promotion of Aβ proliferation could increase the ability for a plaque to form as there is the availability to encourage the process. The applica‐ tion of a potential BACE1 inhibitor is rudimentary to the prevention of this disease because of the potential to bypass the amyloidogenic pathway. With minimal Aβ production the pressure released on the natural inflammatory defences would allow for the metabolism of impeding plaques.

γ-secretase inhibitors have already been progressing to phase III clinical trials which seek to influence the production of Aβ but maintain a number of important consequences that sur‐ round this process [159]. The implication of the Notch signalling pathway, important in ax‐ on myelination and apoptosis, could be more detrimental than plaque formation because of the regulatory purpose in which it serves [112].

Merck have developed the MK-8931 inhibitor, which is currently undergoing phase II clini‐ cal trials [160]. It has already shown an ability to reduce Aβ40, Aβ42 and sAPPβ levels, is well tolerated *in vivo* and shows minimal adverse reactions, which is encouraging for the <450 Da compounds. Eli Lilly and AstraZeneca are also involved with BACE1 inhibitor clinical trials, which will increase competition once results come to fruition. The opposing mentality be‐ hind BACE1 inhibitors, as small <450 Da compounds, could be the answer to years of at‐ tempts. The crossing of the barrier is not a major obstacle for this method but show alternative hurdles of selectivity and bioavailability. This has created a race for a pharma‐ ceutically able inhibitor.

Alternatively, the membrane transporters concept for the advantage of allowing the transfer of a range of different inhibitors, in a variety of doses, could produce a platform for a num‐ ber of different brain based diseases without being overly invasive. The BBB is an ominous obstacle for a number of the earlier peptidic inhibitors, whilst showing a lot of promising pharmaceutical attributes. The combination of the two technologies could provide the differ‐ ence required for drug penetration and selectivity.

Once an inhibitor has been potentiated, the focus can move into research looking at plaque clearing and neurogenesis that will aid regeneration of the AD brain. One option is the utiliza‐ tion of stem cells to replace lost brain mass by utilizing multi-potent adult neural stem cells found in the subgranular and subventricular zones. The CNS has the ability to regenerate a number of neuronal cell lines with astrocytes, oligodendrocytes, and functional neurons [161]. By utilizing these cell lines the brain could be "rebuilt" to maintain the structures affected by the plaque formation. However, this would be implicated by numerous ethical challenges.

### **7. Conclusions**

research is required for the use in human studies but there is anticipation for its use against

There are a number of exciting directions that future research will take to determine a safe and effective treatment for AD. The regulation of internal pathways for the natural manage‐ ment of BACE1 activity is of key interest as it involves the crossroad from compound based, active site mimics. The recent study identifying the sAPPα as a BACE1 modulator could mo‐ tivate the investigation of the broader pathway rather than focussing on the enzyme itself [98]. Pathway regulation could reduce side effects and associated pathologies because of the

The implication of Aβ signalling in plaque formation is another concept for investigation for the possibility of a feedback system. The promotion of Aβ proliferation could increase the ability for a plaque to form as there is the availability to encourage the process. The applica‐ tion of a potential BACE1 inhibitor is rudimentary to the prevention of this disease because of the potential to bypass the amyloidogenic pathway. With minimal Aβ production the pressure released on the natural inflammatory defences would allow for the metabolism of

γ-secretase inhibitors have already been progressing to phase III clinical trials which seek to influence the production of Aβ but maintain a number of important consequences that sur‐ round this process [159]. The implication of the Notch signalling pathway, important in ax‐ on myelination and apoptosis, could be more detrimental than plaque formation because of

Merck have developed the MK-8931 inhibitor, which is currently undergoing phase II clini‐ cal trials [160]. It has already shown an ability to reduce Aβ40, Aβ42 and sAPPβ levels, is well tolerated *in vivo* and shows minimal adverse reactions, which is encouraging for the <450 Da compounds. Eli Lilly and AstraZeneca are also involved with BACE1 inhibitor clinical trials, which will increase competition once results come to fruition. The opposing mentality be‐ hind BACE1 inhibitors, as small <450 Da compounds, could be the answer to years of at‐ tempts. The crossing of the barrier is not a major obstacle for this method but show alternative hurdles of selectivity and bioavailability. This has created a race for a pharma‐

Alternatively, the membrane transporters concept for the advantage of allowing the transfer of a range of different inhibitors, in a variety of doses, could produce a platform for a num‐ ber of different brain based diseases without being overly invasive. The BBB is an ominous obstacle for a number of the earlier peptidic inhibitors, whilst showing a lot of promising pharmaceutical attributes. The combination of the two technologies could provide the differ‐

the aggregation of Aβ.

242 Neurodegenerative Diseases

impeding plaques.

ceutically able inhibitor.

**6. Future perspectives**

broad spectrum enzymes that envelope amyloidogenesis.

the regulatory purpose in which it serves [112].

ence required for drug penetration and selectivity.

The onset of AD is relatively unknown, but early stages of the disease are met with the accu‐ mulation of amyloid plaques. The amyloidogenic pathway, mediated by the proteolytic cleavage of APP, is the major focus for the alleviation of AD. BACE1, the defining enzyme of this process, is responsible for the cleavage of the Aβ fragment. The determinant of fragment length, 38-42 amino acids, is coordinated by γ-secretase. The focus is placed specifically on fragments Aβ40 and Aβ42 because of the influence they maintain over plaque formation and an increased affinity for aggregation.

Whilst there is an indication that γ-secretase maintains the ability to determine the severity of plaque formation, there are a number of reasons to avoid inhibition, mainly because of its involvement in the Notch pathway and the extreme consequences which brings to light the cost outweighing the means. This is a hallmark of plaque formation as the enzymes in‐ volved have a number of substrates in different pathways that do not involve AD. This mo‐ tivates the notion of regulation, as opposed to complete inhibition, to aid the process alleviating majority of the proteolysis.

The recent association between the cleaved product of APP by α-secretase, sAPPα, having an influence on the amyloidogenic pathway adds another perspective to AD and the mecha‐ nisms relating to brain homeostasis. It prompts the idea of maintaining normal processing as opposed to initiating inhibition. The BACE1 cleavage of APP is not a response, or trigger, to AD progression but is fundamental in Aβ production, which implies that normal conser‐ vation is a natural process and regulation can be achieved with an external stimulus.

A large obstacle with potential BACE1 inhibitors is the ability to maintain the pharmaceuti‐ cal properties between *in vitro* and *in vivo* testing, namely the BBB. The transfer between the Tight junctions of brain endothelium and the neuronal cells has defined the way BACE1 in‐ hibitors are engaged. The change from basic peptidic structures to that of small <450 Da chemicals has solved one hurdle but has inadvertently created others. The original *in vitro* studies of peptidic inhibitors maintained positive selectivity for BACE1 but were too bulky for transport. The application of a BACE1 inhibitor, whatever structure it is formed from, will be rudimentary in treating and alleviating the devastating prognosis of AD in the years to come.

#### **Author details**

Justin Read and Cenk Suphioglu

NeuroAllergy Research Laboratory (NARL), School of Life and Environmental Sciences, Deakin University, Geelong, Victoria, Australia

[12] Fountoulakis M, Kossida S. Proteomics-driven Progress in Neurodegeneration Re‐

Dropping the BACE: Beta Secretase (BACE1) as an Alzheimer's Disease Intervention Target

http://dx.doi.org/10.5772/53603

245

[13] Cai H, Wang Y, McCarthy D, Wen H, Borchelt DR, Price DL, et al. BACE1 is the Ma‐ jor β-secretase for Generation of Aβ Peptides by Neurons. Nature Neuroscience.

[14] Spillantini MG, Goedert M. Tau Protein Pathology in Neurodegenerative Diseases.

[15] Lebouvier T, Scales TME, Williamson R, Noble W, Duyckaerts C, Hanger DP, et al. The Microtubule-Associated Protein Tau is Also Phosphorylated on Tyrosine. Jour‐

[16] Selkoe DJ. Alzheimer Disease: Mechanistic Understanding Predicts Novel Therapies.

[17] Eikelenboom P, Hack C, Rozemuller J, Stam F. Complement Activation in Amyloid Plaques in Alzheimer's Dementia. Virchows Archiv B Cell Pathology Zell-pathologie.

[18] Tuppo EE, Arias HR. The Role of Inflammation in Alzheimer's Disease. The Interna‐

[19] Wolfe MS. When Loss is Gain: Reduced Presenilin Proteolytic Function Leads to In‐

[20] Arendt T. Synaptic Degeneration in Alzheimer's Disease. Acta Neuropathologica.

[21] Francis PT. Glutamatergic Systems in Alzheimer's Disease. International Journal of

[22] Nixon RA, Wegiel J, Kumar A, Yu WH, Peterhoff C, Cataldo A, et al. Extensive In‐ volvement of Autophagy in Alzheimer Disease: An Immuno-Electron Microscopy Study. Journal of Neuropathology & Experimental Neurology. 2005;64(2):113-22. [23] Martins IJ, Berger T, Sharman MJ, Verdile G, Fuller SJ, Martins RN. Cholesterol Me‐ tabolism and Transport in the Pathogenesis of Alzheimer's Disease. Journal of Neu‐

[24] Bibel M, Barde Y-A. Neurotrophins: Key Regulators of Cell Fate and Cell Shape in the Vertebrate Nervous System. Genes & Development. 2000;14(23):2919-37.

[25] Morfini GA, Burns M, Binder LI, Kanaan NM, LaPointe N, Bosco DA, et al. Axonal Transport Defects in Neurodegenerative Diseases. The Journal of Neuroscience.

tional Journal of Biochemistry & Cell Biology. 2005;37(2):289-305.

creased Aβ42/Aβ40. EMBO Reports. 2007;8(2):136-40.

Geriatric Psychiatry. 2003;18(S1):S15-S21.

rochemistry. 2009;111(6):1275-308.

2009;29(41):12776-86.

search. Electrophoresis. 2006;27(8):1556-73.

Trends in Neurosciences. 1998;21(10):428-33.

nal of Alzheimer's Disease. 2009;18(1):1-9.

Annals of Internal Medicine. 2004;140(8):627-38.

2001;4(3):233-4.

1988;56(1):259-62.

2009;118(1):167-79.

#### **References**


[12] Fountoulakis M, Kossida S. Proteomics-driven Progress in Neurodegeneration Re‐ search. Electrophoresis. 2006;27(8):1556-73.

**Author details**

244 Neurodegenerative Diseases

**References**

Justin Read and Cenk Suphioglu

2006;9(12):1520-5.

2005;280(24):23009-17.

Deakin University, Geelong, Victoria, Australia

NeuroAllergy Research Laboratory (NARL), School of Life and Environmental Sciences,

[1] Hu X, Hicks CW, He W, Wong P, Macklin WB, Trapp BD, et al. Bace1 Modulates Myelination in the Central and Peripheral Nervous System. Nature Neuroscience.

[2] Wong H-K, Sakurai T, Oyama F, Kaneko K, Wada K, Miyazaki H, et al. β Subunits of Voltage-gated Sodium Channels Are Novel Substrates of β-Site Amyloid Precursor Protein-cleaving Enzyme (BACE1) and γ-Secretase. Journal of Biological Chemistry.

[4] Brun A, Englund E. Regional Pattern of Degeneration in Alzheimer's Disease: Neuro‐

[5] Brookmeyer R, Johnson E, Ziegler-Graham K, Arrighi HM. Forecasting the Global Burden of Alzheimer's Disease. Alzheimer's and Dementia. 2007;3(3):186-91.

[6] WHO. Dementia: a public health priority. Geneva: World Health Organization and Alzheimer's Disease International. http://www.who.int/mental\_health/publications/

[7] Rice DP, Fillit HM, Max W, Knopman DS, Lloyd JR, Duttagupta S. Prevalence, Costs, and Treatment of Alzheimer's Disease and Related Dementia: a Managed Care Per‐

[8] Malone DC, McLaughlin TP, Wahl PM, Leibman C, Arrighi M, Cziraky MJ, et al. Burden of Alzheimer's Disease and Association With Negative Health Outcomes.

[9] Wenk GL. Neuropathologic Changes in Alzheimer's Disease. The Journal of Clinical

[10] Sultzer DL, Brown CV, Mandelkern MA, Mahler ME, Mendez MF, Chen ST, et al. Delusional Thoughts and Regional Frontal/Temporal Cortex Metabolism in Alzheim‐

[11] Bruen PD, McGeown WJ, Shanks MF, Venneri A. Neuroanatomical Correlates of Neuropsychiatric Symptoms in Alzheimer's Disease. Brain. 2008;131(9):2455-63.

[3] Choi DW. Excitotoxic cell death. Journal of Neurobiology. 1992;23(9):1261-76.

nal Loss and Histopathological Grading. Histopathology. 1981;5:549-64.

dementia\_report\_2012/en/index.html. (accessed 7 July 2012).

The American Journal of Managed Care. 2009;15(8):481-8.

er's Disease. American Journal Of Psychiatry. 2003;160:341-9.

Psychiatry. 2003;64 Suppl 9:7-10.

spective. The American Journal of Managed Care. 2001;7(8):809-18.


[26] Hardy J, Duff K, Hardy KG, Perez-Tur J, Hutton M. Genetic Dissection of Alzheim‐ er's Disease and Related Dementias: Amyloid and its Relationship to Tau. Nature Neuroscience. 1998;1(5):355-8.

[38] Müller T, Concannon CG, Ward MW, Walsh CM, Tirniceriu AL, Tribl F, et al. Modu‐ lation of Gene Expression and Cytoskeletal Dynamics by the Amyloid Precursor Pro‐ tein Intracellular Domain (AICD). Molecular Biology of the Cell. 2007;18(1):201-10.

Dropping the BACE: Beta Secretase (BACE1) as an Alzheimer's Disease Intervention Target

http://dx.doi.org/10.5772/53603

247

[39] Dulin F, Léveillé F, Ortega JB, Mornon J-P, Buisson A, Callebaut I, et al. p3 Peptide, a Truncated Form of Aβ Devoid of Synaptotoxic Effect, Does Not Assemble Into Solu‐

[40] Walsh DM, Klyubin I, Fadeeva JV, Cullen WK, Anwyl R, Wolfe MS, et al. Naturally Secreted Oligomers of Amyloidβ Protein Potently Inhibit Hippocampal Long-term

[41] De Strooper B. Aph-1, Pen-2, and Nicastrin with Presenilin Generate an Active γ-Sec‐

[42] Takasugi N, Tomita T, Hayashi I, Tsuruoka M, Niimura M, Takahashi Y, et al. The Role of Presenilin Cofactors in the γ-secretase Complex. Nature. 2003;422(6930):

[43] LaVoie MJ, Fraering PC, Ostaszewski BL, Ye W, Kimberly WT, Wolfe MS, et al. As‐ sembly of the γ-Secretase Complex Involves Early Formation of an Intermediate Sub‐ complex of Aph-1 and Nicastrin. Journal of Biological Chemistry. 2003;278(39):

[44] Wolfe M, Xia W, Ostaszewski B, Diehl T, Kimberly W, Selkoe D. Two Transmem‐ brane Aspartates in Presenilin-1 Required for Presenilin Endoproteolysis and γ-sec‐

[45] Levy-Lahad E, Wasco W, Poorkaj P, Romano D, Oshima J, Pettingell W, et al. Candi‐ date Gene for the Chromosome 1 Familial Alzheimer's Disease Locus. Science.

[46] Francis R, McGrath G, Zhang J, Ruddy DA, Sym M, Apfeld J, et al. aph-1 and pen-2 Are Required for Notch Pathway Signaling, γ-Secretase Cleavage of βAPP, and Pre‐

[47] Tanzi RE, Bertram L. Twenty Years of the Alzheimer's Disease Amyloid Hypothesis:

[48] Suzuki N, Cheung T, Cai X, Odaka A, Otvos L, Eckman C, et al. An Increased Per‐ centage of Long Amyloid Beta Protein Secreted by Familial Amyloid Beta Protein

[49] Burdick D, Soreghan B, Kwon M, Kosmoski J, Knauer M, Henschen A, et al. Assem‐ bly and Aggregation Properties of Synthetic Alzheimer's A4/beta Amyloid Peptide

senilin Protein Accumulation. Developmental Cell. 2002;3(1):85-97.

Precursor (Beta APP717) Mutants. Science. 1994;264(5163):1336-40.

Analogs. Journal of Biological Chemistry. 1992;267(1):546-54.

ble Oligomers. FEBS Letters. 2008;582(13):1865-70.

Potentiation in vivo. Nature. 2002;416(6880):535-9.

retase Complex. Neuron. 2003;38(1):9-12.

retase Activity. Nature. 1999;398(6727):513-7.

A Genetic Perspective. Cell. 2005;120(4):545-55.

438-41.

37213-22.

1995;269(5226):973-7.


[38] Müller T, Concannon CG, Ward MW, Walsh CM, Tirniceriu AL, Tribl F, et al. Modu‐ lation of Gene Expression and Cytoskeletal Dynamics by the Amyloid Precursor Pro‐ tein Intracellular Domain (AICD). Molecular Biology of the Cell. 2007;18(1):201-10.

[26] Hardy J, Duff K, Hardy KG, Perez-Tur J, Hutton M. Genetic Dissection of Alzheim‐ er's Disease and Related Dementias: Amyloid and its Relationship to Tau. Nature

[27] Varani L, Hasegawa M, Spillantini MG, Smith MJ, Murrell JR, Ghetti B, et al. Struc‐ ture of Tau Exon 10 Splicing Regulatory Element RNA and Destabilization by Muta‐ tions of Frontotemporal Dementia and Parkinsonism Linked to Chromosome 17.

[28] Glenner GG, Wong CW. Alzheimer's Disease: Initial Report of the Purification and Characterization of a Novel Cerebrovascular Amyloid Protein. Biochemical and Bio‐

[29] Tanzi R, Gusella J, Watkins P, Bruns G, St George-Hyslop P, Van Keuren M, et al. Amyloid Beta Protein Gene: cDNA, mRNA Distribution, and Genetic Linkage Near

[30] Kang J, Lemaire H-G, Unterbeck A, Salbaum JM, Masters CL, Grzeschik K-H, et al. The Precursor of Alzheimer's Disease Amyloid Aβ Protein Resembles a Cell-surface

[31] Lee J, Retamal C, Cuitiño L, Caruano-Yzermans A, Shin J-E, van Kerkhof P, et al. Adaptor Protein Sorting Nexin 17 Regulates Amyloid Precursor Protein Trafficking and Processing in the Early Endosomes. Journal of Biological Chemistry.

[32] Jorissen E, Prox J, Bernreuther C, Weber S, Schwanbeck R, Serneels L, et al. The Dis‐ integrin/Metalloproteinase ADAM10 Is Essential for the Establishment of the Brain

[33] Selkoe DJ. The Molecular Pathology of Alzheimer's Disease. Neuron. 1991;6(4):

[34] Vassar R, Bennett BD, Babu-Khan S, Kahn S, Mendiaz EA, Denis P, et al. β-Secretase Cleavage of Alzheimer's Amyloid Precursor Protein by the Transmembrane Aspartic

[35] Kuhn P-H, Wang H, Dislich B, Colombo A, Zeitschel U, Ellwart JW, et al. ADAM10 is the Physiologically Relevant, Constitutive α-secretase of the Amyloid Precursor Pro‐

[36] Alves da Costa C, Sunyach C, Pardossi-Piquard R, Sévalle J, Vincent B, Boyer N, et al. Presenilin-Dependent γ-Secretase-Mediated Control of p53-Associated Cell Death in

[37] Madeira A, Pommet J-M, Prochiantz A, Allinquant B. SET Protein (TAF1β, I2PP2A) is Involved in Neuronal Apoptosis Induced by an Amyloid Precursor Protein Cyto‐

tein in Primary Neurons. The EMBO Journal. 2010;29(17):3020-32.

plasmic Subdomain. The FASEB Journal. 2005;19(13):1905-07.

Alzheimer's Disease. The Journal of Neuroscience. 2006;26(23):6377-85.

Proceedings of the National Academy of Sciences. 1999;96(14):8229-34.

physical Research Communications. 1984;120(3):885-90.

the Alzheimer Locus. Science. 1987;235(4791):880-4.

Cortex. The Journal of Neuroscience. 2010;30(14):4833-44.

Protease BACE. Science. 1999;286(5440):735-41.

Receptor. Nature. 1987;325(6106):733-6.

2008;283(17):11501-8.

487-98.

Neuroscience. 1998;1(5):355-8.

246 Neurodegenerative Diseases


[50] Sato T, Dohmae N, Qi Y, Kakuda N, Misonou H, Mitsumori R, et al. Potential Link between Amyloid β-Protein 42 and C-terminal Fragment γ 49–99 of β-Amyloid Pre‐ cursor Protein. Journal of Biological Chemistry. 2003;278(27):24294-301.

[63] Rezaie P, Male D. Mesoglia & Microglia-A Historical Review of the Concept of Mon‐ onuclear Phagocytes Within the Central Nervous System. Journal of the History of

Dropping the BACE: Beta Secretase (BACE1) as an Alzheimer's Disease Intervention Target

http://dx.doi.org/10.5772/53603

249

[64] Yan SD, Roher A, Schmidt AM, Stern DM. Cellular Cofactors for Amyloid β-Peptide-Induced Cell Stress: Moving from Cell Culture to in vivo. The American Journal of

[65] Yan SD, Chen X, Fu J, Chen M, Zhu H, Roher A, et al. RAGE and Amyloid-β Peptide

[66] Tan J, Town T, Paris D, Mori T, Suo Z, Crawford F, et al. Microglial Activation Re‐ sulting from CD40-CD40L Interaction After β-Amyloid Stimulation. Science.

[67] Mrak RE, Griffin WST. Glia and Their Cytokines in Progression of Neurodegenera‐

[68] Walker DG, Lue LF. Investigations with Cultured Human Microglia on Pathogenic Mechanisms of Alzheimer's Disease and Other Neurodegenerative Diseases. Journal

[69] Bard F, Cannon C, Barbour R, Burke R-L, Games D, Grajeda H, et al. Peripherally Administered Antibodies Against Amyloid β-peptide Enter the Central Nervous Sys‐ tem and Reduce Pathology in a Mouse Model of Alzheimer Disease. Nature Medi‐

[70] Majumdar A, Chung H, Dolios G, Wang R, Asamoah N, Lobel P, et al. Degradation of Fibrillar Forms of Alzheimer's Amyloid β-peptide by Macrophages. Neurobiology

[71] Banati RB, Gehrmann J, Czech C, Mönning U, Jones LL, König G, et al. Early and Rapid de novo Synthesis of Alzheimer βA4-Amyloid Precursor Protein (APP) in Ac‐

[72] Shi J, Yang SH, Stubley L, Day AL, Simpkins JW. Hypoperfusion Induces Overex‐ pression of β-amyloid Precursor Protein mRNA in a Focal Ischemic Rodent Model.

[73] Ciallella JR, Ikonomovic MD, Paljug WR, Wilbur YI, Dixon CE, Kochanek PM, et al. Changes in Expression of Amyloid Precursor Protein and Interleukin-1beta After Ex‐ perimental Traumatic Brain Injury in Rats. Journal of Neurotrauma. 2002;19(12):

[74] Guglielmotto M, Monteleone D, Giliberto L, Fornaro M, Borghi R, Tamagno E, et al. Amyloid-β₄₂ Activates the Expression of BACE1 Through the JNK Pathway. Journal

Neurotoxicity in Alzheimer's Disease. Nature. 1996;382(6593):685-91.

the Neurosciences. 2002;11(4):325.

Pathology. 1999;155(5):1403-11.

tion. Neurobiology of Aging. 2005;26(3):349-54.

of Neuroscience Research. 2005;81(3):412-25.

tivated Microglia. Glia. 1993;9(3):199-210.

of Alzheimer's Disease. 2011;27(4):871-83.

1999;286(5448):2352-5.

cine. 2000;6(8):916-9.

of Aging. 2008;29(5):707-15.

Brain Research. 2000;853(1):1-4.

1555-67.


[63] Rezaie P, Male D. Mesoglia & Microglia-A Historical Review of the Concept of Mon‐ onuclear Phagocytes Within the Central Nervous System. Journal of the History of the Neurosciences. 2002;11(4):325.

[50] Sato T, Dohmae N, Qi Y, Kakuda N, Misonou H, Mitsumori R, et al. Potential Link between Amyloid β-Protein 42 and C-terminal Fragment γ 49–99 of β-Amyloid Pre‐

[51] Zou K, Kim D, Kakio A, Byun K, Gong J-S, Kim J, et al. Amyloid β-protein (Aβ)1–40 Protects Neurons from Damage Induced by Aβ1–42 in Culture and in Rat Brain.

[52] Duering M, Grimm MOW, Grimm HS, Schröder J, Hartmann T. Mean Age of Onset in Familial Alzheimer's Disease is Determined by Amyloid Beta 42. Neurobiology of

[53] Coles M, Bicknell W, Watson AA, Fairlie DP, Craik DJ. Solution Structure of Amy‐ loid β-Peptide(1−40) in a Water−Micelle Environment. Is the Membrane-Spanning

[54] Crescenzi O, Tomaselli S, Guerrini R, Salvadori S, D'Ursi AM, Temussi PA, et al. Sol‐ ution Structure of the Alzheimer Amyloid β-peptide (1–42) in an Apolar Microenvir‐

[55] Zhang S, Iwata K, Lachenmann MJ, Peng JW, Li S, Stimson ER, et al. The Alzheimer's Peptide Aβ Adopts a Collapsed Coil Structure in Water. Journal of Structural Biolo‐

[56] Barrow C, Zagorski M. Solution Structures of Beta Peptide and its Constituent Frag‐

[57] Ohyagi Y, Asahara H, Chui D-H, Tsuruta Y, Sakae N, Miyoshi K, et al. Intracellular Aβ42 Activates p53 Promoter: a Pathway to Neurodegeneration in Alzheimer's Dis‐

[58] Hartley DM, Walsh DM, Ye CP, Diehl T, Vasquez S, Vassilev PM, et al. Protofibrillar Intermediates of Amyloid β-Protein Induce Acute Electrophysiological Changes and Progressive Neurotoxicity in Cortical Neurons. The Journal of Neuroscience.

[59] Yankner B, Dawes L, Fisher S, Villa-Komaroff L, Oster-Granite M, Neve R. Neurotox‐ icity of a Fragment of the Amyloid Precursor Associated with Alzheimer's Disease.

[60] Shen J, Bronson RT, Chen DF, Xia W, Selkoe DJ, Tonegawa S. Skeletal and CNS De‐

[61] De Strooper B, Saftig P, Craessaerts K, Vanderstichele H, Guhde G, Annaert W, et al. Deficiency of Presenilin-1 Inhibits the Normal Cleavage of Amyloid Precursor Pro‐

[62] Wyss-Coray T, Mucke L. Inflammation in Neurodegenerative Disease—A Double-

fects in Presenilin-1-Deficient Mice. Cell. 1997;89(4):629-39.

ments: Relation to Amyloid Deposition. Science. 1991;253(5016):179-82.

Domain Where We Think It Is? Biochemistry. 1998;37(31):11064-77.

onment. European Journal of Biochemistry. 2002;269(22):5642-8.

cursor Protein. Journal of Biological Chemistry. 2003;278(27):24294-301.

Journal of Neurochemistry. 2003;87(3):609-19.

Aging. 2005;26(6):785-8.

248 Neurodegenerative Diseases

gy. 2000;130(2–3):130-41.

1999;19(20):8876-84.

Science. 1989;245(4916):417-20.

tein. Nature. 1998;391(6665):387-90.

Edged Sword. Neuron. 2002;35(3):419-32.

ease. The FASEB Journal. 2004;19(2):255-57.


[75] Davies C, Tournier C. Exploring the Function of the JNK (c-Jun N-terminal Kinase) Signalling Pathway in Physiological and Pathological Processes to Design Novel Therapeutic Strategies. Biochemical Society Transactions 2012;40(1):85-9.

[86] Walter J, Fluhrer R, Hartung B, Willem M, Kaether C, Capell A, et al. Phosphoryla‐ tion Regulates Intracellular Trafficking of β-Secretase. Journal of Biological Chemis‐

Dropping the BACE: Beta Secretase (BACE1) as an Alzheimer's Disease Intervention Target

http://dx.doi.org/10.5772/53603

251

[87] Shimizu H, Tosaki A, Kaneko K, Hisano T, Sakurai T, Nukina N. Crystal Structure of an Active Form of BACE1, an Enzyme Responsible for Amyloid β Protein Produc‐

[88] Pasternak SH, Bagshaw RD, Guiral M, Zhang S, Ackerley CA, Pak BJ, et al. Preseni‐ lin-1, Nicastrin, Amyloid Precursor Protein, and γ-Secretase Activity are Co-localized in the Lysosomal Membrane. Journal of Biological Chemistry. 2003;278(29):26687-94.

[89] Yan R, Munzner JB, Shuck ME, Bienkowski MJ. BACE2 Functions as an Alternative α-Secretase in Cells. Journal of Biological Chemistry. 2001;276(36):34019-27.

[90] Lingwood D, Simons K. Lipid Rafts As a Membrane-Organizing Principle. Science.

[91] Lin X, Koelsch G, Wu S, Downs D, Dashti A, Tang J. Human Aspartic Protease Mem‐ apsin 2 Cleaves the β-secretase Site of β-amyloid Precursor Protein. Proceedings of

[92] Zhao J, Fu Y, Yasvoina M, Shao P, Hitt B, O'Connor T, et al. β-Site Amyloid Precursor Protein Cleaving Enzyme 1 Levels Become Elevated in Neurons around Amyloid Plaques: Implications for Alzheimer's Disease Pathogenesis. The Journal of Neuro‐

[93] Coulson DTR, Beyer N, Quinn JG, Brockbank S, Hellemans J, Irvine GB, et al. BACE1 mRNA Expression in Alzheimer's Disease Postmortem Brain Tissue. Journal Of Alz‐

[94] Davies BA, Lee JRE, Oestreich AJ, Katzmann DJ. Membrane Protein Targeting to the

[95] Kang EL, Cameron AN, Piazza F, Walker KR, Tesco G. Ubiquitin Regulates GGA3 mediated Degradation of BACE1. Journal of Biological Chemistry. 2010;285(31):

[96] Koh YH, von Arnim CAF, Hyman BT, Tanzi RE, Tesco G. BACE Is Degraded via the Lysosomal Pathway. Journal of Biological Chemistry. 2005;280(37):32499-504.

[97] Sannerud R, Declerck I, Peric A, Raemaekers T, Menendez G, Zhou L, et al. ADP Ri‐ bosylation Factor 6 (ARF6) Controls Amyloid Precursor Protein (APP) Processing by Mediating the Endosomal Sorting of BACE1. Proceedings of the National Academy

[98] Obregon D, Hou H, Deng J, Giunta B, Tian J, Darlington D, et al. Soluble Amyloid Precursor Protein-α Modulates β-secretase Activity and Amyloid-β Generation. Na‐

tion. Molecular and Cellular Biology. 2008;28(11):3663-71.

the National Academy of Sciences. 2000;97(4):1456-60.

MVB/Lysosome. Chemical Reviews. 2009;109(4):1575-86.

try. 2001;276(18):14634-41.

2010;327(5961):46-50.

science. 2007;27(14):3639-49.

24108-19.

heimer's Disease: JAD. 2010;22(4):1111-22.

of Sciences. 2011;108(34):E559–E68.

ture Communicationa. 2012;3:777.


[86] Walter J, Fluhrer R, Hartung B, Willem M, Kaether C, Capell A, et al. Phosphoryla‐ tion Regulates Intracellular Trafficking of β-Secretase. Journal of Biological Chemis‐ try. 2001;276(18):14634-41.

[75] Davies C, Tournier C. Exploring the Function of the JNK (c-Jun N-terminal Kinase) Signalling Pathway in Physiological and Pathological Processes to Design Novel

[76] Benjannet S, Elagoz A, Wickham L, Mamarbachi M, Munzer JS, Basak A, et al. Posttranslational Processing of β-Secretase (β-Amyloid-converting Enzyme) and its Ecto‐

[77] Creemers JWM, Ines Dominguez D, Plets E, Serneels L, Taylor NA, Multhaup G, et al. Processing of β-Secretase by Furin and Other Members of the Proprotein Conver‐

[78] Shi X-P, Chen E, Yin K-C, Na S, Garsky VM, Lai M-T, et al. The Pro Domain of β-Secretase Does Not Confer Strict Zymogen-like Properties but Does Assist Proper Folding of the Protease Domain. Journal of Biological Chemistry. 2001;276(13):

[79] Haniu M, Denis P, Young Y, Mendiaz EA, Fuller J, Hui JO, et al. Characterization of Alzheimer's β-Secretase Protein BACE. Journal of Biological Chemistry. 2000;275(28):

[80] Hong L, Koelsch G, Lin X, Wu S, Terzyan S, Ghosh AK, et al. Structure of the Pro‐ tease Domain of Memapsin 2 (β-Secretase) Complexed with Inhibitor. Science.

[81] Charlwood J, Dingwall C, Matico R, Hussain I, Johanson K, Moore S, et al. Character‐ ization of the Glycosylation Profiles of Alzheimer's β-Secretase Protein Asp-2 Ex‐ pressed in a Variety of Cell Lines. Journal of Biological Chemistry. 2001;276(20):

[82] Hussain I, Powell D, Howlett DR, Tew DG, Meek TD, Chapman C, et al. Identifica‐ tion of a Novel Aspartic Protease (Asp 2) as β-secretase. Molecular and Cellular Neu‐

[83] Sinha S, Anderson JP, Barbour R, Basi GS, Caccaveffo R, Davis D, et al. Purification and Cloning of Amyloid Precursor Protein β-secretase from Human Brain. Nature.

[84] Yan R, Blenkowski MJ, Shuck ME, Miao H, Tory MC, Pauley AM, et al. Membraneanchored Aspartyl Protease with Alzheimer's Disease β-secretase Activity. Nature.

[85] Lin X, Koelsch G, Wu S, Downs D, Dashti A, Tang J. Human Aspartic Protease Mem‐ apsin 2 Cleaves the β-secretase Site of β-amyloid Precursor Protein. Proceedings of the National Academy of Sciences of the United States of America. 2000;97(4):

Therapeutic Strategies. Biochemical Society Transactions 2012;40(1):85-9.

domain Shedding. Journal of Biological Chemistry. 2001;276(14):10879-87.

tase Family. Journal of Biological Chemistry. 2001;276(6):4211-7.

10366-73.

250 Neurodegenerative Diseases

21099-106.

16739-48.

2000;290(5489):150-3.

rosciences. 1999;14(6):419-27.

1999;402(6761):537-40.

1999;402(6761):533-7.

1456-60.


[99] Sun X, Wang Y, Qing H, Christensen MA, Liu Y, Zhou W, et al. Distinct Transcrip‐ tional Regulation and Function of the Human BACE2 and BACE1 Genes. The FASEB Journal. 2005;19(7):739-49.

[112] Louvi A, Artavanis-Tsakonas S. Notch Signalling in Vertebrate Neural Development.

Dropping the BACE: Beta Secretase (BACE1) as an Alzheimer's Disease Intervention Target

http://dx.doi.org/10.5772/53603

253

[113] Grüninger-Leitch F, Schlatter D, Küng E, Nelböck P, Döbeli H. Substrate and Inhibi‐ tor Profile of BACE (β-Secretase) and Comparison with Other Mammalian Aspartic

[114] Nguyen J-T, Hamada Y, Kimura T, Kiso Y. Design of Potent Aspartic Protease Inhibi‐ tors to Treat Various Diseases. Archiv der Pharmazie. 2008;341(9):523-35.

[115] Hansen RA, Gartlehner G, Webb AP, Morgan LC, Moore CG, Jonas DE. Efficacy and Safety of Donepezil, Galantamine, and Rivastigmine for the Treatment of Alzheim‐ er's Disease: A Systematic Review and Meta-analysis. Dove Medical Press. 2008;3(2):

[116] Ghosh AK, Kumaragurubaran N, Hong L, Koelsh G, Tang J. Memapsin 2 (Beta-Secre‐ tase) Inhibitors: Drug Development. Current Alzheimer Research. 2008;5(2):121-31.

[117] Román GC, Kalaria RN. Vascular Determinants of Cholinergic Deficits in Alzheimer Disease and Vascular Dementia. Neurobiology of Aging. 2006;27(12):1769-85.

