**Meet the editor**

Dr Chang did his BSc training in Biochemistry in Hong Kong. Then he furthered his MPhil training in neurochemistry and neuroanatomy at the same university. He received a research scholarship by German Academic Exchange Service (DAAD) to pursue his doctoral training in neurophysiology and clinical neuroscience at the University of Munich, Germany. Afterwards, he

continued his postdoctoral training in neuropharmacology and molecular neuroscience at NIH, USA. He has published over 80 peer-reviewed papers and book chapters in neurodegeneration, neuroimmunology and drug discovery in these areas. Dr Chang is now the Scientific Advisory Board member in International AD/PD Symposium, Scientific Review Committee for Alzheimer Association in USA, editorial board member in many different journals including Journal of Alzheimer's Disease. Dr. Chang's research interest is to investigate molecular mechanisms of neurodegeneration in Alzheimer's disease, Parkinson's disease, ALS, glaucoma and aging-related macular degeneration (AMD) so that pharmacological targets can be found.

Contents

**Preface IX** 

Garth F. Hall

Chapter 2 **Roles of Microtubules in** 

**Part 1 Toxic Proteins and Disturbance of** 

Chapter 1 **What is the Link Between Protein** 

**Neuronal Functions in Neurodegeneration 1** 

**Propagation in Neurodegenerative Disease? 3** 

Kentaro Yomogida, Shumi Yoshida-Yamamoto and Hiroshi Doi

**Aggregation and Interneuronal Lesion** 

**Maintenance of Nerve Cell Networks 35** 

Chapter 3 **Oxidative Stress and Neurodegenerative Disease 53**  Selva Rivas-Arancibia, Cesar Gallegos-Ríos,

Nancy Gomez-Crisostomo, Ever Ferreira-Garcidueñas,

**Modulation by Palladium** α**-Lipoic Acid Complex 89**  Chirakkal V. Krishnan, Merrill Garnett and Frank Antonawich

**and Treatment of Neurodegenerative Diseases 127** 

**Histones H3 and H4 in Human Neuronal Cells 141** 

Rodrigo Valenzuela B. and Alfonso Valenzuela B.

Nadia Sadli, Nayyar Ahmed, M. Leigh Ackland, Andrew Sinclair, Colin J. Barrow and Cenk Suphioglu

**Part 2 Oxidative Stress and Neurodegeneration 51** 

Dulce Flores Briseño, Luz Navarro and Erika Rodríguez-Martínez

Chapter 4 **Free Radicals in Neurodegenerative Diseases:** 

Chapter 5 **Docosahexaenoic Acid (DHA), in the Prevention** 

Chapter 6 **Effect of Zinc and DHA on Expression Levels and Post-Translational Modifications of** 

### Contents

#### **Preface XIII**


Andrew Sinclair, Colin J. Barrow and Cenk Suphioglu


Contents VII

Chapter 16 **Can VEGF-B Be Used to Treat** 

Chapter 17 **Power of a Metabonomic Approach to**

Céline Domange, Alain Paris,

Chapter 19 **Quantification of Volumetric Changes of** 

Niyazi Acer, Ahmet Tuncay Turgut, Yelda Özsunar and Mehmet Turgut

Chapter 20 **Acid-Sensing Ion Channels in Neurodegenerative** 

**Part 6 Prevention, Protection and** 

Chapter 18 **Extract of** *Achillea fragrantissima*

Chapter 21 **Genome Profiling and Potential** 

**Neurodegenerative Diseases? 387** 

Henri Schroeder and Nathalie Priymenko

**Monitoring of Neurodegeneration 433** 

**Downregulates ROS Production and Protects** 

Anat Elmann, Alona Telerman, Sharon Mordechay, Hilla Erlank, Miriam Rindner, Rivka Ofir and Elie Beit-Yannai

**Brain in Neurodegenerative Diseases Using** 

**Diseases: Potential Therapeutic Target 477** Chu Xiang-Ping, Wang John Q. and Xiong Zhi-Gang

Luca Lovrečić, Aleš Maver and Borut Peterlin

**Using a Portable Wireless Sensor Device 541** Paul Bustamante, Gonzalo Solas and Karol Grandez

Chapter 22 **Immunization with Neural-Derived Peptides as a** 

Humberto Mestre and Antonio Ibarra

Chapter 23 **Neurodegenerative Disease Monitoring** 

**Biomarkers in Neurodegenerative Disorders 503** 

**Potential Therapy in Neurodegenerative Diseases 519** 

**Magnetic Resonance Imaging and Stereology 453** 

**Astrocytes from Oxidative-Stress-Induced Cell Death 435**

Xuri Li, Anil Kumar, Chunsik Lee, Zhongshu Tang, Yang Li, Pachiappan Arjunan, Xu Hou and Fan Zhang

**Investigate an Unknown Nervous Disease 407**

	- **Part 4 Roles of Glial Cells in Neurodegeneration 255**

#### **Part 5 Hormonal Control and Metabolism in Neurodegeneration 347**

Chapter 15 **Hormonal Signaling Systems of the Brain in Diabetes Mellitus 349**  Alexander Shpakov, Oksana Chistyakova, Kira Derkach and Vera Bondareva


VI Contents

Chapter 7 **Free Radicals, Neuronal Death and Neuroprotection 165** Diana Gallego, Manuel Rojas and Camilo Orozco

**Part 3 Intracellular Signaling of Neurodegeneration 199** 

Chapter 8 **Neuropathological Disorders and Calcium Independent** 

Chapter 9 **ASK1 and Its Role in Neurodegenerative Diseases 217** 

Chapter 10 **Role of Connexin Hemichannels in Neurodegeneration 235** Juan A. Orellana, Christian Giaume and Juan C. Sáez

Julie Allyson and Guy Massicotte

Emmanuel Sturchler, Daniel Feurstein, Patricia McDonald and Derek Duckett

**Part 4 Roles of Glial Cells in Neurodegeneration 255**

George E. Barreto, Janneth Gonzalez, Francisco Capani and Ludis Morales

**Interactions in Neurodegeneration:** 

Alla B. Salmina, Marina M. Petrova,

**Neurodegenerative Diseases 301**

**on Neurodegenerative Diseases 323**  Anne-Marie Miller, Brian F. Deighan,

**Metabolism in Neurodegeneration 347** 

**of the Brain in Diabetes Mellitus 349**  Alexander Shpakov, Oksana Chistyakova,

Kira Derkach and Vera Bondareva

Yvonne Nolan and Marina A. Lynch

Eric Downer, Anthony Lyons, Petra Henrich-Noack,

Chapter 12 **Alteration of Neuron-Glia** 

Chapter 13 **Microglia, Calcification and** 

**Part 5 Hormonal Control and** 

Chapter 15 **Hormonal Signaling Systems**

Chapter 14 **Analysis of the Impact of CD200** 

Chapter 11 **Role of Astrocytes in Neurodegenerative Diseases 257** 

Tatyana E. Taranushenko, Semen V. Prokopenko, Natalia A. Malinovskaya, Olesya S. Okuneva,

**Molecular Biomarkers and Therapeutic Strategy 273** 

Alyona I. Inzhutova, Andrei V. Morgun and Alexander A. Fursov

Jose M. Vidal-Taboada, Nicole Mahy and Manuel J. Rodríguez

**Forms of Phospholipase A2 Activities in the Brain 201** 

Preface

We have different forms of neurodegenerative diseases. Most of them are agingassociated such as Alzheimer's disease (AD), Parkinson's disease (PD), glaucoma, aging-related macular degeneration (AMD) and aging-related hearing loss. Some are related to genetic such as Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and Frontotemporal dementia. Some are related to autoimmune responses such as multiple sclerosis (MS). Although the primary reasons for different neurodegenerative diseases are very different and occur in different regions of the central nervous system (CNS), the degenerative processes share high similarity. Therefore, prevention and protective measures are very similar. This is the reason why

The first section of this book discusses toxic proteins in neurodegenerative diseases. Mis-folded/unfolded proteins escaped from regulatory control of chaperons usually create lots of problems to neurons. At early state, those aggregated proteins may block axonal transport by impairing the dynamic of cytoskeletal proteins. One should note that axonal transport along the track, microtubules, are extremely important for neurons. At late state, aggregated proteins choke ubiquitin-proteasomes system (UPS)

Having set the general landscape of neurodegenerative processes, the key factor to promote the processes is oxidative stress. Therefore, a whole section is dedicated to oxidative stress. We have chapters to discuss how and why oxidative stress contributes to neurodegeneration. We have also chapters to discuss how docosahexaenoic acid (DHA) elicits neuroprotection and current therapeutic methods to reduce oxidative stress. It is hope that reduction of oxidative stress can delay and

Then, how different stresses activate different signaling pathways to inform neurons to undergo the death pathways is discussed. It is apparently a vast variety of different signaling pathways leading to neurodegeneration. We collect chapters to discuss phospholipase A2 and apoptosis-signaling kinase-1 (ASK1) in this section. These two signaling pathways are partly activated by oxidative stress. We have a chapter to

discuss how connexin hemichannels contribute to degenerative processes.

and may also impair autophagic-lysosomal pathway to degrade them.

we can collect different chapters together in this book.

prevent progression of neurodegeneration.

### Preface

We have different forms of neurodegenerative diseases. Most of them are agingassociated such as Alzheimer's disease (AD), Parkinson's disease (PD), glaucoma, aging-related macular degeneration (AMD) and aging-related hearing loss. Some are related to genetic such as Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and Frontotemporal dementia. Some are related to autoimmune responses such as multiple sclerosis (MS). Although the primary reasons for different neurodegenerative diseases are very different and occur in different regions of the central nervous system (CNS), the degenerative processes share high similarity. Therefore, prevention and protective measures are very similar. This is the reason why we can collect different chapters together in this book.

The first section of this book discusses toxic proteins in neurodegenerative diseases. Mis-folded/unfolded proteins escaped from regulatory control of chaperons usually create lots of problems to neurons. At early state, those aggregated proteins may block axonal transport by impairing the dynamic of cytoskeletal proteins. One should note that axonal transport along the track, microtubules, are extremely important for neurons. At late state, aggregated proteins choke ubiquitin-proteasomes system (UPS) and may also impair autophagic-lysosomal pathway to degrade them.

Having set the general landscape of neurodegenerative processes, the key factor to promote the processes is oxidative stress. Therefore, a whole section is dedicated to oxidative stress. We have chapters to discuss how and why oxidative stress contributes to neurodegeneration. We have also chapters to discuss how docosahexaenoic acid (DHA) elicits neuroprotection and current therapeutic methods to reduce oxidative stress. It is hope that reduction of oxidative stress can delay and prevent progression of neurodegeneration.

Then, how different stresses activate different signaling pathways to inform neurons to undergo the death pathways is discussed. It is apparently a vast variety of different signaling pathways leading to neurodegeneration. We collect chapters to discuss phospholipase A2 and apoptosis-signaling kinase-1 (ASK1) in this section. These two signaling pathways are partly activated by oxidative stress. We have a chapter to discuss how connexin hemichannels contribute to degenerative processes.

#### XIV Preface

The processes of neurodegeneration are greatly affected by neighboring glial cells. Activation of astrocytes into gliosis or morphological changes of microglia can serve as pathological markers to inform cerebral inflammation. How microglia affected by calcium will be discussed. More importantly, how activation of microglia is regulated by CD200 is also discussed in this chapter.

Neurodegeneration is a result of multi-factorial factors contributing the fate of neurons. Among these factors, increasing lines of study pay attention to hormonal control and metabolism of brain cells or even the whole body metabolism. Vascular endothelia growth factor-B (VEGF-B) has recently been found to be a potential neuroprotective agent. We have a chapter to discuss its recent development.

The last section of this chapter integrates prevention, protection and monitoring of neurodegenerative diseases. We have chapter to discuss a natural product for prevention. Two chapters discuss how brain volume and acid-sensing channels help us to monitor degenerative processes. We have a chapter to discuss immunization. More importantly, we have a chapter to introduce how medical device helps monitoring disease progression.

Collectively, this book can give us a comprehensive overview of neurodegeneration from the processes, prevention, protection and monitoring. It is hope that we can collect a wide scope of neurodegeneration in a book to illustrate the principle of neurodegeneration. I am very pleased to be the editor of this book.

**Raymond Chuen-Chung Chang, PhD** 

Assistant Professor and Laboratory Head Laboratory of Neurodegenerative Diseases Department of Anatomy The University of Hong Kong Alzheimer's Disease Research Network Research Centre of Heart, Brain, Hormone and Healthy Aging LKS Faculty of Medicine The University of Hong Kong Pokfulam, Hong Kong SAR, China

## **Part 1**

**Toxic Proteins and Disturbance of Neuronal Functions in Neurodegeneration** 

**1** 

Garth F. Hall

*USA* 

**What is the Link Between Protein** 

**Aggregation and Interneuronal Lesion** 

*Department of Biological Sciences, University of Massachusetts Lowell,* 

**Propagation in Neurodegenerative Disease?** 

During the past 10-15 years it has become clear that most major neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, ALS, tauopathies, prion diseases and trinucleotide repeat diseases – henceforth to be referred to collectively as AANDs) share cellular and systemic features that suggest a common underlying mechanism of pathogenesis. At the cellular level, our understanding of the common aspects of AAND pathogenesis can be most simply summarized in terms of the downstream consequences of uncontrollable protein oligomerization and aggregation in postmitotic cells. The aggregated proteins block or disrupt normal proteosomal turnover and autophagy and become abnormally modified over time, generating toxicity via multiple pathways (mitochondrial damage, increased intracellular Ca++, caspase activation etc.) eventually leading to neurodegeneration and neuron death. This hypothesis is consistent with a key genetic similarity between these diseases – e.g. that familial forms are typically caused by autosomal dominant mutations that favor aggregation (in the case of asyn, tau, PrP and SOD1) or formation (in the case of APP and CAG repeat sequences) of disease-specific, aggregationprone proteins. These similarities have suggested to many that a single central defect (i.e. the failure of normal protein folding) lies at the heart of most or all of the diseases listed above, and has led to them being categorized be some as "protein misfolding diseases". While the importance of aggregate formation (and its attendant cellular dysfunctions) in each of these diseases is well established and has been intensively studied, our understanding of the intercellular and systemic aspects of these diseases is less detailed. That said, enough has been learned about their roles in neuronal biology and pathobiology and in the neuropathogenesis of AANDs to generate a general consensus that AAND development is 1) not cell autonomous and 2) that AANDs have another common hallmark– the progressive involvement of synaptically connected regions of the CNS over time in disease-specific patterns. Furthermore, it has become clear that important synergistic interactions between specific aggregation-prone proteins (tau and asyn (83), PrP and APP/Abeta (134), PrP and tau (216), PrP and asyn (95) may occur at both at the cellular and interneuronal level that affect the pathogenesis of specific AANDs. However, while neurofibrillary lesions develop according to characteristic, disease-specific sequences between highly interconnected regions of the brain in some AANDs (e.g. AD, tauopathies

**1. Introduction** 

### **What is the Link Between Protein Aggregation and Interneuronal Lesion Propagation in Neurodegenerative Disease?**

Garth F. Hall

*Department of Biological Sciences, University of Massachusetts Lowell, USA* 

#### **1. Introduction**

During the past 10-15 years it has become clear that most major neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, ALS, tauopathies, prion diseases and trinucleotide repeat diseases – henceforth to be referred to collectively as AANDs) share cellular and systemic features that suggest a common underlying mechanism of pathogenesis. At the cellular level, our understanding of the common aspects of AAND pathogenesis can be most simply summarized in terms of the downstream consequences of uncontrollable protein oligomerization and aggregation in postmitotic cells. The aggregated proteins block or disrupt normal proteosomal turnover and autophagy and become abnormally modified over time, generating toxicity via multiple pathways (mitochondrial damage, increased intracellular Ca++, caspase activation etc.) eventually leading to neurodegeneration and neuron death. This hypothesis is consistent with a key genetic similarity between these diseases – e.g. that familial forms are typically caused by autosomal dominant mutations that favor aggregation (in the case of asyn, tau, PrP and SOD1) or formation (in the case of APP and CAG repeat sequences) of disease-specific, aggregationprone proteins. These similarities have suggested to many that a single central defect (i.e. the failure of normal protein folding) lies at the heart of most or all of the diseases listed above, and has led to them being categorized be some as "protein misfolding diseases".

While the importance of aggregate formation (and its attendant cellular dysfunctions) in each of these diseases is well established and has been intensively studied, our understanding of the intercellular and systemic aspects of these diseases is less detailed. That said, enough has been learned about their roles in neuronal biology and pathobiology and in the neuropathogenesis of AANDs to generate a general consensus that AAND development is 1) not cell autonomous and 2) that AANDs have another common hallmark– the progressive involvement of synaptically connected regions of the CNS over time in disease-specific patterns. Furthermore, it has become clear that important synergistic interactions between specific aggregation-prone proteins (tau and asyn (83), PrP and APP/Abeta (134), PrP and tau (216), PrP and asyn (95) may occur at both at the cellular and interneuronal level that affect the pathogenesis of specific AANDs. However, while neurofibrillary lesions develop according to characteristic, disease-specific sequences between highly interconnected regions of the brain in some AANDs (e.g. AD, tauopathies

What is the Link Between Protein Aggregation and

delineate individual AANDs from one another (63, 59, 109, 116).

that partly overlaps those of the other ANDDs (illustrated in Figure 1).

Interneuronal Lesion Propagation in Neurodegenerative Disease? 5

regions concerned with olfaction, spatial localization and episodic memory formation and consolidation (transentorhinal, entorhinal, pyriform cortices), functions that are typically compromised in the earliest clinical (and even preclinical) phases of AD. This is followed by the progressive involvement of limbic and paralimbic centers including the hippocampus, adjacent allocortical regions of the medial temporal lobe (e.g. subiculum), the insula and anterior cingulate cortex. Again, these neuropathological changes match the development of AD symptoms quite closely, with changes in emotional processing and short term memory becoming evident by the time AD can be recognized as such in the clinic, together with the onset of cognitive changes. The most prominently affected limbic centers are strongly interconnected with one another synaptically as well as functionally (203), as would be necessary for lesion propagaion via transsynaptic toxicity transfer. The areas affected in this "limbic stage" of mild AD make up only a small proportion of the brain by volume (24), but make and receive major inputs to and from large neocortical regions that become involved in later (isocortical) stages of AD, which could account for the sudden expansion of AD neurofibrillary lesions at the onset of Braak Stage 5 (24, 202). Although some regions of the brain (e.g. the primary sensory and motor cortices) are almost never involved significantly in AD despite being strongly interconnected with highly vulnerable limbic centers, it seems likely that this is due to cell specific or even connectivity-specific factors (8) that may

The progressive involvement of synaptically interconnected brain regions seen in AD is mirrored in non-AD tauopathies such as frontotemporal dementia (FTD), Pick's Disease (PiD) progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) and involve some of the same parts of the brain (prefrontal/frontal cortex temporal cortex, insula) although the areas of initial involvement are different from AD and from each other (9, 10, 11, 63). Similarly, Parkinson's Disease (PD) and PD-associated dementing syndromes such as Lewy Body Disease (LBD) share a common set of vulnerable loci (dopaminergic neurons in the substantia nigra, brainstem autonomic nuclei, olfactory bulb and neocortical loci) but vary significantly in the initial nidus of vulnerability and the degree of involvement of other parts of the brain (48**,** recently reviewed in 3, 50, 59). Also, the progression of Lewy Body containing lesions in LBD and PD differs significantly from that seen in AD in that it is not tightly linked to overall clinical or neuropathological severity (28). Overall, significant overlap between the areas vulnerable to synucleinopathies with those involved in early stages of AD (nucleus of Meynert, olfactory bulb, various isocortical loci) and in non-AD tauopathies (basal ganglia, isocortex). Familial prion diseases (fatal familial insomnia, Creutzfeld Jacob disease (CJD), Gerstmann-Schenker-Straussler syndrome) show a similar pattern (lesion evolution via a subset of synaptically connected areas from characteristic initiation loci) with a common set of vulnerable loci (thalamus, neocortex, ANS, cerebellum)

Another distinctive feature of AANDs as a group is the manifestation of each syndrome in both sporadic and familial forms, with exonic or intronic mutations in a specific aggregation-prone protein being sufficient to generate a (usually) dominant allele capable of replicating all aspects of the (usually more common) sporadic disease with high penetrance (197). Perhaps the most interesting aspects of this pattern are a) the degree of similarity between sporadic and familial disease forms, and b) the greater tendency of sporadic, but not familial, disease forms to show asymmetrical development, especially in non AD tauopathies (59, 143). These emphasize the importance of both selective vulnerability and synaptic connectivity as common factors in these diseases, and is consistent with the

and LBD), the mechanisms by which the tendency toward aggregate formation is propagated between neurons as the disease progresses remains unclear, as does the degree to which such mechanisms contribute to disease pathogenesis as a whole. Similarly, there is still a gap between what we now know about the normal (mostly as monomer) and toxic (mostly as oligomers and aggregates) functions of each of these proteins at the cellular level. We know a good deal about the factors that favor AAND oligomerization, but very little about how oligomerization actually occurs in human disease. In particular, we have no real idea how these factors might 1) interact synergistically to drive cytotoxicity and degeneration and 2) are related to the mechanisms by which interneuronal toxicity is propagated between neurons in different parts of the brain. This review will attempt to integrate relatively recent findings about the interactions between the 3 most widely studied of these proteins (i.e. tau, alpha synuclein and the prion protein) both with each other and with cellular mechanisms associated with unconventional protein secretion into a framework that will account for common pathogenic features of these diseases and suggest future avenues of inquiry. For the sake of clarity, the discussion will be focused on asyn, tau and PrP and their interactions with APP/Abeta, and will omit a detailed consideration of other diseases that may have similar pathogenetic features (e.g ALS, Huntington's disease) and associated aggregation-prone proteins (SOD1, polyglutamine expansions, TDP-43, FUS), except when these become relevant to the discussion of general mechanisms. It will be guided by the example of PrP misprocessing and prion diseases, where the key link between intracellular protein aggregation, interneuronal transfer and the spread of neurofibrillary lesions through the brain has already been definitively established and which provides hints as to where to look for similar links in other AANDs.

#### **Overview of common neuropathological and genetic aspects of AAND pathogenesis at the cellular and systemic levels**

The predominant focus of basic research over the past 2 decades into the pathogenesis of all of the major AANDs has been on 1) the mechanisms responsible for protein aggregate formation and 2) the nature of cytotoxic changes that accompany and result from the aggregation of each of the proteins being discussed. As a consequence, aggregationassociated events and downstream consequences of aggregation such as the failure of protein turnover mechanisms in long-lived postmitotic cells such as neurons are among the best-characterized cytopathological features of neurodegenerative diseases. This work has generated a broad consensus that aggregation causes the failure of normal protein turnover mechanisms and the consequent development of abnormal toxic routes of protein disposal are central pathogenic events of the degenerative diseases that afflict the human central nervous system as it ages. Common toxic elements downstream of protein aggregation in AANDs include: 1) aggregation associated damage to protein turnover mechanisms, 2) mitochondrial dysfunction and or maldistribution leading to apoptosis-associated changes due to low ATP, generation of oxidative stress and abnormal Ca++ fluxes and 3) aggregatemediated sequestration of normal proteins resulting in a loss of the normal function associated with sequestered proteins.

The classic example of a neuropathogenesis pattern suggestive of lesion spread in AANDs (outside of prion diseases) is provided by Alzheimer's Disease (AD). Ever since the seminal studies of Heiko and Eva Braak (27), it has been apparent that the neurofibrillary degenerative changes of AD develop in a characteristic sequence that closely follows the clinical progression of symptoms (11, 203). The earliest changes occur in specific limbic

and LBD), the mechanisms by which the tendency toward aggregate formation is propagated between neurons as the disease progresses remains unclear, as does the degree to which such mechanisms contribute to disease pathogenesis as a whole. Similarly, there is still a gap between what we now know about the normal (mostly as monomer) and toxic (mostly as oligomers and aggregates) functions of each of these proteins at the cellular level. We know a good deal about the factors that favor AAND oligomerization, but very little about how oligomerization actually occurs in human disease. In particular, we have no real idea how these factors might 1) interact synergistically to drive cytotoxicity and degeneration and 2) are related to the mechanisms by which interneuronal toxicity is propagated between neurons in different parts of the brain. This review will attempt to integrate relatively recent findings about the interactions between the 3 most widely studied of these proteins (i.e. tau, alpha synuclein and the prion protein) both with each other and with cellular mechanisms associated with unconventional protein secretion into a framework that will account for common pathogenic features of these diseases and suggest future avenues of inquiry. For the sake of clarity, the discussion will be focused on asyn, tau and PrP and their interactions with APP/Abeta, and will omit a detailed consideration of other diseases that may have similar pathogenetic features (e.g ALS, Huntington's disease) and associated aggregation-prone proteins (SOD1, polyglutamine expansions, TDP-43, FUS), except when these become relevant to the discussion of general mechanisms. It will be guided by the example of PrP misprocessing and prion diseases, where the key link between intracellular protein aggregation, interneuronal transfer and the spread of neurofibrillary lesions through the brain has already been definitively established and which provides hints

**Overview of common neuropathological and genetic aspects of AAND pathogenesis at** 

The predominant focus of basic research over the past 2 decades into the pathogenesis of all of the major AANDs has been on 1) the mechanisms responsible for protein aggregate formation and 2) the nature of cytotoxic changes that accompany and result from the aggregation of each of the proteins being discussed. As a consequence, aggregationassociated events and downstream consequences of aggregation such as the failure of protein turnover mechanisms in long-lived postmitotic cells such as neurons are among the best-characterized cytopathological features of neurodegenerative diseases. This work has generated a broad consensus that aggregation causes the failure of normal protein turnover mechanisms and the consequent development of abnormal toxic routes of protein disposal are central pathogenic events of the degenerative diseases that afflict the human central nervous system as it ages. Common toxic elements downstream of protein aggregation in AANDs include: 1) aggregation associated damage to protein turnover mechanisms, 2) mitochondrial dysfunction and or maldistribution leading to apoptosis-associated changes due to low ATP, generation of oxidative stress and abnormal Ca++ fluxes and 3) aggregatemediated sequestration of normal proteins resulting in a loss of the normal function

The classic example of a neuropathogenesis pattern suggestive of lesion spread in AANDs (outside of prion diseases) is provided by Alzheimer's Disease (AD). Ever since the seminal studies of Heiko and Eva Braak (27), it has been apparent that the neurofibrillary degenerative changes of AD develop in a characteristic sequence that closely follows the clinical progression of symptoms (11, 203). The earliest changes occur in specific limbic

as to where to look for similar links in other AANDs.

**the cellular and systemic levels** 

associated with sequestered proteins.

regions concerned with olfaction, spatial localization and episodic memory formation and consolidation (transentorhinal, entorhinal, pyriform cortices), functions that are typically compromised in the earliest clinical (and even preclinical) phases of AD. This is followed by the progressive involvement of limbic and paralimbic centers including the hippocampus, adjacent allocortical regions of the medial temporal lobe (e.g. subiculum), the insula and anterior cingulate cortex. Again, these neuropathological changes match the development of AD symptoms quite closely, with changes in emotional processing and short term memory becoming evident by the time AD can be recognized as such in the clinic, together with the onset of cognitive changes. The most prominently affected limbic centers are strongly interconnected with one another synaptically as well as functionally (203), as would be necessary for lesion propagaion via transsynaptic toxicity transfer. The areas affected in this "limbic stage" of mild AD make up only a small proportion of the brain by volume (24), but make and receive major inputs to and from large neocortical regions that become involved in later (isocortical) stages of AD, which could account for the sudden expansion of AD neurofibrillary lesions at the onset of Braak Stage 5 (24, 202). Although some regions of the brain (e.g. the primary sensory and motor cortices) are almost never involved significantly in AD despite being strongly interconnected with highly vulnerable limbic centers, it seems likely that this is due to cell specific or even connectivity-specific factors (8) that may delineate individual AANDs from one another (63, 59, 109, 116).

The progressive involvement of synaptically interconnected brain regions seen in AD is mirrored in non-AD tauopathies such as frontotemporal dementia (FTD), Pick's Disease (PiD) progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) and involve some of the same parts of the brain (prefrontal/frontal cortex temporal cortex, insula) although the areas of initial involvement are different from AD and from each other (9, 10, 11, 63). Similarly, Parkinson's Disease (PD) and PD-associated dementing syndromes such as Lewy Body Disease (LBD) share a common set of vulnerable loci (dopaminergic neurons in the substantia nigra, brainstem autonomic nuclei, olfactory bulb and neocortical loci) but vary significantly in the initial nidus of vulnerability and the degree of involvement of other parts of the brain (48**,** recently reviewed in 3, 50, 59). Also, the progression of Lewy Body containing lesions in LBD and PD differs significantly from that seen in AD in that it is not tightly linked to overall clinical or neuropathological severity (28). Overall, significant overlap between the areas vulnerable to synucleinopathies with those involved in early stages of AD (nucleus of Meynert, olfactory bulb, various isocortical loci) and in non-AD tauopathies (basal ganglia, isocortex). Familial prion diseases (fatal familial insomnia, Creutzfeld Jacob disease (CJD), Gerstmann-Schenker-Straussler syndrome) show a similar pattern (lesion evolution via a subset of synaptically connected areas from characteristic initiation loci) with a common set of vulnerable loci (thalamus, neocortex, ANS, cerebellum) that partly overlaps those of the other ANDDs (illustrated in Figure 1).

Another distinctive feature of AANDs as a group is the manifestation of each syndrome in both sporadic and familial forms, with exonic or intronic mutations in a specific aggregation-prone protein being sufficient to generate a (usually) dominant allele capable of replicating all aspects of the (usually more common) sporadic disease with high penetrance (197). Perhaps the most interesting aspects of this pattern are a) the degree of similarity between sporadic and familial disease forms, and b) the greater tendency of sporadic, but not familial, disease forms to show asymmetrical development, especially in non AD tauopathies (59, 143). These emphasize the importance of both selective vulnerability and synaptic connectivity as common factors in these diseases, and is consistent with the

What is the Link Between Protein Aggregation and

discussed futher below.

Interneuronal Lesion Propagation in Neurodegenerative Disease? 7

misprocessing pathway that can also lead to the spread of tau–based lesions in the same overall pattern, rather than recruiting tau directly. Such a pathway might involve the mislocalization of proteins important in the maintenance of neuronal polarity from the axon to the soma and dendrites via a failure of hnnRP-mediated mRNA localization. This possibility is particularly intriguing since a) hnnRP interactions with the 3' UTR of the mRNA encoding tau have been shown to be responsible for both tau localization to the axon (12, 146) and the generation of neuronal polarity (147) and b) the neuropathology of AD and non-AD tauopathies suggests that polarity loss plays a role in tauopathy pathogenesis (107, 1741). Overall, the common neuropathological and genetic features of AANDs involving tau, asyn and PrP are largely consistent with the existence of a common lesion propagation mechanism (or several closely related mechanisms) that involves direct interneuronal

The prototypic (and most extreme) example of an aggregation-prone protein that propagates its misfolded state at the protein, cellular and even organismal level is of course the prion protein (PrP), the misfolding of which mediates a class of mostly rare neurological degenerative diseases (transmissable spongiform encephalopathies) of humans and other mammals, the best known of which are CJD, scrapie, and kuru. Due to the pioneering work of Tikva Alper, Carlton Gadjusek and (particularly and most recently) Stanley Prusiner and co-workers over the past 50 years, and after rigorous verification by often highly skeptical investigators, there is now a general consensus that the so called "Prion hypothesis" proposed by Prusiner 30 years ago has correctly predicted key peculiarities of prion disease transmission such as the effect of PrP knockouts (31) and thus correctly describes the pathogenesis of these diseases (reviewed in 3, 49, 175). The Prion Hypothesis states that individual molecules of a single, widely expressed protein (the prion protein, or PrP) becomes misfolded and misprocessed in a manner that makes it adopt a neurotoxic conformation (PrPSc), but more importantly, permits it to transmit this conformation on to other prion proteins in the normal (PrPC) conformation. The peculiar and controversial history of prion biology thus provides us with a highly verified example of how the misprocessing of an aggregation-prone protein into a toxic form can result in the interneuronal propagation of a protein with self regenerating, neurotoxic characteristics, and thus effect the spreading of neurofibrillary lesions to adjacent, presynaptic or postsynaptic neurons. The likely relevance of PrP misprocessing mechanisms to the pathogenesis of tauopathies, synucleinopathies, and other AANDs is further underscored by recent demonstrations that immensely subtle differences in PrP misprocessing and PrPSc structure appear to mediate the distinctive clinical and neuropathological manifestations of the various prion diseases (18, 40, 49, 168). In addition, recent studies of the normal cellular functions of PrPC suggest that it is involved in the function of the actin-rich subcortical cytoskeleton and its interactions with microtubules, cellular membrane trafficking, cell adhesion and signal transduction in a variety of cell types (reviewed in 3, 53). In neurons, PrPC appears to play a critical (if subtle) role in synaptic plasticity and most interestingly, in the propagation of HIV infection in the CNS (149, 180). The similarities in the cellular function, localization and misprocessing of PrP, APP/Abeta, asyn and tau identify likely points of interaction between these proteins, and synergy in their misprocessing, which are

transfer of a toxic factor between adjacent and transsynaptic neurons. **Linking aggregation to lesion spreading – The case of the prion protein** 

intriguing relationship between acquired, sporadic and familial forms of prion diseases such as Creutzfeld-Jacob disease (CJD), where the point of origin is clearly different in each case, but common aspects of vulnerability and synaptic connectivity are sufficient to generate a common clinical presentation (CJD), despite the presence of characteristic differences in lesion form (175). A similar relationship may hold between certain non-AD tauopathies and clinically identical diseases (both called FTDP or FTDP-17) involving loss of function changes in RNA-binding proteins (TDP-43, progranin) involved in the localization and translation of cytoskeletal proteins (hnnRPs), including tau and neurofilament proteins (147). Here, TDP-43 and or progranin may be activating downstream elements of a common

Fig. 1. Schematic illustrating the relationship between the characteristically vulnerable regions in AD, (green) nonAD tauopathies (pink), familial prion diseases (yellow) and synucleinopathies (blue). Regions typically involved in multiple AAND disease classes are shown in overlapping areas. Individual syndromes from all of these diseases eventually involve polymodal (associative) isocortical areas and thus cause dementia, even though severe cognitive changes may be absent or develop very late in other members of each group (e.g. Parkinson's Disease, fatal familial insomnia). Vulnerable areas in familial and sporadic forms of each AAND are identical, with familial syndromes beginning earlier and progressing faster than sporadic ones. Characteristic areas of vulnerability for frontotemporal syndromes (FTDP-U) that involve TDP-43 and FUS rather than tau aggregates and acquired forms of prion disease (vCJD, kuru) are virtually identical to non AD tauopathies (pink) and familial prion diseases (yellow), respectively, possibly owing to the presence of "prion like" motifs in these proteins (53). ANS: autonomic nervous sytem, MFB: medial forebrain bundle/nucleus of Meynert, GP: globus pallidus

intriguing relationship between acquired, sporadic and familial forms of prion diseases such as Creutzfeld-Jacob disease (CJD), where the point of origin is clearly different in each case, but common aspects of vulnerability and synaptic connectivity are sufficient to generate a common clinical presentation (CJD), despite the presence of characteristic differences in lesion form (175). A similar relationship may hold between certain non-AD tauopathies and clinically identical diseases (both called FTDP or FTDP-17) involving loss of function changes in RNA-binding proteins (TDP-43, progranin) involved in the localization and translation of cytoskeletal proteins (hnnRPs), including tau and neurofilament proteins (147). Here, TDP-43 and or progranin may be activating downstream elements of a common

Fig. 1. Schematic illustrating the relationship between the characteristically vulnerable regions in AD, (green) nonAD tauopathies (pink), familial prion diseases (yellow) and synucleinopathies (blue). Regions typically involved in multiple AAND disease classes are shown in overlapping areas. Individual syndromes from all of these diseases eventually involve polymodal (associative) isocortical areas and thus cause dementia, even though severe cognitive changes may be absent or develop very late in other members of each group (e.g. Parkinson's Disease, fatal familial insomnia). Vulnerable areas in familial and sporadic forms of each AAND are identical, with familial syndromes beginning earlier and

progressing faster than sporadic ones. Characteristic areas of vulnerability for frontotemporal syndromes (FTDP-U) that involve TDP-43 and FUS rather than tau aggregates and acquired forms of prion disease (vCJD, kuru) are virtually identical to non AD tauopathies (pink) and familial prion diseases (yellow), respectively, possibly owing to the presence of "prion like" motifs in these proteins (53). ANS: autonomic nervous sytem,

MFB: medial forebrain bundle/nucleus of Meynert, GP: globus pallidus

misprocessing pathway that can also lead to the spread of tau–based lesions in the same overall pattern, rather than recruiting tau directly. Such a pathway might involve the mislocalization of proteins important in the maintenance of neuronal polarity from the axon to the soma and dendrites via a failure of hnnRP-mediated mRNA localization. This possibility is particularly intriguing since a) hnnRP interactions with the 3' UTR of the mRNA encoding tau have been shown to be responsible for both tau localization to the axon (12, 146) and the generation of neuronal polarity (147) and b) the neuropathology of AD and non-AD tauopathies suggests that polarity loss plays a role in tauopathy pathogenesis (107, 1741). Overall, the common neuropathological and genetic features of AANDs involving tau, asyn and PrP are largely consistent with the existence of a common lesion propagation mechanism (or several closely related mechanisms) that involves direct interneuronal transfer of a toxic factor between adjacent and transsynaptic neurons.

#### **Linking aggregation to lesion spreading – The case of the prion protein**

The prototypic (and most extreme) example of an aggregation-prone protein that propagates its misfolded state at the protein, cellular and even organismal level is of course the prion protein (PrP), the misfolding of which mediates a class of mostly rare neurological degenerative diseases (transmissable spongiform encephalopathies) of humans and other mammals, the best known of which are CJD, scrapie, and kuru. Due to the pioneering work of Tikva Alper, Carlton Gadjusek and (particularly and most recently) Stanley Prusiner and co-workers over the past 50 years, and after rigorous verification by often highly skeptical investigators, there is now a general consensus that the so called "Prion hypothesis" proposed by Prusiner 30 years ago has correctly predicted key peculiarities of prion disease transmission such as the effect of PrP knockouts (31) and thus correctly describes the pathogenesis of these diseases (reviewed in 3, 49, 175). The Prion Hypothesis states that individual molecules of a single, widely expressed protein (the prion protein, or PrP) becomes misfolded and misprocessed in a manner that makes it adopt a neurotoxic conformation (PrPSc), but more importantly, permits it to transmit this conformation on to other prion proteins in the normal (PrPC) conformation. The peculiar and controversial history of prion biology thus provides us with a highly verified example of how the misprocessing of an aggregation-prone protein into a toxic form can result in the interneuronal propagation of a protein with self regenerating, neurotoxic characteristics, and thus effect the spreading of neurofibrillary lesions to adjacent, presynaptic or postsynaptic neurons. The likely relevance of PrP misprocessing mechanisms to the pathogenesis of tauopathies, synucleinopathies, and other AANDs is further underscored by recent demonstrations that immensely subtle differences in PrP misprocessing and PrPSc structure appear to mediate the distinctive clinical and neuropathological manifestations of the various prion diseases (18, 40, 49, 168). In addition, recent studies of the normal cellular functions of PrPC suggest that it is involved in the function of the actin-rich subcortical cytoskeleton and its interactions with microtubules, cellular membrane trafficking, cell adhesion and signal transduction in a variety of cell types (reviewed in 3, 53). In neurons, PrPC appears to play a critical (if subtle) role in synaptic plasticity and most interestingly, in the propagation of HIV infection in the CNS (149, 180). The similarities in the cellular function, localization and misprocessing of PrP, APP/Abeta, asyn and tau identify likely points of interaction between these proteins, and synergy in their misprocessing, which are discussed futher below.

What is the Link Between Protein Aggregation and

**aggregation and intercellular transfer General molecular and cellular considerations** 

175, 215).

Interneuronal Lesion Propagation in Neurodegenerative Disease? 9

The abnormal and irreversible oligomerization and/or aggregation of specific proteins (e.g. tau, asyn, PrP) is the central common feature in AAND cytopathogenesis and by itself accounts for many of the other common cellular features of these diseases (a good review of the subject can be found in 196). Familial AANDs are typically induced by intronic, autosomal dominant mutations that either directly favor aggregation (tau, asyn, PrP), favor events that lead to generation of the aggregation-prone form of the protein (e.g. cleavage, abnormal association with other proteins, abnormal glycosylation or phosphorylation), or both (tau, PrP) (3, 50, 202). Exceptions to the autosomal dominant pattern include recessive mutations responsible for loss of function effects in protein turnover pathways (e,g, parkin 126). These genetics suggest that AAND pathology is due to a gain of function leading to aggregate formation and downstream toxicity involving the poisoning or overloading of proteasomal or autophagy-based protein turnover. A common structural feature among these proteins relevant to their tendency to aggregate is the co-existence in each one of a "core" domain which can form beta sheet interactions plus at least one other domain that inhibits this tendency, resulting in a balance between a normal conformation (rich in alpha helix or "random coil") conformation and an abnormal beta sheet-rich conformation that favors aggregation (50, 5, 156). Key common features in the cellular functions of tau, asyn and PrP include interaction with both chaperone proteins and with signal transduction elements, which might be expected of proteins capable of both aggregation and transcellular movement, respectively. Moreover, all three proteins are frequently associated with cellular membranes under normal conditions, especially in synapses (29, 71, 76, 148, 212, 213, 233) where they interact with APP (an integral membrane protein) and/or Abeta (93, 171), and are substrates for lipid raft-associated Srk family tyrosine kinases (e.g. fyn - 95, 137, 188, syk - 136 and abl - 37). In particular, the luminal localization of each protein in endosomes and/or trafficking vesicles associated with unconventional secretion (35, 78, 140, 142), reviewed in 215), and the interactions (in some cases copolymerization) that can occur between them (83, 134, 171, 216, 217) make endosomal pathways a highly plausible candidate site that might mediate the synergistic misprocessing of these proteins. An endosome-mediated common misprocessing pathway is also consistent with the availability of templating polyanionic ligands such as membrane-associated proteoglycans favoring further aggregation and toxicity (51, 52, 91, 106, 111), and the ready diversion of endocytosed proteins to unconventional secretion pathways (68, 70, 733, 102, 116, 124, 140,

**Tau, asyn and PrP are all "switch" proteins that alternate between 2 states based on regulated charge/charge modifications.** Under normal circumstances, asyn, tau and PrP function as soluble monomers that interact extensively with other proteins in the both in the cytosol and in association with cellular membranes. Soluble PrPC and asyn contain alpha helical and random coil domains, and take up a predominantly random coil conformation in aqueous solution (186, 219, 231). In cells, tau normally extends along microtubules**,** where it stabilizes them by preventing classic dynamic instability via binding to them at multiple sites in its aggregation prone-microtubule binding domain (MTBR) (33). Tau:MT binding is itself dynamic (186), and tau interacts with fyn kinase, actin and protein chaperones via loci

**2. Common structural/functional features of AAND proteins favoring** 


Table 1. Comparison of the pathobiological characteristics of 4 aggregation-prone proteins responsible for most aggregation-associated neurodegenerative diseases (AANDs) in humans The table summarizes aspects of disease-associated misprocessing of 4 aggregationprone proteins (amyloid precursor protein/beta amyloid (APP/Abeta) tau, alpha synuclein (asyn) and prion (PrP)) discussed in the text that are relevant to both aggregate formation and lesion propagation in major human neurodegenerative diseases (Alzheimer's Disease (AD) Down's Syndrome (DS), Pick's Disease (PiD), progressive supernuclear palsy (PSP), corticobasal degeneration (CBD), Parkinson's disease (PD), Lewy Body disease (LBD), Creutzfeld-Jakob disease (CJD), Gerstmann Straussler Schenker disease (GSS), fatal familial insomnia (FFI), kuru and variant CJD (vCJD). \*publication in review (185)

#### **2. Common structural/functional features of AAND proteins favoring aggregation and intercellular transfer**

#### **General molecular and cellular considerations**

8 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

Table 1. Comparison of the pathobiological characteristics of 4 aggregation-prone proteins responsible for most aggregation-associated neurodegenerative diseases (AANDs) in humans The table summarizes aspects of disease-associated misprocessing of 4 aggregationprone proteins (amyloid precursor protein/beta amyloid (APP/Abeta) tau, alpha synuclein (asyn) and prion (PrP)) discussed in the text that are relevant to both aggregate formation and lesion propagation in major human neurodegenerative diseases (Alzheimer's Disease (AD) Down's Syndrome (DS), Pick's Disease (PiD), progressive supernuclear palsy (PSP), corticobasal degeneration (CBD), Parkinson's disease (PD), Lewy Body disease (LBD), Creutzfeld-Jakob disease (CJD), Gerstmann Straussler Schenker disease (GSS), fatal familial

insomnia (FFI), kuru and variant CJD (vCJD). \*publication in review (185)

The abnormal and irreversible oligomerization and/or aggregation of specific proteins (e.g. tau, asyn, PrP) is the central common feature in AAND cytopathogenesis and by itself accounts for many of the other common cellular features of these diseases (a good review of the subject can be found in 196). Familial AANDs are typically induced by intronic, autosomal dominant mutations that either directly favor aggregation (tau, asyn, PrP), favor events that lead to generation of the aggregation-prone form of the protein (e.g. cleavage, abnormal association with other proteins, abnormal glycosylation or phosphorylation), or both (tau, PrP) (3, 50, 202). Exceptions to the autosomal dominant pattern include recessive mutations responsible for loss of function effects in protein turnover pathways (e,g, parkin 126). These genetics suggest that AAND pathology is due to a gain of function leading to aggregate formation and downstream toxicity involving the poisoning or overloading of proteasomal or autophagy-based protein turnover. A common structural feature among these proteins relevant to their tendency to aggregate is the co-existence in each one of a "core" domain which can form beta sheet interactions plus at least one other domain that inhibits this tendency, resulting in a balance between a normal conformation (rich in alpha helix or "random coil") conformation and an abnormal beta sheet-rich conformation that favors aggregation (50, 5, 156). Key common features in the cellular functions of tau, asyn and PrP include interaction with both chaperone proteins and with signal transduction elements, which might be expected of proteins capable of both aggregation and transcellular movement, respectively. Moreover, all three proteins are frequently associated with cellular membranes under normal conditions, especially in synapses (29, 71, 76, 148, 212, 213, 233) where they interact with APP (an integral membrane protein) and/or Abeta (93, 171), and are substrates for lipid raft-associated Srk family tyrosine kinases (e.g. fyn - 95, 137, 188, syk - 136 and abl - 37). In particular, the luminal localization of each protein in endosomes and/or trafficking vesicles associated with unconventional secretion (35, 78, 140, 142), reviewed in 215), and the interactions (in some cases copolymerization) that can occur between them (83, 134, 171, 216, 217) make endosomal pathways a highly plausible candidate site that might mediate the synergistic misprocessing of these proteins. An endosome-mediated common misprocessing pathway is also consistent with the availability of templating polyanionic ligands such as membrane-associated proteoglycans favoring further aggregation and toxicity (51, 52, 91, 106, 111), and the ready diversion of endocytosed proteins to unconventional secretion pathways (68, 70, 733, 102, 116, 124, 140, 175, 215).

**Tau, asyn and PrP are all "switch" proteins that alternate between 2 states based on regulated charge/charge modifications.** Under normal circumstances, asyn, tau and PrP function as soluble monomers that interact extensively with other proteins in the both in the cytosol and in association with cellular membranes. Soluble PrPC and asyn contain alpha helical and random coil domains, and take up a predominantly random coil conformation in aqueous solution (186, 219, 231). In cells, tau normally extends along microtubules**,** where it stabilizes them by preventing classic dynamic instability via binding to them at multiple sites in its aggregation prone-microtubule binding domain (MTBR) (33). Tau:MT binding is itself dynamic (186), and tau interacts with fyn kinase, actin and protein chaperones via loci

What is the Link Between Protein Aggregation and

traumatic head injury in AD (151, 178).

propagation entirely (16, reviewed in 209).

Interneuronal Lesion Propagation in Neurodegenerative Disease? 11

their environment that may favor templating interactions and oligomer formation (67) Hyperphosphorylation, cleavage and aggregation of wild type tau isoforms can be induced simply by increasing the concentration of protein that is not MT-associated and thus vulnerable to misfolding (reviewed by 13, 203), causing the release of tau to the cytosol. This kind of release likely accompanies Abeta or axotomy-induced MT loss (32, 101), and thus could account for some of the dependence of tau misprocessing on Abeta generation and

While aggregate formation is a central event in the misprocessing of aggregation-prone proteins that drive AAND pathogenesis, it remains unclear how it is connected to the diversion of these proteins into the unconventional secretion pathways that might account for the interneuronal transmission of neurofibrillary lesions that appears to occur in these diseases. One possibly relevant property common to tau, asyn and PrP is their tendency to associate with membranes (29, 40, 53, 67, 173, 230, 233) and bind to membrane associated molecules such as HSPGs and fatty acids (44, 222, 225, 232). HSPG binding favors oligomer and fibril formation (52, 91, 120, 225) and may facilitate interactions with APP, which also interacts with HSPGs in cholesterol rich microdomains (lipid rafts 64, 193). Such interactions seem to be favored in AAND pathogenesis, since APP, tau and asyn colocalize with HSPGs in AAND neuropathological lesions (51, 59, 109 197). HSPGs may facilitate interactions between asyn and tau (both localized to elements on the inside of the membrane) and PrPC, which is typically found on the exterior surface attached via a GPI anchor (163, 232) and may themselves mediate transcellular protein movement, as has been suggested by studies of morphogen movement during *Drosophila* development (166), possibly by trapping interacting proteins in the extracellular space (232). Raft-associated interactions appear to be important in disease-associated misprocessing of tau, asyn and PrP mediated via fyn (131, 138, 188, 221), in aggregation (195, 230) and in disruption of signal transduction pathways in CNS dendrites (108, 117). Lipid association also drives oligomer and filament formation of Abeta, tau and PrP (44, 208, 221). In a very recent study by Binder and colleagues (170), a mAb specific for tau oligomer identified the presence of arachidonic acid as one of the requirements for early oligomer formation in cell culture. Similarly, the presence of membrane anchors and raft localization motifs plays an essential role in the development of characteristic lesion morphology of PrP-mediated disease (40); the removal of the GPI anchor has been shown to produce novel syndromes in transgenic models (43), while the removal of all of the multiple raft localization motifs on PrP blocks lesion formation and

The relationship between asyn misprocessing and membrane localization in AANDs may be more complex than that of PrP and tau, since some, but not all disease-inducing mutations block raft-asyn association (75). Like PrP and tau, asyn is localized to lipid raft microdomains in presynaptic terminals, where it accumulates in dystrophic neurites associated with Parkinson's Disease and Lewy body dementia (81). Similarly, asyn fibrillization is favored by interactions with unsaturated fatty acids (173) but unlike tau, this is inhibited by saturated fatty acids (233). A particularly intriguing recent finding by Fang et. al. demonstrated a direct link between oligomerization and unconventional secretion in a study showing that higher-order oligomerization can drive exosome-mediated secretion of a wide variety of oligomerized proteins (70). This is particularly interesting given that tau, asyn and PrP are all substrates for fyn and related raft-associated srk tyrosine kinases (136, 138, 188), and that such interactions are associated with AAND pathogenesis (19, 110) and have potentially

that overlap the MTBR when not bound to MTs (95, 107, 189). Monomeric asyn exists in both membrane-associated and cytosolic loci, and like tau, can bind to both actin and tubulin (4). As with tau, disease-causing mutations in asyn cause it to preferentially bind to membraneassociated proteins (69). Both membrane and MT-associated asyn have been found to aggregate (4, 139), in some cases forming clusters of microvesicles (195). PrP possesses an aggregation-prone domain (octopeptide repeat) that appears to be oligomerized reversibly during endocytosis. Unlike tau, it also possesses a separate N terminal MT-binding domain (231). All three proteins possess-aggregation-prone domains via which they aggregate resulting in a significant increase in beta sheet structure (156, 187, 231). Deletion analyses of all three proteins show that the removal or inactivation of non-aggregating domains (the N terminus of PrP, the tau N and C termini, the asyn C terminus) may tip this balance toward aggregate formation (1, 38, 112, 226). Post-translational regulation of each protein via phosphorylation may also do this (5, 41, 42, 80), either because it blocks the binding of the aggregation-prone domain to its normal cellular ligand, thereby permitting self assembly (156), or by favoring conversion of soluble oligomer to insoluble higher-order aggregates (187). Familial disease mutations may mimic these changes (13, 66, 80) as well. Overall, while tau, asyn and PrP are capable of aggregate formation and normally interact with both MTs and membrane associated components, the details of how oligomer formation and membrane association is related to normal function vary considerably. A key common feature relevant to the appearance of gain-of-function properties leading to interneuronal propagation in AANDs is the existence of self-binding/assembly capable and assemblyinhibiting domains in each protein that are normally balanced in favor of monomeric functions. This can thus act as a "switch" between normal and abnormal processing pathways which may be mutated to favor oligomer formation in familial AANDs, or alternatively, be "flipped" by derangement of regulatory elements (e.g. kinase/phosphatase and protease activities) that induce these posttranslational processing events in sporadic AAND pathogenesis.

**Protein misprocessing in AANDs becomes irreversible and opens processing pathways associated with cellular membranes.** A key feature of almost all AANDs involving tau, asyn and PrP is that they can occur as both familial and sporadic syndromes, which suggests that a common AAND pathogenesis mechanism must involve self-regenerating alteration in cellular function that is largely irreversible. Initial stages of oligomerization (e.g. dimerization) are most likely insufficient to do this, since all 3 proteins are normally found in a variety of reversible folding states, including low level oligomers, and are ligands for membraneassociated signal transduction kinases that reversibly oligomerize downstream elements (205). However, the binding of these proteins to templating ligands is likely to create higherorder oligomers that could become subject to irreversible structural changes such as proteolytic cleavage (90, 226, 234) and covalent crosslinking (62, 118, 161, 192). The nature of ligands shown to be capable of doing this currently includes 1) the proteins themselves, in the case of PrPSc (175) and mutant asyn (227), 2) polyanions such as heparan sulfate proteoglycans (HSPGs) (106, 225) or RNA (57, 119) and 3) other aggregation-prone proteins (83, 93, 95, 216). Other effectively irreversible changes in the cellular environment may be produced by downstream toxic effects of the initial aggregates, such as protease activation (7, 169), possibly aided by ionophore formation (39, 84, 133), or the recruitment of monomers into existing toxic aggregates via sequestration (6, 120). Endocytosed proteins that bind to the membrane via charge-charge interactions will undergo an acidification of

that overlap the MTBR when not bound to MTs (95, 107, 189). Monomeric asyn exists in both membrane-associated and cytosolic loci, and like tau, can bind to both actin and tubulin (4). As with tau, disease-causing mutations in asyn cause it to preferentially bind to membraneassociated proteins (69). Both membrane and MT-associated asyn have been found to aggregate (4, 139), in some cases forming clusters of microvesicles (195). PrP possesses an aggregation-prone domain (octopeptide repeat) that appears to be oligomerized reversibly during endocytosis. Unlike tau, it also possesses a separate N terminal MT-binding domain (231). All three proteins possess-aggregation-prone domains via which they aggregate resulting in a significant increase in beta sheet structure (156, 187, 231). Deletion analyses of all three proteins show that the removal or inactivation of non-aggregating domains (the N terminus of PrP, the tau N and C termini, the asyn C terminus) may tip this balance toward aggregate formation (1, 38, 112, 226). Post-translational regulation of each protein via phosphorylation may also do this (5, 41, 42, 80), either because it blocks the binding of the aggregation-prone domain to its normal cellular ligand, thereby permitting self assembly (156), or by favoring conversion of soluble oligomer to insoluble higher-order aggregates (187). Familial disease mutations may mimic these changes (13, 66, 80) as well. Overall, while tau, asyn and PrP are capable of aggregate formation and normally interact with both MTs and membrane associated components, the details of how oligomer formation and membrane association is related to normal function vary considerably. A key common feature relevant to the appearance of gain-of-function properties leading to interneuronal propagation in AANDs is the existence of self-binding/assembly capable and assemblyinhibiting domains in each protein that are normally balanced in favor of monomeric functions. This can thus act as a "switch" between normal and abnormal processing pathways which may be mutated to favor oligomer formation in familial AANDs, or alternatively, be "flipped" by derangement of regulatory elements (e.g. kinase/phosphatase and protease activities) that induce these posttranslational processing events in sporadic

**Protein misprocessing in AANDs becomes irreversible and opens processing pathways associated with cellular membranes.** A key feature of almost all AANDs involving tau, asyn and PrP is that they can occur as both familial and sporadic syndromes, which suggests that a common AAND pathogenesis mechanism must involve self-regenerating alteration in cellular function that is largely irreversible. Initial stages of oligomerization (e.g. dimerization) are most likely insufficient to do this, since all 3 proteins are normally found in a variety of reversible folding states, including low level oligomers, and are ligands for membraneassociated signal transduction kinases that reversibly oligomerize downstream elements (205). However, the binding of these proteins to templating ligands is likely to create higherorder oligomers that could become subject to irreversible structural changes such as proteolytic cleavage (90, 226, 234) and covalent crosslinking (62, 118, 161, 192). The nature of ligands shown to be capable of doing this currently includes 1) the proteins themselves, in the case of PrPSc (175) and mutant asyn (227), 2) polyanions such as heparan sulfate proteoglycans (HSPGs) (106, 225) or RNA (57, 119) and 3) other aggregation-prone proteins (83, 93, 95, 216). Other effectively irreversible changes in the cellular environment may be produced by downstream toxic effects of the initial aggregates, such as protease activation (7, 169), possibly aided by ionophore formation (39, 84, 133), or the recruitment of monomers into existing toxic aggregates via sequestration (6, 120). Endocytosed proteins that bind to the membrane via charge-charge interactions will undergo an acidification of

AAND pathogenesis.

their environment that may favor templating interactions and oligomer formation (67) Hyperphosphorylation, cleavage and aggregation of wild type tau isoforms can be induced simply by increasing the concentration of protein that is not MT-associated and thus vulnerable to misfolding (reviewed by 13, 203), causing the release of tau to the cytosol. This kind of release likely accompanies Abeta or axotomy-induced MT loss (32, 101), and thus could account for some of the dependence of tau misprocessing on Abeta generation and traumatic head injury in AD (151, 178).

While aggregate formation is a central event in the misprocessing of aggregation-prone proteins that drive AAND pathogenesis, it remains unclear how it is connected to the diversion of these proteins into the unconventional secretion pathways that might account for the interneuronal transmission of neurofibrillary lesions that appears to occur in these diseases. One possibly relevant property common to tau, asyn and PrP is their tendency to associate with membranes (29, 40, 53, 67, 173, 230, 233) and bind to membrane associated molecules such as HSPGs and fatty acids (44, 222, 225, 232). HSPG binding favors oligomer and fibril formation (52, 91, 120, 225) and may facilitate interactions with APP, which also interacts with HSPGs in cholesterol rich microdomains (lipid rafts 64, 193). Such interactions seem to be favored in AAND pathogenesis, since APP, tau and asyn colocalize with HSPGs in AAND neuropathological lesions (51, 59, 109 197). HSPGs may facilitate interactions between asyn and tau (both localized to elements on the inside of the membrane) and PrPC, which is typically found on the exterior surface attached via a GPI anchor (163, 232) and may themselves mediate transcellular protein movement, as has been suggested by studies of morphogen movement during *Drosophila* development (166), possibly by trapping interacting proteins in the extracellular space (232). Raft-associated interactions appear to be important in disease-associated misprocessing of tau, asyn and PrP mediated via fyn (131, 138, 188, 221), in aggregation (195, 230) and in disruption of signal transduction pathways in CNS dendrites (108, 117). Lipid association also drives oligomer and filament formation of Abeta, tau and PrP (44, 208, 221). In a very recent study by Binder and colleagues (170), a mAb specific for tau oligomer identified the presence of arachidonic acid as one of the requirements for early oligomer formation in cell culture. Similarly, the presence of membrane anchors and raft localization motifs plays an essential role in the development of characteristic lesion morphology of PrP-mediated disease (40); the removal of the GPI anchor has been shown to produce novel syndromes in transgenic models (43), while the removal of all of the multiple raft localization motifs on PrP blocks lesion formation and propagation entirely (16, reviewed in 209).

The relationship between asyn misprocessing and membrane localization in AANDs may be more complex than that of PrP and tau, since some, but not all disease-inducing mutations block raft-asyn association (75). Like PrP and tau, asyn is localized to lipid raft microdomains in presynaptic terminals, where it accumulates in dystrophic neurites associated with Parkinson's Disease and Lewy body dementia (81). Similarly, asyn fibrillization is favored by interactions with unsaturated fatty acids (173) but unlike tau, this is inhibited by saturated fatty acids (233). A particularly intriguing recent finding by Fang et. al. demonstrated a direct link between oligomerization and unconventional secretion in a study showing that higher-order oligomerization can drive exosome-mediated secretion of a wide variety of oligomerized proteins (70). This is particularly interesting given that tau, asyn and PrP are all substrates for fyn and related raft-associated srk tyrosine kinases (136, 138, 188), and that such interactions are associated with AAND pathogenesis (19, 110) and have potentially

What is the Link Between Protein Aggregation and

discussion below).