[118] Hellström-Lindahl E, Moore H, Nordberg A. Increased Levels of Tau Protein in SH-SY5Y Cells After Treatment with Cholinesterase Inhibitors and Nicotinic Agonists.

[119] Chalmers K, Wilcock G, Vinters H, Perry E, Perry R, Ballard C, et al. Cholinesterase Inhibitors may Increase Phosphorylated Tau in Alzheimer's Disease. Journal of Neu‐

[120] Danysz W, Parsons C, MÖbius H-J, StÖffler A, Quack G. Neuroprotective and Symp‐ tomatological Action of Memantine Relevant for Alzheimer's Disease — a Unified Glutamatergic Hypothesis on the Mechanism of Action. Neurotoxicity Research.

[121] Orrego F, Villanueva S. The Chemical Nature of the Main Central Excitatory Trans‐ mitter: A Critical Appraisal Based Upon Release Studies and Synaptic Vesicle Locali‐

[122] Lipton SA, Rosenberg PA. Excitatory Amino Acids as a Final Common Pathway for Neurologic Disorders. New England Journal of Medicine. 1994;330(9):613-22.

[123] Luo X, Yan R. Inhibition of BACE1 for Therapeutic use in Alzheimer's Disease. Inter‐ national Journal of Clinical and Experimental Pathology. 2010;3(6):618-28.

[124] Guo T, Hobbs DW. Development of BACE1 Inhibitors for Alzheimer's Disease. Cur‐

[125] Baxter EW, Conway KA, Kennis L, Bischoff F, Mercken MH, De Winter HL, et al. 2- Amino-3,4-dihydroquinazolines as Inhibitors of BACE-1 (β-Site APP Cleaving En‐

Proteases. Journal of Biological Chemistry. 2002;277(7):4687-93.

Nature Reviews Neuroscience. 2006;7(2):93-102.

Journal of Neurochemistry. 2000;74(2):777-84.

zation. Neuroscience. 1993;56(3):539-55.

rent Medicinal Chemistry. 2006;13(15):1811-29.

rology. 2009;256(5):717-20.

2000;2(2):85-97.

211-25.


[112] Louvi A, Artavanis-Tsakonas S. Notch Signalling in Vertebrate Neural Development. Nature Reviews Neuroscience. 2006;7(2):93-102.

[99] Sun X, Wang Y, Qing H, Christensen MA, Liu Y, Zhou W, et al. Distinct Transcrip‐ tional Regulation and Function of the Human BACE2 and BACE1 Genes. The FASEB

[100] Farzan M, Schnitzler CE, Vasilieva N, Leung D, Choe H. BACE2, a β-secretase Ho‐ molog, Cleaves at the β Site and Within the Amyloid-β Region of the Amyloid-β Pre‐ cursor Protein. Proceedings of the National Academy of Sciences.2000;97(17):9712-7.

[101] Luo Y, Bolon B, Damore MA, Fitzpatrick D, Liu H, Zhang J, et al. BACE1 (β-secre‐ tase) Knockout Mice do not Acquire Compensatory Gene Expression Changes or De‐

[102] Bennett BD, Babu-Khan S, Loeloff R, Louis J-C, Curran E, Citron M, et al. Expression Analysis of BACE2 in Brain and Peripheral Tissues. Journal of Biological Chemistry.

[103] von Arnim CAF, Kinoshita A, Peltan ID, Tangredi MM, Herl L, Lee BM, et al. The Low Density Lipoprotein Receptor-related Protein (LRP) is a Novel β-Secretase

[104] Li Q, Südhof TC. Cleavage of Amyloid-β Precursor Protein and Amyloid-β Precur‐ sor-like Protein by BACE 1. Journal of Biological Chemistry. 2004;279(11):10542-50.

[105] Lichtenthaler SF, Dominguez D-i, Westmeyer GG, Reiss K, Haass C, Saftig P, et al. The Cell Adhesion Protein P-selectin Glycoprotein Ligand-1 is a Substrate for the As‐

partyl Protease BACE1. Journal of Biological Chemistry. 2003;278(49):48713-9.

[106] Kuhn P-H, Marjaux E, Imhof A, De Strooper B, Haass C, Lichtenthaler SF. Regulated Intramembrane Proteolysis of the Interleukin-1 Receptor II by α-, β-, and γ-secretase.

[107] Hemming ML, Elias JE, Gygi SP, Selkoe DJ. Identification of β-Secretase (BACE1)

[108] Dominguez D, Tournoy J, Hartmann D, Huth T, Cryns K, Deforce S, et al. Phenotypic and Biochemical Analyses of BACE1- and BACE2-deficient Mice. Journal of Biologi‐

[109] Hitt B, Jaramillo T, Chetkovich D, Vassar R. BACE1-/- Mice Exhibit Seizure Activity that does not Correlate with Sodium Channel Level or Axonal Localization. Molecu‐

[110] Siman R, Reaume AG, Savage MJ, Trusko S, Lin Y-G, Scott RW, et al. Presenilin-1 P264L Knock-In Mutation: Differential Effects on Aβ Production, Amyloid Deposi‐ tion, and Neuronal Vulnerability. The Journal of Neuroscience. 2000;20(23):8717-26.

[111] Flood DG, Reaume AG, Dorfman KS, Lin Y-G, Lang DM, Trusko SP, et al. FAD Mu‐ tant PS-1 Gene-Targeted Mice: Increased Aβ42 and Aβ Deposition Without APP

Overproduction. Neurobiology of Aging. 2002;23(3):335-48.

Substrates Using Quantitative Proteomics. PLoS ONE. 2009;4(12):e8477.

Journal of Biological Chemistry. 2007;282(16):11982-95.

cal Chemistry. 2005;280(35):30797-806.

lar Neurodegeneration. 2010;5(1):31.

(BACE1) Substrate. Journal of Biological Chemistry. 2005;280(18):17777-85.

velop Neural Lesions Over Time. Neurobiology of Disease. 2003;14(1):81-8.

Journal. 2005;19(7):739-49.

252 Neurodegenerative Diseases

2000;275(27):20647-51.


zyme):  Use of Structure Based Design to Convert a Micromolar Hit into a Nanomolar Lead. Journal of Medicinal Chemistry. 2007;50(18):4261-4.

[137] Ghersi-Egea JF, Leninger-Muller B, Suleman G, Siest G, Minn A. Localization of Drug-metabolizing Enzyme Activities to Blood-Brain Interfaces and Circumventricu‐

Dropping the BACE: Beta Secretase (BACE1) as an Alzheimer's Disease Intervention Target

http://dx.doi.org/10.5772/53603

255

[138] Meyer RP, Gehlhaus M, Knoth R, Volk B. Expression and Function of Cytochrome p450 in Brain Drug Metabolism. Current Drug Metabolism. 2007;8(4):297-306. [139] Bauer B, Hartz AMS, Lucking JR, Yang X, Pollack GM, Miller DS. Coordinated Nu‐ clear Receptor Regulation of the Efflux Transporter, Mrp2, and the Phase-II Metabo‐ lizing Enzyme, GST[pi], at the Blood-brain Barrier. Journal of Cerebral Blood Flow

[140] Fischer H, Gottschlich R, Seelig A. Blood-Brain Barrier Permeation: Molecular Pa‐ rameters Governing Passive Diffusion. Journal of Membrane Biology. 1998;165(3):

[141] Träuble H. The Movement of Molecules Across Lipid Membranes: A Molecular

[142] Nadal A, Fuentes E, Pastor J, McNaughton PA. Plasma Albumin is a Potent Trigger of Calcium Signals and DNA Synthesis in Astrocytes. Proceedings of the National

[143] Pardridge WM. Blood–brain Barrier Delivery. Drug Discovery Today. 2007;12(1–2):

[144] Tuszynski MH, Thal L, Pay M, Salmon DP, U HS, Bakay R, et al. A Phase 1 Clinical Trial of Nerve Growth Factor Gene Therapy for Alzheimer Disease. Nature Medi‐

[145] Bodor N, Prokai L, Wu W, Farag H, Jonalagadda S, Kawamura M, et al. A Strategy for Delivering Peptides into the Central Nervous System by Sequential Metabolism.

[146] Kastin AJ, Akerstrom V, Hackler L. Food Deprivation Decreases Blood Galanin-like Peptide and its Rapid Entry into the Brain. Neuroendocrinology. 2001;74(6):423-32.

[147] Simpson IA, Carruthers A, Vannucci SJ. Supply and Demand in Cerebral Energy Me‐ tabolism: the Role of Nutrient Transporters. Journal of Cerebral Blood Flow Metabo‐

[148] Duffy KR, Pardridge WM. Blood-brain Barrier Transcytosis of Insulin in Developing

[149] Fishman JB, Rubin JB, Handrahan JV, Connor JR, Fine RE. Receptor-mediated Trans‐ cytosis of Transferrin Across the Blood-brain Barrier. Journal of Neuroscience Re‐

[150] Reinhardt RR, Bondy CA. Insulin-like Growth Factors Cross the Blood-brain Barrier.

Academy of Sciences of the United States of America. 1995;92(5):1426-30.

lar Organs. Journal of Neurochemistry. 1994;62(3):1089-96.

Theory. Journal of Membrane Biology. 1971;4(1):193-208.

Metabolism. 2008;28(6):1222-34.

201-11.

54-61.

cine. 2005;11(5):551-5.

Science. 1992;257(5077):1698-700.

Rabbits. Brain Research. 1987;420(1):32-8.

lism. 2007;27(11):1766-91.

search. 1987;18(2):299-304.

Endocrinology. 1994;135(5):1753-61.


[137] Ghersi-Egea JF, Leninger-Muller B, Suleman G, Siest G, Minn A. Localization of Drug-metabolizing Enzyme Activities to Blood-Brain Interfaces and Circumventricu‐ lar Organs. Journal of Neurochemistry. 1994;62(3):1089-96.

zyme):  Use of Structure Based Design to Convert a Micromolar Hit into a

[126] Cole DC, Manas ES, Stock JR, Condon JS, Jennings LD, Aulabaugh A, et al. Acylgua‐ nidines as Small-Molecule β-Secretase Inhibitors. Journal of Medicinal Chemistry.

[127] Edwards PD, Albert JS, Sylvester M, Aharony D, Andisik D, Callaghan O, et al. Ap‐ plication of Fragment-Based Lead Generation to the Discovery of Novel, Cyclic Ami‐ dine β-Secretase Inhibitors with Nanomolar Potency, Cellular Activity, and High

[128] Hussain I, Hawkins J, Harrison D, Hille C, Wayne G, Cutler L, et al. Oral Administra‐ tion of a Potent and Selective Non-peptidic BACE-1 Inhibitor Decreases β-cleavage of Amyloid Precursor Protein and Amyloid-β Production in vivo. Journal of Neuro‐

[129] Iserloh U, Pan J, Stamford AW, Kennedy ME, Zhang Q, Zhang L, et al. Discovery of an Orally Efficaceous 4-phenoxypyrrolidine-based BACE-1 Inhibitor. Bioorganic and

[130] Kortum SW, Benson TE, Bienkowski MJ, Emmons TL, Prince DB, Paddock DJ, et al. Potent and Selective Isophthalamide S2 Hydroxyethylamine Inhibitors of BACE1. Bi‐

[131] Marcin LR, Higgins MA, Zusi FC, Zhang Y, Dee MF, Parker MF, et al. Synthesis and SAR of Indole-and 7-azaindole-1,3-dicarboxamide Hydroxyethylamine Inhibitors of

[132] Stachel SJ, Coburn CA, Steele TG, Jones KG, Loutzenhiser EF, Gregro AR, et al. Struc‐ ture-Based Design of Potent and Selective Cell-Permeable Inhibitors of Human β-Sec‐

[133] Stachel SJ, Coburn CA, Sankaranarayanan S, Price EA, Pietrak BL, Huang Q, et al. Macrocyclic Inhibitors of β-Secretase:  Functional Activity in an Animal Model. Jour‐

[134] Tung JS, Davis DL, Anderson JP, Walker DE, Mamo S, Jewett N, et al. Design of Sub‐ strate-Based Inhibitors of Human β-Secretase. Journal of Medicinal Chemistry.

[135] Armulik A, Genove G, Mae M, Nisancioglu MH, Wallgard E, Niaudet C, et al. Peri‐

[136] Brightman MW, Reese TS. Junctions Between Intinately Apposed Cell Membranes in

cytes Regulate the Blood-brain Barrier. Nature. 2010;468(7323):557-61.

the Verterbrate Brain. The Journal of Cell Biology. 1969;40(3):648-77.

oorganic and Medicinal Chemistry Letters. 2007;17(12):3378-83.

BACE-1. Bioorganic & Medicinal Chemistry Letters. 2011;21(1):537-41.

retase (BACE-1). Journal of Medicinal Chemistry. 2004;47(26):6447-50.

Ligand Efficiency. Journal of Medicinal Chemistry. 2007;50(24):5912-25.

Nanomolar Lead. Journal of Medicinal Chemistry. 2007;50(18):4261-4.

2006;49(21):6158-61.

254 Neurodegenerative Diseases

chemistry. 2007;100(3):802-9.

Medicinal Chemistry Letters. 2008;18(1):418-22.

nal of Medicinal Chemistry. 2006;49(21):6147-50.

2001;45(2):259-62.


[151] Tadayoni BM, Friden PM, Walus LR, Musso GF. Synthesis, in vitro Kinetics and in vivo Studies on Protein Conjugates of AZT: Evaluation as a Transport System to In‐ crease Brain Delivery. Bioconjugate Chemistry. 1993;4(2):139-45.

**Chapter 11**

**QSAR Analysis of Purine-Type and Propafenone-Type**

Therapy for central nervous system (CNS) diseases requires drugs that can cross the bloodbrain barrier (BBB) (Cheng et al, 2010). BBB not only maintains the homeostasis of the CNS, but also refuses many potentially important diagnostic and therapeutic agents from entering into the brain (Chen et al, 2009). The pathogenesis of Alzheimer's disease (AD) senile plaque and neurofibrillary tangle lesions putatively involves a compromised BBB (Jevnes & Provias, 2011), whichprotectsthebrainagainstendogenousandexogenouscompoundsandplaysanimportant part in the maintenance of the microenvironment of the brain (Vogelgesang et al, 2011). The ability of drug permeating across BBB becomes critical in the development of new medicines, especially in the design of new drugs which are active in brain tissue. In particular, the importance of brain-to-blood transport of brain-derived metabolites across the BBB has gained increasing attention as a potential mechanism in the pathogenesis of neurodegenerative disorders such as Parkinson's disease (Bartels, 2011) and AD characterized by the aberrant polymerization and accumulation of specific misfolded proteins, particularly β-amyloid (Aβ), a neuropathological hallmark of AD. P-glycoprotein (P-gp or MDR1/ABCB1) is a 170-kDa transmembraneprotein widely expressedfrom the epithelial cells ofthe intestine, liver, kidney, placenta, uterus, andtestis to endothelial cells oftheBBB(Gottesman&Pastan, 1993).It belongs to the ABC (ATP-binding cassette) transporter family and serves to pump exogenous substan‐ ces out of the cells (Suresh et al, 1999). The domain topology of P-gp consist of two homolo‐ gous halves each consist a transmembrane domain preceding a cytosolic nucleotide binding domain. Each transmembrane domain is composed of six transmembrane α-helix segments involved in efflux as well as in drug binding (Kast et al, 1996). The ABC transport protein Pgp, a major component of the BBB, mediates the efflux of Aβ from the brain as well as a major

> © 2013 Yang and Chen; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Substrates of P-Glycoprotein Targeting β-Amyloid**

**Clearance**

Jie Yang and Jie Chen

http://dx.doi.org/10.5772/54975

**1. Introduction**

Additional information is available at the end of the chapter


## **QSAR Analysis of Purine-Type and Propafenone-Type Substrates of P-Glycoprotein Targeting β-Amyloid Clearance**

Jie Yang and Jie Chen

[151] Tadayoni BM, Friden PM, Walus LR, Musso GF. Synthesis, in vitro Kinetics and in vivo Studies on Protein Conjugates of AZT: Evaluation as a Transport System to In‐

[152] Penichet ML, Kang Y-S, Pardridge WM, Morrison SL, Shin S-U. An Antibody-Avidin Fusion Protein Specific for the Transferrin Receptor Serves as a Delivery Vehicle for Effective Brain Targeting: Initial Applications in Anti-HIV Antisense Drug Delivery

[153] Huwyler J, Wu D, Pardridge WM. Brain Drug Delivery of Small Molecules Using Im‐ munoliposomes. Proceedings of the National Academy of Sciences. 1996;93(24):

[154] Doi A, Kawabata S, Iida K, Yokoyama K, Kajimoto Y, Kuroiwa T, et al. Tumor-specif‐ ic Targeting of Sodium Borocaptate (BSH) to Malignant Glioma by Transferrin-PEG Liposomes: a Modality for Boron Neutron Capture Therapy. Journal of Neuro-Oncol‐

[155] Bosi S, Da Ros T, Spalluto G, Prato M. Fullerene Derivatives: an Attractive Tool for Biological Applications. European Journal of Medicinal Chemistry. 2003;38(11–12):

[156] Basso AS, Frenkel D, Quintana FJ, Costa-Pinto FA, Petrovic-Stojkovic S, Puckett L, et al. Reversal of Axonal Loss and Disability in a Mouse Model of Progressive Multiple

[157] Huang SS, Tsai SK, Chih CL, Chiang L-Y, Hsieh HM, Teng CM, et al. Neuroprotec‐ tive Effect of Hexasulfobutylated C60 on Rats Subjected to Focal Cerebral Ischemia.

[158] Yang DY, Wang MF, Chen IL, Chan YC, Lee MS, Cheng FC. Systemic Administration of a Water-soluble Hexasulfonated C60 (FC4S) Reduces Cerebral Ischemia-induced

[159] Henley DB, May PC, Dean RA, Siemers ER. Development of Semagacestat (LY450139), a Functional γ-secretase Inhibitor, for the Treatment of Alzheimer's Dis‐

[160] Forman M, Tseng J, Palcza J, Leempoels J, Ramael S, Krishna G, et al. The Novel BACE Inhibitor MK-8931 Dramatically Lowers CSF Aβ Peptides in Healthy Subjects: Results from a Rising Single Dose Study (PL02.004) 64th American Academy of Neu‐

[161] Ming GL, Song H. Adult Neurogenesis in the Mammalian Central Nervous System.

Sclerosis. The Journal of Clinical Investigation. 2008;118(4):1532-43.

Infarct Volume in Gerbils. Neuroscience Letters. 2001;311(2):121-4.

ease. Expert Opinion on Pharmacotherapy. 2009;10(10):1657-64.

rology Annual Meeting; 2012; New Orleans: Neurology.

Annual Review of Neuroscience. 2005;28:223-50.

Free Radical Biology and Medicine. 2001;30(6):643-9.

crease Brain Delivery. Bioconjugate Chemistry. 1993;4(2):139-45.

to the Brain. The Journal of Immunology. 1999;163(8):4421-6.

14164-9.

256 Neurodegenerative Diseases

913-23.

ogy. 2008;87(3):287-94.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54975

### **1. Introduction**

Therapy for central nervous system (CNS) diseases requires drugs that can cross the bloodbrain barrier (BBB) (Cheng et al, 2010). BBB not only maintains the homeostasis of the CNS, but also refuses many potentially important diagnostic and therapeutic agents from entering into the brain (Chen et al, 2009). The pathogenesis of Alzheimer's disease (AD) senile plaque and neurofibrillary tangle lesions putatively involves a compromised BBB (Jevnes & Provias, 2011), whichprotectsthebrainagainstendogenousandexogenouscompoundsandplaysanimportant part in the maintenance of the microenvironment of the brain (Vogelgesang et al, 2011). The ability of drug permeating across BBB becomes critical in the development of new medicines, especially in the design of new drugs which are active in brain tissue. In particular, the importance of brain-to-blood transport of brain-derived metabolites across the BBB has gained increasing attention as a potential mechanism in the pathogenesis of neurodegenerative disorders such as Parkinson's disease (Bartels, 2011) and AD characterized by the aberrant polymerization and accumulation of specific misfolded proteins, particularly β-amyloid (Aβ), a neuropathological hallmark of AD. P-glycoprotein (P-gp or MDR1/ABCB1) is a 170-kDa transmembraneprotein widely expressedfrom the epithelial cells ofthe intestine, liver, kidney, placenta, uterus, andtestis to endothelial cells oftheBBB(Gottesman&Pastan, 1993).It belongs to the ABC (ATP-binding cassette) transporter family and serves to pump exogenous substan‐ ces out of the cells (Suresh et al, 1999). The domain topology of P-gp consist of two homolo‐ gous halves each consist a transmembrane domain preceding a cytosolic nucleotide binding domain. Each transmembrane domain is composed of six transmembrane α-helix segments involved in efflux as well as in drug binding (Kast et al, 1996). The ABC transport protein Pgp, a major component of the BBB, mediates the efflux of Aβ from the brain as well as a major

© 2013 Yang and Chen; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

factor in mediating resistance to brain entry by numerous exogenous chemicals, including therapeutic pharmaceuticals (Bendayan et al, 2002). P-gp plays a role in the etiology of AD through the clearance of Aβ from the brain. Some drugs, such as rifampicin, dexamethasone, caffeine, verapamil, hyperforin, β-estradiol and pentylenetetrazole, were able to improve the efflux of Aβ from the cells via P-gp up-regulation (Abuznait et al, 2011). Meanwhile, some compounds have been shown to reverse the P-gp mediated multidrug resistance (MDR), includingverapamil,adriamycin, cyclosporin,anddexverapamil(Kothandanetal,2011).Harta et al (2010) have shown that up-regulate P-gp in the early stages of AD has the potential to increase Aβ clearance from the brain and reduce Aβ brain accumulation by a transgenic mouse model of AD (human amyloid precursor protein-overexpressing mice). Abuznait et al (2011) have also elucidated the impact of P-gp up-regulation on the clearance of Aβ, which indicat‐ ed targeting Aβ clearance via P-gp up-regulation effective in slowing or halting the progres‐ sion of AD and the possibility of P-gp as a potential therapeutic target for AD.

pounds, indole alkaloids, cyclic peptides and macrolide compounds, flayanoids and miscel‐ laneous compounds (Wang et al, 2003), which mostly share common structural features, such as aromatic ring structures and high lipophilicity. Some of them possess MDR reversing activity. But only a small number of them have entered clinical study and classification of candidate drugs as substrates or inhibitors of the carrier protein is of crucial importance in

QSAR Analysis of Purine-Type and Propafenone-Type Substrates of P-Glycoprotein Targeting β-Amyloid Clearance

http://dx.doi.org/10.5772/54975

259

On the other hand, the prerequisite to cure neurological disorders is that the drug distribution in CNS can reach effectively therapeutic concentrations (Chen et al, 2009). Usually, the high BBB penetration is needed for drugs that activate in brain. The molecule negotiating the BBB must go through cellular membranes comprising of a lipid bilayer. Until now, it is widely accepted that interaction of compounds with P-gp is a complex process and at this time the details of its mechanism of action are still the subject on hot debate. Although the experimental analysis of drug permeability is essential but the procedure of experiment is time consuming and complicated, a theoretical model of drug permeability is effective to give predictions. Membrane-interaction (MI)-QSAR (quantitative structure-activity relationship) method is a structure-based design methodology combined with classic intramolecular QSAR analysis to model chemically and structurally diverse compounds interacting with cellular membranes. Our modified MI-QSAR method that combines QSAR with solute-membrane-water complex simulating the BBB environment is more close to the body condition than MI-QSAR and possesses higher ability to predict organic compounds across BBB (Chen & Yang, 2006). Before we construct any QSAR models, several things should be consider seriously first. There are several critical assumptions that can influence validity and correctness of any QSAR study as follows: the same mechanism of action of all studied analogs; a comparable manner of their binding to the receptor; correlation of binding to the interaction energies; correlation of measured biological activities to the binding affinities (Kubinyi, 1995). All the accuracy answer and research based on the questions above may guarantee that proper and reliable relation‐ ships are obtained. However, in case of MDR modulators different mechanisms and different binding sites may be involved. Several screening assays can help in the identification of substrates and inhibitors although they have both advantages and drawbacks, such as cytotoxity assays (Wiese & Pajeva, 2001), inhibition of efflux assays (Stouch & Gudrmundsson,

2001), P-gp-ATPase activation assays, and drug transport assays (Taub et al, 2005).

The goal of a QSAR study is to find a means of predicting the activity of a new compound. If possible, a desirable goal is the understanding of the biology and chemistry that give rise to that activity and the consequential possibility of reengineering the compound to remove or enhance that activity. One successful example is the transformation of nalidixic acid with the help of QSAR into an important family of drug: the quinolone carboxylates, such as norflox‐ acin, fleroxacin, ciprofloxacin, and levofloxacin (Alka, 2003). Since the method was established in the 1960s, QSAR equations have been used to describe the biological activities of thousands of different drugs and drug candidates (Kuo et al, 2004). The method definitely provides a more accurate way to synthesize or filtrate the new chemical compounds. At last, the final destination is to degrade the cost of research and manufacture. To date, so many methods have been used in QSAR study and some of them have got successful results. There are general

drug development (Wang et al, 2005).

P-gp at the BBB functions as an active efflux pump by extruding a substrate from the brain, which is important for maintaining loco-regional homeostasis in the brain and protection against toxic compounds (Bartels, 2011). P-gp is also discovered in various resistant tumor cells and expressed widely in many normal tissues and plays a very important role in drug ADME-Tox (absorption, distribution, metabolism, excretion, and toxicity). MDR is a matter of growing concern in chemotherapy. Cells which express the multidrug resistance phenotype can overexpress efflux transporters after exposure to a single agent. As a result, these cells become resistant to the selective agent and cross-resistant to a broad spectrum of structurally and functionally dissimilar drugs. The drug efflux pump P-gp has been shown to promote MDR in tumors as well as to influence ADME properties of drug candidates (Jabben et al, 2012). Pgp is expressed at the BBB, the blood-cerebrospinal fluid barrier, and the intestinal barrier, thus modulating the absorption and excretion of xenobiotics across these barriers. P-gp and its ligands (substrates and inhibitors) are therefore extensively studied both with respect to reversing MDR in tumors and for modifying ADME-Tox properties of drug candidates, such as CNS active agents (Jabben et al, 2012). P-gp possesses broad substrate specificity and substrates include members of many clinically important therapeutic drug classes, including anti-HIV protease inhibitors, calcium channel blockers used in the treatment of angina, hypertension, antibiotics and cancer chemotherapeutics (Stouch & Gudmundsson, 2002). In this active efflux process, energy originating from ATP hydrolysis is directly consumed. Because of such a wide distribution of P-gp, so if a drug such as quinidine or verapamil inhibits the function of P-gp, it will also inhibit the excretion of digoxin by P-gp leading to increased plasma levels and toxicity due to digoxin. It is believed to be an important protective mecha‐ nism against environmental toxins (Martin, 2004). Since the function of P-gp always results in lack of intracellular levels of the drug necessary for effective therapy, the overexpression of Pgp in certain malignant cells is always associated with MDR phenotype (Sharom, 1997). Although recently low resolution structure of P-gp is obtained, its physiological function and mechanisms of MDR modulation are still not very clear (Li et al, 2005). It is well known that a large number of structurally and functionally diverse compounds act as substrates or modu‐ lators of P-gp, including calcium and sodium channel blockers, calmodulin antagonists and structural analogues, protein kinase C inhibitors, steroidal and structurally related com‐ pounds, indole alkaloids, cyclic peptides and macrolide compounds, flayanoids and miscel‐ laneous compounds (Wang et al, 2003), which mostly share common structural features, such as aromatic ring structures and high lipophilicity. Some of them possess MDR reversing activity. But only a small number of them have entered clinical study and classification of candidate drugs as substrates or inhibitors of the carrier protein is of crucial importance in drug development (Wang et al, 2005).

factor in mediating resistance to brain entry by numerous exogenous chemicals, including therapeutic pharmaceuticals (Bendayan et al, 2002). P-gp plays a role in the etiology of AD through the clearance of Aβ from the brain. Some drugs, such as rifampicin, dexamethasone, caffeine, verapamil, hyperforin, β-estradiol and pentylenetetrazole, were able to improve the efflux of Aβ from the cells via P-gp up-regulation (Abuznait et al, 2011). Meanwhile, some compounds have been shown to reverse the P-gp mediated multidrug resistance (MDR), includingverapamil,adriamycin, cyclosporin,anddexverapamil(Kothandanetal,2011).Harta et al (2010) have shown that up-regulate P-gp in the early stages of AD has the potential to increase Aβ clearance from the brain and reduce Aβ brain accumulation by a transgenic mouse model of AD (human amyloid precursor protein-overexpressing mice). Abuznait et al (2011) have also elucidated the impact of P-gp up-regulation on the clearance of Aβ, which indicat‐ ed targeting Aβ clearance via P-gp up-regulation effective in slowing or halting the progres‐

P-gp at the BBB functions as an active efflux pump by extruding a substrate from the brain, which is important for maintaining loco-regional homeostasis in the brain and protection against toxic compounds (Bartels, 2011). P-gp is also discovered in various resistant tumor cells and expressed widely in many normal tissues and plays a very important role in drug ADME-Tox (absorption, distribution, metabolism, excretion, and toxicity). MDR is a matter of growing concern in chemotherapy. Cells which express the multidrug resistance phenotype can overexpress efflux transporters after exposure to a single agent. As a result, these cells become resistant to the selective agent and cross-resistant to a broad spectrum of structurally and functionally dissimilar drugs. The drug efflux pump P-gp has been shown to promote MDR in tumors as well as to influence ADME properties of drug candidates (Jabben et al, 2012). Pgp is expressed at the BBB, the blood-cerebrospinal fluid barrier, and the intestinal barrier, thus modulating the absorption and excretion of xenobiotics across these barriers. P-gp and its ligands (substrates and inhibitors) are therefore extensively studied both with respect to reversing MDR in tumors and for modifying ADME-Tox properties of drug candidates, such as CNS active agents (Jabben et al, 2012). P-gp possesses broad substrate specificity and substrates include members of many clinically important therapeutic drug classes, including anti-HIV protease inhibitors, calcium channel blockers used in the treatment of angina, hypertension, antibiotics and cancer chemotherapeutics (Stouch & Gudmundsson, 2002). In this active efflux process, energy originating from ATP hydrolysis is directly consumed. Because of such a wide distribution of P-gp, so if a drug such as quinidine or verapamil inhibits the function of P-gp, it will also inhibit the excretion of digoxin by P-gp leading to increased plasma levels and toxicity due to digoxin. It is believed to be an important protective mecha‐ nism against environmental toxins (Martin, 2004). Since the function of P-gp always results in lack of intracellular levels of the drug necessary for effective therapy, the overexpression of Pgp in certain malignant cells is always associated with MDR phenotype (Sharom, 1997). Although recently low resolution structure of P-gp is obtained, its physiological function and mechanisms of MDR modulation are still not very clear (Li et al, 2005). It is well known that a large number of structurally and functionally diverse compounds act as substrates or modu‐ lators of P-gp, including calcium and sodium channel blockers, calmodulin antagonists and structural analogues, protein kinase C inhibitors, steroidal and structurally related com‐

sion of AD and the possibility of P-gp as a potential therapeutic target for AD.

258 Neurodegenerative Diseases

On the other hand, the prerequisite to cure neurological disorders is that the drug distribution in CNS can reach effectively therapeutic concentrations (Chen et al, 2009). Usually, the high BBB penetration is needed for drugs that activate in brain. The molecule negotiating the BBB must go through cellular membranes comprising of a lipid bilayer. Until now, it is widely accepted that interaction of compounds with P-gp is a complex process and at this time the details of its mechanism of action are still the subject on hot debate. Although the experimental analysis of drug permeability is essential but the procedure of experiment is time consuming and complicated, a theoretical model of drug permeability is effective to give predictions. Membrane-interaction (MI)-QSAR (quantitative structure-activity relationship) method is a structure-based design methodology combined with classic intramolecular QSAR analysis to model chemically and structurally diverse compounds interacting with cellular membranes. Our modified MI-QSAR method that combines QSAR with solute-membrane-water complex simulating the BBB environment is more close to the body condition than MI-QSAR and possesses higher ability to predict organic compounds across BBB (Chen & Yang, 2006). Before we construct any QSAR models, several things should be consider seriously first. There are several critical assumptions that can influence validity and correctness of any QSAR study as follows: the same mechanism of action of all studied analogs; a comparable manner of their binding to the receptor; correlation of binding to the interaction energies; correlation of measured biological activities to the binding affinities (Kubinyi, 1995). All the accuracy answer and research based on the questions above may guarantee that proper and reliable relation‐ ships are obtained. However, in case of MDR modulators different mechanisms and different binding sites may be involved. Several screening assays can help in the identification of substrates and inhibitors although they have both advantages and drawbacks, such as cytotoxity assays (Wiese & Pajeva, 2001), inhibition of efflux assays (Stouch & Gudrmundsson, 2001), P-gp-ATPase activation assays, and drug transport assays (Taub et al, 2005).

The goal of a QSAR study is to find a means of predicting the activity of a new compound. If possible, a desirable goal is the understanding of the biology and chemistry that give rise to that activity and the consequential possibility of reengineering the compound to remove or enhance that activity. One successful example is the transformation of nalidixic acid with the help of QSAR into an important family of drug: the quinolone carboxylates, such as norflox‐ acin, fleroxacin, ciprofloxacin, and levofloxacin (Alka, 2003). Since the method was established in the 1960s, QSAR equations have been used to describe the biological activities of thousands of different drugs and drug candidates (Kuo et al, 2004). The method definitely provides a more accurate way to synthesize or filtrate the new chemical compounds. At last, the final destination is to degrade the cost of research and manufacture. To date, so many methods have been used in QSAR study and some of them have got successful results. There are general methods used in the literatures these years, such as multiple linear regression (MLR) method, partial least square regression (PLSR) (Li et al, 2005), MI-QSAR analysis (Chen & Yang, 2006), and three-dimension (3D) QSAR (Cramer et al, 1988), and artificial neural network (ANN) (Chen et al, 2006). In order to get more accurate results and QSAR models, we have used two different analyses: MLR and PLSR. Moreover, we focus on constructing theoretical models of the interaction between organic compounds and P-gp as well as the predictive models of bloodbrain barrier partitioning of organic compounds on the basis of QSAR analysis and MI-QSAR analysis.

**No. Structure in vitro reversal fold**

N N N N HN

N N N N HN

N N N N HN

N N N N NH N H2 C CH2

N N N N HN

> N N N N HN

N N N N HN

> N N N N NH

> N N N N HN

N NH CH2

N NH CH2

<sup>N</sup> NH SO2

N NH CH2

N NH CH2 O2 S N CH3

N CH3

O CH3

N NH CH2

N NH CH2

N NH CH2 O2 S N CH3

A1

A2

A3

A4

A5

A6

A7

A8

A9

**reversion (MDR ratio)**

QSAR Analysis of Purine-Type and Propafenone-Type Substrates of P-Glycoprotein Targeting β-Amyloid Clearance

50 171 A19

78 278 A20

75 238 A21

53 236 A22

236 160 A23

93 208 A24

30 120 A26

57 75 A27

Cl 124 102 A25

**P388/ VCR-20** **No. Structure in vitro reversal fold**

**KB-A1 P388/VCR-20 KB-A1**

N N N N

N H <sup>N</sup> <sup>H</sup> N CH2 F F

N N N N

N H <sup>N</sup> <sup>H</sup> N CH2 O2S N CH3

N N N N

N N N N

N H <sup>N</sup> <sup>H</sup> N CH2

N N N N

N H <sup>N</sup> <sup>H</sup> N CH2

N N N N

N H N NH

F

N N N N

N N CH

F

N NH CH2

F

N N CH

N N N N HN

> N N N N HN

F

N H

N H <sup>N</sup> <sup>H</sup> N CH2

O

**reversion (MDR ratio)**

261

36 49

http://dx.doi.org/10.5772/54975

35 113

CH3 133 200

193 189

24 142

13 6

24 9

<sup>O</sup> 84 406

Cl 70 214

### **2. Materials and methods**

#### **2.1. P-glycoprotein ligands**

**Building of some compounds** 36 purine derivatives were selected and used in QSAR analysis (table 1) (Dhainaut et al, 1996). These compounds were divided into two sets: the training set and the test set. The study of the MDR-reversing properties of these derivatives was carried out in vitro on P388/VCR-20 cells, a murine leukemia cell line whose resistance was induced by vincristine (VCR), and KB-A1 cells, a human epidermoid carcinoma cell line whose resistance was induced by adriamycin (ADR). The compounds were tested at four concentra‐ tions (0.5−5 µM) in association with VCR (P388/VCR-20 cells) or ADR (KB-A1 cells). In this test, MDR ratio in P388/VDR-20 and KB-A1 in vitro was used as biological activity for the

whole dataset, namely *MDRratio* <sup>=</sup> *IC*50(*CD*) *IC*50(*CD* <sup>+</sup> mod) . Here "CD" is the abbreviation for cytotoxic

drug (such as VCR and ADR) in cytotoxity assays, and "mod" means modulators. It is defined as ratio between the IC50 values (concentration that inhibits the growth of MDR cells by 50%) of the cytotoxic agent in absence and presence of relatively nontoxic concentration of the modifier (Wiese & Pajeva, 2001). Most often the IC50 for several concentration of a cytotoxic drug is evaluated in the presence and absence of a nontoxic concentration of a P-gp modifier. In this assay modulators that interact with P-gp and thus, reduce the efflux of the cytotoxic compounds, will increase the apparent toxicity of the cytotoxic compound. It is important to keep in mind that it is based on a general assessment of cytotoxicity and thus may account for more then one acting mechanism in the resistant cells used (Stouch & Gudmundsso,. 2001). Furthermore, it is well known that the MDR ratio for any given compound can vary greatly depending on the cell type used for the assay as well as the intrinsic cytotoxicity of the compounds used. The data is also dependent on the concentration of the P-gp substrates or modulators used in the studies (Ford et al, 1990).

Similarly, another 21 propafenone analogs were selected from the literature of Diethart Schmid et al (1999) and used in QSAR analysis (table 2). In this test Ka of P-gp ATPase in the adria‐ mycin-resistant subline CCRF ADR5000 was used as biological activity for the whole dataset (Schmid et al, 1999). The assays were performed based on the colorimetric determination of inorganic phosphate released by the hydrolysis of ATP. Table 2 shows all the structures and the experimental biological activity value.

QSAR Analysis of Purine-Type and Propafenone-Type Substrates of P-Glycoprotein Targeting β-Amyloid Clearance http://dx.doi.org/10.5772/54975 261

methods used in the literatures these years, such as multiple linear regression (MLR) method, partial least square regression (PLSR) (Li et al, 2005), MI-QSAR analysis (Chen & Yang, 2006), and three-dimension (3D) QSAR (Cramer et al, 1988), and artificial neural network (ANN) (Chen et al, 2006). In order to get more accurate results and QSAR models, we have used two different analyses: MLR and PLSR. Moreover, we focus on constructing theoretical models of the interaction between organic compounds and P-gp as well as the predictive models of bloodbrain barrier partitioning of organic compounds on the basis of QSAR analysis and MI-QSAR

**Building of some compounds** 36 purine derivatives were selected and used in QSAR analysis (table 1) (Dhainaut et al, 1996). These compounds were divided into two sets: the training set and the test set. The study of the MDR-reversing properties of these derivatives was carried out in vitro on P388/VCR-20 cells, a murine leukemia cell line whose resistance was induced by vincristine (VCR), and KB-A1 cells, a human epidermoid carcinoma cell line whose resistance was induced by adriamycin (ADR). The compounds were tested at four concentra‐ tions (0.5−5 µM) in association with VCR (P388/VCR-20 cells) or ADR (KB-A1 cells). In this test, MDR ratio in P388/VDR-20 and KB-A1 in vitro was used as biological activity for the

drug (such as VCR and ADR) in cytotoxity assays, and "mod" means modulators. It is defined as ratio between the IC50 values (concentration that inhibits the growth of MDR cells by 50%) of the cytotoxic agent in absence and presence of relatively nontoxic concentration of the modifier (Wiese & Pajeva, 2001). Most often the IC50 for several concentration of a cytotoxic drug is evaluated in the presence and absence of a nontoxic concentration of a P-gp modifier. In this assay modulators that interact with P-gp and thus, reduce the efflux of the cytotoxic compounds, will increase the apparent toxicity of the cytotoxic compound. It is important to keep in mind that it is based on a general assessment of cytotoxicity and thus may account for more then one acting mechanism in the resistant cells used (Stouch & Gudmundsso,. 2001). Furthermore, it is well known that the MDR ratio for any given compound can vary greatly depending on the cell type used for the assay as well as the intrinsic cytotoxicity of the compounds used. The data is also dependent on the concentration of the P-gp substrates or

Similarly, another 21 propafenone analogs were selected from the literature of Diethart Schmid et al (1999) and used in QSAR analysis (table 2). In this test Ka of P-gp ATPase in the adria‐ mycin-resistant subline CCRF ADR5000 was used as biological activity for the whole dataset (Schmid et al, 1999). The assays were performed based on the colorimetric determination of inorganic phosphate released by the hydrolysis of ATP. Table 2 shows all the structures and

*IC*50(*CD* <sup>+</sup> mod) . Here "CD" is the abbreviation for cytotoxic

analysis.

260 Neurodegenerative Diseases

**2. Materials and methods**

whole dataset, namely *MDRratio* <sup>=</sup> *IC*50(*CD*)

modulators used in the studies (Ford et al, 1990).

the experimental biological activity value.

**2.1. P-glycoprotein ligands**



**No. Structure Ka(μM/L) LogP No. Structure Ka(μM/L) LogP**

QSAR Analysis of Purine-Type and Propafenone-Type Substrates of P-Glycoprotein Targeting β-Amyloid Clearance

O N OH OCH3

O OH N N F

O

OH O OH N

O

OH <sup>O</sup> <sup>N</sup>

OCH3 <sup>O</sup> <sup>N</sup>

O N OH O CH3

> O N OH OH

O

O OH N OH

<sup>O</sup> <sup>N</sup> OH

1.53 4.3

http://dx.doi.org/10.5772/54975

263

1.47 4.93

7.64 4.25

10.5 2.38

12.8 3.94

<sup>N</sup> <sup>F</sup>4.15 4.93

<sup>N</sup> <sup>F</sup> 0.55 5.2

OH 12.2 4.52

OH 2.26 4.88

3.34 3.39 A45

5.3 3.62 A46

2.59 3.67 A47

122 1.42 A48

<sup>N</sup> <sup>F</sup> 0.36 4.93 A49

<sup>N</sup> <sup>F</sup> 6.13 2.67 A50

<sup>O</sup> 120 0.94 A51

<sup>O</sup> 18.5 2.54 A52

1.01 3.98 A53

**Table 2.** The structures and Ka values and LogP of 18 propafenone analogs in the training/test sets.

A36 <sup>O</sup>

A37

A38

A39

A40

A41

A42

A43

A44

OH O

O OH O N CH3 CH3

O OH O N

O N OH O CH3

O N OH O

O N OH O CH3

> O N OH O CH3

O N OH O

O N OH O

OH

CH3

Note: Ratio of IC50 (cytotoxic alone (VCR for P388/VCR-20, ADR for KB-A1 cells))/IC50 (cytotoxic + modulator) (1μM in association with VCR or 2.5 μM in association with ADR) (Dhainaut et al, 1996).

**Table 1.** The structures and MDR ratios of 35 purine derivatives in the training/test sets.

QSAR Analysis of Purine-Type and Propafenone-Type Substrates of P-Glycoprotein Targeting β-Amyloid Clearance http://dx.doi.org/10.5772/54975 263

**No. Structure in vitro reversal fold**

CH3 CH3

A10

A11

A12

A13

A14

A15

A16

A17

A18

N N N N HN

262 Neurodegenerative Diseases

N N N N HN

N N N N HN

N N N N HN

N N N N HN

N N N N HN

> N N N N HN

> N N N N HN

> N N N N HN

N NH CH2

N NH CH2

N NH CH2

N NH CH2

N NH CH2

<sup>N</sup> NH CH2 OCH3

N NH

N NH

N NH CH2

O

O

**reversion (MDR ratio)**

108 136 A28

15 83 A30

78 272 A31

CH3 56 147 A32

CH3 75 152 A33

51 209 A34

70 171 A35

Note: Ratio of IC50 (cytotoxic alone (VCR for P388/VCR-20, ADR for KB-A1 cells))/IC50 (cytotoxic + modulator) (1μM in

129 156

association with VCR or 2.5 μM in association with ADR) (Dhainaut et al, 1996).

**Table 1.** The structures and MDR ratios of 35 purine derivatives in the training/test sets.

<sup>O</sup> 37 44 A29

**P388/ VCR-20** **No. Structure in vitro reversal fold**

N CH3 Cl

**KB-A1 P388/VCR-20 KB-A1**

<sup>N</sup> NH SO2

N NH CH2

N N N N HN

> N N N HN

N N N N HN

N NH H2 C C

N N N N

N N N N NH

N N N N HN

> N N N N N HN

N H <sup>N</sup> <sup>H</sup> N CH2

N NH CH2 F F

N NH H2 <sup>C</sup> NH O

N NH CH2

N N N N HN

N N CH

F

F

**reversion (MDR ratio)**

57 68

108 723

27 370

288 210

59 121

71 499

13 264

3 5


**Table 2.** The structures and Ka values and LogP of 18 propafenone analogs in the training/test sets.

Finally, all two-dimensional structures of these compounds mentioned above were construct‐ ed using the chemical drawing software ChemDraw 8.0 and prepared for the next calculation.

With the aid of Chemoffice Chem3D Ultra 8.0 and Hyperchem 7.5, we calculated the following descriptors by the procedure in detail below: (1) Draw the structures in ChemDraw 8.0; (2) Change structures to 3D by Chem3D; (3) Considering our chosen compounds, minimize the energy of the molecule based on molecular mechanism MM2 Force Field (Because under the

QSAR Analysis of Purine-Type and Propafenone-Type Substrates of P-Glycoprotein Targeting β-Amyloid Clearance

the number of atoms.). We have chosen the job type as minimize energy to minimum RMS (root mean square) Gradient of 0.100 (the default value of 0.100 is a reasonable compromise between accuracy and speed). (4) Under the menu of Analyze-compute properties, select the

**QSAR models** QSAR model of some purine derivatives (table 1) are achieved by partial sum of squares for regression with software SPSS 10.0. Some biological activity data are so large or small that the group of data cannot form a normal school, which is very important in lineal regression, and will surely degrade the accuracy of QSAR equations. So we discarded several data out of the normal school and some without necessary descriptors value. A training set of 26 structurally diverse purine derivatives are measured is used to construct QSAR models. The QSAR models are optimized using MLR fitting and stepwise method (Eq.1-Eq.5). A test set of five compounds is evaluated using the QSAR models as part of a validation process. Take MDR ratio in vitro in P388/VDR cell lines as dependent variable and molecule descriptors as independent variable. With the aid of Virtual Computational Chemistry Laboratory

Similarly, a training set of 18 structurally diverse propafenone analogs (table 2) are measured is used to construct QSAR models. The QSAR models are optimized using MLR fitting and stepwise method (Eq.7-Eq.11). Another QSAR modeling was constructed by PLSR (Eg. 12). A test set of five compounds is evaluated using the QSAR models as part of a validation process.

**Building of some compounds** 37 organic compounds (Abraham et al, 1995; Abraham et al, 1997) were elected, composed a train set, and another 8 organic compounds were acted as a test set (table 3). The dependent variable used in this predictive model is the logarithm of the BBB partition coefficient, log BB = log (Cbrain / Cblood), where Cbrain is the concentration of the test compound in the brain, and Cblood is the concentration of the test compound in blood. Experi‐ mental values of log BB published to date lie approximately between -2.00 to +1.04. Com‐ pounds with log BB values of > 0.30 are readily distributed to the brain whereas compounds with values < -1.00 are poorly distributed to the brain. Building of all these compounds was performed on a PC computer using the Build modules of the commercial software packages Hyperchem 7.5. First, the geometry of these compounds was opitimized using the Amber 94 force field in gas state. Second, they were placed at a periodic solvent box whose volume was X=16Å, Y=10Å, Z=18Å, which included 96 water molecules. Here, temperature is 300ºK and pressure is 1 standard atmosphere. Then, the compounds in water were minimized by the above method. Third, the compounds in water were simulated by Monte Carlo method and

, where N is

265

http://dx.doi.org/10.5772/54975

MM2 force field, the time required for performing computations increases as N2

properties to calculate and get every descriptor value of each compound.

software (Wang et al, 2005), QSAR modeling was constructed by PLSR (Eg. 6).

**2.2. Blood-brain-barrier**

minimized by the above method.

**Calculation of some descriptors** Molecular descriptors are "numbers that characterize a specific aspect of the molecular structure" (Karelson, 2000). There are a great number of molecular descriptors that can be used in QSAR studies in the structure parameterization form, which include physicochemical properties (such as hydrophobicity, aqueous solubili‐ ty, molecular electronegativity, and molecular refractivity), quantum chemical parameters (i.g. atomic charges, energies of HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital)) (Karelson & Lobanov, 1996), topological indexes (i.e. molecular connectivity indexes) (Ponce et al, 2004), and other three-dimentional (3D) descriptors. Molecular descriptors were mostly calculated by the commercial software packages Chemoffice Chem3D Ultra 8.0, which included molecular mechanism parameters (Bending Energy (Ebend), Stretch-Bend Energy (Estretch), Torsion Energy (Etorsion), Total Energy (Etotal), van der Waals Energy (EVDW), etc), quantum chemistry parameters (i.e. Electronic Energy (Eelectronic), HOMO Energy (EHOMO) and LUMO Energy (ELUMO)), hydrophobic param‐ eters (such as Clog P), stereo parameters (eg. Es, Balaban Index (BI), Connolly Accessible Area (CAA), Molecular Weight (MW), Shape Attribute (ShA), Total Connectivity (Tcon), and Wiener Index (WI)), thermodynamic parameters, including Henry's Law Constant (H), Hydration Energy (Ehyd), Logarithm of partition coefficient in n-octanol/water (LogP), Molar Refractivity (MR), and molecular polar surface area (PSA). PSA is defined as the surface area (Å2 ) occupied by polar atoms, usually oxygen, nitrogen and hydrogen attached to them, which will restrict molecule penetration into the membranes (Chen et al, 2009). The other properties involved in number of hydrogen bond acceptor (NBA) and number of hydro‐ gen bond donor (NBD).

The energy parameters root in the results of molecular mechanism and molecular dynamics. The total energy of a system expressed as follows (Iyer et al 2002): *Etotal* =*Evalence* + *Ecrossterm* + *Enonbone*.

Here, the valence interactions includes bond stretching (bond), valence angle bending (angle), dihedral angle torsion (torsion), and inversion, also called out-of-plane interactions (oop) terms, which are part of nearly all forcefields for covalent systems. A Urey-Bradley term (UB) may be used to account for interactions between atom pairs involved in 1-3 configurations (i.e., atoms bound to a common atom): *Evalence* =*Ebond* + *Eangle* + *Etorsion* + *Eoop* + *EUP*. Modern (secondgeneration) forcefields generally achieve higher accuracy by including cross terms to account for such factors as bond or angle distortions caused by nearby atoms. Crossterms can include the following terms: stretch-stretch, stretch-bend-stretch, bend-bend, torsion-stretch, torsionbend-bend, bend-torsion-bend, stretch-torsion-stretch. The interaction energy between nonbonded atoms is accounted by van der Waals (VDW), electrostatic (Coulomb), and hydrogen bond (hbond) terms in some older forcefields. *Enon*−*bond* = *EVDW* + *ECoulomb* + *Ehbond* . Restraints that can be added to an energy expression include distance, angle, torsion, and inversion restraints. Restraints are useful if you, for example, are interested in only part of a structure for information on restraints and their implementation and use, and also the documentation for the particular simulation engine.

With the aid of Chemoffice Chem3D Ultra 8.0 and Hyperchem 7.5, we calculated the following descriptors by the procedure in detail below: (1) Draw the structures in ChemDraw 8.0; (2) Change structures to 3D by Chem3D; (3) Considering our chosen compounds, minimize the energy of the molecule based on molecular mechanism MM2 Force Field (Because under the MM2 force field, the time required for performing computations increases as N2 , where N is the number of atoms.). We have chosen the job type as minimize energy to minimum RMS (root mean square) Gradient of 0.100 (the default value of 0.100 is a reasonable compromise between accuracy and speed). (4) Under the menu of Analyze-compute properties, select the properties to calculate and get every descriptor value of each compound.

**QSAR models** QSAR model of some purine derivatives (table 1) are achieved by partial sum of squares for regression with software SPSS 10.0. Some biological activity data are so large or small that the group of data cannot form a normal school, which is very important in lineal regression, and will surely degrade the accuracy of QSAR equations. So we discarded several data out of the normal school and some without necessary descriptors value. A training set of 26 structurally diverse purine derivatives are measured is used to construct QSAR models. The QSAR models are optimized using MLR fitting and stepwise method (Eq.1-Eq.5). A test set of five compounds is evaluated using the QSAR models as part of a validation process. Take MDR ratio in vitro in P388/VDR cell lines as dependent variable and molecule descriptors as independent variable. With the aid of Virtual Computational Chemistry Laboratory software (Wang et al, 2005), QSAR modeling was constructed by PLSR (Eg. 6).

Similarly, a training set of 18 structurally diverse propafenone analogs (table 2) are measured is used to construct QSAR models. The QSAR models are optimized using MLR fitting and stepwise method (Eq.7-Eq.11). Another QSAR modeling was constructed by PLSR (Eg. 12). A test set of five compounds is evaluated using the QSAR models as part of a validation process.

#### **2.2. Blood-brain-barrier**

Finally, all two-dimensional structures of these compounds mentioned above were construct‐ ed using the chemical drawing software ChemDraw 8.0 and prepared for the next calculation. **Calculation of some descriptors** Molecular descriptors are "numbers that characterize a specific aspect of the molecular structure" (Karelson, 2000). There are a great number of molecular descriptors that can be used in QSAR studies in the structure parameterization form, which include physicochemical properties (such as hydrophobicity, aqueous solubili‐ ty, molecular electronegativity, and molecular refractivity), quantum chemical parameters (i.g. atomic charges, energies of HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital)) (Karelson & Lobanov, 1996), topological indexes (i.e. molecular connectivity indexes) (Ponce et al, 2004), and other three-dimentional (3D) descriptors. Molecular descriptors were mostly calculated by the commercial software packages Chemoffice Chem3D Ultra 8.0, which included molecular mechanism parameters (Bending Energy (Ebend), Stretch-Bend Energy (Estretch), Torsion Energy (Etorsion), Total Energy (Etotal), van der Waals Energy (EVDW), etc), quantum chemistry parameters (i.e. Electronic Energy (Eelectronic), HOMO Energy (EHOMO) and LUMO Energy (ELUMO)), hydrophobic param‐ eters (such as Clog P), stereo parameters (eg. Es, Balaban Index (BI), Connolly Accessible Area (CAA), Molecular Weight (MW), Shape Attribute (ShA), Total Connectivity (Tcon), and Wiener Index (WI)), thermodynamic parameters, including Henry's Law Constant (H), Hydration Energy (Ehyd), Logarithm of partition coefficient in n-octanol/water (LogP), Molar Refractivity (MR), and molecular polar surface area (PSA). PSA is defined as the surface area

) occupied by polar atoms, usually oxygen, nitrogen and hydrogen attached to them, which will restrict molecule penetration into the membranes (Chen et al, 2009). The other properties involved in number of hydrogen bond acceptor (NBA) and number of hydro‐

The energy parameters root in the results of molecular mechanism and molecular dynamics. The total energy of a system expressed as follows (Iyer et al 2002):

Here, the valence interactions includes bond stretching (bond), valence angle bending (angle), dihedral angle torsion (torsion), and inversion, also called out-of-plane interactions (oop) terms, which are part of nearly all forcefields for covalent systems. A Urey-Bradley term (UB) may be used to account for interactions between atom pairs involved in 1-3 configurations (i.e.,

generation) forcefields generally achieve higher accuracy by including cross terms to account for such factors as bond or angle distortions caused by nearby atoms. Crossterms can include the following terms: stretch-stretch, stretch-bend-stretch, bend-bend, torsion-stretch, torsionbend-bend, bend-torsion-bend, stretch-torsion-stretch. The interaction energy between nonbonded atoms is accounted by van der Waals (VDW), electrostatic (Coulomb), and hydrogen bond (hbond) terms in some older forcefields. *Enon*−*bond* = *EVDW* + *ECoulomb* + *Ehbond* . Restraints that can be added to an energy expression include distance, angle, torsion, and inversion restraints. Restraints are useful if you, for example, are interested in only part of a structure for information on restraints and their implementation and use, and also the documentation

+ *EUP*. Modern (second-

atoms bound to a common atom): *Evalence* =*Ebond* + *Eangle* + *Etorsion* + *Eoop*

(Å2

gen bond donor (NBD).

264 Neurodegenerative Diseases

*Etotal* =*Evalence* + *Ecrossterm* + *Enonbone*.

for the particular simulation engine.

**Building of some compounds** 37 organic compounds (Abraham et al, 1995; Abraham et al, 1997) were elected, composed a train set, and another 8 organic compounds were acted as a test set (table 3). The dependent variable used in this predictive model is the logarithm of the BBB partition coefficient, log BB = log (Cbrain / Cblood), where Cbrain is the concentration of the test compound in the brain, and Cblood is the concentration of the test compound in blood. Experi‐ mental values of log BB published to date lie approximately between -2.00 to +1.04. Com‐ pounds with log BB values of > 0.30 are readily distributed to the brain whereas compounds with values < -1.00 are poorly distributed to the brain. Building of all these compounds was performed on a PC computer using the Build modules of the commercial software packages Hyperchem 7.5. First, the geometry of these compounds was opitimized using the Amber 94 force field in gas state. Second, they were placed at a periodic solvent box whose volume was X=16Å, Y=10Å, Z=18Å, which included 96 water molecules. Here, temperature is 300ºK and pressure is 1 standard atmosphere. Then, the compounds in water were minimized by the above method. Third, the compounds in water were simulated by Monte Carlo method and minimized by the above method.


**No Structure LogBB No Structure LogBB**

QSAR Analysis of Purine-Type and Propafenone-Type Substrates of P-Glycoprotein Targeting β-Amyloid Clearance

0.44 B33 H3C

0.14 B34 H3C

0.22 B35 <sup>F</sup>

H3C -0.06 B36 CH3 0.37


1.01 T6 <sup>H</sup>

0.90 T7 H3C CH3

F F F

Cl 0.35

http://dx.doi.org/10.5772/54975

OH -0.16

0.93

267

0.27

0.34

0.04

0.76


Br

Br F F F

Cl

H HH

H3C CH3 O


similar to Stouch's research results (Bassolino-Klimas et al,

<sup>O</sup> OH -0.02 B32

B13

B14

B15

B16

B17

B18

B19

N

<sup>O</sup> <sup>H</sup> N N S

O NH

O NH

NN H3C

> N N NH2

H3C O

H3C

CH3

CH3

**Table 3.** The structures and LogBB values of some compounds in the training/test sets.

H3C N H3C

T1 CH3

T2 H3C CH3

T3 CH3

T4 3C OH CH3

area of each lipid molecule 64Å2

N S

N O

NH O

0.25

**Test set**

0.00 T8

**Molecular modeling of a DMPC monolayer membrane complex with a layer of water.** A model of dimyristoylphosphatidylcholine (DMPC) monolayer membrane was constructed using the software Material Studio, and minimized for 200 steps with the smart minimizer. The DMPC monolayer membrane was composed of 25 DMPC molecules (5×5×1). Here, the parameter of the single crystal of DMPC was a=8Å, b=8Å, and *γ* =96.0 , which resulted average

1993). Moreover, we add a layer of water (40×40×10) including 529 water molecules to the

N

N

N

QSAR Analysis of Purine-Type and Propafenone-Type Substrates of P-Glycoprotein Targeting β-Amyloid Clearance http://dx.doi.org/10.5772/54975 267

**No Structure LogBB No Structure LogBB Training set**




0.11 B24



0.49 B25 CH3






N H3C H3C

N

CH3 HO CH3

H3C H3C OH

H3C

CH3

CH3 H3C H3C

F F Cl

> Cl Cl Cl

Cl F O F F F F

F F O CH2

H HH H HH

0.85

0.03

0.37



0.97

1.04

0.08

0.40

0.24

0.13

OH -0.16

B1

B3

B4

B6

B7

B8

B9

B10

B11

B12

B2 <sup>N</sup>

266 Neurodegenerative Diseases

NH2 2N NH S N

> H NNO HN

> > NH N HN O

N H N N H

N Cl

O H3C

N <sup>N</sup> HN H2N

> NH2 H2N N S N

> H2N NH2 N S N

H2N NH2 N S N

N

N

N CH3 H3C

N HN Cl

NN

CH3 N CH3 N

NH2

HN CH3 O

<sup>O</sup> <sup>H</sup> N CH3 O

<sup>O</sup> <sup>H</sup> N O

O

N CH3

N C H 3

O CH3

O S

H3C N H3C

H3C <sup>N</sup> CH3 O S

B5 <sup>H</sup>


**Table 3.** The structures and LogBB values of some compounds in the training/test sets.

**Molecular modeling of a DMPC monolayer membrane complex with a layer of water.** A model of dimyristoylphosphatidylcholine (DMPC) monolayer membrane was constructed using the software Material Studio, and minimized for 200 steps with the smart minimizer. The DMPC monolayer membrane was composed of 25 DMPC molecules (5×5×1). Here, the parameter of the single crystal of DMPC was a=8Å, b=8Å, and *γ* =96.0 , which resulted average area of each lipid molecule 64Å2 similar to Stouch's research results (Bassolino-Klimas et al, 1993). Moreover, we add a layer of water (40×40×10) including 529 water molecules to the polar side of the DMPC monolayer membrane. Figure 1 showed the dominant conformation of B1 compound colored by atom-type in water. The red box denotes the water solvent box defined in Monte Carlo simulation.

used to construct QSAR models. The QSAR models were optimized using MLR fitting and stepwise method by the SPSS software (Eq.1-Eq.5). A test set of 5 compounds (compound A27- A31) was evaluated using the models as part of a validation process (figure 2 upper, Table 5).

QSAR Analysis of Purine-Type and Propafenone-Type Substrates of P-Glycoprotein Targeting β-Amyloid Clearance

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269

Meanwhile, take MDR ratio in vitro in P388/VDR cell lines as dependent variable and molecule descriptors as independent variable. With the aid of Virtual Computational Chemistry Laboratory software (http://vcclab.org) (Wang et al, 2005), construct QSAR modeling by PLSR (Eg.6, figure 2 down). Table 6 shows the calculated descriptors mentioned above and the result

**Figure 2.** Comparison of the experimental MDR values with the corresponding predicted MDR values. Upper: MDR value in KB-A1/ADR cell lines (blue rhombic dots); MDR as predicted by Eg.4 MLR model (red square dots) and by Eg.5 MLR model (yellow triangle dots) for all the molecules of the training and test set. Down: MDR value in P388/VDR cell lines (blue rhombic dots); MDR as predicted by the method of PLSR (Eg. 6) (red square dots) for all the molecules of the training and test set. The rhombic dots represented the experimental values (P388) and the predicted values of

6.537 7.16

37.830 48.862 0.49

*LogMDR* =- + - *LogMR ShA* 9

*LogMDR* =- + <sup>2</sup>*LogMR* (1)

(2)

= 27; R= 0.445; F=6.187

of predicted value was in Table 5.

MDR, respectively.

N

N= 27; R= 0.889; F=45.415

**Figure 1.** The dominant conformation of B1 compound colored by atom-type in water. The red box denotes the water solvent box defined in Monte Carlo simulation.

**Molecular dynamic simulation of a small molecule complex with DMPC-water model.** A DMPC molecule at the center of the above DMPC monolayer membrane complex with a layer of water was replaced with an organic compound to form a solute-membrane-water complex. The center organic compound was inserted at three different positions in the DMPC-water model before the start of each of the three corresponding molecular dynamics simulation. Molecular dynamic simulation of the complex was performed for 1000 steps by Discover module with Materials Studios, using Compass force field. Here, the three-dimensional volume was restricted to a border of X=40Å, Y=40Å, Z=91.76Å, and *γ* =96.0 .

**QSAR model of BBB partitioning of some compounds.** MI-QSAR model of some organic compounds through BBB are achieved by partial sum of squares for regression with software SPSS. A training set of 37 structurally diverse compounds whose BBB partition coefficients are measured is used to construct QSAR models. Molecular dynamics simulations are used to determine the explicit interaction of each test compound with a model of DMPC monolayer membrane complex with a layer of water. An additional set of intramolecular solute descrip‐ tors are computed and considered in the trial pool of descriptors for building MI-QSAR models. The QSAR models are optimized using multidimensional linear regression fitting and stepwise method. A test set of eight compounds is evaluated using the MI-QSAR models as part of a validation process.

#### **3. Results**

#### **3.1. QSAR analysis based on MDR ratio in P388/VDR-20 and KB-A1 in vitro**

Take MDR ratio in vitro in KB-A1/ADR cell lines as dependent variable and molecule descrip‐ tors as independent variable. A training set of 26 structurally diverse compounds (Table 4) was used to construct QSAR models. The QSAR models were optimized using MLR fitting and stepwise method by the SPSS software (Eq.1-Eq.5). A test set of 5 compounds (compound A27- A31) was evaluated using the models as part of a validation process (figure 2 upper, Table 5).

polar side of the DMPC monolayer membrane. Figure 1 showed the dominant conformation of B1 compound colored by atom-type in water. The red box denotes the water solvent box

**Figure 1.** The dominant conformation of B1 compound colored by atom-type in water. The red box denotes the water

**Molecular dynamic simulation of a small molecule complex with DMPC-water model.** A DMPC molecule at the center of the above DMPC monolayer membrane complex with a layer of water was replaced with an organic compound to form a solute-membrane-water complex. The center organic compound was inserted at three different positions in the DMPC-water model before the start of each of the three corresponding molecular dynamics simulation. Molecular dynamic simulation of the complex was performed for 1000 steps by Discover module with Materials Studios, using Compass force field. Here, the three-dimensional

**QSAR model of BBB partitioning of some compounds.** MI-QSAR model of some organic compounds through BBB are achieved by partial sum of squares for regression with software SPSS. A training set of 37 structurally diverse compounds whose BBB partition coefficients are measured is used to construct QSAR models. Molecular dynamics simulations are used to determine the explicit interaction of each test compound with a model of DMPC monolayer membrane complex with a layer of water. An additional set of intramolecular solute descrip‐ tors are computed and considered in the trial pool of descriptors for building MI-QSAR models. The QSAR models are optimized using multidimensional linear regression fitting and stepwise method. A test set of eight compounds is evaluated using the MI-QSAR models as

volume was restricted to a border of X=40Å, Y=40Å, Z=91.76Å, and *γ* =96.0 .

**3.1. QSAR analysis based on MDR ratio in P388/VDR-20 and KB-A1 in vitro**

Take MDR ratio in vitro in KB-A1/ADR cell lines as dependent variable and molecule descrip‐ tors as independent variable. A training set of 26 structurally diverse compounds (Table 4) was

defined in Monte Carlo simulation.

268 Neurodegenerative Diseases

solvent box defined in Monte Carlo simulation.

part of a validation process.

**3. Results**

Meanwhile, take MDR ratio in vitro in P388/VDR cell lines as dependent variable and molecule descriptors as independent variable. With the aid of Virtual Computational Chemistry Laboratory software (http://vcclab.org) (Wang et al, 2005), construct QSAR modeling by PLSR (Eg.6, figure 2 down). Table 6 shows the calculated descriptors mentioned above and the result of predicted value was in Table 5.

**Figure 2.** Comparison of the experimental MDR values with the corresponding predicted MDR values. Upper: MDR value in KB-A1/ADR cell lines (blue rhombic dots); MDR as predicted by Eg.4 MLR model (red square dots) and by Eg.5 MLR model (yellow triangle dots) for all the molecules of the training and test set. Down: MDR value in P388/VDR cell lines (blue rhombic dots); MDR as predicted by the method of PLSR (Eg. 6) (red square dots) for all the molecules of the training and test set. The rhombic dots represented the experimental values (P388) and the predicted values of MDR, respectively.

$$\begin{aligned} \text{LogMDR} &= -6.537 + 7.162 \,\text{LogMR} \\ \text{N=27; R=0.445; F=6.187} \end{aligned} \tag{1}$$

$$\begin{aligned} \text{LogMDR} &= -37.830 + 48.862 \,\text{LogMR} - 0.499 \,\text{ShA} \\ \text{N=27; R=0.889; F=45.415} \end{aligned} \tag{2}$$

$$\begin{aligned} \text{LogMDR} &= -35.816 + 52.416 \text{LogMR} - 0.717 \times \text{ShA} + 6.612 \times 10^{-7} \text{ BI} \\ \text{N=27; R=0.919; F=41.442} \end{aligned} \tag{3}$$

**No. MDR ratio (KB-A1)**

**No. MDR (P388)**

**Pred MDR** **Predictive values of MDR ratio No. MDR**

A29 723 125.69 411.51 400.76 323.36 248.59

**Table 5.** The experimental values and the predictive values of MDR ratio of these compounds.

**LogP MR EVDW ShA WI No. MDR**

**(P388)**

A1 50 43.66 3.33 15.85 32.21 37.03 5476 A18 129 62.87 2.13 15.90 27.08 37.03 5476 A2 78 40.71 2.59 15.10 21.84 35.08 4872 A19 36 29.48 1.61 15.13 21.63 37.03 5585 A3 75 53.15 1.14 16.63 24.83 40.02 6522 A20 70 73.87 0.76 17.12 25.52 41.02 6855 A4 53 55.39 2.21 13.91 20.16 32.03 3916 A21 35 54.41 1.9 13.82 20.17 32.03 3874 A6 93 86.84 2.13 15.83 32.75 37.03 5476 A24 24 24.57 2.13 15.39 23.42 36.03 4855 A8 30 47.64 2.28 15.85 25.21 37.03 5476 A25 13 11.32 1.71 14.21 22.20 35.03 4487 A9 57 79.51 1.29 17.56 26.03 42.02 7353 A26 24 11.44 1.71 14.21 20.92 35.03 4538 A10 108 138.69 0.99 17.40 29.44 41.02 6935 A27 84 58.01 1.41 15.54 26.05 37.03 5476

**Pred MDR** **LogP MR EVDW ShA WI**

**ratio (KB-A1)**

**Eq.1 Eq.2 Eq.3 Eq.4 Eq.5 Eq.1 Eq.2 Eq.3 Eq.4 Eq.5**

**Training set** A1 171 114.32 215.61 200.33 123.15 180.87 A14 147 151.94 151.04 171.02 173.75 144.79 A2 278 80.79 200.54 305.22 260.52 228.39 A15 152 150.27 140.09 157.75 159.16 130.87 A3 238 161.08 71.37 78.37 79.12 92.55 A16 209 115.64 233.15 217.87 209.32 197.83 A4 236 44.84 113.11 178.94 196.99 213.61 A17 171 115.64 233.15 217.87 209.32 193.94 A5 160 150.27 140.09 157.75 168.14 148.66 A18 156 116.97 252.09 236.91 229.26 219.10 A6 208 113.02 199.36 184.18 174.42 159.21 A19 49 81.98 22.31 33.79 29.47 25.56 A7 102 163.01 77.39 67.24 80.67 105.63 A20 214 198.44 93.91 119.73 132.46 165.76 A8 120 114.32 215.61 200.33 180.88 153.75 A21 113 42.69 80.90 121.41 145.60 174.56 A9 75 237.62 101.82 179.28 149.94 146.81 A22 200 150.27 140.09 149.67 160.44 139.02 A10 136 222.49 204.96 297.19 322.30 315.82 A23 189 113.02 199.36 176.36 167.80 160.14 A11 44 79.86 58.80 41.66 49.61 53.08 A24 142 92.42 159.30 116.68 117.52 112.54 A12 83 92.42 159.30 121.35 121.70 115.60 A25 6 52.12 10.08 9.16 8.45 8.02 A13 272 53.80 124.41 185.32 190.50 197.66 A26 9 52.12 10.08 9.53 8.76 8.16 **Testset** A27 406 99.21 81.97 70.98 80.61 81.67 A30 370 243.10 375.08 504.47 282.78 192.47 A28 68 163.01 77.39 67.24 80.67 106.16 A31 210 114.32 215.61 191.82 183.83 174.21

QSAR Analysis of Purine-Type and Propafenone-Type Substrates of P-Glycoprotein Targeting β-Amyloid Clearance

**Predictive values of MDR ratio**

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271

$$\begin{aligned} \text{LogMDR} &= -38.791 + 56.923 \,\text{LogMR} - 0.769 \,\text{ShA} + 5.897 \times 10^{-7} \,\text{BI} - 0.159 \,\text{LogP} \\ \text{N} &= 27; \text{ R} = 0.927; \text{ F} = 33.504 \end{aligned} \tag{4}$$

$$\begin{aligned} \text{LogMDR} &= -42.192 + 61.818 \text{LogMR} - \\ -0.801 \text{ShA} + 4.791 \times 10^{-7} \text{BI} - 0.369 \text{LogP} + 3.595 \times 10^{-2} E\_{hyd} \\ \text{N=27; R=0.936; F=29.749} \end{aligned}$$

2 4 2 7.611 3.138 10 0.245 N=30; Q =0 0.495 .465 0.509 8.8 10 0 02 *LogMDR LogP MR E ShA VDW WI* - - =+´ - + - +´ (6)


**Table 4.** The molecular descriptors of some compounds related to MDR ratios in the training/test sets

<sup>7</sup> 35.816 52.416 0.717 6

<sup>7</sup> 38.791 56.923 0.769 5.897 10 0.15

*ShA BI LogP Ehyd*

7 2


2 4


0.495

3.595 10

**(kcal/mol) No. Log MR ShA BI LogP**

*LogMDR LogMR ShA BI* - =- + - ´ + ´

*LogMDR LogMR ShA BI LogP* -

=- + - + ´ -

.612

10

0.509 8.8 10

9

(3)

(4)

(6)

**Ehyd (kcal/mol)**

N= 27; R=

270 Neurodegenerative Diseases

N 27; R 0.927; F 33.