Interneuronal Lesion Propagation in Neurodegenerative Disease? 13

Abeta in association with endosomes, since tau binds to and may modulate the activity of presenilin 1, an intrinsic membrane protein which serves as the gamma secretase responsible for completing the cleavage of APP to Abeta (207), and is the site of most mutations responsible for autosomal dominant familial AD. Similarly, PrPC is normally endocytosed via a raft specific, flotillin2/clathrin dependent pathway (204), and it has been suggested that the conversion of PrPC to PrPSc, like APP cleavage to Abeta, occurs during endosome formation. There is indeed some evidence that PrP conversion to misfolded PrPSc forms can increase the misprocessing of APP by increasing the activity of the so-called beta secretase, which cleaves APP to a extracellularly released fragment and a "C99" transmembrane domain (14). Asyn interactions with APP have also been shown to greatly increase the level of Abeta secretion from PC12 cells (121). Conversely, the observation that Abeta activates the srk family kinase Abl resulting in tau phosphorylation at sites crucial to disease-associated tau aggregation (34), is also consistent with the possibility that Abeta-

**AAND-associated protein misprocessing may favor exosomal secretion by damaging autophagy-mediated protein turnover mechanisms.** It has long been suspected that alterations in protein turnover mechanisms play a significant role in the cytopathogenesis of AANDs. Under normal conditions, much of the proteolytic turnover of small cytosolic proteins such as tau, asyn and very likely PrP as well is accomplished via the ubiquitin/proteosome pathway (88, 181, 218). The aggregation of these proteins blocks this pathway, apparently due to the steric limitations of the proteosome, resulting in the ubiquitination of tau and Asyn aggregates typically seen in AANDs (158, 220). This provokes the upregulation of the macroautophagy (or simply autophagy) pathway, producing endosomal and lysosomal hypertrophy (35, 36, 165, 167) presumably due to the diversion of proteosome-mediated turnover of AAND associated proteins to the autophagy pathway. It is now becoming clear that aberrant autophagy pathway function is a general phenomenon in AANDs, and increasingly appears that autophagy pathway insufficiency rather than overactivity is the key cytopathological factor (105, 220), reviewed in (153). Since autophagy can function to remove cytosolic debris from cells via lysosomes as well as recycle cytosolic components, this may provide a secretion route for aggregated or misprocessed proteins in AANDs, especially if lysosome-mediated proteolysis is compromised (see Figure 2). Specific inhibition of autophagy combined with tau overexpression results in tau aggregate formation even in cultured neuronal cells, with tau aggregates (104) and toxic cleavage fragments (129) accumulating in lysosomal compartments. Blockade of normal retrograde axonal transport of lysosomes in AD (23) or by specific mutation (178) appears to inhibit autolysosome function indirectly by preventing amphisome-lysosome fusion in the soma, which may favor secretion by diverting incompletely degraded cytoskeletal material into exosomal secretion pathways (Figure 2). Such secretion has been described as "exophagy" in yeast (2). It is quite possible that this kind of diversion into exosomal secretion pathways may apply generally in AANDs, as autophagy disruption also occurs to a significant extent in association with asyn, Abeta, and PrPSc-positive lesions in AANDs (154, 164). Moreover, the tendency of AAND associated proteins to disrupt retrograde transport of autophagosomes (229) could very well promote exosomal secretion of these proteins from ectopic locations in the distal axons, providing a mechanism for the long distance lesion propagation seen in AD (203) and other AANDs (9-11) - see further

induced tau misprocessing may occur in the context of endosome formation.

self-regenerative features (i.e. by activating both the tyrosine kinase and its substrate 179). Such activation can result in fyn-mediated endocytosis via the caveolar pathway (204) or direct release of microvesicles to the extracellular space mediated via the SH4 domain of fyn (or other srk kinases) (34, 210). Regulation of endocytosis and exocytosis in neuronal growth cones by srk family kinases regulates endothelial apical endocytosis (77) and has been described in the marine snail Aplysia (223) suggesting that this is an evolutionarily conserved role for fyn-like Srk family kinases in diverse tissues. Developmental programs requiring high levels of localized membrane addition (e.g. neurite outgrowth) are dependent on the local presence of both srk family kinases and aggregation-prone proteins such as tau (20, 21) asyn (17) or Abeta (172) and are often abnormally reactivated in AANDs (26, 108, 174).

#### **3. Cytopathological features linking aggregation and secretion in ANDDs**

We have discussed the generation of abnormal tau, asyn and PrP oligomers as the most likely proximate cause of neurodegeneration in AANDs and proposed a common set of membrane-associated ligands for these proteins (e.g. HSPGs, signal transduction pathway kinases, fatty acids) which might mediate common aspects of their misprocessing, including their oligomerization, cellular colocalization and diversion into unconventional secretion pathways. Several features peculiar to neuronal AAND pathobiology that seem particularly likely to be important are discussed below.

#### **Misprocessing of APP to Abeta 1-42 in early endosomes**

So far, this discussion has focused the discussion on tau, asyn and PrP as aggregation-prone proteins immediately responsible for downstream neurotoxicity, and has ignored the contribution of aberrant APP misprocessing to Abeta in AAND pathogenesis, despite its well established importance in the pathogenesis of AD in particular (32, 94). However, it has now generally regarded as established that APP misprocessing to Abeta is the initiating event in the pathological cascade leading to AD, even if much of the proximate cytotoxicity driving neurodegeneration is mediated by tau (87, 125, 177, 180). The high cholesterol environment of rafts appears to be necessary for AAND associated misprocessing both in cell culture and in *in situ* AD models (64, 120, 198, 208). Furthermore, Abeta production and toxicity appears to play an important role in AANDs involving asyn and PrP as well as tau (48, 58, 134, 164, 198**)**. Most important for the present analysis is the major site of Abeta production from APP – the early endosome. Endosomal production of Abeta 1-42 RNAi experiments have shown that APP endocytosis requires the raft marker flotillin2 in neurons, and furthermore, that misprocessing of wild type APP to Abeta 1-42 is blocked by inhibition of endocytosis (191), as is the secretion of Abeta to the extracellular space (46). APP is recruited to rafts by the raft-associated tyrosine kinase fyn **(**155**)**, where its interactions with tau, asyn and PrP may play a role in both oligomerization and raft patching **(**163**)** leading to secretion of these proteins via either endocytosis and eventually exosome-mediated release **(**68, 70, 73, 176, 185**)**, or microvesicle shedding **(**145, 163). This similarity should result in extensive opportunities for co-oligomerization between tau, asyn and possibly PrP in endosomal processing, resulting in diversion of oligomerized proteins to the exosome pathway – schematized in Figure 3.

**AAND-associated proteins interact with APP in lipid rafts and may affect A beta production.** There is some reason to believe that tau may influence APP misprocessing to

self-regenerative features (i.e. by activating both the tyrosine kinase and its substrate 179). Such activation can result in fyn-mediated endocytosis via the caveolar pathway (204) or direct release of microvesicles to the extracellular space mediated via the SH4 domain of fyn (or other srk kinases) (34, 210). Regulation of endocytosis and exocytosis in neuronal growth cones by srk family kinases regulates endothelial apical endocytosis (77) and has been described in the marine snail Aplysia (223) suggesting that this is an evolutionarily conserved role for fyn-like Srk family kinases in diverse tissues. Developmental programs requiring high levels of localized membrane addition (e.g. neurite outgrowth) are dependent on the local presence of both srk family kinases and aggregation-prone proteins such as tau (20, 21) asyn (17) or Abeta (172) and are often abnormally reactivated in AANDs (26, 108,

**3. Cytopathological features linking aggregation and secretion in ANDDs** 

We have discussed the generation of abnormal tau, asyn and PrP oligomers as the most likely proximate cause of neurodegeneration in AANDs and proposed a common set of membrane-associated ligands for these proteins (e.g. HSPGs, signal transduction pathway kinases, fatty acids) which might mediate common aspects of their misprocessing, including their oligomerization, cellular colocalization and diversion into unconventional secretion pathways. Several features peculiar to neuronal AAND pathobiology that seem particularly

So far, this discussion has focused the discussion on tau, asyn and PrP as aggregation-prone proteins immediately responsible for downstream neurotoxicity, and has ignored the contribution of aberrant APP misprocessing to Abeta in AAND pathogenesis, despite its well established importance in the pathogenesis of AD in particular (32, 94). However, it has now generally regarded as established that APP misprocessing to Abeta is the initiating event in the pathological cascade leading to AD, even if much of the proximate cytotoxicity driving neurodegeneration is mediated by tau (87, 125, 177, 180). The high cholesterol environment of rafts appears to be necessary for AAND associated misprocessing both in cell culture and in *in situ* AD models (64, 120, 198, 208). Furthermore, Abeta production and toxicity appears to play an important role in AANDs involving asyn and PrP as well as tau (48, 58, 134, 164, 198**)**. Most important for the present analysis is the major site of Abeta production from APP – the early endosome. Endosomal production of Abeta 1-42 RNAi experiments have shown that APP endocytosis requires the raft marker flotillin2 in neurons, and furthermore, that misprocessing of wild type APP to Abeta 1-42 is blocked by inhibition of endocytosis (191), as is the secretion of Abeta to the extracellular space (46). APP is recruited to rafts by the raft-associated tyrosine kinase fyn **(**155**)**, where its interactions with tau, asyn and PrP may play a role in both oligomerization and raft patching **(**163**)** leading to secretion of these proteins via either endocytosis and eventually exosome-mediated release **(**68, 70, 73, 176, 185**)**, or microvesicle shedding **(**145, 163). This similarity should result in extensive opportunities for co-oligomerization between tau, asyn and possibly PrP in endosomal processing, resulting in diversion of oligomerized proteins to the exosome

**AAND-associated proteins interact with APP in lipid rafts and may affect A beta production.** There is some reason to believe that tau may influence APP misprocessing to

174).

likely to be important are discussed below.

pathway – schematized in Figure 3.

**Misprocessing of APP to Abeta 1-42 in early endosomes** 

Abeta in association with endosomes, since tau binds to and may modulate the activity of presenilin 1, an intrinsic membrane protein which serves as the gamma secretase responsible for completing the cleavage of APP to Abeta (207), and is the site of most mutations responsible for autosomal dominant familial AD. Similarly, PrPC is normally endocytosed via a raft specific, flotillin2/clathrin dependent pathway (204), and it has been suggested that the conversion of PrPC to PrPSc, like APP cleavage to Abeta, occurs during endosome formation. There is indeed some evidence that PrP conversion to misfolded PrPSc forms can increase the misprocessing of APP by increasing the activity of the so-called beta secretase, which cleaves APP to a extracellularly released fragment and a "C99" transmembrane domain (14). Asyn interactions with APP have also been shown to greatly increase the level of Abeta secretion from PC12 cells (121). Conversely, the observation that Abeta activates the srk family kinase Abl resulting in tau phosphorylation at sites crucial to disease-associated tau aggregation (34), is also consistent with the possibility that Abetainduced tau misprocessing may occur in the context of endosome formation.

**AAND-associated protein misprocessing may favor exosomal secretion by damaging autophagy-mediated protein turnover mechanisms.** It has long been suspected that alterations in protein turnover mechanisms play a significant role in the cytopathogenesis of AANDs. Under normal conditions, much of the proteolytic turnover of small cytosolic proteins such as tau, asyn and very likely PrP as well is accomplished via the ubiquitin/proteosome pathway (88, 181, 218). The aggregation of these proteins blocks this pathway, apparently due to the steric limitations of the proteosome, resulting in the ubiquitination of tau and Asyn aggregates typically seen in AANDs (158, 220). This provokes the upregulation of the macroautophagy (or simply autophagy) pathway, producing endosomal and lysosomal hypertrophy (35, 36, 165, 167) presumably due to the diversion of proteosome-mediated turnover of AAND associated proteins to the autophagy pathway. It is now becoming clear that aberrant autophagy pathway function is a general phenomenon in AANDs, and increasingly appears that autophagy pathway insufficiency rather than overactivity is the key cytopathological factor (105, 220), reviewed in (153). Since autophagy can function to remove cytosolic debris from cells via lysosomes as well as recycle cytosolic components, this may provide a secretion route for aggregated or misprocessed proteins in AANDs, especially if lysosome-mediated proteolysis is compromised (see Figure 2). Specific inhibition of autophagy combined with tau overexpression results in tau aggregate formation even in cultured neuronal cells, with tau aggregates (104) and toxic cleavage fragments (129) accumulating in lysosomal compartments. Blockade of normal retrograde axonal transport of lysosomes in AD (23) or by specific mutation (178) appears to inhibit autolysosome function indirectly by preventing amphisome-lysosome fusion in the soma, which may favor secretion by diverting incompletely degraded cytoskeletal material into exosomal secretion pathways (Figure 2). Such secretion has been described as "exophagy" in yeast (2). It is quite possible that this kind of diversion into exosomal secretion pathways may apply generally in AANDs, as autophagy disruption also occurs to a significant extent in association with asyn, Abeta, and PrPSc-positive lesions in AANDs (154, 164). Moreover, the tendency of AAND associated proteins to disrupt retrograde transport of autophagosomes (229) could very well promote exosomal secretion of these proteins from ectopic locations in the distal axons, providing a mechanism for the long distance lesion propagation seen in AD (203) and other AANDs (9-11) - see further discussion below).

What is the Link Between Protein Aggregation and

formation.

Interneuronal Lesion Propagation in Neurodegenerative Disease? 15

This schematic illustrates how an aggregation-prone cytosolic protein with alternative membrane-associated ligands (in this case tau and fyn, respectively) might become aberrantly included in one of several possible vesicle trafficking pathways leading to unconventional release if it is released from its normal cytosolic ligand (microtubules) due to disease-associated conditions, which include hyperphosphorylation and microtubule loss, and which can be mimicked by overexpression (142). While tau is shown in this figure, the exosomal secretion pathways for Asyn, A beta, and PrP appear to be similar, especially since misprocessing of each of these proteins favors membrane-associated misprocessing (1) in association with the activation of autophagy (2) combined with disruption of downstream autophagic mechanisms that are necessary for the complete degradation of proteins in the autophagosome (3). While the secretion mechanism that has been identified for any of these proteins is nominally the "classic" exosomal pathway, marked by the presence of exosome– enriched proteins (e.g. Alix), it is likely that exosome secretion occurs via a number of closely related pathways that are associated to a greater or lesser degree with macroautophagy and lysosome-mediated protein turnover. Some of these pathways are indistinguishable from (or even included in) the "classical" exosome pathway (which does not involve lysosomal processing) and can be identified only via the identification of autophagosomal marker proteins (e.g cleaved LC3 (LC3II), cytoskeletal/mitochondrial proteins (COX, tubulins) and/or lysosomal markers (LAMP2, cathepsins) copurifed with exosomal/MVB markers and the AAND-associated protein in question. Involvement of autophagy-associated mechanisms to form a hybrid "exophagy" pathway (2) is particularly likely if misprocessing is associated with aggregate-induced impairment of autophagy, as occurs in AANDs. Secretion pathways are elaborated from Abrahamsen et. al. (2) and Nickel (163). (1) microvesicle shedding– this pathway is driven by srk kinase activity and oligomermediated "patching", but does not involve endocytosis, (2) endosome recycling pathway, (3) classic exosome pathway, (4) non-exosomal autophagosome dumping (commonly seen with tau overexpression models), (5-6) "exophagy" pathways either without autophagolysosomal

**Unconventional secretion may be linked to axonal transport and neuronal polarity defects caused by AAND-associated protein misprocessing.** Another attractive area to look for common links between AAND associated aggregation and secretion of tau, PrP, asyn and APP is that of axonal transport and axonal identity. Each of these proteins is normally axonally localized (22, 127, 150), and the misprocessing of each protein has been shown to disrupt axoplasmic transport in AANDs and AAND models, (157, 162, 199, 200, reviewed in 183), while disruption of dynein/dynactin mediated transport produces a phenocopy of AAND-like syndromes (132). General abnormalities in axonal transport are likely relevant to common neuropathological characteristics of AANDs, such as the anterograde and retrograde propagation of lesions between distant areas of the brain and the disproportionate involvement of large neurons, presumably due to their inherently increased vulnerability to

The reported nature of the disruptions of axonal transport has most often involved the obstruction of axonal transport and accompanied by neurodegeneration via what may be effectively an axotomy syndrome related to synapse loss and growth factor deprivation (157, 195) However, the more interesting possibility, at least with respect to lesion propagation, is that misprocessed tau, asyn or PrP could be itself aberrantly transported along the axon in ways that could account for disease-specific features of AANDs. There is a great deal of circumstantial and correlative evidence in favor of a major role for axonal

mitochondrial misdistribution and growth factor deprivation (160, 190, 206).

Fig. 2. Overview of possible secretion routes for AAND-associated proteins based on current literature Unconventional secretion has now been demonstrated for tau, asyn, PrP and Abeta in various model systems

Fig. 2. Overview of possible secretion routes for AAND-associated proteins based on current literature Unconventional secretion has now been demonstrated for tau, asyn, PrP and Abeta

in various model systems

This schematic illustrates how an aggregation-prone cytosolic protein with alternative membrane-associated ligands (in this case tau and fyn, respectively) might become aberrantly included in one of several possible vesicle trafficking pathways leading to unconventional release if it is released from its normal cytosolic ligand (microtubules) due to disease-associated conditions, which include hyperphosphorylation and microtubule loss, and which can be mimicked by overexpression (142). While tau is shown in this figure, the exosomal secretion pathways for Asyn, A beta, and PrP appear to be similar, especially since misprocessing of each of these proteins favors membrane-associated misprocessing (1) in association with the activation of autophagy (2) combined with disruption of downstream autophagic mechanisms that are necessary for the complete degradation of proteins in the autophagosome (3). While the secretion mechanism that has been identified for any of these proteins is nominally the "classic" exosomal pathway, marked by the presence of exosome– enriched proteins (e.g. Alix), it is likely that exosome secretion occurs via a number of closely related pathways that are associated to a greater or lesser degree with macroautophagy and lysosome-mediated protein turnover. Some of these pathways are indistinguishable from (or even included in) the "classical" exosome pathway (which does not involve lysosomal processing) and can be identified only via the identification of autophagosomal marker proteins (e.g cleaved LC3 (LC3II), cytoskeletal/mitochondrial proteins (COX, tubulins) and/or lysosomal markers (LAMP2, cathepsins) copurifed with exosomal/MVB markers and the AAND-associated protein in question. Involvement of autophagy-associated mechanisms to form a hybrid "exophagy" pathway (2) is particularly likely if misprocessing is associated with aggregate-induced impairment of autophagy, as occurs in AANDs. Secretion pathways are elaborated from Abrahamsen et. al. (2) and Nickel (163). (1) microvesicle shedding– this pathway is driven by srk kinase activity and oligomermediated "patching", but does not involve endocytosis, (2) endosome recycling pathway, (3) classic exosome pathway, (4) non-exosomal autophagosome dumping (commonly seen with tau overexpression models), (5-6) "exophagy" pathways either without autophagolysosomal formation.

**Unconventional secretion may be linked to axonal transport and neuronal polarity defects caused by AAND-associated protein misprocessing.** Another attractive area to look for common links between AAND associated aggregation and secretion of tau, PrP, asyn and APP is that of axonal transport and axonal identity. Each of these proteins is normally axonally localized (22, 127, 150), and the misprocessing of each protein has been shown to disrupt axoplasmic transport in AANDs and AAND models, (157, 162, 199, 200, reviewed in 183), while disruption of dynein/dynactin mediated transport produces a phenocopy of AAND-like syndromes (132). General abnormalities in axonal transport are likely relevant to common neuropathological characteristics of AANDs, such as the anterograde and retrograde propagation of lesions between distant areas of the brain and the disproportionate involvement of large neurons, presumably due to their inherently increased vulnerability to mitochondrial misdistribution and growth factor deprivation (160, 190, 206).

The reported nature of the disruptions of axonal transport has most often involved the obstruction of axonal transport and accompanied by neurodegeneration via what may be effectively an axotomy syndrome related to synapse loss and growth factor deprivation (157, 195) However, the more interesting possibility, at least with respect to lesion propagation, is that misprocessed tau, asyn or PrP could be itself aberrantly transported along the axon in ways that could account for disease-specific features of AANDs. There is a great deal of circumstantial and correlative evidence in favor of a major role for axonal

What is the Link Between Protein Aggregation and

containing these proteins in AANDs (15, 162, 212).

associated with damage to axonal transport and identity mechanisms.

misprocessing and the cytopathogenesis of AANDs is summarized in Figure 3.

The aggregation of the AAND-associated proteins tau, asyn, PrP and APP/Abeta appears to be triggered by one or more post-translational events (cleavage/phosphorylation/ glycosylation) that redistribute charges so as to change the predominant secondary structure from an unfolded/alpha helical pattern to a beta pleated sheet pattern. This change is associated with and driven by familial disease mutations, and may also be favored by the interaction with hydrophobic elements in cellular membranes and/or the binding of perimembranous polyanions (e.g. HSPGs), raising the interesting (and heretofore largely ignored) possibility that aggregate formation in AANDs may depend at least in part on interactions with cellular membranes. The relationship between membrane associated

**4. Summary and conclusions** 

Interneuronal Lesion Propagation in Neurodegenerative Disease? 17

caused by multiple concussions (CTE) poses for the development of neurodegenerative disease, AD in particular (152). Torsion and stretching injury to the brain resulting in occult axotomy of long tracts in the CNS is a major pathological feature in CTE (212), and can occur very close to the soma of the axotomized neuron without killing it (194). Such injury results in the accumulation of axonally transported asyn, APP, PrP and in some cases tau at the proximal axon stump of injured neurons that are reminiscent of axonal swellings

Studies in lower vertebrate (98, 99, 101) and mammalian (45, 144, 182) systems have consistently suggested that polarity loss induced by proximal axotomy could be a mechanism capable of linking axonal injury and the development of AAND-like neuropathology. Proximal axotomy induces ectopic axonlike sprouting (98, 182), the aberrant phosphorylation and missorting of cytoskeletal proteins (99, 100) and thus reproduces key aspects of AD neuritic pathology (26, 107, 174). Missorting of axonal elements such as tau can produce ADlike loss of function degenerative changes in the axon such as synapse loss (54) as well as somatodendritic hyperphosphorylated tau accumulation, which it does even at low levels of overexpression in murine transgenics (30, 86). Interestingly, tau induced neuropathology in tauopathy models produces a number of toxic changes in the dendrites that might shed light on the link between tau misprocessing and interneuronal tau transfer. Tau expression in models causes progressive dendritic degeneration (101) and has specific effects on dendritic MT number (103) and function (61) that resemble both AD pathology (27, 151) and the effects of proximal axotomy (72, 182 , 200, 101). A recent result of particular interest in this context is the recent demonstration by Ittner and co-workers (117) that ectopically localized dendritic tau mediates Abeta toxicity in a transgenic mouse tauopathy model. This finding highlights the possibility that Abeta-mediated tau misprocessing might be initiated by the aberrant juxtaposition of (normally axonal) tau with membrane-associated signal transduction partners that are present in dendrites, causing abnormalities in tau processing that lead to aggregation and eventually secretion, possibly via interactions with synaptic Abeta (71, 135). The dependence of neuronal polarization and axonal outgrowth on normal interactions between tau and localized membrane-associated tyrosine kinases (20, 21, 55) and the sensitivity of dendritic integrity to disruption of dendritic signal transduction pathways by mislocalized PrP (115) suggests that the relocalization of key proteins in AANDs might be a generally applicable mechanism in the misprocessing of AAND proteins by which normal cellular functions and interactions are replaced by abnormal ones by missorting events

transport of vesicle-associated pathogens within the CNS, which closely resembles the movement of infectious prions within the brain (3, 53, 215). Interneuronal movement of HIV has recently been shown to involve PrPC mediation (181) and the binding of a raft-localizing domain that also mediates Abeta and PrPSc localization to rafts (149), lending direct support to the operation of this mechanism in AANDs. The transfer of PrPSc from the gut to the CNS in diseases such as kuru and vCJD involves passage through lymphatic tissues where intercellular movement of both proteins and viral particles occurs via exosomes (215) the unconventional secretion pathway common to asyn, Abeta, PrP and tau (68, 73, 176, 185). Each of these proteins is associated with axonally transported vesicles (71, 76, 123, 127, 140, 141, 150, 230), sometimes in colocalization with (71) or functionally linked with one another (134) in synapses. Moreover, exosome release of PrP has recently been tied to synaptic function with specific neurotransmitters (135), illustrating one mechanism by which specific anterograde and or retrograde pathways might be targeted. The possible operation of common a "prion like" propagation of vesicle-associated misprocessed protein in AAND pathogenesis is further strengthened by the demonstrations that Abeta toxicity can be propagated from the peritoneal cavity to the CNS in a manner similar to ingested prions (65), and that vesicle-associated tau can be dendritically transported and secreted in an *in situ* tauopathy model (123, 141). Finally, numerous studies of LBD, AD and CJD pathology in human patients and/or disease models have now documented the selective colocalization of axonally transported tau and asyn in dystrophic neurites associated with neurofibrillary lesions (neuritic plaques) produced by APP and/or PrP based amyloids (81, 82, 109) suggesting that synergistic interactions associated with vesicle formation (presumably during endocytosis or endosomal processing) may play a role in the lesion overlap and risk synergy so often seen in AAND neuropathology and epidemiology.

#### **Is polarity loss connected to the misprocessing and secretion of tau and other AANDassociated proteins?**

Another aspect of axonal function that is of particular relevance to tauopathies and AD, but may well be involved in any or all of the AANDs under discussion, is the selective effect of tau misprocessing on axonal identity, process outgrowth and synaptic connectivity in AD and non-AD tauopathies. Tau is normally axonally localized in neurons (22) and plays a well-established role in axonal outgrowth (20, 34, 60, 235, reviewed in 91) and in the generation of axonal identity in at least some CNS neuron types (21, 34). Much of this developmental activity of tau involves interactions with the plasma membrane and signal transduction elements rather than MTs (20, 115, 235), and appears to be partly recapitulated in AD and tauopathy pathogenesis with the outgrowth of axonlike processes (neuropil threads). Another aspect of AD pathogenesis that reflects developmental tau function is the loss of neuronal polarity seen in the neuropathology of AD and non-AD tauopathies, which is manifested in a) the progressive movement of tau from the axons to the somatodendritic compartment with the development of neurofibrillary pathology (15, 89) and b) the origination of many tau-positive neuropil threads from the dendrites of neurons in AD (107, 174).

The link between AAND neuropathology and polarity loss accounts for important neuropathological and etiological peculiarities of AD, including: a) the mislocalization and trapping of signal transduction elements essential to the establishment of axonal identity and neuronal polarity, such as CRMP-2 (159, 228) and PAR1/MARK kinase (21), and b) the greatly increased risk (up to 19 fold) that traumatic brain injury (TBI) and chronic injury

transport of vesicle-associated pathogens within the CNS, which closely resembles the movement of infectious prions within the brain (3, 53, 215). Interneuronal movement of HIV has recently been shown to involve PrPC mediation (181) and the binding of a raft-localizing domain that also mediates Abeta and PrPSc localization to rafts (149), lending direct support to the operation of this mechanism in AANDs. The transfer of PrPSc from the gut to the CNS in diseases such as kuru and vCJD involves passage through lymphatic tissues where intercellular movement of both proteins and viral particles occurs via exosomes (215) the unconventional secretion pathway common to asyn, Abeta, PrP and tau (68, 73, 176, 185). Each of these proteins is associated with axonally transported vesicles (71, 76, 123, 127, 140, 141, 150, 230), sometimes in colocalization with (71) or functionally linked with one another (134) in synapses. Moreover, exosome release of PrP has recently been tied to synaptic function with specific neurotransmitters (135), illustrating one mechanism by which specific anterograde and or retrograde pathways might be targeted. The possible operation of common a "prion like" propagation of vesicle-associated misprocessed protein in AAND pathogenesis is further strengthened by the demonstrations that Abeta toxicity can be propagated from the peritoneal cavity to the CNS in a manner similar to ingested prions (65), and that vesicle-associated tau can be dendritically transported and secreted in an *in situ* tauopathy model (123, 141). Finally, numerous studies of LBD, AD and CJD pathology in human patients and/or disease models have now documented the selective colocalization of axonally transported tau and asyn in dystrophic neurites associated with neurofibrillary lesions (neuritic plaques) produced by APP and/or PrP based amyloids (81, 82, 109) suggesting that synergistic interactions associated with vesicle formation (presumably during endocytosis or endosomal processing) may play a role in the lesion overlap and risk

synergy so often seen in AAND neuropathology and epidemiology.

**associated proteins?** 

174).

**Is polarity loss connected to the misprocessing and secretion of tau and other AAND-**

Another aspect of axonal function that is of particular relevance to tauopathies and AD, but may well be involved in any or all of the AANDs under discussion, is the selective effect of tau misprocessing on axonal identity, process outgrowth and synaptic connectivity in AD and non-AD tauopathies. Tau is normally axonally localized in neurons (22) and plays a well-established role in axonal outgrowth (20, 34, 60, 235, reviewed in 91) and in the generation of axonal identity in at least some CNS neuron types (21, 34). Much of this developmental activity of tau involves interactions with the plasma membrane and signal transduction elements rather than MTs (20, 115, 235), and appears to be partly recapitulated in AD and tauopathy pathogenesis with the outgrowth of axonlike processes (neuropil threads). Another aspect of AD pathogenesis that reflects developmental tau function is the loss of neuronal polarity seen in the neuropathology of AD and non-AD tauopathies, which is manifested in a) the progressive movement of tau from the axons to the somatodendritic compartment with the development of neurofibrillary pathology (15, 89) and b) the origination of many tau-positive neuropil threads from the dendrites of neurons in AD (107,

The link between AAND neuropathology and polarity loss accounts for important neuropathological and etiological peculiarities of AD, including: a) the mislocalization and trapping of signal transduction elements essential to the establishment of axonal identity and neuronal polarity, such as CRMP-2 (159, 228) and PAR1/MARK kinase (21), and b) the greatly increased risk (up to 19 fold) that traumatic brain injury (TBI) and chronic injury caused by multiple concussions (CTE) poses for the development of neurodegenerative disease, AD in particular (152). Torsion and stretching injury to the brain resulting in occult axotomy of long tracts in the CNS is a major pathological feature in CTE (212), and can occur very close to the soma of the axotomized neuron without killing it (194). Such injury results in the accumulation of axonally transported asyn, APP, PrP and in some cases tau at the proximal axon stump of injured neurons that are reminiscent of axonal swellings containing these proteins in AANDs (15, 162, 212).

Studies in lower vertebrate (98, 99, 101) and mammalian (45, 144, 182) systems have consistently suggested that polarity loss induced by proximal axotomy could be a mechanism capable of linking axonal injury and the development of AAND-like neuropathology. Proximal axotomy induces ectopic axonlike sprouting (98, 182), the aberrant phosphorylation and missorting of cytoskeletal proteins (99, 100) and thus reproduces key aspects of AD neuritic pathology (26, 107, 174). Missorting of axonal elements such as tau can produce ADlike loss of function degenerative changes in the axon such as synapse loss (54) as well as somatodendritic hyperphosphorylated tau accumulation, which it does even at low levels of overexpression in murine transgenics (30, 86). Interestingly, tau induced neuropathology in tauopathy models produces a number of toxic changes in the dendrites that might shed light on the link between tau misprocessing and interneuronal tau transfer. Tau expression in models causes progressive dendritic degeneration (101) and has specific effects on dendritic MT number (103) and function (61) that resemble both AD pathology (27, 151) and the effects of proximal axotomy (72, 182 , 200, 101). A recent result of particular interest in this context is the recent demonstration by Ittner and co-workers (117) that ectopically localized dendritic tau mediates Abeta toxicity in a transgenic mouse tauopathy model. This finding highlights the possibility that Abeta-mediated tau misprocessing might be initiated by the aberrant juxtaposition of (normally axonal) tau with membrane-associated signal transduction partners that are present in dendrites, causing abnormalities in tau processing that lead to aggregation and eventually secretion, possibly via interactions with synaptic Abeta (71, 135). The dependence of neuronal polarization and axonal outgrowth on normal interactions between tau and localized membrane-associated tyrosine kinases (20, 21, 55) and the sensitivity of dendritic integrity to disruption of dendritic signal transduction pathways by mislocalized PrP (115) suggests that the relocalization of key proteins in AANDs might be a generally applicable mechanism in the misprocessing of AAND proteins by which normal cellular functions and interactions are replaced by abnormal ones by missorting events associated with damage to axonal transport and identity mechanisms.

#### **4. Summary and conclusions**

The aggregation of the AAND-associated proteins tau, asyn, PrP and APP/Abeta appears to be triggered by one or more post-translational events (cleavage/phosphorylation/ glycosylation) that redistribute charges so as to change the predominant secondary structure from an unfolded/alpha helical pattern to a beta pleated sheet pattern. This change is associated with and driven by familial disease mutations, and may also be favored by the interaction with hydrophobic elements in cellular membranes and/or the binding of perimembranous polyanions (e.g. HSPGs), raising the interesting (and heretofore largely ignored) possibility that aggregate formation in AANDs may depend at least in part on interactions with cellular membranes. The relationship between membrane associated misprocessing and the cytopathogenesis of AANDs is summarized in Figure 3.

What is the Link Between Protein Aggregation and

and unconventional secretion.

Interneuronal Lesion Propagation in Neurodegenerative Disease? 19

Hypothetical scheme by which the initial misfolding of AAND-associated proteins (tau asyn, PrP and Abeta) produces intracellular aggregates and other typical AAND cytopathological features in combination with the propagation of this pathology to adjacent, presynaptic and postsynaptic neurons. AAND cytopoathology is produced via a combination of pathological gain of function and loss of function toxicity pathways as indicated. Recent evidence for a common membrane associated misprocessing route that causes the diversion of endocytosed proteins into abnormal vesicle trafficking pathways is highlighted, as it links oligomeri formation with interneuronal transfer and offers multiple opportunities for the colocalization and synergistic interaction (e.g. co-oligomerization) between AANDs at the cellular level necessary to explain the clinical and neuropathological evidence for synergy between AANDs. The classical cytosolic route for aggregate formation is also shown. Novel relationships suggested by recent studies (peach - see text for discussion) that account for key common and/or specific AAND features and could be fruitful foci of future research include links between a) axonal damage, protein mislocation due to polarity loss, and aberrant toxic interactions with dendritic signalling pathways and b) membrane-associated oligomerization and aggregate formation are shown as well, as c) the possible link between damage to axonal transport (failure of normal autophagosome/lysosome colocalization)

Current evidence indicates that initial protein misprocessing in AANDs becomes irreversible due to cleavage and/or crosslinking events that are favored by and occur during the oligomerization/aggregation process and that novel emergent pathological interactions due to polymerization eventually become dominant in the affected neuron, leading both to the dysfunction and death of the aggregate-containing neuron and the spreading of the aggregation tendency to other neurons, where the degenerative cycle is repeated. The retrograde and/or anterograde transfer of membrane associated, oligomerized, toxic protein to other neurons involves axonal propagation of endosome-derived vesicles via transport mechanisms that may have been altered by aggregate-mediated toxicity. Lesion spreading occurs either 1) via a toxic consequence of aberrant neuronal function, such as the loss of transneuronal trophic factor transmission or the increased generation of toxic byproducts of degeneration, or 2) via the actual transfer of misprocessed proteins from one neuron to another. Evidence supporting the latter possibility (that lesion spread occurs via actual protein transfer in AANDs) has accumulated recently, as specific secretion, uptake, transfer and interneuronal toxicity transfer has now been observed for each of these proteins (47, 57, 73, 74, 75, 85, 123, 124, 128, 135, 140 - summarized in Table 1) and a common unconventional secretion pathway (i.e. exosome-mediated secretion) has been identified for PrP and Abeta (73, 176) and (quite recently) asyn and tau (68, 185). A hypothetical common misprocessing

The focus of this discussion has been on the shared characteristics of tau, asyn, PrP and Abeta that could allow each to a) associate with signal transduction elements in membrane raft domains and b) interact and oligomerize in association with elements capable of driving endocytosis (HSPGs, each other, possibly RNA, possibly via acidification driven chargecharge interactions) under circumstances which allow entry to exosomal secretion pathways, possibly via modifications induced in protein turnover mechanisms (autophagy) by aggregate toxicity. In particular, I have focused on whether this hypothesis is consistent with the now voluminous evidence that AANDs involving tau, asyn, PrP and APP misprocessing overlap one another in their etiology and pathogenesis, and whether and how well this hypothesized common link between aggregation and lesion propagation accounts for the

pathway for these proteins in AANDs is schematized in Figure 3.

Fig. 3. Summary of common cellular misprocessing pathways linking aggregation and interneuronal transfer of AAND-associated proteins

Fig. 3. Summary of common cellular misprocessing pathways linking aggregation and

interneuronal transfer of AAND-associated proteins

Hypothetical scheme by which the initial misfolding of AAND-associated proteins (tau asyn, PrP and Abeta) produces intracellular aggregates and other typical AAND cytopathological features in combination with the propagation of this pathology to adjacent, presynaptic and postsynaptic neurons. AAND cytopoathology is produced via a combination of pathological gain of function and loss of function toxicity pathways as indicated. Recent evidence for a common membrane associated misprocessing route that causes the diversion of endocytosed proteins into abnormal vesicle trafficking pathways is highlighted, as it links oligomeri formation with interneuronal transfer and offers multiple opportunities for the colocalization and synergistic interaction (e.g. co-oligomerization) between AANDs at the cellular level necessary to explain the clinical and neuropathological evidence for synergy between AANDs. The classical cytosolic route for aggregate formation is also shown. Novel relationships suggested by recent studies (peach - see text for discussion) that account for key common and/or specific AAND features and could be fruitful foci of future research include links between a) axonal damage, protein mislocation due to polarity loss, and aberrant toxic interactions with dendritic signalling pathways and b) membrane-associated oligomerization and aggregate formation are shown as well, as c) the possible link between damage to axonal transport (failure of normal autophagosome/lysosome colocalization) and unconventional secretion.

Current evidence indicates that initial protein misprocessing in AANDs becomes irreversible due to cleavage and/or crosslinking events that are favored by and occur during the oligomerization/aggregation process and that novel emergent pathological interactions due to polymerization eventually become dominant in the affected neuron, leading both to the dysfunction and death of the aggregate-containing neuron and the spreading of the aggregation tendency to other neurons, where the degenerative cycle is repeated. The retrograde and/or anterograde transfer of membrane associated, oligomerized, toxic protein to other neurons involves axonal propagation of endosome-derived vesicles via transport mechanisms that may have been altered by aggregate-mediated toxicity. Lesion spreading occurs either 1) via a toxic consequence of aberrant neuronal function, such as the loss of transneuronal trophic factor transmission or the increased generation of toxic byproducts of degeneration, or 2) via the actual transfer of misprocessed proteins from one neuron to another. Evidence supporting the latter possibility (that lesion spread occurs via actual protein transfer in AANDs) has accumulated recently, as specific secretion, uptake, transfer and interneuronal toxicity transfer has now been observed for each of these proteins (47, 57, 73, 74, 75, 85, 123, 124, 128, 135, 140 - summarized in Table 1) and a common unconventional secretion pathway (i.e. exosome-mediated secretion) has been identified for PrP and Abeta (73, 176) and (quite recently) asyn and tau (68, 185). A hypothetical common misprocessing pathway for these proteins in AANDs is schematized in Figure 3.

The focus of this discussion has been on the shared characteristics of tau, asyn, PrP and Abeta that could allow each to a) associate with signal transduction elements in membrane raft domains and b) interact and oligomerize in association with elements capable of driving endocytosis (HSPGs, each other, possibly RNA, possibly via acidification driven chargecharge interactions) under circumstances which allow entry to exosomal secretion pathways, possibly via modifications induced in protein turnover mechanisms (autophagy) by aggregate toxicity. In particular, I have focused on whether this hypothesis is consistent with the now voluminous evidence that AANDs involving tau, asyn, PrP and APP misprocessing overlap one another in their etiology and pathogenesis, and whether and how well this hypothesized common link between aggregation and lesion propagation accounts for the

What is the Link Between Protein Aggregation and

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**2** 

**Roles of Microtubules in** 

*Department of Food Science and Nutrition, School of Human Environmental Sciences,* 

*Mukogawa Women's University* 

*Nishinomia Japan* 

**Maintenance of Nerve Cell Networks** 

Kentaro Yomogida, Shumi Yoshida-Yamamoto and Hiroshi Doi

Various higher brain functions such as reflex, memory, emotion, imagination and so on, are supported by complicated neuronal networks. To keep the precise connections of the wires is very important for the central nerve functions. The discovery of neural stem cell provided us many clues to understand the mechanism of neural networking. Now, we know that the networking neurons and the supportive neuroglia cells are yielded from the neural stem cells by regulation of several specific bHLH transcription factors (Sakamoto. M., et al., 2003, Liu, Y. et al., 2004, Parras, C.M. et al., 2002). In these processes, the networking cells project axons to connect the dendrite of counterpart cells precisely. Since the connections between differentiated nerve cells must be kept for the functions, the morphological disruptions lead to some neural disorders. Recent brilliant studies about the microtubule dynamics enhance our understandings of the mechanism of neural network

During early neural development, neural stem cells transform from neuroepithelial cells into radial glial cells (Hatakeyama, J. et al., 2004). The radial glial cell in ventricular zone, projected a long radial glial process to cerebral membrane, self-renews and produces an immature neuron (Miyata T., et al., 2004). The immature neuron transforms into multipolar cell with many process containing actin fibers. Cyclin-dependent kinase 5 (Cdk5) regulates the formation of these process and the transform of multipolar cell into bipolar locomotion cell having a leading process (Kawauchi, T., et al., 2006). The bipolar locomotion cells move to the precise layer along the radial glial fiber, and differentiate into mature networking neurons. To construct an ordered six layer structure of mammalian cerebral cortex, the locomotion of these neural stem cell linage cells is strictly regulated by Reelin signal pathway affecting microtubule dynamics (Liu, J.S., 2011). So, some disorders of the microtubule

**1.1 Neural networking and neural stem cell during neural development** 

regulation can cause structural errors of neural network development.

**1. Introduction** 

**Recent topics of neural networking studies** 

maintenance and the disorders.


### **Roles of Microtubules in Maintenance of Nerve Cell Networks**

Kentaro Yomogida, Shumi Yoshida-Yamamoto and Hiroshi Doi

*Department of Food Science and Nutrition, School of Human Environmental Sciences, Mukogawa Women's University Nishinomia Japan* 

#### **1. Introduction**

34 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

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affects bilayer structure, stability, and fibril formation. *J. Biol. Chem.* 278 (41): 40186–

E, Novak M. Truncated tau from sporadic Alzheimer's disease suffices to drive

#### **Recent topics of neural networking studies**

Various higher brain functions such as reflex, memory, emotion, imagination and so on, are supported by complicated neuronal networks. To keep the precise connections of the wires is very important for the central nerve functions. The discovery of neural stem cell provided us many clues to understand the mechanism of neural networking. Now, we know that the networking neurons and the supportive neuroglia cells are yielded from the neural stem cells by regulation of several specific bHLH transcription factors (Sakamoto. M., et al., 2003, Liu, Y. et al., 2004, Parras, C.M. et al., 2002). In these processes, the networking cells project axons to connect the dendrite of counterpart cells precisely. Since the connections between differentiated nerve cells must be kept for the functions, the morphological disruptions lead to some neural disorders. Recent brilliant studies about the microtubule dynamics enhance our understandings of the mechanism of neural network maintenance and the disorders.

#### **1.1 Neural networking and neural stem cell during neural development**

During early neural development, neural stem cells transform from neuroepithelial cells into radial glial cells (Hatakeyama, J. et al., 2004). The radial glial cell in ventricular zone, projected a long radial glial process to cerebral membrane, self-renews and produces an immature neuron (Miyata T., et al., 2004). The immature neuron transforms into multipolar cell with many process containing actin fibers. Cyclin-dependent kinase 5 (Cdk5) regulates the formation of these process and the transform of multipolar cell into bipolar locomotion cell having a leading process (Kawauchi, T., et al., 2006). The bipolar locomotion cells move to the precise layer along the radial glial fiber, and differentiate into mature networking neurons. To construct an ordered six layer structure of mammalian cerebral cortex, the locomotion of these neural stem cell linage cells is strictly regulated by Reelin signal pathway affecting microtubule dynamics (Liu, J.S., 2011). So, some disorders of the microtubule regulation can cause structural errors of neural network development.

Roles of Microtubules in Maintenance of Nerve Cell Networks 37

J.B., and Borisy, G.G., 1975), and calcium inhibits microtubule assembly. The assembly kinetics of the microtubule protein is altered by the ionic strength, temperature, and magnesium ion but not by the pH (Barton, J.S., et al., 1987). Timasheff and Grisham have reviewed in detail an *in vitro* assembly process from tubulin and the mechanism of microtubule assembly

On the growth of microtubules, Mitchison and Kirshner (1984) proposed a behavior called dynamic instability. Horio and Hotani (1986) confirmed alternate phases of growth and

Post-translational modifications of tubulin building generate functional diversity of microtubules. Hammond et al. (2008) have reported that tubulin modifications influence microtubule-associated proteins, such as severing proteins, plus-end tracking proteins, and molecular motors. In this way, tubulin modifications play an important role in regulating microtubule properties, such as stability and structure, as well as microtubule-based functions, such as ciliary beating, cell division, and intracellular tracking (Hammond, J.H., et

Tubulin used in our experiments was prepared from bovine brain by the modified procedure of Lee et al. (Weisenberg, R.C., and Timasheff, S.N., 1970; Lee, J.C., et al., 1973; Na, G.C. and Timasheff, S.N., 1981). Microtubule assembly was monitored by turbidity at 350 nm using a spectrophotometer with a recorder. GTP hydrolysis accompanies microtubule formation. GTP bound at an exchangeable site is hydrolyzed. GTPase activity was evaluated by the measurement of GDP produced using HPLC with an ODS column (Seckler, R., et al., 1990). We examined the effects of the magnesium ion on microtubule assembly and the GTPase activity of tubulin. GTPase activity was clearly observed at a 2 mM magnesium ion concentration, while the formation of microtubules under the same conditions was not observed (Doi, H., et al., 1991). Microtubule assembly and GTPase activity were examined in the presence of 0.1 mM calcium ion as well. GTPase activity was apparently observed at 2min after heating at 37 °C, while there was no turbidity. The results described above indicate that the GTPase activity of tubulin occurs before microtubule assembly. The facts support the results of O'Brien et al. (O'Brien, E.T., et al., 1987) rather

**3. Some evidence of nerve cell dysfunction caused by the microtubules** 

Here, we present some evidence of nerve cell dysfunction caused by the microtubules disorder. Our series of experiments using a neural cell line PC12 demonstrated that the oxidative damage of microtubules causes the morphological abnormality cell (Yamanaka,

In neurons, microtubules play a variety of roles in brain function. As in many other cells, microtubules form organized structures within a cell that can act as structural scaffolds. With respect to specific for neuron, microtubules have three functions. First, the stabilization of microtubules is sufficient to induce axon formation during neuronal development, and

(Timasheff, S.N., and Grisham, L.M., 1980).

than those of Carlier (Carlier, M.-F., 1982).

**3.1 Function of microtubules in neuronal cells** 

al., 2008).

**disorder** 

Y., et al., 2008).

shrinkage of microtubule assembly using a light optical technique.

**2.3 Relationship between microtubule assembly and GTP hydrolysis** 

#### **1.2 Maintenance of neural network in adult hippocampus**

In adult hippocampus, it was shown that neurogenesis also occurs constantly (Eriksson, PS. et al. 1998). Like early neural development, these new neurons are produced from radial glia cells (Fukuda, S., et al., 2003). Since the neurogenesis and activity-dependent synaptic plasticity are accelerated by long term learned behavior (Bruel-Jungernab, E., et al., 2006), it can participate in functional remodeling of neural networks during the formation of memories. A recent interesting study indicates microtubule transport systems in the dendrites play important roles in maintenance of the synaptic plasticity (Okada, D., et al., 2009). It suggests that the healthy microtubule kinetics is needed to maintenance the neural networking during the formation of memories. What microtubule is all about?

#### **2. The kinetics of microtubules and cell functions**

#### **2.1 Function of microtubules**

The cytoskeleton is the essential infrastructure of all cells; it consists of microtubules, actin microfilament, and intermediate filaments. Microtubules are a major component of the cytoskeleton and form a highly organized network of intermingled filaments in eukaryotic cells. Microtubules are important components of several subcellular structures, including the mitotic apparatus, cilia, flagella, and neurons. Microtubules are fundamentally composed of a protein called tubulin. Tubulin is made of α- and β-tubulin. The molecular weight of each is about 50 kDa. There are many microtubule-associated proteins (MAPs) (Wade, R.H., 2009) in addition to the tau protein, which contributes to the formation of microtubules. The tau protein is enriched in axons. Two types of high-molecular-weight MAPs (200-300 kDa) and the lower-molecular-weight ones (~55 kDa) have been isolated from the brain. For example, MAP2 is found in the cell body and dendrites. In addition, microtubules interact with many proteins, including motor proteins, such as kinesin and dynein.

Microtubules play many roles in cellular processes, such as cell division, cell motility, and morphogenesis, and they are required for brain function. Purich and Kristofferson (1984) have reviewed microtubule assembly. Wade has described the function of the cell division of microtubules in detail (Wade, R.H., 2009). The motor proteins kinesin and dynein use microtubules as pathways for transport and are also involved in cell division. Microtubules organize the spatial distribution of organelles. Actin and microtubule cytoskeletons determine cell shape and polarity during morphogenesis and promote stable cell-cell and cell-matrix adhesions through their interactions with cadherins and integrins, respectively (Hall, A., 2009).

#### **2.2 Polymerization of tubulin: Microtubule assembly**

Tubulin is widely distributed in eukaryotic cells, and the specific self-assembly of tubulin results in microtubule formation. Microtubules are hollow tubes approximately 25nm in diameter. Tubulin is composed of two subunits of α- and β-tubulins that bind one mole of guanosine triphosphate (GTP) each. GTP binding to α-tubulin is present at the nonexchangeable site in α-tubulin, and that binding to β-tubulin is at an exchangeable site in βtubulin. Some reports have focused on microtubule assembly kinetics (Detrich, et al., 1985; Barton, J.S., et al., 1987; Caplow, M., and Shanks, J., 1990). The polymerization mechanism of tubulin is fundamentally due to the polymer self-assembly theory of Oosawa and Kasai (1962). Magnesium is required for tubulin polymerization (Weisenberg, R.C., 1972; Olmsted

In adult hippocampus, it was shown that neurogenesis also occurs constantly (Eriksson, PS. et al. 1998). Like early neural development, these new neurons are produced from radial glia cells (Fukuda, S., et al., 2003). Since the neurogenesis and activity-dependent synaptic plasticity are accelerated by long term learned behavior (Bruel-Jungernab, E., et al., 2006), it can participate in functional remodeling of neural networks during the formation of memories. A recent interesting study indicates microtubule transport systems in the dendrites play important roles in maintenance of the synaptic plasticity (Okada, D., et al., 2009). It suggests that the healthy microtubule kinetics is needed to maintenance the neural

The cytoskeleton is the essential infrastructure of all cells; it consists of microtubules, actin microfilament, and intermediate filaments. Microtubules are a major component of the cytoskeleton and form a highly organized network of intermingled filaments in eukaryotic cells. Microtubules are important components of several subcellular structures, including the mitotic apparatus, cilia, flagella, and neurons. Microtubules are fundamentally composed of a protein called tubulin. Tubulin is made of α- and β-tubulin. The molecular weight of each is about 50 kDa. There are many microtubule-associated proteins (MAPs) (Wade, R.H., 2009) in addition to the tau protein, which contributes to the formation of microtubules. The tau protein is enriched in axons. Two types of high-molecular-weight MAPs (200-300 kDa) and the lower-molecular-weight ones (~55 kDa) have been isolated from the brain. For example, MAP2 is found in the cell body and dendrites. In addition, microtubules interact

Microtubules play many roles in cellular processes, such as cell division, cell motility, and morphogenesis, and they are required for brain function. Purich and Kristofferson (1984) have reviewed microtubule assembly. Wade has described the function of the cell division of microtubules in detail (Wade, R.H., 2009). The motor proteins kinesin and dynein use microtubules as pathways for transport and are also involved in cell division. Microtubules organize the spatial distribution of organelles. Actin and microtubule cytoskeletons determine cell shape and polarity during morphogenesis and promote stable cell-cell and cell-matrix adhesions through their interactions with cadherins and integrins, respectively (Hall, A.,

Tubulin is widely distributed in eukaryotic cells, and the specific self-assembly of tubulin results in microtubule formation. Microtubules are hollow tubes approximately 25nm in diameter. Tubulin is composed of two subunits of α- and β-tubulins that bind one mole of guanosine triphosphate (GTP) each. GTP binding to α-tubulin is present at the nonexchangeable site in α-tubulin, and that binding to β-tubulin is at an exchangeable site in βtubulin. Some reports have focused on microtubule assembly kinetics (Detrich, et al., 1985; Barton, J.S., et al., 1987; Caplow, M., and Shanks, J., 1990). The polymerization mechanism of tubulin is fundamentally due to the polymer self-assembly theory of Oosawa and Kasai (1962). Magnesium is required for tubulin polymerization (Weisenberg, R.C., 1972; Olmsted

networking during the formation of memories. What microtubule is all about?

with many proteins, including motor proteins, such as kinesin and dynein.

**2.2 Polymerization of tubulin: Microtubule assembly** 

**1.2 Maintenance of neural network in adult hippocampus** 

**2. The kinetics of microtubules and cell functions** 

**2.1 Function of microtubules** 

2009).

J.B., and Borisy, G.G., 1975), and calcium inhibits microtubule assembly. The assembly kinetics of the microtubule protein is altered by the ionic strength, temperature, and magnesium ion but not by the pH (Barton, J.S., et al., 1987). Timasheff and Grisham have reviewed in detail an *in vitro* assembly process from tubulin and the mechanism of microtubule assembly (Timasheff, S.N., and Grisham, L.M., 1980).

On the growth of microtubules, Mitchison and Kirshner (1984) proposed a behavior called dynamic instability. Horio and Hotani (1986) confirmed alternate phases of growth and shrinkage of microtubule assembly using a light optical technique.

Post-translational modifications of tubulin building generate functional diversity of microtubules. Hammond et al. (2008) have reported that tubulin modifications influence microtubule-associated proteins, such as severing proteins, plus-end tracking proteins, and molecular motors. In this way, tubulin modifications play an important role in regulating microtubule properties, such as stability and structure, as well as microtubule-based functions, such as ciliary beating, cell division, and intracellular tracking (Hammond, J.H., et al., 2008).

#### **2.3 Relationship between microtubule assembly and GTP hydrolysis**

Tubulin used in our experiments was prepared from bovine brain by the modified procedure of Lee et al. (Weisenberg, R.C., and Timasheff, S.N., 1970; Lee, J.C., et al., 1973; Na, G.C. and Timasheff, S.N., 1981). Microtubule assembly was monitored by turbidity at 350 nm using a spectrophotometer with a recorder. GTP hydrolysis accompanies microtubule formation. GTP bound at an exchangeable site is hydrolyzed. GTPase activity was evaluated by the measurement of GDP produced using HPLC with an ODS column (Seckler, R., et al., 1990). We examined the effects of the magnesium ion on microtubule assembly and the GTPase activity of tubulin. GTPase activity was clearly observed at a 2 mM magnesium ion concentration, while the formation of microtubules under the same conditions was not observed (Doi, H., et al., 1991). Microtubule assembly and GTPase activity were examined in the presence of 0.1 mM calcium ion as well. GTPase activity was apparently observed at 2min after heating at 37 °C, while there was no turbidity. The results described above indicate that the GTPase activity of tubulin occurs before microtubule assembly. The facts support the results of O'Brien et al. (O'Brien, E.T., et al., 1987) rather than those of Carlier (Carlier, M.-F., 1982).

#### **3. Some evidence of nerve cell dysfunction caused by the microtubules disorder**

Here, we present some evidence of nerve cell dysfunction caused by the microtubules disorder. Our series of experiments using a neural cell line PC12 demonstrated that the oxidative damage of microtubules causes the morphological abnormality cell (Yamanaka, Y., et al., 2008).

#### **3.1 Function of microtubules in neuronal cells**

In neurons, microtubules play a variety of roles in brain function. As in many other cells, microtubules form organized structures within a cell that can act as structural scaffolds. With respect to specific for neuron, microtubules have three functions. First, the stabilization of microtubules is sufficient to induce axon formation during neuronal development, and

Roles of Microtubules in Maintenance of Nerve Cell Networks 39

(A) (B)

neuronal-like cells within 3days

**3.3 The oxidative damage to PC12 cells** 

system may be useful as a model for AD brain cells.

Fig. 1. Phase contrast microscopic observation of PC12 cells (A)before differentiation (B) after differentiation. For differentiation, the undiffereantiated cells were treated with 100 ng/mL NGF. NGF induced the apparent morphological transformation of PC12 cells into

Many studies have demonstrated that lipid peroxidants are present in the AD brain (Keller. J. N., and Mattson, M. D., 1998; Markesbery, W. R., and Carney, J. M., 1999;). We tried to verify whether lipid peroxidation was induced in PC12 cells by exogenously added phosphatidylcholine hydroperoxides (PCOOH) which is a primary product of lipid peroxidation. Lipid peroxidation was measured according to the method of Hedley and Chow (1992), which utilizes time-resolved flow cytometry. Table 1 shows the fluorescence of undifferentiated and differentiated cells before and after exposure to PCOOH for 24 or 48 h. Compared with that of undifferentiated cells, the fluorescence of differentiated cells was significantly decreased in the presence of 100 μM PCOOH for 48 h (P < 0.05). The fluorescence of undifferentiated cells exposed to the same concentration of PCOOH was slightly but not significantly affected. These results suggest that PCOOH induces membrane lipid peroxidation in PC12 cells before and after differentiation. The levels of peroxidation were higher in the membranes of differentiated cells than in those of undifferentiated cells. It is likely that differentiated cells are more sensitive to oxidative stress. Considering that lipid peroxidation was certainly induced in differentiated PC12 cells, this experimental

Conc. of PCOOH Before Differentiation (%) After Differentiation (%) (μM) 24h 48h 24h 48h 0 100 100 100 100 100 88.8 79.2 84.9 63.8\*

Table 1. Relative fluorescent intensity of *cis*-parinaric acid bound by the cell membrane

Neurites consist mainly of microtubules, whose function is significantly based on the ability of tubulin to polymerize and depolymerize. To examine the effect of PCOOH on microtubule formation from tubulin, we measured the GTPase activity of PC12 cells. GTPase activity is an indicator of microtubule formation and, therefore, provides the degree of microtubule assembly (O'Brien, E.T. et al., 1987; Seckler, R., et al., 1990; Doi, H., et al.,

they act as signal molecules for initial neuronal polarization (Witte H et al., 2008). Second, the development of dendritic spines that are major sites of excitatory synaptic input is regulated by microtubules (Gu, J., et al., 2008). Third, microtubules participate in the trafficking of synaptic cargo molecules that are essential for synapse formation, function, and plasticity. Cargos are transported between axons and dendrites mediated by motor proteins moving along microtubules to their plus or minus ends (Hirokawa N and Takemura, R., 2005). The motor proteins are the minus-end directed dynein and plus-end directed kinesins (Schliwa, M., 2003, Vale, R.D., 2003). On the other hand, several studies have shown the importance of the actin-based transport mechanism at excitatory synapses. Actin, which is abundant in highly dynamic structures, such as growth cones and dendritic spines, receives the cargo following passage of the microtubules. Neuronal transmission is achieved partly by collaboration of both microtubules and actins.

As reported above, it is clear that microtubules play an essential role in neuronal development, function, and transmission. Disruption of neuronal microtubules means functional failure of brain. Indeed, microtubule dysfunction and impairment of neurotransmission were observed in neurodegenerative diseases, such as Alzheimer's disease (refer to Chapter 4) and Parkinson's disease. The Alzheimer brain is characterized by the presence of aberrant amyloid plaques, neurofibrillary tangles, and alpha-synuclein. Neurofibrillary tangles are composed of paired helical filaments made from abnormally formed tau protein. In the normal brain, tau binds to microtubules and, thereby, stabilizes neuron structure and promotes tubulin assembly into microtubules. However, hyperphosphorylation of tau is assumed to be the cause of the formation of paired helical filaments; namely, it could result in the self-assembly of tangles of paired helical filaments and straight filaments. α-Synuclein is a microtubule-associated protein (MAP) that is colocalized with tubulin in Lewy's bodies. The deposition of α-synuclein as fibrillary aggregates in neurons or glial cells is observed in a Lewy variant of Alzheimer's disease (Spillantini, M.G., et al., 1997) and Parkinson's disease (Lücking, C.B. and Brice, A., 2000). It has been reported that α-synuclein could promote tubulin polymerization in microtubules (Alim MA et al., 2004), whereas other studies have indicated that α-synuclein inhibits tubulin polymerization (Chen L et al., 2007, Zhou RM et al., 2010).

Cumulative evidence suggests that neurodegenerative diseases are associated with neuronal cytoskeletal alterations. These findings suggest that elucidating the biology of the cytoskeleton could be a target for drug therapy.

#### **3.2 The PC12 cells as a model for neurite outgrowth**

To study the behavior of the neuronal microtubules, PC12 would be an appropriate cultured cell line. It can enable us to conduct a visual assessment of neurite behavior from formation to disruption.

The adrenal pheochromocytoma (PC12) cell line has been well studied as a model for neurite outgrowth. It was originally isolated from a tumor in the adrenal medulla of a rat in 1976 (Greene, L. A., and Tischler, A. S., 1976). One of the main characteristics of PC12 cells is to differentiate into sympathetic neuron-like phenotypes in response to nerve growth factor (NGF) (Figure 1A, 1B). The mechanism of NGF-induced neuronal differentiation has not been fully elucidated; however, it has been reported that the regulator of G-protein signaling (RGS) proteins associates TrkA with activated signaling proteins of the Ras/pErk1/2 pathway (Willard, M.D., et al., 2007, Nusser, N., et al., 2002).

they act as signal molecules for initial neuronal polarization (Witte H et al., 2008). Second, the development of dendritic spines that are major sites of excitatory synaptic input is regulated by microtubules (Gu, J., et al., 2008). Third, microtubules participate in the trafficking of synaptic cargo molecules that are essential for synapse formation, function, and plasticity. Cargos are transported between axons and dendrites mediated by motor proteins moving along microtubules to their plus or minus ends (Hirokawa N and Takemura, R., 2005). The motor proteins are the minus-end directed dynein and plus-end directed kinesins (Schliwa, M., 2003, Vale, R.D., 2003). On the other hand, several studies have shown the importance of the actin-based transport mechanism at excitatory synapses. Actin, which is abundant in highly dynamic structures, such as growth cones and dendritic spines, receives the cargo following passage of the microtubules. Neuronal transmission is

As reported above, it is clear that microtubules play an essential role in neuronal development, function, and transmission. Disruption of neuronal microtubules means functional failure of brain. Indeed, microtubule dysfunction and impairment of neurotransmission were observed in neurodegenerative diseases, such as Alzheimer's disease (refer to Chapter 4) and Parkinson's disease. The Alzheimer brain is characterized by the presence of aberrant amyloid plaques, neurofibrillary tangles, and alpha-synuclein. Neurofibrillary tangles are composed of paired helical filaments made from abnormally formed tau protein. In the normal brain, tau binds to microtubules and, thereby, stabilizes neuron structure and promotes tubulin assembly into microtubules. However, hyperphosphorylation of tau is assumed to be the cause of the formation of paired helical filaments; namely, it could result in the self-assembly of tangles of paired helical filaments and straight filaments. α-Synuclein is a microtubule-associated protein (MAP) that is colocalized with tubulin in Lewy's bodies. The deposition of α-synuclein as fibrillary aggregates in neurons or glial cells is observed in a Lewy variant of Alzheimer's disease (Spillantini, M.G., et al., 1997) and Parkinson's disease (Lücking, C.B. and Brice, A., 2000). It has been reported that α-synuclein could promote tubulin polymerization in microtubules (Alim MA et al., 2004), whereas other studies have indicated that α-synuclein inhibits tubulin polymerization (Chen L et al., 2007,

Cumulative evidence suggests that neurodegenerative diseases are associated with neuronal cytoskeletal alterations. These findings suggest that elucidating the biology of the cytoskeleton

To study the behavior of the neuronal microtubules, PC12 would be an appropriate cultured cell line. It can enable us to conduct a visual assessment of neurite behavior from formation

The adrenal pheochromocytoma (PC12) cell line has been well studied as a model for neurite outgrowth. It was originally isolated from a tumor in the adrenal medulla of a rat in 1976 (Greene, L. A., and Tischler, A. S., 1976). One of the main characteristics of PC12 cells is to differentiate into sympathetic neuron-like phenotypes in response to nerve growth factor (NGF) (Figure 1A, 1B). The mechanism of NGF-induced neuronal differentiation has not been fully elucidated; however, it has been reported that the regulator of G-protein signaling (RGS) proteins associates TrkA with activated signaling proteins of the

Ras/pErk1/2 pathway (Willard, M.D., et al., 2007, Nusser, N., et al., 2002).

achieved partly by collaboration of both microtubules and actins.

Zhou RM et al., 2010).

to disruption.

could be a target for drug therapy.