== =

2

.465

0

**No. Log MR ShA BI LogP**

A29 1.21 37.03 2662570 2.75 -15.55

**Table 4.** The molecular descriptors of some compounds related to MDR ratios in the training/test sets

N=30; Q =0

0.919; F=41

N= 27; R= 0.936; F=29.74

7.611 3.138 10 0.245

.442

504

42.192 61.818 0.801 4.791 10 0.369

=- + -

*LogMDR LogMR*

9

02 *LogMDR LogP MR E ShA VDW WI* - - =+´ - + - +´

**Ehyd**

**Training set** A1 1.20 37.03 2662570 3.33 -3.56 A14 1.22 39.02 3358755 1.48 -19.64 A2 1.18 35.03 2440928 2.59 -13.71 A15 1.22 39.02 3358755 1.48 -19.71 A3 1.22 40.02 3649082 1.14 -16.23 A16 1.20 37.03 2662570 2.13 -15.99 A4 1.14 32.03 1669953 2.21 -13.54 A17 1.202 37.03 2662570 2.13 -16.23 A5 1.22 39.02 3358755 1.33 -19.71 A18 1.202 37.03 2662570 2.13 -15.95 A6 1.20 37.03 2662570 2.13 -16.22 A19 1.18 37.03 3091919 1.61 -15.9 A7 1.22 40.02 3491392 0.76 -17.67 A20 1.23 41.02 4008723 0.76 -17.36 A8 1.20 37.03 2662570 2.28 -16.3 A21 1.14 32.03 1651352 1.9 -13.79 A9 1.24 42.02 4491514 1.29 -16.34 A22 1.22 39.02 3324212 1.33 -20.06 A10 1.24 41.02 4055919 0.99 -19.77 A23 1.20 37.03 2634052 2.13 -15.77 A11 1.18 36.03 2271976 1.41 -17.73 A24 1.19 36.03 2246188 2.13 -16.15 A12 1.19 36.03 2271976 2.13 -16.17 A25 1.15 35.03 2244801 1.71 -14.88 A13 1.15 33.03 1900460 2.28 -13.57 A26 1.15 35.03 2271261 1.71 -15.03 **Test set** A27 1.19 37.03 2662570 1.41 -18.09 A30 1.25 41.02 3977672 2.98 -13.52 A28 1.22 40.02 3491392 0.76 -17.61 A31 1.20 37.03 2634052 2.13 -15.95


**Table 5.** The experimental values and the predictive values of MDR ratio of these compounds.



N= 16; R= 0.860; F=39 2.424 0.

N= 16; R= 0.900; F=27.676

11

N= 16; R= 0.914; F=20.2

N= 16; R= 0.928; F=17. 1

N= 16; R=0.945; F=16.832

2

0

**(eV)**

N=18, Q =0.710

**No. LogP ShA MR EHOMO**

3.612 0.285 0.07

*LogKa* =- - *LogP ShA* 32

2.573 0.480 0.285 0

51 *LogKa* =- - + *LogP ShA MR* 51

7.313 0.752 0.647 1.642 0.60

10.021 0.875 1.044 2.263 0.673 6.734 10 *HOMO LogKa LogP ShA MR E WI* - = - - + + +´

3.662 0.279 4.71 10 1.223

**Table 7.** The molecular descriptors of some compounds related to ATPase in the training/test sets.

*HOMO LogKa LogP MW <sup>E</sup>* - - = - -´ + ´

A36 3.39 21.04 9.254 -9.14 1366 312.41 A45 4.3 26.04 11.42 -9.17 2345 383.53 A37 3.62 24.04 10.55 -9.20 1949 355.48 A46 4.93 32.03 13.27 -8.24 4689 462.57 A38 3.67 25.04 10.84 -9.16 2172 367.49 A47 5.2 32.03 13.38 -8.19 4329 464.58 A39 1.42 18.05 7.86 -9.12 920 277.37 A48 4.25 26.04 11.45 -9.24 2607 383.53 A40 4.93 32.03 13.27 -8.16 4329 462.57 A49 4.52 26.04 11.59 -8.94 2367 385.55 A41 2.67 25.04 10.29 -8.15 2244 372.44 A50 4.88 27.03 12.06 -8.94 2550 399.58 A42 0.94 19.05 8.01 -9.15 1050 293.37 A51 2.38 26.04 10.99 -9.09 2400 383.49 A43 2.54 25.04 10.52 -9.20 2172 369.46 A52 3.94 25.04 10.95 -9.05 2172 369.51 A44 3.98 32.03 13.50 -9.16 4227 459.59 A53 4.93 32.03 13.27 -8.19 4509 462.57

.

8

*HOMO LogKa* =- - + + *LogP ShA MR E* (10)

3 2

**WI MW No. LogP ShA MR EHOMO**

10

*LogKa* = - *LogP* (7)

.6

5

(8)

273

(9)

(11)

(12)

**WI MW**

4

http://dx.doi.org/10.5772/54975

**(eV)**

4

QSAR Analysis of Purine-Type and Propafenone-Type Substrates of P-Glycoprotein Targeting β-Amyloid Clearance

84 74

**Table 6.** Comparison of experimental value of MDR ratio with predicted value of MDR ratio by PLSR.

#### **3.2. QSAR analysis based on Ka of ATPase in CCRF ADR5000 cell lines**

Similarly, take Ka of ATPase in CCRF ADR5000 cell lines as dependent variable and molecule descriptors as independent variable. We construct QSAR models using two methods, MLD method (Eq.7-Eq.11) and PLSR method (Eg.12) (see figure 3).

**Figure 3.** Comparison of the experimental Ka value (blue rhombic dots) with the corresponding predicted Ka as pre‐ dicted by Eg.11 MLR model (red square dats) and by Eg.12 PLSR model (yellow triangle dots) for all the molecules of the training and test.

A training set of 16 structurally diverse compounds was used to construct QSAR models. All the molecular descriptors were calculated as Table 7. The QSAR models were optimized using MLR fitting and stepwise method. A test set of 2 compounds was evaluated using the models as part of a validation process. Table 8 displays the comparison of the experiment and prediction value.

QSAR Analysis of Purine-Type and Propafenone-Type Substrates of P-Glycoprotein Targeting β-Amyloid Clearance http://dx.doi.org/10.5772/54975 273

**No. MDR (P388)**

272 Neurodegenerative Diseases

the training and test.

prediction value.

**Pred MDR** **LogP MR EVDW ShA WI No. MDR**

**Table 6.** Comparison of experimental value of MDR ratio with predicted value of MDR ratio by PLSR.

**3.2. QSAR analysis based on Ka of ATPase in CCRF ADR5000 cell lines**

method (Eq.7-Eq.11) and PLSR method (Eg.12) (see figure 3).

A11 37 30.21 1.41 15.08 24.04 36.03 4909 A28 57 35.88 0.76 16.66 23.78 40.02 6244 A12 15 27.61 2.13 15.39 23.512 36.03 4909 A29 108 49.03 2.75 16.06 25.95 37.03 5476 A13 78 87.23 2.28 14.27 29.12 33.03 4216 A30 27 35.79 2.98 17.61 26.49 41.02 6804 A14 56 96.24 1.48 16.50 28.19 39.02 6288 A32 59 30.18 1.83 15.13 22.08 37.03 5642 A15 75 89.20 1.48 16.47 27.54 39.02 6288 A33 71 73.84 0.86 15.79 27.62 38.03 5822 A16 51 54.90 2.13 15.88 25.61 37.03 5476 A34 13 31.95 2.58 15.64 25.36 37.03 5476 A17 70 54.43 2.13 15.88 25.49 37.03 5476 A35 3 12.22 1.71 14.21 20.40 35.03 4589

Similarly, take Ka of ATPase in CCRF ADR5000 cell lines as dependent variable and molecule descriptors as independent variable. We construct QSAR models using two methods, MLD

**Figure 3.** Comparison of the experimental Ka value (blue rhombic dots) with the corresponding predicted Ka as pre‐ dicted by Eg.11 MLR model (red square dats) and by Eg.12 PLSR model (yellow triangle dots) for all the molecules of

A training set of 16 structurally diverse compounds was used to construct QSAR models. All the molecular descriptors were calculated as Table 7. The QSAR models were optimized using MLR fitting and stepwise method. A test set of 2 compounds was evaluated using the models as part of a validation process. Table 8 displays the comparison of the experiment and

**(P388)**

**Pred MDR** **LogP MR EVDW ShA WI**

$$\begin{aligned} \text{LogKa} &= 2.424 - 0.484 \,\text{Log}P\\ \text{N} &= 16; \text{ R} = 0.860; \text{ F} = 39.748 \end{aligned} \tag{7}$$

$$\begin{aligned} \text{LogKa} &= 3.612 - 0.285 \text{Log}P - 0.0732 \text{Sha}A\\ \text{N} &= 16; \text{ R} = 0.900; \text{ F=27.676} \end{aligned} \tag{8}$$

$$\begin{aligned} \text{LogKa} &= 2.573 - 0.480 \text{Log P} - 0.285 \text{Sh}A + 0.651 MR \\ \text{N} &= 16; \text{R} = 0.914; \text{F} = 20.251 \end{aligned} \tag{9}$$

$$\begin{aligned} \text{LogKa} &= 7.313 - 0.752 \,\text{Log}P - 0.647 ShA + 1.642 MR + 0.605 E\_{\text{avoo}} \\ \text{N=16; R=0.928; F=17.111} \end{aligned} \tag{10}$$

4 N= 16; R=0.945; F=16.832 10.021 0.875 1.044 2.263 0.673 6.734 10 *HOMO LogKa LogP ShA MR E WI* - = - - + + +´ (11)

$$\begin{aligned} \text{LogKa} &= 3.662 - 0.279 \text{Log P} - 4.71 \times 10^{-3} \,\text{MW} + 1.223 \times 10^{-2} \,\text{E}\_{\text{HOMO}} \\ \text{N=18, Q}^2 &= 0.7100 \end{aligned} \tag{12}$$


**Table 7.** The molecular descriptors of some compounds related to ATPase in the training/test sets.


2 2 <sup>7</sup> log 6.262 10 1.36 10 0.205 log 7.11 10 0.185

log 6.580 10 1.21 10 0.206 log 7.77 10 0.197

log 8.730 10 1.04 10 0.222 log 9.60 10 0.183


*stretch BB PSA C P BI E*

2 2 7

2 2 7

= ´ -´ + -´ - +

Here, n means the number of compounds in a training set, *R* means the correlative coefficient, and *S* means the standard residual error. Log BB = log (Cbrain/Cblood). PSA means the total polar surface area of a molecule. CLogP and BI display calculated LogP and connective index of molecular average total distance (relative covalent radius), respectively. They come from CS calculation. ΔEtotal and ΔEtorsion are related to interaction between an organic compound and membrane-water model. The total energy and the torsion energy of the DMPC monolayer membrane complex with a layer of water are -340.7589 and -1724.4164 (Kcal/mol), respectively. ΔEtotal is the change in the total potential energy of the solute-membrane-water complex


*BB PSA C P BI E*

= ´ -´ + -´ - +


*BB PSA C P BI E*

== = (16)

QSAR Analysis of Purine-Type and Propafenone-Type Substrates of P-Glycoprotein Targeting β-Amyloid Clearance

*stretch*

http://dx.doi.org/10.5772/54975

*stretch*

(17)

275

(18)

37 0.938 0.264

*nR S*

3


== =

*nR S*

*nR S*

== =

37 0.947 0.248

1.364 10 2.68 10 37 0.955 0.232

+ ´D -´D

*E*

*total*

3 3 2

**No PSA (Å2) ClogP BI(Å) Estretch (Kcal/**


*E E*

*total torsion*

comparing with that of the membrane-water model and so is ΔEtorsion.

**mol)**

**Training set** B1 78.90 1.20 12378 -1.35503 -298.2972 -1713.1146 42.46 11.30 B2 94.00 1.99 1101758 -0.15595 -406.0803 -1789.8084 -65.32 -65.39 B3 73.00 3.80 1738650 -1.48472 -256.3021 -1703.1425 84.46 21.27 B4 87.00 1.63 1346396 -1.39112 -302.7543 -1841.5635 38.00 -117.15 B5 39.00 1.02 41807 0.58131 -226.3773 -1734.7452 114.38 -10.33 B6 26.80 3.23 305770 -0.09264 -228.2923 -1679.4604 112.47 44.96 B7 88.80 1.01 58510 0.71038 -279.0781 -1671.3414 61.68 53.07 B8 76.60 2.80 62216 -0.38334 -309.2981 -1654.6730 31.46 69.74

**Etotal a (Kcal/ mol)**

**Etorsion a (Kcal/ mol)**

**ΔEtotal b (Kcal/ mol)**

**ΔEtorsion <sup>b</sup> (Kcal/mol)**

1.330 10

+ ´D

**Table 8.** Comparison the experimental values with the predictive values of Ka of these compounds.

#### **3.3. QSAR analysis based on blood-brain barrier partitioning of organic compounds**

37 organic compounds of training set and 8 compounds of test set are built and minimized, dissolved in liquid, and are optimized by Monte Carlo method and molecular mechanism, finally the dominant conformation of these compounds are obtained. Molecular modeling of a small molecule complex with the membrane-water model reveals that the energy of an organic compound inserted at the middle position in the DMPC model with a layer of water is lower than that of the other two positions. MI-QSAR analysis has been used to develop predictive models of some organic compounds through BBB, in part, simulating the interaction of an organic compound with the phospholipide-rich regions of cellular membranes sur‐ rounding by a layer of water. Molecular descriptors of compounds in a training set and a test set are listed in Table 9. Six QSAR equations were constructed based on Table 9 and were listed as follows.

$$\begin{aligned} \log BB &= 0.552 - 1.73 \times 10^{-2} \text{ PSA} \\ m &= 37 \qquad R = 0.835 \qquad S = 0.398 \end{aligned} \tag{13}$$

$$\begin{aligned} \log BB &= 0.229 - 1.70 \times 10^{-2} PSA + 0.131 \text{C} \log P \\ m &= 37 \quad R = 0.878 \quad S = 0.352 \end{aligned} \tag{14}$$

$$\begin{aligned} \text{V} \log \text{BB} &= 4.965 \times 10^{-2} - 1.28 \times 10^{-2} \text{PSA} + 0.211 \text{C} \log P - 6.40 \times 10^{-7} \text{BI} \\ \text{m} &= 37 \quad \text{R} = 0.924 \quad \text{S} = 0.285 \end{aligned} \tag{15}$$

$$\begin{aligned} \text{mg } \text{BB} &= 6.262 \times 10^{-2} - 1.36 \times 10^{-2} \text{PSA} + 0.205 \text{C} \log P - 7.11 \times 10^{-7} \text{BI} - 0.185 \text{E}\_{\text{stretch}} \\ \text{m} &= 37 \quad \text{R} = 0.938 \quad \text{S} = 0.264 \end{aligned} \tag{16}$$

**No.**

as follows.

**Ka**

274 Neurodegenerative Diseases

**(μM/L) Predictive values of Ka No.**

Eg.7 Eg.8 Eg.9 Eg.10 Eg.11 Eg.12 Eg.7 Eg.8 Eg.9 Eg.10 Eg.11 Eg.12

**Table 8.** Comparison the experimental values with the predictive values of Ka of these compounds.

**3.3. QSAR analysis based on blood-brain barrier partitioning of organic compounds**

<sup>2</sup> log 0.552 1.73 10

<sup>2</sup> log 0.229 1.70 10 0.131 log

2 2 <sup>7</sup> log 4.965 10 1.28 10 0.211 log 6.40 10


*nR S*

37 0.878 0.352

*nR S*

37 0.924 0.285

*nR S*

37 0.835 0.398 *BB PSA*

*BB PSA C P*

*BB PSA C P BI*


== = (13)

== = (14)

== = (15)


37 organic compounds of training set and 8 compounds of test set are built and minimized, dissolved in liquid, and are optimized by Monte Carlo method and molecular mechanism, finally the dominant conformation of these compounds are obtained. Molecular modeling of a small molecule complex with the membrane-water model reveals that the energy of an organic compound inserted at the middle position in the DMPC model with a layer of water is lower than that of the other two positions. MI-QSAR analysis has been used to develop predictive models of some organic compounds through BBB, in part, simulating the interaction of an organic compound with the phospholipide-rich regions of cellular membranes sur‐ rounding by a layer of water. Molecular descriptors of compounds in a training set and a test set are listed in Table 9. Six QSAR equations were constructed based on Table 9 and were listed

A36 3.34 6.07 12.75 9.37 6.43 6.14 13.57 A45 1.53 2.20 3.02 3.32 2.69 2.08 3.50 A37 5.30 4.70 6.62 7.10 6.19 5.60 7.33 A46 1.47 1.09 0.73 0.52 0.48 0.81 1.02 A38 2.59 4.44 5.41 5.36 3.98 3.06 6.24 A47 0.55 0.81 0.61 0.46 0.50 0.53 0.84 A39 122 54.54 76.92 73.09 90.64 160.46 70.37 A48 7.64 2.33 3.13 3.83 3.33 4.22 3.60 A40 0.36 1.09 0.73 0.52 0.53 0.51 1.02 A49 12.20 1.72 2.62 3.39 4.98 5.00 2.99 A41 6.13 13.54 10.43 7.17 11.79 7.23 11.56 A50 2.26 1.15 1.75 2.37 3.50 3.28 2.04 A42 120.00 93.12 89.09 81.21 80.47 99.37 80.44 A51 10.50 18.71 10.66 14.57 16.6 13.20 12.02 A43 18.50 15.65 11.36 11.73 8.26 5.56 12.59 A52 12.80 3.29 4.53 4.74 4.57 3.91 5.15 A44 1.01 3.15 1.36 2.10 1.66 2.13 1.88 A53 4.15 1.09 0.73 0.52 0.51 0.65 1.02

**Ka**

**(μM/L) Predictive values of Ka**

$$\begin{aligned} \log{BB} &= 6.580 \times 10^{-2} - 1.21 \times 10^{-2} PSA + 0.206 C \log{P} - 7.77 \times 10^{-7} BI - 0.197 E\_{stretch} + \\ &+ 1.330 \times 10^{-3} \Delta E\_{total} \\ &= 37 \quad R = 0.947 \quad S = 0.248 \end{aligned} \tag{17}$$

$$\begin{aligned} \log{BB} &= 8.730 \times 10^{-2} - 1.04 \times 10^{-2} PSA + 0.222C \log{P} - 9.60 \times 10^{-7} BI - 0.183E\_{stretch} + \\ &+ 1.364 \times 10^{-3} \Delta E\_{total} - 2.68 \times 10^{-3} \Delta E\_{torsion} \\ \ln{n} &= 37 \quad R = 0.955 \quad S^2 = 0.232 \end{aligned} \tag{18}$$

Here, n means the number of compounds in a training set, *R* means the correlative coefficient, and *S* means the standard residual error. Log BB = log (Cbrain/Cblood). PSA means the total polar surface area of a molecule. CLogP and BI display calculated LogP and connective index of molecular average total distance (relative covalent radius), respectively. They come from CS calculation. ΔEtotal and ΔEtorsion are related to interaction between an organic compound and membrane-water model. The total energy and the torsion energy of the DMPC monolayer membrane complex with a layer of water are -340.7589 and -1724.4164 (Kcal/mol), respectively. ΔEtotal is the change in the total potential energy of the solute-membrane-water complex comparing with that of the membrane-water model and so is ΔEtorsion.



**No PSA (Å2) ClogP BI(Å) Estretch (Kcal/**

**mol)**

**Training set** B36 0.00 2.64 2050 -0.02344 -220.3940 -1681.1548 120.36 43.26 B37 0.00 2.63 712 -0.00002 -231.5752 -1722.2582 109.18 2.16 **Test set** T1 22.70 0.321 712 0.00000 -274.7201 -1713.7409 66.04 10.68 T2 0.00 3.738 1838 0.00000 -225.6308 -1716.6234 115.13 7.79 T3 0.00 4.267 4150 0.00000 -331.3754 -1700.6397 9.38 23.78 T4 11.30 0.870 791 0.00000 -181.5954 -1700.8447 159.16 23.57 T5 0.00 4.397 4650 0.00000 -404.2903 -1741.2420 -63.53 -16.83 T6 0.00 1.103 0 0.00000 -282.9386 -1746.1889 57.82 -21.77 T7 0.00 3.339 791 0.00063 -271.9174 -1681.9440 68.84 42.47 T8 22.70 -0.208 213 0.00000 -364.8884 -1695.3605 -24.13 29.06

QSAR Analysis of Purine-Type and Propafenone-Type Substrates of P-Glycoprotein Targeting β-Amyloid Clearance

Note: a Etotal and Etorsion mean the total energy and the torsion energy of the complex with an organic compound and DMPC monolayer membrane. b The total energy and the torsion energy of the DMPC monolayer membrane are -340.758901 and -1724.416387 (Kcal/mol). ΔEtotal and ΔEtorsion are the residues between a complex of an organic

With the increase of the variable from one to six, the relativity of QSAR equation is also improved, and the predictive ability of the model is enhanced. Eq.18 is most significant, which means that the capability of an organic compound through BBB depends upon PSA, ClogP, BI, Estretch, ΔEtotal, and ΔEtorsion. Moreover, the potential of an organic compound through BBB is directly proportional to ClogP and ΔEtotal, but inversely proportional to PSA, BI, Estretch, and ΔEtorsion. The observed and predicted log BB values of the training set compounds are listed in Table 10. Figure 4 shows the comparison of the experimental log BB values for all the molecules of the training set with the corresponding predicted log BB as predicted by Eg.17 and -18 MI-QSAR models. Compound B18 in the training set is predicted to have a much higher log BB than observed, and this molecule has also been identified as an outlier in other studies (Iyer

A test set of eight solute compounds was constructed as one way to attempt to validate the QSAR models given by six equations mentioned. The test set compounds were selected so as to span almost the entire range in BBB partitioning. The observed and predicted log BB values for this test set are given in Table 10 and plotted in Figure 4 (right). It seems to suggest that Eg.

compound with DMPC monolayer membrane and the DMPC monolayer membrane.

**Table 9.** The molecular descriptors of the compounds related to BBB in the training/test sets

et al, 2002). Protonation of the molecule could account for its low log BB value.

17and -18 QSAR models could predict log BB for other compounds in drug design.

**Etotal a (Kcal/ mol)**

**Etorsion a (Kcal/ mol)**

**ΔEtotal b (Kcal/ mol)**

http://dx.doi.org/10.5772/54975

**ΔEtorsion <sup>b</sup> (Kcal/mol)** 277


Note: a Etotal and Etorsion mean the total energy and the torsion energy of the complex with an organic compound and DMPC monolayer membrane. b The total energy and the torsion energy of the DMPC monolayer membrane are -340.758901 and -1724.416387 (Kcal/mol). ΔEtotal and ΔEtorsion are the residues between a complex of an organic compound with DMPC monolayer membrane and the DMPC monolayer membrane.

**Table 9.** The molecular descriptors of the compounds related to BBB in the training/test sets

**No PSA (Å2) ClogP BI(Å) Estretch (Kcal/**

276 Neurodegenerative Diseases

**mol)**

**Training set** B9 104.40 1.77 83798 -0.35599 -313.4237 -1639.9898 27.34 84.43 B10 108.80 2.00 193593 -0.52172 -548.5593 -1640.9214 -207.80 83.49 B11 47.90 2.51 352512 -0.09496 -312.1226 -1656.7465 28.64 67.67 B12 45.20 4.27 779210 0.00479 -163.8011 -1716.3101 176.96 8.11 B13 38.50 2.61 158640 -0.09491 -170.3338 -1716.7159 170.43 7.70 B14 40.00 4.28 431722 -1.30506 -247.0951 -1748.0241 93.66 -23.61 B15 39.20 5.88 766256 0.09911 -289.2825 -1735.4004 51.48 -10.98 B16 54.90 5.14 766256 -0.14215 -181.0636 -1743.6068 159.70 -19.19 B17 18.80 0.62 20863 0.18071 -331.7044 -1695.6999 9.05 28.72 B18 46.70 0.27 20264 -1.36843 -209.4697 -1644.6752 131.29 79.74 B19 44.10 2.80 190375 -2.97778 -311.9182 -1713.8942 28.84 10.52 B20 5.40 4.85 210631 -0.06079 -235.7250 -1704.3399 105.03 20.08 B21 0.00 -0.47 4 0.00000 -407.3194 -1729.3793 -66.56 -4.96 B22 0.00 2.14 972 -0.00009 -239.8807 -1675.1827 100.88 49.23 B23 23.40 0.07 213 0.00000 -160.1278 -1672.3898 180.63 52.03 B24 22.60 0.69 712 0.00000 -319.0674 -1742.6968 21.69 -18.28 B25 0.00 3.74 1899 0.00067 -282.3721 -1751.6193 58.39 -27.20 B26 0.00 3.61 1661 0.00000 -285.7132 -1731.9518 55.05 -7.54 B27 0.00 1.43 1661 -0.00008 -238.7249 -1731.3090 102.03 -6.89 B28 0.00 2.48 633 0.00003 -291.5583 -1725.7370 49.20 -1.32 B29 11.60 2.46 21380 -0.00005 -418.0323 -1682.7138 -77.27 41.70 B30 24.40 -0.24 47 0.00000 -329.3150 -1704.6187 11.44 19.80 B31 10.70 1.27 7864 -0.00002 -253.3453 -1747.7044 87.41 -23.29 B32 0.00 2.37 7322 -0.00003 -268.8335 -1714.2486 71.93 10.17 B33 0.00 3.31 931 0.02567 -353.8395 -1739.7672 -13.08 -15.35 B34 24.40 -0.24 47 0.00000 -187.4520 -1720.5500 153.31 3.87 B35 0.00 1.93 7322 -0.00003 -177.4875 -1728.8621 163.27 -4.45

**Etotal a (Kcal/ mol)**

**Etorsion a (Kcal/ mol)**

**ΔEtotal b (Kcal/ mol)**

**ΔEtorsion <sup>b</sup> (Kcal/mol)**

> With the increase of the variable from one to six, the relativity of QSAR equation is also improved, and the predictive ability of the model is enhanced. Eq.18 is most significant, which means that the capability of an organic compound through BBB depends upon PSA, ClogP, BI, Estretch, ΔEtotal, and ΔEtorsion. Moreover, the potential of an organic compound through BBB is directly proportional to ClogP and ΔEtotal, but inversely proportional to PSA, BI, Estretch, and ΔEtorsion. The observed and predicted log BB values of the training set compounds are listed in Table 10. Figure 4 shows the comparison of the experimental log BB values for all the molecules of the training set with the corresponding predicted log BB as predicted by Eg.17 and -18 MI-QSAR models. Compound B18 in the training set is predicted to have a much higher log BB than observed, and this molecule has also been identified as an outlier in other studies (Iyer et al, 2002). Protonation of the molecule could account for its low log BB value.

> A test set of eight solute compounds was constructed as one way to attempt to validate the QSAR models given by six equations mentioned. The test set compounds were selected so as to span almost the entire range in BBB partitioning. The observed and predicted log BB values for this test set are given in Table 10 and plotted in Figure 4 (right). It seems to suggest that Eg. 17and -18 QSAR models could predict log BB for other compounds in drug design.


**Figure 4.** Comparison of the experimental log BB values (blue rhombic dots) for all the molecules of the training sets (upper) or the test set (down) to the corresponding predicted log BB as predicted by Eg.17 MI-QSAR model (red

QSAR Analysis of Purine-Type and Propafenone-Type Substrates of P-Glycoprotein Targeting β-Amyloid Clearance

http://dx.doi.org/10.5772/54975

279

We have built some predictive models of MDR, Ka and BBB partitioning of organic compounds by simulating the interaction of modulators or drugs interact with P-gp and/or of an organic compound with the phospholipide-rich regions of cellular membranes. As we know in the introduction part, modulators or drugs interact with P-gp and thus reduce the efflux of the cytotoxic compounds will increase the apparent toxicity of the cytotoxic compounds. It is very important to keep in mind that it is based on a general assessment of cytotoxicity and thus may account for more than one acting mechanism in the resistant cells used. So there are many uncertainty factors in the MDR ratio assay method and it is also convinced by our linear regression models. Our research results using two different statistic methods, MLR and PLSR, have revealed that the QSAR equation was also improved and the predictive ability of the models was enhanced with the increase of the variable. Eg.5 is built on KB-A1 cell line with a cytotoxic compound of 2.5µM ADR while Eg.6 is based on P388/VDR-20 cell line with 1.5µM VCR. Here, most of the models gave satisfactory cross-validated Q2 above 0.500, conventional R above 0.800 and less SE values indicating their proper predictive ability. Significant differ‐ ences between values were examined using two-tailed paired T test provided by SPSS. All the results were considered not significant if P<0.05. Eg.5 model is the most significant and indicated that the capability of P-gp modulators interacted with P-gp depends upon MR, BI, Ehyd, ShA, and LogP. The former three display positive contributions to the MDR activity of P-

square dots) and by Eg.18 MI-QSAR model (yellow triangle dots).

**4. Discussion**

**Table 10.** The experimental values and the predictive values of LogBB of these compounds.

QSAR Analysis of Purine-Type and Propafenone-Type Substrates of P-Glycoprotein Targeting β-Amyloid Clearance http://dx.doi.org/10.5772/54975 279

**Figure 4.** Comparison of the experimental log BB values (blue rhombic dots) for all the molecules of the training sets (upper) or the test set (down) to the corresponding predicted log BB as predicted by Eg.17 MI-QSAR model (red square dots) and by Eg.18 MI-QSAR model (yellow triangle dots).

#### **4. Discussion**

**No LogBB**

278 Neurodegenerative Diseases

**Predictive Value of logBB**

B19 0.25 -0.21 -0.15 -0.05 0.45 0.59 0.62

**No LogBB**

**Eg.13 Eg.14 Eg.15 Eg.16 Eg.17 Eg.18 Eg.13 Eg.14 Eg.15 Eg.16 Eg.17 Eg.18**

**Training set** B1 -0.04 -0.81 -0.95 -0.71 -0.52 -0.33 -0.20 B20 0.85 0.46 0.77 0.87 0.85 0.99 1.01 B2 -2.00 -1.07 -1.11 -1.44 -1.56 -1.57 -1.39 B21 0.03 0.55 0.17 -0.05 -0.03 -0.12 -0.10 B3 -1.30 -0.71 -0.51 -1.20 -1.11 -0.98 -1.17 B22 0.37 0.55 0.51 0.50 0.50 0.64 0.57 B4 -1.06 -0.95 -1.04 -1.58 -1.49 -1.37 -1.13 B23 -0.15 0.15 -0.16 -0.23 -0.24 0.04 -0.03 B5 0.11 -0.12 -0.30 -0.26 -0.40 -0.19 -0.06 B24 -0.17 0.16 -0.06 -0.09 -0.10 -0.04 0.08 B6 0.49 0.09 0.20 0.19 0.16 0.34 0.28 B25 0.97 0.55 0.72 0.84 0.83 0.91 1.07 B7 -1.17 -0.98 -1.15 -0.91 -1.11 -0.90 -0.86 B26 1.04 0.55 0.70 0.81 0.80 0.88 0.98 B8 -0.18 -0.77 -0.71 -0.38 -0.38 -0.22 -0.22 B27 0.08 0.55 0.42 0.35 0.35 0.49 0.56 B9 -1.15 -1.25 -1.31 -0.97 -0.99 -0.79 -0.81 B28 0.40 0.55 0.55 0.57 0.57 0.64 0.71 B10 -1.57 -1.33 -1.36 -1.05 -1.05 -1.16 -1.20 B29 0.24 0.35 0.35 0.41 0.39 0.31 0.27 B11 -0.46 -0.28 -0.26 -0.26 -0.31 -0.21 -0.32 B30 -0.16 0.13 -0.22 -0.31 -0.32 -0.26 -0.26 B12 -0.24 -0.23 0.02 -0.13 -0.23 0.03 0.04 B31 0.13 0.37 0.21 0.18 0.17 0.31 0.43 B13 -0.02 -0.11 -0.08 0.01 -0.02 0.26 0.34 B32 0.35 0.55 0.54 0.55 0.54 0.64 0.68 B14 0.44 -0.14 0.11 0.17 0.33 0.51 0.64 B33 0.93 0.55 0.66 0.75 0.74 0.73 0.84 B15 0.14 -0.13 0.33 0.30 0.17 0.26 0.33 B34 -0.16 0.13 -0.22 -0.31 -0.32 -0.07 -0.02 B16 0.22 -0.40 -0.03 -0.06 -0.15 0.10 0.22 B35 0.27 0.55 0.48 0.45 0.45 0.68 0.74 B17 -0.06 0.23 -0.01 -0.07 -0.11 -0.07 -0.09 B36 0.37 0.55 0.57 0.61 0.61 0.77 0.72 B18 -1.40 -0.26 -0.53 -0.50 -0.28 -0.02 -0.14 B37 0.34 0.55 0.57 0.60 0.60 0.75 0.81

**Test set** T1 -0.08 0.16 -0.11 -0.17 -0.18 -0.06 -0.02 T5 0.81 0.55 0.81 0.97 0.96 0.88 1.02 T2 1.01 0.55 0.72 0.84 0.83 0.99 1.05 T6 0.04 0.55 0.37 0.28 0.29 0.37 0.47 T3 0.90 0.55 0.79 0.95 0.93 0.95 0.98 T7 0.76 0.55 0.67 0.75 0.75 0.84 0.81 T4 0.00 0.36 0.15 0.09 0.09 0.32 0.32 T8 -0.15 0.16 -0.18 -0.28 -0.29 -0.28 -0.31

**Table 10.** The experimental values and the predictive values of LogBB of these compounds.

**Predictive Value of logBB**

We have built some predictive models of MDR, Ka and BBB partitioning of organic compounds by simulating the interaction of modulators or drugs interact with P-gp and/or of an organic compound with the phospholipide-rich regions of cellular membranes. As we know in the introduction part, modulators or drugs interact with P-gp and thus reduce the efflux of the cytotoxic compounds will increase the apparent toxicity of the cytotoxic compounds. It is very important to keep in mind that it is based on a general assessment of cytotoxicity and thus may account for more than one acting mechanism in the resistant cells used. So there are many uncertainty factors in the MDR ratio assay method and it is also convinced by our linear regression models. Our research results using two different statistic methods, MLR and PLSR, have revealed that the QSAR equation was also improved and the predictive ability of the models was enhanced with the increase of the variable. Eg.5 is built on KB-A1 cell line with a cytotoxic compound of 2.5µM ADR while Eg.6 is based on P388/VDR-20 cell line with 1.5µM VCR. Here, most of the models gave satisfactory cross-validated Q2 above 0.500, conventional R above 0.800 and less SE values indicating their proper predictive ability. Significant differ‐ ences between values were examined using two-tailed paired T test provided by SPSS. All the results were considered not significant if P<0.05. Eg.5 model is the most significant and indicated that the capability of P-gp modulators interacted with P-gp depends upon MR, BI, Ehyd, ShA, and LogP. The former three display positive contributions to the MDR activity of P- gp, suggesting that the MDR activity increase accordingly with MR increase. The latter two displays negative contribution to the MDR activity of P-gp.

larger its value of LogBB is. It displays that a small molecule tight combining with the mem‐ brane-water complex leads to increase its value of LogBB. And the relationship would sug‐ gest that as the solute becomes more flexible within the membrane-water complex, the greater would be its log BB value, which is in agreement with the research results of Iyer M et

QSAR Analysis of Purine-Type and Propafenone-Type Substrates of P-Glycoprotein Targeting β-Amyloid Clearance

http://dx.doi.org/10.5772/54975

281

Several non-MI-QSAR computational models to describe and predict BBB partitioning have been reported that includes other descriptors besides PSA and ClogP (Lombardo et al, 1996; Keseru & Molnar, 2001; Crivori et al, 2000). An alternative, complementary approach to BBB partitioning prediction uses MI-QSAR analysis developed by Iyer M et al (2002). Their research results show that BBB partitioning of an organic compound depend upon PSA, CLogP, and the conformational flexibility of the compounds as well as the strength of their "binding" to the model biologic membrane. The MI-QSAR models indicate that BBB partitioning process can be reliably described for structurally diverse molecules provided interactions of the molecule with the phospholipide-rich regions of cellular membranes are explicitly considered. An extension of these approaches that combines QSAR with solute-membrane-water complex has been developed by us, which is addition of a layer of water on the hydrophilic side of DMPC monolayer membrane in our research. And so, it is more analogous to the truth BBB environment. Our results reveal that the distribution of organic molecules through BBB was not only influenced by the properties of organic solutes, but also related to the property of the solute-membrane-water complex. The former involves the polarity, hydrophobic, size, and conformational freedom degree of organic molecules. The latter deals with the strength of an organic molecule combined with BBB membrane and the structural changeability of a solutemembrane-water complex. Furthermore, the capability of a small molecule across BBB is mainly related to four physicochemical factors, which depend on the relative polarity of a small molecule, the molecular volume, the strength of a small molecule combined with DMPC-water model, and the changeability of the structure of a solute-membrane-water complex. The relative polarity of a small molecule includes two parameters, namely PSA and ClogP. The QSAR model shows that less polarity and more hydrophobic molecules relatively easily pass through BBB and enter brain to cure. The molecular volume involves one parameter, namely BI. The strength of a small molecule combined with DMPC monolayer membrane complex with a layer of water involves one parameter, namely ΔEtorsion. The changeability of the structure of a complex between a small molecule and the membrane-water complex includes one parameter, namely ΔEtotal. The reason for the change of total energy is that small molecule across BBB membrane leads to the change of the structure of the solute-membrane-water complex. The more the changeability of the complex structure is, the more the change value

of total energy is, and the more easily a small molecule penetrates BBB.

Cerebral clearance of Aβ is considered to occur via elimination across BBB, as well as proteo‐ lytic degradation. Attenuation of its elimination is likely to result in increased cerebral Aβ deposition, which may facilitate progression of AD (Ohtsuki et al, 2010). P-gp detoxifies cells by exporting hundreds of chemically unrelated toxins but has been implicated in MDR in the treatment of cancers. Substrate promiscuity is a hallmark of P-gp activity, thus a structural description of poly-specific drug-binding is important for the rational design of anti-amyloid accumulation drugs, anticancer drugs and MDR inhibitors. The x-ray structure of apo P-gp at

al (2002).

On the other hand, we have built the predicted models for Ka of ATPase of some compounds using the same statistic methods in order to get a more accurate model. Both models, Eg.11 by MLR and Eg.12 by PLSR, point out that LogP and EHOMO are both important parameters with the affinity for and simulation of the P-gp ATPase. LogP is negative related with the activity of P-gp ATPase, suggesting that the ATPase activity also decrease with the increase of LogP. Figure 3 showed that molecular A39 and A42 have higher Ka value of ATPase and is a departure from other compounds. This may be because they have lower lipophilicity, which is supported by the research results of Diethart Schmid et al (1999). Another significant descriptor EHOMO is positive related with the activity of P-gp ATPase.

In another aspect, BBB partitioning is mainly found to depend upon two parameters, namely PSA and ClogP. With the increase of the variable, the relativity of QSAR equation is also im‐ proved, and the predictive ability of the model is enhanced, especially Eq.18 most significant. Moreover, the BBB partitioning measures of the test set compounds were predicted with the same accuracy as the compounds of the training set. The family of these QSAR models reveal that the capability of BBB partitioning of an organic compound focus on six significant fea‐ tures, which are PSA, ClogP, BI, Estretch, ΔEtotal, and ΔEtorsion (Eg. 18). Obviously, two of the six descriptors of the QSAR models have positive regression coefficients and the other four de‐ scriptors have negative regression coefficients. The potential of an organic compound through BBB is directly proportional to ClogP and ΔEtotal, but inversely proportional to PSA, BI, Estretch, and ΔEtorsion. Moreover, PSA descriptor is found as a dominant descriptor in these QSAR models, which related to the aqueous solubility of the solute compound along with a direct lipophilicity descriptor (Clark et al, 1999). When the value of PSA of a molecule lessens within the range from 0 to 108.80 Å2 , its value of LogBB will increase. This is consistent with the experimental results that the more polarity it possesses, the more difficultly a molecule enters the hydrophobic environment of BBB (Stouch, 1993). BI is the connective index of mo‐ lecular average total distance, which pertains to the volume parameter. Our research result points it out that with the accretion of its bulk, a molecule more and more difficultly across through BBB by diffusion. However, the value of LogBB of a molecule increases with the in‐ crease of ClogP. It means that the hydrophobic molecule can pass through BBB more easily than the hydrophilic molecule does, which is supported by the experimental results (Kalis‐ zan & Markuszewski, 1996). The presence of Estretch descriptor suggests that with the decrease of the stretch-bend energy of a molecule, its value of LogBB increases. Two of the descriptors, found in the log BB QSAR models (Eg.17 and Eq.18), reflect the behavior of the solute in the membrane and the entire membrane-solute complex. Along with the meaning mentioned, ΔEtotal is equivalent to the change in average total potential energy between the triple member complex and the double member complex. Similarly, ΔEtorsion is the difference between the di‐ hedral torsion energy of the triple complex and that of the double complex. Here, the more the change value of ΔEtotal is, the more its value of LogBB increases. This is because small mol‐ ecule across BBB membrane leads to the change of the structure of the complex. The more changeability of the structure results in greater change of total potential energy, while the ac‐ cretion of the energy change is the important cause of the increase of the capability of a small molecule through BBB. On the contrary, the less the difference of the torsion energy is, the larger its value of LogBB is. It displays that a small molecule tight combining with the mem‐ brane-water complex leads to increase its value of LogBB. And the relationship would sug‐ gest that as the solute becomes more flexible within the membrane-water complex, the greater would be its log BB value, which is in agreement with the research results of Iyer M et al (2002).

gp, suggesting that the MDR activity increase accordingly with MR increase. The latter two

On the other hand, we have built the predicted models for Ka of ATPase of some compounds using the same statistic methods in order to get a more accurate model. Both models, Eg.11 by MLR and Eg.12 by PLSR, point out that LogP and EHOMO are both important parameters with the affinity for and simulation of the P-gp ATPase. LogP is negative related with the activity of P-gp ATPase, suggesting that the ATPase activity also decrease with the increase of LogP. Figure 3 showed that molecular A39 and A42 have higher Ka value of ATPase and is a departure from other compounds. This may be because they have lower lipophilicity, which is supported by the research results of Diethart Schmid et al (1999). Another significant

In another aspect, BBB partitioning is mainly found to depend upon two parameters, namely PSA and ClogP. With the increase of the variable, the relativity of QSAR equation is also im‐ proved, and the predictive ability of the model is enhanced, especially Eq.18 most significant. Moreover, the BBB partitioning measures of the test set compounds were predicted with the same accuracy as the compounds of the training set. The family of these QSAR models reveal that the capability of BBB partitioning of an organic compound focus on six significant fea‐ tures, which are PSA, ClogP, BI, Estretch, ΔEtotal, and ΔEtorsion (Eg. 18). Obviously, two of the six descriptors of the QSAR models have positive regression coefficients and the other four de‐ scriptors have negative regression coefficients. The potential of an organic compound through BBB is directly proportional to ClogP and ΔEtotal, but inversely proportional to PSA, BI, Estretch, and ΔEtorsion. Moreover, PSA descriptor is found as a dominant descriptor in these QSAR models, which related to the aqueous solubility of the solute compound along with a direct lipophilicity descriptor (Clark et al, 1999). When the value of PSA of a molecule lessens

the experimental results that the more polarity it possesses, the more difficultly a molecule enters the hydrophobic environment of BBB (Stouch, 1993). BI is the connective index of mo‐ lecular average total distance, which pertains to the volume parameter. Our research result points it out that with the accretion of its bulk, a molecule more and more difficultly across through BBB by diffusion. However, the value of LogBB of a molecule increases with the in‐ crease of ClogP. It means that the hydrophobic molecule can pass through BBB more easily than the hydrophilic molecule does, which is supported by the experimental results (Kalis‐ zan & Markuszewski, 1996). The presence of Estretch descriptor suggests that with the decrease of the stretch-bend energy of a molecule, its value of LogBB increases. Two of the descriptors, found in the log BB QSAR models (Eg.17 and Eq.18), reflect the behavior of the solute in the membrane and the entire membrane-solute complex. Along with the meaning mentioned, ΔEtotal is equivalent to the change in average total potential energy between the triple member complex and the double member complex. Similarly, ΔEtorsion is the difference between the di‐ hedral torsion energy of the triple complex and that of the double complex. Here, the more the change value of ΔEtotal is, the more its value of LogBB increases. This is because small mol‐ ecule across BBB membrane leads to the change of the structure of the complex. The more changeability of the structure results in greater change of total potential energy, while the ac‐ cretion of the energy change is the important cause of the increase of the capability of a small molecule through BBB. On the contrary, the less the difference of the torsion energy is, the

, its value of LogBB will increase. This is consistent with

displays negative contribution to the MDR activity of P-gp.

280 Neurodegenerative Diseases

descriptor EHOMO is positive related with the activity of P-gp ATPase.

within the range from 0 to 108.80 Å2

Several non-MI-QSAR computational models to describe and predict BBB partitioning have been reported that includes other descriptors besides PSA and ClogP (Lombardo et al, 1996; Keseru & Molnar, 2001; Crivori et al, 2000). An alternative, complementary approach to BBB partitioning prediction uses MI-QSAR analysis developed by Iyer M et al (2002). Their research results show that BBB partitioning of an organic compound depend upon PSA, CLogP, and the conformational flexibility of the compounds as well as the strength of their "binding" to the model biologic membrane. The MI-QSAR models indicate that BBB partitioning process can be reliably described for structurally diverse molecules provided interactions of the molecule with the phospholipide-rich regions of cellular membranes are explicitly considered. An extension of these approaches that combines QSAR with solute-membrane-water complex has been developed by us, which is addition of a layer of water on the hydrophilic side of DMPC monolayer membrane in our research. And so, it is more analogous to the truth BBB environment. Our results reveal that the distribution of organic molecules through BBB was not only influenced by the properties of organic solutes, but also related to the property of the solute-membrane-water complex. The former involves the polarity, hydrophobic, size, and conformational freedom degree of organic molecules. The latter deals with the strength of an organic molecule combined with BBB membrane and the structural changeability of a solutemembrane-water complex. Furthermore, the capability of a small molecule across BBB is mainly related to four physicochemical factors, which depend on the relative polarity of a small molecule, the molecular volume, the strength of a small molecule combined with DMPC-water model, and the changeability of the structure of a solute-membrane-water complex. The relative polarity of a small molecule includes two parameters, namely PSA and ClogP. The QSAR model shows that less polarity and more hydrophobic molecules relatively easily pass through BBB and enter brain to cure. The molecular volume involves one parameter, namely BI. The strength of a small molecule combined with DMPC monolayer membrane complex with a layer of water involves one parameter, namely ΔEtorsion. The changeability of the structure of a complex between a small molecule and the membrane-water complex includes one parameter, namely ΔEtotal. The reason for the change of total energy is that small molecule across BBB membrane leads to the change of the structure of the solute-membrane-water complex. The more the changeability of the complex structure is, the more the change value of total energy is, and the more easily a small molecule penetrates BBB.

Cerebral clearance of Aβ is considered to occur via elimination across BBB, as well as proteo‐ lytic degradation. Attenuation of its elimination is likely to result in increased cerebral Aβ deposition, which may facilitate progression of AD (Ohtsuki et al, 2010). P-gp detoxifies cells by exporting hundreds of chemically unrelated toxins but has been implicated in MDR in the treatment of cancers. Substrate promiscuity is a hallmark of P-gp activity, thus a structural description of poly-specific drug-binding is important for the rational design of anti-amyloid accumulation drugs, anticancer drugs and MDR inhibitors. The x-ray structure of apo P-gp at 3.8 angstroms reveals an internal cavity of approximately 6000 angstroms cubed with a 30 angstrom separation of the two nucleotide-binding domains. Two additional P-gp structures with cyclic peptide inhibitors demonstrate distinct drug-binding sites in the internal cavity capable of stereoselectivity that is based on hydrophobic and aromatic interactions. Apo and drug-bound P-gp structures have portals open to the cytoplasm and the inner leaflet of the lipid bilayer for drug entry. The inward-facing conformation represents an initial stage of the transport cycle that is competent for drug binding (Aller et al, 2009). Currently, P-gp is identificated as an energy-dependent pump, ATPase activity as an assay in itself is possibly problematical cause it is based upon one assumption that drug-induced ATP hydrolysis reflects transport by the transporter (Stouch & Gudmundsson, 2001). There may be many ways in which this activity could be altered, including direct action on the ATP binding domain. Scientists once observed some compounds such as daunomycin and vinblastine inhibit ATPase activity, but increase in others, suggesting that modulation of ATPase activity is highly dependent on experimental conditions and may not correlate well with the ability of P-gp to transport the drug (Ambudkar et al, 1992; Shapiro & Ling, 1994; Doige et al, 1993). The work of Litman et al was one of the few studies suggesting that affinity between drugs and ATPase activity has no correlation to LogP, but Surface Area (Litman et al, 1997). Because of the less comparability of molecular structures in a training set, our QSAR equation possesses universal significance. However, the precision of QSAR equation is so low that there is still a distance to its application. So a series of organic compounds with similar structures are chosen and consist of a training set, thus the precision of QSAR simulation is largely increased, while the prediction of the analogues through BBB is greatly improved.

Additionally, our constructed MI-QSAR model indicates that the distribution of organic molecules through BBB was not only influenced by organic solutes themselves, but also related to the properties of the solute-membrane water complex, namely interactions of the molecule with the phospholipide-rich regions of cellular membranes. Moreover, our results reveal that the ability of organic molecules permeating across BBB is proportional to LogP but inversely proportional to PSA (see Eg.13 to 18), which is consistent with the research results of Chen and co-worker (2009), namely the increasing PSA decreased LogBB rapidly while LogP positively related to LogBB. It indicates that molecules with higher lipophilic will be partitioned into the lipid bilayer more easily with more chances to penetrate BBB, supported by the research result of Wang et al (2003), namely a large number of structurally and functionally diverse com‐ pounds as substrates or modulators of P-gp mostly sharing common structural features, such as aromatic ring structures and high lipophilicity. PSA of CNS active drug should be lower

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(Chen et al, 2009), while the penetration through the BBB is optimal for LogP value

In comparison with the ability of organic molecules permeating across BBB, P-gp binding or MDR-reversal activity of compounds has a negative correlation with LogP. There are two reasons for this phenomenon. Firstly, the compounds with higher liposolubility are more vulnerable to cytochrome P450 metabolism, leading to faster clearance (Waterhouse, 2003). P450 enzymes (CYP450s) catalyze the metabolism of a wide variety of endogenous and

**Scheme 1.** Flowchart for QSAR analysis of some substrates of P-glycoprotein targeting β-amyloid clearance.

than 90 Å2

in the range 1.5–2.7 (Norinder & Haeberlein, 2002).

#### **5. Conclusion**

P-gp is involved in MDR and in neurodegenerative disorders such as Parkinson disease, AD and epilepsy. The xenobiotic efflux pump P-gp limits intracellular drug accumulation by active extrusion of compounds out of cells. P-gp mediates the efflux of Aβ from the brain together with mediating MDR, while P-gp transports neutral or positively-charged hydrophobic substrates with consuming energy from ATP hydrolysis. We have built up theoretical models of the interaction between organic compounds and P-gp and compounds with the affinity for and simulation of the P-gp ATPase. The interaction between compounds and p-gp (P-gp binding or MDR-reversal activity of compounds) is found to depend on LogP, LogMR, and ShA of compounds it transports, which proportional to LogMR while inversely proportional to LogP and ShA (see Eg.1 to Eg.5). Until now we have not convinced that ATPase activity of P-gp is well correlated with the ability of P-gp to transport the drugs. However, our constructed model based on the analogies of purine and propafenone analogs suggests that the enzyme hydrolysis of these compounds largely depends on LogP, MR, ShA, MW and EHOMO, especially positive related to MR but negative to LogP and ShA (see Eg.7 to Eq.11). This shows that the P-gp binding capacity of these compounds shares common characteristics with their ATPase hydrolysis, namely their hydrophobic parameters (such as log P) and steric parameters (eg. MW, ShA, MR, and WI).

Additionally, our constructed MI-QSAR model indicates that the distribution of organic molecules through BBB was not only influenced by organic solutes themselves, but also related to the properties of the solute-membrane water complex, namely interactions of the molecule with the phospholipide-rich regions of cellular membranes. Moreover, our results reveal that the ability of organic molecules permeating across BBB is proportional to LogP but inversely proportional to PSA (see Eg.13 to 18), which is consistent with the research results of Chen and co-worker (2009), namely the increasing PSA decreased LogBB rapidly while LogP positively related to LogBB. It indicates that molecules with higher lipophilic will be partitioned into the lipid bilayer more easily with more chances to penetrate BBB, supported by the research result of Wang et al (2003), namely a large number of structurally and functionally diverse com‐ pounds as substrates or modulators of P-gp mostly sharing common structural features, such as aromatic ring structures and high lipophilicity. PSA of CNS active drug should be lower than 90 Å2 (Chen et al, 2009), while the penetration through the BBB is optimal for LogP value in the range 1.5–2.7 (Norinder & Haeberlein, 2002).

3.8 angstroms reveals an internal cavity of approximately 6000 angstroms cubed with a 30 angstrom separation of the two nucleotide-binding domains. Two additional P-gp structures with cyclic peptide inhibitors demonstrate distinct drug-binding sites in the internal cavity capable of stereoselectivity that is based on hydrophobic and aromatic interactions. Apo and drug-bound P-gp structures have portals open to the cytoplasm and the inner leaflet of the lipid bilayer for drug entry. The inward-facing conformation represents an initial stage of the transport cycle that is competent for drug binding (Aller et al, 2009). Currently, P-gp is identificated as an energy-dependent pump, ATPase activity as an assay in itself is possibly problematical cause it is based upon one assumption that drug-induced ATP hydrolysis reflects transport by the transporter (Stouch & Gudmundsson, 2001). There may be many ways in which this activity could be altered, including direct action on the ATP binding domain. Scientists once observed some compounds such as daunomycin and vinblastine inhibit ATPase activity, but increase in others, suggesting that modulation of ATPase activity is highly dependent on experimental conditions and may not correlate well with the ability of P-gp to transport the drug (Ambudkar et al, 1992; Shapiro & Ling, 1994; Doige et al, 1993). The work of Litman et al was one of the few studies suggesting that affinity between drugs and ATPase activity has no correlation to LogP, but Surface Area (Litman et al, 1997). Because of the less comparability of molecular structures in a training set, our QSAR equation possesses universal significance. However, the precision of QSAR equation is so low that there is still a distance to its application. So a series of organic compounds with similar structures are chosen and consist of a training set, thus the precision of QSAR simulation is largely increased, while the

P-gp is involved in MDR and in neurodegenerative disorders such as Parkinson disease, AD and epilepsy. The xenobiotic efflux pump P-gp limits intracellular drug accumulation by active extrusion of compounds out of cells. P-gp mediates the efflux of Aβ from the brain together with mediating MDR, while P-gp transports neutral or positively-charged hydrophobic substrates with consuming energy from ATP hydrolysis. We have built up theoretical models of the interaction between organic compounds and P-gp and compounds with the affinity for and simulation of the P-gp ATPase. The interaction between compounds and p-gp (P-gp binding or MDR-reversal activity of compounds) is found to depend on LogP, LogMR, and ShA of compounds it transports, which proportional to LogMR while inversely proportional to LogP and ShA (see Eg.1 to Eg.5). Until now we have not convinced that ATPase activity of P-gp is well correlated with the ability of P-gp to transport the drugs. However, our constructed model based on the analogies of purine and propafenone analogs suggests that the enzyme hydrolysis of these compounds largely depends on LogP, MR, ShA, MW and EHOMO, especially positive related to MR but negative to LogP and ShA (see Eg.7 to Eq.11). This shows that the P-gp binding capacity of these compounds shares common characteristics with their ATPase hydrolysis, namely their hydrophobic parameters (such as log P) and steric parameters (eg.

prediction of the analogues through BBB is greatly improved.

**5. Conclusion**

282 Neurodegenerative Diseases

MW, ShA, MR, and WI).

In comparison with the ability of organic molecules permeating across BBB, P-gp binding or MDR-reversal activity of compounds has a negative correlation with LogP. There are two reasons for this phenomenon. Firstly, the compounds with higher liposolubility are more vulnerable to cytochrome P450 metabolism, leading to faster clearance (Waterhouse, 2003). P450 enzymes (CYP450s) catalyze the metabolism of a wide variety of endogenous and

**Scheme 1.** Flowchart for QSAR analysis of some substrates of P-glycoprotein targeting β-amyloid clearance.

exogenous compounds including xenobiotics, drugs, environmental toxins, steroids, and fatty acids. Aminated thioxanthones have recently been reported as P-gp inhibitors as well as its interaction with cytochrome P450 3A4 (CYP3A4), as many substrates of P-glycoprotein and CYP3A4 are common (Palmeira et al, 2012). The second reason is related to the mechanism of P-gp action. According to model proposed by Higgins and Gottesman (1992), after entering into the phospholipid bilayer, compound may interact with P-gp in the inner leaflet of the lipid bilayer. Upon interaction with P-gp, the compound is flipped from the inner leaflet to the outer leaflet of the lipid bilayer. The lipophilic compounds with high LogP enter into cellular membrane easily and intend to retain there, so its opportunity to interact with P-gp increases. The LogP not only offers opportunity to penetrate the lipid bilayer, but also gives favorable contribution to binding with protein, such as P450, P-gp.

[2] Abraham, M. H, Takacs-novak, K, & Mitchell, R. C. (1997). On the partition of am‐ pholytes: Application to blood-brain distribution. *J Pharm Sci* (Mar 1997), 0022-3549,

QSAR Analysis of Purine-Type and Propafenone-Type Substrates of P-Glycoprotein Targeting β-Amyloid Clearance

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285

[3] Abuznait, A. H, Cain, C, Ingram, D, Burk, D, & Kaddoumi, A. (2011). Up-regulation of P-glycoprotein reduces intracellular accumulation of beta amyloid: investigation of P-glycoprotein as a novel therapeutic target for Alzheimer's disease. J Pharm Phar‐

[4] Alka, K. (2003). C-QSAR: a database of 18000 QSARs and associated biological and physical data. *J Comput Aided Mol Des* (Feb-Apr 2003), 1573-4951, 17(2-4), 187-196. [5] Aller, S. G, Yu, J, Ward, A, Weng, Y, Chittaboina, S, Zhuo, R, Harrell, P. M, Trinh, Y. T, Zhang, Q, Urbatsch, I. L, & Chang, G. (2009). Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding. *Science.* (Mar 2009), 0036-8075,

[6] Ambudkar, S. V, Dey, S, Hrycyna, C. A, Ramachandra, M, Pastan, I, & Gottesman, M. M. (1999). Biochemical, cellular, and pharmacological aspects of the multidrug

[7] Ambudkar, S. V, Lelong, I. H, Zhang, J, Cardarelli, C. O, Gottesman, M. M, & Pastan, I. (1992). Partial purification and reconstitution of the human multidrug-resistance pump: characterization of the drug-stimulatable ATP hydrolysis. *Proc. Natl. Acad. Sci.*

[8] Bartels, A. L. (2011). Blood-brain barrier P-glycoprotein function in neurodegenera‐

[9] Bassolino-klimas, D, Alper, H. E, & Stouch, T. R. (1993). Solute diffusion in lipid bi‐ layer membranes: an atomic level study by molecular dynamics simulation. *Biochem‐*

[10] Bendayan, R, Lee, G, & Bendayan, M. (2002). Functional expression and localization of p-glycoprotein at the blood brain barrier. Microsc. Res. Tech., (Jun 2002),

[11] Chen, C, & Yang, J. (2006). MI-QSAR models for prediction of corneal permeability of organic compounds. *Acta Pharmacologica Sinica.* (Feb 2006), 1745-7254, 27(2), 193-204.

[12] Chen, L. J, Lian, G. P, & Han, L. J. (2007). Prediction of human skin permeability us‐ ing artificial neural network (ANN) modeling. *Acta Pharmacologica Sinica,* (Apr 2007),

[13] Chen, Y, Zhu, Q. J, Pan, J, Yang, Y, & Wu, X. P. (2009). A prediction model for bloodbrain barrier permeation and analysis on its parameter biologically. *Comput Methods*

[14] Cheng, Z, Zhang, J, Liu, H, Li, Y, Zhao, Y, & Yang, E. (2010). Central nervous system penetration for small molecule therapeutic agents does not increase in multiple scle‐

transporter. *Annu. Rev. Pharmacol. Toxicol.* (Apr 1999), 0362-1642, 39, 361-398.

86(3), 310-315.

323(5922), 1718-1722.

macol. (Aug 2011), 0022-3573, 63(8), 1111-1118.

*USA* (Sep 1992), 0027-8424, 89(18), 8472-8476.

*istry.* (Nov 1993), 0006-2960, 32(47), 12624-12637.

*Programs Biomed.,* (Sep 2009), 0169-2607, 95(3), 280-287.

0105-9910X., 57(5), 365-380.

1745-7254, 28(4), 591-600.

tive disease. *Curr Pharm Des.* 1381-6128, 17(26), 2771-2777.

In conclusion, the predictive model of BBB partitioning of organic compounds contributes to discovery of some molecules through BBB as potential AD therapeutic drugs. Moreover, the interaction model of P-gp and modulators for treatment of multidrug resistance indicates discovery of some molecules to increase Aβ clearance from the brain and reduce Aβ brain accumulation by regulate BBB P-gp in the early stages of AD. The mechanism suggests new therapeutic strategy in AD.

### **Acknowledgements**

This work was supported by a grant from Basic Scientific Research Expenses of Central University (020814360012), National Key Technology R&D Program (2008BAI51B01) and Specialized Research Fund for the Doctoral Program of Higher Education (20120091110038).

### **Author details**

Jie Yang\* and Jie Chen

\*Address all correspondence to: luckyjyj@sina.com.cn

State Key Laboratory of Pharmaceutical Biotechnology, Life College, Nanjing University, Nanjing, China

### **References**

[1] Abraham, M. H, Chadha, H. S, & Mitchell, R. C. (1995). Hydrogen bonding. 36. De‐ termination of blood-brain barrier distribution using octanol-water partition coeffi‐ cients. *Drug Des Discov* (Nov 1995), 1055-9612, 13(2), 123-131.

[2] Abraham, M. H, Takacs-novak, K, & Mitchell, R. C. (1997). On the partition of am‐ pholytes: Application to blood-brain distribution. *J Pharm Sci* (Mar 1997), 0022-3549, 86(3), 310-315.

exogenous compounds including xenobiotics, drugs, environmental toxins, steroids, and fatty acids. Aminated thioxanthones have recently been reported as P-gp inhibitors as well as its interaction with cytochrome P450 3A4 (CYP3A4), as many substrates of P-glycoprotein and CYP3A4 are common (Palmeira et al, 2012). The second reason is related to the mechanism of P-gp action. According to model proposed by Higgins and Gottesman (1992), after entering into the phospholipid bilayer, compound may interact with P-gp in the inner leaflet of the lipid bilayer. Upon interaction with P-gp, the compound is flipped from the inner leaflet to the outer leaflet of the lipid bilayer. The lipophilic compounds with high LogP enter into cellular membrane easily and intend to retain there, so its opportunity to interact with P-gp increases. The LogP not only offers opportunity to penetrate the lipid bilayer, but also gives favorable

In conclusion, the predictive model of BBB partitioning of organic compounds contributes to discovery of some molecules through BBB as potential AD therapeutic drugs. Moreover, the interaction model of P-gp and modulators for treatment of multidrug resistance indicates discovery of some molecules to increase Aβ clearance from the brain and reduce Aβ brain accumulation by regulate BBB P-gp in the early stages of AD. The mechanism suggests new

This work was supported by a grant from Basic Scientific Research Expenses of Central University (020814360012), National Key Technology R&D Program (2008BAI51B01) and Specialized Research Fund for the Doctoral Program of Higher Education (20120091110038).

State Key Laboratory of Pharmaceutical Biotechnology, Life College, Nanjing University,

[1] Abraham, M. H, Chadha, H. S, & Mitchell, R. C. (1995). Hydrogen bonding. 36. De‐ termination of blood-brain barrier distribution using octanol-water partition coeffi‐

cients. *Drug Des Discov* (Nov 1995), 1055-9612, 13(2), 123-131.

contribution to binding with protein, such as P450, P-gp.

therapeutic strategy in AD.

284 Neurodegenerative Diseases

**Acknowledgements**

**Author details**

Nanjing, China

**References**

and Jie Chen

\*Address all correspondence to: luckyjyj@sina.com.cn

Jie Yang\*


rosis- and Alzheimer's disease-related animal models despite reported blood-brain barrier disruption. *Drug Metab Dispos.* (Aug 2010), 0090-9556, 38(8), 1355-1361.

[26] Kast, C, Canfield, V, Levenson, R, & Gros, P. (1996). Transmembrane organization of mouse P-glycoprotein determined by epitope insertion and immunofluorescence. J

QSAR Analysis of Purine-Type and Propafenone-Type Substrates of P-Glycoprotein Targeting β-Amyloid Clearance

http://dx.doi.org/10.5772/54975

287

[27] Karelson, M. (2000). Molecular Descriptors in QSAR/ QSPR; John Wiley & Sons: New

[28] Karelson, M, Lobanov, V. S, & Katritzky, A. R. (1996). Quantum-chemical descriptors in QSAR/QSPR studies. Chem. Rev. May 9), 0003-6021X., 96(3), 1027-1044.

[29] Kothandan, G, Gadhe, C. G, Madhavan, T, Choi, C. H, & Cho, S. J. (2011). Docking and 3D-QSAR (quantitative structure activity relationship) studies of flavones, the potent inhibitors of p-glycoprotein targeting the nucleotide binding domain. Eur J

[30] Kubinyi, H. (1995). Strategies and recent technologies in drug discovery. Pharmazie.

[31] Kuo, C. L, Assefa, H, Kamath, S, Brzozowski, Z, Slawinski, J, Saczewski, F, Buolam‐ wini, J. K, & Neamati, N. (2004). Application of CoMFA and CoMSIA 3D-QSAR and docking studies in optimization of mercaptobenzenesulfonamides as HIV-1 integrase

[32] Li, Y, Wang, Y. H, Yang, L, Zhang, S. W, Liu, C. H, & Yang, S. L. (2005). Comparison of steroid substrates and inhibitors of P-glycoprotein by 3D-QSAR analysis. J. Mol.

[33] Litman, T, Zeuthen, T, Skovsgaard, T, & Stein, W. D. (1997). Structure-activity rela‐ tionships of P-glycoprotein interacting drugs: kinetic characterization of their effects on ATPase activity, Biochem. Biophys. Acta Aug 1997), 0006-3002, 1361(2), 159-168.

[34] Ma, X. L, Chen, C, & Yang, J. (2005). Predictive model of blood-brain barrier penetra‐ tion of organic compounds. Acta Pharmacologica Sinica. Apr 2005), 1745-7254, 26(4),

[35] Norinder, U, & Haeberlein, M. (2002). Computational approaches to the prediction of the blood-brain distribution. Adv Drug Deliv Rev., (Mar 2002):291-313, 0016-9409X.,

[36] Ohtsuki, S, Ito, S, & Terasaki, T. (2010). Is P-glycoprotein involved in amyloid-β elim‐ ination across the blood-brain barrier in Alzheimer's disease? *Clin Pharmacol Ther.*

[37] Ooms, F, Wouters, J, Collin, S, Durant, F, Jegham, S, & George, P. (1998). Molecular lipophilicity potential by CLIP, a reliable tool for the description of the 3D distribu‐ tion of lipophilicity: application to 3-phenyloxazolidin-2-one, a prototype series of re‐ versible MAOA inhibitors. *Bioorg. Med. Chem. Lett.* (Jun 1998), 0096-0894X., 8(11),

Biol Chem. Apr 1996), 0021-9258, 271(16), 9240-9248.

Med Chem. Sep 2011), 0223-5234, 46(9), 4078-4088.

Struct. Sep 2005), 0166-1280, 733(1-3), 111-118.

(Oct 2010), 0009-9236, 88(4), 443-445.

inhibitors. J Med Chem. Jan 2004), 0223-5234, 47(2), 385-399.

Oct 1995), 0031-7144, 50(10), 647-662.

York. 0-47135-168-7

500-512.

54(3)

1425-1430.


[26] Kast, C, Canfield, V, Levenson, R, & Gros, P. (1996). Transmembrane organization of mouse P-glycoprotein determined by epitope insertion and immunofluorescence. J Biol Chem. Apr 1996), 0021-9258, 271(16), 9240-9248.

rosis- and Alzheimer's disease-related animal models despite reported blood-brain

barrier disruption. *Drug Metab Dispos.* (Aug 2010), 0090-9556, 38(8), 1355-1361.

*Chem. Sci.* (Aug 1988), 0002-7863, 110(18), 5959-5967.

*Pharmacol Sci.* (August 2004), 0165-6147, 25(8), 423-429.

*Pharmacol.* (May 2010), 0002-6895X., 77(5), 715-723.

*Pharmaceutical Res* Nov 2002), 0724-8741, 19(11), 1611-1621.

*Trends. Biol. Sci.*, 0962-8924, 17, 18-21.

0223-5234, 55(7), 3261-3273.

65-72.

286 Neurodegenerative Diseases

1748-1756.

385-427.

389-393.

resistance. *J. Med. Chem.* (Sep 1996), 0223-5234, 39(20), 4099-4108.