**3.2 The PC12 cells as a model for neurite outgrowth** 

Fig. 1. Phase contrast microscopic observation of PC12 cells (A)before differentiation (B) after differentiation. For differentiation, the undiffereantiated cells were treated with 100 ng/mL NGF. NGF induced the apparent morphological transformation of PC12 cells into neuronal-like cells within 3days

#### **3.3 The oxidative damage to PC12 cells**

Many studies have demonstrated that lipid peroxidants are present in the AD brain (Keller. J. N., and Mattson, M. D., 1998; Markesbery, W. R., and Carney, J. M., 1999;). We tried to verify whether lipid peroxidation was induced in PC12 cells by exogenously added phosphatidylcholine hydroperoxides (PCOOH) which is a primary product of lipid peroxidation. Lipid peroxidation was measured according to the method of Hedley and Chow (1992), which utilizes time-resolved flow cytometry. Table 1 shows the fluorescence of undifferentiated and differentiated cells before and after exposure to PCOOH for 24 or 48 h. Compared with that of undifferentiated cells, the fluorescence of differentiated cells was significantly decreased in the presence of 100 μM PCOOH for 48 h (P < 0.05). The fluorescence of undifferentiated cells exposed to the same concentration of PCOOH was slightly but not significantly affected. These results suggest that PCOOH induces membrane lipid peroxidation in PC12 cells before and after differentiation. The levels of peroxidation were higher in the membranes of differentiated cells than in those of undifferentiated cells. It is likely that differentiated cells are more sensitive to oxidative stress. Considering that lipid peroxidation was certainly induced in differentiated PC12 cells, this experimental system may be useful as a model for AD brain cells.


Table 1. Relative fluorescent intensity of *cis*-parinaric acid bound by the cell membrane

Neurites consist mainly of microtubules, whose function is significantly based on the ability of tubulin to polymerize and depolymerize. To examine the effect of PCOOH on microtubule formation from tubulin, we measured the GTPase activity of PC12 cells. GTPase activity is an indicator of microtubule formation and, therefore, provides the degree of microtubule assembly (O'Brien, E.T. et al., 1987; Seckler, R., et al., 1990; Doi, H., et al.,

Roles of Microtubules in Maintenance of Nerve Cell Networks 41

To visualize PCOOH-induced damage to the tubulin, we performed immunofluorescence microscopy using an antibody to monoclonal mouse anti-α-tubulin clone B-5-1-2. Undifferentiated or differentiated cells were individually cultured with 250 μM of PCOOH for 6 h. After that, the cells were stained by the antibody to monoclonal mouse anti-αtubulin clone B-5-1-2 and the antibody to Cy3-conjugated sheep anti-mouse IgG. As shown in the photographs in Figure 4A, the undifferentiated cells looked like grape clusters, and the cell shape was clear. However, after exposure to PCOOH, the cell shape was drastically changed (Figure 4B), becoming too vague to identify. The fluorescence emitted from cells was weakened. PCOOH may have induced cell shape alteration by the degradation of tubulin, which was more marked in differentiated cells than in undifferentiated cells. Although the extended neurites were observed clearly in the absence of PCOOH (Figure 4C), they disappeared when exposed in PCOOH for 6h (Figure 4D). The shape of the small cell was vague, as it was in undifferentiated cells, and the fluorescence emitted from cells became extremely weak. The fact that neurites composed of microtubules are easy to be

injured may account for the higher vulnerability of differentiated cells.

effective strategy to prevent some neurodegenerative diseases.

**neural malnutrition** 

malnutrition.

 (A) (B) (C) (D) Fig. 4. Fluorescence microscopic observation of cells after tubulin antibody staining. Representative fields are shown: undifferentiated cells before (A) and after exposure to PCOOH for 6h (B), and differentiated cells before (C) and after exposure to PCOOH for 6h (D) Furthermore, we tried to verify that the tubulin depolymerization induced by PCOOH could be attenuated by antioxidant. Differentiated cells were cultivated with 5 µM retinol or ascorbic acid beforehand and then exposed to PCOOH. The GTPase activity of cell extracts derived from cells treated with retinol was three-fold higher than that of untreated control cells (Figure 5). Incorporation of antioxidants in cells before exposure to PCOOH protected tubulin depolymerization. This experimental data might lead to the development of an

**4. Ageing of central nerve system and the microtubules disorder caused by** 

As people get older, the brain functions decline in varying degree. Although the causes are still unknown, the neurogenesis in hippocampus is decreased dramatically with ageing (Cameron, HA., et al., 1999). The other hand, we can detect neurofibrillary tangles in aged entorhinal cortex or brain cortex of neurodegenerative disorder. These tangle formation are concerned with aggregation of tau, which is a microtubule binding protein. In this section, we will discuss the factors determining the ageing-related neural functional decline in Alzheimer's disease from the aspect of the axonal microtubules disorder caused by neural

1991). The specific activity of GTPase derived from differentiated cells was significantly decreased in the presence of 50 μM PCOOH (P < 0.01) (Figure 2). In the case of exposure to 100 μM PCOOH, the value was decreased by one-tenth compared to that in the absence of PCOOH. In undifferentiated cells, the specific activity of GTPase decreased by half in the presence of 50 μM PCOOH (Figure 3). The difference in sensitivity might be due to the presence or absence of neurites. Although GTP hydrolysis accompanies the polymerization reaction (Doi, H., et al., 1991), GTP resynthesis does not occur in the reverse reaction of depolymerization (David-Pfeuty, T., et al., 1977). Thus, PCOOH disrupts existing microtubules and inhibits new microtubule formation from tubulin.

Fig. 2. GTPase-specific activity of the differentiated cells incubated with PCOOH at various concentrations for 24 h. The data represent means ± SD, \*\*P < 0.01 compared with the control value

Fig. 3. GTPase-specific activity of the undifferentiated cells in the same condition as that described in Figure 2. The data represent means ± SD, \*P < 0.05 compared with the control value

1991). The specific activity of GTPase derived from differentiated cells was significantly decreased in the presence of 50 μM PCOOH (P < 0.01) (Figure 2). In the case of exposure to 100 μM PCOOH, the value was decreased by one-tenth compared to that in the absence of PCOOH. In undifferentiated cells, the specific activity of GTPase decreased by half in the presence of 50 μM PCOOH (Figure 3). The difference in sensitivity might be due to the presence or absence of neurites. Although GTP hydrolysis accompanies the polymerization reaction (Doi, H., et al., 1991), GTP resynthesis does not occur in the reverse reaction of depolymerization (David-Pfeuty, T., et al., 1977). Thus, PCOOH disrupts existing microtubules

Concn. of PCOOH (μM)

Concn. of PCOOH (μM)

Fig. 3. GTPase-specific activity of the undifferentiated cells in the same condition as that described in Figure 2. The data represent means ± SD, \*P < 0.05 compared with the control

0 50 100

Fig. 2. GTPase-specific activity of the differentiated cells incubated with PCOOH at various concentrations for 24 h. The data represent means ± SD, \*\*P < 0.01 compared with the

0 50 100

and inhibits new microtubule formation from tubulin.

GTPase Specific Activity (nmol/mg/min)

control value

7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0

GTPase Specific Activity (nmol/mg/min)

value

9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 To visualize PCOOH-induced damage to the tubulin, we performed immunofluorescence microscopy using an antibody to monoclonal mouse anti-α-tubulin clone B-5-1-2. Undifferentiated or differentiated cells were individually cultured with 250 μM of PCOOH for 6 h. After that, the cells were stained by the antibody to monoclonal mouse anti-αtubulin clone B-5-1-2 and the antibody to Cy3-conjugated sheep anti-mouse IgG. As shown in the photographs in Figure 4A, the undifferentiated cells looked like grape clusters, and the cell shape was clear. However, after exposure to PCOOH, the cell shape was drastically changed (Figure 4B), becoming too vague to identify. The fluorescence emitted from cells was weakened. PCOOH may have induced cell shape alteration by the degradation of tubulin, which was more marked in differentiated cells than in undifferentiated cells. Although the extended neurites were observed clearly in the absence of PCOOH (Figure 4C), they disappeared when exposed in PCOOH for 6h (Figure 4D). The shape of the small cell was vague, as it was in undifferentiated cells, and the fluorescence emitted from cells became extremely weak. The fact that neurites composed of microtubules are easy to be injured may account for the higher vulnerability of differentiated cells.

Fig. 4. Fluorescence microscopic observation of cells after tubulin antibody staining. Representative fields are shown: undifferentiated cells before (A) and after exposure to PCOOH for 6h (B), and differentiated cells before (C) and after exposure to PCOOH for 6h (D)

Furthermore, we tried to verify that the tubulin depolymerization induced by PCOOH could be attenuated by antioxidant. Differentiated cells were cultivated with 5 µM retinol or ascorbic acid beforehand and then exposed to PCOOH. The GTPase activity of cell extracts derived from cells treated with retinol was three-fold higher than that of untreated control cells (Figure 5). Incorporation of antioxidants in cells before exposure to PCOOH protected tubulin depolymerization. This experimental data might lead to the development of an effective strategy to prevent some neurodegenerative diseases.

#### **4. Ageing of central nerve system and the microtubules disorder caused by neural malnutrition**

As people get older, the brain functions decline in varying degree. Although the causes are still unknown, the neurogenesis in hippocampus is decreased dramatically with ageing (Cameron, HA., et al., 1999). The other hand, we can detect neurofibrillary tangles in aged entorhinal cortex or brain cortex of neurodegenerative disorder. These tangle formation are concerned with aggregation of tau, which is a microtubule binding protein. In this section, we will discuss the factors determining the ageing-related neural functional decline in Alzheimer's disease from the aspect of the axonal microtubules disorder caused by neural malnutrition.

Roles of Microtubules in Maintenance of Nerve Cell Networks 43

exposed by oxidative stress, and then oxidative modifications of lipid in the cell membrane and DNA are introduced. The role of oxidative stress in Alzheimer's disease has been reported in several studies, some of which showed elevated markers of oxidative stress, including lipid oxidation products (Sultana, R., et al., 2006). Oxidized lipid hydroperoxides are a characteristic of neurodegenerative disease, and oxidized lipid by-products were

Hydroperoxides of phospholipid were detected in brain samples from patients with

We have investigated the effect of lipid hydroperoxides on microtubule assembly (Kawakami, M., et al., 1993; Kawakami, M., et al., 1998). Lipid hydroperoxides were prepared from soybean phosphatidylcholine by photosensitized oxidation in methanol, with methylene blue being added to the phosphatidylcholine-methanol solution as a sensitizer (Kawakami, M., et al., 1998). Microtubule formation was inhibited dose-dependently by lipid peroxides. This result suggests the possibility that the interaction between tubulin and lipid peroxides may be the cause of some brain diseases. Matsuyama and Jarvik speculated that microtubule integration was a key to Alzheimer's disease (Matsuyama, S.S. and Jarvik,

Bizzozero et al. (2007) also indicated by *in vitro* experiments that lipid hydroperoxides were most likely responsible for protein oxidation. Lipid peroxidation scavengers, such as butylated hydroxytoluene, prevent the carbonylation of cytoskeletal brain protein-induced

**4.4 The mechanism of tubulin modification by phosphatidylcholine hydroperoxides**  We examined the concentration-dependent effects of phosphatidylcholine hydroperoxides on the ability of tubulin to polymerize into microtubules (Kawakami, M., et al., 2000). The results demonstrated that even very low concentrations of peroxides were sufficient to interfere with tubulin and, therefore, microtubule function. In the fluorescence spectra of tubulin before and after interaction with phosphatidylcholine hydroperoxides, a red shift in the emission maximum was observed. This fact indicates a conformational change upon the reaction, namely, that fluorescent aromatic amino acids become easier to dissolve on reaction with phosphatidylcholine hydroperoxides. The interaction mechanism may be a hydrophobic one because no effect on electric conductivity was observed, indicating that

**4.5 Possibility of recovery of tubulin function deteriorated by lipid hydroperoxides**  The effects of lipid hydroperoxides on microtubule assembly were studied in an *in vitro* assay system, as were the protective effects of vitamin A derivatives (β-carotene, retinal, and retinol). All vitamin A derivatives had the ability to protect against the inhibitory effects of lipid hydroperoxides, presumably owing to their antioxidant activities. This suggests a

Glutathione and cysteine were used as water soluble reductants (Kawakami, M., et al., 1999). Tubulin GTPase activity deteriorated by lipid hydroperoxides was restored by the addition of water soluble reductants as well. These chemicals also have a protective effect on

enriched in the brain with Alzheimer's disease (Yoo, M-H., et al., 2010).

**4.3 Inhibition of microtubule assembly by lipid hydroperoxides** 

glutathione depletion (Bizzozero, O.A., et al., 2007).

modulation of ionic binding was not involved.

mechanism for the ability of vitamin A to inhibit cell ageing.

cellular ageing by the reduction of materials oxidized *in vivo*.

L.F., 1989).

Alzheimer's disease using oxidative lipidomics (Tyurin, V.A., et al., 2008).

Fig. 5. GTPase-specific activity of the differentiated cells incorporated 5 µM retinol or 5µM ascorbic acid before exposure to PCOOH. The data represent means ± SD, §, ¶, #P<0.01 compared with the corresponding control value, a<0.01 compared with the data without PCOOH, b,c<0.05 compared with the data without PCOOH

#### **4.1 Microtubule degeneration and Alzheimer's disease**

Alzheimer's disease (AD) is characterized by neuronal cell death and two kinds of deposits, neurofibrillary tangles (NFT) and senile plaques. Abnormal microtubule-binding tau proteins were isolated from AD by Liu et al. (1991). As is well known, in an AD brain, aberrant accumulation of amyloid-β-protein (Aβ) occurs ahead of the accumulation of paired helical filament in NFT. Imahori and Uchida (1997) observed extensive phosphorylation of tau and programmed cell death in a primary culture of embryonic rat hippocampus with Aβ (Imahori, K., and Uchida, T., 1997). There are several important reports on the phosphorylation of tau protein in AD by the group of Iqbal (Alonso, A.D.C., et al., 1994; Gong, C-X., et al., 1994; Iqbal, K., et al., 1994; Gong, C-X., et al., 1995). Glycogen synthase kinase -3β(GSK-3β) is responsible for most of the abnormal hyperphosphorylation of tau observed in paired helical filaments, which are diagnostic for AD (Imahori, K. and Uchida, T., 1997). The tau protein is a microtubule-associated protein that contributes to the formation of microtubules. It is considered that hyperphosphorylated tau is free from microtubules and induces the destruction of the cytoskeleton.

It is possible that microtubules are related to many neurodegenerative diseases in addition to AD. In the brain with Alzheimer's disease, glycation end products are observed. Microtubule-associated protein T is glycated at the tubulin binding site (Ledesma, et al., 1995). The facts observed in microtubule-associated proteins of tau and T appear to indicate that they play a role in microtubule assembly. Furthermore, microtubule assembly is not likely to take place when tubulin has been modified.

#### **4.2 Lipid hydroperoxides in neurodegenative disease**

A part of the oxygen introduced in cell produces reactive oxygen species as a by-product in an electron transport system because of NADPH-dependent oxidase. Materials in cell are

5μM retinol Control

a

5μM L-ascorbic acid

<sup>b</sup> <sup>c</sup>

Concn. of PCOOH (μM)

a

Fig. 5. GTPase-specific activity of the differentiated cells incorporated 5 µM retinol or 5µM ascorbic acid before exposure to PCOOH. The data represent means ± SD, §, ¶, #P<0.01 compared with the corresponding control value, a<0.01 compared with the data without

Alzheimer's disease (AD) is characterized by neuronal cell death and two kinds of deposits, neurofibrillary tangles (NFT) and senile plaques. Abnormal microtubule-binding tau proteins were isolated from AD by Liu et al. (1991). As is well known, in an AD brain, aberrant accumulation of amyloid-β-protein (Aβ) occurs ahead of the accumulation of paired helical filament in NFT. Imahori and Uchida (1997) observed extensive phosphorylation of tau and programmed cell death in a primary culture of embryonic rat hippocampus with Aβ (Imahori, K., and Uchida, T., 1997). There are several important reports on the phosphorylation of tau protein in AD by the group of Iqbal (Alonso, A.D.C., et al., 1994; Gong, C-X., et al., 1994; Iqbal, K., et al., 1994; Gong, C-X., et al., 1995). Glycogen synthase kinase -3β(GSK-3β) is responsible for most of the abnormal hyperphosphorylation of tau observed in paired helical filaments, which are diagnostic for AD (Imahori, K. and Uchida, T., 1997). The tau protein is a microtubule-associated protein that contributes to the formation of microtubules. It is considered that hyperphosphorylated tau is free from

It is possible that microtubules are related to many neurodegenerative diseases in addition to AD. In the brain with Alzheimer's disease, glycation end products are observed. Microtubule-associated protein T is glycated at the tubulin binding site (Ledesma, et al., 1995). The facts observed in microtubule-associated proteins of tau and T appear to indicate that they play a role in microtubule assembly. Furthermore, microtubule assembly is not

A part of the oxygen introduced in cell produces reactive oxygen species as a by-product in an electron transport system because of NADPH-dependent oxidase. Materials in cell are

PCOOH, b,c<0.05 compared with the data without PCOOH

**4.1 Microtubule degeneration and Alzheimer's disease** 

microtubules and induces the destruction of the cytoskeleton.

likely to take place when tubulin has been modified.

**4.2 Lipid hydroperoxides in neurodegenative disease** 

0 50 100

GTPase Specific Activity (nmol/mg/min)

35.0

30.0

25.0

20.0

15.0

10.0

5.0

0.0

exposed by oxidative stress, and then oxidative modifications of lipid in the cell membrane and DNA are introduced. The role of oxidative stress in Alzheimer's disease has been reported in several studies, some of which showed elevated markers of oxidative stress, including lipid oxidation products (Sultana, R., et al., 2006). Oxidized lipid hydroperoxides are a characteristic of neurodegenerative disease, and oxidized lipid by-products were enriched in the brain with Alzheimer's disease (Yoo, M-H., et al., 2010).

Hydroperoxides of phospholipid were detected in brain samples from patients with Alzheimer's disease using oxidative lipidomics (Tyurin, V.A., et al., 2008).

#### **4.3 Inhibition of microtubule assembly by lipid hydroperoxides**

We have investigated the effect of lipid hydroperoxides on microtubule assembly (Kawakami, M., et al., 1993; Kawakami, M., et al., 1998). Lipid hydroperoxides were prepared from soybean phosphatidylcholine by photosensitized oxidation in methanol, with methylene blue being added to the phosphatidylcholine-methanol solution as a sensitizer (Kawakami, M., et al., 1998). Microtubule formation was inhibited dose-dependently by lipid peroxides. This result suggests the possibility that the interaction between tubulin and lipid peroxides may be the cause of some brain diseases. Matsuyama and Jarvik speculated that microtubule integration was a key to Alzheimer's disease (Matsuyama, S.S. and Jarvik, L.F., 1989).

Bizzozero et al. (2007) also indicated by *in vitro* experiments that lipid hydroperoxides were most likely responsible for protein oxidation. Lipid peroxidation scavengers, such as butylated hydroxytoluene, prevent the carbonylation of cytoskeletal brain protein-induced glutathione depletion (Bizzozero, O.A., et al., 2007).

#### **4.4 The mechanism of tubulin modification by phosphatidylcholine hydroperoxides**

We examined the concentration-dependent effects of phosphatidylcholine hydroperoxides on the ability of tubulin to polymerize into microtubules (Kawakami, M., et al., 2000). The results demonstrated that even very low concentrations of peroxides were sufficient to interfere with tubulin and, therefore, microtubule function. In the fluorescence spectra of tubulin before and after interaction with phosphatidylcholine hydroperoxides, a red shift in the emission maximum was observed. This fact indicates a conformational change upon the reaction, namely, that fluorescent aromatic amino acids become easier to dissolve on reaction with phosphatidylcholine hydroperoxides. The interaction mechanism may be a hydrophobic one because no effect on electric conductivity was observed, indicating that modulation of ionic binding was not involved.

#### **4.5 Possibility of recovery of tubulin function deteriorated by lipid hydroperoxides**

The effects of lipid hydroperoxides on microtubule assembly were studied in an *in vitro* assay system, as were the protective effects of vitamin A derivatives (β-carotene, retinal, and retinol). All vitamin A derivatives had the ability to protect against the inhibitory effects of lipid hydroperoxides, presumably owing to their antioxidant activities. This suggests a mechanism for the ability of vitamin A to inhibit cell ageing.

Glutathione and cysteine were used as water soluble reductants (Kawakami, M., et al., 1999). Tubulin GTPase activity deteriorated by lipid hydroperoxides was restored by the addition of water soluble reductants as well. These chemicals also have a protective effect on cellular ageing by the reduction of materials oxidized *in vivo*.

Roles of Microtubules in Maintenance of Nerve Cell Networks 45

Since neurons require sufficient energy supply for maintaining their high-performance, the lack of energy might damage the microtubule dynamics. As mentioned above, the microtubules disruption can be a trigger of neural degeneration. Further investigation for causes of microtubule disruption in neurons might be contribute for our understanding

This work was supported in part by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (to HD) (No. 20500731 and No. 23500985). In addition, this work was partially supported by the Research Center for Elderly Nutrition

Alim, M.A., Takeda, K., Aizawa, T., Matsubara, M., Asada, A., Saito, T., Kaji, H., Yoshii, M.,

Alonso, A.D.C., Zaidi, T., G-Iqbal, I., and Iqbal, K, (1994) Role of abnormally phosphorylated

Arai, T., Hasegawa, M., Akiyama, H., Ikeda, K., Nonaka, T., Mori, H., Mann., D., Tsuchiya,

Barton, J.S., Vandivort, D.L., Heacock, D.H., Coffman, J.A., and Trygg, K.A., (1987)

Bizzozero, O.A., Reyes, S., Ziegler, J., and Smerjac, S., (2007) Lipid peroxidation scavengers

Bruel-Jungernab, E., Davis, S., Rampon C, Laroche, S., (2006) Long-term potentiation enhances neurogenesis in the adult dentate gyrus. J. Neurosci., 26, 5888-5893 Caplow, M., and Shanks, J., (1990) Mechanism for oscillatory assembly of microtubules. J.

Carlier, M.-F, (1982) Guanosine-5'-triphosphate hydrolysis and tubulin polymerization. Mol.

Chen, L., Jin, J., Davis, J., Zhou, Y., Wang, Y., Liu, J., Lockhart, P.J., and Zhang, J., (2007)

Chiti, F., Dobson, C.M., (2006) Protein misfolding, functional amyloid, and human disease.

Oligomeric alpha-synuclein inhibits tubulin polymerization. Biochem. Biophys.

dementia with Lewy bodies. Acta Neuropathol., 117, 125-136

depletion. Neurochem. Res., 32, 2114-2122.

Biol. Chem., 265, 1414-1418.

Res. Commun. 356, 548-553.

Annu. Rev. Biochem., 75, 333-336

Cell Biochem., 47, 97-113.

Hisanaga, S., and Ueda, K., (2004) Demonstration of a role for alpha-synuclein as a functional microtubule-associated protein. J. Alzheimers Dis. 6, 435-442; discussion

tau in the breakdown of microtubules in Alzheimer disease. Proc. Natl. Acad. Sci.

K., Yoshida, M., Hashizume, Y., Oda, T., (2006) TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem. Biophys. Res. Commun., 351, 602-611 Arai, T., Mackenzie, I.R., Hasegawa, M., Nonoka, T., Niizato, K., Tsuchiya, K., Iritani, S.,

Onaya, M., Akiyama, H., (2009) Phosphorylated TDP-43 in Alzheimer's disease and

Microtubule assembly kinetics. Changes with solution conditions. Biochem. J., 247,

prevent the carbonylation of cytoskeletal brain proteins induced by glutathione

and Development, Mukogawa Women's University (recipients: HD and YK).

neurodegenerative disease.

**6. Acknowledgements** 

443-439.

505-511.

USA, 91, 5562-5566.

**7. References** 

The detection of microtubule assembly-promoting material was tried using tubulin GTPase activity as the assay of microtubule assembly. Kawaguchi, M., et al. (2007) found a peptide with a molecular weight of 1340.8 from Japanese classified barley flour.

#### **4.6 Polymerization and calcium binding to tubulin-colchicine complex**

Calcium plays important roles as a messenger in a signal transaction by changing its concentration. The calcium concentration is continually changing, while the concentration is fundamentally very low in a cell. This means that the change affects the functions of many cell constituents.

Calpain is a neutral cysteine proteinase activated by calcium in cytozol, and it converts p35 to p25 (Lee, M-S., et al., 2000). In the brain of AD patients, p25 is stimulated. P25 induces the activation of cyclin-dependent kinase 5 (CDK5). CDK5 is also a factor for the hyperphosphorylation of tau. Indirubins, which are inhibitors of CDK5/p25, repress cell death (Leclerc, S., et al., 2001).

We are interested in the effect of calcium on tubulin polymerization because calcium is an inhibitor of microtubule assembly. Another reason may be the contribution of calpain, which is regulated by calcium, to AD. Instead of tubulin, the tubulin-colchicine complex was used (Doi, H., et al., 2003a). The high affinity sites of calcium took part in the polymerization of the complex in the GTP state, while the low ones participated in the depolymerization. The complex had 2 high-affinity sites with a dissociation constant of 11.5 x 10-6 M and 16 low-affinity sites with a dissociation constant of 2.27 x 10-4 M in the GTP state. In the case of the GDP state, the dissociation constant of the high-affinity site was 7.2 x 10-6 M, and that of the-low affinity site was not observed. The ultracentrifugal experiment indicated a slightly more compact structure in the GTP state compared with the GDP state. The partial specific volumes of the tubulin-colchicine complex in the state of GTP were 0.739 and 0.744 ml/g in imidazole and BES buffers, respectively (Doi, H., et al., 2000b). The sedimentation coefficient S020.w increased from 5.38 S with no calcium to 5.75 and 6.08 S with calcium concentrations of 0.1 and 0.5 mM, respectively, in the absence of the magnesium ion. In an imidazole buffer, the sedimentation coefficients S020.w were 5.82 and 6.06 S in the presence of 0 and 2 mM MgCl2, respectively. These results indicate that the tubulin-colchicine complex causes the calcium affinity to become low after polymerization with its conformational change. This means that the assembly induces the stability of microtubules from calcium.

#### **5. The microtubules disruption and some neurodegenerative diseases**

Finally, we will discuss the association between the microtubules disorders and other some neurodegenerative diseases. Each neurodegenerative disease has specific aberrant intracellular structures like neurofibrillary tangles of AD (Chiti, F. and Dobson, C.M., 2006). Recently, TRA DNA-binding protein of 43kD (TDP-43) has been spotlighted as a common factor associated with the formation of these aberrant structure (Neumann, M. et al., 2006, Arai, T. et al., 2006, 2009, Fujishiro, H. et al. 2009, Schwab, C. et al., 2008). Although several diseases show only TDP-43 intracellular accumulation, TDP-43 is combined with other protein such as tau in many neurodegenerative diseases. It suggests that TDP-43 is a causal factor of microtubules disruption in these diseases. Although the intrinsic or extrinsic causes of many neurodegenerative diseases have been investigated aggressively, the breakdown of microtubules maintenance system by lack of brain blood flow has not been understand well. Since neurons require sufficient energy supply for maintaining their high-performance, the lack of energy might damage the microtubule dynamics. As mentioned above, the microtubules disruption can be a trigger of neural degeneration. Further investigation for causes of microtubule disruption in neurons might be contribute for our understanding neurodegenerative disease.

#### **6. Acknowledgements**

This work was supported in part by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (to HD) (No. 20500731 and No. 23500985). In addition, this work was partially supported by the Research Center for Elderly Nutrition and Development, Mukogawa Women's University (recipients: HD and YK).

#### **7. References**

44 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

The detection of microtubule assembly-promoting material was tried using tubulin GTPase activity as the assay of microtubule assembly. Kawaguchi, M., et al. (2007) found a peptide

Calcium plays important roles as a messenger in a signal transaction by changing its concentration. The calcium concentration is continually changing, while the concentration is fundamentally very low in a cell. This means that the change affects the functions of many

Calpain is a neutral cysteine proteinase activated by calcium in cytozol, and it converts p35 to p25 (Lee, M-S., et al., 2000). In the brain of AD patients, p25 is stimulated. P25 induces the activation of cyclin-dependent kinase 5 (CDK5). CDK5 is also a factor for the hyperphosphorylation of tau. Indirubins, which are inhibitors of CDK5/p25, repress cell

We are interested in the effect of calcium on tubulin polymerization because calcium is an inhibitor of microtubule assembly. Another reason may be the contribution of calpain, which is regulated by calcium, to AD. Instead of tubulin, the tubulin-colchicine complex was used (Doi, H., et al., 2003a). The high affinity sites of calcium took part in the polymerization of the complex in the GTP state, while the low ones participated in the depolymerization. The complex had 2 high-affinity sites with a dissociation constant of 11.5 x 10-6 M and 16 low-affinity sites with a dissociation constant of 2.27 x 10-4 M in the GTP state. In the case of the GDP state, the dissociation constant of the high-affinity site was 7.2 x 10-6 M, and that of the-low affinity site was not observed. The ultracentrifugal experiment indicated a slightly more compact structure in the GTP state compared with the GDP state. The partial specific volumes of the tubulin-colchicine complex in the state of GTP were 0.739 and 0.744 ml/g in imidazole and BES buffers, respectively (Doi, H., et al., 2000b). The sedimentation coefficient S020.w increased from 5.38 S with no calcium to 5.75 and 6.08 S with calcium concentrations of 0.1 and 0.5 mM, respectively, in the absence of the magnesium ion. In an imidazole buffer, the sedimentation coefficients S020.w were 5.82 and 6.06 S in the presence of 0 and 2 mM MgCl2, respectively. These results indicate that the tubulin-colchicine complex causes the calcium affinity to become low after polymerization with its conformational change. This

with a molecular weight of 1340.8 from Japanese classified barley flour.

cell constituents.

death (Leclerc, S., et al., 2001).

**4.6 Polymerization and calcium binding to tubulin-colchicine complex** 

means that the assembly induces the stability of microtubules from calcium.

**5. The microtubules disruption and some neurodegenerative diseases** 

Finally, we will discuss the association between the microtubules disorders and other some neurodegenerative diseases. Each neurodegenerative disease has specific aberrant intracellular structures like neurofibrillary tangles of AD (Chiti, F. and Dobson, C.M., 2006). Recently, TRA DNA-binding protein of 43kD (TDP-43) has been spotlighted as a common factor associated with the formation of these aberrant structure (Neumann, M. et al., 2006, Arai, T. et al., 2006, 2009, Fujishiro, H. et al. 2009, Schwab, C. et al., 2008). Although several diseases show only TDP-43 intracellular accumulation, TDP-43 is combined with other protein such as tau in many neurodegenerative diseases. It suggests that TDP-43 is a causal factor of microtubules disruption in these diseases. Although the intrinsic or extrinsic causes of many neurodegenerative diseases have been investigated aggressively, the breakdown of microtubules maintenance system by lack of brain blood flow has not been understand well.


Roles of Microtubules in Maintenance of Nerve Cell Networks 47

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**Part 2** 

**Oxidative Stress and Neurodegeneration** 

Zhou, R.M., Huang, Y.X., Li, X.L., Chen, C., Shi, Q., Wang, G.R., Tian, C., Wang, Z.Y., Gao, C., and Dong, X.P., (2010) Molecular interaction of alpha-synuclein with tubulin influences on the polymerization of microtubule in vitro and structure of microtubule in cells. Mol. Biol. Rep. 37, 3183-3192

## **Part 2**

**Oxidative Stress and Neurodegeneration** 

50 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

Zhou, R.M., Huang, Y.X., Li, X.L., Chen, C., Shi, Q., Wang, G.R., Tian, C., Wang, Z.Y., Gao,

microtubule in cells. Mol. Biol. Rep. 37, 3183-3192

C., and Dong, X.P., (2010) Molecular interaction of alpha-synuclein with tubulin influences on the polymerization of microtubule in vitro and structure of

**3** 

*México* 

**Oxidative Stress and** 

*Departamento de Fisiología* 

**Neurodegenerative Disease** 

Selva Rivas-Arancibia, Cesar Gallegos-Ríos,

Nancy Gomez-Crisostomo, Ever Ferreira-Garcidueñas,

*Universidad Nacional Autónoma de México, Facultad de Medicina,* 

Dulce Flores Briseño, Luz Navarro and Erika Rodríguez-Martínez

In an oxidation-reduction balance, the antioxidant and oxidant molecules are in equilibrium in the organism. When a free radical increase causes an increase in the activity of the antioxidant systems, this leads to a state of redox homeostasis. The oxidation-reduction balance loss in the organism, caused by an excess of oxidants or a deficit in the antioxidant system, is defined as an oxidative-stress state, which is characterized by high levels of

The oxidative-stress state has an important role in the development of many degenerative diseases, such as autoimmune disease, cancer, cardiac disease, and diabetes, but it also has a crucial role in the neurodegenerative diseases, such as Alzheimer's (Pan et al., 2011), Parkinson's (Sevcsik et al., 2011), Huntington's (Lee et al., 2011), lateral amyotrophic sclerosis (Zhao et al., 2011), multiple Sclerosis (Witherrick et al., 2010), and other processes

Brain plasticity allows certain mental functions to work normally, e.g the learning and memory process. The synapses that form between the neurons are highly organized and are specific structures that permit fast and highly selective interactions between the cells in response to the constant environmental changes that produce neuroplasticity (Bruel-Jungerman et al., 2011). This allows the cells of the nervous system to be both functional and continuously structurally modified to establish new dendrites and synaptic connections. The brain plasticity process can be altered by oxidative stress, which produces oxidative damage, loss of process, synapse deaths, and alteration in the formation of new cells (Rivas-

The synaptic transmission involves the liberation of neurotransmitters from the presynaptic neurons and their detection by a specific receptor on the surface of the membrane of the postsynaptic neuron. Under conditions of homeostasis, the synaptic plasticity is regulated by changes in the amount of receptors in the postsynaptic membrane, changes in the form and size of the dendrite spines, and kinetic modulation of the protein synthesis and

**1. Introduction** 

reactive species.

Arancibia et al., 2010).

degradation.

related to pathological aging (Flovd et al., 2011).

### **Oxidative Stress and Neurodegenerative Disease**

Selva Rivas-Arancibia, Cesar Gallegos-Ríos, Nancy Gomez-Crisostomo, Ever Ferreira-Garcidueñas, Dulce Flores Briseño, Luz Navarro and Erika Rodríguez-Martínez *Universidad Nacional Autónoma de México, Facultad de Medicina, Departamento de Fisiología México* 

#### **1. Introduction**

In an oxidation-reduction balance, the antioxidant and oxidant molecules are in equilibrium in the organism. When a free radical increase causes an increase in the activity of the antioxidant systems, this leads to a state of redox homeostasis. The oxidation-reduction balance loss in the organism, caused by an excess of oxidants or a deficit in the antioxidant system, is defined as an oxidative-stress state, which is characterized by high levels of reactive species.

The oxidative-stress state has an important role in the development of many degenerative diseases, such as autoimmune disease, cancer, cardiac disease, and diabetes, but it also has a crucial role in the neurodegenerative diseases, such as Alzheimer's (Pan et al., 2011), Parkinson's (Sevcsik et al., 2011), Huntington's (Lee et al., 2011), lateral amyotrophic sclerosis (Zhao et al., 2011), multiple Sclerosis (Witherrick et al., 2010), and other processes related to pathological aging (Flovd et al., 2011).

Brain plasticity allows certain mental functions to work normally, e.g the learning and memory process. The synapses that form between the neurons are highly organized and are specific structures that permit fast and highly selective interactions between the cells in response to the constant environmental changes that produce neuroplasticity (Bruel-Jungerman et al., 2011). This allows the cells of the nervous system to be both functional and continuously structurally modified to establish new dendrites and synaptic connections. The brain plasticity process can be altered by oxidative stress, which produces oxidative damage, loss of process, synapse deaths, and alteration in the formation of new cells (Rivas-Arancibia et al., 2010).

The synaptic transmission involves the liberation of neurotransmitters from the presynaptic neurons and their detection by a specific receptor on the surface of the membrane of the postsynaptic neuron. Under conditions of homeostasis, the synaptic plasticity is regulated by changes in the amount of receptors in the postsynaptic membrane, changes in the form and size of the dendrite spines, and kinetic modulation of the protein synthesis and degradation.

Oxidative Stress and Neurodegenerative Disease 55

superoxide (• O2-) into hydrogen peroxide (H2O2) (McCord & Fridovich, 1969). This finding convincingly proved the importance of the free radical in biological systems. The simplest of the free radicals is the hydrogen atom, with a single proton and a single unpaired electron. The elimination of a hydrogen atom from a biological molecule produces an unpaired electron on the atom or molecule to which the hydrogen atom was originally

The diatomic molecule of oxygen (O2) is regarded as a radical because it has two unpaired electrons, each located in a different orbital, but the two have the same spin. This is why O2 has a relatively low reactivity in contrast with other highly reactive radicals. The radicals can be formed by the loss of a single electron or by the gain of a single electron, each action from some stable molecule. A radical could donate its unpaired electron to another molecule or could also trap an electron from another molecule turning the latter into a free radical. The unpaired electrons increase the chemical reactivity of the molecule. It is the manifestation of the free radical to get to the most energetically stable state through pairing with another electron. Thus, many radical-radical and radical-molecule reactions take place as soon as two molecules of the reaction are found. In addition the molecules would be changed by this type of reaction. The high reactivity of free radicals causes their half-life to

be brief, on the order of milliseconds, varying according to the type of free radical.

In biological systems, free radicals and the intermediate products of the biological metabolism are formed. Both free radicals and metabolites are called reactive species. Those most often found are reactive oxygen species (ROS) and reactive nitrogen species (RNS). There are also reactive iron species (RIS) and reactive copper species (RCS) (Valko et al.,

About 60 years ago, it was not thought that the ROS were part of the biological system reactions because of their high reactivity and low selectivity. More than 90% of the oxygen that enters into the cells is used for the production of energy. The mitochondria produce more than 80% of the adenosine triphosphate (ATP) necessary in animal cells. During this process, four electrons are added to each molecule of O2 resulting in the formation of two molecules of H2O. During the phosphorylation oxidative process, 1% to 5% of the O2 used by the mitochondria via complex I and III (Buetler et al., 2004) escape the respiratory chain to form the superoxide anion. Some of these molecules contain an unpaired electron, thus a free radical. The intermediate products have several levels of reactivity with non free radical species. Different ROS often coexist, and it is difficult to identify the specific species as responsible for a given biological effect. For example, different reactive oxygen species formed from the elimination of the superoxide can participate in different types of reactions during which cellular metabolism can suffer a oxidation or reduction

This is a relatively unreactive species but potentially toxic. It can start reactions that give rise to other reactive ROS. This new anion can be formed as a product of many reactions catalyzed enzymatically, as in flavoprotein reactions (Xanthine oxidase, aldehyde oxidase,

**3. How are the reactive species formed?** 

**3.1 Reactive oxygen species (ROS)** 

bonded.

2007).

process.

**3.1.1 Superoxide anion** 

The reactive species produce oxidation of lipids, proteins, and DNA in the cell, unfolding the proteins. The oxidation of the molecules that form the cell membrane alters its selective permeability, which leads to a loss of osmotic balance.

Smythies (1999) proposed the redox hypothesis of learning and neurocomputing. This hypothesis suggests that redox signals may control a mechanism involved in brain plasticity, in which the growth and elimination of synapses and dendrite spines depend on the redox state. The fate of a synapse depending in part on the redox balance means that if the oxidant-environmental cell produces an oxidative-stressed state, and the reactive oxygen species (ROS) cause elimination of spines. This has been demonstrated in alcoholism and neurodegenerative diseases (Götz et al, 2001). If the cell's environmental are antioxidant, synapses are preserved (Smythies, 1999) and increase the number of synapses, facilitating plastic brain phenomena. The central nervous system (CNS) is especially sensitive to the oxidants because of its high lipid content, high consumption of oxygen, and low levels of antioxidant enzymes. The hippocampus, substantia nigra, and the striatum are particularly vulnerable to oxidative stress (Rivas-Arancibia et al., 2010; Santiago-López et al., 2010). The vulnerability of these structures is probably caused by the neurochemical and metabolic characteristics of neural network. The hippocampus contains neurotransmitters such as acetylcholine and glutamate, and also has the ability to produce new neurons in the dentate gyrus, which make it susceptible to redox changes. This response is in part modulated by oxidative changes and an excess of reactive species block neurogenesis (Rivas-Arancibia et al., 2010). In the substantia nigra and the striatum, the normal metabolism of dopamine involves many oxidative reactions. In a state of redox balance, the dopamine oxidation does not disrupt normal metabolism of dopamine, because oxidized dopamine is converted by a complex series of reactions to neuromelanin. The loss of the redox balance causes oxidation of cytoplasmic dopamine in the presence of transition metals, with the formation of superoxide, hydrogen peroxide, and the hydroxyl radical. The dopaminergic neurons in the substantia nigra are involved in different functions such as learning and memory processes and motor control. With a loss of redox equilibrium, these neurons easily suffer oxidative damage and begin to produce a chain of events, in which the synthesis and metabolic path of dopamine contribute to the increase of the oxidative stress state because of quinone formation, making the nigroestriatal pathway much more vulnerable to damage in comparison to other brain structures (Santiago López et al., 2010).

#### **2. Free radicals, reactive species formation, and cell signaling**

A free radical is a species containing one or more unpaired electrons with the ability to exist independently (Halliwell, 2006, 2007). Free radicals are highly reactive, but despite their chemical reactivity, this reactivity changes over a wide spectrum. The first organic free radical was identified by Gomberg in 1900, the methyl triphenyl radical. More than 50 years ago, free radicals were described in living systems in a classic work by Commoner & Townsed published in Nature (1954). Subsequently, it was proposed that free radicals of oxygen (or reactive species of oxygen), could be formed as products of the enzymatic reactions of cell metabolism. At that time the theory was that the reactive oxygen species (ROS) could be the direct cause of many diseases, including cancer and almost any neurodegenerative process (Harman, 1956). In the 1960s and 1970s, free radicals began to be considered as important elements in biological systems. Among other findings, the enzyme superoxide dismutase (SOD) was discovered that converts the free radical

The reactive species produce oxidation of lipids, proteins, and DNA in the cell, unfolding the proteins. The oxidation of the molecules that form the cell membrane alters its selective

Smythies (1999) proposed the redox hypothesis of learning and neurocomputing. This hypothesis suggests that redox signals may control a mechanism involved in brain plasticity, in which the growth and elimination of synapses and dendrite spines depend on the redox state. The fate of a synapse depending in part on the redox balance means that if the oxidant-environmental cell produces an oxidative-stressed state, and the reactive oxygen species (ROS) cause elimination of spines. This has been demonstrated in alcoholism and neurodegenerative diseases (Götz et al, 2001). If the cell's environmental are antioxidant, synapses are preserved (Smythies, 1999) and increase the number of synapses, facilitating plastic brain phenomena. The central nervous system (CNS) is especially sensitive to the oxidants because of its high lipid content, high consumption of oxygen, and low levels of antioxidant enzymes. The hippocampus, substantia nigra, and the striatum are particularly vulnerable to oxidative stress (Rivas-Arancibia et al., 2010; Santiago-López et al., 2010). The vulnerability of these structures is probably caused by the neurochemical and metabolic characteristics of neural network. The hippocampus contains neurotransmitters such as acetylcholine and glutamate, and also has the ability to produce new neurons in the dentate gyrus, which make it susceptible to redox changes. This response is in part modulated by oxidative changes and an excess of reactive species block neurogenesis (Rivas-Arancibia et al., 2010). In the substantia nigra and the striatum, the normal metabolism of dopamine involves many oxidative reactions. In a state of redox balance, the dopamine oxidation does not disrupt normal metabolism of dopamine, because oxidized dopamine is converted by a complex series of reactions to neuromelanin. The loss of the redox balance causes oxidation of cytoplasmic dopamine in the presence of transition metals, with the formation of superoxide, hydrogen peroxide, and the hydroxyl radical. The dopaminergic neurons in the substantia nigra are involved in different functions such as learning and memory processes and motor control. With a loss of redox equilibrium, these neurons easily suffer oxidative damage and begin to produce a chain of events, in which the synthesis and metabolic path of dopamine contribute to the increase of the oxidative stress state because of quinone formation, making the nigroestriatal pathway much more vulnerable to damage in

permeability, which leads to a loss of osmotic balance.

comparison to other brain structures (Santiago López et al., 2010).

**2. Free radicals, reactive species formation, and cell signaling** 

A free radical is a species containing one or more unpaired electrons with the ability to exist independently (Halliwell, 2006, 2007). Free radicals are highly reactive, but despite their chemical reactivity, this reactivity changes over a wide spectrum. The first organic free radical was identified by Gomberg in 1900, the methyl triphenyl radical. More than 50 years ago, free radicals were described in living systems in a classic work by Commoner & Townsed published in Nature (1954). Subsequently, it was proposed that free radicals of oxygen (or reactive species of oxygen), could be formed as products of the enzymatic reactions of cell metabolism. At that time the theory was that the reactive oxygen species (ROS) could be the direct cause of many diseases, including cancer and almost any neurodegenerative process (Harman, 1956). In the 1960s and 1970s, free radicals began to be considered as important elements in biological systems. Among other findings, the enzyme superoxide dismutase (SOD) was discovered that converts the free radical superoxide (• O2-) into hydrogen peroxide (H2O2) (McCord & Fridovich, 1969). This finding convincingly proved the importance of the free radical in biological systems. The simplest of the free radicals is the hydrogen atom, with a single proton and a single unpaired electron. The elimination of a hydrogen atom from a biological molecule produces an unpaired electron on the atom or molecule to which the hydrogen atom was originally bonded.

The diatomic molecule of oxygen (O2) is regarded as a radical because it has two unpaired electrons, each located in a different orbital, but the two have the same spin. This is why O2 has a relatively low reactivity in contrast with other highly reactive radicals. The radicals can be formed by the loss of a single electron or by the gain of a single electron, each action from some stable molecule. A radical could donate its unpaired electron to another molecule or could also trap an electron from another molecule turning the latter into a free radical. The unpaired electrons increase the chemical reactivity of the molecule. It is the manifestation of the free radical to get to the most energetically stable state through pairing with another electron. Thus, many radical-radical and radical-molecule reactions take place as soon as two molecules of the reaction are found. In addition the molecules would be changed by this type of reaction. The high reactivity of free radicals causes their half-life to be brief, on the order of milliseconds, varying according to the type of free radical.

#### **3. How are the reactive species formed?**

In biological systems, free radicals and the intermediate products of the biological metabolism are formed. Both free radicals and metabolites are called reactive species. Those most often found are reactive oxygen species (ROS) and reactive nitrogen species (RNS). There are also reactive iron species (RIS) and reactive copper species (RCS) (Valko et al., 2007).

#### **3.1 Reactive oxygen species (ROS)**

About 60 years ago, it was not thought that the ROS were part of the biological system reactions because of their high reactivity and low selectivity. More than 90% of the oxygen that enters into the cells is used for the production of energy. The mitochondria produce more than 80% of the adenosine triphosphate (ATP) necessary in animal cells. During this process, four electrons are added to each molecule of O2 resulting in the formation of two molecules of H2O. During the phosphorylation oxidative process, 1% to 5% of the O2 used by the mitochondria via complex I and III (Buetler et al., 2004) escape the respiratory chain to form the superoxide anion. Some of these molecules contain an unpaired electron, thus a free radical. The intermediate products have several levels of reactivity with non free radical species. Different ROS often coexist, and it is difficult to identify the specific species as responsible for a given biological effect. For example, different reactive oxygen species formed from the elimination of the superoxide can participate in different types of reactions during which cellular metabolism can suffer a oxidation or reduction process.

#### **3.1.1 Superoxide anion**

This is a relatively unreactive species but potentially toxic. It can start reactions that give rise to other reactive ROS. This new anion can be formed as a product of many reactions catalyzed enzymatically, as in flavoprotein reactions (Xanthine oxidase, aldehyde oxidase,

Oxidative Stress and Neurodegenerative Disease 57

from chemical reactions such as the autoxidation of ascorbic acid catalyzed by copper

This is the most reactive species, with an average life estimated of about 10-9 seconds (Liochev & Fridovich, 1994). Because of Its high reactivity its chemical action is confined to the vicinity of the site of production. It can be formed in vivo as a result of high-energy radiation (x-ray, gamma ray), which can cause homolytic breakage of water. UV light does not have enough energy to split a water molecule but it can split oxygenated water into two molecules of the hydroxyl radical. At the biological level, the most important hydroxyl

Hydrogen peroxide and the superoxide radical can form the hydroxyl radical by the Haber-

Reaction 5. Hydroxyl radical formation, the Haber-Weiss reaction. This reaction is catalyzed

radical formation is the Fenton reaction (Halliwel & Guteridge, 1992) (reaction 4).

(Korycka-Dahi & Richardson, 1991).

Reaction 2. Reduction of oxygen

**3.1.3 Hydroxyl (•OH)** 

Reaction 3. Dismutation of superoxide anion

Reaction 4. Hydroxyl radical formation. Fenton reaction

Weiss reaction (Wardman, 1996) (Reaction 5):

by metals such as iron or copper

purine oxidase) (Behar et al., 1979; Korycka-Dahi & Richardson, 1981), oxidases and hydroxylases (diamino oxidase, galactose oxidase, cytochrome p450), and also those that can be formed in nonenzymatic reactions of oxygen with cysteine (Sáez et al., 1982) or riboflavin, as happens in the mitochondrial respiratory chain (Boveris, 1972).

Fig. 1. Schematic oxidation-reduction reactions. Note the reduction reactions in a redox balance (left), and oxidation reactions in an oxidative stress condition (right)

Reaction 1. Formation of Superoxide anion

#### **3.1.2 Hydrogen peroxide (H2O2)**

Hydrogen peroxide is not a free radical, but it is a reactive oxygen species that can easily diffuse through the membranes. In biological media it is formed by two pathways; 1) After the direct reduction of the oxygen by two electrons (reaction 2) and 2) By the catalyzation of the superoxide anion with SOD (reaction 3).

Many enzymes produce hydrogen peroxide from oxygen, such as xanthine oxide reductase, superoxide dismutase, glucose oxidase, D-amino acid oxidase, uricase (Battaner et al., 1990; Fridovich, 1986; Janolino & Swaisgood, 1975; Romero-Alvira et al., 1987) and may also result from chemical reactions such as the autoxidation of ascorbic acid catalyzed by copper (Korycka-Dahi & Richardson, 1991).

$$\text{O}\_2 + 2\text{e}^- + 2\text{H}^+ \longrightarrow \overset{\text{лувтерят ратеви}}{\left(\overset{\text{лувтерят ратеви}}{\text{лув}}\right)}$$

Reaction 2. Reduction of oxygen

56 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

purine oxidase) (Behar et al., 1979; Korycka-Dahi & Richardson, 1981), oxidases and hydroxylases (diamino oxidase, galactose oxidase, cytochrome p450), and also those that can be formed in nonenzymatic reactions of oxygen with cysteine (Sáez et al., 1982) or riboflavin,

Fig. 1. Schematic oxidation-reduction reactions. Note the reduction reactions in a redox

Hydrogen peroxide is not a free radical, but it is a reactive oxygen species that can easily diffuse through the membranes. In biological media it is formed by two pathways; 1) After the direct reduction of the oxygen by two electrons (reaction 2) and 2) By the catalyzation of

Many enzymes produce hydrogen peroxide from oxygen, such as xanthine oxide reductase, superoxide dismutase, glucose oxidase, D-amino acid oxidase, uricase (Battaner et al., 1990; Fridovich, 1986; Janolino & Swaisgood, 1975; Romero-Alvira et al., 1987) and may also result

balance (left), and oxidation reactions in an oxidative stress condition (right)

Reaction 1. Formation of Superoxide anion

the superoxide anion with SOD (reaction 3).

**3.1.2 Hydrogen peroxide (H2O2)** 

as happens in the mitochondrial respiratory chain (Boveris, 1972).

Reaction 3. Dismutation of superoxide anion

#### **3.1.3 Hydroxyl (•OH)**

This is the most reactive species, with an average life estimated of about 10-9 seconds (Liochev & Fridovich, 1994). Because of Its high reactivity its chemical action is confined to the vicinity of the site of production. It can be formed in vivo as a result of high-energy radiation (x-ray, gamma ray), which can cause homolytic breakage of water. UV light does not have enough energy to split a water molecule but it can split oxygenated water into two molecules of the hydroxyl radical. At the biological level, the most important hydroxyl radical formation is the Fenton reaction (Halliwel & Guteridge, 1992) (reaction 4).

Reaction 4. Hydroxyl radical formation. Fenton reaction

Hydrogen peroxide and the superoxide radical can form the hydroxyl radical by the Haber-Weiss reaction (Wardman, 1996) (Reaction 5):

Reaction 5. Hydroxyl radical formation, the Haber-Weiss reaction. This reaction is catalyzed by metals such as iron or copper

Oxidative Stress and Neurodegenerative Disease 59

balance is present. However, upon the loss of the redox balance when the concentration of •NO increases, the •NO has indirect effects through metabolites associated with RNS, and they may react with oxygen or the superoxide radical, which occurs during oxidative stress

Nitric oxide can generate the peroxynitrite anion (•ONOO-) by reaction with the superoxide

There are many ways by which organisms are exposed to the effects of oxygen free radicals. Free radicals can be produced through several chemical processes, both within and outside the organism. The same cell is potentially more than one source of production of a free radical. Depending on the origin of its production, the peroxynitrite can be in equilibrium with its conjugate acid (ONOOH). In neutral solution it is a powerful oxidizing agent able to nitrate tyrosine residues, nitrating and oxidizing guanosine, degrade carbohydrates, initiate

The production of •O2- and •NO in vivo is different. The peroxynitrite production always occurs when there is an excess of one or the other (Grisham et al., 1999). Some authors established that both reactions of oxidation and nitration mediated by the peroxynitrite are influenced largely by the relative flow of production of •O2- and •NO (Jourd'Heuil et al., 2001). They also established that the highest rates of oxidation occur with an excess of •NO, producing oxidation through the •OH and from the peroxynitrite •NO2 formed. However, the reaction of peroxynitrite with CO2 is the most important way that the peroxynitrite decomposes in vivo (Lymar & Hurst, 1995), forming the end product •N2O3, which is a

In addition to the reactions of oxidation, the peroxynitrite has the ability to nitrate phenolic compounds under physiological conditions, such as the rings of tyrosine (Goldstein et al., 2000). Tyrosine residues are oxidized by the radical derivatives of the peroxynitrite forming the radical tyrosyl, which in turn reacts with •NO to form 3-nitrotyrosine. The nitration mediated by peroxynitrite in vivo might be inhibited by a relative overproduction of O<sup>2</sup>

because of competition between them by the radical tyrosyl, by which the formation of 3 nitrotyrosine would be inhibited when the rate of formation of •O2- exceeded that of •NO (Goldstein et al., 2000) in the exogenous and endogenous sources (Freeman & Crapo, 1982).

Many antineoplastic agents (Dedon & Goldberg, 1982), such as the adriamycin, bleomycin, daunorubicin, and other antibiotics (Doroshow & Hochstein, 1982) depend on quinoide


lipid peroxidation, and fragment DNA (Beckman & Koppenol, 1994, 1996).

and an inflammatory response (Tweedie et al., 2011).

anion (Gryglewsli et al., 1986; Miles et al., 1996).

Reaction 7. Formation of peroxynitrite anion

**3.1.7 Peroxynitrite (•ONOO-)** 

**4. Free radical production** 

potent nitrating agent.

**4.1 Exogenous ROS production** 

#### **3.1.4 Peroxyl radical (ROO•)**

The peroxyl radicals are probably the most abundant in biological systems, and they are not as reactive as the ROS. They originate from the addition of oxygen to any hydrocarbon radical (reaction 6). This radical has a relatively long half-life (on the order of seconds).

Reaction 6. Production of the peroxyl radical

#### **3.1.5 Oxygen singlet (1 O2)**

This is an excited form of molecular oxygen. It is not a free radical and it is formed in vivo by the action of light on oxygen molecules in the presence of photoactivators, such as riboflavin (Aurand et al., 1977). Its half-life is about 10-6 seconds, depending on the nature of the surrounding matrix. It can interact with other molecules by transferring to them its excitation energy or by chemically combining with them. It can form in the oxidation of NADPH in the microsomes by the activity of several enzymes such as xanthine oxidase, lactoperoxidase, lipoxygenase, and prostaglandin synthetase.

#### **3.1.6 Nitric oxide (•NO)**

It is a lyophilic and water-soluble gas, with an average half-life of 3 to 5 seconds. Enzymatically formed from arginine, its reaction is catalyzed by nitric oxide synthase (NOS). The NOS has three isoforms. The neuronal nitric oxide synthase (nNOS) or type I, the inducible nitric oxide synthase (iNOS) or type II, and the endothelial nitric oxide synthase (eNOS) or type III. They are constitutively expressed. Their activity is regulated by the intracellular concentration of calcium (Bredt et al., 1991). The inducible nitric oxide synthase (iNOS) or type II is expressed in the macrophages when they are stimulated by cytokines, lipopolysaccharides, or other immune substances. It is also is found in other tissues, such as brain tissue and endothelium (MacMicking et al., 1997). Its expression is regulated at both the transcriptional and posttranscriptional level, which involves the transcription by redox signaling as an increase in reactive species and cytokines, such as nuclear factor kappa B (NF-kB) and the MAP kinases (MacMicking et al., 1997). Nitric oxide plays a fundamental role in the regulation of local blood flow, inhibits platelet aggregation, is a neurotransmitter, and is produced by activated macrophages that contribute to the primary immune defense. Another effect of the •NO radical is its ability to react with the iron of intracellular protein, mainly mitochondrial. Most of the enzymes that possess a heme prosthetic group can be inactivated by nitric oxide. Nitric oxide can react with nucleic acids leading to mutations and DNA breakage and it can also cause necrosis (Tang et al., 2011).

The •NO radical has an important antiinflammatory action, and it has the ability to cause cellular and tissue dysfunction by a proinflammatory effect. To understand this double effect it has been proposed that the regulatory and antiinflammatory effects of nitric oxide occur when it has a direct impact on a biological molecule (Grisham et al., 1999), which occurs under physiological conditions in which the production of •NO is low and a redox balance is present. However, upon the loss of the redox balance when the concentration of •NO increases, the •NO has indirect effects through metabolites associated with RNS, and they may react with oxygen or the superoxide radical, which occurs during oxidative stress and an inflammatory response (Tweedie et al., 2011).

#### **3.1.7 Peroxynitrite (•ONOO-)**

58 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

The peroxyl radicals are probably the most abundant in biological systems, and they are not as reactive as the ROS. They originate from the addition of oxygen to any hydrocarbon radical (reaction 6). This radical has a relatively long half-life (on the order of seconds).

This is an excited form of molecular oxygen. It is not a free radical and it is formed in vivo by the action of light on oxygen molecules in the presence of photoactivators, such as riboflavin (Aurand et al., 1977). Its half-life is about 10-6 seconds, depending on the nature of the surrounding matrix. It can interact with other molecules by transferring to them its excitation energy or by chemically combining with them. It can form in the oxidation of NADPH in the microsomes by the activity of several enzymes such as xanthine oxidase,

It is a lyophilic and water-soluble gas, with an average half-life of 3 to 5 seconds. Enzymatically formed from arginine, its reaction is catalyzed by nitric oxide synthase (NOS). The NOS has three isoforms. The neuronal nitric oxide synthase (nNOS) or type I, the inducible nitric oxide synthase (iNOS) or type II, and the endothelial nitric oxide synthase (eNOS) or type III. They are constitutively expressed. Their activity is regulated by the intracellular concentration of calcium (Bredt et al., 1991). The inducible nitric oxide synthase (iNOS) or type II is expressed in the macrophages when they are stimulated by cytokines, lipopolysaccharides, or other immune substances. It is also is found in other tissues, such as brain tissue and endothelium (MacMicking et al., 1997). Its expression is regulated at both the transcriptional and posttranscriptional level, which involves the transcription by redox signaling as an increase in reactive species and cytokines, such as nuclear factor kappa B (NF-kB) and the MAP kinases (MacMicking et al., 1997). Nitric oxide plays a fundamental role in the regulation of local blood flow, inhibits platelet aggregation, is a neurotransmitter, and is produced by activated macrophages that contribute to the primary immune defense. Another effect of the •NO radical is its ability to react with the iron of intracellular protein, mainly mitochondrial. Most of the enzymes that possess a heme prosthetic group can be inactivated by nitric oxide. Nitric oxide can react with nucleic acids leading to mutations and DNA breakage and it can also cause

The •NO radical has an important antiinflammatory action, and it has the ability to cause cellular and tissue dysfunction by a proinflammatory effect. To understand this double effect it has been proposed that the regulatory and antiinflammatory effects of nitric oxide occur when it has a direct impact on a biological molecule (Grisham et al., 1999), which occurs under physiological conditions in which the production of •NO is low and a redox

**3.1.4 Peroxyl radical (ROO•)** 

**3.1.5 Oxygen singlet (1**

**3.1.6 Nitric oxide (•NO)** 

necrosis (Tang et al., 2011).

Reaction 6. Production of the peroxyl radical

**O2)** 

lactoperoxidase, lipoxygenase, and prostaglandin synthetase.

Nitric oxide can generate the peroxynitrite anion (•ONOO-) by reaction with the superoxide anion (Gryglewsli et al., 1986; Miles et al., 1996).

Reaction 7. Formation of peroxynitrite anion

#### **4. Free radical production**

There are many ways by which organisms are exposed to the effects of oxygen free radicals. Free radicals can be produced through several chemical processes, both within and outside the organism. The same cell is potentially more than one source of production of a free radical. Depending on the origin of its production, the peroxynitrite can be in equilibrium with its conjugate acid (ONOOH). In neutral solution it is a powerful oxidizing agent able to nitrate tyrosine residues, nitrating and oxidizing guanosine, degrade carbohydrates, initiate lipid peroxidation, and fragment DNA (Beckman & Koppenol, 1994, 1996).

The production of •O2- and •NO in vivo is different. The peroxynitrite production always occurs when there is an excess of one or the other (Grisham et al., 1999). Some authors established that both reactions of oxidation and nitration mediated by the peroxynitrite are influenced largely by the relative flow of production of •O<sup>2</sup> - and •NO (Jourd'Heuil et al., 2001). They also established that the highest rates of oxidation occur with an excess of •NO, producing oxidation through the •OH and from the peroxynitrite •NO2 formed. However, the reaction of peroxynitrite with CO2 is the most important way that the peroxynitrite decomposes in vivo (Lymar & Hurst, 1995), forming the end product •N2O3, which is a potent nitrating agent.

In addition to the reactions of oxidation, the peroxynitrite has the ability to nitrate phenolic compounds under physiological conditions, such as the rings of tyrosine (Goldstein et al., 2000). Tyrosine residues are oxidized by the radical derivatives of the peroxynitrite forming the radical tyrosyl, which in turn reacts with •NO to form 3-nitrotyrosine. The nitration mediated by peroxynitrite in vivo might be inhibited by a relative overproduction of O<sup>2</sup> -• because of competition between them by the radical tyrosyl, by which the formation of 3 nitrotyrosine would be inhibited when the rate of formation of •O2- exceeded that of •NO (Goldstein et al., 2000) in the exogenous and endogenous sources (Freeman & Crapo, 1982).

#### **4.1 Exogenous ROS production**

Many antineoplastic agents (Dedon & Goldberg, 1982), such as the adriamycin, bleomycin, daunorubicin, and other antibiotics (Doroshow & Hochstein, 1982) depend on quinoide

Oxidative Stress and Neurodegenerative Disease 61

most likely candidates as free radical generators seem to be iron-sulfide centers, whereas complex III has been discussed intensively to determine if they could be a semiquinone (Boveris & Chance, 1973) or cytochrome b (Nohl & Jordan, 1986; Turrens, 2003; Ghouleh et

Both systems of intracellular membranes contain cytochromes P450 and b5, which can oxidize unsaturated fatty acids (Capdevila et al., 1981; Ghouleh et al., 2011) and xenobiotics (Chignell, 1979). The cytochromes P450 and b5 are the most powerful oxidizers in vivo, although they can also act as reducing agents. There are several actions that activate molecular oxygen species (Ghouleh et al., 2011) generating oxygen electrophílics in turn by

Peroxisomes are cellular sources of the production of hydrogen peroxide because of their high concentration in oxidases, none of which uses superoxide as a precursor. These enzymes include the D-aminoacid oxidase, urate oxidase, L-a-hydroxyacidic oxidase, and

Peroxisomal catalase is the enzyme that metabolizes most of the hydrogen peroxide

Free radicals generated extracellularly must cross the plasma membrane before reacting with other cellular components and can then start toxic reactions. Unsaturated fatty acids present in the membrane and transmembrane proteins with oxidizable amino acids are likely to be altered by free radicals. These reactions affect the properties of the membranes by changing their permeability and decreasing the potential of the membranes, making secretory functions stop, and inhibiting metabolic processes in the cells. All this is caused by lipid peroxidation or the oxidation of important structural proteins (Freeman & Crapo,

In the presence of the oxygen, organisms have been forced to develop mechanisms for their protection against the ROS. Antioxidants are biological substances that are able to compete for oxidizable substrates and inhibit oxidation (Halliwell & Gutteridge, 1984). Antioxidant systems can be divided into enzymatic and nonenzymatic (Somogyi et al., 2007). The first are the SOD, glutathione peroxidase, catalase, and thioredoxin. Nonenzymatic types include vitamins, proteins, and amino acids, which are less reactive but in greater concentration in contrast to the enzymatic types, which have a high reactivity with the ROS, but are in lower

Antioxidant systems counteract the activity of the ROS, thus maintaining the oxidationreduction balance. These systems can be endogenous and exogenous. The most important endogenous antioxidant systems are the enzymes superoxide dismutase, catalase, and

generated by the peroxisomes oxidases (Freeman & Crapo, 1982; Frei, 1994).

**4.2.3 Electronic transport of the endoplasmic reticulum and nuclear membrane** 

radicals that can be released into the cell (Dolphin, 1988).

acyl-fatty-Coenzyme A oxidase (Boveris et al., 1973).

**5. Antioxidant systems and loss of redox balance** 

al., 2011).

**systems** 

**4.2.4 Peroxisomes** 

**4.2.5 Plasma membrane** 

1982).

concentrations.

glutathione peroxidase.

groups or joining metals for their activity. Some of the effects of these drugs have been attributed to their ability to reduce oxygen to superoxide, the hydroxyl radical, and hydrogen peroxide. The irradiation of organisms by electromagnetic radiation (x-rays and gamma rays) or by particle radiation (electrons, protons, deuterons, and neutrons) also cause free radicals (Bielsky & Gebieki, 1977).

Environmental factors, such as photochemical air pollutants as ozone, hyperoxia, pesticides, tobacco smoke, solvents, anesthetics, and aromatic hydrocarbons are a source of reactive species. These agents have free radicals, such as in tobacco smoke, or become reactive species with cellular metabolism and detoxification processes (Mason, 1982). An important source of reactive species that deserves special importance is environmental pollution (Searing & Rabinovitch, 2011; Bhalla, 1999) because it has been shown that an oxidation environment, in which we live in polluted cities, is associated with chronic-degenerative diseases. An example is ozone pollution (Bhalla & Gupta, 2000). Studies have shown that ozone pollution causes serious damage to human health and is a determining factor in the progression of neurodegenerative diseases (Zawia et al., 2009; Schwela, 2000). This gas acts to produce ROS in the body, causing an increase in oxidants, increasing the state of oxidative stress in the organism and thus contributing to increase the neurodegenerative process in the patient (Cretu et al., 2010).

#### **4.2 Endogenous ROS production**

Autoxidation of small molecules. There are a variety of soluble components able to produce phosphorylation in the cell, such as thiols, hydroquinone, catecholamines, flavins, and tetrahydropterins. In all these, the superoxide radical is the radical primarily formed by the dioxygen reduction by these molecules (Baccarini, 1978). Hydrogen peroxide is also produced as a byproduct from the disproportionation of the superoxide radical, either spontaneously or enzymatically catalyzed by superoxide dismutase (SOD).

#### **4.2.1 Soluble enzymes and proteins**

 Enzymes, such as xanthine oxide reductase, aldehyde oxidase, flavinprotein dehydrogenase, and tryptophan dioxygenase, generate free radicals during their catalytic cycle (Massey et al., 1989). During ischemia, calcium stimulates the activation of proteases leading to changes in the activation of these enzymes (Warner et al., 2004) causing cell damage and death.

#### **4.2.2 Mitochondrial electronic transport chain**

In healthy tissue, one of the main sources of free radicals are the mitochondria. This is because these organelles are responsible for more than 90% of cellular oxygen consumption and the radicals in biological systems always, ultimately are generated by the metabolism of oxygen by this route.

Most mitochondrial hydrogen peroxide comes from the disproportionation of the superoxide radical (Boveris & Chance, 1973). The generation of the superoxide radical by mitochondria occurs when the conveyors of the respiratory chain, located in the inner mitochondrial membrane, are highly reduced (Turrens & Boveri, 1980).

Four complexes are responsible for electronic transport in the respiratory chain. The production of radicals has been observed in the mitochondria isolated in complex I (Turrens & Boveri, 1980) and in complex III (Boveris & Chance, 1973). For complex I, the most likely candidates as free radical generators seem to be iron-sulfide centers, whereas complex III has been discussed intensively to determine if they could be a semiquinone (Boveris & Chance, 1973) or cytochrome b (Nohl & Jordan, 1986; Turrens, 2003; Ghouleh et al., 2011).

#### **4.2.3 Electronic transport of the endoplasmic reticulum and nuclear membrane systems**

Both systems of intracellular membranes contain cytochromes P450 and b5, which can oxidize unsaturated fatty acids (Capdevila et al., 1981; Ghouleh et al., 2011) and xenobiotics (Chignell, 1979). The cytochromes P450 and b5 are the most powerful oxidizers in vivo, although they can also act as reducing agents. There are several actions that activate molecular oxygen species (Ghouleh et al., 2011) generating oxygen electrophílics in turn by radicals that can be released into the cell (Dolphin, 1988).

#### **4.2.4 Peroxisomes**

60 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

groups or joining metals for their activity. Some of the effects of these drugs have been attributed to their ability to reduce oxygen to superoxide, the hydroxyl radical, and hydrogen peroxide. The irradiation of organisms by electromagnetic radiation (x-rays and gamma rays) or by particle radiation (electrons, protons, deuterons, and neutrons) also

Environmental factors, such as photochemical air pollutants as ozone, hyperoxia, pesticides, tobacco smoke, solvents, anesthetics, and aromatic hydrocarbons are a source of reactive species. These agents have free radicals, such as in tobacco smoke, or become reactive species with cellular metabolism and detoxification processes (Mason, 1982). An important source of reactive species that deserves special importance is environmental pollution (Searing & Rabinovitch, 2011; Bhalla, 1999) because it has been shown that an oxidation environment, in which we live in polluted cities, is associated with chronic-degenerative diseases. An example is ozone pollution (Bhalla & Gupta, 2000). Studies have shown that ozone pollution causes serious damage to human health and is a determining factor in the progression of neurodegenerative diseases (Zawia et al., 2009; Schwela, 2000). This gas acts to produce ROS in the body, causing an increase in oxidants, increasing the state of oxidative stress in the organism and thus contributing to increase the neurodegenerative

Autoxidation of small molecules. There are a variety of soluble components able to produce phosphorylation in the cell, such as thiols, hydroquinone, catecholamines, flavins, and tetrahydropterins. In all these, the superoxide radical is the radical primarily formed by the dioxygen reduction by these molecules (Baccarini, 1978). Hydrogen peroxide is also produced as a byproduct from the disproportionation of the superoxide radical, either

 Enzymes, such as xanthine oxide reductase, aldehyde oxidase, flavinprotein dehydrogenase, and tryptophan dioxygenase, generate free radicals during their catalytic cycle (Massey et al., 1989). During ischemia, calcium stimulates the activation of proteases leading to changes in the activation of these enzymes (Warner et al., 2004) causing cell

In healthy tissue, one of the main sources of free radicals are the mitochondria. This is because these organelles are responsible for more than 90% of cellular oxygen consumption and the radicals in biological systems always, ultimately are generated by the metabolism of

Most mitochondrial hydrogen peroxide comes from the disproportionation of the superoxide radical (Boveris & Chance, 1973). The generation of the superoxide radical by mitochondria occurs when the conveyors of the respiratory chain, located in the inner

Four complexes are responsible for electronic transport in the respiratory chain. The production of radicals has been observed in the mitochondria isolated in complex I (Turrens & Boveri, 1980) and in complex III (Boveris & Chance, 1973). For complex I, the

spontaneously or enzymatically catalyzed by superoxide dismutase (SOD).

mitochondrial membrane, are highly reduced (Turrens & Boveri, 1980).

cause free radicals (Bielsky & Gebieki, 1977).

process in the patient (Cretu et al., 2010).

**4.2 Endogenous ROS production** 

**4.2.1 Soluble enzymes and proteins** 

**4.2.2 Mitochondrial electronic transport chain** 

damage and death.

oxygen by this route.

Peroxisomes are cellular sources of the production of hydrogen peroxide because of their high concentration in oxidases, none of which uses superoxide as a precursor. These enzymes include the D-aminoacid oxidase, urate oxidase, L-a-hydroxyacidic oxidase, and acyl-fatty-Coenzyme A oxidase (Boveris et al., 1973).

Peroxisomal catalase is the enzyme that metabolizes most of the hydrogen peroxide generated by the peroxisomes oxidases (Freeman & Crapo, 1982; Frei, 1994).

#### **4.2.5 Plasma membrane**

Free radicals generated extracellularly must cross the plasma membrane before reacting with other cellular components and can then start toxic reactions. Unsaturated fatty acids present in the membrane and transmembrane proteins with oxidizable amino acids are likely to be altered by free radicals. These reactions affect the properties of the membranes by changing their permeability and decreasing the potential of the membranes, making secretory functions stop, and inhibiting metabolic processes in the cells. All this is caused by lipid peroxidation or the oxidation of important structural proteins (Freeman & Crapo, 1982).

#### **5. Antioxidant systems and loss of redox balance**

In the presence of the oxygen, organisms have been forced to develop mechanisms for their protection against the ROS. Antioxidants are biological substances that are able to compete for oxidizable substrates and inhibit oxidation (Halliwell & Gutteridge, 1984). Antioxidant systems can be divided into enzymatic and nonenzymatic (Somogyi et al., 2007). The first are the SOD, glutathione peroxidase, catalase, and thioredoxin. Nonenzymatic types include vitamins, proteins, and amino acids, which are less reactive but in greater concentration in contrast to the enzymatic types, which have a high reactivity with the ROS, but are in lower concentrations.

Antioxidant systems counteract the activity of the ROS, thus maintaining the oxidationreduction balance. These systems can be endogenous and exogenous. The most important endogenous antioxidant systems are the enzymes superoxide dismutase, catalase, and glutathione peroxidase.

Oxidative Stress and Neurodegenerative Disease 63

peroxidase, which along with the reduced glutathione contributes to the elimination of peroxides. Glutathione (GSH) is a tripeptide compound of glutamic acid, cysteine, and glycine that has many important functions within cells (Fig. 3). Glutathione serves as a reducer, conjugates to drugs to make them more soluble in water, is involved in the transport of amino acids across cell membranes (γ-glutamyl cycle), is a substrate for the peptide-leukotrienes, serves as a cofactor for some enzyme reactions, and as an aid in the

The role of GSH as a reducing agent is important especially in a highly oxidizing environment. The sulfhydryl of GSH can be used to reduce peroxides. The resulting form of oxidized GSH consists of two molecules of disulfide linked together (GSSG). Glutathione reductase uses NADPH as the cofactor to reduce GSSG to two molecules of GSH. Therefore, the pentose phosphate pathway is important to produce the NADPH required for glutathione reductase. Glutathione peroxidase is a selenium-dependent enzyme that catalyzes the reduction of

Oxidized glutathione is reduced by glutathione reductase that uses NADPH (from the pentose phosphate pathway) as an electron donor, thus maintaining the ratio GSH /GSSG (Fig. 4). There are at least three forms of glutathione peroxidase dependent on selenium; an intracellular form, extracellular (GPx-C), or plasma (GPx-P) that has specific activity for phospho-lipoperoxides (GPx-PH), usually associated with the membrane and although its activity is the same, has structural differences. The GPx-C and GPx-P are tetrameric enzymes composed of four identical subunits with each containing a selenium atom attached covalently to a molecule of cysteine. The sequence of amino acids in the subunits of the GPx-C is different from the sequence of the GPx-P. The separate subunits have no catalytic activity. The GPx-PH is a monomer enzyme that also has an atom of selenium and catalytic activity (Stepanik & Ewing, 1993). The GPx-C has higher affinity for H2O2 than for lipoperoxides, and the GPx-P has a similar affinity for the two substrates. The GPx-C and GPx-P are used as substrates for H2O2 and the lipoperoxides. They are not able to use the phospholipoperoxides (PHL-OOH) that are the major substrates for the GPx-PH (Maiorino

H2O2 or lipoperoxide (L-OOH) using the reduced glutathione (GSH).

reorganization of protein bridges.

Fig. 3. Structure of glutathione

et al., 1991).

Fig. 2. Shows an oxygen free radical (1), which in the presence of the enzyme Cu-Zn SOD (2) gives rise to peroxides (3) that react with reduced glutathione (4) and are catalyzed by the enzyme glutathione peroxidase (5) resulting in oxidized glutathione (6) and water (7). The peroxides (3) that can be toxic to the cell are removed. The oxidized glutathione (6) in the presence of glutathione reductase (8) and NADPH (9), which hosts an electron, allows the oxidized glutathione to return to its reduced form

#### **5.1 Endogenous systems 5.1.1 Superoxide dismutase (SOD)**

The catalytic role of the SOD was discovered by McCord and Fridovich in 1969. The SOD is an enzyme that catalyzes the reduction of the superoxide anion, which is produced in the body as the resulting product of oxidative phosphorylation, either derived from UV radiation or during inflammation, by transforming the superoxide anion into a product such as hydrogen peroxide that is metabolized easily to water by glutathione peroxidase (GPx) and catalase (CAT).The SOD is present in different forms, such as copper-zinc SOD and manganese SOD (Mn-SOD). The Cu-Zn SOD is found in the cytosol and the cell membrane, has a molecular mass of 32 kDa with two identical subunits. The Mn-SOD is located in the mitochondrial matrix (Grisham et al., 1999; Halliwell & Gutteridge, 1989; Ohno et al., 1994) and has a molecular mass of 88 kDa with four identical subunits (Ohno et al., 1994). It acts as a first line of defense in the detoxification of the superoxide anion and seems to be involved in processes of tumor removal or cellular differentiation.

#### **5.1.2 The glutathione antioxidant system**

The glutathione antioxidant system is formed by reduced glutathione and the activity of the enzyme glutathione reductase that reduces the oxidized glutathione and glutathione

Fig. 2. Shows an oxygen free radical (1), which in the presence of the enzyme Cu-Zn SOD (2) gives rise to peroxides (3) that react with reduced glutathione (4) and are catalyzed by the enzyme glutathione peroxidase (5) resulting in oxidized glutathione (6) and water (7). The peroxides (3) that can be toxic to the cell are removed. The oxidized glutathione (6) in the presence of glutathione reductase (8) and NADPH (9), which hosts an electron, allows the

The catalytic role of the SOD was discovered by McCord and Fridovich in 1969. The SOD is an enzyme that catalyzes the reduction of the superoxide anion, which is produced in the body as the resulting product of oxidative phosphorylation, either derived from UV radiation or during inflammation, by transforming the superoxide anion into a product such as hydrogen peroxide that is metabolized easily to water by glutathione peroxidase (GPx) and catalase (CAT).The SOD is present in different forms, such as copper-zinc SOD and manganese SOD (Mn-SOD). The Cu-Zn SOD is found in the cytosol and the cell membrane, has a molecular mass of 32 kDa with two identical subunits. The Mn-SOD is located in the mitochondrial matrix (Grisham et al., 1999; Halliwell & Gutteridge, 1989; Ohno et al., 1994) and has a molecular mass of 88 kDa with four identical subunits (Ohno et al., 1994). It acts as a first line of defense in the detoxification of the superoxide anion and seems to be involved

The glutathione antioxidant system is formed by reduced glutathione and the activity of the enzyme glutathione reductase that reduces the oxidized glutathione and glutathione

oxidized glutathione to return to its reduced form

in processes of tumor removal or cellular differentiation.

**5.1.2 The glutathione antioxidant system** 

**5.1 Endogenous systems** 

**5.1.1 Superoxide dismutase (SOD)** 

peroxidase, which along with the reduced glutathione contributes to the elimination of peroxides. Glutathione (GSH) is a tripeptide compound of glutamic acid, cysteine, and glycine that has many important functions within cells (Fig. 3). Glutathione serves as a reducer, conjugates to drugs to make them more soluble in water, is involved in the transport of amino acids across cell membranes (γ-glutamyl cycle), is a substrate for the peptide-leukotrienes, serves as a cofactor for some enzyme reactions, and as an aid in the reorganization of protein bridges.

Fig. 3. Structure of glutathione

The role of GSH as a reducing agent is important especially in a highly oxidizing environment. The sulfhydryl of GSH can be used to reduce peroxides. The resulting form of oxidized GSH consists of two molecules of disulfide linked together (GSSG). Glutathione reductase uses NADPH as the cofactor to reduce GSSG to two molecules of GSH. Therefore, the pentose phosphate pathway is important to produce the NADPH required for glutathione reductase. Glutathione peroxidase is a selenium-dependent enzyme that catalyzes the reduction of H2O2 or lipoperoxide (L-OOH) using the reduced glutathione (GSH).

Oxidized glutathione is reduced by glutathione reductase that uses NADPH (from the pentose phosphate pathway) as an electron donor, thus maintaining the ratio GSH /GSSG (Fig. 4). There are at least three forms of glutathione peroxidase dependent on selenium; an intracellular form, extracellular (GPx-C), or plasma (GPx-P) that has specific activity for phospho-lipoperoxides (GPx-PH), usually associated with the membrane and although its activity is the same, has structural differences. The GPx-C and GPx-P are tetrameric enzymes composed of four identical subunits with each containing a selenium atom attached covalently to a molecule of cysteine. The sequence of amino acids in the subunits of the GPx-C is different from the sequence of the GPx-P. The separate subunits have no catalytic activity. The GPx-PH is a monomer enzyme that also has an atom of selenium and catalytic activity (Stepanik & Ewing, 1993). The GPx-C has higher affinity for H2O2 than for lipoperoxides, and the GPx-P has a similar affinity for the two substrates. The GPx-C and GPx-P are used as substrates for H2O2 and the lipoperoxides. They are not able to use the phospholipoperoxides (PHL-OOH) that are the major substrates for the GPx-PH (Maiorino et al., 1991).

Oxidative Stress and Neurodegenerative Disease 65

Reaction 9. Destruction of hydrogen peroxide by catalase

of two enzymes.

conditions.

**5.2 Exogenous systems** 

Reaction 10. Catalytic reaction of the enzyme catalase on hydrogen peroxide

and selenium. The last is a cofactor for the enzyme glutathione peroxidase.

In the peroxidative reaction the enzyme can be used as donors of hydrogen to methanol, ethanol, formic acid, and formaldehyde. This reaction can be with monomers, dimers, and tetramers. Glutathione peroxidase (GPx) and glutathione reductase (GRd) are part of an antioxidant system (GPx-GRd), and catalase (SOD-CAT). It has been observed that both systems fail to act simultaneously. The CAT acts in the presence of high concentrations of H2O2, and the GPx at low concentrations, which shows an inverse correlation in the activity

Antioxidant vitamins, along with glutathione, comprise a group of reducing agents able to donate electrons to oxidized species such as free radicals and the lipoperoxides, thus neutralizing their destructive oxidative potential (Chao et al., 2002).The most significant exogenous antioxidant systems are vitamins A, C, and E, and some metals such as copper

Vitamin A. It can be derived from retinol of animal origin and comes from different plant carotenes. The main sources of vitamin A are fish liver oils, liver of mammals, and milk. In plants it exists in the form of carotene (provitamin). It has an important role in vision. In the form of retinoic acid, vitamin A is effective in the treatment of acne and other skin

Vitamin E is a substituted lipid isoprenoid of the tocopherol family. Its biologically active form is D-alpha tocopherol, whose phenolic hydroxyl is responsible for the antioxidant effects. Vitamin B12 is plentiful in the yolk of eggs, whole milk, the offal of mammals, and fish oils. It is essential for humans (Mayes, 1997). The activity of vitamin E is one of the first barriers against the peroxidation of the polyunsaturated fatty acids. Mitochondrial, endoplasmic reticulum, and plasma membrane phospholipids have affinities to alphatocopherol, so it is highly concentrated in these sites (Nenzil et al., 2001). Tocopherols act by interrupting free radical chain reactions because of their ability to transfer a phenolic hydrogen to a peroxide free radical. Vitamin E can be in the form of phenoxy or phenoxyl free radical, in unreversible intermediate reactions that presuppose the transformation of the vitamin to its final harmless products. Tocopherols and selenium act synergistically

Fig. 4. (a) Reduced glutathione structure (GSH). (b) Oxidized glutathione structure (GSSG)

#### **5.1.3 Catalase**

Catalase (CAT) or hydrogen peroxide oxidoreductase is one of the more abundant enzymes in nature and is widely distributed in the human body. Its activity varies depending on the tissue, highest in the liver and kidneys, lowest in connective tissue and the lining, and practically nonexistent in the nervous tissue. At the cellular level it is located in the mitochondria and peroxisomes, except in erythrocytes, where it is located in the cytosol. This enzyme is a tetrameric metaloprotein of four identical subunits that are held together by noncovalent interactions. Each subunit contains a prosthetic group of protoporphyrin IX. Catalase is involved in the destruction of hydrogen peroxide generated during cellular metabolism. It has two features; the catalytic and the peroxidative. Both can be represented by reaction 9.

The general reaction covers the substrate reduction taking hydrogen atoms from a donor, and the products are the reduced substrate and the oxidized donor. In the catalytic reaction, the donor is another molecule of H2O2. This reaction can only be accomplished by the enzyme in its tetrameric form.

(a)

(b) Fig. 4. (a) Reduced glutathione structure (GSH). (b) Oxidized glutathione structure (GSSG)

Catalase (CAT) or hydrogen peroxide oxidoreductase is one of the more abundant enzymes in nature and is widely distributed in the human body. Its activity varies depending on the tissue, highest in the liver and kidneys, lowest in connective tissue and the lining, and practically nonexistent in the nervous tissue. At the cellular level it is located in the mitochondria and peroxisomes, except in erythrocytes, where it is located in the cytosol. This enzyme is a tetrameric metaloprotein of four identical subunits that are held together by noncovalent interactions. Each subunit contains a prosthetic group of protoporphyrin IX. Catalase is involved in the destruction of hydrogen peroxide generated during cellular metabolism. It has two features; the catalytic and the peroxidative. Both can be represented

The general reaction covers the substrate reduction taking hydrogen atoms from a donor, and the products are the reduced substrate and the oxidized donor. In the catalytic reaction, the donor is another molecule of H2O2. This reaction can only be accomplished by the

**5.1.3 Catalase** 

by reaction 9.

enzyme in its tetrameric form.

Reaction 9. Destruction of hydrogen peroxide by catalase

Reaction 10. Catalytic reaction of the enzyme catalase on hydrogen peroxide

In the peroxidative reaction the enzyme can be used as donors of hydrogen to methanol, ethanol, formic acid, and formaldehyde. This reaction can be with monomers, dimers, and tetramers. Glutathione peroxidase (GPx) and glutathione reductase (GRd) are part of an antioxidant system (GPx-GRd), and catalase (SOD-CAT). It has been observed that both systems fail to act simultaneously. The CAT acts in the presence of high concentrations of H2O2, and the GPx at low concentrations, which shows an inverse correlation in the activity of two enzymes.

#### **5.2 Exogenous systems**

Antioxidant vitamins, along with glutathione, comprise a group of reducing agents able to donate electrons to oxidized species such as free radicals and the lipoperoxides, thus neutralizing their destructive oxidative potential (Chao et al., 2002).The most significant exogenous antioxidant systems are vitamins A, C, and E, and some metals such as copper and selenium. The last is a cofactor for the enzyme glutathione peroxidase.

Vitamin A. It can be derived from retinol of animal origin and comes from different plant carotenes. The main sources of vitamin A are fish liver oils, liver of mammals, and milk. In plants it exists in the form of carotene (provitamin). It has an important role in vision. In the form of retinoic acid, vitamin A is effective in the treatment of acne and other skin conditions.

Vitamin E is a substituted lipid isoprenoid of the tocopherol family. Its biologically active form is D-alpha tocopherol, whose phenolic hydroxyl is responsible for the antioxidant effects. Vitamin B12 is plentiful in the yolk of eggs, whole milk, the offal of mammals, and fish oils. It is essential for humans (Mayes, 1997). The activity of vitamin E is one of the first barriers against the peroxidation of the polyunsaturated fatty acids. Mitochondrial, endoplasmic reticulum, and plasma membrane phospholipids have affinities to alphatocopherol, so it is highly concentrated in these sites (Nenzil et al., 2001). Tocopherols act by interrupting free radical chain reactions because of their ability to transfer a phenolic hydrogen to a peroxide free radical. Vitamin E can be in the form of phenoxy or phenoxyl free radical, in unreversible intermediate reactions that presuppose the transformation of the vitamin to its final harmless products. Tocopherols and selenium act synergistically

Oxidative Stress and Neurodegenerative Disease 67

of cytokines during inflammatory processes, the participation in cell signaling (Stone & Yang, 2006; Biniert et al., 2006), and the mechanisms of second messengers (Smythies, 1999;

The ROS are characterized by their dual nature, which depends on the redox state of the organism. In a balanced oxidation-reduction reaction, the main effects of the ROS in the cell are through their actions in signaling pathways. These oxidant signals can easily be offset through antioxidant systems, To restablish a redox equilibrium, the ROS causes the expression of antioxidant enzymes and related defense mechanisms. At low concentrations the ROS are involved in many physiological functions. It has been suggested that the main effects of the ROS in cells are through interactions they have with different signaling pathways and not by their direct action on macromolecules (Maher, 2000). Both phenomena do coexist. There is evidence that living organisms have not only adapted to coexist with free radicals but they also have generated several mechanisms for using free radicals in

Infectious diseases possibly were a mechanism of natural selection in the early stages of human civilization. The ROS participate directly in the defense mechanism against infections. They are part of a respiratory burst and are important modulators of the inflammatory response. Resident glia in the brain in normal situations are produced by ROS and its role in the brain is to counter cell damage. In addition, they participate in other functions, such as the regulation of vascular tone, the monitoring of the oxygen pressure, and the expansion of signal transduction. Signal transduction mediated by reactive species regulates the response to oxidative stress, which keeps the redox balance within homeostatic limits (Droge, 2002). For all this, the homeostatic regulation of the oxidation-reduction balance has a special importance of keeping the delicate balance between the adaptive advantages of the biological use of free radicals and their harmful effects. One of the most important discoveries is that the ROS can regulate gene expression of several bacterial genes, which is generated by H2O2 (Christman et al., 1985). For mammals, small amounts of •O2- and H2O2 increase the production of interleukin 2 (IL-2), which is an important factor of lymphocyte growth, possibly as a response to the activation of the nuclear factor kappa B

The term "redox signaling" is used to describe a process of regulation, that involves processes of oxidation-reduction. This type of signaling is used by a wide range of microorganisms, including bacteria, and its most common use by the cell is the generation of antioxidant defenses to restore the original state of redox homeostasis after a temporary exposure to the ROS (Droge, 2002). The interaction of various components of antioxidant systems (Mendiratta et al., 1998a, 1998b) is effective for the recycling of components and is sufficient to cope with the stress caused by the ROS for long periods in the life of an organism (Soberman, 2003). Aging and particularly inflammatory, chronic diseases cause an alteration in the maintenance of redox state, which causes mechanisms of progressively

As mentioned, the ROS generate cellular events, such as the activation of the pathway of the MAP. These consist of four subfamilies sensitive to the ROS and are identified as kinases regulated by extracellular signaling (ERK1-2), kinase c-Jun NH2 - terminal (JNK), and kinase p38 kinase big MAP type 1 (BMK1 or ERK5). Each family has its trigger mechanism and then modulates specific cellular functions (Suzaki et al., 2002). We will discuss the physiological role of the ROS and the path of the BMK1 or ERK5 kinase in neuronal cells. There is evidence, found in experiments in PC12 cells, of different intracellular signaling

different physiological functions (Halliwell & Gutterdge, 2007; Kirkwood, 2005).

(NFκB) that occurs in the presence of the ROS (Schreck et al., 1992).

Chiarugi & Fiaschi, 2007).

aggravated pathology.

allowing the organism to have its antioxidant activity (Hoenyet et al., 2005). Selenium is required for the normal pancreatic function (Rayman, 2000), which is necessary for the proper digestion of lipids. Though it is known that the levels of vitamin E are correlated with the ability to digest and absorb lipids. Because of its hydrophobic nature a deficiency of tocopherols is found in processes such as hepatic cholestasis and cystic fibrosis or bowel resections. Recent work shows the close relationship of the increase in the requirement of vitamin E and selenium with the intake of unsaturated fatty acids, aging, and the degenerative diseases such as atherosclerosis (Penn et al., 2003), Alzheimer's disease (Butterfield et al., 2002), or prostatic carcinoma (Thomas, 2004).

Vitamin C or L-ascorbic acid is a derivative of glucose. It is essential in the human diet. It is a lactone, in which the hydroxyl associated with the double bond groups function as agents with a high reducing potential, allowing it to participate in the direct reduction of oxygen, thus functioning as a donor substrate in the reactions of the peroxidases (Mayes, 1997). The mechanism of action of this vitamin yields a higher antioxidant level because it includes the inhibition of the formation of the superoxide radical or nitrosamines during digestion. In addition, it is the agent that reduces the phenoxy radical formed during vitamin E activity (Chao et al., 2002). Vitamins C and E are classified as antioxidant switches because they act by stopping the formation of free radical chain reactions (Shite et al., 2001), trapping them and reducing them, unlike the preventive antioxidants (which include peroxidase enzymes) to prevent the initiation of the sequence of reactions. Tocopherols work in an environment of high oxygen partial pressure, whereas beta-carotene works at low O2 partial pressures.

Fig. 5. Action of tocopherol on lipid peroxides and regeneration in the presence of ascorbate and reduced glutathione

#### **6. Role of the reactive species in cellular signaling**

The mechanisms of oxidation-reduction and free radicals play an important role in cell physiology (Kovacik & Wells, 2006), from the renewal of membranes, cellular plastic phenomena, cell migration, synthesis and release of some hormones, increase in transcription

allowing the organism to have its antioxidant activity (Hoenyet et al., 2005). Selenium is required for the normal pancreatic function (Rayman, 2000), which is necessary for the proper digestion of lipids. Though it is known that the levels of vitamin E are correlated with the ability to digest and absorb lipids. Because of its hydrophobic nature a deficiency of tocopherols is found in processes such as hepatic cholestasis and cystic fibrosis or bowel resections. Recent work shows the close relationship of the increase in the requirement of vitamin E and selenium with the intake of unsaturated fatty acids, aging, and the degenerative diseases such as atherosclerosis (Penn et al., 2003), Alzheimer's disease

Vitamin C or L-ascorbic acid is a derivative of glucose. It is essential in the human diet. It is a lactone, in which the hydroxyl associated with the double bond groups function as agents with a high reducing potential, allowing it to participate in the direct reduction of oxygen, thus functioning as a donor substrate in the reactions of the peroxidases (Mayes, 1997). The mechanism of action of this vitamin yields a higher antioxidant level because it includes the inhibition of the formation of the superoxide radical or nitrosamines during digestion. In addition, it is the agent that reduces the phenoxy radical formed during vitamin E activity (Chao et al., 2002). Vitamins C and E are classified as antioxidant switches because they act by stopping the formation of free radical chain reactions (Shite et al., 2001), trapping them and reducing them, unlike the preventive antioxidants (which include peroxidase enzymes) to prevent the initiation of the sequence of reactions. Tocopherols work in an environment of high oxygen partial pressure, whereas beta-carotene works at low O2 partial pressures.

Fig. 5. Action of tocopherol on lipid peroxides and regeneration in the presence of ascorbate

The mechanisms of oxidation-reduction and free radicals play an important role in cell physiology (Kovacik & Wells, 2006), from the renewal of membranes, cellular plastic phenomena, cell migration, synthesis and release of some hormones, increase in transcription

**6. Role of the reactive species in cellular signaling** 

and reduced glutathione

(Butterfield et al., 2002), or prostatic carcinoma (Thomas, 2004).

of cytokines during inflammatory processes, the participation in cell signaling (Stone & Yang, 2006; Biniert et al., 2006), and the mechanisms of second messengers (Smythies, 1999; Chiarugi & Fiaschi, 2007).

The ROS are characterized by their dual nature, which depends on the redox state of the organism. In a balanced oxidation-reduction reaction, the main effects of the ROS in the cell are through their actions in signaling pathways. These oxidant signals can easily be offset through antioxidant systems, To restablish a redox equilibrium, the ROS causes the expression of antioxidant enzymes and related defense mechanisms. At low concentrations the ROS are involved in many physiological functions. It has been suggested that the main effects of the ROS in cells are through interactions they have with different signaling pathways and not by their direct action on macromolecules (Maher, 2000). Both phenomena do coexist. There is evidence that living organisms have not only adapted to coexist with free radicals but they also have generated several mechanisms for using free radicals in different physiological functions (Halliwell & Gutterdge, 2007; Kirkwood, 2005).

Infectious diseases possibly were a mechanism of natural selection in the early stages of human civilization. The ROS participate directly in the defense mechanism against infections. They are part of a respiratory burst and are important modulators of the inflammatory response. Resident glia in the brain in normal situations are produced by ROS and its role in the brain is to counter cell damage. In addition, they participate in other functions, such as the regulation of vascular tone, the monitoring of the oxygen pressure, and the expansion of signal transduction. Signal transduction mediated by reactive species regulates the response to oxidative stress, which keeps the redox balance within homeostatic limits (Droge, 2002). For all this, the homeostatic regulation of the oxidation-reduction balance has a special importance of keeping the delicate balance between the adaptive advantages of the biological use of free radicals and their harmful effects. One of the most important discoveries is that the ROS can regulate gene expression of several bacterial genes, which is generated by H2O2 (Christman et al., 1985). For mammals, small amounts of •O2- and H2O2 increase the production of interleukin 2 (IL-2), which is an important factor of lymphocyte growth, possibly as a response to the activation of the nuclear factor kappa B (NFκB) that occurs in the presence of the ROS (Schreck et al., 1992).

The term "redox signaling" is used to describe a process of regulation, that involves processes of oxidation-reduction. This type of signaling is used by a wide range of microorganisms, including bacteria, and its most common use by the cell is the generation of antioxidant defenses to restore the original state of redox homeostasis after a temporary exposure to the ROS (Droge, 2002). The interaction of various components of antioxidant systems (Mendiratta et al., 1998a, 1998b) is effective for the recycling of components and is sufficient to cope with the stress caused by the ROS for long periods in the life of an organism (Soberman, 2003). Aging and particularly inflammatory, chronic diseases cause an alteration in the maintenance of redox state, which causes mechanisms of progressively aggravated pathology.

As mentioned, the ROS generate cellular events, such as the activation of the pathway of the MAP. These consist of four subfamilies sensitive to the ROS and are identified as kinases regulated by extracellular signaling (ERK1-2), kinase c-Jun NH2 - terminal (JNK), and kinase p38 kinase big MAP type 1 (BMK1 or ERK5). Each family has its trigger mechanism and then modulates specific cellular functions (Suzaki et al., 2002). We will discuss the physiological role of the ROS and the path of the BMK1 or ERK5 kinase in neuronal cells. There is evidence, found in experiments in PC12 cells, of different intracellular signaling

Oxidative Stress and Neurodegenerative Disease 69

of BMK1 (Yan, 1999) and this is able to phosphorylate Bad, a protein involved in the signaling of apoptosis. The scaffold 14-3-3 protein binds to Bad when it is phosphorylated and stops it from having proapoptotic activity. The nonphosphorylated Bad is able to travel to the mitochondria, causing the release of cytochrome C, a crucial step for the activation of caspase 3 (Xinchun et al., 2004). This evidence can suggest that the protective role and

The inflammatory response is a natural and important process in the repair of tissues and a fundamental mechanism of defense of the organism against infections and harmful agents. When the inflammatory response is not limited a process of chronic inflammation is established. In the animal model of oxidative stress, caused by exposure to ozone on healthy animals, chronic oxidative stress is able to cause an inflammatory response and dysregulation of the same answer (Rivas-Arancibia et al., 2010). It is widely reported that chronic inflammation produces ROS that lead to a state of oxidative stress. This change in the redox balance, causing activation of the signaling pathways of the cell, causes a perpetually inflammatory state. Maintaining the redox balance is important for cell signaling and adequate transcriptional activity (Chung et al., 2009). The ROS and other reactive species regulate the expression of proinflammatory cytokines, such as TNFα, IL-1β, IL-6, and IL-8, and the cell-adhesion molecules, such as the adhesion intercellular-1 (ICAM-1) and E-selectin molecules. Several biological states cause an increase in the amount of proinflammatory cytokines. Aging causes an increase in the levels of cytokines such as TNF-α, IL-1β, IL-6, gamma interferon (IFNγ), the beta-transforming growth factor (TGFβ), and acute phase proteins (Bruunsgaard et al., 2003). High levels of IL-6 have been associated with neuronal atrophy and chronic inflammatory states such as diabetes type 2 and atherosclerosis (Devaux et al., 1997; Willette et al., 2010). The C-reactive protein (CRP) levels increase with ageing, and high levels of chronic diseases are found associated with ageing,

Activation of transcription factors that are sensitive to redox signals generate the production of inflammatory mediators, such as interleukin-1β (IL-1β), Interleukin-6 (IL-6), necrosis tumor-α factor (TNF-α), cycle-oxygenase-2 (COX-2), lipoxygenase (LOX), and inducible nitric oxide synthase (iNOS), and the cell adhesion molecules(CAMs) (VCAM-1, ICAM-1, and P- and E-selectin) (Salminen & Kaarniranta, 2010). NF-κB is a transcription factor activated by a wide variety of stimuli, such as oxidative stress, infection, and inflammation. This activation plays a key role in regulating the immune response (Salminen & Kaarniranta, 2010). NF-κB is composed of a heterodimericprotein complex containing a DNA-binding domain and a domain of acidic transactivation formed by the heterodimer polypeptides RelA-p65 and p50. Normally NF-κB is linked to a protein of the IκB family in cytoplasm, which inhibits its activation (Baldwin, 1996). When IκB is degraded, the NF-κB is freed and causes its translocation to the nucleus, where the NF-κB can bind to a promoter and start the transcription of specific genes that encode for proinflammatory mediators. Activation of tNF-kB is usually transient, but chronic activation produces changes in the inflammatory response. Protein-generated NF-kB and COX-2, TNF-α,e, and IL-1β and IL-6 are also potent activators of the same pathway, creating a vicious cycle (Handel et al., 1995, Fisher et al., 1996 ). There is evidence showing that aging increases the degradation of

antiapoptotic action of BMK1-ERK5 is generated by the ROS (Liu et al., 2003).

**7. Inflammation response and oxidative stress** 

heart disease, and Alzheimer's disease (Ridker et al., 2001).

steps identified in the path of the MAP kinases, where the ROS are involved (Suzaki et al., 2002).

The activation of the pathway of the BKM1 stimulated by the ROS depends on the presence of c-Src, a protein kinase encoded by the gene Src, and that becomes involved in the internalization of the signals to the nucleus through the phosphorylation of other proteins, including second messengers. When the ROS present in the cells interact with the c-Src, this causes activation of the kinase, which in turn has an immediate effect on the activation of the BMK1 through key intermediates to continue the internalization of the signal initiated by the ROS. Below is shown the cascade of the MEKK3 and MEK5 kinases (Suzaki et al., 2002). The BMK1-ERK5 possesses a amino acid motif TEY of physiological importance because it is on this site where atoms join with the phosphorous kinase MEK5 (Lee et al., 1995; Zhou et al., 1995). This, when phosphorylated, acquires the ability to cause the activation of the MEF2C and MEF2A, both belonging to the family of MEF2 transcription factors, and to cause the translocation at the nucleus, which is involved in the expression of genes of c-Jun and c-Fos, themselves part of the family of the AP1 (Silva, 2001). Components of this family have a common binding site on the DNA that results in the expression of neuropeptides and neurotrophins, synthesis and expression of receptors to various ligands, activation of transcription factors, the synthesis of various enzymes involved in the production of neurotransmitters, such as thyroxine hydroxylase, a limiting enzyme in the production of catecholamines and the formation and polymerization of proteins to the cytoskeleton (Silva, 2001). In endothelial cells, oxidative stress is involved in the formation

Fig. 6. Shows the action of reactive species on cellular signaling. In this example we can observ the effects of ROS on the AP1 family mediated by the pathway of MEKK3

steps identified in the path of the MAP kinases, where the ROS are involved (Suzaki et al.,

The activation of the pathway of the BKM1 stimulated by the ROS depends on the presence of c-Src, a protein kinase encoded by the gene Src, and that becomes involved in the internalization of the signals to the nucleus through the phosphorylation of other proteins, including second messengers. When the ROS present in the cells interact with the c-Src, this causes activation of the kinase, which in turn has an immediate effect on the activation of the BMK1 through key intermediates to continue the internalization of the signal initiated by the ROS. Below is shown the cascade of the MEKK3 and MEK5 kinases (Suzaki et al., 2002). The BMK1-ERK5 possesses a amino acid motif TEY of physiological importance because it is on this site where atoms join with the phosphorous kinase MEK5 (Lee et al., 1995; Zhou et al., 1995). This, when phosphorylated, acquires the ability to cause the activation of the MEF2C and MEF2A, both belonging to the family of MEF2 transcription factors, and to cause the translocation at the nucleus, which is involved in the expression of genes of c-Jun and c-Fos, themselves part of the family of the AP1 (Silva, 2001). Components of this family have a common binding site on the DNA that results in the expression of neuropeptides and neurotrophins, synthesis and expression of receptors to various ligands, activation of transcription factors, the synthesis of various enzymes involved in the production of neurotransmitters, such as thyroxine hydroxylase, a limiting enzyme in the production of catecholamines and the formation and polymerization of proteins to the cytoskeleton (Silva, 2001). In endothelial cells, oxidative stress is involved in the formation

MEKK3

ROS

MEK5

AP1 Family

MEF2C MEF2A

Fig. 6. Shows the action of reactive species on cellular signaling. In this example we can observ the effects of ROS on the AP1 family mediated by the pathway of MEKK3

c-Fos

c-Jun

 Cell nucleus BMK1 ERK5 Cell cycle progression

● ● ● ● ● ● Neuronal Survival

Production of neuropeptides

Cytoskeletal protein binding

Neurotrophins Receptor expression Transcription factors Enzymes synthesis for the neurotransmitters

2002).

Bad

Bad

14-3-3

Caspase 3

14-3-3

of BMK1 (Yan, 1999) and this is able to phosphorylate Bad, a protein involved in the signaling of apoptosis. The scaffold 14-3-3 protein binds to Bad when it is phosphorylated and stops it from having proapoptotic activity. The nonphosphorylated Bad is able to travel to the mitochondria, causing the release of cytochrome C, a crucial step for the activation of caspase 3 (Xinchun et al., 2004). This evidence can suggest that the protective role and antiapoptotic action of BMK1-ERK5 is generated by the ROS (Liu et al., 2003).

#### **7. Inflammation response and oxidative stress**

The inflammatory response is a natural and important process in the repair of tissues and a fundamental mechanism of defense of the organism against infections and harmful agents. When the inflammatory response is not limited a process of chronic inflammation is established. In the animal model of oxidative stress, caused by exposure to ozone on healthy animals, chronic oxidative stress is able to cause an inflammatory response and dysregulation of the same answer (Rivas-Arancibia et al., 2010). It is widely reported that chronic inflammation produces ROS that lead to a state of oxidative stress. This change in the redox balance, causing activation of the signaling pathways of the cell, causes a perpetually inflammatory state. Maintaining the redox balance is important for cell signaling and adequate transcriptional activity (Chung et al., 2009). The ROS and other reactive species regulate the expression of proinflammatory cytokines, such as TNFα, IL-1β, IL-6, and IL-8, and the cell-adhesion molecules, such as the adhesion intercellular-1 (ICAM-1) and E-selectin molecules. Several biological states cause an increase in the amount of proinflammatory cytokines. Aging causes an increase in the levels of cytokines such as TNF-α, IL-1β, IL-6, gamma interferon (IFNγ), the beta-transforming growth factor (TGFβ), and acute phase proteins (Bruunsgaard et al., 2003). High levels of IL-6 have been associated with neuronal atrophy and chronic inflammatory states such as diabetes type 2 and atherosclerosis (Devaux et al., 1997; Willette et al., 2010). The C-reactive protein (CRP) levels increase with ageing, and high levels of chronic diseases are found associated with ageing, heart disease, and Alzheimer's disease (Ridker et al., 2001).

Activation of transcription factors that are sensitive to redox signals generate the production of inflammatory mediators, such as interleukin-1β (IL-1β), Interleukin-6 (IL-6), necrosis tumor-α factor (TNF-α), cycle-oxygenase-2 (COX-2), lipoxygenase (LOX), and inducible nitric oxide synthase (iNOS), and the cell adhesion molecules(CAMs) (VCAM-1, ICAM-1, and P- and E-selectin) (Salminen & Kaarniranta, 2010). NF-κB is a transcription factor activated by a wide variety of stimuli, such as oxidative stress, infection, and inflammation. This activation plays a key role in regulating the immune response (Salminen & Kaarniranta, 2010). NF-κB is composed of a heterodimericprotein complex containing a DNA-binding domain and a domain of acidic transactivation formed by the heterodimer polypeptides RelA-p65 and p50. Normally NF-κB is linked to a protein of the IκB family in cytoplasm, which inhibits its activation (Baldwin, 1996). When IκB is degraded, the NF-κB is freed and causes its translocation to the nucleus, where the NF-κB can bind to a promoter and start the transcription of specific genes that encode for proinflammatory mediators. Activation of tNF-kB is usually transient, but chronic activation produces changes in the inflammatory response. Protein-generated NF-kB and COX-2, TNF-α,e, and IL-1β and IL-6 are also potent activators of the same pathway, creating a vicious cycle (Handel et al., 1995, Fisher et al., 1996 ). There is evidence showing that aging increases the degradation of

Oxidative Stress and Neurodegenerative Disease 71

Like other cells, nerve cells use ATP as a source of energy for biochemical processes involved in various cell functions, and produce ROS as a result of oxidative phosphorylation. The electrical excitability and structural changes, coupled with the synaptic complexity of neurons yields unusual demands in cellular systems that produce or respond to ATP and ROS. Mitochondria in axons and presynaptic terminals provide for sources of ATP to pump ions that are concentrated in these structures to quickly restore the subsequent ion gradients for depolarization and neurotransmitter release. Mitochondria also play a role in the regulation of synaptic functions because of their ability to regulate calcium levels and the

Neurons in the brain are highly vulnerable to metabolic changes so that a mitochondrial disorder, which causes a decrease in the production of ATP, represents a clear threat to the viability of the neurons and glial cells, the functionality of neural networks, and consequently the normal functions of the brain can be changed. The alteration in the regulation of calcium levels by the failures of the mitochondrial buffer and the release of mitochondria-bound calcium contributes to a severe injury of brain tissue in response to excitotoxicity by glutamate, oxidative stress, or metabolic damage such as trauma. Similarly, an abnormal increase in the generation of ROS by the mitochondria also puts at risk cell viability because many shock-mechanism absorbers might be overwhelmed (Kann & Kovács, 2007).The result is an oxidative damage to the structural and regulatory proteins of the cell membranes that modulate the redox state, and can lead to abnormal activity in various ionic channels. Another event that puts cell viability at risk is the formation of the mitochondrial permeability-transition pore (mPTP), which occurs in response to a mitochondrial overload of calcium in the presence of high levels of ROS. The mitochondrial permeability-transition pore is characterized by an increase in nonspecific permeability in the inner mitochondrial membrane, loss of membrane potential, a possible rupture of the outer membrane, and a severe mitochondrial swelling. When the opening of the mPTP is transitory, the release of cytochrome C from the intermembranal space can activate the caspase cascade that leads to apoptosis. If the opening of the mPTP is prolonged, the mitochondrial content is reduced by quickly causing necrosis (Kann & Kovács, 2007).

Fig. 7. Electronic microphotography that shows the effects of an oxidative stress state caused by ozone exposure on the neuron mitochondria of the rat hippocampus (30.000x). Observe the loss of the external mitochondrial membrane and damage of the mitochondrial crests

production of ROS (Mattson & Liu, 2002).

after exposure to ozone (right)

NF-kB because of phosphorylation of IKB by NIK-IKK and MAPKs (Kim et al., 2000). There are other transcription factors involved in the inflammatory response. The family of the forkhead box O (FOXO) is evolutionarily conserved and integrated by FOXO1, FOXO3a, FOXO4, and FOXO6 in mammals (Van der Heide et al., 2004). The FOXO activation causes the transcription of genes involved in the regulation of the cell-cycle metabolism, cell death, and resistance to oxidative stress (Hedrick, 2009). The activation of these factors of transcription is regulated by growth factors through the phosphorylation of protein kinase B (PKB) (also known as Akt). This formation of phosphoinositide 3-kinase (PI3K) leads to the translocation of FOXO in the cytoplasm to the nucleus (Salih & Brunet, 2008). Both proteinkinase PI3K and Akt are able to mediate many signals of cell survival through inhibition of apoptosis processes (Lawlor and Alessi, 2001). However, little is known about how PI3K-Akt regulates levels of the ROS in cells. FOXO1 reduces the degree of oxidative stress increasing the amount of mRNA coding for Mn-SOD and catalase (Burgering & Medema, 2003). FOXO3a and FOXO4 protect quiescent cells in vitro from oxidative stress. FOXO3a directly activates the transcription of antioxidant enzymes Mn-SOD, catalase, and peroxiredoxin 3 (Prx3) (Marinkovic et al., 2007). This suggests that FOXO also has an important role in the redox balance. Both PI3K and Akt protein kinase are able to mediate many signals of cell survival through inhibition of apoptosis processes (Lawlor & Alessi, 2001).

The hypothesis of molecular inflammation can facilitate a better understanding of the aging process and related diseases such as dementia, cancer, osteoporosis, gingivitis, and vascular diseases.

#### **8. Loss of the redox balance**

The free radicals interact with other cell components, such as proteins, DNA, and lipids, to form multiple catabolic products. An example of these is lipid peroxidation resulting in lipid hydroperoxides and aldehydes that interact with the sulfhydryl groups of proteins causing the loss of protein functionality and thus perpetuating cell damage. The increased levels of calcium and nitric oxide stimulates the production of inflammatory interleukins causing gliosis and increasing the state of oxidative stress. This causes damage and cell death (Sugaya et al., 1998; Ryter et al., 2007), thus establishing a cycle through a chain of oxidative reactions that involve both neurons and glia. These are involved in the maintenance of the damage that extends into adjacent tissue cells.

#### **8.1 The role of mitochondria in oxidative stress**

Mitochondria play a critical role in maintaining cellular homeostasis. This organelle is an important cellular source of energy in producing ATP. In addition they maintain the intracellular levels of calcium within appropriate ranges to mediate cell signaling and control neuronal excitability and synaptic function. In the brain there is a metabolic coupling between vascular substrates, providing oxygen and glucose and the metabolic needs of the brain tissue, formed by neurons and glia alike (Foster et al., 2006).The sequence of events that occurs after neural stimulation includes an initial decrease of oxygen in areas of high demand for this gas (for example, those first stimulated) and a large further increase of oxygen associated with a wide field of arterial vasodilatation. These events are closely related to mitochondrial activity through the production of H2O2 as a signaling molecule (Foster et al., 2006).

NF-kB because of phosphorylation of IKB by NIK-IKK and MAPKs (Kim et al., 2000). There are other transcription factors involved in the inflammatory response. The family of the forkhead box O (FOXO) is evolutionarily conserved and integrated by FOXO1, FOXO3a, FOXO4, and FOXO6 in mammals (Van der Heide et al., 2004). The FOXO activation causes the transcription of genes involved in the regulation of the cell-cycle metabolism, cell death, and resistance to oxidative stress (Hedrick, 2009). The activation of these factors of transcription is regulated by growth factors through the phosphorylation of protein kinase B (PKB) (also known as Akt). This formation of phosphoinositide 3-kinase (PI3K) leads to the translocation of FOXO in the cytoplasm to the nucleus (Salih & Brunet, 2008). Both proteinkinase PI3K and Akt are able to mediate many signals of cell survival through inhibition of apoptosis processes (Lawlor and Alessi, 2001). However, little is known about how PI3K-Akt regulates levels of the ROS in cells. FOXO1 reduces the degree of oxidative stress increasing the amount of mRNA coding for Mn-SOD and catalase (Burgering & Medema, 2003). FOXO3a and FOXO4 protect quiescent cells in vitro from oxidative stress. FOXO3a directly activates the transcription of antioxidant enzymes Mn-SOD, catalase, and peroxiredoxin 3 (Prx3) (Marinkovic et al., 2007). This suggests that FOXO also has an important role in the redox balance. Both PI3K and Akt protein kinase are able to mediate many signals of cell survival through inhibition of apoptosis processes (Lawlor & Alessi,

The hypothesis of molecular inflammation can facilitate a better understanding of the aging process and related diseases such as dementia, cancer, osteoporosis, gingivitis, and vascular

The free radicals interact with other cell components, such as proteins, DNA, and lipids, to form multiple catabolic products. An example of these is lipid peroxidation resulting in lipid hydroperoxides and aldehydes that interact with the sulfhydryl groups of proteins causing the loss of protein functionality and thus perpetuating cell damage. The increased levels of calcium and nitric oxide stimulates the production of inflammatory interleukins causing gliosis and increasing the state of oxidative stress. This causes damage and cell death (Sugaya et al., 1998; Ryter et al., 2007), thus establishing a cycle through a chain of oxidative reactions that involve both neurons and glia. These are involved in the maintenance of the

Mitochondria play a critical role in maintaining cellular homeostasis. This organelle is an important cellular source of energy in producing ATP. In addition they maintain the intracellular levels of calcium within appropriate ranges to mediate cell signaling and control neuronal excitability and synaptic function. In the brain there is a metabolic coupling between vascular substrates, providing oxygen and glucose and the metabolic needs of the brain tissue, formed by neurons and glia alike (Foster et al., 2006).The sequence of events that occurs after neural stimulation includes an initial decrease of oxygen in areas of high demand for this gas (for example, those first stimulated) and a large further increase of oxygen associated with a wide field of arterial vasodilatation. These events are closely related to mitochondrial activity through the production of H2O2 as a signaling molecule

2001).

diseases.

(Foster et al., 2006).

**8. Loss of the redox balance** 

damage that extends into adjacent tissue cells.

**8.1 The role of mitochondria in oxidative stress** 

Like other cells, nerve cells use ATP as a source of energy for biochemical processes involved in various cell functions, and produce ROS as a result of oxidative phosphorylation. The electrical excitability and structural changes, coupled with the synaptic complexity of neurons yields unusual demands in cellular systems that produce or respond to ATP and ROS. Mitochondria in axons and presynaptic terminals provide for sources of ATP to pump ions that are concentrated in these structures to quickly restore the subsequent ion gradients for depolarization and neurotransmitter release. Mitochondria also play a role in the regulation of synaptic functions because of their ability to regulate calcium levels and the production of ROS (Mattson & Liu, 2002).

Neurons in the brain are highly vulnerable to metabolic changes so that a mitochondrial disorder, which causes a decrease in the production of ATP, represents a clear threat to the viability of the neurons and glial cells, the functionality of neural networks, and consequently the normal functions of the brain can be changed. The alteration in the regulation of calcium levels by the failures of the mitochondrial buffer and the release of mitochondria-bound calcium contributes to a severe injury of brain tissue in response to excitotoxicity by glutamate, oxidative stress, or metabolic damage such as trauma. Similarly, an abnormal increase in the generation of ROS by the mitochondria also puts at risk cell viability because many shock-mechanism absorbers might be overwhelmed (Kann & Kovács, 2007).The result is an oxidative damage to the structural and regulatory proteins of the cell membranes that modulate the redox state, and can lead to abnormal activity in various ionic channels. Another event that puts cell viability at risk is the formation of the mitochondrial permeability-transition pore (mPTP), which occurs in response to a mitochondrial overload of calcium in the presence of high levels of ROS. The mitochondrial permeability-transition pore is characterized by an increase in nonspecific permeability in the inner mitochondrial membrane, loss of membrane potential, a possible rupture of the outer membrane, and a severe mitochondrial swelling. When the opening of the mPTP is transitory, the release of cytochrome C from the intermembranal space can activate the caspase cascade that leads to apoptosis. If the opening of the mPTP is prolonged, the mitochondrial content is reduced by quickly causing necrosis (Kann & Kovács, 2007).

Fig. 7. Electronic microphotography that shows the effects of an oxidative stress state caused by ozone exposure on the neuron mitochondria of the rat hippocampus (30.000x). Observe the loss of the external mitochondrial membrane and damage of the mitochondrial crests after exposure to ozone (right)

Oxidative Stress and Neurodegenerative Disease 73

biomolecules and the activity of antioxidant systems clearly determine the redox state in which an individual is found, e.g. we can find high levels of oxidized lipids or proteins, but these can be accompanied by an increase in the activity of the SOD and glutathione peroxidase, or an increase in the levels of reduced glutathione. This indicates that oxidative stress is compensated for because the increase of the prooxidants is accompanied by an increase in the antioxidant systems, which leads to a balanced redox system and tissue changes that are reversible. If the oxidation of the biomolecules increases and there is a decrease in the activity of antioxidant systems, we can then infer that there is a loss of redox balance that produces a state of oxidative stress. This is important to define because many experimental models do not consider these effects and the results are often contradictory. As an example of the models that used ozone, the administration of high doses of this gas in animals may cause a strong antioxidant response and then the increase in the levels of antioxidants has a repair effect on the organism. However, in Wistar rats more than 2-years old, this response causes a severe neuronal and endothelial damage because older animals have a decreased antioxidant activity level in a chronic oxidatively stressed state, and this

Another important factor is the dose and exposure time. In healthy young animals exposed to low doses of ozone for 4-h daily for a prolonged time, a chronic oxidative stress state is generated that causes a process of progressive neurodegeneration. This degenerative process becomes irreversible after 30 days of exposure to this gas. Though animals are no longer exposed to ozone the damage continues to make progress. The progressive neurodegeneration process is shown in figures 8,9,10 in which oxidative stress, depending on the time of exposure to ozone, increases the immunoreactivity to p53 and the

translocation of p53 to the nucleus, indicating an increase in cell death by apoptosis.

p53 control ozone ozone ozone

Fig. 8. The effect of oxidative stress on P53 immunoreactivity caused by chronic exposure to low ozone doses for different times (15, 30, and 60 days) in different brain structures (striatum, hippocampus, and substatia nigra). Note immunoreactivity increases in the

15 days 30 days 60 days

also occurs in chronic-degenerative diseases.

stratium

hippocampus CA3

> substancia nigra

nucleus as a function of the time of exposure to ozone

#### **9. Oxidative stress state and neurodegenerative process in an animal model**

#### **9.1 Ozone as a model for oxidative stress**

Various methods have been used to deal with the study of oxidative stress and its biological significance in the organism. This ranges from biochemistry, cell culture, and animal models to clinical studies. Ozone exposure causes the generation of ROS (Chen & Qu, 1997; Kennedy et al., 1992; Pryor, 1994; Pryor & Church, 1991; Romieu et al., 1998, Saintot et al., 1999) and the formation of relatively stable products (Bocci, 2006; Pryor et al.,1995) able to oxidize DNA, proteins (Kanofsky & Sima, 1993), and lipid membranes (Postlethwait et al., 1998), which if they are not offset causes damage and cell death. In the epithelial lining of the lung, the fluid is characterized by high concentrations of antioxidants, mainly ascorbic acid and glutathione (GSH) (Bocci, 2006). To react with these antioxidants a portion of the inhaled ozone is destroyed. The pulmonary antioxidant defenses are able to neutralize the damage, depending on the dose and exposure time, but when they are overwhelmed a chain of chemical reactions begins that leads to the formation of ROS, caused by secondary exposure to ozone. The ROS pass into the blood, and through the bloodstream reach all the organism, producing a state of widespread oxidative stress (Rivas-Arancibia et al., 2000, 2003). The mechanism of toxicity of ozone is explained as a cascade of reactions (Pryor et al., 1995) in which inhaled ozone reacts with molecules in the fluid of the epithelial lining producing ROS and toxic byproducts, which in turn are able to cause other reactions in the blood. This cascade of reactions is responsible for the toxic effects of ozone both in the lung microenvironment and throughout the body (Ballinger et al., 2005; Bocci, 2006; Pryor et al., 1995). Although the majority of the studies on the effects of oxides of carbon, sulphur, nitrogen, and ozone were made in animals, they indicate that damage may be caused in humans when air pollution increases. Oxidative stress caused by acute or prolonged exposure to ozone causes alterations in the brain plasticity that are manifested by the deficit in the learning processes, memory, and motor activity behavior (Rivas-Arancibia et al., 2000; Dorado-Martínez et al., 2001). Exposure to low doses of ozone over a long time causes a process of progressive neurodegeneration (Angoa-Pérez et al., 2006; Pereyra-Muñoz et al., 2006, Rivas-Arancibia 2010).

Brain tissue is most vulnerable to oxidative damage caused by its high consumption of oxygen, a high metabolic rate, and low levels of antioxidant enzymes, such as SOD, glutathione peroxidase, and catalase. A large increase of lipid peroxidate levels is caused by an increase in ROS, because of the brain's high content of polyunsaturated fatty acids that are highly susceptible to oxidation. Different brain structures show differences in their response to oxidative damage (Hermida-Ameijeiras et al., 2004).

There is clear evidence that air pollution causes an oxidizing environment for humans. High levels of contamination in highly populated cities are correlated with the rise of a number of pathologies, such as autoimmune, degenerative, and neurodegenerative diseases. When using a model of oxidative stress, produced by ozone exposure to low doses (0.25 ppm) for 4 hours daily for different times (7, 15, 30, 60, and 90 days), healthy animals developed a process of progressive neurodegeneration that depends on the exposure time (Angoa-Perez et al., 2006; Pereyra-Muñoz et al., 2006; Rivas-Arancibia et al., 2010, Santiago-Lopez et al., 2010).