[15] Cramer, R. D, Patterson, D. E, & Bunce, J. D. (1988). Comparative molecular field analysis (CoMFA). 1. Effect of shape on binding of steroids to carrier proteins. *J. Am.*

[16] Dhainaut, A, Regnier, G, Tizot, A, Pierre, A, Leonce, S, Guilbaud, N, Kraus-berthier, L, & Atassi, G. (1996). New purines and purine analogs as modulators of multidrug

[17] Doige, C. A, Yu, X, & Sharom, F. J. (1993). The effects of lipids and detergents on AT‐ Pase-active P-glycoprotein. *Biochim. Biophys. Acta* (Feb 1993), 0006-3002, 1146(1),

[18] Ford, J. M, Bruggemann, E. P, Pastan, I, Gottesman, M. M, & Hait, W. N. (1990). Cel‐ lular and biochemical characterization of thioxanthenes for revesal of multidrug re‐ sistance in human and murine cell lines. *Cancer Res.* (Mar 1990), 0008-5472, 50(6),

[19] Fromm, M. F. (2004). Importance of P-glycoprotein at blood-tissue barriers. *Trends*

[20] Gottesman, M. M, & Pastan, I. (1993). Biochemistry of Multidrug Resistance Mediat‐ ed by the Multidrug Transporter, *Annu Rev Biochem* No. (July 1993), 0066-4154, 62,

[21] Hartz, A. M, Miller, D. S, & Bauer, B. (2010). Restoring blood-brain barrier P-glyco‐ protein reduces brain amyloid-beta in a mouse model of Alzheimer's disease. *Mol*

[22] Higgins, C. F, & Gottesman, M. M. (1992). Is the multidrug transporter a flippase?

[23] Iyer, M, Mishra, R, Han, Y, & Hopfinger, A. J. (2002). Predicting Blood-Brain Barrier Partitioning of Organic Molecules Using Membrane-Interaction QSAR Analysis.

[24] Jabeen, I, Pleban, K, Rinner, U, Chiba, P, & Ecker, G. F. (2012). Structure-activity rela‐ tionships, ligand efficiency, and lipophilic efficiency profiles of benzophenone-type inhibitors of the multidrug transporter p-glycoprotein. J. Med. Chem. Apr 2012),

[25] Jeynes, B, & Provias, J. (2011). An investigation into the role of P-glycoprotein in Alz‐ heimer's disease lesion pathogenesis. Neurosci Lett. Jan 2011), 0168-0102, 487(3),


[38] Palmeira, A, Sousa, E, Fernandes, M. X, Pinto, M. M, & Vasconcelos, M. H. (2012). Multidrug resistance reversal effects of aminated thioxanthones and interaction with cytochrome P450 3A4. J Pharm Pharm Sci., (Jan 2012), 1482-1826, 15(1), 31-45.

[50] Wiese, M, & Pajeva, I. K. (2001). Structure-Activity Relationships of Multidrug Resist‐

QSAR Analysis of Purine-Type and Propafenone-Type Substrates of P-Glycoprotein Targeting β-Amyloid Clearance

http://dx.doi.org/10.5772/54975

289

ance Reversers. *Curr. Med. Chem.* (May 2001), 0929-8673, 8(6), 685-713.


[38] Palmeira, A, Sousa, E, Fernandes, M. X, Pinto, M. M, & Vasconcelos, M. H. (2012). Multidrug resistance reversal effects of aminated thioxanthones and interaction with

[39] Ponce, Y. M, Garit, J. A, Torrens, F, Zaldivar, V. R, & Castro, E. A. (2004). Atom, atom-type, and total linear indices of the "molecular pseudograph's atom adjacency matrix": application to QSPR/QSAR studies of organic compounds. Molecules (Dec.

[40] Schmid, D, Ecker, G, Kopp, S, Hitzler, M, & Chiba, P. (1999). Structure-activity rela‐ tionship studies of propafenone analogs cased on P-glycoprotein ATPase activity

[41] Shapiro, A. B, & Ling, V. (1994). ATPase activity of purified and reconstituted P-gly‐ coprotein from Chinese hamster ovary cells. *J. Biol. Chem.* (Feb 1994), 0021-9258,

[42] Sharom, F. J. (1997). The P-Glycoprotein Efflux Pump: How Does it Transport Drugs?

[43] Stouch, T. R, & Gudmundsson, O. (2002). Progress in understanding the structureactivity relationships of P-glycoprotein. *Adv Drug Deliv Rev.* (Mar 2002), 0016-9409X.,

[44] Taub, M. E, Podila, L, Ely, D, & Almeida, I. (2005). Functional assessment of multiple P-glycoprotein (P-gp) probe substrates: influence of cell line and modulator concen‐ tration on P-gp activity. *Drug Metab Dispos.* (Nov 2005), 0090-9556, 33(11), 1679-1687.

[45] Vogelgesang, S, Jedlitschky, G, Brenn, A, & Walker, L. C. (2011). The role of the ATPbinding cassette transporter P-glycoprotein in the transport of β-amyloid across the

[46] Wang, R. B, Kuo, C. L, Lien, L. L, & Lien, E. J. (2003). Structure-activity relationship: analyses of p-glycoprotein substrates and inhibitors. *J Clin Pharm Ther.* (Jun 2003),

[47] Wang, Y. H, Li, Y, Yang, S. L, & Yang, L. (2005). An in silico approach for screening flavonoids as P-glycoprotein inhibitors based on a Bayesian-regularized neural net‐

[48] Wang, Y. H, Li, Y, Yang, S. L, & Yang, L. (2005). Classification of Substrates and In‐ hibitors of P-Glycoprotein Using Unsupervised Machine Learning Approach. *J.*

[49] Waterhouse, R. N. (2003). Determination of lipophilicity and its use as a predictor of blood-brain barrier penetration of molecular imaging agents. *Mol Imaging Biol.*, (Nov-

work. *J Comput Aided Mol Des.* (Mar 2005), 0092-0654X., 19(3), 137-147.

*Chem. Inf. Model.* (May 2005), 1549-9596, 45(3), 750-757.

blood-brain barrier. *Curr Pharm Des.17*1381-6128(26), 2778-2786.

*J*. *Membr. Biol*. (Dec 1997), 0022-2631, 160(3), 161-175.

measurements. *Biochem Pharmacol.* (Nov 1999), 0300-5127, 58(9), 1447-1456.

cytochrome P450 3A4. J Pharm Pharm Sci., (Jan 2012), 1482-1826, 15(1), 31-45.

31), 1420-3049, 9(12), 1100-1123.

269(5), 3745-3754.

288 Neurodegenerative Diseases

54(3), 315-328.

0269-4727, 28(3), 203-228.

Dec 2003), 1536-1632, 5(6), 376-389.

**Chapter 12**

**Therapeutic Interventions in Alzheimer Disease**

Alzheimer disease (AD), was first recognized in the early 1900's by Alois Alzheimer, a German psychiatrist and neuro pathologist and named after him (Fig.1). Auguste Deter, in 1902 is a reported patient of Dr. Alois (Fig.2). AD is the most common form of dementia affecting millions of the geriatric population worldwide, mostly those above 65-85 yrs of age. Women are more commonly affected than man [1]. Alzheimer currently afflicts about 5.2 million Americans and with the rapid escalation of the prevalence of the disease, the figure is expected to double by 2020. According to WHO, there are about 18 million people worldwide with AD, the figure will be projected to nearly double by 2025 to 34 million. Developing countries like India and China will be among the countries worst hit by AD due to ageing of the population and likely some genetic factors. In 2000, India had 3.5 million Alzheimer patients, however with the fast graying of population and growth rate being fastest in the 80+ segment of the society, the number of Alzheimer patients have been growing at a phenomenal rate [2]. This neurodegenerative fatal brain disorder generally begins in late life and disease progression is gradual and continuous, the longevity of a patient is about 8-10 yrs after symptoms appear. The disease conditions range from mild, moderate to severe; in mild conditions patients have some functional impairments, in moderate conditions there's a dependence on care givers for some important daily activities, in severe conditions there is complete neuronal and memory loss, motor impairment making the patient absolutely dependent on care givers. Age related

behavioral changes and symptoms of Alzheimer should not be confused.

Typical clinical symptoms of Alzheimer include: signs of progressive memory loss disturbing dailyactivities,difficultyincompletingdailyactivities,poorjudgment,visionproblems,sudden changes in mood and personality, self withdrawal from hobbies or social contacts, loss of cognition, loss of coordination, etc. Ageing is the greatest risk factor of AD though Alzheimer is not a normal part of aging since people below 65yrs of age can also develop AD referred to as 'younger or early onset' [1,2]. The differences between a normal aged brain and the brain of an

and reproduction in any medium, provided the original work is properly cited.

© 2013 Mitra and Dey; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Analava Mitra and Baishakhi Dey

http://dx.doi.org/10.5772/54915

**1. Introduction**

Additional information is available at the end of the chapter

### **Therapeutic Interventions in Alzheimer Disease**

Analava Mitra and Baishakhi Dey

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54915

### **1. Introduction**

Alzheimer disease (AD), was first recognized in the early 1900's by Alois Alzheimer, a German psychiatrist and neuro pathologist and named after him (Fig.1). Auguste Deter, in 1902 is a reported patient of Dr. Alois (Fig.2). AD is the most common form of dementia affecting millions of the geriatric population worldwide, mostly those above 65-85 yrs of age. Women are more commonly affected than man [1]. Alzheimer currently afflicts about 5.2 million Americans and with the rapid escalation of the prevalence of the disease, the figure is expected to double by 2020. According to WHO, there are about 18 million people worldwide with AD, the figure will be projected to nearly double by 2025 to 34 million. Developing countries like India and China will be among the countries worst hit by AD due to ageing of the population and likely some genetic factors. In 2000, India had 3.5 million Alzheimer patients, however with the fast graying of population and growth rate being fastest in the 80+ segment of the society, the number of Alzheimer patients have been growing at a phenomenal rate [2]. This neurodegenerative fatal brain disorder generally begins in late life and disease progression is gradual and continuous, the longevity of a patient is about 8-10 yrs after symptoms appear. The disease conditions range from mild, moderate to severe; in mild conditions patients have some functional impairments, in moderate conditions there's a dependence on care givers for some important daily activities, in severe conditions there is complete neuronal and memory loss, motor impairment making the patient absolutely dependent on care givers. Age related behavioral changes and symptoms of Alzheimer should not be confused.

Typical clinical symptoms of Alzheimer include: signs of progressive memory loss disturbing dailyactivities,difficultyincompletingdailyactivities,poorjudgment,visionproblems,sudden changes in mood and personality, self withdrawal from hobbies or social contacts, loss of cognition, loss of coordination, etc. Ageing is the greatest risk factor of AD though Alzheimer is not a normal part of aging since people below 65yrs of age can also develop AD referred to as 'younger or early onset' [1,2]. The differences between a normal aged brain and the brain of an

© 2013 Mitra and Dey; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Figure 1.** Dr. Alois Alzheimer(1864-1915)

**Figure 2.** Auguste Deter (1902), patient of Dr. Alois

AlzheimerpatienthavebeendepictedinFig.3.OtherprominentriskfactorsinADincludefamily history, heredity (genetics), environmental factors. A genetic factor in late onset AD is ApoE ε4. The gene for apolipoprotein E (ApoE) on chromosome 19 has gained much recent attention in thepathogenesis ofAD.ApoEis aproteinmodulator ofphospholipidtransportthatmighthave a role in synaptic remodeling. ApoE has three common alleles, ApoE ε2, ε3, and ε4, which are expressed in varying amounts in the normal person. It is the ApoE ε4 genotype that is associat‐ ed with the risk of AD. Everyone inherits one form of APoE gene from each parent; those who receive ApoE ε4 gene are at the risk of developing AD than those who receive ApoE ε2, ApoE ε3. Individuals who inherit two ApoE ε4 genes are at even higher risk; however inheriting one or two copies of genes does not gurantee that the individual will develop Alzheimer [3,4,5]. Moderate andsevere headtrauma,traumatic brain injuries are associated with increasedrisk of AD.Headinjuryresultinginlossofconsciousnessorposttraumaticamnesialastingabout30mins is associatedwithtwice the risk ofdevelopingAD; severeheadinjurieshave about 4.5 foldrisks. Mildcognitiveimpairment(MCI)hasanestablishedlinkwithADinwhichapersonhasproblems with memory, language, and some essential cognitive ability severe enough to be noticeable to others and shown upon cognitive tests but not severe enough to interfere with the daily life. However there is no clear explanation on the fact that why some people with MCI develops dementia and in some cases it is not. The overall health of the heart and blood vessels shows a close linkagewiththebrainhealthsince abrainisnourishedbythe richnetworkofbloodvessels and a healthy heart pumps nutrient and oxygen rich blood to these vessels. Cardiovascular diseases,highbloodpressure,hypertensions,type2diabetes,cholesterolemia,obesity, smoking habits, and physical inactivity potentially increases the risk of AD [6-9].

#### **2. Patho physiology of Alzheimer**

The human brain consists of 100 millions of neurons connected to each other through synapses forming communication network; each cells entitled to perform their own duties relating to memory, thinking, smell, taste, emotions etc. In case of AD these brain cells can't perform their duties. Patho physiology of Alzheimer disease being complex and multi factorial shows important pathological changes in brain like accumulation of amyloid cerebral plaques and neurofibrillary tangles of abnormal insoluble 'tau' protein (Fig.6,8,9). AD is also considered a 'tauopathy' due to abnormal aggregation of the tau protein. Every neuron has a cytoskeleton, an internal support structure partly made up of structures called microtubules. These micro‐ tubules act like tracks, guiding nutrients and molecules from the body of the cell to the ends of the axon and back. A protein called *tau* stabilizes the microtubules when phosphorylated, and is therefore called a microtubule-associated protein. In AD, tau undergoes chemical

**Figure 3.** Alzheimer disease diagram, normal aged brain (left), Alzheimer brain (right)

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293

**Figure 4.** Shrinkage of Brain tissue in Alzheimer disease

**Figure 3.** Alzheimer disease diagram, normal aged brain (left), Alzheimer brain (right)

**Figure 4.** Shrinkage of Brain tissue in Alzheimer disease

AlzheimerpatienthavebeendepictedinFig.3.OtherprominentriskfactorsinADincludefamily history, heredity (genetics), environmental factors. A genetic factor in late onset AD is ApoE ε4. The gene for apolipoprotein E (ApoE) on chromosome 19 has gained much recent attention in thepathogenesis ofAD.ApoEis aproteinmodulator ofphospholipidtransportthatmighthave a role in synaptic remodeling. ApoE has three common alleles, ApoE ε2, ε3, and ε4, which are expressed in varying amounts in the normal person. It is the ApoE ε4 genotype that is associat‐ ed with the risk of AD. Everyone inherits one form of APoE gene from each parent; those who receive ApoE ε4 gene are at the risk of developing AD than those who receive ApoE ε2, ApoE ε3. Individuals who inherit two ApoE ε4 genes are at even higher risk; however inheriting one or two copies of genes does not gurantee that the individual will develop Alzheimer [3,4,5]. Moderate andsevere headtrauma,traumatic brain injuries are associated with increasedrisk of AD.Headinjuryresultinginlossofconsciousnessorposttraumaticamnesialastingabout30mins is associatedwithtwice the riskofdevelopingAD; severeheadinjurieshave about 4.5 foldrisks. Mildcognitiveimpairment(MCI)hasanestablishedlinkwithADinwhichapersonhasproblems with memory, language, and some essential cognitive ability severe enough to be noticeable to others and shown upon cognitive tests but not severe enough to interfere with the daily life. However there is no clear explanation on the fact that why some people with MCI develops dementia and in some cases it is not. The overall health of the heart and blood vessels shows a close linkagewiththebrainhealthsince abrainisnourishedbythe richnetworkofbloodvessels and a healthy heart pumps nutrient and oxygen rich blood to these vessels. Cardiovascular diseases,highbloodpressure,hypertensions,type2diabetes,cholesterolemia,obesity, smoking

habits, and physical inactivity potentially increases the risk of AD [6-9].

The human brain consists of 100 millions of neurons connected to each other through synapses forming communication network; each cells entitled to perform their own duties relating to

**2. Patho physiology of Alzheimer**

**Figure 1.** Dr. Alois Alzheimer(1864-1915)

292 Neurodegenerative Diseases

**Figure 2.** Auguste Deter (1902), patient of Dr. Alois

memory, thinking, smell, taste, emotions etc. In case of AD these brain cells can't perform their duties. Patho physiology of Alzheimer disease being complex and multi factorial shows important pathological changes in brain like accumulation of amyloid cerebral plaques and neurofibrillary tangles of abnormal insoluble 'tau' protein (Fig.6,8,9). AD is also considered a 'tauopathy' due to abnormal aggregation of the tau protein. Every neuron has a cytoskeleton, an internal support structure partly made up of structures called microtubules. These micro‐ tubules act like tracks, guiding nutrients and molecules from the body of the cell to the ends of the axon and back. A protein called *tau* stabilizes the microtubules when phosphorylated, and is therefore called a microtubule-associated protein. In AD, tau undergoes chemical

**Figure 5.** Normal homeostasis of Alzheimer Disease

changes, becoming hyperphosphorylated; it then begins to pair with other threads, creating neurofibrillary tangles and disintegrating the neuron's transport system [10,11].

As per the amyloid hypothesis, the neuropathologic hallmarks of AD are neuritic plaques and neurofibrillary tangles, consisting of hyper-phosphorylated microtubule-associated protein called 'tau' and extracellular amyloid plaques. The main component of amyloid plaques in AD is amyloid β (Aβ) peptide (38–43 amino acids) which is a proteolytic by-product from the amyloid precursor protein (APP) generated by the sequential β-secretase and γ-secretase cleavage (Fig.7). Research data have been shown that oligomeric Aβ species (smallest of which are dimers) isolated from AD brains are the most synaptotoxic forms. Aggregated amyloid fibrils, which are believed to be the toxic form of the protein disrupts the cell's calcium ion homeostasis, induces programmed cell death or apoptosis (Fig.7). It is also known that Aβ selectively builds up in the mitochondria in the cells of Alzheimer's-affected brains, and it also inhibits certain enzyme functions and the utilization of glucose by neurons [3,11-13].

neural systems and composed of paired helical filaments of hyperphosphorylated microtubule-associated tau protein that may cause disruption of normal cytoskeletal architecture with subsequent neuronal cell death. Other pathological alterations commonly seen in the brains of AD patients include neuropil threads, granulovacuolar degeneration, and amyloid angiopathy. Amyloid angiopathy is a distinct vascular lesion found in many AD brains, consisting of amyloid deposition in the walls of small-to medium-sized cortical and leptome‐ ningeal arteries. As a result of the deposits, the involved vessels may become compromised

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**Figure 6.** Normal Neuron (left), Neurofibrillary tangles, Amyloid plaques in Alzheimer (right)

**Figure 7.** Enzyme action on APP (Amyloid precursor protein) and its fragmentation

Inflammatory processes and cytokines play a controversial role in the pathology of Alzheimer's disease. Inflammation is a general marker of tissue damage in any disease, and may be either

with resultant hemorrhage [14-17].

The brain of a Alzheimer patient shows marked histo-pathophysiological changes like, widened sulci and shrinkage of the gyri (Fig.10). In the great majority of cases, every part of the cerebral cortex is involved; however, the occipital pole is often relatively spared. The cortical ribbon may be thinned and ventricular dilatation apparent, especially in the temporal horn, due to atrophy of the amygdala and hippocampus. Microscopically, there is significant loss of neurons, in addition to shrinkage of large cortical neurons, synapses, in association with shrinkage of the dendritic arbor of large neurons, is thought to be the critical pathological substrate (Fig.4). Neurofibrillary tangles are the other characteristic neurohistopathologic hallmarks seen in AD. These tangles found inside the neurons, concentrated in vulnerable

**Figure 6.** Normal Neuron (left), Neurofibrillary tangles, Amyloid plaques in Alzheimer (right)

changes, becoming hyperphosphorylated; it then begins to pair with other threads, creating

As per the amyloid hypothesis, the neuropathologic hallmarks of AD are neuritic plaques and neurofibrillary tangles, consisting of hyper-phosphorylated microtubule-associated protein called 'tau' and extracellular amyloid plaques. The main component of amyloid plaques in AD is amyloid β (Aβ) peptide (38–43 amino acids) which is a proteolytic by-product from the amyloid precursor protein (APP) generated by the sequential β-secretase and γ-secretase cleavage (Fig.7). Research data have been shown that oligomeric Aβ species (smallest of which are dimers) isolated from AD brains are the most synaptotoxic forms. Aggregated amyloid fibrils, which are believed to be the toxic form of the protein disrupts the cell's calcium ion homeostasis, induces programmed cell death or apoptosis (Fig.7). It is also known that Aβ selectively builds up in the mitochondria in the cells of Alzheimer's-affected brains, and it also

neurofibrillary tangles and disintegrating the neuron's transport system [10,11].

**Figure 5.** Normal homeostasis of Alzheimer Disease

294 Neurodegenerative Diseases

inhibits certain enzyme functions and the utilization of glucose by neurons [3,11-13].

The brain of a Alzheimer patient shows marked histo-pathophysiological changes like, widened sulci and shrinkage of the gyri (Fig.10). In the great majority of cases, every part of the cerebral cortex is involved; however, the occipital pole is often relatively spared. The cortical ribbon may be thinned and ventricular dilatation apparent, especially in the temporal horn, due to atrophy of the amygdala and hippocampus. Microscopically, there is significant loss of neurons, in addition to shrinkage of large cortical neurons, synapses, in association with shrinkage of the dendritic arbor of large neurons, is thought to be the critical pathological substrate (Fig.4). Neurofibrillary tangles are the other characteristic neurohistopathologic hallmarks seen in AD. These tangles found inside the neurons, concentrated in vulnerable

**Figure 7.** Enzyme action on APP (Amyloid precursor protein) and its fragmentation

neural systems and composed of paired helical filaments of hyperphosphorylated microtubule-associated tau protein that may cause disruption of normal cytoskeletal architecture with subsequent neuronal cell death. Other pathological alterations commonly seen in the brains of AD patients include neuropil threads, granulovacuolar degeneration, and amyloid angiopathy. Amyloid angiopathy is a distinct vascular lesion found in many AD brains, consisting of amyloid deposition in the walls of small-to medium-sized cortical and leptome‐ ningeal arteries. As a result of the deposits, the involved vessels may become compromised with resultant hemorrhage [14-17].

Inflammatory processes and cytokines play a controversial role in the pathology of Alzheimer's disease. Inflammation is a general marker of tissue damage in any disease, and may be either secondary to tissue damage in AD or a marker of an immunological response. Some studies have demonstrated the presence of activated microglia, a marker of the brain's immune response. Alterations in the distribution of different neurotrophic factors and in the expression of their receptors such as the brain derived neurotrophic factor (BDNF) have been found in AD. Microglia have been shown to be significantly activated in AD brains and localized at sites of amyloid deposition. Early activation of microglia in early AD pathogenesis has been shown to be beneficial in scavenging and clearing toxic Aβ from the brain. Peri vascular macrophages like CD163 (hemoglobin-haptoglobin scavenger receptor) and CD206 (mannose receptor) are antigen-presenting phagocytic cells located in outer aspects of blood vessels within the brain, have shown to respond to CNS inflammation. Results of clinical trials have shown that, bloodderived macrophages from AD patients were shown to be less effective at phagocytosing Aβ compared with cells derived from non-demented control patients. Microglial and peri vascular macrophages may play role in clearing Aβ from the brain and in enhancing AD-related inflammation [18-20].

**Figure 9.** Changes in Tau protein & disintegration of microtubules in brain cells

**Figure 10.** Histopathologic image of senile plaques in Alzheimer Brain

Vascular dysfunction has a critical role in AD. Results of epidemiological and pathological studies have demonstrated positive links between cerebro vascular disorders and AD. For example a person with severe atherosclerosis is at a threefold increased risk of developing AD or vascular dementia. Due to reduced cerebral blood flow in AD there are abnormal cholinergic innervations of intra cerebral blood vessels leading to brain hypo perfusion. Results of recent investigations have shown that, the upregulation of two transcription factors myocardin (MYOCD) and serum response factor (SRF) in AD lead to arterial hypercontractility potenti‐ ating reduced cerebral blood flow. Other vascular anatomical defects observed in AD include

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**Figure 8.** Neuro-fibrillary tangle with hyperphosphorylated Tau Protein(high resolution)

**Figure 9.** Changes in Tau protein & disintegration of microtubules in brain cells

secondary to tissue damage in AD or a marker of an immunological response. Some studies have demonstrated the presence of activated microglia, a marker of the brain's immune response. Alterations in the distribution of different neurotrophic factors and in the expression of their receptors such as the brain derived neurotrophic factor (BDNF) have been found in AD. Microglia have been shown to be significantly activated in AD brains and localized at sites of amyloid deposition. Early activation of microglia in early AD pathogenesis has been shown to be beneficial in scavenging and clearing toxic Aβ from the brain. Peri vascular macrophages like CD163 (hemoglobin-haptoglobin scavenger receptor) and CD206 (mannose receptor) are antigen-presenting phagocytic cells located in outer aspects of blood vessels within the brain, have shown to respond to CNS inflammation. Results of clinical trials have shown that, bloodderived macrophages from AD patients were shown to be less effective at phagocytosing Aβ compared with cells derived from non-demented control patients. Microglial and peri vascular macrophages may play role in clearing Aβ from the brain and in enhancing AD-related

**Figure 8.** Neuro-fibrillary tangle with hyperphosphorylated Tau Protein(high resolution)

inflammation [18-20].

296 Neurodegenerative Diseases

**Figure 10.** Histopathologic image of senile plaques in Alzheimer Brain

Vascular dysfunction has a critical role in AD. Results of epidemiological and pathological studies have demonstrated positive links between cerebro vascular disorders and AD. For example a person with severe atherosclerosis is at a threefold increased risk of developing AD or vascular dementia. Due to reduced cerebral blood flow in AD there are abnormal cholinergic innervations of intra cerebral blood vessels leading to brain hypo perfusion. Results of recent investigations have shown that, the upregulation of two transcription factors myocardin (MYOCD) and serum response factor (SRF) in AD lead to arterial hypercontractility potenti‐ ating reduced cerebral blood flow. Other vascular anatomical defects observed in AD include atrophy and irregularities of arterioles and capillaries, increase in collagen IV, heparin sulfate proteoglycans and laminin deposition in the basement membrane, disruption of the basement membrane, reduced total micro vascular density, occasional swelling of astrocytic endfeets, and extensive degeneration of the endothelium during the disease progression [21-24].

Decreased cerebral blood flow (CBF) has a negative impact on the protein synthesis necessary for memory and learning, and may eventually lead to neuritic injury and neuronal death. Moreover due to cerebral hypo perfusion amyloid β-peptide (Aβ) clearance across the blood– brain barrier (BBB) will be impaired leading to accumulation of Aβ on cerebral blood vessels and brain parenchyma causing cerebral amyloid angiopathy (CAA), which is associated with cognitive decline and is another significant factor in the pathogenesis of AD. CAA can severely disrupt the integrity of the blood vessel wall resulting in micro or macro intra cerebral bleedings that exacerbates neurodegenerative process and inflammatory response and may lead to hemorrhagic stroke. Cerebral amyloid angiopathy (CAA) with Aβ deposits in the vascular smooth muscle cell layer is a major pathological threat to the neurovascular unit in AD [13,17,20,21].

**Figure 11.** Cascade of events in Alzheimer Disease

[12,25,27-30].

disease is depicted in Fig.11.

**3. Diagnosis of Alzheimer**

Fromthegeneticpointofviewithasbeenfoundthatthedistributionof cerebrovascularamyloid in AD varies with apoE genotype and specifically the increasing dose of apoE4 alleles has been associatedwithincreasedCAA.Understandingthecellularandmolecularmechanismbywhich apoE genotype influences the pathogenicity of the disease process in AD individuals can act as important targets in developing new therapeutic interventions and diagnostic aids for AD.

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Interactions of Aβ with human Vascular Smooth Muscle Cells has been found to significantly increase the activation of matrix metalloproteinase 2 (MMP2) via increasing the mRNA expres‐ sion of membrane type 1 (MT1)-MMP, the primary MMP2 activator at the cell surface. MMP9 specifically has been found in postmortem AD tissue in significant amounts. Activated MMP9 can degrade basement membranes, extracellular matrix proteins and tight junction proteins subsequentlydamagingthe integrityoftheBBBandpotentiallyleadingtospontaneous cerebral hemorrhages. Similarly high levels of ROS in AD may damage proteins essentials for impor‐ tant neurovascular mechanisms. The breakdown of the BBB may in turn disrupt the normal transport of nutrients, vitamins and electrolytes across the BBB, which are essential for proper neuronal functioning. Therefore, therapies that reduce ROS, MMP2, and MMP9, or that block RAGE-Aβ interaction may offer potentially useful strategies to correct BBB dysfunction in AD

Research data of epidemiological studies have shown controversial results as regards the association between APOE polymorphism and the rate of progression of cognitive decline in AD after onset. Some reports have suggested that homozygous APOE *ε*4 patients have more rapid cognitive and functional decline following clinical disease onset, but an MRI study on a large cognitively normal population showed rate of volume decrease of entorhinal cortex and hippocampus suggesting potential development. Thus though the role of APOE in the predisposition of AD is well established, still further studies are needed to understand the possible association of APOE with rate of AD progression. The cascade of events in Alzheimer

Alzheimer's disease is usually diagnosed clinically from the patient history, collateral history from relatives, and clinical observations, based on the presence of characteristic neurological

Research evidences have suggested that imbalances between Aβ production and clearance from the brain cause accumulation of Aβ in the wall of cerebral vessels and in the brains of AD individuals. Aβ that is produced both in the brain and periphery by a number of different cell types is transported across the BBB via receptor-mediated transcytosis; the key receptors being involved are: RAGE (Receptor for Advanced Glycation End products) that transports Aβ from the blood into the brain and LRP (low-density lipoprotein receptor related protein-1) that is the major cell surface Aβ clearance receptor that transports Aβ out of the brain across the BBB and promotes Aβ clearance on VSMC. Aβ is not only cleared from the brain interstitial fluid (ISF) as a soluble peptide, but can also be transported by its chaperone proteins in the ISF, such as apolipoprotein E (apoE), apolipoprotein J, and α2-macroglobulin. Apart from direct Aβ clearance into the blood, alternative perivascular route for the clearance of Aβ in the human brain also exists. The pulsation force of cerebral blood vessels has been proposed to drive an Aβ drainage route along the perivascular spaces. Vessel constriction or stiffening reduces the pulsatile blood flow which in turn reduces Aβ clearance along the perivascular spaces leading to increase in Aβ deposition in the arterial wall of AD patients [20-24].

ApoE is a reactive apolipoprotein that exists in 3 iso forms (apoE2, apoE3, and apoE4) in humans has a major function in the transport of lipids and cholesterol in our body. Individuals who carry at least one apoE4 allele have great chances to develop AD. ApoE, an Aβ chaperone protein is found to be associated with impaired transport of Aβ across the BBB. Free Aβ can be rapidly cleared from the brain mainly via LRP or RAGE receptor mediated transcytosis, but Aβ-ApoE complexes (mostly apoE4-Aβ complexes in contrast to apoE2-Aβ and apoE3-Aβ complexes) are cleared by the very low-density lipoprotein receptor at a much slower rate, causing Aβ retention in the brain. Transport of Aβ via the receptor for advanced glycation endproducts (RAGE) across the BBB provides a major source of Aβ that can deposit in the brain and can directly lead to neuroinflammation by activating nuclear factor-κB mediated secretion of pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin 6 that may reduce the BBB potency [13,17,24-29].

**Figure 11.** Cascade of events in Alzheimer Disease

atrophy and irregularities of arterioles and capillaries, increase in collagen IV, heparin sulfate proteoglycans and laminin deposition in the basement membrane, disruption of the basement membrane, reduced total micro vascular density, occasional swelling of astrocytic endfeets, and extensive degeneration of the endothelium during the disease progression [21-24].

Decreased cerebral blood flow (CBF) has a negative impact on the protein synthesis necessary for memory and learning, and may eventually lead to neuritic injury and neuronal death. Moreover due to cerebral hypo perfusion amyloid β-peptide (Aβ) clearance across the blood– brain barrier (BBB) will be impaired leading to accumulation of Aβ on cerebral blood vessels and brain parenchyma causing cerebral amyloid angiopathy (CAA), which is associated with cognitive decline and is another significant factor in the pathogenesis of AD. CAA can severely disrupt the integrity of the blood vessel wall resulting in micro or macro intra cerebral bleedings that exacerbates neurodegenerative process and inflammatory response and may lead to hemorrhagic stroke. Cerebral amyloid angiopathy (CAA) with Aβ deposits in the vascular smooth muscle cell layer is a major pathological threat to the neurovascular unit in

Research evidences have suggested that imbalances between Aβ production and clearance from the brain cause accumulation of Aβ in the wall of cerebral vessels and in the brains of AD individuals. Aβ that is produced both in the brain and periphery by a number of different cell types is transported across the BBB via receptor-mediated transcytosis; the key receptors being involved are: RAGE (Receptor for Advanced Glycation End products) that transports Aβ from the blood into the brain and LRP (low-density lipoprotein receptor related protein-1) that is the major cell surface Aβ clearance receptor that transports Aβ out of the brain across the BBB and promotes Aβ clearance on VSMC. Aβ is not only cleared from the brain interstitial fluid (ISF) as a soluble peptide, but can also be transported by its chaperone proteins in the ISF, such as apolipoprotein E (apoE), apolipoprotein J, and α2-macroglobulin. Apart from direct Aβ clearance into the blood, alternative perivascular route for the clearance of Aβ in the human brain also exists. The pulsation force of cerebral blood vessels has been proposed to drive an Aβ drainage route along the perivascular spaces. Vessel constriction or stiffening reduces the pulsatile blood flow which in turn reduces Aβ clearance along the perivascular

spaces leading to increase in Aβ deposition in the arterial wall of AD patients [20-24].

may reduce the BBB potency [13,17,24-29].

ApoE is a reactive apolipoprotein that exists in 3 iso forms (apoE2, apoE3, and apoE4) in humans has a major function in the transport of lipids and cholesterol in our body. Individuals who carry at least one apoE4 allele have great chances to develop AD. ApoE, an Aβ chaperone protein is found to be associated with impaired transport of Aβ across the BBB. Free Aβ can be rapidly cleared from the brain mainly via LRP or RAGE receptor mediated transcytosis, but Aβ-ApoE complexes (mostly apoE4-Aβ complexes in contrast to apoE2-Aβ and apoE3-Aβ complexes) are cleared by the very low-density lipoprotein receptor at a much slower rate, causing Aβ retention in the brain. Transport of Aβ via the receptor for advanced glycation endproducts (RAGE) across the BBB provides a major source of Aβ that can deposit in the brain and can directly lead to neuroinflammation by activating nuclear factor-κB mediated secretion of pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin 6 that

AD [13,17,20,21].

298 Neurodegenerative Diseases

Fromthegeneticpointofviewithasbeenfoundthatthedistributionof cerebrovascularamyloid in AD varies with apoE genotype and specifically the increasing dose of apoE4 alleles has been associatedwithincreasedCAA.Understandingthecellularandmolecularmechanismbywhich apoE genotype influences the pathogenicity of the disease process in AD individuals can act as important targets in developing new therapeutic interventions and diagnostic aids for AD.

Interactions of Aβ with human Vascular Smooth Muscle Cells has been found to significantly increase the activation of matrix metalloproteinase 2 (MMP2) via increasing the mRNA expres‐ sion of membrane type 1 (MT1)-MMP, the primary MMP2 activator at the cell surface. MMP9 specifically has been found in postmortem AD tissue in significant amounts. Activated MMP9 can degrade basement membranes, extracellular matrix proteins and tight junction proteins subsequentlydamagingthe integrityoftheBBBandpotentiallyleadingtospontaneous cerebral hemorrhages. Similarly high levels of ROS in AD may damage proteins essentials for impor‐ tant neurovascular mechanisms. The breakdown of the BBB may in turn disrupt the normal transport of nutrients, vitamins and electrolytes across the BBB, which are essential for proper neuronal functioning. Therefore, therapies that reduce ROS, MMP2, and MMP9, or that block RAGE-Aβ interaction may offer potentially useful strategies to correct BBB dysfunction in AD [12,25,27-30].

Research data of epidemiological studies have shown controversial results as regards the association between APOE polymorphism and the rate of progression of cognitive decline in AD after onset. Some reports have suggested that homozygous APOE *ε*4 patients have more rapid cognitive and functional decline following clinical disease onset, but an MRI study on a large cognitively normal population showed rate of volume decrease of entorhinal cortex and hippocampus suggesting potential development. Thus though the role of APOE in the predisposition of AD is well established, still further studies are needed to understand the possible association of APOE with rate of AD progression. The cascade of events in Alzheimer disease is depicted in Fig.11.

### **3. Diagnosis of Alzheimer**

Alzheimer's disease is usually diagnosed clinically from the patient history, collateral history from relatives, and clinical observations, based on the presence of characteristic neurological and neuropsychological features and the absence of alternative conditions. Advanced medical imaging technologies like computed tomography (CT) or magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT) or positron emission tomography (PET) can be used to study the cerebral histo pathophysiological conditions of Alzheimer's disease. The Alzheimer's Disease and Related Disorders Association (now known as the Alzheimer's Association) have set up certain criteria for AD diagnosis which points to eight cognitive domains that are most commonly impaired in AD—memory, language, perceptual skills, attention, constructive abilities, orientation, problem solving and functional abilities. The presence of such symptoms should be confirmed by neuropsychological testing for a clinical diagnosis of possible or probable AD. A histopathologic confirmation including a microscopic examination of brain tissue is required for a definitive diagnosis. In addition to histopathologic confirmation, definite AD requires the clinical finding of dementia as deter‐ mined by the Mini-Mental State Examination (MMSE) or other standardized neuropsycho‐ logical testing; the examination must demonstrate deficits in two or more areas of cognition, with progressive memory loss in the absence of delirium. Assessment of intellectual function‐ ing including memory testing can further characterize the state of the disease. The diagnosis can be confirmed with very high accuracy post-mortem when brain material is available and can be examined histologically. Neuropsychological screening tests can help in the diagnosis of AD. In the tests, people are instructed to copy drawings similar to the one shown in the picture, remember words, read, and subtract serial numbers. Neuropsychological tests such as the mini-mental state examination (MMSE), are widely used to evaluate the cognitive impairments needed for diagnosis. More comprehensive test arrays are necessary for high reliability of results, particularly in the earliest stages of the disease. Neurological examination in early AD will usually provide normal results, except for obvious cognitive impairment, which may not differ from that resulting from other diseases processes, including other causes of dementia. Interviews of family members and informations from caregivers about the patient's daily living abilities, the person's mental function are very important in this regard. Psychological tests for depression are also employed, since depression can either be concurrent with AD, an early sign of cognitive impairment, or even the cause [3,4,22].

with mild cognitive impairment will develop Alzheimer's disease within two years. Volumet‐ ric MRI can detect changes in the size of brain regions and measure the atropy in those regions in order to study the progress of AD. Amyloid imaging used in conjunction with other markers

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Resultsofroutinelaboratorytestsonchemistrypanels,bloodcounts,metabolicpanels(e.g.,TSH) spinalfluidanalyses,andinflammatorymarkersareallfoundtobewithinnormallimitsinpatients withAD.Electroencephalographic(EEG)recordingsareusuallynormalorshowdiffuseslowing in later stages of the disease. However it has been found that in AD patients it have been found that the levels of glutamate, creatinine, myo-inositol, N-acetyl aspartate are decreased as compared to normal people. Both decrease in N-acetyl aspartate/Creatinine ratio and decrease in hippocampal glutamate may be an early indicator of AD. Monitoring the level of blood dehydroepiandrosterone (DHEA) variations in response to an oxidative stress could be a useful proxy test for AD since experimental research data have shown that the subjects with Mild Cognitive Impairment did not have a DHEA variation, while the healthy controls did [3,4,8]. Another important marker of the disease is the analysis of cerebrospinal fluid for amyloid beta or tau proteins, both total tau protein and phosphorylated tau181P protein concentrations which predicts the onset of AD with a sensitivity of 94-100%. When used in conjunction with existing neuro imaging techniques, doctors can identify people with significant memory loss who are already developing the disease. Spinal fluid tests are commercially available, unlike the latest

The differential diagnosis for AD is extensive and includes a multitude of neurodegenerative diseases that are associated with the development of dementia including Pick's disease, Lewy body disease, and other diseases such as vascular dementia and Creutzfeldt-Jakob disease. Most of these entities can be differentiated from AD by the clinical history and a careful examination. However, the challenge lies to test new hypotheses and not just to continue descriptive studies using better tools, technologies, increased parameters in CSF analysis and routine lab testing's, and more descriptions about the amount of amyloid in the brain [3,4].

can serve as an important diagnostic tool [3,4].

neuro imaging technology.

**Figure 12.** PET scan in the brain of Alzheimer patient (loss of function in temporal lobe)

With the advancements in imaging technology, computed tomography (CT) and magnetic resonance imaging (MRI) can be used to study the cortical atrophy, disproportionate volume loss in the medial temporal lobe structures and also normal age related changes that may be present at the early onset of disease. Functional imaging studies like the positron emission tomography (PET) and single-photon emission computed tomography (SPECT) scans, can be used as a diagnostic tool, which demonstrate hypometabolism and hypoperfusion, respec‐ tively, in the temporal-parietal regions bilaterally; for neuro imaging to confirm a diagnosis of Alzheimer's in conjunction with evaluations involving mental status examination (Fig.12). In a person already having dementia, SPECT appears to be superior in differentiating Alzheimer's disease from other possible causes, compared with the usual attempts employing mental testing and medical history analysis. Amyloid burden imaging compounds are under devel‐ opment. A new technique known as PiB PET has been developed that uses carbon-11 PET scanning for directly and clearly imaging beta-amyloid deposits in vivo using a tracer that binds selectively to the A-beta deposits with high levels of accuracy in predicting which people with mild cognitive impairment will develop Alzheimer's disease within two years. Volumet‐ ric MRI can detect changes in the size of brain regions and measure the atropy in those regions in order to study the progress of AD. Amyloid imaging used in conjunction with other markers can serve as an important diagnostic tool [3,4].

**Figure 12.** PET scan in the brain of Alzheimer patient (loss of function in temporal lobe)

and neuropsychological features and the absence of alternative conditions. Advanced medical imaging technologies like computed tomography (CT) or magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT) or positron emission tomography (PET) can be used to study the cerebral histo pathophysiological conditions of Alzheimer's disease. The Alzheimer's Disease and Related Disorders Association (now known as the Alzheimer's Association) have set up certain criteria for AD diagnosis which points to eight cognitive domains that are most commonly impaired in AD—memory, language, perceptual skills, attention, constructive abilities, orientation, problem solving and functional abilities. The presence of such symptoms should be confirmed by neuropsychological testing for a clinical diagnosis of possible or probable AD. A histopathologic confirmation including a microscopic examination of brain tissue is required for a definitive diagnosis. In addition to histopathologic confirmation, definite AD requires the clinical finding of dementia as deter‐ mined by the Mini-Mental State Examination (MMSE) or other standardized neuropsycho‐ logical testing; the examination must demonstrate deficits in two or more areas of cognition, with progressive memory loss in the absence of delirium. Assessment of intellectual function‐ ing including memory testing can further characterize the state of the disease. The diagnosis can be confirmed with very high accuracy post-mortem when brain material is available and can be examined histologically. Neuropsychological screening tests can help in the diagnosis of AD. In the tests, people are instructed to copy drawings similar to the one shown in the picture, remember words, read, and subtract serial numbers. Neuropsychological tests such as the mini-mental state examination (MMSE), are widely used to evaluate the cognitive impairments needed for diagnosis. More comprehensive test arrays are necessary for high reliability of results, particularly in the earliest stages of the disease. Neurological examination in early AD will usually provide normal results, except for obvious cognitive impairment, which may not differ from that resulting from other diseases processes, including other causes of dementia. Interviews of family members and informations from caregivers about the patient's daily living abilities, the person's mental function are very important in this regard. Psychological tests for depression are also employed, since depression can either be concurrent

300 Neurodegenerative Diseases

with AD, an early sign of cognitive impairment, or even the cause [3,4,22].

With the advancements in imaging technology, computed tomography (CT) and magnetic resonance imaging (MRI) can be used to study the cortical atrophy, disproportionate volume loss in the medial temporal lobe structures and also normal age related changes that may be present at the early onset of disease. Functional imaging studies like the positron emission tomography (PET) and single-photon emission computed tomography (SPECT) scans, can be used as a diagnostic tool, which demonstrate hypometabolism and hypoperfusion, respec‐ tively, in the temporal-parietal regions bilaterally; for neuro imaging to confirm a diagnosis of Alzheimer's in conjunction with evaluations involving mental status examination (Fig.12). In a person already having dementia, SPECT appears to be superior in differentiating Alzheimer's disease from other possible causes, compared with the usual attempts employing mental testing and medical history analysis. Amyloid burden imaging compounds are under devel‐ opment. A new technique known as PiB PET has been developed that uses carbon-11 PET scanning for directly and clearly imaging beta-amyloid deposits in vivo using a tracer that binds selectively to the A-beta deposits with high levels of accuracy in predicting which people

Resultsofroutinelaboratorytestsonchemistrypanels,bloodcounts,metabolicpanels(e.g.,TSH) spinalfluidanalyses,andinflammatorymarkersareallfoundtobewithinnormallimitsinpatients withAD.Electroencephalographic(EEG)recordingsareusuallynormalorshowdiffuseslowing in later stages of the disease. However it has been found that in AD patients it have been found that the levels of glutamate, creatinine, myo-inositol, N-acetyl aspartate are decreased as compared to normal people. Both decrease in N-acetyl aspartate/Creatinine ratio and decrease in hippocampal glutamate may be an early indicator of AD. Monitoring the level of blood dehydroepiandrosterone (DHEA) variations in response to an oxidative stress could be a useful proxy test for AD since experimental research data have shown that the subjects with Mild Cognitive Impairment did not have a DHEA variation, while the healthy controls did [3,4,8].

Another important marker of the disease is the analysis of cerebrospinal fluid for amyloid beta or tau proteins, both total tau protein and phosphorylated tau181P protein concentrations which predicts the onset of AD with a sensitivity of 94-100%. When used in conjunction with existing neuro imaging techniques, doctors can identify people with significant memory loss who are already developing the disease. Spinal fluid tests are commercially available, unlike the latest neuro imaging technology.

The differential diagnosis for AD is extensive and includes a multitude of neurodegenerative diseases that are associated with the development of dementia including Pick's disease, Lewy body disease, and other diseases such as vascular dementia and Creutzfeldt-Jakob disease. Most of these entities can be differentiated from AD by the clinical history and a careful examination. However, the challenge lies to test new hypotheses and not just to continue descriptive studies using better tools, technologies, increased parameters in CSF analysis and routine lab testing's, and more descriptions about the amount of amyloid in the brain [3,4].

### **4. Therapeutic interventions in Alzheimer**

Virtually there are no proven modalities for cure of Alzheimer's disease; however there are treatment regimens that may improve symptoms and may even delay their progression in the early and middle stages of the disease, allowing patients to maintain certain daily functions for longer. Various country- or region-specific, evidence-based guidelines have been devel‐ oped for the treatment of Alzheimer's disease. These make recommendations that vary according to available resources, funding practices, and local practice. In general, however, the guidelines provide recommendations regarding psychiatric management, psychosocial treatments, and the treatment of specific target symptoms. There are relatively few diseases that have been successfully prevented or even controlled without an understanding of specific etiology of the disease. Apart from the first line and second line FDA recommended synthetic drugs of choice in treating AD, some of the preventive strategies proved to be beneficial in AD include treatment of hypertension, omega fatty acid supplementation, physical activity and cognitive engagement.

agonists mimic ACh on postsynaptic end terminal receptors, M2 and M3 receptor antagonists regulate ACh release via negative feedback, nicotinic agonists which would enhance ACh

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AChE inhibitors are mostly well tolerated by the patients; however common side effects include nausea, vomiting, loss of appetite, increased frequency of bowel movements etc. Tacrine, the first FDA approved AD-drug, which inhibits AChE reversibly in a non-competi‐ tive manner is no longer in use due to severe side effects (hepatotoxicity) and short biological half life. Donepezil hydrochloride is the second drug of choice approved by USFDA for treatment of mild to moderate AD. This drug is a centrally acting, reversible and noncompetitive AChE inhibitor having an N-benzylpiperidine and an indanone moiety which shows longer and more selective action. However it also suffers from the side effects of GI

N

disturbances, nausea, vomiting, headache etc [31,33].

**Figure 13.** Donepezil hydrochloride

H3Co <sup>O</sup>

H3Co - HCl

Galantamine reversibly inhibits AChE in a competitive manner and also acts on nicotinic acetylcholine receptors, beneficial for cognitive and non-cognitive AD symptoms. Results of clinical trials have reported this drug to be 50 times more potent against human AChE than butyrylcholinesterase at therapeutic doses. With escalations in drug doses some of the notable adverse effects include vomiting, nausea, diarrhoea etc. Rivastigmine is a reversible carbamate AChE inhibitor that interacts preferentially with acetylcholinesterase G1 with high brain selectivity; this drug has been approved in at least 40 countries around the world. Rivastigmine has the ability to inhibit the activity of butyrylcholinesterase. It binds to both the esteratic and ionic locations of AChE but dissociates at a much slower rate than AChE. Metrifonate, a precursor to the active pseudo irreversible AChE inhibitor DDVP (2,2-dichlorovinyl dimethyl phosphate) rapidly enters the brain with a longer plasma half life than donepezil but shows side effects of diarrhea and muscular cramps and hence could not achieve the market due to muscular weakness. There are certain AChE inhibitors of natural origin finding its use in AD. Physostigmine, a parasympathomimetic plant alkaloid isolated from the seeds of *Physostigma venenosum* have the ability to cross the BBB, having role in cholinergic transmission, can stimulate indirectly both nicotinic and muscarinic receptors. However physostigmine also inhibits another enzyme butyrylcholinesterase which has a role in AD and some of the adverse effects of this drug like nausea, vomiting, headache, diarrhea are attributed to its inhibitory actions on butyrylcholinesterase. Despite of the advantage to cross the BBB, the short half life, narrow margin of therapeutic index has restricted its potentiality. Galanthamine is an alkaloid isolated from *Galanthus nivalis* with competitive reversible AChE inhibitory activity. Galanth‐ amine shows dual mechanism of action, AChE inhibition and allosteric modulation of nicotinic acetyl choline receptors. This drug has 10 fold selectivity for AChE than butyrylcholinesterase. Alpha-7 nicotinic acetylcholine receptors have a role in beta-amyloid mediated neurotoxiciy

release.

### **5. Conventional therapeutic regimen in Alzheimer**

From the point of conventional approach, major six classes of drugs are included in the treatment of AD: Acetylcholinesterase inhibitors (AChE-I), N-methyl-D-aspartate (NMDA) receptor antagonists, monoamine oxidase (MAO) inhibitors, antioxidants, metal chelators, anti-inflammatory drugs. AChE inhibitors are the first line agents for the treatment of mild to moderate AD. FDA approved five prescription drugs to control the symptoms of AD include: Donepezil, Galantamine, Rivastigmine, Tacrine among the AChE inhibitors and Memantine coming under NMDA receptor antagonists. However tacrine have been withdrawn due to the hepato toxicity effects. AChE inhibitors are prescribed to treat symptoms relating to memory, thinking, language, judgment and other thought processes. Cholinesterase inhibitors primar‐ ily act by increasing the levels of acetylcholine, the chemical messenger involved in memory, judgment and other thought processes. In AD the cells producing or using ACh are destroyed and thus less amount of ACh is available to carry messages. ACh is produced from acetyl-CoA and choline by cholineacetyltransferase which is released into the synaptic cleft and hydro‐ lyzed by the actions of AChE to choline and acetic acid. This choline is reutilized in ACh synthesis. In the early stages of AD, the activity of AChE is found to be increased in the neuritic plaques and neurofibrillary tangles that accelerate aggregations of beta-amyloid. AChE inhibitors reversibly bind and block the activity of the enzyme acetyl cholinesterase that degrades ACh. AChE inhibitors block the actions of AChE thereby facilitating ACh neuro‐ transmission and reducing beta-amyloid burden [31-34].

Basing on the cholinergic theory, different classes of drugs have been developed to enhance cholinergic deficit in AD patients. Amongst them, AChE inhibitors block the activity of AChE enzyme to improve cognitive function, choline precursors like phosphatidylcholine improves the bioavailability of choline, ACh releasers enhance the release of ACh, M1 and M3 receptor agonists mimic ACh on postsynaptic end terminal receptors, M2 and M3 receptor antagonists regulate ACh release via negative feedback, nicotinic agonists which would enhance ACh release.

AChE inhibitors are mostly well tolerated by the patients; however common side effects include nausea, vomiting, loss of appetite, increased frequency of bowel movements etc. Tacrine, the first FDA approved AD-drug, which inhibits AChE reversibly in a non-competi‐ tive manner is no longer in use due to severe side effects (hepatotoxicity) and short biological half life. Donepezil hydrochloride is the second drug of choice approved by USFDA for treatment of mild to moderate AD. This drug is a centrally acting, reversible and noncompetitive AChE inhibitor having an N-benzylpiperidine and an indanone moiety which shows longer and more selective action. However it also suffers from the side effects of GI disturbances, nausea, vomiting, headache etc [31,33].

**Figure 13.** Donepezil hydrochloride

**4. Therapeutic interventions in Alzheimer**

**5. Conventional therapeutic regimen in Alzheimer**

transmission and reducing beta-amyloid burden [31-34].

cognitive engagement.

302 Neurodegenerative Diseases

Virtually there are no proven modalities for cure of Alzheimer's disease; however there are treatment regimens that may improve symptoms and may even delay their progression in the early and middle stages of the disease, allowing patients to maintain certain daily functions for longer. Various country- or region-specific, evidence-based guidelines have been devel‐ oped for the treatment of Alzheimer's disease. These make recommendations that vary according to available resources, funding practices, and local practice. In general, however, the guidelines provide recommendations regarding psychiatric management, psychosocial treatments, and the treatment of specific target symptoms. There are relatively few diseases that have been successfully prevented or even controlled without an understanding of specific etiology of the disease. Apart from the first line and second line FDA recommended synthetic drugs of choice in treating AD, some of the preventive strategies proved to be beneficial in AD include treatment of hypertension, omega fatty acid supplementation, physical activity and

From the point of conventional approach, major six classes of drugs are included in the treatment of AD: Acetylcholinesterase inhibitors (AChE-I), N-methyl-D-aspartate (NMDA) receptor antagonists, monoamine oxidase (MAO) inhibitors, antioxidants, metal chelators, anti-inflammatory drugs. AChE inhibitors are the first line agents for the treatment of mild to moderate AD. FDA approved five prescription drugs to control the symptoms of AD include: Donepezil, Galantamine, Rivastigmine, Tacrine among the AChE inhibitors and Memantine coming under NMDA receptor antagonists. However tacrine have been withdrawn due to the hepato toxicity effects. AChE inhibitors are prescribed to treat symptoms relating to memory, thinking, language, judgment and other thought processes. Cholinesterase inhibitors primar‐ ily act by increasing the levels of acetylcholine, the chemical messenger involved in memory, judgment and other thought processes. In AD the cells producing or using ACh are destroyed and thus less amount of ACh is available to carry messages. ACh is produced from acetyl-CoA and choline by cholineacetyltransferase which is released into the synaptic cleft and hydro‐ lyzed by the actions of AChE to choline and acetic acid. This choline is reutilized in ACh synthesis. In the early stages of AD, the activity of AChE is found to be increased in the neuritic plaques and neurofibrillary tangles that accelerate aggregations of beta-amyloid. AChE inhibitors reversibly bind and block the activity of the enzyme acetyl cholinesterase that degrades ACh. AChE inhibitors block the actions of AChE thereby facilitating ACh neuro‐

Basing on the cholinergic theory, different classes of drugs have been developed to enhance cholinergic deficit in AD patients. Amongst them, AChE inhibitors block the activity of AChE enzyme to improve cognitive function, choline precursors like phosphatidylcholine improves the bioavailability of choline, ACh releasers enhance the release of ACh, M1 and M3 receptor

Galantamine reversibly inhibits AChE in a competitive manner and also acts on nicotinic acetylcholine receptors, beneficial for cognitive and non-cognitive AD symptoms. Results of clinical trials have reported this drug to be 50 times more potent against human AChE than butyrylcholinesterase at therapeutic doses. With escalations in drug doses some of the notable adverse effects include vomiting, nausea, diarrhoea etc. Rivastigmine is a reversible carbamate AChE inhibitor that interacts preferentially with acetylcholinesterase G1 with high brain selectivity; this drug has been approved in at least 40 countries around the world. Rivastigmine has the ability to inhibit the activity of butyrylcholinesterase. It binds to both the esteratic and ionic locations of AChE but dissociates at a much slower rate than AChE. Metrifonate, a precursor to the active pseudo irreversible AChE inhibitor DDVP (2,2-dichlorovinyl dimethyl phosphate) rapidly enters the brain with a longer plasma half life than donepezil but shows side effects of diarrhea and muscular cramps and hence could not achieve the market due to muscular weakness. There are certain AChE inhibitors of natural origin finding its use in AD. Physostigmine, a parasympathomimetic plant alkaloid isolated from the seeds of *Physostigma venenosum* have the ability to cross the BBB, having role in cholinergic transmission, can stimulate indirectly both nicotinic and muscarinic receptors. However physostigmine also inhibits another enzyme butyrylcholinesterase which has a role in AD and some of the adverse effects of this drug like nausea, vomiting, headache, diarrhea are attributed to its inhibitory actions on butyrylcholinesterase. Despite of the advantage to cross the BBB, the short half life, narrow margin of therapeutic index has restricted its potentiality. Galanthamine is an alkaloid isolated from *Galanthus nivalis* with competitive reversible AChE inhibitory activity. Galanth‐ amine shows dual mechanism of action, AChE inhibition and allosteric modulation of nicotinic acetyl choline receptors. This drug has 10 fold selectivity for AChE than butyrylcholinesterase. Alpha-7 nicotinic acetylcholine receptors have a role in beta-amyloid mediated neurotoxiciy and since galanthamine can modulate nicotinic acetylcholine receptors it is suggested to prevent beta-amyloid mediated neurotoxicity. Huperzine A, an alkaloid drug isolated from club moss (*Huperzia serrata*) is claimed to show neuroprotective properties with significant improvements in cognitive function and results of clinical trials have shown this drug to be free from unexpected toxicities. This drug has attracted the attention of the scientists due to its strong AChE activities; (-)-huperzine A, a natural isomer have shown strongest dose dependent inhibitory activity against AChE, in comparison to commercially available syn‐ thetic drugs like donepezil, tacrine etc [31-33].

Memantine is an uncompetitive low-to-moderate affinity *N*-methyl-D-aspartate (NMDA) receptor antagonist that regulates glutamate activity. Glutamate plays an essential role in learning and memory by triggering NMDA receptors to let a controlled amount of calcium into the nerve cell. This calcium creates the chemical environment required for information storage. Excess glutamate on the other hand, over stimulates NMDA receptors so that they allow too much calcium into nerve cells leading to disruption and death of these cells. Memantine protects cells against excess glutamate by partially blocking NMDA receptors. It's a recently FDA approved NMDA antagonist for the treatment of cognition in moderate to severe Alzheimer's disease, having a half life period between 3-7 h and free from harsh adverse side effects. Memantine can be administered with AChE inhibitors. However FDA did not approve Memantine for mild AD. Results of clinical trials have shown small but statistically significant improvements in mental functions and ability to perform daily activities in AD patients [34,35].

dementia associated with AD and shows behavioral improvements. However the proper mechanism of action is not yet revealed. Transition metals like copper and zinc are found to be in high concentrations in the neo-cortical regions of the AD patients and these metals are mostly aggregated in the neurotic plaques potentiating beta amyloid aggregation and neuro‐ toxicity. Clioquinol chelates with these metals and reduces the beta amyloid aggregation in

N R N CH3

OH

CH3

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CH3

H2N

**Figure 15.** Memantine

**Figure 16.** Rivastigmine

**Figure 17.** Galanthamine

From the pathology of Alzheimer it is clear that several neurotransmitter systems, especially those regulating dopamine, serotonin, acetylcholine has a role in it. With the dysfunction of the dopaminergic system, there is increase in activity of type B monoamine oxidase, the enzyme responsible for degradation of dopamine. The increase in monoamine oxidase level is prominent in the brain platelets contributing to the severity of dementia. The drug Selegiline reversibly inhibits MAO-B; moreover it has a potent anti oxidative effect over the neurons of the brain and also protect against glutamate-receptor-mediated toxicity. Apart from MAO inhibition selegiline has a potent action in the recovery of damaged neurons involving a number of mechanisms like stimulation of neurite outgrowth, stimulation of gene expression in pre apoptotic neurons or stimulation of cytokine biosynthesis. Rasagiline, structurally similar to selegiline also shows neuro protective activities and is found to be ten times more

the brain, thus finding use as a therapeutic target in treating Alzheimer [35].

H3C <sup>N</sup> <sup>O</sup>

H3CO

CH3

O

O

**Figure 14.** AChE inhibitors (a) Tacrine (b) Donepezil (c) Galantamine (d) Rivastigmine (e) Metrifonate (f) Huperzine

Several metal species like iron, zinc, copper, aluminum are reported to induce beta amyloid aggregation and neurotoxicity in the brain of AD patients; results of structural evidences showing interrelations between aluminum and Abeta, presence of iron and zinc in abnormal high concentrations in AD patients have triggered the idea of using metal chelators as therapeutic targets in the treatment of AD. Desferrioxamine (DFO) and Clioquinol are clinically proven metal chelators used to treat AD patients. DFO chelates with metal ions or aluminium and reduces its neocortical concentrations thereby delaying the progression of

**Figure 15.** Memantine

and since galanthamine can modulate nicotinic acetylcholine receptors it is suggested to prevent beta-amyloid mediated neurotoxicity. Huperzine A, an alkaloid drug isolated from club moss (*Huperzia serrata*) is claimed to show neuroprotective properties with significant improvements in cognitive function and results of clinical trials have shown this drug to be free from unexpected toxicities. This drug has attracted the attention of the scientists due to its strong AChE activities; (-)-huperzine A, a natural isomer have shown strongest dose dependent inhibitory activity against AChE, in comparison to commercially available syn‐

Memantine is an uncompetitive low-to-moderate affinity *N*-methyl-D-aspartate (NMDA) receptor antagonist that regulates glutamate activity. Glutamate plays an essential role in learning and memory by triggering NMDA receptors to let a controlled amount of calcium into the nerve cell. This calcium creates the chemical environment required for information storage. Excess glutamate on the other hand, over stimulates NMDA receptors so that they allow too much calcium into nerve cells leading to disruption and death of these cells. Memantine protects cells against excess glutamate by partially blocking NMDA receptors. It's a recently FDA approved NMDA antagonist for the treatment of cognition in moderate to severe Alzheimer's disease, having a half life period between 3-7 h and free from harsh adverse side effects. Memantine can be administered with AChE inhibitors. However FDA did not approve Memantine for mild AD. Results of clinical trials have shown small but statistically significant improvements in mental functions and ability to perform daily activities in AD

N

Cl

Cl

**Figure 14.** AChE inhibitors (a) Tacrine (b) Donepezil (c) Galantamine (d) Rivastigmine (e) Metrifonate (f) Huperzine

Several metal species like iron, zinc, copper, aluminum are reported to induce beta amyloid aggregation and neurotoxicity in the brain of AD patients; results of structural evidences showing interrelations between aluminum and Abeta, presence of iron and zinc in abnormal high concentrations in AD patients have triggered the idea of using metal chelators as therapeutic targets in the treatment of AD. Desferrioxamine (DFO) and Clioquinol are clinically proven metal chelators used to treat AD patients. DFO chelates with metal ions or aluminium and reduces its neocortical concentrations thereby delaying the progression of

P O

O

O

N

H2N

O

(c)

(f)

O

OH

N O

H

O

thetic drugs like donepezil, tacrine etc [31-33].

patients [34,35].

304 Neurodegenerative Diseases

(a)

NH2

O

(b)

O

Cl

O

Cl

Cl

(e)

O P OH <sup>O</sup>

N

N

O N

<sup>O</sup> (d)

**Figure 16.** Rivastigmine

**Figure 17.** Galanthamine

dementia associated with AD and shows behavioral improvements. However the proper mechanism of action is not yet revealed. Transition metals like copper and zinc are found to be in high concentrations in the neo-cortical regions of the AD patients and these metals are mostly aggregated in the neurotic plaques potentiating beta amyloid aggregation and neuro‐ toxicity. Clioquinol chelates with these metals and reduces the beta amyloid aggregation in the brain, thus finding use as a therapeutic target in treating Alzheimer [35].

From the pathology of Alzheimer it is clear that several neurotransmitter systems, especially those regulating dopamine, serotonin, acetylcholine has a role in it. With the dysfunction of the dopaminergic system, there is increase in activity of type B monoamine oxidase, the enzyme responsible for degradation of dopamine. The increase in monoamine oxidase level is prominent in the brain platelets contributing to the severity of dementia. The drug Selegiline reversibly inhibits MAO-B; moreover it has a potent anti oxidative effect over the neurons of the brain and also protect against glutamate-receptor-mediated toxicity. Apart from MAO inhibition selegiline has a potent action in the recovery of damaged neurons involving a number of mechanisms like stimulation of neurite outgrowth, stimulation of gene expression in pre apoptotic neurons or stimulation of cytokine biosynthesis. Rasagiline, structurally similar to selegiline also shows neuro protective activities and is found to be ten times more

**Figure 18.** (a) Desferrioxamine (b) Ferrioxamine B (c) Clioquinol (d) Clioquinol Zn complex (e) Clioquinol Cu complex

O

O

O

N

N

N+

H Cl-

O O

(e)

(d)

N

O

O OH

O

boxy phenyl) porphyrin (d) Idebenone (e) Dehydroevodiamine (f) Mn-salen

of drugs have not found routine use in AD treatment.

(b)

N H

H

O

O

(c)

HO

HO

(f)

**Figure 20.** Antioxidants: (a)alpha tocopherol (b) Melatonin (c) Manganese (ш) beta-octabromo-meso tetrakis (4-car‐

In AD, neuronal destruction is due to inflammation around Abeta plaques. Drugs under the NSAID groups have anti-inflammatory actions, and found to inhibit cyclooxygenase-1 and cyclooxygenase-2 which are responsible for the oxidation of arachidonic acids to prostaglan‐ dins; however due to adverse effects of some NSAIDs on cardiovascular systems, these group

N

Br

Br

O

**N**

**O X R R**

**N Mn O** Br Br

Therapeutic Interventions in Alzheimer Disease

http://dx.doi.org/10.5772/54915

N Mn N

N

Br Br

O

OH

OH

307

Br

O

Br

(a) OH

active in inhibition of MAO-B. The propargylamine moiety in rasagiline is responsible for neuro protective activities. The drug Ladostigil, is the result of combination of active com‐ pounds from rasagiline (MAO-B inhibitor, neuroprotector) and rivastigmine (AChE inhibitor) thus finding application as a effective therapeutic agent in treating AD [33-35].

**Figure 19.** Monoamine oxidase inhibitors (a) Selegiline (b) Rasagiline

Oxidative damage to neurons has a significant role in the pathogenesis of AD; hence antioxi‐ dant therapy to prevent oxidative injury can be effective in preventing or retarding the progress of AD. Extracts from *Ginko biloba* named as Egb761 have been found to show cognitive improvement in AD patients and in those with multi-infarct dementia. Similarly Melatonin is found to have antiamyloidogenic activities and is found to reduce neuronal damage caused by reactive oxygen species in AD patients. Antioxidant therapy with vitamin E though reported in some cases but it does not improve cognitive impairment and its therapeutic use has not been established. Clinical trials with Idebenone, a co-enzyme Q10 analog have been found to attenuate Abeta-induced neurotoxicity and cognitive impairments. Dehydroevodi‐ amine hydrochloride (DHED), a compound extracted from *Evodia rutaecarpa* showed AChE inhibitory activities, DHED protects neurons against hydrogen peroxide and glutamate. Moreover DHED decreases reactive oxygen species production and cell death induced by Abeta and carboxyterminal peptides of amyloid precursor proteins, thus improving cognitive impairments in AD. Metalloporphyrin antioxidants have been found to delay neuronal death resulting from increased mitochondrial oxidative stress; Mn-salen complexes have been found to be efficacious against oxidative stress [35].

active in inhibition of MAO-B. The propargylamine moiety in rasagiline is responsible for neuro protective activities. The drug Ladostigil, is the result of combination of active com‐ pounds from rasagiline (MAO-B inhibitor, neuroprotector) and rivastigmine (AChE inhibitor)

**Figure 18.** (a) Desferrioxamine (b) Ferrioxamine B (c) Clioquinol (d) Clioquinol Zn complex (e) Clioquinol Cu complex

(b)

Oxidative damage to neurons has a significant role in the pathogenesis of AD; hence antioxi‐ dant therapy to prevent oxidative injury can be effective in preventing or retarding the progress of AD. Extracts from *Ginko biloba* named as Egb761 have been found to show cognitive improvement in AD patients and in those with multi-infarct dementia. Similarly Melatonin is found to have antiamyloidogenic activities and is found to reduce neuronal damage caused by reactive oxygen species in AD patients. Antioxidant therapy with vitamin E though reported in some cases but it does not improve cognitive impairment and its therapeutic use has not been established. Clinical trials with Idebenone, a co-enzyme Q10 analog have been found to attenuate Abeta-induced neurotoxicity and cognitive impairments. Dehydroevodi‐ amine hydrochloride (DHED), a compound extracted from *Evodia rutaecarpa* showed AChE inhibitory activities, DHED protects neurons against hydrogen peroxide and glutamate. Moreover DHED decreases reactive oxygen species production and cell death induced by Abeta and carboxyterminal peptides of amyloid precursor proteins, thus improving cognitive impairments in AD. Metalloporphyrin antioxidants have been found to delay neuronal death resulting from increased mitochondrial oxidative stress; Mn-salen complexes have been found

N

H

Cu

thus finding application as a effective therapeutic agent in treating AD [33-35].

N

(a)

306 Neurodegenerative Diseases

to be efficacious against oxidative stress [35].

**Figure 19.** Monoamine oxidase inhibitors (a) Selegiline (b) Rasagiline

In AD, neuronal destruction is due to inflammation around Abeta plaques. Drugs under the NSAID groups have anti-inflammatory actions, and found to inhibit cyclooxygenase-1 and cyclooxygenase-2 which are responsible for the oxidation of arachidonic acids to prostaglan‐ dins; however due to adverse effects of some NSAIDs on cardiovascular systems, these group of drugs have not found routine use in AD treatment.

associations between PUFA and central nervous system activity. Levels of n-3 PUFA have been associated with monoaminergic neurotransmitter levels. There are indications that PUFA are involved in the synthesis and activities of brain peptides, which are involved in modulating the activities of neurotransmitters. Evidence points to the role of eicosanoids in healthy brain functioning, and phospholipid membranes in neural cell signaling. Results of animal studies have shown that, n-3 deficiency is found to reduce phosphatidylserine (PS) levels in the brain, which is thought to play an important function in neural signaling activities. In alcoholics, DHA deficiency has predicted reduced 5-hydroxyindoleacetic acid (5-HIAA) concentrations in cerebrospinal fluid, an indicator of low serotonin turnover rate in the frontal cortex. Studies have further indicated that n-3 PUFA may affect receptor properties or activation of signal transduction by receptors. Electrical impulse conduction is dependent on the exchange of ions through the cell membrane, which relies on the fluidity and physiological structure of cell membranes. Furthermore, n-3 PUFA are also thought to influence gene expression of a range of enzymes required for important neural functions including synaptic transduction, ion channel formation, energy metabolism and formulation of proteins vital for brain development and function. Regular delivery of oxygen and nutrients via the blood is also vital for optimal brain function, and psychopathology is associated with both reduced cerebral blood flow and transportation of glucose, the brain's primary energy source, to brain regions as required. In this regard, n-3 PUFA are associated with production of nitric oxide, as well as anti-inflam‐ matory and vasodilatory eicosanoids (notably PGI2), and are known to assist in endothelialdependent vasodilatation. They have also been associated with substantially increased transport of glucose across the blood-brain barrier. Therefore, it is also possible that their primary influence on brain function includes improved cerebral blood flow and blood-brain barrier integrity. This idea is supported by the fact that there's a high co-morbidity between cardiovascular disease and psychopathology, having a common underlying vascular pathol‐ ogy which can be mediated by lifestyle factors such as suboptimal levels of n-3 PUFA [36].