The increase in the levels of oxidized lipids, proteins, carbohydrates, and nucleic acids are used as indicators of the state of oxidative stress. Levels of antioxidant enzymes and their activity are used as indicators of antioxidant capacity. The determination of oxidized

**9. Oxidative stress state and neurodegenerative process in an animal model** 

Various methods have been used to deal with the study of oxidative stress and its biological significance in the organism. This ranges from biochemistry, cell culture, and animal models to clinical studies. Ozone exposure causes the generation of ROS (Chen & Qu, 1997; Kennedy et al., 1992; Pryor, 1994; Pryor & Church, 1991; Romieu et al., 1998, Saintot et al., 1999) and the formation of relatively stable products (Bocci, 2006; Pryor et al.,1995) able to oxidize DNA, proteins (Kanofsky & Sima, 1993), and lipid membranes (Postlethwait et al., 1998), which if they are not offset causes damage and cell death. In the epithelial lining of the lung, the fluid is characterized by high concentrations of antioxidants, mainly ascorbic acid and glutathione (GSH) (Bocci, 2006). To react with these antioxidants a portion of the inhaled ozone is destroyed. The pulmonary antioxidant defenses are able to neutralize the damage, depending on the dose and exposure time, but when they are overwhelmed a chain of chemical reactions begins that leads to the formation of ROS, caused by secondary exposure to ozone. The ROS pass into the blood, and through the bloodstream reach all the organism, producing a state of widespread oxidative stress (Rivas-Arancibia et al., 2000, 2003). The mechanism of toxicity of ozone is explained as a cascade of reactions (Pryor et al., 1995) in which inhaled ozone reacts with molecules in the fluid of the epithelial lining producing ROS and toxic byproducts, which in turn are able to cause other reactions in the blood. This cascade of reactions is responsible for the toxic effects of ozone both in the lung microenvironment and throughout the body (Ballinger et al., 2005; Bocci, 2006; Pryor et al., 1995). Although the majority of the studies on the effects of oxides of carbon, sulphur, nitrogen, and ozone were made in animals, they indicate that damage may be caused in humans when air pollution increases. Oxidative stress caused by acute or prolonged exposure to ozone causes alterations in the brain plasticity that are manifested by the deficit in the learning processes, memory, and motor activity behavior (Rivas-Arancibia et al., 2000; Dorado-Martínez et al., 2001). Exposure to low doses of ozone over a long time causes a process of progressive neurodegeneration (Angoa-Pérez et al., 2006; Pereyra-Muñoz et al.,

Brain tissue is most vulnerable to oxidative damage caused by its high consumption of oxygen, a high metabolic rate, and low levels of antioxidant enzymes, such as SOD, glutathione peroxidase, and catalase. A large increase of lipid peroxidate levels is caused by an increase in ROS, because of the brain's high content of polyunsaturated fatty acids that are highly susceptible to oxidation. Different brain structures show differences in their

There is clear evidence that air pollution causes an oxidizing environment for humans. High levels of contamination in highly populated cities are correlated with the rise of a number of pathologies, such as autoimmune, degenerative, and neurodegenerative diseases. When using a model of oxidative stress, produced by ozone exposure to low doses (0.25 ppm) for 4 hours daily for different times (7, 15, 30, 60, and 90 days), healthy animals developed a process of progressive neurodegeneration that depends on the exposure time (Angoa-Perez et al., 2006; Pereyra-Muñoz et al., 2006; Rivas-Arancibia et al., 2010, Santiago-Lopez et al.,

The increase in the levels of oxidized lipids, proteins, carbohydrates, and nucleic acids are used as indicators of the state of oxidative stress. Levels of antioxidant enzymes and their activity are used as indicators of antioxidant capacity. The determination of oxidized

response to oxidative damage (Hermida-Ameijeiras et al., 2004).

**9.1 Ozone as a model for oxidative stress** 

2006, Rivas-Arancibia 2010).

2010).

biomolecules and the activity of antioxidant systems clearly determine the redox state in which an individual is found, e.g. we can find high levels of oxidized lipids or proteins, but these can be accompanied by an increase in the activity of the SOD and glutathione peroxidase, or an increase in the levels of reduced glutathione. This indicates that oxidative stress is compensated for because the increase of the prooxidants is accompanied by an increase in the antioxidant systems, which leads to a balanced redox system and tissue changes that are reversible. If the oxidation of the biomolecules increases and there is a decrease in the activity of antioxidant systems, we can then infer that there is a loss of redox balance that produces a state of oxidative stress. This is important to define because many experimental models do not consider these effects and the results are often contradictory. As an example of the models that used ozone, the administration of high doses of this gas in animals may cause a strong antioxidant response and then the increase in the levels of antioxidants has a repair effect on the organism. However, in Wistar rats more than 2-years old, this response causes a severe neuronal and endothelial damage because older animals have a decreased antioxidant activity level in a chronic oxidatively stressed state, and this also occurs in chronic-degenerative diseases.

Another important factor is the dose and exposure time. In healthy young animals exposed to low doses of ozone for 4-h daily for a prolonged time, a chronic oxidative stress state is generated that causes a process of progressive neurodegeneration. This degenerative process becomes irreversible after 30 days of exposure to this gas. Though animals are no longer exposed to ozone the damage continues to make progress. The progressive neurodegeneration process is shown in figures 8,9,10 in which oxidative stress, depending on the time of exposure to ozone, increases the immunoreactivity to p53 and the translocation of p53 to the nucleus, indicating an increase in cell death by apoptosis.

Fig. 8. The effect of oxidative stress on P53 immunoreactivity caused by chronic exposure to low ozone doses for different times (15, 30, and 60 days) in different brain structures (striatum, hippocampus, and substatia nigra). Note immunoreactivity increases in the nucleus as a function of the time of exposure to ozone

Oxidative Stress and Neurodegenerative Disease 75

Neurodegenerative diseases (diseases in which nerve cells degenerate and die) have a variety of symptoms, can affect different parts of the brain, and the causes are multifactorial and still are not entirely clear. All of them have in common the altered mitochondrial function, increased oxidative damage, presence of abnormal aggregates of proteins and proteasomes, alteration in the metabolism of iron, and changes and dysregulation of inflammation and exitotoxicity. All these form a vicious cycle and can initiate cell death and quickly recruit other cells in its destructive purpose. Oxidized proteins are usually removed by the proteasomes. Inhibition of the proteasomes by a redox state alteration leads to an accumulation of abnormal proteins and ROS production. The ROS-producing agents can initiate neurodegeneration because the ROS causes damaged mitochondria, producing an increase in the Ca2+, and inhibiting the function of the proteasomes. The iron in several areas of the brain increases with age and with other metals promotes oxidation, and with this the

Alzheimer's disease is characterized by the pathogenic presence of intracellular tangles of tau protein containing hyperphosphorylated and extracellular senile plaques formed primarily by β-amyloid oligomers. Different scenarios have been proposed that explain the causes involved in the development of the disease; one is oxidative stress. There are several studies suggesting that accumulation of free radicals in excess formed during normal metabolism is able to cause oxidation of proteins, DNA and RNA, lipid peroxidation, and modification of sugars, thus generating massive neuronal death in the hippocampus, associated with parts of the neocortex (Praticò, 2008). The formation of senile plaques is caused by the intracellular and extracellular accumulation of insoluble beta amyloid in the brain. The peptide beta amyloid is generated by the splitting of the amyloid precursor protein (APP) that involves the enzymes alpha, beta, and gamma secretases (Rajendran, 2008). There are multiple forms of oligomerization that can be found in the beta amyloid peptide. This peptide can play various physiological and pathological roles depending on

Fig. 12. Microphotography that shows the effects of a chronic, oxidative-stress state on the expression of the insoluble form of β-amyloid 1-42 immunoreactivity in a healthy rat

**10. Neurodegenerative diseases** 

aggregation of various proteins.

**10.1 Oxidative stress and Alzheimer's disease** 

exposed to low ozone doses for 4 h daily for 90 days

This result shows that oxidative stress by itself is able to produce damage and neuronal death, which is accompanied by the loss of regulation of the inflammatory response and by changes in astrocytes and microglia.

We can therefore conclude that oxidative stress caused by ozone produces a state of progressive neurodegeneration, which is characterized by neuronal death, changes in the microglia, loss of regulation of the inflammatory response, and loss of the ability of brain to be repaired.

Fig. 9. Double micrograph that shows the effects of oxidative stress on astrocytes (green) and microglia (red) in the rat hippocampus exposed chronically to low ozone doses. Control (A) 30 days (B) 60 days (C), and 90 days (D) of ozone exposure (40x). Note that oxidative stress causes morphological changes in astrocytes and phenotypic changes in microglia

Fig. 10. Micrograph that shows the effects of oxidative stress on the hippocampal neurons of the rat exposed to ozone for 30 days. Control (A) and 30 days of ozone exposure (B) (100x)

#### **10. Neurodegenerative diseases**

74 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

This result shows that oxidative stress by itself is able to produce damage and neuronal death, which is accompanied by the loss of regulation of the inflammatory response and by

We can therefore conclude that oxidative stress caused by ozone produces a state of progressive neurodegeneration, which is characterized by neuronal death, changes in the microglia, loss of regulation of the inflammatory response, and loss of the ability of brain to

Fig. 9. Double micrograph that shows the effects of oxidative stress on astrocytes (green) and microglia (red) in the rat hippocampus exposed chronically to low ozone doses. Control (A) 30 days (B) 60 days (C), and 90 days (D) of ozone exposure (40x). Note that oxidative stress causes morphological changes in astrocytes and phenotypic changes in microglia

Fig. 10. Micrograph that shows the effects of oxidative stress on the hippocampal neurons of the rat exposed to ozone for 30 days. Control (A) and 30 days of ozone exposure (B) (100x)

**A B**

changes in astrocytes and microglia.

be repaired.

Neurodegenerative diseases (diseases in which nerve cells degenerate and die) have a variety of symptoms, can affect different parts of the brain, and the causes are multifactorial and still are not entirely clear. All of them have in common the altered mitochondrial function, increased oxidative damage, presence of abnormal aggregates of proteins and proteasomes, alteration in the metabolism of iron, and changes and dysregulation of inflammation and exitotoxicity. All these form a vicious cycle and can initiate cell death and quickly recruit other cells in its destructive purpose. Oxidized proteins are usually removed by the proteasomes. Inhibition of the proteasomes by a redox state alteration leads to an accumulation of abnormal proteins and ROS production. The ROS-producing agents can initiate neurodegeneration because the ROS causes damaged mitochondria, producing an increase in the Ca2+, and inhibiting the function of the proteasomes. The iron in several areas of the brain increases with age and with other metals promotes oxidation, and with this the aggregation of various proteins.

#### **10.1 Oxidative stress and Alzheimer's disease**

Alzheimer's disease is characterized by the pathogenic presence of intracellular tangles of tau protein containing hyperphosphorylated and extracellular senile plaques formed primarily by β-amyloid oligomers. Different scenarios have been proposed that explain the causes involved in the development of the disease; one is oxidative stress. There are several studies suggesting that accumulation of free radicals in excess formed during normal metabolism is able to cause oxidation of proteins, DNA and RNA, lipid peroxidation, and modification of sugars, thus generating massive neuronal death in the hippocampus, associated with parts of the neocortex (Praticò, 2008). The formation of senile plaques is caused by the intracellular and extracellular accumulation of insoluble beta amyloid in the brain. The peptide beta amyloid is generated by the splitting of the amyloid precursor protein (APP) that involves the enzymes alpha, beta, and gamma secretases (Rajendran, 2008). There are multiple forms of oligomerization that can be found in the beta amyloid peptide. This peptide can play various physiological and pathological roles depending on

Fig. 12. Microphotography that shows the effects of a chronic, oxidative-stress state on the expression of the insoluble form of β-amyloid 1-42 immunoreactivity in a healthy rat exposed to low ozone doses for 4 h daily for 90 days

Oxidative Stress and Neurodegenerative Disease 77

pathogenesis may involve two processes; damage from a specific disease or that combined with damage associated with normal aging. The PD is the result of neurodegeneration in specific areas of the brain (substantia nigra, pars compact, and putamen) resulting in a decrease of dopamine (Cui, 2004). Factors involving dopamine, neuromelanin, increase in the deposits of iron in the substantia nigra, a decrease of ferritin and glutathione (GSH), a deficiency in the role of the complex I mitochondrial respiratory chain, mitochondrial dysfunction and excitotoxicity may be the cause or result of the ROS. Toxins such as paraquat, MPTP, and rotenone have proven to increase the risk of PD in humans. Studies with animal models and cells reveal the oxidative and inflammatory properties of these toxins, and their ability to activate glial cells that subsequently destroy the neighboring dopaminergic neurons. The activity of the complex I mitochondria is deficient in the substance nigra (SN) in PD and can be associated with a genetic abnormality or be the result of oxidative stress. The postulate of a defect in the mitochondrial DNA is still uncertain. It has been shown in the culture of dopaminergic cells that the decrease of glutathione, from the selective loss in the activity of the mitochondrial complex I is an important feature of PD.

Dopamine (DA) is a catecholamine that plays an important role in the human brain as an inhibitory neurotransmitter, particularly involved in the regulation of motor function. It is synthesized in the nerve terminals from tyrosine, the precursor of the amino acid dopaminergic neurons. The synthesis begins with the formation of L-DOPA, through the action of tyrosine hydroxylase and the biopteridines. The former enzyme is the limiting enzyme in the synthesis of dopamine. The activity is strictly controlled. L-DOPA is metabolized to form DA by an aromatic amino acid decarboxylase. In the nerve terminals, the DA is stored in synaptic vesicles with an acid content because it prevents the autoxidation of the DA until it is released. The action ends with the uptake of DA by a membrane transporter and the subsequent reuse or catabolism by the enzyme monoaminooxidase (MAO) or the catechol-o-methyltransferase (COMT). Within the brain, dopaminergic systems are involved in processes of motivation, learning, memory, and motor control. It is estimated that dopamine is > 80% of the total content of catecholamines in the brain. The greatest risk of the DA is that this catechol group oxidizes easily through a process that involves the transfer of an electron to oxygen. Thus, this oxidation results in the formation of an superoxide anion, hydrogen peroxide, hydroxyl radical, and other ROS that are able to generate a state of oxidative stress and start a process of neurodegeneration.

Mechanisms through which the DA stimulates the production of ROS have been proposed. These depend on the presence or absence of enzyme mediators. It is known that the DA of the SN and the striatum (STR) is deaminated by the enzyme MAO located in the outer membrane of the mitochondrion. This reaction has resulted in the production of a superoxide radical, hydroxyl radical, and hydrogen peroxide (Graham, 1978). Proposed mechanisms through which the DA stimulates the production of ROS depend on the

The endogenous dopamine-derived N-methyl(R)salsolinol is one of the most studied derivatives of DA for two reasons. It is present in the human brain and can easily become a neurotoxin able to cause cell death. It has been proposed that this compound can be formed

**10.2.1 Dopaminergic system** 

**10.2.2 Dopamine as a source of ROS in the CNS** 

presence or absence of enzyme mediators.

the path of its formation. The beta amyloid may deposit in specific regions of brain as amyloid plates that form. Break up of the APP is in two phases; a nonamyloidogenic pathway and an amyloidogenic pathway (Rajendran, 2008). In the nonamyloidogenic pathway, the alpha secretase cleaves in the position of the amino acid 83 from the side carboxyl terminal producing a long ectodominion amino (N) - terminal (sAPPα). The result of this process is the formation of C83, which is retained by the membrane to be cut by the gamma secretase forming short fragments of p3. The breakdown by the alpha secretase occurs in the region of beta amyloid.

The amyloidogenic pathway is an alternate way of rupturing of the APP that leads to the generation of beta amyloid. This path is caused by the beta secretase that cuts in the amino acid 99 allowing the release of sAPPβ in the extracellular space. Subsequently the rupture of this fragment between residue 38 and 43 by γ-secretase releases an intact peptide Aβ. The full length of the β-amyloid peptide is 40 residues (Aβ40), with 10% a variant of 42 residues (Aβ42). This latter variant is more hydrophobic and easily causes formation of fibrils and is the form of this peptide which predominates in beta amyloid plaques (Rajendran, 2008; Tillement et al., 2010).

Lower levels of the intracellular β-amyloid peptide produce the internalization of the amyloid precursor protein. This internalization is mediated by a low density receptorrelated lipoprotein 1B (LRP1B), one of the members of the LDL family, This receptor typically joins the precursor protein of amyloid in the plasma membrane to prevent the internalization of the beta amyloid peptide by reducing its production. The failure of these mechanisms and the association of the tau protein causes the internalization of extracellular protein neurons, which gives rise to the production and outsourcing of the insoluble beta amyloid isoform.

Synthesis of soluble β-amyloid is altered during this phase and increases the synthesis of the insoluble, unfolded β-amyloid as part of the insoluble plates of the β-amyloid. The loss of redox homeostasis, both endogenous or exogenous, produces a state of chronic oxidative stress that increases the production of ROS and RNS, causes a reduced expression or activity of antioxidant systems, accelerates ageing, and plays a key role in the pathogenesis and the course of Alzheimer's disease by the altering of many signaling metabolic pathways in the cell by promoting mutations or altering the postransductional mechanisms. The chronic disruption of the oxidation-reduction balance, causes bad protein folding, products of advanced glycosylation, overload of peroxidation of saturated fatty acids (hydroxynonenal, HNE) (Liu, 2008), oxidation of cholesterol, disturbances in the insulin receptor to cause insulin resistance, and oxidation of LDL receptors involved in the reentry of peptide or the APP (Liu, 2008). We can infer that Alzheimer's is the final manifestation of a series of oxidative alterations of metabolism, which involve different biomolecules, in which the loss of the oxide-reduction balance plays a decisive role in the formation of the phosphorylated tau protein and insoluble beta amyloid .

#### **10.2 Environmental toxics and Parkinson's disease**

The initiation and development of the Parkinson disease's (PD) is still uncertain. The pathophysiology is complex and multifactorial and often differs among affected individuals. A large number of studies have provided evidence that loss of redox regulation contributes to all forms of PD, but it has not yet been determined whether the ROS are a primary event or a consequence of the pathogenic factors. An overproduction of ROS is unquestionably an important mediator in cell death in PD (Berg, 2004). It has been suggested that the PD

the path of its formation. The beta amyloid may deposit in specific regions of brain as amyloid plates that form. Break up of the APP is in two phases; a nonamyloidogenic pathway and an amyloidogenic pathway (Rajendran, 2008). In the nonamyloidogenic pathway, the alpha secretase cleaves in the position of the amino acid 83 from the side carboxyl terminal producing a long ectodominion amino (N) - terminal (sAPPα). The result of this process is the formation of C83, which is retained by the membrane to be cut by the gamma secretase forming short fragments of p3. The breakdown by the alpha secretase

The amyloidogenic pathway is an alternate way of rupturing of the APP that leads to the generation of beta amyloid. This path is caused by the beta secretase that cuts in the amino acid 99 allowing the release of sAPPβ in the extracellular space. Subsequently the rupture of this fragment between residue 38 and 43 by γ-secretase releases an intact peptide Aβ. The full length of the β-amyloid peptide is 40 residues (Aβ40), with 10% a variant of 42 residues (Aβ42). This latter variant is more hydrophobic and easily causes formation of fibrils and is the form of this peptide which predominates in beta amyloid plaques (Rajendran, 2008;

Lower levels of the intracellular β-amyloid peptide produce the internalization of the amyloid precursor protein. This internalization is mediated by a low density receptorrelated lipoprotein 1B (LRP1B), one of the members of the LDL family, This receptor typically joins the precursor protein of amyloid in the plasma membrane to prevent the internalization of the beta amyloid peptide by reducing its production. The failure of these mechanisms and the association of the tau protein causes the internalization of extracellular protein neurons, which gives rise to the production and outsourcing of the insoluble beta

Synthesis of soluble β-amyloid is altered during this phase and increases the synthesis of the insoluble, unfolded β-amyloid as part of the insoluble plates of the β-amyloid. The loss of redox homeostasis, both endogenous or exogenous, produces a state of chronic oxidative stress that increases the production of ROS and RNS, causes a reduced expression or activity of antioxidant systems, accelerates ageing, and plays a key role in the pathogenesis and the course of Alzheimer's disease by the altering of many signaling metabolic pathways in the cell by promoting mutations or altering the postransductional mechanisms. The chronic disruption of the oxidation-reduction balance, causes bad protein folding, products of advanced glycosylation, overload of peroxidation of saturated fatty acids (hydroxynonenal, HNE) (Liu, 2008), oxidation of cholesterol, disturbances in the insulin receptor to cause insulin resistance, and oxidation of LDL receptors involved in the reentry of peptide or the APP (Liu, 2008). We can infer that Alzheimer's is the final manifestation of a series of oxidative alterations of metabolism, which involve different biomolecules, in which the loss of the oxide-reduction balance plays a decisive role in the formation of the phosphorylated

The initiation and development of the Parkinson disease's (PD) is still uncertain. The pathophysiology is complex and multifactorial and often differs among affected individuals. A large number of studies have provided evidence that loss of redox regulation contributes to all forms of PD, but it has not yet been determined whether the ROS are a primary event or a consequence of the pathogenic factors. An overproduction of ROS is unquestionably an important mediator in cell death in PD (Berg, 2004). It has been suggested that the PD

occurs in the region of beta amyloid.

tau protein and insoluble beta amyloid .

**10.2 Environmental toxics and Parkinson's disease** 

Tillement et al., 2010).

amyloid isoform.

pathogenesis may involve two processes; damage from a specific disease or that combined with damage associated with normal aging. The PD is the result of neurodegeneration in specific areas of the brain (substantia nigra, pars compact, and putamen) resulting in a decrease of dopamine (Cui, 2004). Factors involving dopamine, neuromelanin, increase in the deposits of iron in the substantia nigra, a decrease of ferritin and glutathione (GSH), a deficiency in the role of the complex I mitochondrial respiratory chain, mitochondrial dysfunction and excitotoxicity may be the cause or result of the ROS. Toxins such as paraquat, MPTP, and rotenone have proven to increase the risk of PD in humans. Studies with animal models and cells reveal the oxidative and inflammatory properties of these toxins, and their ability to activate glial cells that subsequently destroy the neighboring dopaminergic neurons. The activity of the complex I mitochondria is deficient in the substance nigra (SN) in PD and can be associated with a genetic abnormality or be the result of oxidative stress. The postulate of a defect in the mitochondrial DNA is still uncertain. It has been shown in the culture of dopaminergic cells that the decrease of glutathione, from the selective loss in the activity of the mitochondrial complex I is an important feature of PD.

#### **10.2.1 Dopaminergic system**

Dopamine (DA) is a catecholamine that plays an important role in the human brain as an inhibitory neurotransmitter, particularly involved in the regulation of motor function. It is synthesized in the nerve terminals from tyrosine, the precursor of the amino acid dopaminergic neurons. The synthesis begins with the formation of L-DOPA, through the action of tyrosine hydroxylase and the biopteridines. The former enzyme is the limiting enzyme in the synthesis of dopamine. The activity is strictly controlled. L-DOPA is metabolized to form DA by an aromatic amino acid decarboxylase. In the nerve terminals, the DA is stored in synaptic vesicles with an acid content because it prevents the autoxidation of the DA until it is released. The action ends with the uptake of DA by a membrane transporter and the subsequent reuse or catabolism by the enzyme monoaminooxidase (MAO) or the catechol-o-methyltransferase (COMT). Within the brain, dopaminergic systems are involved in processes of motivation, learning, memory, and motor control. It is estimated that dopamine is > 80% of the total content of catecholamines in the brain. The greatest risk of the DA is that this catechol group oxidizes easily through a process that involves the transfer of an electron to oxygen. Thus, this oxidation results in the formation of an superoxide anion, hydrogen peroxide, hydroxyl radical, and other ROS that are able to generate a state of oxidative stress and start a process of neurodegeneration.

#### **10.2.2 Dopamine as a source of ROS in the CNS**

Mechanisms through which the DA stimulates the production of ROS have been proposed. These depend on the presence or absence of enzyme mediators. It is known that the DA of the SN and the striatum (STR) is deaminated by the enzyme MAO located in the outer membrane of the mitochondrion. This reaction has resulted in the production of a superoxide radical, hydroxyl radical, and hydrogen peroxide (Graham, 1978). Proposed mechanisms through which the DA stimulates the production of ROS depend on the presence or absence of enzyme mediators.

The endogenous dopamine-derived N-methyl(R)salsolinol is one of the most studied derivatives of DA for two reasons. It is present in the human brain and can easily become a neurotoxin able to cause cell death. It has been proposed that this compound can be formed

Oxidative Stress and Neurodegenerative Disease 79

polymerization process eventually leads to the generation of a dark pigment called neuromelanin. The dark appearance of the SN is caused by the presence of this pigment containing products derived from the oxidation of the cysteinyl-DA. When the autoxidation of the DA takes place in the presence of L-cysteine, the DA-o-Quinone undergoes a nucleophilic attack by the thiol of the amino acid group to form cysteinyl-DA. This differs from normal oxidation of the DA to form neuromelanin (Hermida-Ameijeiras et al., 2004).

In a balanced oxidation-reduction system, reactive species have an effect as signaling or a regulator of both the glia and neurons in internal signaling pathways, act as regulators of the immune response, which includes inflammatory response, and as regulators of the cell cycle, neuroplasticity, and metabolism. The short-term loss of the oxidation-reduction balance causes an increase in the activity of antioxidant systems to counteract the oxidative stimulus. The endogenous increase of the antioxidant systems play a restorative role of the organism. An enlightening physiological example of this is the repairing role of exercise in chronic degenerative diseases, which is explained by a rise in free radicals as a consequence

The loss of the redox balance (as shown in the model of oxidative stress caused by ozone) implies a loss of regulation of the inflammatory response, which then causes a reparative and self-limiting response. This becomes a perpetual response, a vicious cycle, in which there is mitochondrial failure that leads to a lack of ATP, an increase in the state of oxidative stress, loss of regulation of inflammatory markers, blocking of antioxidant systems, inability to synthesize new proteins, disorders of the proteasome, accumulation of misfolded proteins, and the conformational change in key receptors involved in metabolism and cell signaling. All of them, established slowly over time, can produce chronic degenerative diseases and neurodegenerative diseases as a manifestation of a series of alterations caused by multiple factors, including that of the establishment of a state of chronic-oxidative stress. This plays a key role in the loss of the regulation of cell signaling, of the different responses that lead to neuronal death and loss of brain repair and the altering of the process of neurogenesis. All these are clinically manifested into neurodegenerative diseases long after the vicious cycle has started. The discovery of early oxidative markers specific to each neurodegenerative disease can allow an early diagnosis and break the vicious cycle established by oxidative stress. This can be seen occurring in the near future for the

Acknowledgment to Dirección General de Apoyo al Personal Académico (IN219511-3 to S R-A). The authors thank Gabino Borgonio-Perez for his invaluable technical support.

Angoa-Pérez, M. Jiang, H. Rodriguez, A. Lemini, C. Levine, RA. Rivas-Arancibia, S. (2006).

Estrogen counteracts ozone-induced oxidative stress and nigral neuronal death.

**11. Conclusions** 

of an increase in endogenous antioxidant systems.

treatment and detection of these diseases.

Thanks to Dr. Ellis Glazier for editing this English-language text.

*Neuroreport*, 24, 17, 6, pp. 629-33.

**12. Acknowledgment** 

**13. References** 

by an enzymatic pathway that involves a synthase or a nonenzymatic pathway by the condensation of the DA with acetaldehyde (Naoi et al., 1996).Another derivative of the DA is the tetrahydropapaveroline (THP), which is obtained from enzymatic catabolism. The THP by itself is able to cause necrosis in neuroblastom cells and is related to the pathogenesis of Parkinson's disease. Derivatives of the metabolism of DA act as proneurotoxins in the development of Parkinson's disease. It is known that certain components of tobacco smoke may react with these proneurotoxins preventing its activation. This may explain the beneficial effect of smoking on the incidence of Parkinson's disease (Hermida-Ameijeiras et al., 2004).

#### **10.2.3 The autoxidation of dopamine**

Another mechanism through which DA can contribute to the formation of ROS is its spontaneous autoxidation. The DA is a molecule of the catechol group that can easily oxidize nonenzymatically to form a series of electrochemical type quinoide species. The initial step in the oxidation of the DA involves a reaction with molecular oxygen to form two molecules of the superoxide anion and DA-o-quinone. The formation of the superoxide anions during the autoxidation of the DA leads to the production of hydrogen peroxide by the dismutation of superoxide. The DA-o-quinone then undergoes an intramolecular clyclization to form 5,6-dihydroxiquinoline, which is subsequently oxidized by the DA-o-quinone to form dopaminochrome. This compound undergoes a rearrangement to form 5,6-dihydroxyindole, which in turn is oxidized to an indole quinone. The next

Fig. 13. Showing the effect of oxidative stress on the pathological process and the pathway that is followed depending on the regulation or nonregulation of the inflammatory responses

polymerization process eventually leads to the generation of a dark pigment called neuromelanin. The dark appearance of the SN is caused by the presence of this pigment containing products derived from the oxidation of the cysteinyl-DA. When the autoxidation of the DA takes place in the presence of L-cysteine, the DA-o-Quinone undergoes a nucleophilic attack by the thiol of the amino acid group to form cysteinyl-DA. This differs from normal oxidation of the DA to form neuromelanin (Hermida-Ameijeiras et al., 2004).

#### **11. Conclusions**

78 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

by an enzymatic pathway that involves a synthase or a nonenzymatic pathway by the condensation of the DA with acetaldehyde (Naoi et al., 1996).Another derivative of the DA is the tetrahydropapaveroline (THP), which is obtained from enzymatic catabolism. The THP by itself is able to cause necrosis in neuroblastom cells and is related to the pathogenesis of Parkinson's disease. Derivatives of the metabolism of DA act as proneurotoxins in the development of Parkinson's disease. It is known that certain components of tobacco smoke may react with these proneurotoxins preventing its activation. This may explain the beneficial effect of smoking on the incidence of Parkinson's disease (Hermida-Ameijeiras et

Another mechanism through which DA can contribute to the formation of ROS is its spontaneous autoxidation. The DA is a molecule of the catechol group that can easily oxidize nonenzymatically to form a series of electrochemical type quinoide species. The initial step in the oxidation of the DA involves a reaction with molecular oxygen to form two molecules of the superoxide anion and DA-o-quinone. The formation of the superoxide anions during the autoxidation of the DA leads to the production of hydrogen peroxide by the dismutation of superoxide. The DA-o-quinone then undergoes an intramolecular clyclization to form 5,6-dihydroxiquinoline, which is subsequently oxidized by the DA-o-quinone to form dopaminochrome. This compound undergoes a rearrangement to form 5,6-dihydroxyindole, which in turn is oxidized to an indole quinone. The next

Pathological process

Fig. 13. Showing the effect of oxidative stress on the pathological process and the pathway that is followed depending on the regulation or nonregulation of the inflammatory

Oxidative signals changes

Oxidative stress state

Antioxidant systems fail

Progressive Neurodegenerative process

> Degenerative diseases

Cellular death

Autoimmune diseases

Inflammatory response dysregulation

al., 2004).

ROS INCREASES

> Infection diseases

Inflammatory response regulation

Homeostasis Recovery

responses

**10.2.3 The autoxidation of dopamine** 

In a balanced oxidation-reduction system, reactive species have an effect as signaling or a regulator of both the glia and neurons in internal signaling pathways, act as regulators of the immune response, which includes inflammatory response, and as regulators of the cell cycle, neuroplasticity, and metabolism. The short-term loss of the oxidation-reduction balance causes an increase in the activity of antioxidant systems to counteract the oxidative stimulus. The endogenous increase of the antioxidant systems play a restorative role of the organism. An enlightening physiological example of this is the repairing role of exercise in chronic degenerative diseases, which is explained by a rise in free radicals as a consequence of an increase in endogenous antioxidant systems.

The loss of the redox balance (as shown in the model of oxidative stress caused by ozone) implies a loss of regulation of the inflammatory response, which then causes a reparative and self-limiting response. This becomes a perpetual response, a vicious cycle, in which there is mitochondrial failure that leads to a lack of ATP, an increase in the state of oxidative stress, loss of regulation of inflammatory markers, blocking of antioxidant systems, inability to synthesize new proteins, disorders of the proteasome, accumulation of misfolded proteins, and the conformational change in key receptors involved in metabolism and cell signaling. All of them, established slowly over time, can produce chronic degenerative diseases and neurodegenerative diseases as a manifestation of a series of alterations caused by multiple factors, including that of the establishment of a state of chronic-oxidative stress. This plays a key role in the loss of the regulation of cell signaling, of the different responses that lead to neuronal death and loss of brain repair and the altering of the process of neurogenesis. All these are clinically manifested into neurodegenerative diseases long after the vicious cycle has started. The discovery of early oxidative markers specific to each neurodegenerative disease can allow an early diagnosis and break the vicious cycle established by oxidative stress. This can be seen occurring in the near future for the treatment and detection of these diseases.

#### **12. Acknowledgment**

Acknowledgment to Dirección General de Apoyo al Personal Académico (IN219511-3 to S R-A). The authors thank Gabino Borgonio-Perez for his invaluable technical support. Thanks to Dr. Ellis Glazier for editing this English-language text.

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**4** 

*USA* 

**Free Radicals in Neurodegenerative Diseases:** 

Chirakkal V. Krishnan1,2, Merrill Garnett1 and Frank Antonawich1

*1Garnett McKeen Laboratory, Inc, Bohemia;* 

*2Department of Chemistry, Stony Brook University;* 

**Modulation by Palladium α-Lipoic Acid Complex** 

Our research is based on the need for modern medicine to develop a safe and nontoxic product with a wide spectrum of uses. We strongly believe that one of the best ways to achieve this is to have a product that participates actively in most of the roles played by the mitochondria for optimal cellular function. Mitochondria are ubiquitous, and taking care of mitochondria is similar to taking care of all the parts leading to greater achievements than

Oxidative stress is caused by the chemical imbalance between reactive oxygen species (ROS) production and their breakdown by antioxidants. Over-abundance of ROS has been found during neuronal development, as well as in numerous neuropathological conditions. A

Oxidative stress and mitochondrial dysfunction have been closely associated in many subcellular, cellular, animal, and human studies of both acute brain injury such as ischemia and stroke and neurodegenerative processes such as Parkinson's, Alzheimer's and Huntington's. While the oxidative stress occurs chronically in Alzheimer's disease, it is more acute in ischemic reperfusion injury. The consequences of mitochondrial dysfunction include DNA and protein damage, lipid peroxidation, disruption of the mitochondrial permeability transition, Ca2+ homeostasis, and triggering apoptosis. It is essential to have a healthy mitochondria contributing substantially to the physical, mental, and emotional elements needed to support the well being of patients suffering from brain injury or neurodegenerative

Energy metabolism, calcium regulation, and apoptosis-signaling pathways are the major roles of mitochondria. Energy requirements dictate the number of mitochondria in a cell [Beattie, 2002; Nagley et. al., 2010]. Cardiac and skeletal muscles, the brain, and the liver have the most mitochondria because of their high metabolic activities. These cells are also exposed to the most oxidative stress because the source of free radical production is also the mitochondria. Due to low levels of antioxidants in neurons, they are intrinsically illequipped to defend against an increase in oxidative stress. Glial cells including astrocytes

Our search for an extremely safe (up to 40 mL/day, 0.037 M aqueous solution) and nontoxic therapeutic agent resulted in the development of a novel redox molecule, "Palladium α-Lipoic Acid Complex" that is active in mitochondrial cellular metabolism and other

play a supplementary role in antioxidant defense of neurons [Higgins et. al., 2010].

predominant feature of neuronal injury is the onset of oxidative stress.

**1. Introduction** 

diseases.

the sum of the parts [Krishnan et. al., 2011].


### **Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex**

Chirakkal V. Krishnan1,2, Merrill Garnett1 and Frank Antonawich1

*1Garnett McKeen Laboratory, Inc, Bohemia; 2Department of Chemistry, Stony Brook University; USA* 

#### **1. Introduction**

88 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

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> Our research is based on the need for modern medicine to develop a safe and nontoxic product with a wide spectrum of uses. We strongly believe that one of the best ways to achieve this is to have a product that participates actively in most of the roles played by the mitochondria for optimal cellular function. Mitochondria are ubiquitous, and taking care of mitochondria is similar to taking care of all the parts leading to greater achievements than the sum of the parts [Krishnan et. al., 2011].

> Oxidative stress is caused by the chemical imbalance between reactive oxygen species (ROS) production and their breakdown by antioxidants. Over-abundance of ROS has been found during neuronal development, as well as in numerous neuropathological conditions. A predominant feature of neuronal injury is the onset of oxidative stress.

> Oxidative stress and mitochondrial dysfunction have been closely associated in many subcellular, cellular, animal, and human studies of both acute brain injury such as ischemia and stroke and neurodegenerative processes such as Parkinson's, Alzheimer's and Huntington's. While the oxidative stress occurs chronically in Alzheimer's disease, it is more acute in ischemic reperfusion injury. The consequences of mitochondrial dysfunction include DNA and protein damage, lipid peroxidation, disruption of the mitochondrial permeability transition, Ca2+ homeostasis, and triggering apoptosis. It is essential to have a healthy mitochondria contributing substantially to the physical, mental, and emotional elements needed to support the well being of patients suffering from brain injury or neurodegenerative diseases.

> Energy metabolism, calcium regulation, and apoptosis-signaling pathways are the major roles of mitochondria. Energy requirements dictate the number of mitochondria in a cell [Beattie, 2002; Nagley et. al., 2010]. Cardiac and skeletal muscles, the brain, and the liver have the most mitochondria because of their high metabolic activities. These cells are also exposed to the most oxidative stress because the source of free radical production is also the mitochondria. Due to low levels of antioxidants in neurons, they are intrinsically illequipped to defend against an increase in oxidative stress. Glial cells including astrocytes play a supplementary role in antioxidant defense of neurons [Higgins et. al., 2010].

> Our search for an extremely safe (up to 40 mL/day, 0.037 M aqueous solution) and nontoxic therapeutic agent resulted in the development of a novel redox molecule, "Palladium α-Lipoic Acid Complex" that is active in mitochondrial cellular metabolism and other

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 91

chain reactions and damage to proteins, polysaccharides, and DNA. The steady state

Another beneficial aspect of free radicals is that they participate with leukocytes in phagocytosis, the engulfing and destruction of particulate matter and bacteria. Leukocytes contain the enzymes of the hexose-monophosphate shunt, glycolysis, citric acid cycle, and respiratory enzymes. Phagocytosis requires a lot of energy, which is obtained from glucose by glycolysis and also by the hexose-monophosphate shunt. The role of this shunt is to produce hydrogen peroxide from superoxide free radical, which is used in the phagocytotic

Oxidative damage to many biological molecules compromise the viability of cells. The

Antioxidants or physiologic reducing agents get oxidized by donating electrons to free radicals. The relative abilities of antioxidants to donate electrons and free radicals to accept

The rate of a reaction cannot be predicted from redox potentials. However the direction of a reaction, decided by the free energy of the reaction, can be predicted from redox potentials. A positive voltage for the net reaction predicts the spontaneity of the reaction. Examples of one-electron and two-electron reduction potentials of reactions of biological interest are easily available [Buettner, 1993; Voet D. & Voet J. G., 1995; Krishnan et.al., 2011]. Positive voltage indicates that vitamin E is spontaneously regenerated by ascorbate or vitamin C.


The criteria often used to evaluate the antioxidant potential as well as preventive or therapeutic applications of a compound are 1) specificity of free radical quenching, 2) metal chelating ability, 3) interaction with other antioxidants, 4) effects on gene expression, 5) absorption and bioavailability, 6) concentration in tissues, cells, and extracellular fluid,

ROS generation, which increases with increasing stress conditions, is characteristic for all tissues and cells. The interaction of molecular oxygen with biological molecules is not energetically favored because of the unique electronic configuration of molecular oxygen. Molecular oxygen, a diatomic molecule, in its normal or ground state is in its triplet state,

molecular orbitals. Most organic molecules cannot react with this spin-forbidden triplet oxygen because of their singlet configurations with antiparallel electron spins. By adding energy, the triplet oxygen can form two types of excited singlet oxygen, 1O2, 1∑+g with the two electrons of opposite spins in two separate molecular orbitals or 1∆g with the two electrons of opposite spins occupying one molecular orbital leaving the other molecular orbital empty. The former is too short lived from a biological point of view. A two electron interaction with molecular oxygen is thus not possible without a spin inversion because it will result in parallel spins in the same orbital, which is spin-forbidden. Thus the preferable interaction is reduction of oxygen by addition of one electron at a time. This process leads to the production of oxygen radicals that can cause cellular damage. When one of the two unpaired electrons is excited and changes its spin, the resulting high energy singlet oxygen with two electrons and opposite spins in the two orbitals is capable of two-electron interactions. The initial step or one electron reduction of oxygen requires energy. The subsequent reduction reactions with appropriate electron donors can proceed spontaneously.

g]. It has two electrons of parallel spins singly occupied in its two π\* antibonding

and 7) location (in aqueous or membrane domains or in both) [Packer et. al., 1995].

process. Thus the free radicals produced in this process are beneficial [Singh, 2006].

electrons are a function of their reduction or redox potentials, measured in volts.

results of this free radical mischief have been assessed [Sies, 1986].

<sup>i</sup><sup>−</sup> and H2O2 are ~ 10-10 M and 5 x 10-9 M respectively [Dröge, 2002].

concentrations of O2

3O2 [3∑-

functions. The selection of the naturally occurring coenzyme, α-lipoic acid, as our ligand was based on its safety as well as its redox, antioxidant, and fatty acid properties. After selecting the ligand that plays a critical role in biological energy metabolism and numerous other functions, we wanted to tweak the properties of the ligand by complexing it with a metal that is safe and has very high catalytic and electronic properties. After numerous investigations with a variety of metals, the final selection was made to use palladium.

The properties of the resulting palladium α-lipoic acid complex were remarkable in many ways and have been reviewed recently [Krishnan et. al., 2011]. Briefly, this complex enhances the enzymatic activities of Krebs cycle enzymes, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase and mitochondrial respiratory enzymes, complex I, complex II, complex III, and complex IV. These enzymatic activity enhancements by the metal complex were, in general, much greater than that of the ligand, α-lipoic acid. Coupling this increase in the efficiency of the aerobic metabolic cascade with its powerful antioxidant properties, such as scavenging of free radicals, lowering lipid peroxidation, increasing the levels of glutathione, glutathione peroxidase, manganese superoxide dismutase, and catalase, gave us a powerful weapon to combat fatigue associated with numerous mitochondrial abnormalities. The complex also modulates mitochondrial dysfunction, acts as a prophylactic for neuronal protection from transient ischemic attack, repairs DNA damage resulting from radiation, acts as a prophylactic for protection from radiation, and improves the quality of life. The electronic properties corresponding to tunnel diode behavior and the therapeutic ability/potential of this complex may be exploited in its applications for combating brain injury resulting from transient ischemic attack, death of neurons and other progressive loss of structure or function of neurons associated with diseases such as Parkinson's and Alzheimer's.

#### **2. Oxygen (the source of free radicals) and antioxidants**

The appearance of oxygen in the atmosphere is associated with a great expansion of the varieties and numbers of higher living forms. Oxygen is the source for the emergence of respiratory metabolism and energy efficiency. It is also the source of free radicals such as hydroxyl and superoxide. Oxygen's imprint on earth's metabolic evolution, the effect of oxygen on biochemical networks, and the evolution of complex life have been reviewed [Falkowski, 2006; Raymond & Segré, 2006].

Oxygen is the most abundant element in the earth's mantle. Its limited solubility in water (48.9 mL of oxygen at 1 atm pressure in 1 liter water at 0°C) makes aquatic life possible [Pauling, 1970]. Ordinary oxygen consists of diatomic molecules with an unusual electronic structure. Instead of having a double bond between the two atoms in molecular oxygen in the ground state, only one shared pair is formed leaving two unshared electrons. This makes the molecule a diradical and paramagnetic. Liquid oxygen exhibits a pale blue color.

A free radical is a highly reactive species with an unpaired electron. It can be a neutral species such as hydroxyl, HO<sup>i</sup> **,** or a charged negative ion (anion) such as superoxide, O2 <sup>i</sup>−, or a charged positive ion (cation) such as the guanine radical. An unpaired electron is shown as a dot after the symbol (example: HO<sup>i</sup> ). Being good oxidizing agents, free radicals can remove an electron from other materials and in that process get reduced with the pairing of the unpaired electron. They often participate in chain reactions producing new free radicals. Small fluctuations in the steady state concentrations of free radicals play a significant role in intracellular signaling. Uncontrolled increases in the production of these radicals lead to

functions. The selection of the naturally occurring coenzyme, α-lipoic acid, as our ligand was based on its safety as well as its redox, antioxidant, and fatty acid properties. After selecting the ligand that plays a critical role in biological energy metabolism and numerous other functions, we wanted to tweak the properties of the ligand by complexing it with a metal that is safe and has very high catalytic and electronic properties. After numerous investigations with a variety of metals, the final selection was made to use palladium. The properties of the resulting palladium α-lipoic acid complex were remarkable in many ways and have been reviewed recently [Krishnan et. al., 2011]. Briefly, this complex enhances the enzymatic activities of Krebs cycle enzymes, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase and mitochondrial respiratory enzymes, complex I, complex II, complex III, and complex IV. These enzymatic activity enhancements by the metal complex were, in general, much greater than that of the ligand, α-lipoic acid. Coupling this increase in the efficiency of the aerobic metabolic cascade with its powerful antioxidant properties, such as scavenging of free radicals, lowering lipid peroxidation, increasing the levels of glutathione, glutathione peroxidase, manganese superoxide dismutase, and catalase, gave us a powerful weapon to combat fatigue associated with numerous mitochondrial abnormalities. The complex also modulates mitochondrial dysfunction, acts as a prophylactic for neuronal protection from transient ischemic attack, repairs DNA damage resulting from radiation, acts as a prophylactic for protection from radiation, and improves the quality of life. The electronic properties corresponding to tunnel diode behavior and the therapeutic ability/potential of this complex may be exploited in its applications for combating brain injury resulting from transient ischemic attack, death of neurons and other progressive loss of structure or

function of neurons associated with diseases such as Parkinson's and Alzheimer's.

The appearance of oxygen in the atmosphere is associated with a great expansion of the varieties and numbers of higher living forms. Oxygen is the source for the emergence of respiratory metabolism and energy efficiency. It is also the source of free radicals such as hydroxyl and superoxide. Oxygen's imprint on earth's metabolic evolution, the effect of oxygen on biochemical networks, and the evolution of complex life have been reviewed

Oxygen is the most abundant element in the earth's mantle. Its limited solubility in water (48.9 mL of oxygen at 1 atm pressure in 1 liter water at 0°C) makes aquatic life possible [Pauling, 1970]. Ordinary oxygen consists of diatomic molecules with an unusual electronic structure. Instead of having a double bond between the two atoms in molecular oxygen in the ground state, only one shared pair is formed leaving two unshared electrons. This makes

A free radical is a highly reactive species with an unpaired electron. It can be a neutral

a charged positive ion (cation) such as the guanine radical. An unpaired electron is shown as a dot after the symbol (example: HO<sup>i</sup> ). Being good oxidizing agents, free radicals can remove an electron from other materials and in that process get reduced with the pairing of the unpaired electron. They often participate in chain reactions producing new free radicals. Small fluctuations in the steady state concentrations of free radicals play a significant role in intracellular signaling. Uncontrolled increases in the production of these radicals lead to

**,** or a charged negative ion (anion) such as superoxide, O2

<sup>i</sup>−, or

the molecule a diradical and paramagnetic. Liquid oxygen exhibits a pale blue color.

**2. Oxygen (the source of free radicals) and antioxidants** 

[Falkowski, 2006; Raymond & Segré, 2006].

species such as hydroxyl, HO<sup>i</sup>

chain reactions and damage to proteins, polysaccharides, and DNA. The steady state concentrations of O2 <sup>i</sup><sup>−</sup> and H2O2 are ~ 10-10 M and 5 x 10-9 M respectively [Dröge, 2002].

Another beneficial aspect of free radicals is that they participate with leukocytes in phagocytosis, the engulfing and destruction of particulate matter and bacteria. Leukocytes contain the enzymes of the hexose-monophosphate shunt, glycolysis, citric acid cycle, and respiratory enzymes. Phagocytosis requires a lot of energy, which is obtained from glucose by glycolysis and also by the hexose-monophosphate shunt. The role of this shunt is to produce hydrogen peroxide from superoxide free radical, which is used in the phagocytotic process. Thus the free radicals produced in this process are beneficial [Singh, 2006].

Oxidative damage to many biological molecules compromise the viability of cells. The results of this free radical mischief have been assessed [Sies, 1986].

Antioxidants or physiologic reducing agents get oxidized by donating electrons to free radicals. The relative abilities of antioxidants to donate electrons and free radicals to accept electrons are a function of their reduction or redox potentials, measured in volts.

The rate of a reaction cannot be predicted from redox potentials. However the direction of a reaction, decided by the free energy of the reaction, can be predicted from redox potentials. A positive voltage for the net reaction predicts the spontaneity of the reaction. Examples of one-electron and two-electron reduction potentials of reactions of biological interest are easily available [Buettner, 1993; Voet D. & Voet J. G., 1995; Krishnan et.al., 2011]. Positive voltage indicates that vitamin E is spontaneously regenerated by ascorbate or vitamin C.


The criteria often used to evaluate the antioxidant potential as well as preventive or therapeutic applications of a compound are 1) specificity of free radical quenching, 2) metal chelating ability, 3) interaction with other antioxidants, 4) effects on gene expression, 5) absorption and bioavailability, 6) concentration in tissues, cells, and extracellular fluid, and 7) location (in aqueous or membrane domains or in both) [Packer et. al., 1995].

ROS generation, which increases with increasing stress conditions, is characteristic for all tissues and cells. The interaction of molecular oxygen with biological molecules is not energetically favored because of the unique electronic configuration of molecular oxygen. Molecular oxygen, a diatomic molecule, in its normal or ground state is in its triplet state, 3O2 [3∑ g]. It has two electrons of parallel spins singly occupied in its two π\* antibonding molecular orbitals. Most organic molecules cannot react with this spin-forbidden triplet oxygen because of their singlet configurations with antiparallel electron spins. By adding energy, the triplet oxygen can form two types of excited singlet oxygen, 1O2, 1∑+g with the two electrons of opposite spins in two separate molecular orbitals or 1∆g with the two electrons of opposite spins occupying one molecular orbital leaving the other molecular orbital empty. The former is too short lived from a biological point of view. A two electron interaction with molecular oxygen is thus not possible without a spin inversion because it will result in parallel spins in the same orbital, which is spin-forbidden. Thus the preferable interaction is reduction of oxygen by addition of one electron at a time. This process leads to the production of oxygen radicals that can cause cellular damage. When one of the two unpaired electrons is excited and changes its spin, the resulting high energy singlet oxygen with two electrons and opposite spins in the two orbitals is capable of two-electron interactions. The initial step or one electron reduction of oxygen requires energy. The subsequent reduction reactions with appropriate electron donors can proceed spontaneously.

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 93

the SOD is to increase the rate of the reaction to that of a diffusion controlled process

The spontaneous decomposition of superoxide, but not the catalytic decomposition, produces singlet oxygen instead of the normal molecular oxygen [Klotz, 2002]. Singlet oxygen is produced by stimulated neutrophils in vivo. Superoxide produced by NADPH catalysis spontaneously dismutates to H2O2 and singlet O2. Also the catalytic reaction of myeloperoxidase with H2O2 and Cl<sup>−</sup> produces hypochlorite, which reacts with more H2O2

Hydrogen peroxide is removed by GSHPx, at the expense of glutathione or γ-glutamylcysteinylglycine (GSH), and by catalase. Selenium (as selenocysteine) is a cofactor of GSHPx [Beattie, 2002]. GSHPx is located in both the mitochondrial and cytosolic compartments of the cell. Hydrogen peroxide and organic hydroperoxides in the cytosol are also destroyed by GSHPx. Catalase is highest in peroxisomes and it is less in cytosol and

GSHPx

Glutathione or, GSH is a major nonenzymatic antioxidant. The aqueous compartments of cells and their organelles usually contain millimolar levels of GSH. It is the cell's primary preventative antioxidant. It can react with various highly oxidizing species such as HO<sup>i</sup>

GSHPx GSSG +O O +GSSG 2 2

SOD and GSH provide an excellent natural combination for cellular antioxidant defense by

while the mitochondrial respiration keeps O2 about 0 to 10 µM in the cell. Therefore, 99% of

Lipid (probably a polyunsaturated fatty acid) peroxidation is a free radical chain reaction process. Superoxide is a mediator in oxidative chain reactions. It is known to initiate as well as terminate this process. Overproduction of superoxide initiates the chain reaction by

3 2 O Ferritin-Fe O Ferritin Fe 2 2

Lipid-O lipid-H Lipid lipid-OH + →+ i i (15)

obvious. The normal GSH to GSSG ratio in erythrocytes is 100:1 [Beattie, 2002].

Catalase

and produce H2O, ROH, or ROOH and GS<sup>i</sup>

radical can react rapidly with GSH, most efficiently via GS-

formed should react with GSH to make GSSG and O2

mobilizing iron from the tissue protein ferritin [McCord, 1998].

strong reducing species. It produces O2

<sup>i</sup><sup>−</sup> and HO<sup>i</sup>

2 2 22 2 2H +O O H O O +− − *SOD* + ⎯⎯⎯→ + i i (9)

2 2 <sup>2</sup> 2GSH+H O GSSG 2H O ⎯⎯⎯⎯→ + (10)

2 2 2 2 2H O ⎯⎯⎯⎯→2H O + O (11)

<sup>i</sup><sup>−</sup> and glutathione disulfide, GSSG, by reaction with

i i − − ⎯⎯⎯⎯→ (12)

respectively. The intracellular concentration of GSH is about 1 mM

<sup>−</sup> + + + →+ + <sup>i</sup> (13)

<sup>2</sup> - 3 Fe lipid-OOH(lipidhydroperoxide) Lipid-O OH Fe <sup>+</sup> <sup>+</sup> + → + + <sup>i</sup> (14)

,

(glutathiyl radical). Glutathiyl

to make GSSG**.**-, which is a very

<sup>i</sup><sup>−</sup> . Thus the importance of SOD is

[Turrens, 2003].

producing singlet O2, H2O and Cl<sup>−</sup> .

mitochondria [Beattie, 2002].

RO<sup>i</sup>

GS**.**

oxygen.

removing O2

or ROO<sup>i</sup>

One must wonder at this stage whether nature has given this unique electron configuration for normal molecular oxygen purposefully or not, recognizing not the liability of free radicals but instead their usefulness.

Oxygen undergoes a series of progressive one electron reduction reactions, 2-5. The hydroxyl radical has a very short half life (10-9 s) with the highest rate constant with target molecules [Sies, 1993]. It reacts practically at the site of generation. It is one of the strongest oxidizing agents in nature (redox potential of 2310 mV). It is undoubtedly the most dangerous, with its well known involvement in lipid peroxidation of cell membranes and generation of other toxic radicals. The formation of HO<sup>i</sup> is catalyzed by transition metals in a reduced state. The resulting oxidized metal is reduced back by O2 <sup>i</sup><sup>−</sup> and helps the formation of HO<sup>i</sup> repeatedly.

$$\text{O}\_2 + \text{e}^- \rightarrow \text{O}\_2^{\prime-} \text{(superoxide anion)}\tag{2}$$

$$\text{H}\_2\text{O}\_2^- + \text{e}^- + 2\text{H}^+ \rightarrow \text{H}\_2\text{O}\_2 \text{ (hydrogen peroxide)}\tag{3}$$

$$\text{CH}\_2\text{O}\_2 + \text{e}^- \rightarrow \text{OH}^- + \text{HO}^\* \text{ (hydroxyl radical)}\tag{4}$$

$$\rm{HO}^\* + \rm{e}^- + \rm{H}^+ \rightarrow \rm{H}\_2\rm{O} \tag{5}$$

Superoxide ion is the precursor of most reactive oxygen species. It can act both as a reducing agent or reductant (for Fe3+) and as an oxidizing agent or oxidant for catecholamines. To minimize production of free radical chain reactions such as reactions (6) and (7), metal ions are sequestered under physiological conditions by proteins.

$$\text{O}\_2^{\text{-}} + \text{Fe}^{3+} \rightarrow \text{O}\_2 + \text{Fe}^{2+} \tag{6}$$

$$\text{H}\_2\text{O}\_2 + \text{Fe}^{2+} + \text{H}^+ \rightarrow \text{HO}^\cdot + \text{Fe}^{3+} + \text{H}\_2\text{O} \text{ (classical Fenton reaction)}\tag{7}$$

Adding reactions (6) and (7) gives the Haber-Weiss reaction (8) which is catalyzed by metal ions.

$$\rm H\_2O\_2 + O\_2^{\cdot -} + H^+ \rightarrow HO^\cdot + O\_2 + H\_2O \tag{8}$$

Damage to both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) may result in mutations. Nonspecific binding of Fe2+ to DNA may result in the formation of HO<sup>i</sup> (reaction 7) that attack individual bases and cause strand breaks.

Strategies of antioxidant defense in terms of prevention, intervention, and repair have been elegantly summarized [Sies, 1993]. Cells employ enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSHPx), and catalase to scavenge free radicals. These enzymes are known as preventive antioxidants because they eliminate the species involved in the initiation of free radical chain reactions. SOD has three isoforms: Copper/Zinc SOD, Manganese SOD, and extracellular SOD. A very high rate of production of ~ 300 nmol superoxide/min/mg protein has been reported for a reaction of cyrtochromeb5 reductase using NADH as an electron donor [Starkov & Wallace, 2006]. The superoxide anion shown in reaction (2) and released by the mitochondria undergoes the dismutation reaction either spontaneously or catalytically(reaction 9) producing hydrogen peroxide and O2. The role of

One must wonder at this stage whether nature has given this unique electron configuration for normal molecular oxygen purposefully or not, recognizing not the liability of free

Oxygen undergoes a series of progressive one electron reduction reactions, 2-5. The hydroxyl radical has a very short half life (10-9 s) with the highest rate constant with target molecules [Sies, 1993]. It reacts practically at the site of generation. It is one of the strongest oxidizing agents in nature (redox potential of 2310 mV). It is undoubtedly the most dangerous, with its well known involvement in lipid peroxidation of cell membranes and generation of other toxic radicals. The formation of HO<sup>i</sup> is catalyzed by transition metals in

HO e H H O2

Superoxide ion is the precursor of most reactive oxygen species. It can act both as a reducing agent or reductant (for Fe3+) and as an oxidizing agent or oxidant for catecholamines. To minimize production of free radical chain reactions such as reactions (6) and (7), metal ions

3 2 O Fe O Fe 2 2

Adding reactions (6) and (7) gives the Haber-Weiss reaction (8) which is catalyzed by metal

H O O H HO +O H O 22 2 2 2

Damage to both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) may result in mutations. Nonspecific binding of Fe2+ to DNA may result in the formation of HO<sup>i</sup> (reaction

Strategies of antioxidant defense in terms of prevention, intervention, and repair have been elegantly summarized [Sies, 1993]. Cells employ enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSHPx), and catalase to scavenge free radicals. These enzymes are known as preventive antioxidants because they eliminate the species involved in the initiation of free radical chain reactions. SOD has three isoforms: Copper/Zinc SOD, Manganese SOD, and extracellular SOD. A very high rate of production of ~ 300 nmol superoxide/min/mg protein has been reported for a reaction of cyrtochromeb5 reductase using NADH as an electron donor [Starkov & Wallace, 2006]. The superoxide anion shown in reaction (2) and released by the mitochondria undergoes the dismutation reaction either spontaneously or catalytically(reaction 9) producing hydrogen peroxide and O2. The role of

<sup>−</sup> <sup>−</sup> + → <sup>i</sup> (superoxide anion) (2)

− − +→ + <sup>i</sup> (hydroxyl radical) (4)

− + <sup>i</sup> ++ → (5)

<sup>−</sup> + + + →+ <sup>i</sup> (6)

<sup>−</sup> <sup>+</sup> + +→ + i i (8)

+ + <sup>+</sup> + +→ + + <sup>i</sup> (classical Fenton reaction) (7)

−− + <sup>i</sup> ++ → (hydrogen peroxide) (3)

<sup>i</sup><sup>−</sup> and helps the

a reduced state. The resulting oxidized metal is reduced back by O2

Oe O 2 2

O e 2H H O 2 2 <sup>2</sup>

H O e OH HO 2 2

are sequestered under physiological conditions by proteins.

7) that attack individual bases and cause strand breaks.

2 3 H O Fe H HO Fe H O 2 2 <sup>2</sup>

radicals but instead their usefulness.

formation of HO<sup>i</sup> repeatedly.

ions.

the SOD is to increase the rate of the reaction to that of a diffusion controlled process [Turrens, 2003].

$$\mathrm{2H}^+ \mathrm{+O}\_2^{\cdot-} + \mathrm{O}\_2^{\cdot-} \xrightarrow{\mathrm{SO}D} \mathrm{H}\_2\mathrm{O}\_2 + \mathrm{O}\_2 \tag{9}$$

The spontaneous decomposition of superoxide, but not the catalytic decomposition, produces singlet oxygen instead of the normal molecular oxygen [Klotz, 2002]. Singlet oxygen is produced by stimulated neutrophils in vivo. Superoxide produced by NADPH catalysis spontaneously dismutates to H2O2 and singlet O2. Also the catalytic reaction of myeloperoxidase with H2O2 and Cl<sup>−</sup> produces hypochlorite, which reacts with more H2O2 producing singlet O2, H2O and Cl<sup>−</sup> .

Hydrogen peroxide is removed by GSHPx, at the expense of glutathione or γ-glutamylcysteinylglycine (GSH), and by catalase. Selenium (as selenocysteine) is a cofactor of GSHPx [Beattie, 2002]. GSHPx is located in both the mitochondrial and cytosolic compartments of the cell. Hydrogen peroxide and organic hydroperoxides in the cytosol are also destroyed by GSHPx. Catalase is highest in peroxisomes and it is less in cytosol and mitochondria [Beattie, 2002].

$$2\text{GSH} + \text{H}\_2\text{O}\_2 \xrightarrow{\text{GSHP}} \text{GSSG} + 2\text{H}\_2\text{O} \tag{10}$$

$$2\text{H}\_2\text{O}\_2 \xrightarrow{\text{Catalyst}} 2\text{H}\_2\text{O} + \text{O}\_2\tag{11}$$

Glutathione or, GSH is a major nonenzymatic antioxidant. The aqueous compartments of cells and their organelles usually contain millimolar levels of GSH. It is the cell's primary preventative antioxidant. It can react with various highly oxidizing species such as HO<sup>i</sup> , RO<sup>i</sup> or ROO<sup>i</sup> and produce H2O, ROH, or ROOH and GS<sup>i</sup> (glutathiyl radical). Glutathiyl radical can react rapidly with GSH, most efficiently via GS to make GSSG**.**-, which is a very strong reducing species. It produces O2 <sup>i</sup><sup>−</sup> and glutathione disulfide, GSSG, by reaction with oxygen.

$$\text{GSSG}^{\ast-}\text{+O}\_2 \xrightarrow{\text{GSHP}} \text{O}\_2^{\ast-}\text{+GSSG} \tag{12}$$

SOD and GSH provide an excellent natural combination for cellular antioxidant defense by removing O2 <sup>i</sup><sup>−</sup> and HO<sup>i</sup> respectively. The intracellular concentration of GSH is about 1 mM while the mitochondrial respiration keeps O2 about 0 to 10 µM in the cell. Therefore, 99% of GS**.** formed should react with GSH to make GSSG and O2 <sup>i</sup><sup>−</sup> . Thus the importance of SOD is obvious. The normal GSH to GSSG ratio in erythrocytes is 100:1 [Beattie, 2002].