Therapeutic Interventions in Alzheimer Disease

http://dx.doi.org/10.5772/54915

309

The mechanisms by which ω-3 fatty acids could interfere in AD patho physiologic features are not clear, but since anti-inflammatory effects are an important property of these fish oils rich in PUFA, they are applicable for AD also. Epidemiologic evidences have shown that admin‐ istration of ω-3 fatty acid in patients with mild to moderate AD did not delay the rate of cognitive decline according to the MMSE or the cognitive portion of the Alzheimer Disease Assessment Scale but, positive effects were observed in a small group of patients with very mild AD. Increased intake of the ω-3 polyunsaturated fatty acids (primarily eicosapentaenoic acid (EPA), 20:5ω-3, and docosahexaenoic acid (DHA), 22:6ω-3) may be beneficial in reducing risk for AD. Increased fish consumption and diets supplemented with omega-3 fatty acids are found to exhibit a protective effect, cognitive improvement and enhancement of learning abilities. Animal studies on transgenic mouse model of AD with DHA-enriched diets signifi‐ cantly reduced total β-amyloid by 70% when compared with diets low in DHA or control chow

diets [ext-link ext-link-type="bibr" rid="REF-NOC60062-9 REF-NOC60062-10"/].

potentiality of the ω-3 fatty acids in halting initial progression of the disease [36].

However these findings cannot serve as a basis for general recommendations for treatment of AD with dietary DHA-rich fish oil preparations. Rather, larger cohort's studies in patients with mild cognitive impairment, including those at risk for AD are needed to further explore the

**Figure 21.** NSAIDs (a) Ibuprofen (b) Aspirin (c) Naproxen (d) Flubiprofen

### **6. Omega fatty acids in treating Alzheimer**

Polyunsaturated fatty acids or PUFA have significant biological roles in cellular structure and function. PUFA are the key components of phospholipids, comprising cellular and intracel‐ lular membranes. They govern the growth and vitality through oxidation (metabolism of food to produce energy required for cellular processes), chemical activities and transportation. In addition to being the structural materials for bio-membranes, PUFA are required for generat‐ ing and propagating electrical impulses; synthesis of eicosanoids, important signaling hormones with numerous complex functions. Amongst its wide range of actions include antiinflammatory, anti-thrombotic and vasodilatory properties, balancing and counteracting proinflammatory, vasoconstrictor actions of eicosanoids; the cardiovascular benefits of n-3 PUFA are largely attributed to these eicosanoid properties, and at the same time having significant roles in brain function. Polyunsaturated fatty acids like alpha linoleic acids (ALA), linolenic acids (LA), eicosapentaenoic acids (EPA) or docosahexaenoic acids (DHA) are not synthesized in the body and hence must be supplied in the diet [36].

Lipids constitute approximately sixty percent of the dry weight of the brain. DHA and Arachidonic acids (AA) are the most highly concentrated PUFAs present in neural phospho‐ lipids, including sub cellular membranes. DHA is particularly concentrated at neural synapses, retina, brain and nervous system. Though there is a predominance of omega-6 fatty acids in circulation, in contrast to omega-3 fatty acids, however DHA predominates in these vital structures. The amount of DHA levels in the neural phospholipids depend on the amount of dietary intake rich in omega-3 fatty acids. Clinical studies have shown that insufficient omega-3 PUFA during early neural development shows decreased DHA content in the brain.

The n-3 PUFAs have significant biological mechanisms in brain function. Neurotransmitters such as dopamine and serotonin have a role in mental illness, research data have focused on associations between PUFA and central nervous system activity. Levels of n-3 PUFA have been associated with monoaminergic neurotransmitter levels. There are indications that PUFA are involved in the synthesis and activities of brain peptides, which are involved in modulating the activities of neurotransmitters. Evidence points to the role of eicosanoids in healthy brain functioning, and phospholipid membranes in neural cell signaling. Results of animal studies have shown that, n-3 deficiency is found to reduce phosphatidylserine (PS) levels in the brain, which is thought to play an important function in neural signaling activities. In alcoholics, DHA deficiency has predicted reduced 5-hydroxyindoleacetic acid (5-HIAA) concentrations in cerebrospinal fluid, an indicator of low serotonin turnover rate in the frontal cortex. Studies have further indicated that n-3 PUFA may affect receptor properties or activation of signal transduction by receptors. Electrical impulse conduction is dependent on the exchange of ions through the cell membrane, which relies on the fluidity and physiological structure of cell membranes. Furthermore, n-3 PUFA are also thought to influence gene expression of a range of enzymes required for important neural functions including synaptic transduction, ion channel formation, energy metabolism and formulation of proteins vital for brain development and function. Regular delivery of oxygen and nutrients via the blood is also vital for optimal brain function, and psychopathology is associated with both reduced cerebral blood flow and transportation of glucose, the brain's primary energy source, to brain regions as required. In this regard, n-3 PUFA are associated with production of nitric oxide, as well as anti-inflam‐ matory and vasodilatory eicosanoids (notably PGI2), and are known to assist in endothelialdependent vasodilatation. They have also been associated with substantially increased transport of glucose across the blood-brain barrier. Therefore, it is also possible that their primary influence on brain function includes improved cerebral blood flow and blood-brain barrier integrity. This idea is supported by the fact that there's a high co-morbidity between cardiovascular disease and psychopathology, having a common underlying vascular pathol‐ ogy which can be mediated by lifestyle factors such as suboptimal levels of n-3 PUFA [36].

O

O

O

(b)

O

OH

F

O

OH

(a)

308 Neurodegenerative Diseases

O

OH

(c) (d)

**Figure 21.** NSAIDs (a) Ibuprofen (b) Aspirin (c) Naproxen (d) Flubiprofen

**6. Omega fatty acids in treating Alzheimer**

in the body and hence must be supplied in the diet [36].

O

Polyunsaturated fatty acids or PUFA have significant biological roles in cellular structure and function. PUFA are the key components of phospholipids, comprising cellular and intracel‐ lular membranes. They govern the growth and vitality through oxidation (metabolism of food to produce energy required for cellular processes), chemical activities and transportation. In addition to being the structural materials for bio-membranes, PUFA are required for generat‐ ing and propagating electrical impulses; synthesis of eicosanoids, important signaling hormones with numerous complex functions. Amongst its wide range of actions include antiinflammatory, anti-thrombotic and vasodilatory properties, balancing and counteracting proinflammatory, vasoconstrictor actions of eicosanoids; the cardiovascular benefits of n-3 PUFA are largely attributed to these eicosanoid properties, and at the same time having significant roles in brain function. Polyunsaturated fatty acids like alpha linoleic acids (ALA), linolenic acids (LA), eicosapentaenoic acids (EPA) or docosahexaenoic acids (DHA) are not synthesized

Lipids constitute approximately sixty percent of the dry weight of the brain. DHA and Arachidonic acids (AA) are the most highly concentrated PUFAs present in neural phospho‐ lipids, including sub cellular membranes. DHA is particularly concentrated at neural synapses, retina, brain and nervous system. Though there is a predominance of omega-6 fatty acids in circulation, in contrast to omega-3 fatty acids, however DHA predominates in these vital structures. The amount of DHA levels in the neural phospholipids depend on the amount of dietary intake rich in omega-3 fatty acids. Clinical studies have shown that insufficient omega-3 PUFA during early neural development shows decreased DHA content in the brain. The n-3 PUFAs have significant biological mechanisms in brain function. Neurotransmitters such as dopamine and serotonin have a role in mental illness, research data have focused on

OH

The mechanisms by which ω-3 fatty acids could interfere in AD patho physiologic features are not clear, but since anti-inflammatory effects are an important property of these fish oils rich in PUFA, they are applicable for AD also. Epidemiologic evidences have shown that admin‐ istration of ω-3 fatty acid in patients with mild to moderate AD did not delay the rate of cognitive decline according to the MMSE or the cognitive portion of the Alzheimer Disease Assessment Scale but, positive effects were observed in a small group of patients with very mild AD. Increased intake of the ω-3 polyunsaturated fatty acids (primarily eicosapentaenoic acid (EPA), 20:5ω-3, and docosahexaenoic acid (DHA), 22:6ω-3) may be beneficial in reducing risk for AD. Increased fish consumption and diets supplemented with omega-3 fatty acids are found to exhibit a protective effect, cognitive improvement and enhancement of learning abilities. Animal studies on transgenic mouse model of AD with DHA-enriched diets signifi‐ cantly reduced total β-amyloid by 70% when compared with diets low in DHA or control chow diets [ext-link ext-link-type="bibr" rid="REF-NOC60062-9 REF-NOC60062-10"/].

However these findings cannot serve as a basis for general recommendations for treatment of AD with dietary DHA-rich fish oil preparations. Rather, larger cohort's studies in patients with mild cognitive impairment, including those at risk for AD are needed to further explore the potentiality of the ω-3 fatty acids in halting initial progression of the disease [36].

#### **7. In silico drug design in Alzheimer**

Though a number of FDA approved drugs are currently available for the treatment of Alzheimer but there are no such effective treatments to stop the insidious nerve cell death process once the disease begins. Available drugs can manage and ease some of the symptoms of the disease but the progression of the disease can in no way be slowed down by these treatments. In AD patients there is very less production of neurotransmitter acetylcholine due to the progressive damage of the cells producing acetylcholine. Most of the FDA approved drugs aim to prevent the breakdown of acetylcholine by inhibiting the enzyme acetyl choli‐ nesterase. But in AD due to rapid destruction of nerve cells, the acetylcholine produced even though protected from further breakdown but the amount produced is significantly insuffi‐ cient to transmit messages between the brain cells. This fact necessitates further new drug development.

b-APP is available from PDB (Protein Data Bank) with ID 1RW6. Soft wares like PASS, CASTp provides information about the active site for b-APP. Enzyme modeling can be done using packages like Swiss Model, Modeller etc. Specific interactions of these lead compounds with the active site of b-APP can be studied by docking programs like DOCK, AUTODOCK etc. The most effective protease enzyme with minimum energy conformation can be identified by the above procedure which will act as the potential drug in treating Alzheimer. Use of computa‐ tional docking techniques help to explore a large number of compounds, study the binding characteristics of 'hits' and is found to be effective in reducing the time and monetary expen‐

Therapeutic Interventions in Alzheimer Disease

http://dx.doi.org/10.5772/54915

311

Virtually at this point of time there is no cure for Alzheimer. But apart from therapeutic interventions, attempts can be done to manage the disease and treat the symptoms by the care givers in a non-pharmacologic manner. Along with medications, physical exercise, social involvement as well as proper nutrition are essential in treating the symptoms of AD. The goal of non-pharmacologic treatment in AD though sounds simple but clinically remains a challenge where the care giver has a vital role to play; first thing is to provide a calm structured environment where the comfort, dignity of the afflicted person is maintained and the patient remains functioning as long as possible [39-41]. For AD patients the environment should be so arranged that maximize use of cognitive capacities of AD patients that are intact and compensate for those cognitive capacities that decline. Treatment in a non-pharmacologic manner aims to improve the quality of life to treat the disease symptoms. It is not a simple task for a care giver to increase functional independence, reduce the need for psychoactive medications, prolong life, reduce the need for restraints, reduce acute hospital admissions, reduce depression and improve morale. Alzheimer begins in the medial temporal lobe and spreads to other parts of the brain slowly destroying parietal, temporal and frontal lobes, cingulated cortex, hippocampus, amygdale, damaging the tempero-parieto-occipital associa‐ tion cortex leading to memory dysfunction, emotional disturbances, personality changes, visual, language and movement disorders. Due to damages in the frontal lobes AD patients will have difficulty in performing daily tasks; with the gradual progression of the disease hallucinations, delusions, paranoia, agitation, panic and denial are seen among the afflicted. Among the non-pharmacologic treatment domains include: properly mapped physical environment that removes fear and promotes safety, induce a sense of positive attitude or emotion among the afflicted persons by the care giver while helping in daily activities by helping them to perform the task but not to do the total task for them and make them even more dependent; change negative emotions and promote feelings of purpose and accomplish‐ ment. While treating AD patients in non-pharmacologic manner one of the major corner stone is how we understand the afflicted person and help him to understand himself [42-44]. The care giver must change his/her behavior & environment in order to change the behavior of the patient. A person in middle stage of AD should never be attempted to bring back to the realistic world but reduce his fear in all possible stages and help him to move into his sense of the

diture as associated with traditional drug development techniques [38].

**8. Non-pharmacological approach**

In contrast to traditional drug discovery, "Rational Drug Design'' has been found to be a more deterministic approach where the first necessary step is the identification of the molecular target critical to the disease process and then determine the molecular structure of the target molecule. In-silico drug design approach makes use of Computer Assisted Drug Design (CADD) tools to find a ligand (putative drug) that will interact with a receptor which represents the 'target site'. A ligand can bind to the receptor either by hydrophobic, electrostatic or hydrogen bonding interactions and solvation energies of the ligand and receptor sites are the important facts to be considered in this case.

The basics behind the pathology of AD is the presence of neuritic plaques containing amyloidβ-peptide (Aβ) and intra neuronal accumulation of tubule associated 'tau' protein. In order to target therapeutic strategies basing on molecular mechanisms for AD, new drug entities can be developed that will act directly on the Aβ or the amyloid precursor protein (APP) processing which may include vaccination with Aβ peptide, Aβ passive immunization. However clinical trials with a vaccine made of synthetic Aβ 1-42 showed the lateral development of encephalitis and hence the trial was put on hold though the concept exists. Passive immunization can be obtained by monoclonal antibodies against Aβ. New anti-amyloid agents to prevent fibrilli‐ zations can be designed by detailed characterizations of the proto fibrils and fibril formations. Another lucrative approach is to target the APP processing where the three major enzymes are to be targeted: alpha secretase, beta secretase and gamma secretase, the basic aim is to increase the alpha cleavage or to decrease the beta and gamma secretase activities. Nerve growth factors and neurotrophines can also act as important therapeutic targets. Growth factor gene therapy (though under clinical trial) where patient's fibroblasts transfected with NFG and transplanted to brain are expected to be effective in case of severe AD [37].

In in -silico drug design approach, computational docking techniques can be used to develop an effective drug to prevent the progression of Alzheimer disease. Here the 'TARGET' is the amyloid precursor protein (APP) and the 'LEAD COMPOUND' is the protease enzyme. By the aid of database search tools like FASTA, BLAST in Swissport, proteins having protease activity for b-APP can be identified. Next step is to identify the protease enzyme that cleaves the b-APP so as to prevent the formation of beta-amyloid peptide. Structural informations on b-APP is available from PDB (Protein Data Bank) with ID 1RW6. Soft wares like PASS, CASTp provides information about the active site for b-APP. Enzyme modeling can be done using packages like Swiss Model, Modeller etc. Specific interactions of these lead compounds with the active site of b-APP can be studied by docking programs like DOCK, AUTODOCK etc. The most effective protease enzyme with minimum energy conformation can be identified by the above procedure which will act as the potential drug in treating Alzheimer. Use of computa‐ tional docking techniques help to explore a large number of compounds, study the binding characteristics of 'hits' and is found to be effective in reducing the time and monetary expen‐ diture as associated with traditional drug development techniques [38].

### **8. Non-pharmacological approach**

**7. In silico drug design in Alzheimer**

important facts to be considered in this case.

development.

310 Neurodegenerative Diseases

Though a number of FDA approved drugs are currently available for the treatment of Alzheimer but there are no such effective treatments to stop the insidious nerve cell death process once the disease begins. Available drugs can manage and ease some of the symptoms of the disease but the progression of the disease can in no way be slowed down by these treatments. In AD patients there is very less production of neurotransmitter acetylcholine due to the progressive damage of the cells producing acetylcholine. Most of the FDA approved drugs aim to prevent the breakdown of acetylcholine by inhibiting the enzyme acetyl choli‐ nesterase. But in AD due to rapid destruction of nerve cells, the acetylcholine produced even though protected from further breakdown but the amount produced is significantly insuffi‐ cient to transmit messages between the brain cells. This fact necessitates further new drug

In contrast to traditional drug discovery, "Rational Drug Design'' has been found to be a more deterministic approach where the first necessary step is the identification of the molecular target critical to the disease process and then determine the molecular structure of the target molecule. In-silico drug design approach makes use of Computer Assisted Drug Design (CADD) tools to find a ligand (putative drug) that will interact with a receptor which represents the 'target site'. A ligand can bind to the receptor either by hydrophobic, electrostatic or hydrogen bonding interactions and solvation energies of the ligand and receptor sites are the

The basics behind the pathology of AD is the presence of neuritic plaques containing amyloidβ-peptide (Aβ) and intra neuronal accumulation of tubule associated 'tau' protein. In order to target therapeutic strategies basing on molecular mechanisms for AD, new drug entities can be developed that will act directly on the Aβ or the amyloid precursor protein (APP) processing which may include vaccination with Aβ peptide, Aβ passive immunization. However clinical trials with a vaccine made of synthetic Aβ 1-42 showed the lateral development of encephalitis and hence the trial was put on hold though the concept exists. Passive immunization can be obtained by monoclonal antibodies against Aβ. New anti-amyloid agents to prevent fibrilli‐ zations can be designed by detailed characterizations of the proto fibrils and fibril formations. Another lucrative approach is to target the APP processing where the three major enzymes are to be targeted: alpha secretase, beta secretase and gamma secretase, the basic aim is to increase the alpha cleavage or to decrease the beta and gamma secretase activities. Nerve growth factors and neurotrophines can also act as important therapeutic targets. Growth factor gene therapy (though under clinical trial) where patient's fibroblasts transfected with NFG

and transplanted to brain are expected to be effective in case of severe AD [37].

In in -silico drug design approach, computational docking techniques can be used to develop an effective drug to prevent the progression of Alzheimer disease. Here the 'TARGET' is the amyloid precursor protein (APP) and the 'LEAD COMPOUND' is the protease enzyme. By the aid of database search tools like FASTA, BLAST in Swissport, proteins having protease activity for b-APP can be identified. Next step is to identify the protease enzyme that cleaves the b-APP so as to prevent the formation of beta-amyloid peptide. Structural informations on Virtually at this point of time there is no cure for Alzheimer. But apart from therapeutic interventions, attempts can be done to manage the disease and treat the symptoms by the care givers in a non-pharmacologic manner. Along with medications, physical exercise, social involvement as well as proper nutrition are essential in treating the symptoms of AD. The goal of non-pharmacologic treatment in AD though sounds simple but clinically remains a challenge where the care giver has a vital role to play; first thing is to provide a calm structured environment where the comfort, dignity of the afflicted person is maintained and the patient remains functioning as long as possible [39-41]. For AD patients the environment should be so arranged that maximize use of cognitive capacities of AD patients that are intact and compensate for those cognitive capacities that decline. Treatment in a non-pharmacologic manner aims to improve the quality of life to treat the disease symptoms. It is not a simple task for a care giver to increase functional independence, reduce the need for psychoactive medications, prolong life, reduce the need for restraints, reduce acute hospital admissions, reduce depression and improve morale. Alzheimer begins in the medial temporal lobe and spreads to other parts of the brain slowly destroying parietal, temporal and frontal lobes, cingulated cortex, hippocampus, amygdale, damaging the tempero-parieto-occipital associa‐ tion cortex leading to memory dysfunction, emotional disturbances, personality changes, visual, language and movement disorders. Due to damages in the frontal lobes AD patients will have difficulty in performing daily tasks; with the gradual progression of the disease hallucinations, delusions, paranoia, agitation, panic and denial are seen among the afflicted. Among the non-pharmacologic treatment domains include: properly mapped physical environment that removes fear and promotes safety, induce a sense of positive attitude or emotion among the afflicted persons by the care giver while helping in daily activities by helping them to perform the task but not to do the total task for them and make them even more dependent; change negative emotions and promote feelings of purpose and accomplish‐ ment. While treating AD patients in non-pharmacologic manner one of the major corner stone is how we understand the afflicted person and help him to understand himself [42-44]. The care giver must change his/her behavior & environment in order to change the behavior of the patient. A person in middle stage of AD should never be attempted to bring back to the realistic world but reduce his fear in all possible stages and help him to move into his sense of the world. Due to sensory impairment and lack of receptive and expressive language abilities nonverbal perceptual inputs which replace words like familiar music, known food smell, and known touch may be used and afflicted can be communicated with a look, tone or a hug to induce a feeling of care and safety. In case of negative behavior and aggression the triggering agent should be identified and eliminated. The ultimate outcome of such a treatment approach is to slow the rate of disease progression, delay institutionalization, improve the quality of life and reduce the need for medication [39]. Below in fig.22 a specially designed room is shown for sensory integration therapy.

with this deadly disease. Fortunately, AD and dementia research is now at a progressing stage. Availability of improved medical imaging technologies like CT scan, MRI, PET, use of biomarkers helps in early diagnosis of the disease, its progression and severity. Furthermore, pharmacological and non-pharmacological therapies could be directly tested both to prevent and delay the progression of amyloid in the brain and effects on brain morphology and cognitive decline. Pharmacological therapies that could delay the onset of dementia for several years could result in a substantial reduction in the prevalence of AD because the patients will die of another cause before they develop AD. A second and far more important approach will be the application of these new technologies to understand the etiology of AD. The identifi‐ cation of specific etiological factors is much more likely in the long term to have a major impact on the incidence, prevalence, and disability because of AD and dementia. Proper understand‐ ing the etiology of the disease is essential to develop hypothetical treatment approaches which can be further clinically established. Basing on in-silico drug design approaches it is essential to understand the molecular mechanisms of APP processing, role of folate and homocysteine in neuronal homeostasis. Main target should be to develop novel therapeutic agents via cost effective, eco-friendly methodologies. Along with specific drug therapy, lifestyle interventions and environmental variables are also to be targeted to reduce the incidence of AD. However in case of new drug research in Alzheimer, ability to utilize the technologies, clinical skills and

Therapeutic Interventions in Alzheimer Disease

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313

financial resources to support research studies are of vital importance.

and Baishakhi Dey

\*Address all correspondence to: analavamitra@gmail.com

School of Medical Science and Technology, IIT Kharagpur, India

[1] Basics of Alzheimer diseaseAlzheimer association (2011).

[2] Alzheimer Disease facts and FiguresAlzheimer's & Dementia. Alzheimer association

[3] Yamasaki, T, Muranaka, H, Kaseda, Y, Mimori, Y, & Tobimatsu, S. Understanding the pathophysiology of Alzheimer disease and mild cognitive impairment: A mini review on fMRI and ERP studies. Neurology Research International (2011). , 20(12),

[4] Gary, S. W. Barry RPV, Buckholtz PP, Dekosky NS, Ferris ST et al. Diagnosis and treatment of Alzheimer disease and related disorders: consensus statement of the American Association for Geriatric Psychiatry, the Alzheimer Association and the

**Author details**

Analava Mitra\*

**References**

1-10.

(2011). , 7(2), 1-68.

**Figure 22.** Specially designed room for sensory integration therapy (Snoezelen); an emotion oriented psychosocial in‐ tervention for people with dementia

#### **9. Conclusions**

There is an increasing emergency in finding a prevention or treatment for AD and dementia because of the aging of the populations and realizing the severity and complications associated with this deadly disease. Fortunately, AD and dementia research is now at a progressing stage. Availability of improved medical imaging technologies like CT scan, MRI, PET, use of biomarkers helps in early diagnosis of the disease, its progression and severity. Furthermore, pharmacological and non-pharmacological therapies could be directly tested both to prevent and delay the progression of amyloid in the brain and effects on brain morphology and cognitive decline. Pharmacological therapies that could delay the onset of dementia for several years could result in a substantial reduction in the prevalence of AD because the patients will die of another cause before they develop AD. A second and far more important approach will be the application of these new technologies to understand the etiology of AD. The identifi‐ cation of specific etiological factors is much more likely in the long term to have a major impact on the incidence, prevalence, and disability because of AD and dementia. Proper understand‐ ing the etiology of the disease is essential to develop hypothetical treatment approaches which can be further clinically established. Basing on in-silico drug design approaches it is essential to understand the molecular mechanisms of APP processing, role of folate and homocysteine in neuronal homeostasis. Main target should be to develop novel therapeutic agents via cost effective, eco-friendly methodologies. Along with specific drug therapy, lifestyle interventions and environmental variables are also to be targeted to reduce the incidence of AD. However in case of new drug research in Alzheimer, ability to utilize the technologies, clinical skills and financial resources to support research studies are of vital importance.

### **Author details**

world. Due to sensory impairment and lack of receptive and expressive language abilities nonverbal perceptual inputs which replace words like familiar music, known food smell, and known touch may be used and afflicted can be communicated with a look, tone or a hug to induce a feeling of care and safety. In case of negative behavior and aggression the triggering agent should be identified and eliminated. The ultimate outcome of such a treatment approach is to slow the rate of disease progression, delay institutionalization, improve the quality of life and reduce the need for medication [39]. Below in fig.22 a specially designed room is shown

**Figure 22.** Specially designed room for sensory integration therapy (Snoezelen); an emotion oriented psychosocial in‐

There is an increasing emergency in finding a prevention or treatment for AD and dementia because of the aging of the populations and realizing the severity and complications associated

for sensory integration therapy.

312 Neurodegenerative Diseases

tervention for people with dementia

**9. Conclusions**

Analava Mitra\* and Baishakhi Dey

\*Address all correspondence to: analavamitra@gmail.com

School of Medical Science and Technology, IIT Kharagpur, India

#### **References**


American Geriatrics Society. Journal of the American Medical Association (1997). , 278(16), 1-9.

central nervous system. Journal of Cerebral Blood Flow and Metabolism (2007). , 27,

Therapeutic Interventions in Alzheimer Disease

http://dx.doi.org/10.5772/54915

315

[18] Corder, E. H, Saunders, A. M, Strittmatter, W. J, et al. Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families. Science. (1993).

[19] Deane, R, Bell, R. D, Sagare, A, & Zlokovic, B. Z. Clearance of amyloid-beta peptide across the blood-brain barrier: implications for therapies in Alzheimer's disease. CNS

[20] Deane, R. Du Yan S, Submamaryan RK, et al. RAGE mediates amyloid-beta peptide transport across the blood-brain barrier and accumulation in brain. Nature Medicine

[21] Deane, R, Wu, Z, Sagare, A, et al. LRP/amyloid beta-peptide interaction mediates dif‐ ferential brain efflux of Abeta isoforms. Neuron. (2004). Doi:10.1016/j.neuron.

[22] Drzezga, A, Lautenschlager, N, Siebner, H, et al. Cerebral metabolic changes accom‐ panying conversion of mild cognitive impairment into Alzheimer's disease: a PET follow-up study. European Journal of Nuclear Medicine and Molecular Imaging

[23] Ervin, J. F, Pannell, C, Szymanski, M, Welsh-bohmer, K, Schmechel, D. E, & Hulette, C. M. Vascular smooth muscle actin is reduced in Alzheimer disease brain: a quanti‐ tative analysis. Journal of Neuropathology and Experimental Neurology (2004). , 63,

[24] Grabowski, T. J, Cho, H. S, Vonsattel, J. P, Rebeck, G. W, & Greenberg, S. M. Novel amyloid precursor protein mutation in an Iowa family with dementia and severe cer‐ ebral amyloid angiopathy. Annals of Neurology (2001). Doi:10.1002/ana.1009., 49,

[25] Jung, S. S, Zhang, W, & Van Nostrand, W. E. Pathogenic A beta induces the expres‐ sion and activation of matrix metalloproteinase-2 in human cerebrovascular smooth muscle cells. Journal of Neurochemistry (2003). Doi:10.1046/j.1471-4159.2003.01745.x.,

[26] Parks, J. K, Smith, T. S, Trimmer, P. A, & Bennett, J. P. Jr, Parker WD., Jr Neurotoxic Abeta peptides increase oxidative stress in vivo through NMDA-receptor and nitricoxide-synthase mechanisms, and inhibit complex IV activity and induce a mitochon‐ drial permeability transition in vitro. Journal of Neurochemistry (2001). Doi:10.1046/j.

[27] Preston, S. D, Steart, P. V, Wilkinson, A, Nicoll, J. A, & Weller, R. O. Capillary and arterial cerebral amyloid angiopathy in Alzheimer's disease: defining the perivascu‐

909-918.

Doi:10.1126/science.8346443., 261, 921-923.

(2003). Doi:s00259-003-1194-1., 30, 1104-1113.

1471-4159.2001.00112.x., 76, 1050-1056.

(2003). Doi:nm890., 9, 907-913.

2004.07.017., 43, 333-344.

735-741.

697-705.

85, 1208-1215.

and Neurological Disorders- Drug Targets (2009). Doi:


central nervous system. Journal of Cerebral Blood Flow and Metabolism (2007). , 27, 909-918.

[18] Corder, E. H, Saunders, A. M, Strittmatter, W. J, et al. Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families. Science. (1993). Doi:10.1126/science.8346443., 261, 921-923.

American Geriatrics Society. Journal of the American Medical Association (1997). ,

[5] Verghese, P. B, Castellano, J. M, & Holtzmann, D. M. Roles of Apolipoprotein E in Alzheimer's disease and Other Neurological Disorders. Lancet Neurology (2011). ,

[6] Kuller, L. H, & Lopez, O. L. Dementia and Alzheimer's disease: A new direction. The 2010 Jay L. Foster Memorial Lecture. Alzheimer's Dement (2011). Doi:j.jalz.

[7] Guidelines for Alzheimer Disease ManagementCalifornia Workgroup on Guidelines

[8] Winslow, B. T, Onysko, M. K, Stob, C. M, & Hazlewood, K. A. Treatment of Alzheim‐

[9] Alzheimer's disease and Dementia-A Growing ChallengeNational Academy on an

[10] Bell, R. D, & Zloklovic, B. V. Neurovascular mechanisms and blood brain barrier dis‐ order in Alzheimer's disease. Acta Neuropathologica (2009). Doi:s00401-009-0522-3,

[11] Alonzo, N. C, Hyman, B. T, Rebeck, G. W, & Greenberg, S. M. Progression of cerebral amyloid angiopathy: accumulation of amyloid-beta40 in affected vessels. Journal of

[12] Asahina, M, Yoshiyama, Y, & Hattori, T. Expression of matrix metalloproteinase-9 and urinary-type plasminogen activator in Alzheimer's disease brain. Clinical Neu‐

[13] Attems, J, Jellinger, K. A, & Lintner, F. Alzheimer's disease pathology influences se‐ verity and topographical distribution of cerebral amyloid angiopathy. Acta Neuropa‐

[14] Attems, J, Quass, M, Jellinger, K. A, & Lintner, F. Topographical distribution of cere‐ bral amyloid angiopathy and its effect on cognitive decline are influenced by Alz‐ heimer disease pathology. Journal of Neurological Sciences (2007). Doi:10.1016/j.jns.

[15] Bailey, T. L, Rivara, C. B, Rocher, A. B, & Hof, P. R. The nature and effects of cortical microvascular pathology in aging and Alzheimer's disease. Neurological Research

[16] Begley, D. J, & Brightman, M. W. Structural and functional aspects of the blood-brain

[17] Bell, R. D, Sagare, A. P, Friedman, A. E, et al. Transport pathways for clearance of human Alzheimer's amyloid beta-peptide and apolipoproteins E and J in the mouse

Neuropathology and Experimental Neurology (1998). Doi:, 57, 353-359.

thologica (2005). Doi:s00401-005-1064-y., 110, 222-231.

barrier. Progress in Drug Research (2003). , 61, 39-78.

er Disease. American Family Physician website www.aafp.org/afp.

278(16), 1-9.

314 Neurodegenerative Diseases

10(3), 241-252.

2011.05.901, 7(5), 540-550.

Aging Society (2000).

ropathology (2001). , 20, 60-63.

2007.01.013., 257, 49-55.

(2004). Doi:, 26, 573-578.

118(1), 103-113.

for Alzheimer's disease Management (2008).


lar route for the elimination of amyloid beta from the human brain. Neuropathology and Applied Neurobiology (2003). Doi:10.1046/j.1365-2990.2003.00424.x., 29, 106-117.

[41] Martichuski, D. K, & Bell, P. A. Treating excess disabilities in special care units: A Re‐ view of interventions. The American Journal of Alzheimer's Care and related disor‐

Therapeutic Interventions in Alzheimer Disease

http://dx.doi.org/10.5772/54915

317

[42] Reisberg, B, Kenowsky, S, & Franssen, E. Souren LEM, Shulman E, Steinberg G, Ar‐ onstein Z, Auer S. Slowing the progression of Alzheimer Disease: Towards a Science

[43] Morton, I, & Bleathman, C. The effectiveness of validation therapy in dementia: a pi‐

[44] Sclan, S, Saillon, A, Franssen, E, et al. The Behavioral Pathology in Alzheimer's dis‐ ease Rating Scales (BEHAVE-AD): Reliability and Analysis of symptom category

lot study. International Journal of Geriatric Psychiatry (1991). , 6-327.

scores. International journal of Geriatric Psychiatry (1996). , 11-819.

ders and Research (1993). , 5-8.

of Alzheimer Disease care. Caring (1996). , 2(2), 2-4.


[41] Martichuski, D. K, & Bell, P. A. Treating excess disabilities in special care units: A Re‐ view of interventions. The American Journal of Alzheimer's Care and related disor‐ ders and Research (1993). , 5-8.

lar route for the elimination of amyloid beta from the human brain. Neuropathology and Applied Neurobiology (2003). Doi:10.1046/j.1365-2990.2003.00424.x., 29, 106-117.

[28] Rosenberg, G. A. Matrix metalloproteinases and their multiple roles in neurodege‐ nerative diseases. Lancet Neurology (2009). Doi:S1474-4422(09)70016-X., 8, 205-216.

[29] Sagare, A, Deane, R, Bell, R. D, et al. Clearance of amyloid-beta by circulating lipo‐

[30] Ushijima, Y, Okuyama, C, Mori, S, Kubota, T, Nakai, T, & Nishimura, T. Regional cerebral blood flow in Alzheimer's disease: Comparison between short term and long term donepezil therapy. Annals of Nuclear Medicine (2006). , 20(6), 425-429. [31] Sugimoto, H, Yamanishi, Y, Limura, Y, & Kawakami, Y. Donepezil hydrochloride (E2020) and other Acetylcholinesterase Inhibitors. Current Medicinal Chemistry

[32] Orhan, G, Orhan, I, Oztekin, N. S, Fikri, A. K, & Sener, B. Contemporary Anticholi‐ nesterase Pharmaceuticals of Natural Origin and their Synthetic Analogues for the Treatment of Alzheimer Disease. Recent Patents on CNS Drug Discovery (2009). , 4,

[33] Woodruff Pak DS, Vogel RW, Wenk GL. Mecamylamine Interactions with Galanta‐ mine and Donepezil: Effects on Learning, Acetylcholinesterase and Nicotinic Acetyl‐

[34] Sugimoto, H. Structure-activity relationships of acetylcholinesterase inhibitors: Do‐ nepezil hydrochloride for the treatment of Alzheimer's disease. Journal of Pure and

[35] Suh, W. H, Suslick, K. S, & Suh, Y. H. Therapeutic agents for Alzheimer Disease. Cur‐ rent Medicinal Chemistry-Central Nervous System Agents (2005). , 5-259.

[36] Sinn, N, & Milte, C. Howe PRC. Oiling the Brain: A Review of Randomized Control‐ led Trials of Omega-3 Fatty Acids in Psychopathology across the Lifespan. Nutrients

[37] Religa, D, & Winblad, B. Therapeutic strategies for Alzheimer Disease based on new

[38] Kandavelmani, A. Alzheimer Disease: In silico Drug Design-An Endeavour. Bioinfor‐

[39] Zeisel, J, & Raia, P. Non-pharmacological Treatment for Alzheimer Disease: A mindbrain approach. American Journal of Alzheimer Disease and Other Dementias

[40] Weiner, M. F, & Gray, K. F. Balancing Psychosocial and Psychopharmacologic meas‐ ures in Alzheimer Disease. The American Journal of Alzheimer's Care and Related

molecular mechanisms. Acta Neurobiol Express (2003). , 63-393.

choline Receptors. Neuroscience (2003). , 117-439.

Applied Chemistry (1999). , 71(11), 2031-2037.

protein receptors. Nature Medicine (2007). Doi:nm1635., 13, 1029-1031.

(2000). , 7-303.

(2010). , 2(2), 128-170.

(2000). , 15(6), 331-340.

matics India (2005). , 3(2), 25-28.

Disorders and Research (1994). , 9(4), 6-12.

43-51.

316 Neurodegenerative Diseases


**Section 3**

**Pathopysiological Aspects of Other**

**Neurodegenerative Diseases**