Lipid (probably a polyunsaturated fatty acid) peroxidation is a free radical chain reaction process. Superoxide is a mediator in oxidative chain reactions. It is known to initiate as well as terminate this process. Overproduction of superoxide initiates the chain reaction by mobilizing iron from the tissue protein ferritin [McCord, 1998].

$$\text{O}\_2^{\cdot -} + \text{Fermi-Fe}^{\cdot +} \rightarrow \text{O}\_2 + \text{Fermi} + \text{Fe}^{2+} \tag{13}$$

$$\text{Fe}^{2+} + \text{liquid-COOH} \text{(liquidhydroxperoxide)} \rightarrow \text{Liquid-O}^{\cdot} + \text{OH}^{\cdot} + \text{Fe}^{3+} \tag{14}$$

$$\text{Lipid-} \text{O'} + \text{liquid-} \text{H} \rightarrow \text{Liquid'} + \text{liquid-OH} \tag{15}$$

$$\text{O}\_2^{\cdot -} + \text{Fe}^{\cdot +} \rightarrow \text{O}\_2 + \text{Fe}^{2+} \tag{16}$$

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 95

Vitamin E, and vitamin C, cooperate to protect lipids and lipid structures against peroxidation. Also vitamin C regenerates vitamin E (reaction 1), thereby permitting vitamin E to function again as a free radical chain breaking antioxidant. Other antioxidants such as ubiquinols and GSH also regenerate vitamin E and help to maintain its concentration (< 0.1 nmol/mg membrane) in spite of a very high lipid peroxyl radical generation rate of 1-5

H2O2 in its liquid state, is more strongly associated by hydrogen bonding than pure water. The dipole moment of hydrogen peroxide is 2.1 Debye units compared to 1.84 Debye units for water. This should make ion-dipole interactions stronger with H2O2 than with H2O. The relative interactions of Na+-H2O and Na+-H2O2 as well as Ca2+-H2O and Ca2+-H2O2 and interactions of these ions with both the solvents depend on their relative concentrations. A dilute aqueous solution of hydrogen peroxide is more acidic than water [Cotton &

The following equilibria suggest that H2O2 is a strong oxidizing agent in both acidic and

H O 2H 2e 2H O E 1.77V 2 2 <sup>2</sup>

O 2H 2e H O E 0.68V 2 2 <sup>2</sup>


The enzyme, monoamine oxidase, located in the outer mitochondrial membrane of mammalian tissues, catalyzes the oxidation of biogenic amines and produces H2O2. Other pro-oxidant enzymes include nitric oxide synthases, cyclooxygenases, xanthine dehydrogenase, xanthine oxidase, NADPH oxidase, and myeloperoxidase. Rodent heart and brain can produce H2O2 at a rate of 0.5-3 nmol/min/mg mitochondrial protein, which corresponds to about 5-20% of their total oxygen consumption [Starkov & Wallace, 2006]. Hydrogen peroxide and superoxide have been implicated as mediators of vascular and functional changes in hypertension [Tabet et. al., 2004]. They increase vascular contraction, stimulate vascular smooth muscle cell growth, and induce inflammatory responses that are

Hydrogen peroxide plays a dual role [Lázaro, 2007]. Cancer cells produce high amounts of H2O2. These increased levels of H2O2 result in DNA alterations, cell proliferation, apoptosis resistance, metastasis, angiogenesis, and hypoxia inducible factor 1(HIF-1) activation. Activation of HIF-1 plays crucial roles in apoptosis resistance, invasion/metastasis and angiogenesis. Many human cancers have over expressed HIF-1. On the other hand, hydrogen peroxide also induces apoptosis in cancer cells selectively and the activity of

Hydrogen peroxide is less reactive than superoxide and is relatively more stable. It crosses the membrane lipid bilayer through aquaporins [Singh, 2006]. With increasing concentrations

12

+ − + += =<sup>D</sup> (25)

+ − + += =<sup>D</sup> (26)

− − + += =<sup>D</sup> (27)

2 2 <sup>2</sup> 20 C H O H HO K 1.5 10 <sup>+</sup> − − = + <sup>D</sup> = × (24)

nmol/mg membrane protein per minute [Packer et.al., 1995].

characteristic features of small arteries in hypertension.

many anticancer drugs is mediated, at least in part by H2O2.

**3. Hydrogen peroxide (H2O2)** 

Wilkinson, 1972].

basic solutions.

Chain propagation reactions follow.

$$\text{Lipid}"+\text{O}\_2 \rightarrow \text{Lipid-OO'}\tag{17}$$

$$\text{Lipid-CO}^{\prime} + \text{Lipid-H} \rightarrow \text{Liquid-COOH} + \text{Liquid}^{\prime} \tag{18}$$

If alkoyl (lipid-O )<sup>i</sup> or dioxyl (Lipid-OO )<sup>i</sup> radicals are scavenged by O2 <sup>i</sup><sup>−</sup> , the chain reaction would be terminated.

$$\text{Liipid-O}^{\cdot} + \text{O}\_{2}^{\bullet-} \text{+H}^{+} \rightarrow \text{Lipid-OH} + \text{O}\_{2} \tag{19}$$

$$\text{Lipid-CO}^{\bullet} + \text{O}\_{2}^{\bullet -} \text{+H}^{\bullet} \rightarrow \text{Lipid-COOH} \text{+O}\_{2} \tag{20}$$

Superoxide, in moderate concentrations, initiates lipid peroxidation as well as terminates. Over scavenging of O2 <sup>i</sup><sup>−</sup> by over expressed SOD limits the termination process. At intermediate concentrations, the SOD is able to suppress the lipid peroxidation process and O2 <sup>i</sup><sup>−</sup> concentration is sufficient enough to terminate the chain. Thus it has been found that for a certain level of oxidative stress there is an optimum concentration of SOD [McCord, 1998].

The rate constant values for the bis-allylic hydrogen atom abstraction from polyunsaturated lipids and for the addition to the double bond were found to be the same for hydroxyl radical (109), alkoxyl radical (106) and peroxyl radical (102) M-1s-1 [Takahashi & Niki, 1998].

Non-enzymatic antioxidants include water soluble vitamin C (ascorbic acid) and lipid soluble vitamin E. Ascorbic acid, or ascorbate anion at biological pH is also a cofactor in several biosynthetic pathways including the enzyme prolylhydroxylase, which modifies the polypeptide collagen precursor to facilitate the formation of collagen fibers.

Due to resonance, the ascorbate radical has a long half life of 1 second. Ascorbate can be oxidized in two successive one-electron steps to ascorbate free radical and dehydroascorbic acid respectively.

$$\text{Ascorbate anion - e}^{\cdot} \longrightarrow \text{Ascorbate free radical} \tag{21}$$

$$\text{Ascorbate free radical - e}^{\cdot} \longrightarrow \text{dehydroasorbcic acid} \tag{22}$$

The oxidation of ascorbate to the radical and dehydroascorbic acid can easily be reversed by the enzyme systems that use NADH or NADPH.

$$\text{NADH} + 2\text{Assorbance}^{\circ} \longrightarrow \text{NAD}^{+} + 2\text{Assorbance} \text{ [E = 597 mV]} \tag{23}$$

Vitamin E has eight different related homologues, the most abundant being α-tocopherol. The dynamics of this antioxidant, that acts only in lipid domains and quenches lipid peroxyl radicals as well as its numerous other functions, not related to its antioxidant property, such as inhibition of cell proliferation, platelet aggregation, and protein kinase C and 5-lipoxygenase inhibition have been reviewed, raising the possibility that the nonantioxidant mechanisms may contribute to many of the effects previously attributed to antioxidant functions [Ricciarelli et. al., 2002; Niki & Noguchi, 2004].

Vitamin E, and vitamin C, cooperate to protect lipids and lipid structures against peroxidation. Also vitamin C regenerates vitamin E (reaction 1), thereby permitting vitamin E to function again as a free radical chain breaking antioxidant. Other antioxidants such as ubiquinols and GSH also regenerate vitamin E and help to maintain its concentration (< 0.1 nmol/mg membrane) in spite of a very high lipid peroxyl radical generation rate of 1-5 nmol/mg membrane protein per minute [Packer et.al., 1995].

#### **3. Hydrogen peroxide (H2O2)**

H2O2 in its liquid state, is more strongly associated by hydrogen bonding than pure water. The dipole moment of hydrogen peroxide is 2.1 Debye units compared to 1.84 Debye units for water. This should make ion-dipole interactions stronger with H2O2 than with H2O. The relative interactions of Na+-H2O and Na+-H2O2 as well as Ca2+-H2O and Ca2+-H2O2 and interactions of these ions with both the solvents depend on their relative concentrations. A dilute aqueous solution of hydrogen peroxide is more acidic than water [Cotton &

Wilkinson, 1972].

94 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

<sup>−</sup> + + + →+ <sup>i</sup> (16)

i i (17)

2 2 lipid-O O +H Lipid-OH+O − + i i + → (19)

2 2 lipid-OO O +H Lipid-OOH+O <sup>−</sup> i i + → (20)

<sup>i</sup><sup>−</sup> by over expressed SOD limits the termination process. At



<sup>i</sup><sup>−</sup> , the chain reaction

3 2 O Fe O Fe 2 2

Lipid O Lipid-OO + →<sup>2</sup>

+

Superoxide, in moderate concentrations, initiates lipid peroxidation as well as terminates.

intermediate concentrations, the SOD is able to suppress the lipid peroxidation process and

The rate constant values for the bis-allylic hydrogen atom abstraction from polyunsaturated lipids and for the addition to the double bond were found to be the same for hydroxyl radical (109), alkoxyl radical (106) and peroxyl radical (102) M-1s-1 [Takahashi & Niki, 1998]. Non-enzymatic antioxidants include water soluble vitamin C (ascorbic acid) and lipid soluble vitamin E. Ascorbic acid, or ascorbate anion at biological pH is also a cofactor in several biosynthetic pathways including the enzyme prolylhydroxylase, which modifies the

Due to resonance, the ascorbate radical has a long half life of 1 second. Ascorbate can be oxidized in two successive one-electron steps to ascorbate free radical and dehydroascorbic

The oxidation of ascorbate to the radical and dehydroascorbic acid can easily be reversed by

Vitamin E has eight different related homologues, the most abundant being α-tocopherol. The dynamics of this antioxidant, that acts only in lipid domains and quenches lipid peroxyl radicals as well as its numerous other functions, not related to its antioxidant property, such as inhibition of cell proliferation, platelet aggregation, and protein kinase C and 5-lipoxygenase inhibition have been reviewed, raising the possibility that the nonantioxidant mechanisms may contribute to many of the effects previously attributed to

[ ] - NADH 2Ascorbate NAD 2Ascorbate monoanion E 597 mV <sup>+</sup> + ⎯⎯→ + = <sup>i</sup> (23)

polypeptide collagen precursor to facilitate the formation of collagen fibers.

antioxidant functions [Ricciarelli et. al., 2002; Niki & Noguchi, 2004].

the enzyme systems that use NADH or NADPH.

<sup>i</sup><sup>−</sup> concentration is sufficient enough to terminate the chain. Thus it has been found that for a certain level of oxidative stress there is an optimum concentration of SOD [McCord,

If alkoyl (lipid-O )<sup>i</sup> or dioxyl (Lipid-OO )<sup>i</sup> radicals are scavenged by O2

Lipid-OO Lipid-H Lipid-OOH+Lipid i i + → (18)

Chain propagation reactions follow.

would be terminated.

Over scavenging of O2

O2

1998].

acid respectively.

$$\text{H}\_2\text{O}\_2 = \text{H}^+ + \text{HO}\_2^- \qquad \text{K}\_{20^\circ \text{C}} = 1.5 \times 10^{-12} \tag{24}$$

The following equilibria suggest that H2O2 is a strong oxidizing agent in both acidic and basic solutions.

$$\text{CH}\_2\text{O}\_2 + 2\text{H}^+ + 2\text{e}^- = 2\text{H}\_2\text{O} \quad \text{E}^\circ = 1.77\text{V} \tag{25}$$

$$\text{H}\_2\text{O}\_2 + 2\text{H}^+ + 2\text{e}^- = \text{H}\_2\text{O}\_2 \quad \text{E}^\circ = 0.68\text{V} \tag{26}$$

$$\text{'} \text{HO} \text{''} + \text{H}\_2\text{O} + 2\text{e}^- = \text{''} \text{OH'} \quad \text{E}^\circ = 0.87 \text{V} \tag{27}$$

The enzyme, monoamine oxidase, located in the outer mitochondrial membrane of mammalian tissues, catalyzes the oxidation of biogenic amines and produces H2O2. Other pro-oxidant enzymes include nitric oxide synthases, cyclooxygenases, xanthine dehydrogenase, xanthine oxidase, NADPH oxidase, and myeloperoxidase. Rodent heart and brain can produce H2O2 at a rate of 0.5-3 nmol/min/mg mitochondrial protein, which corresponds to about 5-20% of their total oxygen consumption [Starkov & Wallace, 2006].

Hydrogen peroxide and superoxide have been implicated as mediators of vascular and functional changes in hypertension [Tabet et. al., 2004]. They increase vascular contraction, stimulate vascular smooth muscle cell growth, and induce inflammatory responses that are characteristic features of small arteries in hypertension.

Hydrogen peroxide plays a dual role [Lázaro, 2007]. Cancer cells produce high amounts of H2O2. These increased levels of H2O2 result in DNA alterations, cell proliferation, apoptosis resistance, metastasis, angiogenesis, and hypoxia inducible factor 1(HIF-1) activation. Activation of HIF-1 plays crucial roles in apoptosis resistance, invasion/metastasis and angiogenesis. Many human cancers have over expressed HIF-1. On the other hand, hydrogen peroxide also induces apoptosis in cancer cells selectively and the activity of many anticancer drugs is mediated, at least in part by H2O2.

Hydrogen peroxide is less reactive than superoxide and is relatively more stable. It crosses the membrane lipid bilayer through aquaporins [Singh, 2006]. With increasing concentrations

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 97

However its intracellular concentration is maintained at ~ 4-10 μM by superoxide dismutase. Copper, zinc superoxide dismutase is ~ 0.5% of total soluble proteins in brain. Physiological levels of NO<sup>i</sup> binds to cytochrome c oxidase leading to a competitive and reversible inhibition of mitochondrial respiration [Radi et. al., 2002]. Large levels of NO<sup>i</sup> in

The half-life of ONOO- is 0.05 – 1 s [Sies, 1993]. Mitochondrial scavenging systems for

powerful oxidizing and nitrating agent. It reacts with tyrosine in proteins and produces nitrotyrosines. Nitration of the structural proteins, neurofilaments and actin disrupts the filament assembly leading to major pathological consequences in myocardial ischemia,

The steady state concentrations of NO<sup>i</sup> and - ONOO in liver are ~ 36 nM and 2.2 nM respectively based on the assumption of 20 μM intramitochondrial O2 concentration. But there are claims that O2 concentration is only 3 μM and not 20 μM [Turrens, 2003]. If this is true the steady state concentrations will be much lower. After cerebral ischemia, NO concentration increases 10-100-fold in a few minutes to 2-4 μM[Beckman & Koppenol, 1996]. Nitric oxide can penetrate the lipid bilayer and diffuse rapidly and isotropically through most tissues without any significant reaction or consumption. The rapid diffusion of nitric oxide between cells allows it to modulate, 1) synaptic plasticity in neurons, 2) the oscillatory behavior of neuronal networks, 3) blood flow, and 4) thrombosis [Beckman & Koppenol, 1996]. Since it reacts with oxy hemoglobin and is destroyed, it cannot be transported

 Nitric oxide diffuses and concentrates in the hydrophobic core of low density lipoprotein (LDL) and inhibits its oxidation [Rubbo et. al., 2002]. On the other hand peroxynitrite is involved in LDL oxidation. Since vascular cells are rich sources of superoxide, peroxynitrite formation is also facilitated. Thus the development of atherosclerosis may be intimately

Both peroxynitrite and singlet oxygen are involved in activating mitogen-activated protein (MAP) kinases that respond to extracellular stimuli such as mitogen, osmotic stress, and proinflammatory cytokines and regulate various activities such as gene expression, mitosis,

Mitochondria has unique roles; production of adenosine triphosphate (ATP) by cellular respiration, production of ROS, the distribution/redistribution of Ca2+ pools within cells,

In the absence of mitochondria, or with mitochondrial dysfunction, ATP is produced by an alternative pathway, anaerobic glycolysis. However, this conversion of glucose to pyruvate is not efficient and produces only 2 molecules of ATP compared to 36 molecules of ATP produced by normal glucose oxidation. The pyruvate and the fatty acids are transported into the mitochondrial matrix. There they are broken down into the acetyl group on acetyl

connected to the interactions of these two nitrogen species with LDL.

differentiation, proliferative cell survival, and apoptosis [Klotz, 2002].

coenzyme A (acetyl-CoA or acetyl-SCoA) and then fed into the Krebs cycle.

and control of apoptosis or programmed cell death.

and ONOO--derived radicals such as carbonate (CO3**.**-) and nitrogen dioxide radicals

radicals significantly affect the mitochondrial integrity. Peroxynitrite is a very

<sup>i</sup> ) are cytochrome c oxidase, GSH and ubiquinol [Radi et. al., 2002]. Superoxide and

<sup>i</sup><sup>−</sup> . In aerobic metabolism about 1-5% of oxygen is reduced to superoxide.

<sup>i</sup><sup>−</sup> from complex I and the consequent formation

compete SOD for O2

of more ONOO-.

through the vasculature.

**5. Mitochondria** 

ONOO-

ONOO-

( NO2

mitochondria promote formation of more O2

distressed lung, and amyotropic lateral sclerosis.

of cellular H2O2, its function gradually changes from cell signaling, to cell malignant transformation, and to cell death [Lázaro, 2007]. The mystery surrounding the different roles of hydrogen peroxide may be solved, at least partially, by looking at its electronic properties. Our impedance data suggest that the electronic properties (and consequent circuits) of H2O2 are dependent on its concentration. Our data also suggest that one has to take a serious look at another important contribution of H2O2, the preferential solvation of ions and its biological consequences [Krishnan et.al., 2011] .

The beneficial aspect of H2O2 in cell signaling is emerging. Neurons and brain macrophages produce O2 <sup>i</sup><sup>−</sup> in pathological situations and the H2O2 produced from O2 <sup>i</sup><sup>−</sup> increases gap junctional communication in astrocytes [Rouach et. al., 2004]. Examples of signaling processes include the over-oxidation of the cysteine in peroxiredoxins from the cysteine sulfenic acid to cysteine sulfinic acid, and the over-oxidation of methionine residues in proteins to methionine sulfoxide [Sies, 1986; Wood et. al., 2003].

Embryonic and fetal growth are facilitated by a certain amount of redox imbalance or oxidative stress [Maiorino & Ursini, 2002; Dennery, 2010]. These investigations detail the level of oxygen and antioxidant status at the first, second and third trimester of pregnancy. Low levels of H2O2 and superoxide produced by human sperm are also crucial for the capacitation process that allows the sperm to penetrate the zona pellucida of the ovum. At low, moderate, and highly oxidative states, proliferation, differentiation, and apoptosis or necrosis respectively are favored suggesting the different functions of ROS depending on their concentrations.

#### **4. Reactive nitrogen species**

Nitric oxide, NO**.** , is a neutral free radical with a half life of the order of seconds [Beckman & Koppenol, 1996; Blokhina & Fagerstedt, 2006]. Three types of nitric oxide synthases have been described in mammalian cells: neuronal (nNOS), endothelial (eNOS) and an inducible (immunological) (iNOS). The first two are under the control of Ca2+-calmodulin. The enzyme catalyzes oxygen dependent conversion of L-arginine to citrulline:

$$\text{L}-\text{argin} + \text{NADPH} + \text{O}\_2 \xrightarrow{\text{NOS}} \text{citrumline} + \text{NO} + \text{NADP}^+ \tag{28}$$

Mitochondrial NOS distinct from the ones given above has also been reported [Elfering et. al., 2002]. NO<sup>i</sup> can be converted to other reactive nitrogen species such as nitrosonium cation (NO+), nitroxyl anion (NO ) <sup>−</sup> or peroxynitrite, - ONOO with distinct chemical reactivity and physical properties. Some of the physiological effects may be mediated through the intermediate formation of S-nitroso-cysteine or S-nitroso-glutathione [Dröge, 2002].

ROS are produced at complex I and complex III during respiration. Superoxide ion produced at the mitochondria reacts with NO<sup>i</sup> to produce peroxynitrite. The rate of this reaction is controlled by the rate of diffusion of the two reactants.

$$\mathrm{NO}^{\bullet} + \mathrm{O}\_{2}^{\bullet -} \xrightarrow{\mathrm{NOS}} \mathrm{ONOO}^{\bullet} \tag{29}$$

The reaction rate for the formation of - ONOO is 6.7 x 109 M-1 s-1. This is ~ 6 times faster than the scavenging of superoxide by copper, zinc superoxide dismutase. Inducible nitric oxide synthase, when expressed, can make substantial amounts of nitric oxide and this will outcompete SOD for O2 <sup>i</sup><sup>−</sup> . In aerobic metabolism about 1-5% of oxygen is reduced to superoxide. However its intracellular concentration is maintained at ~ 4-10 μM by superoxide dismutase. Copper, zinc superoxide dismutase is ~ 0.5% of total soluble proteins in brain.

Physiological levels of NO<sup>i</sup> binds to cytochrome c oxidase leading to a competitive and reversible inhibition of mitochondrial respiration [Radi et. al., 2002]. Large levels of NO<sup>i</sup> in mitochondria promote formation of more O2 <sup>i</sup><sup>−</sup> from complex I and the consequent formation of more ONOO-.

The half-life of ONOO is 0.05 – 1 s [Sies, 1993]. Mitochondrial scavenging systems for ONOO and ONOO--derived radicals such as carbonate (CO3 **.**-) and nitrogen dioxide radicals ( NO2 <sup>i</sup> ) are cytochrome c oxidase, GSH and ubiquinol [Radi et. al., 2002]. Superoxide and ONOO radicals significantly affect the mitochondrial integrity. Peroxynitrite is a very powerful oxidizing and nitrating agent. It reacts with tyrosine in proteins and produces nitrotyrosines. Nitration of the structural proteins, neurofilaments and actin disrupts the filament assembly leading to major pathological consequences in myocardial ischemia, distressed lung, and amyotropic lateral sclerosis.

The steady state concentrations of NO<sup>i</sup> and - ONOO in liver are ~ 36 nM and 2.2 nM respectively based on the assumption of 20 μM intramitochondrial O2 concentration. But there are claims that O2 concentration is only 3 μM and not 20 μM [Turrens, 2003]. If this is true the steady state concentrations will be much lower. After cerebral ischemia, NO concentration increases 10-100-fold in a few minutes to 2-4 μM[Beckman & Koppenol, 1996].

Nitric oxide can penetrate the lipid bilayer and diffuse rapidly and isotropically through most tissues without any significant reaction or consumption. The rapid diffusion of nitric oxide between cells allows it to modulate, 1) synaptic plasticity in neurons, 2) the oscillatory behavior of neuronal networks, 3) blood flow, and 4) thrombosis [Beckman & Koppenol, 1996]. Since it reacts with oxy hemoglobin and is destroyed, it cannot be transported through the vasculature.

 Nitric oxide diffuses and concentrates in the hydrophobic core of low density lipoprotein (LDL) and inhibits its oxidation [Rubbo et. al., 2002]. On the other hand peroxynitrite is involved in LDL oxidation. Since vascular cells are rich sources of superoxide, peroxynitrite formation is also facilitated. Thus the development of atherosclerosis may be intimately connected to the interactions of these two nitrogen species with LDL.

Both peroxynitrite and singlet oxygen are involved in activating mitogen-activated protein (MAP) kinases that respond to extracellular stimuli such as mitogen, osmotic stress, and proinflammatory cytokines and regulate various activities such as gene expression, mitosis, differentiation, proliferative cell survival, and apoptosis [Klotz, 2002].

#### **5. Mitochondria**

96 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

of cellular H2O2, its function gradually changes from cell signaling, to cell malignant transformation, and to cell death [Lázaro, 2007]. The mystery surrounding the different roles of hydrogen peroxide may be solved, at least partially, by looking at its electronic properties. Our impedance data suggest that the electronic properties (and consequent circuits) of H2O2 are dependent on its concentration. Our data also suggest that one has to take a serious look at another important contribution of H2O2, the preferential solvation of

The beneficial aspect of H2O2 in cell signaling is emerging. Neurons and brain macrophages

junctional communication in astrocytes [Rouach et. al., 2004]. Examples of signaling processes include the over-oxidation of the cysteine in peroxiredoxins from the cysteine sulfenic acid to cysteine sulfinic acid, and the over-oxidation of methionine residues in

Embryonic and fetal growth are facilitated by a certain amount of redox imbalance or oxidative stress [Maiorino & Ursini, 2002; Dennery, 2010]. These investigations detail the level of oxygen and antioxidant status at the first, second and third trimester of pregnancy. Low levels of H2O2 and superoxide produced by human sperm are also crucial for the capacitation process that allows the sperm to penetrate the zona pellucida of the ovum. At low, moderate, and highly oxidative states, proliferation, differentiation, and apoptosis or necrosis respectively are favored suggesting the different functions of ROS depending on

Koppenol, 1996; Blokhina & Fagerstedt, 2006]. Three types of nitric oxide synthases have been described in mammalian cells: neuronal (nNOS), endothelial (eNOS) and an inducible (immunological) (iNOS). The first two are under the control of Ca2+-calmodulin. The

Mitochondrial NOS distinct from the ones given above has also been reported [Elfering et. al., 2002]. NO<sup>i</sup> can be converted to other reactive nitrogen species such as nitrosonium cation (NO+), nitroxyl anion (NO ) <sup>−</sup> or peroxynitrite, - ONOO with distinct chemical reactivity and physical properties. Some of the physiological effects may be mediated through the intermediate formation of S-nitroso-cysteine or S-nitroso-glutathione [Dröge,

ROS are produced at complex I and complex III during respiration. Superoxide ion produced at the mitochondria reacts with NO<sup>i</sup> to produce peroxynitrite. The rate of this

NOS - NO O ONOO <sup>2</sup>

The reaction rate for the formation of - ONOO is 6.7 x 109 M-1 s-1. This is ~ 6 times faster than the scavenging of superoxide by copper, zinc superoxide dismutase. Inducible nitric oxide synthase, when expressed, can make substantial amounts of nitric oxide and this will out-

enzyme catalyzes oxygen dependent conversion of L-arginine to citrulline:

reaction is controlled by the rate of diffusion of the two reactants.

, is a neutral free radical with a half life of the order of seconds [Beckman &

NOS . L arg <sup>2</sup> inine NADPH O citrulline NO NADP<sup>+</sup> − + + ⎯⎯⎯→ ++ (28)

<sup>−</sup> i i + ⎯⎯⎯→ (29)

<sup>i</sup><sup>−</sup> increases gap

<sup>i</sup><sup>−</sup> in pathological situations and the H2O2 produced from O2

ions and its biological consequences [Krishnan et.al., 2011] .

proteins to methionine sulfoxide [Sies, 1986; Wood et. al., 2003].

produce O2

their concentrations.

Nitric oxide, NO**.**

2002].

**4. Reactive nitrogen species** 

Mitochondria has unique roles; production of adenosine triphosphate (ATP) by cellular respiration, production of ROS, the distribution/redistribution of Ca2+ pools within cells, and control of apoptosis or programmed cell death.

In the absence of mitochondria, or with mitochondrial dysfunction, ATP is produced by an alternative pathway, anaerobic glycolysis. However, this conversion of glucose to pyruvate is not efficient and produces only 2 molecules of ATP compared to 36 molecules of ATP produced by normal glucose oxidation. The pyruvate and the fatty acids are transported into the mitochondrial matrix. There they are broken down into the acetyl group on acetyl coenzyme A (acetyl-CoA or acetyl-SCoA) and then fed into the Krebs cycle.

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 99

producing ATP, both ∆pH and ∆ψ as well as NADH/NAD+ ratio are low and ROS

Superoxide production occurs on the outer mitochondrial membrane, in the matrix, and on both sides of the innermitochondrial membrane [Turrens, 2003]. The highly reducing intra-mitochondrial environment has reduced coenzymes and prosthetic groups of flavoproteins, iron-sulfur clusters (in Complex I) and ubisemiquinones (Complex III) that thermodynamically favor one electron reduction of molecular oxygen to produce O2

whether the mitochondria is respiring or not. For example, in the absence of ADP, the proton movement through ATP synthase stops, protons build up and cause a slowdown of electron flow and thus creating a more reduced State IV respiration state. This reduced state and increasing concentration of O2 will increase the one electron reduction process of

Uptake of high concentrations of Ca2+ into mitochondria, declined ATP, and increased ROS production endanger the health of the mitochondria and the mitochondria resort into a destructive mode by opening the mitochondrial permeability transition pore and consequently

Since different organs can rely on mitochondrial energy to different extents, mitochondrial defects can cause organ-specific phenotypes. The organ system most reliant on mitochondrial energy is the central nervous system. The consequences of mitochondrial dysfunction are numerous and include oxidative stress, loss of cellular Ca2+ homeostasis, promotion of apoptosis, and metabolic failure. Hence, evidence continues to accrue implicating mitochondrial dysfunction in the etiology of a number of neurodegenerative

In transient ischemia, a lack of oxygen and glucose delivery compromise the integrity of aerobic metabolism, while reperfusion potentiates injury via the generation of free radicals. Superoxide, nitric oxide and peroxynitrite production in the brain is increased during reperfusion following 30 minutes of global ischemia. In patients with Parkinson's disease,

oxidative stress and consequent mitochondrial damage resulting in mutations. Evidence for mitochondrial dysfunction in Alzheimer's disease pathogenesis comes from impaired activities of three key Krebs Cycle enzyme complexes and reduced respiratory chain complex I, III, and IV activity observed in postmortem Alzheimer's disease brain, and oxidative damage to both mtDNA and nDNA. It may be possible for mtDNA mutations to disrupt the normal electron flow and seriously affect energy production. Oxidative damage and the resulting serious consequences have been extensively reviewed recently [Singh, 2006]. Compared to nDNA, mtDNA is far more susceptible to mutations due to their being present in a highly oxidative environment, a lack of protective histones and limited repair

During the production of ATP in the cell, about 85% of oxygen is consumed by the

<sup>i</sup><sup>−</sup> production will increase.

<sup>i</sup><sup>−</sup> production. When the mitochondria is actively

<sup>i</sup><sup>−</sup> varies depending on the organ and

. These radicals and their reactions cause

<sup>i</sup><sup>−</sup> , may be produced from about 4% of all oxygen

<sup>i</sup><sup>−</sup> in the heart and lung is Complex III, it seems to be Complex I

<sup>i</sup><sup>−</sup> .

substrates dictate the amount of O2

in the brain [Turrens, 2003]. Also the production of O2

releasing cytochrome c and initiating programmed cell death.

**6. Mitochondrial dysfunction in neurodegenerative diseases** 

conditions such as Parkinson's, Alzheimer's, and transient ischemia.

excess Fe2+ can reduce peroxide and produce HO<sup>i</sup>

capacity [Carew & Huang, 2002; Singh, 2006].

mitochondria. Superoxide radical, O2

production is also low.

While the major source of O2

oxygen and the rate of O2

The ATP has a half-life of seconds to minutes depending on the cell where it is being continuously hydrolyzed and regenerated. Our average normal consumption and regeneration rate of ATP is ~3mol (1.5 kg) h-1. During strenuous activity this rate increases by an order of magnitude [Voet D. & Voet J. G., 1995].

Oxygen deprivation rapidly deteriorates brain cells because ATP is available for only a few seconds [Voet D. & Voet J. G., 1995]. In muscles and nerve cells, ATP has high turnover rates and phosphocreatine acts as its reservoir.

$$\text{ATP} + \text{Creatine} = \text{Phosphocreating} + \text{ADP} \tag{30}$$

This is an energy consuming reaction under standard conditions and is close to equilibrium under normal intracellular concentrations. At resting state, high ATP shifts the equilibrium to the right. High metabolic activity shifts the equilibrium to the left due to low ATP.

The breakdown of carbohydrates, lipids, and proteins produce the acetyl group of the common intermediate, acetyl-CoA. A series of consecutive enzymatic reactions of Krebs cycle, the electron transport chain (ETC), and oxidative phosphorylation then converts acetyl-CoA into CO2 and H2O. The net result is the transfer of electrons from the oxidative substrates to molecular oxygen to generate water, CO2 and ATP.

In oxidative phosphorylation, a series of coupled reactions are involved in the transport of electrons through complexes 1-IV in the inner mitochondrial membrane. The entry point of electrons from the high energy molecules NADH and FADH2 is at complex I and complex II respectively. With the help of a variety of enzymes, a series of coupled redox reactions drive the transport of electrons through these complexes. A proton gradient across the inner mitochondrial membrane is created during this process when protons are pumped out of the matrix at complexes I, III, and IV. The electrochemical gradient consisting of a pH gradient (∆pH) and an electrical potential (∆ψ) drive the ATP synthesis from ADP as the protons re-enter the matrix through the ATP synthase (complex V).

Mitochondrial DNA (mtDNA) harboring their own genome with their own transcription, translation, and machinery for protein synthesis was discovered in the early 1960s. The codes for electron transport chain complexes, I, II, III, IV, and V for nuclear DNA (nDNA) are 36, 4, 10, 10, and 14 protein subunits and for mtDNA, 7, 0, 1, 3, and 2 subunits respectively [Carew & Huang, 2002]. Complex II is encoded by nDNA only. The mtDNA genome also encodes 22 mitochondrial tRNAs that are required for protein synthesis and 2 rRNAs that are essential for translation of mtDNA transcripts. The human mtDNA is a supercoiled, double-stranded molecule containing 16,569 base pairs [Chan, 2006; Carew & Huang, 2002].

A dynamic structural network consisting of about 70% stationary and 30% mobile mitochondria meets the energy demands of axons. The speed of the mobile mitochondria is ~ 1 μm/s [Kiryu-Seo et. al., 2010]. Mitochondrial fission and fusion, probable mitochondrial biogenesis within axons, and the transport of mitochondria to and from neuronal soma determine the content of mitochondria within axons.

Under highly reduced state of ETC, excess electrons at complex I produce O2 <sup>i</sup><sup>−</sup> in the mitochondrial matrix. This is reduced by the matrix MnSOD to H2O2. To a limited extent complex III also produces O2 <sup>i</sup><sup>−</sup> and is released into the mitochondrial intermembrane space where it is converted to H2O2 by Cu/ZnSOD. The presence of Fe2+ readily converts the H2O2 to the dangerous hydroxyl radical (reaction 7). The potential at the inner mitochondrial membrane, the pH in the matrix, local O2 concentration and the nature of the

The ATP has a half-life of seconds to minutes depending on the cell where it is being continuously hydrolyzed and regenerated. Our average normal consumption and regeneration rate of ATP is ~3mol (1.5 kg) h-1. During strenuous activity this rate increases

Oxygen deprivation rapidly deteriorates brain cells because ATP is available for only a few seconds [Voet D. & Voet J. G., 1995]. In muscles and nerve cells, ATP has high turnover rates

This is an energy consuming reaction under standard conditions and is close to equilibrium under normal intracellular concentrations. At resting state, high ATP shifts the equilibrium

The breakdown of carbohydrates, lipids, and proteins produce the acetyl group of the common intermediate, acetyl-CoA. A series of consecutive enzymatic reactions of Krebs cycle, the electron transport chain (ETC), and oxidative phosphorylation then converts acetyl-CoA into CO2 and H2O. The net result is the transfer of electrons from the oxidative

In oxidative phosphorylation, a series of coupled reactions are involved in the transport of electrons through complexes 1-IV in the inner mitochondrial membrane. The entry point of electrons from the high energy molecules NADH and FADH2 is at complex I and complex II respectively. With the help of a variety of enzymes, a series of coupled redox reactions drive the transport of electrons through these complexes. A proton gradient across the inner mitochondrial membrane is created during this process when protons are pumped out of the matrix at complexes I, III, and IV. The electrochemical gradient consisting of a pH gradient (∆pH) and an electrical potential (∆ψ) drive the ATP synthesis from ADP as the

Mitochondrial DNA (mtDNA) harboring their own genome with their own transcription, translation, and machinery for protein synthesis was discovered in the early 1960s. The codes for electron transport chain complexes, I, II, III, IV, and V for nuclear DNA (nDNA) are 36, 4, 10, 10, and 14 protein subunits and for mtDNA, 7, 0, 1, 3, and 2 subunits respectively [Carew & Huang, 2002]. Complex II is encoded by nDNA only. The mtDNA genome also encodes 22 mitochondrial tRNAs that are required for protein synthesis and 2 rRNAs that are essential for translation of mtDNA transcripts. The human mtDNA is a supercoiled, double-stranded molecule containing 16,569 base pairs [Chan, 2006; Carew &

A dynamic structural network consisting of about 70% stationary and 30% mobile mitochondria meets the energy demands of axons. The speed of the mobile mitochondria is ~ 1 μm/s [Kiryu-Seo et. al., 2010]. Mitochondrial fission and fusion, probable mitochondrial biogenesis within axons, and the transport of mitochondria to and from neuronal soma

mitochondrial matrix. This is reduced by the matrix MnSOD to H2O2. To a limited extent

space where it is converted to H2O2 by Cu/ZnSOD. The presence of Fe2+ readily converts the H2O2 to the dangerous hydroxyl radical (reaction 7). The potential at the inner mitochondrial membrane, the pH in the matrix, local O2 concentration and the nature of the

<sup>i</sup><sup>−</sup> and is released into the mitochondrial intermembrane

<sup>i</sup><sup>−</sup> in the

Under highly reduced state of ETC, excess electrons at complex I produce O2

to the right. High metabolic activity shifts the equilibrium to the left due to low ATP.

ATP + Creatine = Phosphocreatine + ADP (30)

by an order of magnitude [Voet D. & Voet J. G., 1995].

substrates to molecular oxygen to generate water, CO2 and ATP.

protons re-enter the matrix through the ATP synthase (complex V).

determine the content of mitochondria within axons.

complex III also produces O2

and phosphocreatine acts as its reservoir.

Huang, 2002].

substrates dictate the amount of O2 <sup>i</sup><sup>−</sup> production. When the mitochondria is actively producing ATP, both ∆pH and ∆ψ as well as NADH/NAD+ ratio are low and ROS production is also low.

Superoxide production occurs on the outer mitochondrial membrane, in the matrix, and on both sides of the innermitochondrial membrane [Turrens, 2003]. The highly reducing intra-mitochondrial environment has reduced coenzymes and prosthetic groups of flavoproteins, iron-sulfur clusters (in Complex I) and ubisemiquinones (Complex III) that thermodynamically favor one electron reduction of molecular oxygen to produce O2 <sup>i</sup><sup>−</sup> . While the major source of O2 <sup>i</sup><sup>−</sup> in the heart and lung is Complex III, it seems to be Complex I in the brain [Turrens, 2003]. Also the production of O2 <sup>i</sup><sup>−</sup> varies depending on the organ and whether the mitochondria is respiring or not. For example, in the absence of ADP, the proton movement through ATP synthase stops, protons build up and cause a slowdown of electron flow and thus creating a more reduced State IV respiration state. This reduced state and increasing concentration of O2 will increase the one electron reduction process of oxygen and the rate of O2 <sup>i</sup><sup>−</sup> production will increase.

Uptake of high concentrations of Ca2+ into mitochondria, declined ATP, and increased ROS production endanger the health of the mitochondria and the mitochondria resort into a destructive mode by opening the mitochondrial permeability transition pore and consequently releasing cytochrome c and initiating programmed cell death.

#### **6. Mitochondrial dysfunction in neurodegenerative diseases**

Since different organs can rely on mitochondrial energy to different extents, mitochondrial defects can cause organ-specific phenotypes. The organ system most reliant on mitochondrial energy is the central nervous system. The consequences of mitochondrial dysfunction are numerous and include oxidative stress, loss of cellular Ca2+ homeostasis, promotion of apoptosis, and metabolic failure. Hence, evidence continues to accrue implicating mitochondrial dysfunction in the etiology of a number of neurodegenerative conditions such as Parkinson's, Alzheimer's, and transient ischemia.

In transient ischemia, a lack of oxygen and glucose delivery compromise the integrity of aerobic metabolism, while reperfusion potentiates injury via the generation of free radicals. Superoxide, nitric oxide and peroxynitrite production in the brain is increased during reperfusion following 30 minutes of global ischemia. In patients with Parkinson's disease, excess Fe2+ can reduce peroxide and produce HO<sup>i</sup> . These radicals and their reactions cause oxidative stress and consequent mitochondrial damage resulting in mutations. Evidence for mitochondrial dysfunction in Alzheimer's disease pathogenesis comes from impaired activities of three key Krebs Cycle enzyme complexes and reduced respiratory chain complex I, III, and IV activity observed in postmortem Alzheimer's disease brain, and oxidative damage to both mtDNA and nDNA. It may be possible for mtDNA mutations to disrupt the normal electron flow and seriously affect energy production. Oxidative damage and the resulting serious consequences have been extensively reviewed recently [Singh, 2006]. Compared to nDNA, mtDNA is far more susceptible to mutations due to their being present in a highly oxidative environment, a lack of protective histones and limited repair capacity [Carew & Huang, 2002; Singh, 2006].

During the production of ATP in the cell, about 85% of oxygen is consumed by the mitochondria. Superoxide radical, O2 <sup>i</sup><sup>−</sup> , may be produced from about 4% of all oxygen

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 101

**Balance of life and death**

↑CMA ↑UPS

**Homeostasis**

↑UPR

**Minor crisis**

**Major crisis**

↑↑ CMA

**Cell death**

↑↑

RONS

UPS

↑↑↑

Fig. 1. Deleterious stress factors and restorative factors in neuronal homeostasis and neuronal death [Adpated from Nagley et. al., 2010]. ∆Ψm, mitochondrial membrane potential; MP, misfolded proteins; RONS, reactive oxygen and nitrogen species; UPS, ubiquitin-proteasome system; UPR, unfolded protein response; CMA, chaperone mediated autophagy; MA, macroautophagy. Proteins failing to fold into native structure produces inactive and usually toxic proteins. Several neurodegenerative and other diseases are believed to result from misfolded proteins (MP). The unfolded protein response (UPR) is activated in response to a stress arising from an accumulation of unfolded or MP in the lumen of the endoplasmic reticulum. UPR attempts initially to restore the normal function of the cell by halting protein translation. They also activate the signaling pathways that lead to increasing the production of molecular chaperones involved in protein folding. If these efforts are not successful, then it induces apoptotic effector proteins. Proteasomes are very large protein complexes whose main function is to degrade unneeded or damaged proteins by proteolysis and thus helping cells to regulate proteins and MP. Ubiquitin, a small protein, is used to tag the proteins that need to be degraded. The overall system of ubiquitination and proteasomal degradation is known as the ubiquitin proteosome system (UPS).

Autophagy, such as micro- and macro-autophagy, refers to the degradation of intracellular components via the lysosome. Chaperone mediated autophagy, CMA, can degrade only certain proteins and not organelles and thus very selective in what it degrades. Also the substrates are translocated across the membrane on a one on one basis instead of engulfing

RONS

MA

↑↑↑

MP

↑↑

ATP

∆Ψm

↑↑↑

∆Ψm

ATP

↑MP

2+

> ↑↑

[Ca ] 2+

↑↑

↑↑↑

↑↑↑

UPR

↓

MP

ATP ↑RONS

↓

↑↑

UPR

↑↑↑

↑↑↑

↑↑↑

UPS

2+ [Ca ]

↑↑

**A**

**B**

**C**

**D**

the substrate in bulk

[Ca ]↑

consumed [Singh, 2006]. Enzymes such as NADPH oxidases, xanthine oxidase, cyclooxygenases, and lipooxygenases also produce ROS. The iron-sulfur cluster in the aconitase enzyme, localized in the matrix space of mitochondria, is oxidized by superoxide and the exposed iron reacts with the peroxide to produce hydroxyl radicals [Singh, 2006]. Also the NO<sup>i</sup> produced within mitochondria by mitochondrial NO synthase produces peroxynitrite [ONOO- ] by reaction with O2 <sup>i</sup><sup>−</sup> . Superoxide radical and ONOO- contribute to substantial mitochondrial damage.

Enzymes such as SOD, GSHPx, catalase, peroxoredoxin, and thioredoxin can inactivate some of the ROS. MnSOD or Cu/ZnSOD converts the O2 <sup>i</sup><sup>−</sup> into H2O2. The active site of cytosolic and extracellular forms of SOD contains Cu/Zn and the mitochondrial form contains Mn [Beattie, 2002]. Oxidative damage is due to the inadequacy of these detoxifying processes.

In aging and neurodegenerative disorders, apart from inherited defects, mitochondrial DNA deletions and point mutations within neurons are well recognized.

Mitochondria is heavily involved in cell death. The various pathways involved in the cell death depend on the type of cellular injury or neurodegeneration. In the core region of stroke necrosis is observed. On the other hand in neurodegenerative diseases such as amyotropic lateral sclerosis, apoptosis markers are observed along with markers of endoplasmic reticulum(ER) stress and autophagy [Nagley et. al., 2010]. Mitochondria influences programmed cell death (type I-apoptotic as well as type III-necrotic). While autophagy routinely turns over various cellular constituents, it is involved in cell death (type II) in some stress conditions. Apart from changing the mitochondrial membrane potential and increasing the production of ROS, the elevation of intracellular and mitochondrial Ca2+ also modulates the process of programmed cell death. The involvement of mitochondria in the multifaceted neuronal death pathways is elegantly demonstrated in 4 steps in Fig. 1 [Nagley et. al., 2010]. Step A illustrates normal physiological conditions where equilibrium between homeostatic and deleterious factors is maintained. In this state of healthy functional neurons, cellular feedback mechanisms help maintain homeostasis. Step B is under conditions of minor stress. The deleterious factors such as reactive oxygen and nitrogen species (RONS) and misfolded proteins (MP) contribute to a decrease in energy production (ATP) and an increase in intracellular Ca2+. Various channels and transporters elevate the Ca2+ in both cytoplasm and mitochondria. Neurons respond to this minor stress by activation of the unfolded protein response (UPR, in its initial, pro-survival phase), ubiquitin proteasome system (UPS), and chaperone-mediated autophagy (CMA). Step C indicates a much greater stress or imbalance due to substantial increases in RONS, MP, and Ca2+. ATP production is substantially less. There is also a substantial decrease in mitochondrial membrane potential. The UPR switches to its destructive mode via induction of apoptotic effector proteins such as C/EBP homologous protein, caspase-12 and c-Jun N-terminal kinase. The UPS also becomes increasingly dysfunctional because of it s inability along with CMA to adequately handle the increased load of MP. This leads to the formation of intracellular aggregates of MP. Thus, deleterious factors overwhelm the cellular homeostatic processes. In spite of the chaperone mediated autophagic process switching to macroautophagy, it cannot adequately handle the load of MP leading to the final step D. There is a strong commitment to death at this level of stress and the cell advances to programmed cell death (type-I, type-II or type-III, or their combinations) [Nagley, 2010].

consumed [Singh, 2006]. Enzymes such as NADPH oxidases, xanthine oxidase, cyclooxygenases, and lipooxygenases also produce ROS. The iron-sulfur cluster in the aconitase enzyme, localized in the matrix space of mitochondria, is oxidized by superoxide and the exposed iron reacts with the peroxide to produce hydroxyl radicals [Singh, 2006]. Also the NO<sup>i</sup> produced within mitochondria by mitochondrial NO synthase produces

Enzymes such as SOD, GSHPx, catalase, peroxoredoxin, and thioredoxin can inactivate

cytosolic and extracellular forms of SOD contains Cu/Zn and the mitochondrial form contains Mn [Beattie, 2002]. Oxidative damage is due to the inadequacy of these detoxifying

In aging and neurodegenerative disorders, apart from inherited defects, mitochondrial DNA

Mitochondria is heavily involved in cell death. The various pathways involved in the cell death depend on the type of cellular injury or neurodegeneration. In the core region of stroke necrosis is observed. On the other hand in neurodegenerative diseases such as amyotropic lateral sclerosis, apoptosis markers are observed along with markers of endoplasmic reticulum(ER) stress and autophagy [Nagley et. al., 2010]. Mitochondria influences programmed cell death (type I-apoptotic as well as type III-necrotic). While autophagy routinely turns over various cellular constituents, it is involved in cell death (type II) in some stress conditions. Apart from changing the mitochondrial membrane potential and increasing the production of ROS, the elevation of intracellular and mitochondrial Ca2+ also modulates the process of programmed cell death. The involvement of mitochondria in the multifaceted neuronal death pathways is elegantly demonstrated in 4 steps in Fig. 1 [Nagley et. al., 2010]. Step A illustrates normal physiological conditions where equilibrium between homeostatic and deleterious factors is maintained. In this state of healthy functional neurons, cellular feedback mechanisms help maintain homeostasis. Step B is under conditions of minor stress. The deleterious factors such as reactive oxygen and nitrogen species (RONS) and misfolded proteins (MP) contribute to a decrease in energy production (ATP) and an increase in intracellular Ca2+. Various channels and transporters elevate the Ca2+ in both cytoplasm and mitochondria. Neurons respond to this minor stress by activation of the unfolded protein response (UPR, in its initial, pro-survival phase), ubiquitin proteasome system (UPS), and chaperone-mediated autophagy (CMA). Step C indicates a much greater stress or imbalance due to substantial increases in RONS, MP, and Ca2+. ATP production is substantially less. There is also a substantial decrease in mitochondrial membrane potential. The UPR switches to its destructive mode via induction of apoptotic effector proteins such as C/EBP homologous protein, caspase-12 and c-Jun N-terminal kinase. The UPS also becomes increasingly dysfunctional because of it s inability along with CMA to adequately handle the increased load of MP. This leads to the formation of intracellular aggregates of MP. Thus, deleterious factors overwhelm the cellular homeostatic processes. In spite of the chaperone mediated autophagic process switching to macroautophagy, it cannot adequately handle the load of MP leading to the final step D. There is a strong commitment to death at this level of stress and the cell advances to programmed cell death (type-I, type-II or type-III, or their combinations)

<sup>i</sup><sup>−</sup> . Superoxide radical and ONOO- contribute to

<sup>i</sup><sup>−</sup> into H2O2. The active site of

] by reaction with O2

deletions and point mutations within neurons are well recognized.

some of the ROS. MnSOD or Cu/ZnSOD converts the O2

peroxynitrite [ONOO-

processes.

[Nagley, 2010].

substantial mitochondrial damage.

**Balance of life and death**

Fig. 1. Deleterious stress factors and restorative factors in neuronal homeostasis and neuronal death [Adpated from Nagley et. al., 2010]. ∆Ψm, mitochondrial membrane potential; MP, misfolded proteins; RONS, reactive oxygen and nitrogen species; UPS, ubiquitin-proteasome system; UPR, unfolded protein response; CMA, chaperone mediated autophagy; MA, macroautophagy. Proteins failing to fold into native structure produces inactive and usually toxic proteins. Several neurodegenerative and other diseases are believed to result from misfolded proteins (MP). The unfolded protein response (UPR) is activated in response to a stress arising from an accumulation of unfolded or MP in the lumen of the endoplasmic reticulum. UPR attempts initially to restore the normal function of the cell by halting protein translation. They also activate the signaling pathways that lead to increasing the production of molecular chaperones involved in protein folding. If these efforts are not successful, then it induces apoptotic effector proteins. Proteasomes are very large protein complexes whose main function is to degrade unneeded or damaged proteins by proteolysis and thus helping cells to regulate proteins and MP. Ubiquitin, a small protein, is used to tag the proteins that need to be degraded. The overall system of ubiquitination and proteasomal degradation is known as the ubiquitin proteosome system (UPS). Autophagy, such as micro- and macro-autophagy, refers to the degradation of intracellular components via the lysosome. Chaperone mediated autophagy, CMA, can degrade only certain proteins and not organelles and thus very selective in what it degrades. Also the substrates are translocated across the membrane on a one on one basis instead of engulfing the substrate in bulk

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 103

other cellular pathways including increased demyelination and inflammation in neurons

While remyelination is extensive in some MS lesions, it is absent or incomplete in others [Zambonin et. al. 2011]. Remyelination helps to restore conduction and helps protect the axons from further inflammation. As an adaptive process or compensatory mechanism, demyelination in the central nervous system causes an increase in the mitochondrial content within axons [Mahad et. al., 2009; Kiryu-Seo et. al., 2010]. This has been attributed to the axons from further inflammation. As an adaptive process or compensatory mechanism, response to the changes in energy needs of axons caused by redistribution of sodium channels. Demyelination compromises the ionic balance and structural integrity of the axons. It results in diffusely expressed Na+ channels with persistent Na+ leakage and forces the need for additional energy to operate the Na+/K+ ATPase pumps. The mitochondria were found to increase in the order of increased energy demand, myelinated, remyelinated and demyelinated axons [Mahad et. al., 2009; Kiryu-Seo et. al., 2010; Zambonin et.al., 2011]. The increase in mitochondrial content (mostly stationary) within remyelinated compared to myelinated axons was attributed to the increase in density of the porin elements. (Porin is a voltage gated anion channel located in the outer membrane of all mitochondria). This increase in mitochondrial content resulted in a corresponding increase in mitochondrial respiratory chain complex IV activity. The change in demyelinated axons was attributed to the change in size. While the number of mobile mitochondria in both remyelinated and myelinated axons were nearly the same, they were much less in demyelinated neurons. An approximately 4 fold increase in mitochondrial content has been observed in chronically demyelinated and non-degenerative axons [Zambonin et. al., 2011]. However, increased mitochondrial content does not necessarily mean more activity. It has been demonstrated that while the mass number may increase in amyloid precursor protein positive segments of demyelinated axons, they appear to harbor mitochondria with complex IV defects [Mahad et. al., 2009]. Within injured axons (non-phosphorylated neurofilaments: SMI32) mitochondrial depletion and decreased complex IV activity was evident, in contrast to chronically demyelinated SMI31 positive axons located in the relatively inactive areas of chronic multiple sclerosis

and tissues that are affected by multiple sclerosis [Mao & Reddy, 2010].

lesions exhibiting a significant increase in complex IV activity and mass [Table 1].

*Structural Changes (fission and fusion imbalance)*

*Abnormal Gene Expresion*

**Mitochondrial Abnormalities in Multiple Sclerosis**

*DNA Defects*

*Abnormal Enzyme Activity*

 *Deficient DNA Repair*

*Increased Free Radicals and Oxidative Damage*

Fig. 3. Mitochondrial abnormalities in multiple sclerosis [adapted from Mao & Reddy, 2010]

#### **7. Multiple sclerosis**

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system. It is associated with demyelination and a variable degree of axonal and neuronal degeneration. Demyelination decreases nerve impulse conduction velocity. Also the axons become vulnerable to inflammatory conditions. The mechanisms of tissue injury and neurodegeneration in MS are still under active investigation. Most MS patients initially experience relapsing-remitting episodes of neurologic deficits that last for six to eight weeks. The initial relapse rate is about 0.3/year. This rate declines progressively with time [Siffrin et. al., 2010]. This is followed by a gradual progression of irreversible neurological impairment or secondary progressive multiple sclerosis (SPMS) [Campbell et. al., 2011]. With advancing disease the observed increase in gray matter atrophy, which is indicative of the loss of neurons and axons, correlates well with the corresponding clinical disability.

Fig. 2. Probable causal factors of multiple sclerosis [adapted from Mao & Reddy, 2010]

Apart from the consistent features, neuroaxonal injury and dysfunction in MS, the vascular aspects of MS, an increased risk for ischemic disease, global cerebral hypoperfusion, and a chronic state of impaired venous drainage are receiving a great deal of attention [D'haeseleer et. al., 2011; Filippi & Rocca, 2011].

The precise causal factors of multiple sclerosis are unknown. However, it is possible that multiple factors [Fig. 2] are involved in causing multiple sclerosis, including DNA defects in nuclear and mitochondrial genomes, viral infection, hypoxia and oxidative stress, lack of sunlight or sufficient levels of vitamin D, and increased macrophages and lymphocytes in the brain [Mao & Reddy, 2010].

Current research has shown that mitochondrial abnormalities are involved in the development and progression of multiple sclerosis [Fig. 3], including: mitochondrial DNA defects, abnormal mitochondrial gene expression, defective mitochondrial enzyme activities, abnormal or deficient mitochondrial DNA repair mechanisms, and mitochondrial dysfunction. Studies suggest that abnormal mitochondrial dynamics (imbalance in mitochondrial fission and fusion) plays a key role in tissues affected by multiple sclerosis. Furthermore, mitochondrial abnormalities and mitochondrial energy failure may impact

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system. It is associated with demyelination and a variable degree of axonal and neuronal degeneration. Demyelination decreases nerve impulse conduction velocity. Also the axons become vulnerable to inflammatory conditions. The mechanisms of tissue injury and neurodegeneration in MS are still under active investigation. Most MS patients initially experience relapsing-remitting episodes of neurologic deficits that last for six to eight weeks. The initial relapse rate is about 0.3/year. This rate declines progressively with time [Siffrin et. al., 2010]. This is followed by a gradual progression of irreversible neurological impairment or secondary progressive multiple sclerosis (SPMS) [Campbell et. al., 2011]. With advancing disease the observed increase in gray matter atrophy, which is indicative of the loss of neurons and axons, correlates well with the corresponding clinical disability.

> *Accumulation of Macrophages and Lymphocytes in the Brain*

Fig. 2. Probable causal factors of multiple sclerosis [adapted from Mao & Reddy, 2010]

Apart from the consistent features, neuroaxonal injury and dysfunction in MS, the vascular aspects of MS, an increased risk for ischemic disease, global cerebral hypoperfusion, and a chronic state of impaired venous drainage are receiving a great deal of attention

The precise causal factors of multiple sclerosis are unknown. However, it is possible that multiple factors [Fig. 2] are involved in causing multiple sclerosis, including DNA defects in nuclear and mitochondrial genomes, viral infection, hypoxia and oxidative stress, lack of sunlight or sufficient levels of vitamin D, and increased macrophages and lymphocytes in

Current research has shown that mitochondrial abnormalities are involved in the development and progression of multiple sclerosis [Fig. 3], including: mitochondrial DNA defects, abnormal mitochondrial gene expression, defective mitochondrial enzyme activities, abnormal or deficient mitochondrial DNA repair mechanisms, and mitochondrial dysfunction. Studies suggest that abnormal mitochondrial dynamics (imbalance in mitochondrial fission and fusion) plays a key role in tissues affected by multiple sclerosis. Furthermore, mitochondrial abnormalities and mitochondrial energy failure may impact

*Multiple*

*Sclerosis*

*Genetic Factors*

> *Reduced Sunlight (↓Vitamin D)*

**7. Multiple sclerosis** 

*Hypoxia and Oxidative Stress*

 *Viral Infection*

[D'haeseleer et. al., 2011; Filippi & Rocca, 2011].

the brain [Mao & Reddy, 2010].

other cellular pathways including increased demyelination and inflammation in neurons and tissues that are affected by multiple sclerosis [Mao & Reddy, 2010].

While remyelination is extensive in some MS lesions, it is absent or incomplete in others [Zambonin et. al. 2011]. Remyelination helps to restore conduction and helps protect the axons from further inflammation. As an adaptive process or compensatory mechanism, demyelination in the central nervous system causes an increase in the mitochondrial content within axons [Mahad et. al., 2009; Kiryu-Seo et. al., 2010]. This has been attributed to the axons from further inflammation. As an adaptive process or compensatory mechanism, response to the changes in energy needs of axons caused by redistribution of sodium channels. Demyelination compromises the ionic balance and structural integrity of the axons. It results in diffusely expressed Na+ channels with persistent Na+ leakage and forces the need for additional energy to operate the Na+/K+ ATPase pumps. The mitochondria were found to increase in the order of increased energy demand, myelinated, remyelinated and demyelinated axons [Mahad et. al., 2009; Kiryu-Seo et. al., 2010; Zambonin et.al., 2011]. The increase in mitochondrial content (mostly stationary) within remyelinated compared to myelinated axons was attributed to the increase in density of the porin elements. (Porin is a voltage gated anion channel located in the outer membrane of all mitochondria). This increase in mitochondrial content resulted in a corresponding increase in mitochondrial respiratory chain complex IV activity. The change in demyelinated axons was attributed to the change in size. While the number of mobile mitochondria in both remyelinated and myelinated axons were nearly the same, they were much less in demyelinated neurons. An approximately 4 fold increase in mitochondrial content has been observed in chronically demyelinated and non-degenerative axons [Zambonin et. al., 2011]. However, increased mitochondrial content does not necessarily mean more activity. It has been demonstrated that while the mass number may increase in amyloid precursor protein positive segments of demyelinated axons, they appear to harbor mitochondria with complex IV defects [Mahad et. al., 2009]. Within injured axons (non-phosphorylated neurofilaments: SMI32) mitochondrial depletion and decreased complex IV activity was evident, in contrast to chronically demyelinated SMI31 positive axons located in the relatively inactive areas of chronic multiple sclerosis lesions exhibiting a significant increase in complex IV activity and mass [Table 1].

Fig. 3. Mitochondrial abnormalities in multiple sclerosis [adapted from Mao & Reddy, 2010]

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 105

Median & (range)

testing; all values represent counted cells/mm2. Active lesions = classical actively demyelinating lesions; slowly expanding lesions = lesions with inactive lesion centre, surrounded by a small rim of activated microglia with recent myelin-degradation products; inactive lesions = lesions without any recent demyelinating activity; NAWM multiple sclerosis = normal-appearing white matter from patients with multiple sclerosis; white matter controls = normal white matter of all controls; APP = amyloid precursor protein reactive axonal spheroids or end bulbs; CD3 = T cells; percentage of E06 positive area = densitometric analysis of area covered by E06 immunoreactivity; E06 axon spheroids = axonal spheroids or end bulbs stained by E06 antibody; HLA-D = class II MHC-positive macrophages/microglia; MDA-2 OG = oligodendrocyte-like cells, immunoreactive for MDA-2; 8-OHdG nuclei = number of cell nuclei containing 8 hydroxy-D-guanosine immunoreactivity. Data show a highly significant accumulation of oxidized DNA and oxidized lipids in active multiple sclerosis lesions in comparison with controls. Oxidized DNA and lipids are predominantly seen in lesions with high T-cell and macrophage infiltrates and with profound microglia activation. (p values not included for brevity).

A recent review details the current immunomodulatory treatments for MS. Other alternatives beyond immune-directed approaches are also speculated in this review [Aktas et. al., 2010]. Increased concentrations of reactive oxygen and nitrogen species found in MS, for example, lead to inhibition of ATP production within the axon. The ATP deficiency leads to loss of Na+/K+ ATPase and collapse of transmembrane ionic gradients. There is also an increase in intracellular Ca2+ levels and a decrease in mRNA levels of mitochondrial genes. At the same time immune –related demyelination also takes place. These results have prompted ion channel homeostasis as a potential therapeutic target to ameliorate the failed

Peroxy nitrite formation at the site of inflammation has been measured using nitrotyrosine as a biochemical marker. Levels of nitrite and nitrate, the stable oxidation products of nitric oxide and peroxynitrite measured in cerebral fluid samples also revealed significantly

higher levels of nitrate during clinical relapses of MS [Cross et. al., 1998].

E06 axon spheroids 1.3 (18.2) 1.2 (121.2) 0.4 (2.3) 0.0 (1.2) 0.00 (0.0) MDA-2 OG 1.6 (25.8) 0.9 (5.4) 0.9 (1.6) 0.4 (9.2) 0.00 (1.6) 8-OHdG nuclei 5.6 (78.1) 9.0 (19.0) 1.6 (6.8) 1.4 (12.0) 0.40 (10.4) APP 44.3 (197.6) 16.1 (40.0) 0.3 (1.5) 0.0 (1.0) 0.00 (0.8) CD3 73.6 (298.8) 51.2 (105.6) 10.0 (69.1) 4.8 (38.4) 9.60 (49.6) HLA-D 324.8 (585.6) 148.3 (398.0) 29.0 (246.5) 99.3 (190.0) 43.10 (114.4) Table 2. Quantification of oxidized lipids and oxidized DNA in different types of multiple sclerosis lesions in comparison with controls [Haider et. al., 2011]. Values represent medians and range (95th percentile range values); P-values are corrected by Bonferroni for multiple

Inactive lesion NAWM MS White matter

Median & (range)

Median & (range)

3.1(26.5) 1.6 (16.7) 0.6 (9.2) 0.8 (17.1) 0.06 (2.9)

controls

Median & (range)

Active lesion Slowly expanding Lesion

Median & (range)

[For details, please see Haider et. al., 2011]

energy metabolism.

Percentage of E06 positive area


Table 1. Quantification of the intensity of complex IV active elements and mitochondrial mass within axons [Mahad et. al., 2009]. The intensity of complex IV active elements represents the difference in densitometric value between background and complex IV active elements in inverted grey scale 100x brightfield images of cytochrome c oxidase or COX histochemistry. The percentage area of porin reactive elements within axons was calculated based on the total area of axonal porin reactive elements in triple labeled (porin, syntaphilin and axonal marker) and area of axons in confocal images. †P = 0.001 (versus NAWM) and ‡P = 0.002 (versus CON). #P<0.001 (versus SMI31 in CON, NAWM and lesion). \*P<0.001 (versus SMI31 in NAWM and CON as well as SMI32 in lesion and APP in lesion). [For details, please see Mahad et. al., 2009]

Mitochondrial injury and subsequent energy failure have been implicated in the pathogenesis of MS [Lu et. al., 2000; Dutta et. al., 2006; Mahad et. al., 2008, 2009; Haider et. al., 2011; Campbell et. al., 2011]. Proteins and DNA in mitochondria are highly vulnerable to free radical damage and consequent mitochondrial injury in MS. The likely candidates involved in tissue injury in MS are the ROS and nitric oxide intermediates. These are produced by activated macrophages and microglia. In the brain tissue of patients with MS, oxidized DNA and oxidized lipids have been detected [Lu et. al., 2000]. Oxidized phospholipids and malondialdehyde (lipid peroxidation-derived structures) data from MS lesions of different activity of patients with acute, relapsing, remitting and progressive disease were found to be concentrated in active MS plaques, in areas known as initial demyelinating lesion or the "prephagocytic" stage of active MS lesions [Haider et. al., 2011]. There was good correlation between inflammation and the extent of DNA and lipid oxidation. Data in table 2 indicate up to a 5 fold increase in the extent of DNA damage (8-OHdG staining) and lipid oxidation (E06 and MDA-2 staining) in active lesions versus inactive lesions, normal white matter in MS patients, and white matter controls. The oxidation is predominantly seen in lesions with high T-cell and macrophage infiltrates and with profound microglial activation (HLA-D staining).

So far the efforts for complete restoration of axonal mitochondria following remyelination have not been successful. Thus the need for preservation of myelinated axons is exemplified by the fact that remyelinated axons have increased energy demand. This may also result in deficient neurons and reach detrimental levels in the long term. Mitochondrial DNA deletions have been found in the neurons in the progressive stage of MS [Campbell et. al., 2011]. The pathological features of MS lesions include demyelination and oligodendrocyte apoptosis, preferential destruction of small- caliber axons, differentiation arrest of oligodendrocyte progenitor cells and remyelination failure, and astrocyte dysfunction [Haider et. al., 2011].


Control(SMI31) NAWM(SMI31) Lesion(APP) Lesion(SMI32) Lesion(SM131)

Brian 17.93±5.89 16.14±6.54 9.85±6.94# 10.26±5.43# 20.10±7.38†, ‡ Spinal cord 16.99±15.49 16.87±12.44 9.01±11.16# 15.93±13.01 23.04±11.07\*

Brian 6.74±.2.82 7.35±4.30 7.93±4.50 4.19±3.95† 11.15±5.23\* Spinal cord 5.93±4.38 3.46±3.92 8.26±7.40 1.64 ±1.39# 13.97±7.23\* Table 1. Quantification of the intensity of complex IV active elements and mitochondrial mass within axons [Mahad et. al., 2009]. The intensity of complex IV active elements represents the difference in densitometric value between background and complex IV active elements in inverted grey scale 100x brightfield images of cytochrome c oxidase or COX histochemistry. The percentage area of porin reactive elements within axons was calculated based on the total area of axonal porin reactive elements in triple labeled (porin, syntaphilin and axonal marker) and area of axons in confocal images. †P = 0.001 (versus NAWM) and ‡P = 0.002 (versus CON). #P<0.001 (versus SMI31 in CON, NAWM and lesion). \*P<0.001 (versus SMI31 in NAWM and CON as well as SMI32 in lesion and APP in lesion). [For details, please see

Mitochondrial injury and subsequent energy failure have been implicated in the pathogenesis of MS [Lu et. al., 2000; Dutta et. al., 2006; Mahad et. al., 2008, 2009; Haider et. al., 2011; Campbell et. al., 2011]. Proteins and DNA in mitochondria are highly vulnerable to free radical damage and consequent mitochondrial injury in MS. The likely candidates involved in tissue injury in MS are the ROS and nitric oxide intermediates. These are produced by activated macrophages and microglia. In the brain tissue of patients with MS, oxidized DNA and oxidized lipids have been detected [Lu et. al., 2000]. Oxidized phospholipids and malondialdehyde (lipid peroxidation-derived structures) data from MS lesions of different activity of patients with acute, relapsing, remitting and progressive disease were found to be concentrated in active MS plaques, in areas known as initial demyelinating lesion or the "prephagocytic" stage of active MS lesions [Haider et. al., 2011]. There was good correlation between inflammation and the extent of DNA and lipid oxidation. Data in table 2 indicate up to a 5 fold increase in the extent of DNA damage (8-OHdG staining) and lipid oxidation (E06 and MDA-2 staining) in active lesions versus inactive lesions, normal white matter in MS patients, and white matter controls. The oxidation is predominantly seen in lesions with high T-cell and macrophage infiltrates and

So far the efforts for complete restoration of axonal mitochondria following remyelination have not been successful. Thus the need for preservation of myelinated axons is exemplified by the fact that remyelinated axons have increased energy demand. This may also result in deficient neurons and reach detrimental levels in the long term. Mitochondrial DNA deletions have been found in the neurons in the progressive stage of MS [Campbell et. al., 2011]. The pathological features of MS lesions include demyelination and oligodendrocyte apoptosis, preferential destruction of small- caliber axons, differentiation arrest of oligodendrocyte progenitor cells and remyelination failure, and astrocyte dysfunction

**Complex IV Intensity** 

Mahad et. al., 2009]

[Haider et. al., 2011].

with profound microglial activation (HLA-D staining).

**Porin Percent Area**


Table 2. Quantification of oxidized lipids and oxidized DNA in different types of multiple sclerosis lesions in comparison with controls [Haider et. al., 2011]. Values represent medians and range (95th percentile range values); P-values are corrected by Bonferroni for multiple testing; all values represent counted cells/mm2. Active lesions = classical actively demyelinating lesions; slowly expanding lesions = lesions with inactive lesion centre, surrounded by a small rim of activated microglia with recent myelin-degradation products; inactive lesions = lesions without any recent demyelinating activity; NAWM multiple sclerosis = normal-appearing white matter from patients with multiple sclerosis; white matter controls = normal white matter of all controls; APP = amyloid precursor protein reactive axonal spheroids or end bulbs; CD3 = T cells; percentage of E06 positive area = densitometric analysis of area covered by E06 immunoreactivity; E06 axon spheroids = axonal spheroids or end bulbs stained by E06 antibody; HLA-D = class II MHC-positive macrophages/microglia; MDA-2 OG = oligodendrocyte-like cells, immunoreactive for MDA-2; 8-OHdG nuclei = number of cell nuclei containing 8 hydroxy-D-guanosine immunoreactivity. Data show a highly significant accumulation of oxidized DNA and oxidized lipids in active multiple sclerosis lesions in comparison with controls. Oxidized DNA and lipids are predominantly seen in lesions with high T-cell and macrophage infiltrates and with profound microglia activation. (p values not included for brevity). [For details, please see Haider et. al., 2011]

A recent review details the current immunomodulatory treatments for MS. Other alternatives beyond immune-directed approaches are also speculated in this review [Aktas et. al., 2010]. Increased concentrations of reactive oxygen and nitrogen species found in MS, for example, lead to inhibition of ATP production within the axon. The ATP deficiency leads to loss of Na+/K+ ATPase and collapse of transmembrane ionic gradients. There is also an increase in intracellular Ca2+ levels and a decrease in mRNA levels of mitochondrial genes. At the same time immune –related demyelination also takes place. These results have prompted ion channel homeostasis as a potential therapeutic target to ameliorate the failed energy metabolism.

Peroxy nitrite formation at the site of inflammation has been measured using nitrotyrosine as a biochemical marker. Levels of nitrite and nitrate, the stable oxidation products of nitric oxide and peroxynitrite measured in cerebral fluid samples also revealed significantly higher levels of nitrate during clinical relapses of MS [Cross et. al., 1998].

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 107

Therapeutic quick intervention may reduce the infarct volume in the penumbra because the irreversible damage occurs relatively slowly [Sims & Muyderman, 2010]. The only embolic or thrombolytic agent used to reverse arterial occlusion within the first 3 h is tissue plasminogen activator (tPA). As an enzyme, it catalyzes the conversion of plasminogen to the enzyme responsible for clot breakdown, plasmin. In ~17 % of ischemic stroke patients spontaneous reversal of occlusion takes place in 6 h and in 40-50% of patients in 4 days. Quick restoration of normal blood flow to this region can result in substantial recovery of energy- related metabolites. However, in the post ischemic tissue, energy requirements are low and the glucose oxidation is due to limitations on the mitochondrial oxidation of pyruvate. This results in a secondary impairment of mitochondrial function and consequent

cell death. Normalcy of function can be maintained in the outermost viable tissue.

vessel, e.g. middle cerebral artery, resulting in primarily apoptotic cell death.

leading to apoptosis and necrosis.

nitric oxide (NO**.**

While focal ischemia is confined to a specific region of the brain, global ischemia encompasses wide areas of the brain tissue. Focal ischemia occurs in a region when a blood clot has occluded in a downstream region of artery in the brain (ischemic stroke). This occlusion or blockage may be caused by thrombosis (a blood clot formed locally obstructing the blood flow) or arterial embolism (obstruction of blood flow due to an embolus from elsewhere in the body). While ischemic strokes are caused by interruption of the blood supply, hemorrhagic strokes are the result of rupture of a blood vessel. Hemorrhagic strokes result in areas of friable tissue, containing areas of both viable and dead tissue. Transient global ischemia involves a brief interruption in blood flow usually in a larger cerebral

More than 80% of all strokes are due to focal ischemia. Unless treated, the occlusion of an artery produces tissue infarction resulting in a loss of all cells including neurons, astrocytes, oligodendrocytes, microglia and endothelial cell [Sims & Muyderman, 2010]. The stroke results in mitochondrial impairment because the blood flow becomes < 20% of the normal and a consequent reduction in glucose and oxygen supplies ensue. This attenuates ATP and reactive oxygen species production as well as apoptosis. Lack of ATP production disrupts the ionic gradients across the plasma membrane. The net result is marked losses of intracellular K+ and a large influx of Ca2+ into cells [Doyle et. al., 2008]. Thus there is heavy involvement of the impaired mitochondria in the development of the tissue injury after ischemic attack, due to modifications in ATP production and other mitochondrial changes

Further, a transient zone in-between the core and penumbra is likely to merge to the core if the cerebral blood flow is not restored early. The damage in this transient zone is a result of the release of cellular contents from those necrotic "core" cells, such as the oxidative enzymes of various organelles i.e. lysozomes and peroxisomes. Eventually this transient zone would contribute to the total infarct volume. The penumbral region is surrounded by a region of viable tissue [Mehta et. al., 2007]. Penumbra varies in size and can be rescued Ischemic cell death is also attributed to abnormal activation of enzymes such as poly-ADP ribose polymerase (PARP) and the caspases. Oxidative stress, which produces free radical

apoptosis in focal ischemia. Peroxynitrite is formed by the reaction of NO**.** with superoxide. Mitochondria are targeted by peroxynitrite and the resulting mitochondrial dysfunction during severe hypoxia-ischemia increases generation of oxygen free radicals. This leads to dysfunction of cellular membrane causing necrosis [Mehta et. al. 2007]. An additional consequence of ischemia involves the dissociation of the electron transport chain within minutes of the insult. Ubiquinone and cytochrome C, which serve as electron shuttles,

) and reactive peroxynitrite (ONOO-), is implicated in both necrosis and

In view of our data with palladium α-lipoic acid formulation (section 10) on the enhanced enzymatic activities of Krebs cycle and electron transport chain enzymes in animals, antioxidant activity and the ability of this formulation to repair DNA, we had decided, as a preliminary step, to investigate its usefulness in ameliorating the fatigue conditions in 15 MS patients. The study is expected to be completed soon.

#### **8. Cerebral ischemia**

An insufficient or reduced blood flow to the brain to meet the metabolic demand will result in cerebral or brain ischemia. The normal cerebral blood flow is ~ 50 to 60 mL/100g/min. Death of brain tissue is a consequence of poor oxygen supply or cerebral hypoxia resulting from the insufficient blood flow. A prototype of brain damage during cerebral ischemia is shown in Fig. 4. [adapted from Mehta et. al., 2007]. Maximum damage occurs as a result of ischemic necrosis (infarction) at the "core" or "focal" tissue region, where the blood flow is < 7 mL/100g/min. Since the cellular integrity is compromised during necrosis, cellular damage repair at the core is extremely hard. The blood flow in the surrounding less-severely ischemic boundary ("penumbral" or "perifocal" tissue) is ~ 7 to 17 mL/100g/min. The penumbra is metabolically active but electrically silent [Mehta et. al., 2007]. More moderate alterations develop in this region because of the near normal glucose use, but the oxidative metabolism is still impaired. Different mechanisms contribute to cell death in the core and penumbra due to differences in the severity of ischemia.

Fig. 4. A prototype brain damage during cerebral ischemia: core, a region where cells undergo necrosis. The region surrounding the core is called ischemic penumbra, a site of delayed mode of cell death (apoptosis) due to availability of ATP. Further, a transient zone in-between the core and penumbra is likely to merge to the core if the cerebral blood flow is not restored early. The penumbral region is surrounded by a region of viable tissue [adapted from Mehta et. al., 2007]

In view of our data with palladium α-lipoic acid formulation (section 10) on the enhanced enzymatic activities of Krebs cycle and electron transport chain enzymes in animals, antioxidant activity and the ability of this formulation to repair DNA, we had decided, as a preliminary step, to investigate its usefulness in ameliorating the fatigue conditions in 15 MS

An insufficient or reduced blood flow to the brain to meet the metabolic demand will result in cerebral or brain ischemia. The normal cerebral blood flow is ~ 50 to 60 mL/100g/min. Death of brain tissue is a consequence of poor oxygen supply or cerebral hypoxia resulting from the insufficient blood flow. A prototype of brain damage during cerebral ischemia is shown in Fig. 4. [adapted from Mehta et. al., 2007]. Maximum damage occurs as a result of ischemic necrosis (infarction) at the "core" or "focal" tissue region, where the blood flow is < 7 mL/100g/min. Since the cellular integrity is compromised during necrosis, cellular damage repair at the core is extremely hard. The blood flow in the surrounding less-severely ischemic boundary ("penumbral" or "perifocal" tissue) is ~ 7 to 17 mL/100g/min. The penumbra is metabolically active but electrically silent [Mehta et. al., 2007]. More moderate alterations develop in this region because of the near normal glucose use, but the oxidative metabolism is still impaired. Different mechanisms contribute to cell death in the core and

Fig. 4. A prototype brain damage during cerebral ischemia: core, a region where cells undergo necrosis. The region surrounding the core is called ischemic penumbra, a site of delayed mode of cell death (apoptosis) due to availability of ATP. Further, a transient zone in-between the core and penumbra is likely to merge to the core if the cerebral blood flow is

not restored early. The penumbral region is surrounded by a region of viable tissue

[adapted from Mehta et. al., 2007]

patients. The study is expected to be completed soon.

penumbra due to differences in the severity of ischemia.

**8. Cerebral ischemia** 

Therapeutic quick intervention may reduce the infarct volume in the penumbra because the irreversible damage occurs relatively slowly [Sims & Muyderman, 2010]. The only embolic or thrombolytic agent used to reverse arterial occlusion within the first 3 h is tissue plasminogen activator (tPA). As an enzyme, it catalyzes the conversion of plasminogen to the enzyme responsible for clot breakdown, plasmin. In ~17 % of ischemic stroke patients spontaneous reversal of occlusion takes place in 6 h and in 40-50% of patients in 4 days. Quick restoration of normal blood flow to this region can result in substantial recovery of energy- related metabolites. However, in the post ischemic tissue, energy requirements are low and the glucose oxidation is due to limitations on the mitochondrial oxidation of pyruvate. This results in a secondary impairment of mitochondrial function and consequent cell death. Normalcy of function can be maintained in the outermost viable tissue.

While focal ischemia is confined to a specific region of the brain, global ischemia encompasses wide areas of the brain tissue. Focal ischemia occurs in a region when a blood clot has occluded in a downstream region of artery in the brain (ischemic stroke). This occlusion or blockage may be caused by thrombosis (a blood clot formed locally obstructing the blood flow) or arterial embolism (obstruction of blood flow due to an embolus from elsewhere in the body). While ischemic strokes are caused by interruption of the blood supply, hemorrhagic strokes are the result of rupture of a blood vessel. Hemorrhagic strokes result in areas of friable tissue, containing areas of both viable and dead tissue. Transient global ischemia involves a brief interruption in blood flow usually in a larger cerebral vessel, e.g. middle cerebral artery, resulting in primarily apoptotic cell death.

More than 80% of all strokes are due to focal ischemia. Unless treated, the occlusion of an artery produces tissue infarction resulting in a loss of all cells including neurons, astrocytes, oligodendrocytes, microglia and endothelial cell [Sims & Muyderman, 2010]. The stroke results in mitochondrial impairment because the blood flow becomes < 20% of the normal and a consequent reduction in glucose and oxygen supplies ensue. This attenuates ATP and reactive oxygen species production as well as apoptosis. Lack of ATP production disrupts the ionic gradients across the plasma membrane. The net result is marked losses of intracellular K+ and a large influx of Ca2+ into cells [Doyle et. al., 2008]. Thus there is heavy involvement of the impaired mitochondria in the development of the tissue injury after ischemic attack, due to modifications in ATP production and other mitochondrial changes leading to apoptosis and necrosis.

Further, a transient zone in-between the core and penumbra is likely to merge to the core if the cerebral blood flow is not restored early. The damage in this transient zone is a result of the release of cellular contents from those necrotic "core" cells, such as the oxidative enzymes of various organelles i.e. lysozomes and peroxisomes. Eventually this transient zone would contribute to the total infarct volume. The penumbral region is surrounded by a region of viable tissue [Mehta et. al., 2007]. Penumbra varies in size and can be rescued

Ischemic cell death is also attributed to abnormal activation of enzymes such as poly-ADP ribose polymerase (PARP) and the caspases. Oxidative stress, which produces free radical nitric oxide (NO**.** ) and reactive peroxynitrite (ONOO-), is implicated in both necrosis and apoptosis in focal ischemia. Peroxynitrite is formed by the reaction of NO**.** with superoxide. Mitochondria are targeted by peroxynitrite and the resulting mitochondrial dysfunction during severe hypoxia-ischemia increases generation of oxygen free radicals. This leads to dysfunction of cellular membrane causing necrosis [Mehta et. al. 2007]. An additional consequence of ischemia involves the dissociation of the electron transport chain within minutes of the insult. Ubiquinone and cytochrome C, which serve as electron shuttles,

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 109

The adenylate energy charge decreases rapidly to ~ 0.4-0.5 during the initial hours and remains above 0.8 after 2 h compared to the normal value in the brain of ~ 0.93. While adenylate kinase catalyses the conversion of some ADP to AMP to meet short term energy needs of the brain, in ischemic tissue, the adenine nucleotide pool is depleted by the conversion of AMP to inosine and hypoxanthine. Phosphocreatine, the short term energy reserve of the brain falls quickly to <30% of normal during ischemia. Phosphocreatine stabilizes to about 70% of normal after ~ 2 h of ischemia. ATP regeneration from ADP is catalyzed by the enzyme creatine kinase. Lack of oxygen forces glucose to go the glycolytic pathway creating a 10-fold increase in lactate and consequent lowering of pH. Of course lack of removal of lactate due to limited blood flow may also be contributing to this accumulation. In addition, restricted blood flow appears to have a greater effect on the delivery of oxygen to the tissues versus glucose, since penumbral glucose levels are either the same or slightly higher, while lactate levels are much higher (but less than in the core). During the first 2 h following reperfusion, phosphocreatine and adenylate energy charge are recovered to >90% of normal compared to ATP values of 50-70%. This resistance of ATP for restoration is attributed to the depletion of the adenine nucleotide pool. In the penumbral tissue, phosphocreatine and adenine nucleotide balance, but not ATP are recovered almost completely within 1 h of reperfusion for ischemic periods of 3 h or longer [Sims & Muyderman, 2010]. The glucose utilization is less in the penumbral region during the first

hour of reperfusion. The lactate, on the other hand, is decreased during this period.

kinase enzyme [Sims & Muyderman, 2010].

irreversible cell dysfunction.

The reduction in ATP production in the ischemic brain may be associated with decreased neuronal activity of the post-ischemic brain as a result of the enzyme AMP-activated protein

It must be mentioned that there is a complete or near complete recovery of mitochondrial respiratory function in core and penumbral tissues within the first hour following reperfusion. This is followed by a secondary deterioration, indicative of the development of

Under normal physiological conditions, the channel within the N-methyl-D-aspartic acid (NMDA) glutamate receptor is blocked in a voltage dependent manner by Mg2+. The triggering of energy deficits and neuronal depolarization are the results of decreased cerebral blood flow. The mild depolarization results in the dislodging of Mg2+ and glutamate, which are consequently released in large amounts to the extracellular space. This leads to an over-activation of the NMDA and AMPA glutamate receptors. Since these receptors regulate Ca2+ ion channels, a calcium ionic imbalance occurs in neurons. This influx of Ca2+, is due to this increase in glutamate release from neurons and astrocytes induced by the ischemia. Apart from the traditional ionotropic glutamate receptors, the influx of Ca2+ is also attributed to some emerging metabotropic and channel mechanisms that include: sodium-calcium exchangers, hemi channels (unopposed half-gap junctions), acid sensing channels, volume-regulated anion channels, nonselective cation channels and signaling cascades that mediate crosstalk between redundant pathways of cell death [Besancon et al., 2008]. This abnormal intracellular accumulation of Ca2+ is involved in the triggering of cell death by up regulation of a wide range of cell death executioners that include ATPases that serve to further deplete energy stores, lipases that damage lipid membranes of organelles and the cell surface itself, proteases that dismantle the cytoarchitecture of the neuron, and DNAses that damage the nucleus [Besancon et. al. 2008]. Bioenergetics of cerebral ischemia (both focal and global) as well as gray and white matter ischemia were recently reviewed from a cellular perspective. A brief summary is given in

translocate from the inner mitochondrial membrane. This is of particular consequence upon restoration of blood flow. While reperfusion limits some damage, oxidative stress is increased under these conditions. It has been found that over expression of Mn2+-superoxide dismutase, which converts superoxide to hydrogen peroxide results in moderate reductions in the size of infarction in temporary ischemia [Sims & Muyderman, 2010]. Addition of the mitochondrial uncoupling agent, dinitrophenol, was found to modulate the Ca2+ content and production of free radicals in the mitochondria of penumbra [Korde et. al., 2005].

The reduced delivery of oxygen and glucose to the tissue in focal ischemia affects the function of the mitochondria. Mitochondrial properties undergo further changes depending on the severity and duration of ischemia and also following reperfusion. Development of cell death pathways depends on the impaired mitochondria's ability to generate ATP.


Table 3. The effects of focal ischemia for up to 2 h and of subsequent reperfusion for 1 h on the content of energy-related metabolites and pathways of energy metabolism [Sims & Muyderman, 2010]. Differences are shown compared to non-ischemic tissue. ↓: decreased to >65%; ↓↓: decreased to between 35% and 65% ; ↓↓↓: decreased to less than 35%; ↑: increased less than four-fold; ↑↑: increased greater than four-fold; N.C.: no significant change. Two symbols indicate findings that differ between published reports. \*, direct evaluation of these properties in severely ischemic tissue may not give reliable information. The magnitude of these reductions is assumed from the large decrease in substrate delivery and large changes in ATP and phosphocreatine content [Sims & Muyderman, 2010]

The changes in energy-related metabolites and in the contributing metabolic pathways in brain tissue in the first 2 h of ischemia and reperfusion for 1 h are summarized in Table 3 [Sims & Muyderman, 2010]. In the core, the ATP and glucose content falls significantly in the first 5 min of occlusion and then ATP stabilizes to ~ 15-30% of normal for at least the first 2 h and then reaches about 50%. The initial rapid decrease is attributed to the major redistribution of ions across the plasma membrane of cells. In view of our admittance data on the H2O2-Ca2+, H2O2-Na+ interactions, we strongly believe that ion-peroxide interactions must also be playing a major part in this process. The adenylate energy charge, a measure of intracellular balance between ATP, ADP, and AMP, is given by:

Adenylate energy charge = {[ATP] + 0.5 [ADP]/ [ATP] + [ADP] + [AMP]} (31)

translocate from the inner mitochondrial membrane. This is of particular consequence upon restoration of blood flow. While reperfusion limits some damage, oxidative stress is increased under these conditions. It has been found that over expression of Mn2+-superoxide dismutase, which converts superoxide to hydrogen peroxide results in moderate reductions in the size of infarction in temporary ischemia [Sims & Muyderman, 2010]. Addition of the mitochondrial uncoupling agent, dinitrophenol, was found to modulate the Ca2+ content and production of free radicals in the mitochondria of penumbra [Korde et. al., 2005]. The reduced delivery of oxygen and glucose to the tissue in focal ischemia affects the function of the mitochondria. Mitochondrial properties undergo further changes depending on the severity and duration of ischemia and also following reperfusion. Development of cell death pathways depends on the impaired mitochondria's ability to generate ATP.

**Focal ischemia Reperfusion** 

ATP ↓↓↓ ↓↓ ↓↓ ↓ Adenylate energy charge ↓↓ ↓ ↓/N.C. N.C. Total adenine nucleotides ↓↓ ↓↓ ↓↓ ↓ Phosphocreatine ↓↓↓ ↓ ↓ N.C. Lactate ↑↑ ↑↑ ↑↑ ↑/N.C. Glucose ↓↓↓ N.C. N.C. N.C.

Glucose use ↓↓↓\* N.C. ↓↓ ↓↓ Oxidative metabolism ↓↓↓\* ↓↓↓ ↓↓ ↓↓

in ATP and phosphocreatine content [Sims & Muyderman, 2010]

intracellular balance between ATP, ADP, and AMP, is given by:

Table 3. The effects of focal ischemia for up to 2 h and of subsequent reperfusion for 1 h on the content of energy-related metabolites and pathways of energy metabolism [Sims & Muyderman, 2010]. Differences are shown compared to non-ischemic tissue. ↓: decreased to >65%; ↓↓: decreased to between 35% and 65% ; ↓↓↓: decreased to less than 35%; ↑: increased less than four-fold; ↑↑: increased greater than four-fold; N.C.: no significant change. Two symbols indicate findings that differ between published reports. \*, direct evaluation of these properties in severely ischemic tissue may not give reliable information. The magnitude of these reductions is assumed from the large decrease in substrate delivery and large changes

The changes in energy-related metabolites and in the contributing metabolic pathways in brain tissue in the first 2 h of ischemia and reperfusion for 1 h are summarized in Table 3 [Sims & Muyderman, 2010]. In the core, the ATP and glucose content falls significantly in the first 5 min of occlusion and then ATP stabilizes to ~ 15-30% of normal for at least the first 2 h and then reaches about 50%. The initial rapid decrease is attributed to the major redistribution of ions across the plasma membrane of cells. In view of our admittance data on the H2O2-Ca2+, H2O2-Na+ interactions, we strongly believe that ion-peroxide interactions must also be playing a major part in this process. The adenylate energy charge, a measure of

Adenylate energy charge = {[ATP] + 0.5 [ADP]/ [ATP] + [ADP] + [AMP]} (31)

**Metabolites** 

**Metabolic activity** 

Core Penumbra Core Penumbra

The adenylate energy charge decreases rapidly to ~ 0.4-0.5 during the initial hours and remains above 0.8 after 2 h compared to the normal value in the brain of ~ 0.93. While adenylate kinase catalyses the conversion of some ADP to AMP to meet short term energy needs of the brain, in ischemic tissue, the adenine nucleotide pool is depleted by the conversion of AMP to inosine and hypoxanthine. Phosphocreatine, the short term energy reserve of the brain falls quickly to <30% of normal during ischemia. Phosphocreatine stabilizes to about 70% of normal after ~ 2 h of ischemia. ATP regeneration from ADP is catalyzed by the enzyme creatine kinase. Lack of oxygen forces glucose to go the glycolytic pathway creating a 10-fold increase in lactate and consequent lowering of pH. Of course lack of removal of lactate due to limited blood flow may also be contributing to this accumulation. In addition, restricted blood flow appears to have a greater effect on the delivery of oxygen to the tissues versus glucose, since penumbral glucose levels are either the same or slightly higher, while lactate levels are much higher (but less than in the core).

During the first 2 h following reperfusion, phosphocreatine and adenylate energy charge are recovered to >90% of normal compared to ATP values of 50-70%. This resistance of ATP for restoration is attributed to the depletion of the adenine nucleotide pool. In the penumbral tissue, phosphocreatine and adenine nucleotide balance, but not ATP are recovered almost completely within 1 h of reperfusion for ischemic periods of 3 h or longer [Sims & Muyderman, 2010]. The glucose utilization is less in the penumbral region during the first hour of reperfusion. The lactate, on the other hand, is decreased during this period.

The reduction in ATP production in the ischemic brain may be associated with decreased neuronal activity of the post-ischemic brain as a result of the enzyme AMP-activated protein kinase enzyme [Sims & Muyderman, 2010].

It must be mentioned that there is a complete or near complete recovery of mitochondrial respiratory function in core and penumbral tissues within the first hour following reperfusion. This is followed by a secondary deterioration, indicative of the development of irreversible cell dysfunction.

Under normal physiological conditions, the channel within the N-methyl-D-aspartic acid (NMDA) glutamate receptor is blocked in a voltage dependent manner by Mg2+. The triggering of energy deficits and neuronal depolarization are the results of decreased cerebral blood flow. The mild depolarization results in the dislodging of Mg2+ and glutamate, which are consequently released in large amounts to the extracellular space. This leads to an over-activation of the NMDA and AMPA glutamate receptors. Since these receptors regulate Ca2+ ion channels, a calcium ionic imbalance occurs in neurons. This influx of Ca2+, is due to this increase in glutamate release from neurons and astrocytes induced by the ischemia. Apart from the traditional ionotropic glutamate receptors, the influx of Ca2+ is also attributed to some emerging metabotropic and channel mechanisms that include: sodium-calcium exchangers, hemi channels (unopposed half-gap junctions), acid sensing channels, volume-regulated anion channels, nonselective cation channels and signaling cascades that mediate crosstalk between redundant pathways of cell death [Besancon et al., 2008]. This abnormal intracellular accumulation of Ca2+ is involved in the triggering of cell death by up regulation of a wide range of cell death executioners that include ATPases that serve to further deplete energy stores, lipases that damage lipid membranes of organelles and the cell surface itself, proteases that dismantle the cytoarchitecture of the neuron, and DNAses that damage the nucleus [Besancon et. al. 2008]. Bioenergetics of cerebral ischemia (both focal and global) as well as gray and white matter ischemia were recently reviewed from a cellular perspective. A brief summary is given in

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 111

Five minutes of carotid artery occlusion was sufficient to hinder the characteristic nesting behavior of gerbils for ~ 3 days. Their nesting behavior was observed to improve significantly after treatment with palladium lipoic acid complex formulation (50 mg/kg every 24h and 30 mg/kg /24h at 24 and 72 hours after ischemia. The lack of nesting behavior at 70 mg/kg-treated animals was attributed to their excessive energy and

It was observed that preventive or prophylactic treatment with 10 mg/kg gerbil (or allometric scaling equivalent of 10 mL-human dosage) offered significant behavioral and

Our studies demonstrate a greater protective effect of palladium α-lipoic acid complex versus α-lipoic acid alone (Cao & Phillis, 1995). Four times more α-lipoic acid and for a longer period of pre-treatment were necessary to obtain morphologiocal protection. Further more immediate administration of palladium α-lipoic acid complex formulation protected over 70%of the CA1 neurons, and administration delayed up to 24 hours after the TIA still offered significant protection (30% of the CA1 pyramidal cells) [Antonawich et. al., 2004].

Alpha- lipoic acid is a very unique and simple biological molecule. It has a carboxylic acid group with a pKa of 4.7. It is ionized at biological pH, and it has a cyclic disulfide or dithiolane ring [Baumgartner et. al., 1996; Patel & Packer, 2008]. It exists intracellularly as the reduced form, (±)-dihydrolipoic acid. Lipoic acid occurs naturally as a coenzyme in both prokaryotic and eukaryotic cells, as well as in plants, and animals including humans. It is

α-Lipoic acid, absorbed intact from the diet, is readily converted into dihydrolipoic acid in many tissues. In the intracellular environment, two or more enzymes reduce the exogenous lipoic acid. The reversible reduction to dihydrolipoic acid is favored by the presence of the ring strain in the 1, 2-dithiolane ring of about 15-25 kJmol-1 [Baumgartner et. al., 1996]. α-Lipoic acid exits as R(+)-and S(-)- enantiomers due to the presence of an asymmetric carbon. The biologically active enantiomer is mostly the former one. Since its first isolation in 1951, numerous investigations have been carried out to decipher the uniqueness of this

Molecular mechanisms and therapeutic potential of α-lipoic acid, a dietary supplement, have been reviewed recently [Shay et. al., 2009]. Lipoic acid can cross the blood-brain barrier. The biological effects of lipoic acid are attributed to its redox property, the antioxidant capacity and the fatty acid properties. There is ample evidence indicating the usefulness of the lipoic acid/dihydrolipoic acid redox couple as a therapeutic agent for diabetes, ischemia-reperfusion injury, heavy metal poisoning, modulator of various inflammatory signaling pathways, age associated cardiovascular, cognitive, and neuromuscular deficits, protection from radiation damage, neurodegeneration, and HIV infection [Packer et. al., 1995; Patel & Packer, 2008; Shay et.al., 2009]. Dihydrolipoic acid can regenerate or recycle the antioxidants CoQ (ubiquinol), vitamins C and E (via glutathione), and glutathione without itself becoming one in the process. Dihydrolipoic acid also prevents lipid peroxidation by regenerating glutathione. [Packer et. al., 1995; Patel & Packer, 2008]. Lipoic acid and dihydrolipoic acid are efficiently transported in and out of both mitochondria and cells. Compared to this, the transport of disulfides such as cystine that is needed in modulating glutathione (GSH) levels in cells is very inefficient. The mitochondrial

consequent ignoring of the nesting material.

morphological improvement from transient global ischemia.

**9. The powerful super-antioxidant,** α**-lipoic acid** 

enzymatically synthesized from octanoic acid in the mitochondrion.

simple but elegant molecule [Reed 2001; Patel & Packer, 2008].

Table 4 [Hertz, 2008]. During the early stages of ischemia, fatal injury is observed for neurons and oligodendrocytes. They are very sensitive to the excitotoxicity of glutamate due to their cell process expression of NMDA receptors and cell body expression of AMPA/kinase receptors. Astrocytes and endothelial cells seem to survive longer. Neurons are damaged from lack of astrocyte support. Axonal injury is due to channel mediated Na+ uptake followed by Na+/Ca2+ exchange.


Table 4. Bioenergetic mechanisms involved in ischemic death of different cell types and constituents [Hertz, 2008]

Studies have demonstrated that ischemic damage may be reduced by blockade of ionotropic glutamate receptors using glutamate receptor antagonists [Mehta et. al., 2007; Besancon et. al., 2008; Doyle et. al., 2008; Sims & Muyderman, 2010]. The most extensively evaluated neuroprotectors that include calcium channel blockers, glutamate antagonists, GABA agonists, antioxidants and radical scavengers, and NO**.** signal down regulator, have been critically reviewed recently [Ginsberg, 2008].

Other neuroprotective approaches involve the use of anti-oxidants. As an example, α-lipoic acid reduced the mortality rate of male Sprague-Dawley rats from 78% to 26% during 24 hours of reperfusion. It was found effective in improving survival and protecting the rat brain against reperfusion injury following cerebral ischemia [Panigrahi et. al., 1996]. In another study rats that received subcutaneous treatment of R-or S-lipoic acid for 2 hours before ischemia significantly reduced the infarct volume [Wolz & Krieglstein, 1996]. Similar results with mice were obtained with 100 mg/kg of lipoic acid given subcutaneously 1.5 hours before ischemia [Clark et. al., 2001]. Transient global ischemia also benefits from pretreatment with α-lipoic acid. Administration of 40 mg/kg for 7 days protected from ischemic damage when gerbils were tested for locomotor behavior and morphological damage to the CA1 region of the hippocampus [Cao & Phillis, 1995].

Animal studies, using adult male Mongolian gerbils, used as controls or treatment group with palladium α-lipoic acid complex formulation (PdLA), demonstrated that acute, post ischemic and prophylactic administration of PdLA limits ischemic damage [Antonawich et. al., 2004]. Following bilateral carotid artery occlusion in the gerbil, the PdLA was administered intraperitoneally (IP) immediately after surgery, then once daily for 3 days. The control group received saline. PdLA treatment significantly protected hippocampal pyramidal cells (CA1) from transient global ischemia at 30, 50, and 70 mg/kg per 24 h.

While a delayed application of the palladium α-lipoic acid complex formulation after 48 hours of ischemic attack had no significant effect in protecting CA 1 cells, a delayed administration after 6 hours of ischemic attack was as good as giving it immediately after ischemic attack in minimizing cell death.

Five minutes of carotid artery occlusion was sufficient to hinder the characteristic nesting behavior of gerbils for ~ 3 days. Their nesting behavior was observed to improve significantly after treatment with palladium lipoic acid complex formulation (50 mg/kg every 24h and 30 mg/kg /24h at 24 and 72 hours after ischemia. The lack of nesting behavior at 70 mg/kg-treated animals was attributed to their excessive energy and consequent ignoring of the nesting material.

It was observed that preventive or prophylactic treatment with 10 mg/kg gerbil (or allometric scaling equivalent of 10 mL-human dosage) offered significant behavioral and morphological improvement from transient global ischemia.

Our studies demonstrate a greater protective effect of palladium α-lipoic acid complex versus α-lipoic acid alone (Cao & Phillis, 1995). Four times more α-lipoic acid and for a longer period of pre-treatment were necessary to obtain morphologiocal protection. Further more immediate administration of palladium α-lipoic acid complex formulation protected over 70%of the CA1 neurons, and administration delayed up to 24 hours after the TIA still offered significant protection (30% of the CA1 pyramidal cells) [Antonawich et. al., 2004].

#### **9. The powerful super-antioxidant,** α**-lipoic acid**

110 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

Table 4 [Hertz, 2008]. During the early stages of ischemia, fatal injury is observed for neurons and oligodendrocytes. They are very sensitive to the excitotoxicity of glutamate due to their cell process expression of NMDA receptors and cell body expression of AMPA/kinase receptors. Astrocytes and endothelial cells seem to survive longer. Neurons are damaged from lack of astrocyte support. Axonal injury is due to channel mediated Na+

Neurons Axons Oligodendrocytes Astrocytes Endothelial

Table 4. Bioenergetic mechanisms involved in ischemic death of different cell types and

Studies have demonstrated that ischemic damage may be reduced by blockade of ionotropic glutamate receptors using glutamate receptor antagonists [Mehta et. al., 2007; Besancon et. al., 2008; Doyle et. al., 2008; Sims & Muyderman, 2010]. The most extensively evaluated neuroprotectors that include calcium channel blockers, glutamate antagonists, GABA

Other neuroprotective approaches involve the use of anti-oxidants. As an example, α-lipoic acid reduced the mortality rate of male Sprague-Dawley rats from 78% to 26% during 24 hours of reperfusion. It was found effective in improving survival and protecting the rat brain against reperfusion injury following cerebral ischemia [Panigrahi et. al., 1996]. In another study rats that received subcutaneous treatment of R-or S-lipoic acid for 2 hours before ischemia significantly reduced the infarct volume [Wolz & Krieglstein, 1996]. Similar results with mice were obtained with 100 mg/kg of lipoic acid given subcutaneously 1.5 hours before ischemia [Clark et. al., 2001]. Transient global ischemia also benefits from pretreatment with α-lipoic acid. Administration of 40 mg/kg for 7 days protected from ischemic damage when gerbils were tested for locomotor behavior and morphological

Animal studies, using adult male Mongolian gerbils, used as controls or treatment group with palladium α-lipoic acid complex formulation (PdLA), demonstrated that acute, post ischemic and prophylactic administration of PdLA limits ischemic damage [Antonawich et. al., 2004]. Following bilateral carotid artery occlusion in the gerbil, the PdLA was administered intraperitoneally (IP) immediately after surgery, then once daily for 3 days. The control group received saline. PdLA treatment significantly protected hippocampal pyramidal cells (CA1) from transient global ischemia at 30, 50, and 70 mg/kg per 24 h. While a delayed application of the palladium α-lipoic acid complex formulation after 48 hours of ischemic attack had no significant effect in protecting CA 1 cells, a delayed administration after 6 hours of ischemic attack was as good as giving it immediately after

Intracellular Na+ increase X X X X X Increased metabolism X ? ? X ? Intracellular Ca2+ increase X X X X ? Mitochondrial damage X X X ? Formation of ROS X X (X) X

cells

signal down regulator, have been

uptake followed by Na+/Ca2+ exchange.

agonists, antioxidants and radical scavengers, and NO**.**

damage to the CA1 region of the hippocampus [Cao & Phillis, 1995].

critically reviewed recently [Ginsberg, 2008].

ischemic attack in minimizing cell death.

constituents [Hertz, 2008]

Alpha- lipoic acid is a very unique and simple biological molecule. It has a carboxylic acid group with a pKa of 4.7. It is ionized at biological pH, and it has a cyclic disulfide or dithiolane ring [Baumgartner et. al., 1996; Patel & Packer, 2008]. It exists intracellularly as the reduced form, (±)-dihydrolipoic acid. Lipoic acid occurs naturally as a coenzyme in both prokaryotic and eukaryotic cells, as well as in plants, and animals including humans. It is enzymatically synthesized from octanoic acid in the mitochondrion.

α-Lipoic acid, absorbed intact from the diet, is readily converted into dihydrolipoic acid in many tissues. In the intracellular environment, two or more enzymes reduce the exogenous lipoic acid. The reversible reduction to dihydrolipoic acid is favored by the presence of the ring strain in the 1, 2-dithiolane ring of about 15-25 kJmol-1 [Baumgartner et. al., 1996]. α-Lipoic acid exits as R(+)-and S(-)- enantiomers due to the presence of an asymmetric carbon. The biologically active enantiomer is mostly the former one. Since its first isolation in 1951, numerous investigations have been carried out to decipher the uniqueness of this simple but elegant molecule [Reed 2001; Patel & Packer, 2008].

Molecular mechanisms and therapeutic potential of α-lipoic acid, a dietary supplement, have been reviewed recently [Shay et. al., 2009]. Lipoic acid can cross the blood-brain barrier. The biological effects of lipoic acid are attributed to its redox property, the antioxidant capacity and the fatty acid properties. There is ample evidence indicating the usefulness of the lipoic acid/dihydrolipoic acid redox couple as a therapeutic agent for diabetes, ischemia-reperfusion injury, heavy metal poisoning, modulator of various inflammatory signaling pathways, age associated cardiovascular, cognitive, and neuromuscular deficits, protection from radiation damage, neurodegeneration, and HIV infection [Packer et. al., 1995; Patel & Packer, 2008; Shay et.al., 2009]. Dihydrolipoic acid can regenerate or recycle the antioxidants CoQ (ubiquinol), vitamins C and E (via glutathione), and glutathione without itself becoming one in the process. Dihydrolipoic acid also prevents lipid peroxidation by regenerating glutathione. [Packer et. al., 1995; Patel & Packer, 2008].

Lipoic acid and dihydrolipoic acid are efficiently transported in and out of both mitochondria and cells. Compared to this, the transport of disulfides such as cystine that is needed in modulating glutathione (GSH) levels in cells is very inefficient. The mitochondrial

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 113

and superoxide radical. The reduction potential for the α-lipoic acid/dihydrolipoic acid couple of -320 mV or -290 mV [Krishnan et. al., 2011] and the GSSG/GSH) couple of -240 mV indicate that dihydrolipoic acid can react with GSSG and regenerate GSH [Packer et. al., 1995]. Thus lipoic acid helps to maintain GSH/GSSG ratio (about 100 to 10,000 times greater than other redox couples such as NAD+/NADH, and NADP+/NADPH), an estimate of

Treatment with lipoic acid increases the GSH levels in human cell lines and primary cells including T cells, erythrocytes, lymphocytes, and glial and neuroblastoma cells.. This is explained by 1) facile transport of lipoic acid into cells, where it is reduced by NADH or NADPH dependent pathways to dihydrolipoic acid. 2) Dihydrolipoic acid is transported back into the extracellular media where it is oxidized by cysteine regenerating lipoic acid and producing cysteine, the limiting substrate on GSH synthesis. 3) Compared to cystine, cysteine is more easily transported into the cell and aids the synthesis of GSH [Patel &

Thus elevated levels of GSH and ascorbic acid, which in turn regenerates vitamin E, are all indicative of lipoic acid acting as an inducer of endogenous antioxidants. It has been reported that lipoic acid is also an effective regulator of signaling pathways and induces synthesis of GSH transcriptionally. Lipoic acid reverses the decline in transcriptional activity

The pharmacokinetics of R-lipoic acid, reviewed recently [Patel & Packer, 2008; Shay et.al., 2009], revealed a plasma level concentration, Cmax, of 1.154 μg/mL from 1 g R-lipoic acid compared to the proposed therapeutic range of 10-20 μg/mL or 50-100 μM (Carlson et al., 2008). A dose of 600-800 mg sodium R-lipoate gave plasma levels of 8-18 μg/mL, which is within the therapeutic range. The upper limit suggested for therapeutic action of 45 μg/mL or 225 µM is reached by a dose of about 1.2 g of racemic-α-lipoic acid. The no adverse observed effect level (NOAEL) of racemic lipoic acid is considered to be 60 mg/kg body mass/day. Therapeutic and energy production applications of this powerful antioxidant

Located within the mitochondrial matrix are lipoic acid requiring enzymes: three α-keto acid dehydrogenase complexes that catalyze the oxidative decarboxylation of α-keto acids such as pyruvate, α-ketoglutarate, and branched chain α-ketoacids [Voet D. and Voet J. G., 1995]. In organisms, hydrogen atom transfer and acyl group transfer take place in the oxidative decarboxylation of α-ketoacids with the aid of α-lipoic acid. The reversible redox reaction between α-lipoic acid and dihydrolipoic acid is thus a very important biochemical reaction. The reversible reduction to dihydrolipoic acid is favored by the presence of the

The multienzyme complex, pyruvate dehydrogenase, consists of three enzymes, pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3) [Voet D. and Voet J. G., 1995]. This enzyme complex participates in five sequential reactions during the conversion of pyruvate to acetyl-CoA. The R- lipoic acid is covalently linked to a ε-amino group of lysine residue via an amide linkage. These lipoic acid

The multienzyme complex, α-ketoglutarate dehydrogenase, also consists of three enzymes, α-ketoglutarate dehydrogenase (E1), dihydrolipoyl transsuccinylase (E2), and dihydrolipoyl dehydrogenase (E3) [Voet D. and Voet J. G., 1995]. The branched chain α-ketoacid dehydrogenase is also a multienzyme complex resembling the other two enzymes

ring strain in the 1,2-dithiolane ring of about 15-25 kJmol-1 [Patel & Packer, 2008].

containing enzymes participate in four out of the five reactions.

redox state, in cells [Patel & Packer, 2008].

of Nrf2 caused by age-related loss of GSH [Suh et. al, 2004].

have been explored extensively [Patel & Packer, 2008].

Packer, 2008].

β-oxidation of lipoic acid has been attributed to its fatty acid properties, similar to that of octanoic acid [Patel & Packer, 2008]. Redox as well as biological antioxidant effects have been attributed to the β-oxidation products of lipoic acid, the oxidized and reduced forms of bisnorlipoic acid and tetranorlipoic acid [Patel & Packer, 2008].


Table 5. Antioxidant activities of lipoic acid (LA) and dihydrolipoic acid (DHLA) [detailed references in Shay et. al., 2009]

The α-lipoic acid/dihydrolipoic acid couple is called a "universal antioxidant" because it fulfills several criteria used to evaluate the antioxidant potential as well as preventive or therapeutic applications of a compound such as specificity of free radical quenching, metal chelating ability, interaction with other antioxidants, effects on gene expression, absorption and bioavailability, concentration in tissues, cells, and extracellular fluid, and location (in aqueous or membrane domains or in both) [Packer et.al., 1995].

The free radical scavenging activities of lipoic acid and dihydrolipoic acid are given in Table 5 [Shay et.al., 2009]. Both are capable of scavenging peroxynitrite, peroxyl radical and hypochlorous acid, but not hydrogen peroxide. There are conflicting data for singlet oxygen and radicals such as hydroxyl, superoxide, and peroxyl.

Questions have been raised regarding the direct-acting antioxidant status of LA/DHLA in vivo based on the invitro data [Shay et.al., 2009]. This is due to the large dosage, 200-600 mg, used for invivo studies and the amounts found in the plasma (both area under the curve and Cmax are in the range of microgram to nanogram levels per mL). The administration of lipoic acid in acid form or salt form, oral or intravenous, with a meal or without meal also contributed to the fluctuations in the data. Also the invitro data cannot imitate the invivo clearance of 98% of LA excretion in the urine within 24 hours.

α-Lipoic acid was found to protect hematopoietic tissues in mice from radiation damage [Ramakrishnan et. al., 1992]. It was also found that α-lipoic acid offered protection from radiation for children affected by the Chernobyl nuclear accident [Korkina et al., 1993]. α-Lipoic acid scavenges hydroxyl radicals but is not effective against hydrogen peroxide

β-oxidation of lipoic acid has been attributed to its fatty acid properties, similar to that of octanoic acid [Patel & Packer, 2008]. Redox as well as biological antioxidant effects have been attributed to the β-oxidation products of lipoic acid, the oxidized and reduced forms of

Scavenged or not by DHLA

and rate constant

and rate constant

Peroxynitrite Yes, 1.4 x 103 M-1 S-1 Yes, 2.5 x 102 M-1 S-1 Nitric oxide No Yes, 3.19 M-1 S-1

Yes, 3.3 x 105 M-1 S-1

Peroxyl radical Yes, 1.8 x 108 M-1 S-1 Yes, 2.3 x 107 M-1 S-1

Table 5. Antioxidant activities of lipoic acid (LA) and dihydrolipoic acid (DHLA) [detailed

The α-lipoic acid/dihydrolipoic acid couple is called a "universal antioxidant" because it fulfills several criteria used to evaluate the antioxidant potential as well as preventive or therapeutic applications of a compound such as specificity of free radical quenching, metal chelating ability, interaction with other antioxidants, effects on gene expression, absorption and bioavailability, concentration in tissues, cells, and extracellular fluid, and location (in

The free radical scavenging activities of lipoic acid and dihydrolipoic acid are given in Table 5 [Shay et.al., 2009]. Both are capable of scavenging peroxynitrite, peroxyl radical and hypochlorous acid, but not hydrogen peroxide. There are conflicting data for singlet oxygen

Questions have been raised regarding the direct-acting antioxidant status of LA/DHLA in vivo based on the invitro data [Shay et.al., 2009]. This is due to the large dosage, 200-600 mg, used for invivo studies and the amounts found in the plasma (both area under the curve and Cmax are in the range of microgram to nanogram levels per mL). The administration of lipoic acid in acid form or salt form, oral or intravenous, with a meal or without meal also contributed to the fluctuations in the data. Also the invitro data cannot imitate the invivo

α-Lipoic acid was found to protect hematopoietic tissues in mice from radiation damage [Ramakrishnan et. al., 1992]. It was also found that α-lipoic acid offered protection from radiation for children affected by the Chernobyl nuclear accident [Korkina et al., 1993]. α-Lipoic acid scavenges hydroxyl radicals but is not effective against hydrogen peroxide

bisnorlipoic acid and tetranorlipoic acid [Patel & Packer, 2008].

Hydroxyl radical Yes, 4.7 x 1010 M-1 S-1 No Yes Yes Superoxide No No

 Yes Singlet oxygen Yes, 1.3 x 108 M-1 S-1 No

 No Yes Hypochlorous acid Yes Yes Hydrogen peroxide No No

aqueous or membrane domains or in both) [Packer et.al., 1995].

and radicals such as hydroxyl, superoxide, and peroxyl.

clearance of 98% of LA excretion in the urine within 24 hours.

Yes

references in Shay et. al., 2009]

Oxidant Scavenged or not by LA

and superoxide radical. The reduction potential for the α-lipoic acid/dihydrolipoic acid couple of -320 mV or -290 mV [Krishnan et. al., 2011] and the GSSG/GSH) couple of -240 mV indicate that dihydrolipoic acid can react with GSSG and regenerate GSH [Packer et. al., 1995]. Thus lipoic acid helps to maintain GSH/GSSG ratio (about 100 to 10,000 times greater than other redox couples such as NAD+/NADH, and NADP+/NADPH), an estimate of redox state, in cells [Patel & Packer, 2008].

Treatment with lipoic acid increases the GSH levels in human cell lines and primary cells including T cells, erythrocytes, lymphocytes, and glial and neuroblastoma cells.. This is explained by 1) facile transport of lipoic acid into cells, where it is reduced by NADH or NADPH dependent pathways to dihydrolipoic acid. 2) Dihydrolipoic acid is transported back into the extracellular media where it is oxidized by cysteine regenerating lipoic acid and producing cysteine, the limiting substrate on GSH synthesis. 3) Compared to cystine, cysteine is more easily transported into the cell and aids the synthesis of GSH [Patel & Packer, 2008].

Thus elevated levels of GSH and ascorbic acid, which in turn regenerates vitamin E, are all indicative of lipoic acid acting as an inducer of endogenous antioxidants. It has been reported that lipoic acid is also an effective regulator of signaling pathways and induces synthesis of GSH transcriptionally. Lipoic acid reverses the decline in transcriptional activity of Nrf2 caused by age-related loss of GSH [Suh et. al, 2004].

The pharmacokinetics of R-lipoic acid, reviewed recently [Patel & Packer, 2008; Shay et.al., 2009], revealed a plasma level concentration, Cmax, of 1.154 μg/mL from 1 g R-lipoic acid compared to the proposed therapeutic range of 10-20 μg/mL or 50-100 μM (Carlson et al., 2008). A dose of 600-800 mg sodium R-lipoate gave plasma levels of 8-18 μg/mL, which is within the therapeutic range. The upper limit suggested for therapeutic action of 45 μg/mL or 225 µM is reached by a dose of about 1.2 g of racemic-α-lipoic acid. The no adverse observed effect level (NOAEL) of racemic lipoic acid is considered to be 60 mg/kg body mass/day. Therapeutic and energy production applications of this powerful antioxidant have been explored extensively [Patel & Packer, 2008].

Located within the mitochondrial matrix are lipoic acid requiring enzymes: three α-keto acid dehydrogenase complexes that catalyze the oxidative decarboxylation of α-keto acids such as pyruvate, α-ketoglutarate, and branched chain α-ketoacids [Voet D. and Voet J. G., 1995]. In organisms, hydrogen atom transfer and acyl group transfer take place in the oxidative decarboxylation of α-ketoacids with the aid of α-lipoic acid. The reversible redox reaction between α-lipoic acid and dihydrolipoic acid is thus a very important biochemical reaction. The reversible reduction to dihydrolipoic acid is favored by the presence of the ring strain in the 1,2-dithiolane ring of about 15-25 kJmol-1 [Patel & Packer, 2008].

The multienzyme complex, pyruvate dehydrogenase, consists of three enzymes, pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3) [Voet D. and Voet J. G., 1995]. This enzyme complex participates in five sequential reactions during the conversion of pyruvate to acetyl-CoA. The R- lipoic acid is covalently linked to a ε-amino group of lysine residue via an amide linkage. These lipoic acid containing enzymes participate in four out of the five reactions.

The multienzyme complex, α-ketoglutarate dehydrogenase, also consists of three enzymes, α-ketoglutarate dehydrogenase (E1), dihydrolipoyl transsuccinylase (E2), and dihydrolipoyl dehydrogenase (E3) [Voet D. and Voet J. G., 1995]. The branched chain α-ketoacid dehydrogenase is also a multienzyme complex resembling the other two enzymes

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 115

the concentration of lipoic acid and the scanning potential, radicals and polymers are formed [Krishnan & Garnett 2011]. Also our data suggest the need for a revalidation of the

Fig. 5. Cyclic voltammogram of 1 mM α-lipoic acid (sodium salt), pH 7.2, scan 2.0 to -2.0 V and back, scan 3, scan rate a and c, 100mV/sec; b. scan rates 1) 400 2) 200 3) 100 4) 50

in a. [Krishnan & Garnett 2011]

**complex** 

mV/sec; **c.** 1) scan 2.0 to -2.0 V and back, 2) scan 1.0 to -0.5V and back; peak 7 same as peak 7

Our electrochemical data also suggested a caution in deciding on the dosage of oral supplements of lipoic acid because of its tendency to form dimers and higher polymers under reducing biological conditions. The pharmacokinetic data mentioned earlier is also

**10. Catalytic, electronic, and therapeutic properties of palladium α-lipoic acid** 

The coordination chemistry of palladium complexes have recently been reviewed with an emphasis on cancer therapy[Gao et. al., 2009]. Even though there are many structural and thermodynamic similarities between the complexes of palladium and platinum, the palladium(II) complexes seem to exhibit biological action very different from those of the

subject to these complexities depending on the dosage chosen for the studies.

reported redox potential of the lipoic acid/dihydrolipoic acid couple.

mentioned above. These three enzymes have the same dihydrolipoyl dehydrogenase and employ the coenzymes thiamine pyrophosphate, lipoamide, FAD and the terminal oxidizing agent NAD+ [Voet D. and Voet J. G., 1995]. The importance of lipoic acid in the energy metabolism is illustrated by these three enzymes.

Disulfide bonds in proteins are formed between the thiol groups of cysteine residues. The other sulfur-containing amino acid, methionine, cannot form disulfide bonds. More aggressive oxidants convert cysteine to the corresponding sulfinic acid and sulfonic acid. A variety of oxidants promote this reaction including air and hydrogen peroxide. Such reactions are thought to proceed via sulfenic acid intermediates.

The oxidized form α-lipoic acid can undergo further oxidation at sulfur or get reduced. This property, which is somewhat similar to an intermediate oxidation state in a transition metal or a nonmetal, makes this molecule very unique compared to other biological molecules. A biological oxidation product of α-lipoic acid (lipoic acid S-oxide or a thiolsulfinate) is known as β-lipoic acid or Protogen-B [Baumgartner et. al., 1996]. It has not been possible to conclusively prove which sulfur is oxidized [Stary et. al. 1975].

The oxidation of α-lipoic acid was found to be a one-electron charge transfer process, pH independent, and an irreversible process [Corduneanu et. al., 2009]. Most voltammetric studies were centered on the oxidation of α-lipoic acid and studies related to its reduction to dihydrolipoic acid were limited [Rogers & Mallett, 1983]. To understand the electrochemical behavior of α-lipoic acid, we had explored potential regions beyond the normal range of the mercury electrode, both on the cathodic side as well as on the anodic side. The advantage in using the mercury electrode is the ease with which we can obtain a fresh consistent surface and drop. We have not observed (visually) any passivation of mercury on the anodic side when lipoic acid was present without any background electrolyte. We had investigated the cathodic side, the normal potential region for mercury working electrode, in much greater detail.

The complexity of the redox process of α-lipoic acid is shown in the cyclic voltammograms in Fig. 5 [Krishnan & Garnett, 2011]. We had assigned the three cathodic peaks to 1) reduction of lipoic acid S-oxide, 2) reduction of lipoic acid to most probably dihydrolipoic acid, and 3) probable reduction of lipoic acid dimers or higher polymers. We have assigned the five anodic peaks to 4) the oxidation product of lipoic acid dimers or higher polymers, 5) oxidation product of dihydrolipoic acid, 6) formation of S-oxide of lipoic acid dimers or higher polymers, 7) formation of lipoic acid S-oxide, and 8) further oxidation of lipoic acid S-oxide/the formation of thiolsulfonates. The peak due to the formation of lipoic acid S-oxide (peak 7) is missing in Figure 3a because of the formation of S-oxide of lipoic acid dimers or higher polymers (peak 6). However, when the formation of lipoic acid polymers is minimized by restricting the scan to less cathodic potentials (to -0.5 V instead of -2.0 V) the formation of S-oxide of lipoic acid dimers or higher polymers is minimized and thus allowing the formation of lipoic acid S-oxide (peak 7) [Krishnan & Garnett, 2011]. Other complexities were expressed at differing scan rates, at higher concentrations and by the presence of electrolytes. The data shown in Fig. 5 are for 1mM α-lipoic acid where solutesolute interactions are minimized and in the absence of electrolytes so that the influence of ion-dipole interactions can be more readily investigated at the double layer.

The molecule, α-lipoic acid, is very unique because of its ability to form a variety of radicals, dimers and higher polymers as well as a variety of lipoic acid S-oxides. This complexity is reflected in its electrochemical redox behavior. Our data demonstrated that depending on

mentioned above. These three enzymes have the same dihydrolipoyl dehydrogenase and employ the coenzymes thiamine pyrophosphate, lipoamide, FAD and the terminal oxidizing agent NAD+ [Voet D. and Voet J. G., 1995]. The importance of lipoic acid in the energy

Disulfide bonds in proteins are formed between the thiol groups of cysteine residues. The other sulfur-containing amino acid, methionine, cannot form disulfide bonds. More aggressive oxidants convert cysteine to the corresponding sulfinic acid and sulfonic acid. A variety of oxidants promote this reaction including air and hydrogen peroxide. Such

The oxidized form α-lipoic acid can undergo further oxidation at sulfur or get reduced. This property, which is somewhat similar to an intermediate oxidation state in a transition metal or a nonmetal, makes this molecule very unique compared to other biological molecules. A biological oxidation product of α-lipoic acid (lipoic acid S-oxide or a thiolsulfinate) is known as β-lipoic acid or Protogen-B [Baumgartner et. al., 1996]. It has not been possible to

The oxidation of α-lipoic acid was found to be a one-electron charge transfer process, pH independent, and an irreversible process [Corduneanu et. al., 2009]. Most voltammetric studies were centered on the oxidation of α-lipoic acid and studies related to its reduction to dihydrolipoic acid were limited [Rogers & Mallett, 1983]. To understand the electrochemical behavior of α-lipoic acid, we had explored potential regions beyond the normal range of the mercury electrode, both on the cathodic side as well as on the anodic side. The advantage in using the mercury electrode is the ease with which we can obtain a fresh consistent surface and drop. We have not observed (visually) any passivation of mercury on the anodic side when lipoic acid was present without any background electrolyte. We had investigated the cathodic side, the normal potential region for mercury working electrode, in much greater

The complexity of the redox process of α-lipoic acid is shown in the cyclic voltammograms in Fig. 5 [Krishnan & Garnett, 2011]. We had assigned the three cathodic peaks to 1) reduction of lipoic acid S-oxide, 2) reduction of lipoic acid to most probably dihydrolipoic acid, and 3) probable reduction of lipoic acid dimers or higher polymers. We have assigned the five anodic peaks to 4) the oxidation product of lipoic acid dimers or higher polymers, 5) oxidation product of dihydrolipoic acid, 6) formation of S-oxide of lipoic acid dimers or higher polymers, 7) formation of lipoic acid S-oxide, and 8) further oxidation of lipoic acid S-oxide/the formation of thiolsulfonates. The peak due to the formation of lipoic acid S-oxide (peak 7) is missing in Figure 3a because of the formation of S-oxide of lipoic acid dimers or higher polymers (peak 6). However, when the formation of lipoic acid polymers is minimized by restricting the scan to less cathodic potentials (to -0.5 V instead of -2.0 V) the formation of S-oxide of lipoic acid dimers or higher polymers is minimized and thus allowing the formation of lipoic acid S-oxide (peak 7) [Krishnan & Garnett, 2011]. Other complexities were expressed at differing scan rates, at higher concentrations and by the presence of electrolytes. The data shown in Fig. 5 are for 1mM α-lipoic acid where solutesolute interactions are minimized and in the absence of electrolytes so that the influence of

ion-dipole interactions can be more readily investigated at the double layer.

The molecule, α-lipoic acid, is very unique because of its ability to form a variety of radicals, dimers and higher polymers as well as a variety of lipoic acid S-oxides. This complexity is reflected in its electrochemical redox behavior. Our data demonstrated that depending on

metabolism is illustrated by these three enzymes.

detail.

reactions are thought to proceed via sulfenic acid intermediates.

conclusively prove which sulfur is oxidized [Stary et. al. 1975].

the concentration of lipoic acid and the scanning potential, radicals and polymers are formed [Krishnan & Garnett 2011]. Also our data suggest the need for a revalidation of the reported redox potential of the lipoic acid/dihydrolipoic acid couple.

Fig. 5. Cyclic voltammogram of 1 mM α-lipoic acid (sodium salt), pH 7.2, scan 2.0 to -2.0 V and back, scan 3, scan rate a and c, 100mV/sec; b. scan rates 1) 400 2) 200 3) 100 4) 50 mV/sec; **c.** 1) scan 2.0 to -2.0 V and back, 2) scan 1.0 to -0.5V and back; peak 7 same as peak 7 in a. [Krishnan & Garnett 2011]

Our electrochemical data also suggested a caution in deciding on the dosage of oral supplements of lipoic acid because of its tendency to form dimers and higher polymers under reducing biological conditions. The pharmacokinetic data mentioned earlier is also subject to these complexities depending on the dosage chosen for the studies.

#### **10. Catalytic, electronic, and therapeutic properties of palladium α-lipoic acid complex**

The coordination chemistry of palladium complexes have recently been reviewed with an emphasis on cancer therapy[Gao et. al., 2009]. Even though there are many structural and thermodynamic similarities between the complexes of palladium and platinum, the palladium(II) complexes seem to exhibit biological action very different from those of the

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 117

The antioxidant status in the liver, kidney and brain of mice after exposure to 2-6 Gy radiation were also measured recently [Menon et. al., 2009]. The results were similar to the ones observed in the heart of rats without radiation. Also an analysis of their blood leukocytes and bone marrow for DNA damage using alkaline single cell gel electrophoresis (alkaline comet assay) revealed DNA repair from the lowering of comet assay parameters, DNA in tail, tail length, tail moment, and olive tail moment. Administration of the complex also reduced the mortality of the mice and also aided recovery from the radiation induced weight loss [Ramachandran et. al., 2010]. DNA repair was also observed in human blood leukocytes with palladium α-lipoic acid complex treatment immediately after exposure to

The mechanism of the superiority of the metal complex compared to that of the ligand is still an unsolved puzzle. Compared to the high oral dose, the available plasma concentration of α-lipoic acid is very small. One possibility for the higher activity of the metal complex is probably due to its increased concentration in the plasma. This needs experimental verification. It is also known that 98% of α-lipoic acid is excreted in urine within 24 hours and since it takes 4-6 weeks for serum and urine clearance of palladium, the previous suggestion seems justified. Another possibility is the chemistry of the transition metal playing a dominant role in the enzymatic activity. The starting material in the synthesis of the palladium α-lipoic acid complex is palladium(II). The final complex is also palladium(II), based on preliminary ESR data. Palladium(II) complexes are diamagnetic. The complex concentration dependent electrochemical characteristics of α-lipoic acid suggest the possibility of free radical formation by one electron reduction under physiological conditions. In such a case the electron spin may be involved in the enzymatic process. The impedance characteristics of the palladium α-lipoic acid as well as that of the α-lipoic acid,

Another possibility for the superiority of the palladium α-lipoic acid complex compared to α-lipoic acid is its ability to form self-assembled structures, such as the one shown in Fig. 6. No self-assembly was observed for sodium lipoate. We want to point out that binding of a lysine residue in the protein to the lipoyl group of E2 in 2-oxoacid dehydrogenases results in a long flexible arm that can oscillate a distance of ~200 Å. This arm is utilized during the catalytic cycle [Patel & Packer, 2008]. It is obvious that the self assembled palladium α-lipoic

The physics and chemistry of non-equilibrium systems have been utilized to understand some of the spatial patterns and temporal patterning observed in biological processes such as bacterial colonies shaped by diffusive instabilities and calcium waves governed by

In homogeneous systems, spiral waves and spatiotemporal phenomena are formed from autocatalytic reactions and diffusion resulting from chemical instabilities. Our data suggest that the propagation of electrical signaling among the packing units and extending to long

We have utilized the technique of electrochemical impedance spectroscopy extensively to probe spatiotemporal phenomena in biological systems. This technique is routinely used to study corrosion and fuel cells. We have used admittance measurements for understanding solute-solvent interactions, "π –way" conduction, ion pair formation, water-structure enforced ion pair formation, potential induced and solvent mediated ion pair formation at the double layer, and semi conduction characteristics of simple biological molecules

nonlinear amplification during intracellular signaling [Levine & Jacob, 2004].

distances by global coupling is viable by such self-assembled systems.

radiation [Menon & Nair, 2011].

described in this section, strongly suggest this possibility.

acid complex can make this process more facile.

toxic platinum complexes. Copper, zinc, and arsenic complexes of α-lipoic acid and palladium α-lipoic acid complexes with 1:1 and 1:2 stoichiometry have been reported [Garnett, 1995a, 1995b; Strasdeit et. al., 1995; Baumgartner et. al., 1996].

Palladium α-lipoic acid complex has demonstrated numerous antitumor activities against various cell lines. Also it was found to halt the growth of glioblastoma in nude mice. Clinical veterinary studies indicated its effectiveness as a complimentary support to chemotherapy. A recent Phase I, dose escalation study, has revealed safety in humans up to 40 mL 0.037 M of this complex with no severe adverse events, and minor adverse events i.e. mild gastrointestinal irritation, and aversion to taste. Washout periods for palladium, monitored in blood serum and urine, ranged from three to seventeen weeks after cessation of the formulation [Krishnan et. al., 2011].

The electrochemical characteristics of α-lipoic acid and palladium α-lipoic acid complex have been explored using glassy carbon and highly oriented pyrolytic graphite electrodes [Corduneanu et al., 2007, 2009]. Palladium complex was found to dissociate at negative potentials with deposition of Pd(0) nanoparticles. The application of a positive potential induced the oxidation of the palladium complex and the formation of a mixed layer of lipoic acid and palladium oxides.

Superior free radical scavenging capacity, as measured by oxygen radical absorbance capacity or ORAC analysis, was observed for palladium α-lipoic acid complex formulation (5.65) compared to vitamin E (1.0, normalized value), vitamin A (1.6), vitamin C (1.12), and α-lipoic acid (1.4) [Krishnan et.al., 2011]. This may be compared to the superior antitumor activities observed for metal complexes compared to that of their ligands [Matesanz et. al., 1999; Maloň et. al., 2001].

The enhanced energy effects observed in gerbils during transient ischemia studies [Antonawich et. al., 2004] prompted us to investigate the influence of palladium α-lipoic acid complex on the activities of enzymes involved in mitochondrial energy production. The activities of four Krebs cycle enzymes, isocitrate dehydrogenase (ICDH), α-ketoglutarate dehydrogenase (α-KGDH), succinate dehydrogenase (SDH), and malate dehydrogenase (MDH) and mitochondrial complexes I, II, III, and IV in aged male albino rats were investigated [Sudheesh et. al., 2009]. The enhanced activity of the metal complex was superior to the activity of lipoic acid, the ligand.

The antioxidant status in the heart of aged male Albino Wistar rats, measured by MnSOD, CAT, and GSHPx, were higher with the palladium lipoic acid treated group than with the α-lipoic acid group [Sudheesh et al., 2010]. This was also true in alloxan induced diabetic rats [Sudheesh et. al. 2011]. Similarly the lipid peroxidation levels were lowered and the GSH levels were increased in the palladium α-lipoic acid treated group. It is not clear at this time whether scavenging some free radicals by either α-lipoic acid or palladium α-lipoic acid complex formulation is connected in any way to the enhanced activities of Krebs cycle and mitochondrial enzymes.

A specific example is illustrated here. The α-ketoglutarate dehydrogenase complex (KGDH) is a critical component of Krebs cycle and of glutamate metabolism. Glutamate is an excitotoxic neurotransmitter. Reactive oxygen species modify the activity of KGDH. It is also known that the activity of KGDH is lower than that of any other enzyme in the brain. Deficiencies in KGDH lead to brain neurological syndromes. Palladium α-lipoic acid complex increases the activity of KGDH and thus helps in the removal of the glutamate.

toxic platinum complexes. Copper, zinc, and arsenic complexes of α-lipoic acid and palladium α-lipoic acid complexes with 1:1 and 1:2 stoichiometry have been reported

Palladium α-lipoic acid complex has demonstrated numerous antitumor activities against various cell lines. Also it was found to halt the growth of glioblastoma in nude mice. Clinical veterinary studies indicated its effectiveness as a complimentary support to chemotherapy. A recent Phase I, dose escalation study, has revealed safety in humans up to 40 mL 0.037 M of this complex with no severe adverse events, and minor adverse events i.e. mild gastrointestinal irritation, and aversion to taste. Washout periods for palladium, monitored in blood serum and urine, ranged from three to seventeen weeks after cessation of the

The electrochemical characteristics of α-lipoic acid and palladium α-lipoic acid complex have been explored using glassy carbon and highly oriented pyrolytic graphite electrodes [Corduneanu et al., 2007, 2009]. Palladium complex was found to dissociate at negative potentials with deposition of Pd(0) nanoparticles. The application of a positive potential induced the oxidation of the palladium complex and the formation of a mixed layer of lipoic

Superior free radical scavenging capacity, as measured by oxygen radical absorbance capacity or ORAC analysis, was observed for palladium α-lipoic acid complex formulation (5.65) compared to vitamin E (1.0, normalized value), vitamin A (1.6), vitamin C (1.12), and α-lipoic acid (1.4) [Krishnan et.al., 2011]. This may be compared to the superior antitumor activities observed for metal complexes compared to that of their ligands [Matesanz et. al.,

The enhanced energy effects observed in gerbils during transient ischemia studies [Antonawich et. al., 2004] prompted us to investigate the influence of palladium α-lipoic acid complex on the activities of enzymes involved in mitochondrial energy production. The activities of four Krebs cycle enzymes, isocitrate dehydrogenase (ICDH), α-ketoglutarate dehydrogenase (α-KGDH), succinate dehydrogenase (SDH), and malate dehydrogenase (MDH) and mitochondrial complexes I, II, III, and IV in aged male albino rats were investigated [Sudheesh et. al., 2009]. The enhanced activity of the metal complex was

The antioxidant status in the heart of aged male Albino Wistar rats, measured by MnSOD, CAT, and GSHPx, were higher with the palladium lipoic acid treated group than with the α-lipoic acid group [Sudheesh et al., 2010]. This was also true in alloxan induced diabetic rats [Sudheesh et. al. 2011]. Similarly the lipid peroxidation levels were lowered and the GSH levels were increased in the palladium α-lipoic acid treated group. It is not clear at this time whether scavenging some free radicals by either α-lipoic acid or palladium α-lipoic acid complex formulation is connected in any way to the enhanced activities of Krebs cycle

A specific example is illustrated here. The α-ketoglutarate dehydrogenase complex (KGDH) is a critical component of Krebs cycle and of glutamate metabolism. Glutamate is an excitotoxic neurotransmitter. Reactive oxygen species modify the activity of KGDH. It is also known that the activity of KGDH is lower than that of any other enzyme in the brain. Deficiencies in KGDH lead to brain neurological syndromes. Palladium α-lipoic acid complex increases the activity of KGDH and thus helps in the removal of the

[Garnett, 1995a, 1995b; Strasdeit et. al., 1995; Baumgartner et. al., 1996].

formulation [Krishnan et. al., 2011].

acid and palladium oxides.

1999; Maloň et. al., 2001].

and mitochondrial enzymes.

glutamate.

superior to the activity of lipoic acid, the ligand.

The antioxidant status in the liver, kidney and brain of mice after exposure to 2-6 Gy radiation were also measured recently [Menon et. al., 2009]. The results were similar to the ones observed in the heart of rats without radiation. Also an analysis of their blood leukocytes and bone marrow for DNA damage using alkaline single cell gel electrophoresis (alkaline comet assay) revealed DNA repair from the lowering of comet assay parameters, DNA in tail, tail length, tail moment, and olive tail moment. Administration of the complex also reduced the mortality of the mice and also aided recovery from the radiation induced weight loss [Ramachandran et. al., 2010]. DNA repair was also observed in human blood leukocytes with palladium α-lipoic acid complex treatment immediately after exposure to radiation [Menon & Nair, 2011].

The mechanism of the superiority of the metal complex compared to that of the ligand is still an unsolved puzzle. Compared to the high oral dose, the available plasma concentration of α-lipoic acid is very small. One possibility for the higher activity of the metal complex is probably due to its increased concentration in the plasma. This needs experimental verification. It is also known that 98% of α-lipoic acid is excreted in urine within 24 hours and since it takes 4-6 weeks for serum and urine clearance of palladium, the previous suggestion seems justified. Another possibility is the chemistry of the transition metal playing a dominant role in the enzymatic activity. The starting material in the synthesis of the palladium α-lipoic acid complex is palladium(II). The final complex is also palladium(II), based on preliminary ESR data. Palladium(II) complexes are diamagnetic. The complex concentration dependent electrochemical characteristics of α-lipoic acid suggest the possibility of free radical formation by one electron reduction under physiological conditions. In such a case the electron spin may be involved in the enzymatic process. The impedance characteristics of the palladium α-lipoic acid as well as that of the α-lipoic acid, described in this section, strongly suggest this possibility.

Another possibility for the superiority of the palladium α-lipoic acid complex compared to α-lipoic acid is its ability to form self-assembled structures, such as the one shown in Fig. 6. No self-assembly was observed for sodium lipoate. We want to point out that binding of a lysine residue in the protein to the lipoyl group of E2 in 2-oxoacid dehydrogenases results in a long flexible arm that can oscillate a distance of ~200 Å. This arm is utilized during the catalytic cycle [Patel & Packer, 2008]. It is obvious that the self assembled palladium α-lipoic acid complex can make this process more facile.

The physics and chemistry of non-equilibrium systems have been utilized to understand some of the spatial patterns and temporal patterning observed in biological processes such as bacterial colonies shaped by diffusive instabilities and calcium waves governed by nonlinear amplification during intracellular signaling [Levine & Jacob, 2004].

In homogeneous systems, spiral waves and spatiotemporal phenomena are formed from autocatalytic reactions and diffusion resulting from chemical instabilities. Our data suggest that the propagation of electrical signaling among the packing units and extending to long distances by global coupling is viable by such self-assembled systems.

We have utilized the technique of electrochemical impedance spectroscopy extensively to probe spatiotemporal phenomena in biological systems. This technique is routinely used to study corrosion and fuel cells. We have used admittance measurements for understanding solute-solvent interactions, "π –way" conduction, ion pair formation, water-structure enforced ion pair formation, potential induced and solvent mediated ion pair formation at the double layer, and semi conduction characteristics of simple biological molecules

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 119

quadrant in the plot. However, if there is inductance, the data will require the first and fourth quadrants. In corrosion studies, the oxides formed at passivation potentials exhibit, quite often, semiconduction characteristics and their impedance data will be in both first and second quadrants. The impedance in the second quadrant may be compared to the negative differential resistance (NDR) observed in the I-V curves of tunnel diodes and some enzymes. Impedance spectra spanning more than two quadrants and especially four quadrants are unusual and are often explained by nonequilibrium phenomena and

The impedance spectra for α-lipoic acid and its modulation by complexing with palladium are shown in Fig. 7. While α-lipoic acid exhibits NDR and shows impedance in only 3 quadrants (chaotic in quadrant 3), the spectra of the metal complex is extended to 4 quadrants and much more smoothly by complexation with palladium. Of course the NDR behavior can be optimized by slightly tweaking the applied potential. This enhancement in NDR behavior may be compared to the enhanced Krebs cycle and mitochondrial enzymatic

We have reason to believe that the self-assembled structure of the complex, by providing a spatial extension of the membrane with much more surface area, may be catalyzing the electron transfer process by enhancing spin coupling. This may account, for example, the enhanced complex I and complex II activities activities of PdLA by 151% and 212% more

Another important aspect of this system is the fact α-lipoic acid is linked to lysine by an amide bond in the multienzyme complexes of pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and branched chain α-ketoglutarate dehydrogenase. Thus both α-lipoic acid and lysine have heavy involvement in the electronic aspects of the enzymatic process.

Oxidative stress is caused by the chemical imbalance between ROS production and their breakdown by antioxidants. Over-abundance of ROS has been found during neuronal

Fig. 7. a) Nyquist plot for 0.0373 M sodium lipoate, -1.15V, pH 7.79, NDR at 4.81Hz. b) Modulation of lipoate impedance by palladium in 0.0373M palladium α-lipoic acid (1:1

complex) in 0.1792 M NaCl, -1.18V, pH 7.78, NDR at 66Hz [Krishnan et. al., 2011]

compared to spatiotemporal oscillations in biological systems.

than that of α-lipoic acid.

**11. Conclusions** 

activities of the palladium α-lipoic acid compared to that of the ligand.

[Krishnan & Garnett, 2006; 2011; Krishnan et al., 2007a,b; 2008a,b,c,d; 2009a,b; 2011]. Simple molecules such as arginine, histidine, lysine, flavin adenine dinucleotide, riboflavin, cysteine, lidocaine hydrochloride, α-lipoic acid, and hydrogen peroxide exhibit negative differential resistance, a characteristic of a tunnel diode.

Fig. 6. Phase microscopy of palladium α-lipoic acid complex, 300X [Krishnan & Garnett, 2006]

In this technique a perturbing sinusoidal voltage E = Eosin(ωt) is applied at angular frequency ω (2πf, where f is the conventional frequency in Hz) to the electrode system consisting of a working electrode, counter electrode and reference electrode. The measurements reported in this chapter were made using and EG & G PARC Model 303A SMDE trielectrode system (mercury working electrode, platinum counter electrode and Ag/AgCl saturated KCl reference electrode) along with Autolab ecochemie. The measurements were carried out in the range 1000Hz to 30 mHz. The amplitude of the sinusoidal perturbation was 10 mV. The response of the applied sinusoidal voltage is analyzed in terms of the resultant current I = Iosin(ωt + Ф), where Ф represents a characteristic phase angle shift. In the plane of Cartesian coordinates, an impedance is expressed by its real (Z′) and imaginary (Z′′) parts. The modulus ⎢Z ⎢and phase angle Ф of **Z**(ω) can be obtained from ⎢Z ⎢= [Z′ 2 + Z′′2]1/2 and Ф = tan-1 [Z′′/Z′], respectively [Macdonald & Johnson, 2005]. Admittance and impedance are interrelated:

$$
\mathbb{Z}'/\mathbb{Y}' = \mathbb{Z}''/\mathbb{Y}'' = (\mathbb{Z}')^2 + (\mathbb{Z}'')^2 = 1/[(\mathbb{Y}')^2 + (\mathbb{Y}'')^2] \tag{32}
$$

Over a frequency bandwidth of interest, there are various ways of representing the impedance spectrum. Most often, the well known Nyquist or Cole-Cole plot (Z′′ as the Y-axis and Z′ as the X-axis for the range of frequencies explored at a fixed potential) and Bode plot (⎢Z ⎢and Ф vs. logω) are employed to represent the data. In simple terms, impedance is like a frequency dependent generalized resistance and admittance is like a frequency dependent conductance. In electrochemistry, the imaginary impedance is almost always capacitive and therefore negative. Majority of impedance data require only the first quadrant in the plot. However, if there is inductance, the data will require the first and fourth quadrants. In corrosion studies, the oxides formed at passivation potentials exhibit, quite often, semiconduction characteristics and their impedance data will be in both first and second quadrants. The impedance in the second quadrant may be compared to the negative differential resistance (NDR) observed in the I-V curves of tunnel diodes and some enzymes. Impedance spectra spanning more than two quadrants and especially four quadrants are unusual and are often explained by nonequilibrium phenomena and compared to spatiotemporal oscillations in biological systems.

The impedance spectra for α-lipoic acid and its modulation by complexing with palladium are shown in Fig. 7. While α-lipoic acid exhibits NDR and shows impedance in only 3 quadrants (chaotic in quadrant 3), the spectra of the metal complex is extended to 4 quadrants and much more smoothly by complexation with palladium. Of course the NDR behavior can be optimized by slightly tweaking the applied potential. This enhancement in NDR behavior may be compared to the enhanced Krebs cycle and mitochondrial enzymatic activities of the palladium α-lipoic acid compared to that of the ligand.

We have reason to believe that the self-assembled structure of the complex, by providing a spatial extension of the membrane with much more surface area, may be catalyzing the electron transfer process by enhancing spin coupling. This may account, for example, the enhanced complex I and complex II activities activities of PdLA by 151% and 212% more than that of α-lipoic acid.

Fig. 7. a) Nyquist plot for 0.0373 M sodium lipoate, -1.15V, pH 7.79, NDR at 4.81Hz. b) Modulation of lipoate impedance by palladium in 0.0373M palladium α-lipoic acid (1:1 complex) in 0.1792 M NaCl, -1.18V, pH 7.78, NDR at 66Hz [Krishnan et. al., 2011]

Another important aspect of this system is the fact α-lipoic acid is linked to lysine by an amide bond in the multienzyme complexes of pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and branched chain α-ketoglutarate dehydrogenase. Thus both α-lipoic acid and lysine have heavy involvement in the electronic aspects of the enzymatic process.

#### **11. Conclusions**

118 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

[Krishnan & Garnett, 2006; 2011; Krishnan et al., 2007a,b; 2008a,b,c,d; 2009a,b; 2011]. Simple molecules such as arginine, histidine, lysine, flavin adenine dinucleotide, riboflavin, cysteine, lidocaine hydrochloride, α-lipoic acid, and hydrogen peroxide exhibit negative

Fig. 6. Phase microscopy of palladium α-lipoic acid complex, 300X [Krishnan & Garnett,

The modulus ⎢Z ⎢and phase angle Ф of **Z**(ω) can be obtained from ⎢Z ⎢= [Z′

In this technique a perturbing sinusoidal voltage E = Eosin(ωt) is applied at angular frequency ω (2πf, where f is the conventional frequency in Hz) to the electrode system consisting of a working electrode, counter electrode and reference electrode. The measurements reported in this chapter were made using and EG & G PARC Model 303A SMDE trielectrode system (mercury working electrode, platinum counter electrode and Ag/AgCl saturated KCl reference electrode) along with Autolab ecochemie. The measurements were carried out in the range 1000Hz to 30 mHz. The amplitude of the sinusoidal perturbation was 10 mV. The response of the applied sinusoidal voltage is analyzed in terms of the resultant current I = Iosin(ωt + Ф), where Ф represents a characteristic phase angle shift. In the plane of Cartesian coordinates, an impedance is expressed by its real (Z′) and imaginary (Z′′) parts.

Ф = tan-1 [Z′′/Z′], respectively [Macdonald & Johnson, 2005]. Admittance and impedance

 Z′/Y′ = Z′′/Y′′ = (Z′)2 + (Z′′)2 = 1/[(Y′)2+ (Y′′)2 (32) Over a frequency bandwidth of interest, there are various ways of representing the impedance spectrum. Most often, the well known Nyquist or Cole-Cole plot (Z′′ as the Y-axis and Z′ as the X-axis for the range of frequencies explored at a fixed potential) and Bode plot (⎢Z ⎢and Ф vs. logω) are employed to represent the data. In simple terms, impedance is like a frequency dependent generalized resistance and admittance is like a frequency dependent conductance. In electrochemistry, the imaginary impedance is almost always capacitive and therefore negative. Majority of impedance data require only the first

2 + Z′′2]1/2 and

differential resistance, a characteristic of a tunnel diode.

2006]

are interrelated:

Oxidative stress is caused by the chemical imbalance between ROS production and their breakdown by antioxidants. Over-abundance of ROS has been found during neuronal

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 121

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development, as well as in numerous neuropathological conditions. Oxidative stress and mitochondrial dysfunction have been closely associated in brain injury such as ischemia and stroke and neurodegenerative processes such as Multiple Sclerosis, Parkinson's, Alzheimer's, and Huntington's.

Lipoic acid was found to be effective in modulating many neurodegenerative disorders. The development of palladium α-lipoic acid complex was intended to augment the properties of this ligand by the catalytic properties of the transition metal. It is formulated to combat mitochondrial dysfunction. The unique electronic properties of palladium modulating the properties of α-lipoic acid appear to be a key to this physiological effectiveness. This is exemplified in our electrochemical impedance spectroscopic studies of α-lipoic acid and palladium α-lipoic acid complex.

Palladium α-lipoic acid complex facilitates aerobic metabolism much more than that of αlipoic acid, by significantly enhancing the enzymatic activity of isocitrate dehydrogenase, αketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase at the Krebs cycle and mitochondrial complexes I, II, III, and IV of the electron transport chain. The electronic properties of palladium also appear to modulate the antioxidant properties of α-lipoic acid in that PdLA enhances the activities of catalase and glutathione peroxidase more than that of α-lipoic acid. The level of GSH also was significantly improved and the level of lipid peroxidation was decreased in the heart mitochondria of aged rats.

PdLA is similar to a multi-spectrum drug. Since it targets the mitochondria it is able to carry out several functions such as combating age-related as well as disease-associated fatigue, and minimizes the effects of ischemic injury. Being a powerful free radical scavenger, it may also be effective in combating death of neurons and other progressive loss of structure or function of neurons caused by free radicals.

PdLA is able to protect from radiation exposure and repair DNA. It also seems to ward off radiation exposure-associated weight loss in mice, possibly protecting susceptible gastrointestinal tract.

#### **12. Acknowledgements**

The area covered in this chapter is vast and warrants citation of numerous publications. The authors regret being unable to cite many publications due to space limitations. The authors wish to express their sincere thanks and gratitude for many fruitful discussions and collaborations in various aspects of the work presented in this chapter: B. Chu, K. K. Janardhanan, T.A. Ajith, C.K.K. Nair, N.P. Sudheesh, A. Menon, and L. Ramachandran.

#### **13. References**


development, as well as in numerous neuropathological conditions. Oxidative stress and mitochondrial dysfunction have been closely associated in brain injury such as ischemia and stroke and neurodegenerative processes such as Multiple Sclerosis, Parkinson's, Alzheimer's,

Lipoic acid was found to be effective in modulating many neurodegenerative disorders. The development of palladium α-lipoic acid complex was intended to augment the properties of this ligand by the catalytic properties of the transition metal. It is formulated to combat mitochondrial dysfunction. The unique electronic properties of palladium modulating the properties of α-lipoic acid appear to be a key to this physiological effectiveness. This is exemplified in our electrochemical impedance spectroscopic studies of α-lipoic acid and

Palladium α-lipoic acid complex facilitates aerobic metabolism much more than that of αlipoic acid, by significantly enhancing the enzymatic activity of isocitrate dehydrogenase, αketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase at the Krebs cycle and mitochondrial complexes I, II, III, and IV of the electron transport chain. The electronic properties of palladium also appear to modulate the antioxidant properties of α-lipoic acid in that PdLA enhances the activities of catalase and glutathione peroxidase more than that of α-lipoic acid. The level of GSH also was significantly improved and the

PdLA is similar to a multi-spectrum drug. Since it targets the mitochondria it is able to carry out several functions such as combating age-related as well as disease-associated fatigue, and minimizes the effects of ischemic injury. Being a powerful free radical scavenger, it may also be effective in combating death of neurons and other progressive loss of structure or

PdLA is able to protect from radiation exposure and repair DNA. It also seems to ward off radiation exposure-associated weight loss in mice, possibly protecting susceptible

The area covered in this chapter is vast and warrants citation of numerous publications. The authors regret being unable to cite many publications due to space limitations. The authors wish to express their sincere thanks and gratitude for many fruitful discussions and collaborations in various aspects of the work presented in this chapter: B. Chu, K. K. Janardhanan, T.A. Ajith, C.K.K. Nair, N.P. Sudheesh, A. Menon, and L. Ramachandran.

Aktas O., Kieseier B. & Hartung, H. P. (2010). Neuroprotection, regeneration and

Antonawich F. J., Fiore S. M., & Welicky L. M. (2004). Regulation of ischemic cell death by

Baumgartner M. R., Schmalle H. & Dubler E. (1996). The Interaction of transition metals

immunomodulation: broadening the therapeutic repertoire in multiple sclerosis.

the lipoic acid-palladium complex, Poly MVA, in gerbils. *Experimental Neurology*,

with the coenzyme α-lipoic acid: synthesis, structure and characterization of copper

level of lipid peroxidation was decreased in the heart mitochondria of aged rats.

and Huntington's.

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**5** 

*Chile* 

**Docosahexaenoic Acid (DHA), in the Prevention** 

The scientific and technological development observed since the late nineteenth century until nowadays has caused a significant increase in the life expectancy of the population, being people over 65 a significant and yearly increasing 15% of the population (Szymański et al., 2010). As result, the increase in the life expectancy has also increased the prevalence of diseases associated to ageing, especially neurodegenerative diseases such as Alzheimer's disease, Multiple sclerosis and Parkinson's disease (Habeck et al., 2010). These diseases, besides its increasing with age, are also associated to the socioeconomic status, work and physical activity, family history and genetic, and in the last two decades, the nutrition has also aroused as a relevant factor (Stampfer, 2006). In this sense, there is general consensus that a healthy diet may prevent the development of many diseases such as obesity, hypertension, diabetes mellitus, stroke, certain kinds of cancers and now neurodegenerative diseases (Massaro et al., 2010). Epidemiological evidences suggest that populations having a significant consumption of fish, a food rich in n-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA), show lower incidence of neurodegenerative diseases (Tully et al, 2003). n-3 LCPUFA, especially docosahexaenoic acid (DHA, C22: 6 Δ 4, 7, 10, 13, 16, 19; n-3), play a fundamental role in the development and preservation of the nervous system, and in the recent years solid evidences of their involvement in the prevention and/or eventually in the treatment of neurodegenerative diseases have appeared (Ikemoto et al., 2001). In neuronal tissue DHA is found in high concentrations, especially in the phospholipids of neuronal and glial membranes. However, as ageing progress and during the development of neurodegenerative diseases, a significant reduction in the DHA content of the brain is produced (Tully et al., 2003), especially in the cortex, cerebellum and hypothalamus, which result in a considerable reduction in the fluidity of neuronal membranes and an alteration of the neuronal homeostasis (Sodeberg et al., 1991; Kalminj et al., 2004). Beyond the effect of DHA at the neuronal membranes, the fatty acid also exerts other protective effects which are mediated by a metabolic derivative named neuroprotectin D-1 (NPD-1) which may protect neurons against oxidative stress, inflammation, disruption of the cytoskeleton and from the activation of apoptotic signaling pathways (Bazan, 2009). NPD-1, formed from DHA, is normally present in the nervous system, especially in the brain, but it is especially relevant

**1. Introduction** 

**and Treatment of Neurodegenerative Diseases** 

Rodrigo Valenzuela B.1 and Alfonso Valenzuela B.2

*1University of Chile, Faculty of Medicine, Nutrition and Dietetic School 2University of Chile, Institute of Nutrition and Food Technology (INTA), Lipid Center and University of Los Andes, Faculty of Medicine; Santiago,* 


### **Docosahexaenoic Acid (DHA), in the Prevention and Treatment of Neurodegenerative Diseases**

Rodrigo Valenzuela B.1 and Alfonso Valenzuela B.2 *1University of Chile, Faculty of Medicine, Nutrition and Dietetic School 2University of Chile, Institute of Nutrition and Food Technology (INTA), Lipid Center and University of Los Andes, Faculty of Medicine; Santiago, Chile* 

#### **1. Introduction**

126 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

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> The scientific and technological development observed since the late nineteenth century until nowadays has caused a significant increase in the life expectancy of the population, being people over 65 a significant and yearly increasing 15% of the population (Szymański et al., 2010). As result, the increase in the life expectancy has also increased the prevalence of diseases associated to ageing, especially neurodegenerative diseases such as Alzheimer's disease, Multiple sclerosis and Parkinson's disease (Habeck et al., 2010). These diseases, besides its increasing with age, are also associated to the socioeconomic status, work and physical activity, family history and genetic, and in the last two decades, the nutrition has also aroused as a relevant factor (Stampfer, 2006). In this sense, there is general consensus that a healthy diet may prevent the development of many diseases such as obesity, hypertension, diabetes mellitus, stroke, certain kinds of cancers and now neurodegenerative diseases (Massaro et al., 2010). Epidemiological evidences suggest that populations having a significant consumption of fish, a food rich in n-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA), show lower incidence of neurodegenerative diseases (Tully et al, 2003). n-3 LCPUFA, especially docosahexaenoic acid (DHA, C22: 6 Δ 4, 7, 10, 13, 16, 19; n-3), play a fundamental role in the development and preservation of the nervous system, and in the recent years solid evidences of their involvement in the prevention and/or eventually in the treatment of neurodegenerative diseases have appeared (Ikemoto et al., 2001). In neuronal tissue DHA is found in high concentrations, especially in the phospholipids of neuronal and glial membranes. However, as ageing progress and during the development of neurodegenerative diseases, a significant reduction in the DHA content of the brain is produced (Tully et al., 2003), especially in the cortex, cerebellum and hypothalamus, which result in a considerable reduction in the fluidity of neuronal membranes and an alteration of the neuronal homeostasis (Sodeberg et al., 1991; Kalminj et al., 2004). Beyond the effect of DHA at the neuronal membranes, the fatty acid also exerts other protective effects which are mediated by a metabolic derivative named neuroprotectin D-1 (NPD-1) which may protect neurons against oxidative stress, inflammation, disruption of the cytoskeleton and from the activation of apoptotic signaling pathways (Bazan, 2009). NPD-1, formed from DHA, is normally present in the nervous system, especially in the brain, but it is especially relevant

Docosahexaenoic Acid (DHA), in the Prevention and Treatment of Neurodegenerative Diseases 129

decreased visual acuity (McNamara & Carlson, 2006). Similar problems have been observed in preterm primates and humans, which may be reversed after n-3 LCPUFA supplementation. DHA intake remains also essential after the end of the brain development. It is required to maintain the normal brain functions, including synaptic plasticity,

**21 15 9 3 COOH**

Due to lack of enzymes necessary for neuronal de novo synthesis of DHA and AA, these fatty acids must be obtained preformed directly from the diet, or be synthesized from their precursors, α-linolenic acid (ALN, C18: 3 Δ 9, 12, 15; n-3) for DHA, and linoleic acid (LA, C18: 2 Δ 9, 12, n-6) for AA (Williard et al., 2001). This synthesis is carried out mainly in the liver and to a lesser extent in the cerebral endothelium by the astrocytes, which may export these fatty acids to the neurons (Lesaet al., 2003, Kalant & Cianflone, 2004). Although is still under discussion how these fatty acids (ALN, LA) can cross the blood-brain barrier, it has been demonstrated that may diffuse through the phospholipids of neuronal membranes (McCann & Ames, 2005). Other evidences suggest that some membrane proteins may facilitate the transport of ALN and/or DHA through the hematoencephalic barrier. One of these transporters has been identified as a caveolin binding protein type or CD36 (Williard et al., 2001 ; Lesa et al., 2003). However, plasma levels of LCPUFA are poorly correlated with the dietary intake of the precursors (Kalant & Cianflone, 2004). In fact, in healthy individuals, Δ5-and Δ6-desaturases, the key enzymes in the conversion process of LA to AA and ALN to DHA, are only induced in the absence of precursors and suppressed when the intake of the precursors (LA and ALN) is sufficient (Kalant & Cianflone, 2004). In contrast, the Δ6-desaturase activity appears to decrease with age, as has been demonstrated in rodent models (Cho et al, 1999; Hrelia et al., 1990). The reduction of the activity of this enzyme could be significantly important during ageing, considering that the elderly shows low tissue levels of DHA, especially when the intake of ALN is chronically low (Strokin et al., 2006; Kalmijn et al., 1997). This situation could lead to profound alterations in the metabolism of the nervous system, especially in the density of the synaptosomes and/or in the release of neurotransmitters, as suggested from studies carried-out in the nematode *Caenorhabditis elegans* deficient for the enzyme Δ6-desaturase (Lesa et al., 2003). DHA is present in the phospholipids of neuronal membrane predominantly at the sn-2 position; therefore the incorporation of DHA in membrane phospholipids depends of the cycle deacylation - reacylation which occurs at the sn-2 position (Serhan et al., 2008). In rodent brains, this cycle has significant activity (Rapoport et al., 2001) and is dependent directly on the specific activities of the enzymes acyl-CoA synthetase (ACS) and phospholipase A2 (PLA2). ACS performs the activation process by binding the fatty acid to CoA, which is an

neurotransmission and visual function (McCann & Ames, 2005).

Fig. 1. Molecular structure of docosahexaenoic acid (DHA)

**CH3**

**3. DHA and brain metabolism** 

in states and/or situations that may compromise the activity, integrity and neuronal viability, as it is the case of neurodegenerative diseases, brain injury by ischemia – reperfusion, leukocyte infiltration and activation of proapoptotic signaling pathways (Bazan, 2009; Belayev et al., 2009). In this context, NPD-1 has anti-inflammatory, antiapoptotic and even neuroregenerative effects, which would help to preserve in general, both the neuronal functioning and the nervous system (Reinoso et al 2008). A significant reduction of the neuronal DHA content is produced during the developing of neurodegenerative diseases. This reduction, which is not only produced by dietary factors, (i.e. low intake), it is also produced by metabolic process such as increased DHA metabolism and/or oxidation (Tully et al., 2003). The greatest evidence about the neuroprotective effect of DHA has been observed in Alzheimer's disease. DHA may suppress the cytotoxic effects of the accumulation of the β-amyloid peptide, being the main mechanism associated to the neuroprotective action of the fatty acid (Bazan, 2009; Reinoso et al., 2008). Facing this evidence, it is reasonable to consider the beneficial effect of increasing the consumption of DHA by eating foods rich in the fatty acid, such as fatty fish or DHA containing supplements. This chapter reviews the neuroprotective effects of DHA in the context of the brain ageing and some neurodegenerative diseases. It is also suggested to promote the consumption of food and/or supplements rich in DHA, as an effective strategy for preserving the brain function during ageing and especially to prevent the incidence, or to delay the onset of neurodegenerative diseases.

#### **2. DHA and brain physiology**

DHA is a LCPUFA with six double bonds, which belong to the series or family of the n-3 polyunsaturated fatty acids (Figure 1). It is relevant that DHA is the most unsaturated fatty acid in our organism and is found specifically concentrated in the brain, retina and sperms of higher animals (Uauy et al., 2001). DHA, when provided by the diet, comes mainly from marine organisms such as fish (fatty or blue species), shellfish, and algae (Horrocks et al., 2004). The first report of a deficiency of n-3 fatty acids was documented in 1982, which described the case of a six years girl, who received parenteral nutrition without the addition of n-3 fatty acids for five months after intestinal surgery (Holman et al., 1982). After the nutritional intervention, the girl presented low plasma DHA levels, dermatitis associated with neurological symptoms including neuropathy, blurred vision and psychological disturbances, which suggested an important role of n-3 LCPUFA, especially DHA, in the nervous system functions. In fact, it is now accepted that DHA is the most important n-3 LCPUFA in the formation of neuronal plasma and synaptosomal membranes (synaptic vesicles), especially in the brain (McNamara & Carlson, 2006). DHA amounts approximately 30-40% of fatty acids of the phospholipids forming the gray matter of the cerebral cortex and retinal photoreceptors (Carlson, 2002). The most important growing of the brain in humans is produced during the third trimester of fetal development and in the first two years of life. It is during these periods that the requirements of n-3 and n-6 LCPUFA are roused considerably, especially the requirements of DHA and arachidonic acid (AA, C20: 4 Δ 5, 8, 11, 14; n-6). Animal studies have shown that the reduced availability of DHA during the perinatal period is associated with deficits in the establishing of neuronal networks, and also with multiple expressions of synaptic pathologies, including deficits in serotonin neurotransmission and alterations in the mesocorticolimbic dopamine pathway, neurocognitive deficits, and a greater anxious behavior, aggression, depression and decreased visual acuity (McNamara & Carlson, 2006). Similar problems have been observed in preterm primates and humans, which may be reversed after n-3 LCPUFA supplementation. DHA intake remains also essential after the end of the brain development. It is required to maintain the normal brain functions, including synaptic plasticity, neurotransmission and visual function (McCann & Ames, 2005).

Fig. 1. Molecular structure of docosahexaenoic acid (DHA)

#### **3. DHA and brain metabolism**

128 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

in states and/or situations that may compromise the activity, integrity and neuronal viability, as it is the case of neurodegenerative diseases, brain injury by ischemia – reperfusion, leukocyte infiltration and activation of proapoptotic signaling pathways (Bazan, 2009; Belayev et al., 2009). In this context, NPD-1 has anti-inflammatory, antiapoptotic and even neuroregenerative effects, which would help to preserve in general, both the neuronal functioning and the nervous system (Reinoso et al 2008). A significant reduction of the neuronal DHA content is produced during the developing of neurodegenerative diseases. This reduction, which is not only produced by dietary factors, (i.e. low intake), it is also produced by metabolic process such as increased DHA metabolism and/or oxidation (Tully et al., 2003). The greatest evidence about the neuroprotective effect of DHA has been observed in Alzheimer's disease. DHA may suppress the cytotoxic effects of the accumulation of the β-amyloid peptide, being the main mechanism associated to the neuroprotective action of the fatty acid (Bazan, 2009; Reinoso et al., 2008). Facing this evidence, it is reasonable to consider the beneficial effect of increasing the consumption of DHA by eating foods rich in the fatty acid, such as fatty fish or DHA containing supplements. This chapter reviews the neuroprotective effects of DHA in the context of the brain ageing and some neurodegenerative diseases. It is also suggested to promote the consumption of food and/or supplements rich in DHA, as an effective strategy for preserving the brain function during ageing and especially to prevent the incidence, or to

DHA is a LCPUFA with six double bonds, which belong to the series or family of the n-3 polyunsaturated fatty acids (Figure 1). It is relevant that DHA is the most unsaturated fatty acid in our organism and is found specifically concentrated in the brain, retina and sperms of higher animals (Uauy et al., 2001). DHA, when provided by the diet, comes mainly from marine organisms such as fish (fatty or blue species), shellfish, and algae (Horrocks et al., 2004). The first report of a deficiency of n-3 fatty acids was documented in 1982, which described the case of a six years girl, who received parenteral nutrition without the addition of n-3 fatty acids for five months after intestinal surgery (Holman et al., 1982). After the nutritional intervention, the girl presented low plasma DHA levels, dermatitis associated with neurological symptoms including neuropathy, blurred vision and psychological disturbances, which suggested an important role of n-3 LCPUFA, especially DHA, in the nervous system functions. In fact, it is now accepted that DHA is the most important n-3 LCPUFA in the formation of neuronal plasma and synaptosomal membranes (synaptic vesicles), especially in the brain (McNamara & Carlson, 2006). DHA amounts approximately 30-40% of fatty acids of the phospholipids forming the gray matter of the cerebral cortex and retinal photoreceptors (Carlson, 2002). The most important growing of the brain in humans is produced during the third trimester of fetal development and in the first two years of life. It is during these periods that the requirements of n-3 and n-6 LCPUFA are roused considerably, especially the requirements of DHA and arachidonic acid (AA, C20: 4 Δ 5, 8, 11, 14; n-6). Animal studies have shown that the reduced availability of DHA during the perinatal period is associated with deficits in the establishing of neuronal networks, and also with multiple expressions of synaptic pathologies, including deficits in serotonin neurotransmission and alterations in the mesocorticolimbic dopamine pathway, neurocognitive deficits, and a greater anxious behavior, aggression, depression and

delay the onset of neurodegenerative diseases.

**2. DHA and brain physiology** 

Due to lack of enzymes necessary for neuronal de novo synthesis of DHA and AA, these fatty acids must be obtained preformed directly from the diet, or be synthesized from their precursors, α-linolenic acid (ALN, C18: 3 Δ 9, 12, 15; n-3) for DHA, and linoleic acid (LA, C18: 2 Δ 9, 12, n-6) for AA (Williard et al., 2001). This synthesis is carried out mainly in the liver and to a lesser extent in the cerebral endothelium by the astrocytes, which may export these fatty acids to the neurons (Lesaet al., 2003, Kalant & Cianflone, 2004). Although is still under discussion how these fatty acids (ALN, LA) can cross the blood-brain barrier, it has been demonstrated that may diffuse through the phospholipids of neuronal membranes (McCann & Ames, 2005). Other evidences suggest that some membrane proteins may facilitate the transport of ALN and/or DHA through the hematoencephalic barrier. One of these transporters has been identified as a caveolin binding protein type or CD36 (Williard et al., 2001 ; Lesa et al., 2003). However, plasma levels of LCPUFA are poorly correlated with the dietary intake of the precursors (Kalant & Cianflone, 2004). In fact, in healthy individuals, Δ5-and Δ6-desaturases, the key enzymes in the conversion process of LA to AA and ALN to DHA, are only induced in the absence of precursors and suppressed when the intake of the precursors (LA and ALN) is sufficient (Kalant & Cianflone, 2004). In contrast, the Δ6-desaturase activity appears to decrease with age, as has been demonstrated in rodent models (Cho et al, 1999; Hrelia et al., 1990). The reduction of the activity of this enzyme could be significantly important during ageing, considering that the elderly shows low tissue levels of DHA, especially when the intake of ALN is chronically low (Strokin et al., 2006; Kalmijn et al., 1997). This situation could lead to profound alterations in the metabolism of the nervous system, especially in the density of the synaptosomes and/or in the release of neurotransmitters, as suggested from studies carried-out in the nematode *Caenorhabditis elegans* deficient for the enzyme Δ6-desaturase (Lesa et al., 2003). DHA is present in the phospholipids of neuronal membrane predominantly at the sn-2 position; therefore the incorporation of DHA in membrane phospholipids depends of the cycle deacylation - reacylation which occurs at the sn-2 position (Serhan et al., 2008). In rodent brains, this cycle has significant activity (Rapoport et al., 2001) and is dependent directly on the specific activities of the enzymes acyl-CoA synthetase (ACS) and phospholipase A2 (PLA2). ACS performs the activation process by binding the fatty acid to CoA, which is an

Docosahexaenoic Acid (DHA), in the Prevention and Treatment of Neurodegenerative Diseases 131

reduced, being even greater this decrease in the population that develops neurodegenerative diseases (Sodeberg et al., 1991). This significant reduction may be caused either, by a lower intake of the fatty acid or of its metabolic precursor and/or by an increased in the cellular utilization of DHA (Jicha & Markesbery, 2010). DHA plays a relevant role in the preservation of both the histology and physiology of the neuronal tissue as the individual ages, by preserving the nervous system functions among which memory and learning are the most remarkable (Lukiw & Bazan, 2008). Several epidemiological studies have strongly established that a higher intake of foods rich in DHA (fatty fish and/or nutritional supplements based on fish oils or microalgae) is highly correlated with a lower risk of developing neurodegenerative diseases (Kalminj et al., 2004; Kalmijn et al., 1997), which is also associated with a clinical history indicating that patients with neurodegenerative disease have significantly lower levels of DHA in plasma and brain

A relevant question about the attributed neuroprotective effects of DHA in ageing and especially against neurodegenerative diseases is referred as how this fatty acid exerts these effects at the molecular level. Their role in the fluidity of the neuronal membrane appears as one of the most relevant attributes (Saiz & Klein, 2001). In fact, up to day the classification of membrane fluidity is based on the level of DHA present in the phospholipids that form the membrane matrix (Stillwell et al., 2005). However, the higher fluidity that confers DHA to neuronal membrane is not sufficient to explain the neuroprotective effects attributed to the fatty acid. As result of multiple investigations, it has been established that acylation of DHA at the sn-2 position in the membrane phospholipids and the activity of PLA-2, are additional features of DHA, by itself, to achieve an additional neuroprotective action of the fatty acid against certain cytotoxic situations, as are neurodegenerative diseases (Stillwell et al., 2005; Brown & London, 2000). It is no casual that DHA is present mainly at the sn-2 position in the phospholipids of neuronal membranes (48% in phosphatidylcholine, 52% in fosfatidilserine and 20% in phosphatidylethanolamine) (Aveldano & Bazan, 1983). It was the discovery of a number of bioactive compounds derived from DHA, called protectins and resolvins, which show cytoprotective properties that open the way to a better understanding of how DHA may exert at the molecular level its neuroprotective actions (Mukherjee et al., 2004). Among these bioactive DHA-derivatives, NPD-1 (protectin D1 or D1 neuroprotectin: 10R, 17Sdihydroxy-docosa-4Z, 7Z, 11E, 13E, 15Z, 19Z-hexaenoic acid), appears the most relevant neuroprotective agent (Serhan et al., 2008). NPD-1 is generated once DHA is released from the phospholipids by the hydrolytic action of PLA-2, where the enzyme 15 lipoxygenase initiate a complex process of lipooxidation, epoxidation and hydrolysis resulting in the formation of NPD-1 (Serhan, 2005). Figure 3 shows a diagram of the formation of NPD-1. NPD-1 may exert its neuroprotective function either through a receptor (as yet unidentified), which may act in an autocrine form and/or NPD-1, once formed, may be diffused to other neurons. The mechanisms involved in the neuroprotection afforded by NPD-1 may include: (i) inhibition of the expression of proinflammatory cytokines (TNFα and IL1β), (ii) inhibition of the generation and neurotoxicity of β-amyloid peptides and Ab42 (iii) increased gene expression of antiapoptotic molecules (Bcl-2 and Bcl-xL), (iv) reduction in the gene expression of proapoptotic molecules (Bax and Bad) and (v) increased neuronal antioxidant potential (Chu Chen & Bazan, 2005). Moreover, inflammatory

(Tully et al., 2003; Sodeberg et al., 1991).

**5. Neuroprotectin D-1 and neuroprotection** 

ATP-dependent reaction. Once activated, the fatty acids can be incorporated into phospholipids. ACS isoenzymes 3, 4 and 6 are specific to LCPUFA, and in the brain the ACS 6 isoform is specific for the acylation of DHA (Marszalek et al., 2005). At present, there is not sufficient background about the type of phospholipase that participate in the release of DHA from phospholipids. However, it has been established that in astrocytes the release of DHA involve a mechanism dependent of 2+Ca but independent of PLA2 (Strokin et al., 2003). The role of PLA2 in neurons has not been clearly demonstrated, but a study in the hippocampus of rats, indicated that the enzyme may be of fundamental importance in the release of DHA in neuronal tissue (Strokin et al., 2006). Figure 2 shows a proposal of how DHA may be incorporated into the phospholipids of neuronal membranes.

Fig. 2. Incorporation of DHA into the phospholipids of neuronal membranes

#### **4. DHA and brain ageing**

The presence of high concentrations of DHA, especially in the phospholipids of neuronal membranes, has been encouraged for more than 30 years the research about the roles of DHA in the nervous system. The evidence has demonstrated that during the embryonic stage and the first years of life, DHA plays a key role in the growth and development of the nervous and visual systems, actively participating in the processes of neurogenesis, neuronal migration, myelination and synaptogenesis (Uauy et al., 2001), thus directly impacting on cognitive development, visual, auditory, and in the memory and learning capabilities (McNamara & Carlson, 2006). As result of these observations, it is now strongly recommended to increase the consumption DHA during the pregnancy and childhood, in order to ensure the proper development of the nervous and visual systems (Uauy et al., 2001). The close relationship between DHA and the developing of the nervous system, encouraged investigators to study what happens with this fatty acid during ageing. It was observed that as the individual ages, the content of DHA in neural tissue is significantly reduced, being even greater this decrease in the population that develops neurodegenerative diseases (Sodeberg et al., 1991). This significant reduction may be caused either, by a lower intake of the fatty acid or of its metabolic precursor and/or by an increased in the cellular utilization of DHA (Jicha & Markesbery, 2010). DHA plays a relevant role in the preservation of both the histology and physiology of the neuronal tissue as the individual ages, by preserving the nervous system functions among which memory and learning are the most remarkable (Lukiw & Bazan, 2008). Several epidemiological studies have strongly established that a higher intake of foods rich in DHA (fatty fish and/or nutritional supplements based on fish oils or microalgae) is highly correlated with a lower risk of developing neurodegenerative diseases (Kalminj et al., 2004; Kalmijn et al., 1997), which is also associated with a clinical history indicating that patients with neurodegenerative disease have significantly lower levels of DHA in plasma and brain (Tully et al., 2003; Sodeberg et al., 1991).

#### **5. Neuroprotectin D-1 and neuroprotection**

130 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

ATP-dependent reaction. Once activated, the fatty acids can be incorporated into phospholipids. ACS isoenzymes 3, 4 and 6 are specific to LCPUFA, and in the brain the ACS 6 isoform is specific for the acylation of DHA (Marszalek et al., 2005). At present, there is not sufficient background about the type of phospholipase that participate in the release of DHA from phospholipids. However, it has been established that in astrocytes the release of DHA involve a mechanism dependent of 2+Ca but independent of PLA2 (Strokin et al., 2003). The role of PLA2 in neurons has not been clearly demonstrated, but a study in the hippocampus of rats, indicated that the enzyme may be of fundamental importance in the release of DHA in neuronal tissue (Strokin et al., 2006). Figure 2 shows a proposal of how

DHA may be incorporated into the phospholipids of neuronal membranes.

Fig. 2. Incorporation of DHA into the phospholipids of neuronal membranes

The presence of high concentrations of DHA, especially in the phospholipids of neuronal membranes, has been encouraged for more than 30 years the research about the roles of DHA in the nervous system. The evidence has demonstrated that during the embryonic stage and the first years of life, DHA plays a key role in the growth and development of the nervous and visual systems, actively participating in the processes of neurogenesis, neuronal migration, myelination and synaptogenesis (Uauy et al., 2001), thus directly impacting on cognitive development, visual, auditory, and in the memory and learning capabilities (McNamara & Carlson, 2006). As result of these observations, it is now strongly recommended to increase the consumption DHA during the pregnancy and childhood, in order to ensure the proper development of the nervous and visual systems (Uauy et al., 2001). The close relationship between DHA and the developing of the nervous system, encouraged investigators to study what happens with this fatty acid during ageing. It was observed that as the individual ages, the content of DHA in neural tissue is significantly

**4. DHA and brain ageing** 

A relevant question about the attributed neuroprotective effects of DHA in ageing and especially against neurodegenerative diseases is referred as how this fatty acid exerts these effects at the molecular level. Their role in the fluidity of the neuronal membrane appears as one of the most relevant attributes (Saiz & Klein, 2001). In fact, up to day the classification of membrane fluidity is based on the level of DHA present in the phospholipids that form the membrane matrix (Stillwell et al., 2005). However, the higher fluidity that confers DHA to neuronal membrane is not sufficient to explain the neuroprotective effects attributed to the fatty acid. As result of multiple investigations, it has been established that acylation of DHA at the sn-2 position in the membrane phospholipids and the activity of PLA-2, are additional features of DHA, by itself, to achieve an additional neuroprotective action of the fatty acid against certain cytotoxic situations, as are neurodegenerative diseases (Stillwell et al., 2005; Brown & London, 2000). It is no casual that DHA is present mainly at the sn-2 position in the phospholipids of neuronal membranes (48% in phosphatidylcholine, 52% in fosfatidilserine and 20% in phosphatidylethanolamine) (Aveldano & Bazan, 1983). It was the discovery of a number of bioactive compounds derived from DHA, called protectins and resolvins, which show cytoprotective properties that open the way to a better understanding of how DHA may exert at the molecular level its neuroprotective actions (Mukherjee et al., 2004). Among these bioactive DHA-derivatives, NPD-1 (protectin D1 or D1 neuroprotectin: 10R, 17Sdihydroxy-docosa-4Z, 7Z, 11E, 13E, 15Z, 19Z-hexaenoic acid), appears the most relevant neuroprotective agent (Serhan et al., 2008). NPD-1 is generated once DHA is released from the phospholipids by the hydrolytic action of PLA-2, where the enzyme 15 lipoxygenase initiate a complex process of lipooxidation, epoxidation and hydrolysis resulting in the formation of NPD-1 (Serhan, 2005). Figure 3 shows a diagram of the formation of NPD-1. NPD-1 may exert its neuroprotective function either through a receptor (as yet unidentified), which may act in an autocrine form and/or NPD-1, once formed, may be diffused to other neurons. The mechanisms involved in the neuroprotection afforded by NPD-1 may include: (i) inhibition of the expression of proinflammatory cytokines (TNFα and IL1β), (ii) inhibition of the generation and neurotoxicity of β-amyloid peptides and Ab42 (iii) increased gene expression of antiapoptotic molecules (Bcl-2 and Bcl-xL), (iv) reduction in the gene expression of proapoptotic molecules (Bax and Bad) and (v) increased neuronal antioxidant potential (Chu Chen & Bazan, 2005). Moreover, inflammatory

Docosahexaenoic Acid (DHA), in the Prevention and Treatment of Neurodegenerative Diseases 133

estimated that about 5% of the population that borders 65 is affected by AD. The prevalence of the disease doubles every 5 years over 65 years (Cummings, 2004), and many studies suggest that almost half the population up to 85 years show symptoms related to the disease (Nussbaum & Ellis, 2003; Forsyth & Ritzline, 1998).The presence of the β-amyloid peptide, which is associated with neurotoxic effects, is one of the characteristic expression of the molecular damage observed in patients (Hardy & Higgins, 1992; Yankner, 1996). Its origin occurs from the degradation (altered or incomplete) of the β-amyloid peptide precursor (APP) (Selkoe, 1994). A significant reduction in DHA levels, both in erythrocytes and in the brain, is observed in AD, specifically in the frontal lobe, and occipital and temporal cortex (Guan et al., 1994). It is also produced a replacement of DHA in phospholipids by saturated fatty acids (SFA) among which myristic acid (14:0), palmitic (16:0) and stearic acid (18:0) are the most frequent (Skinner et al., 1993). Thus, it is likely that changes in the ratio AGS/n-3 and n-6 LCPUFA could alter the neuronal function, especially at the membrane phospholipids, which in turn could result in neurological deficits. The altered fatty acid composition observed in the brains of AD patients could be caused by a deficiency in the LCPUFA transport from blood to the brain. It is remarkable that in patients with certain types of dementia or cognitive impairment it is observed the same reduction in the levels of n-3 LCPUFA, especially DHA (Kyle et al., 1999; Conquer et al., 2000). Interestingly, a decrease of DHA in plasma does not appear to be unique to AD, it is also common in the general cognitive impairment observed in ageing (Catalán et al., 2002). Many studies have demonstrated that a high intake of DHA is associated with a lower risk of AD, and in individuals diagnosed the disease, consumption of DHA result in a decrease in the progression of the characteristic symptoms, especially in relation to the cognitive

In the case of multiple sclerosis (MS), the benefits associated with n-3 LCPUFA, especially DHA, have been shown in both the mental and physical disabilities. Evaluation of patients that has been supplemented with DHA indicates a significant improvement in the symptoms characteristic of the disease (Nussbaum & Ellis, 2003; Shinto et al., 2009). Some of these beneficial effects have been observed even in patients who consume a diet low in fat, but supplemented with n-3 LCPUFA of marine origin (fish oil). However, the evidence regarding a benefic in the progression of MS is not yet fully conclusive (Weinstock-Guttman et al., 2005). Considering the information currently available, it is not yet possible to establish a direct association between the consumption of n-3 LCPUFA and a lower incidence of MS, more studies are required on the issue (Weinstock-Guttman et al., 2005; Marcheselli et al., 2003). A study showed a relationship between reduced risk of this disease and the consumption of fish, but only among women (Nordvik et al., 2000). Currently, most hypotheses about MS suggest that n-3 LCPUFA would provide the molecules needed to rebuild the myelin sheath, which is severely affected in patients with this pathology. Dietary supplementation with n-3 LCPUFA helps to reduce the severity of MS in patients recently diagnosed the pathology and may delay the onset of symptoms. This is especially effective when supplementation is from marine oils along with vitamins and dietary professional counseling (Kelley, 2001). Perhaps the severity of the MS disease can be also reduced by modulating the immune response. Several studies have shown that a reduction in the dietary fat intake and changes in the relationship n-6/n-3 produce changes in the immune

impairment (Barberger-Gateau et al., 2002).

**6.2 DHA and multiple sclerosis** 

cytokines and oxidative stress may activate the synthesis of NPD-1 (Aksenov & Markesbery, 2001).

Fig. 3. Biosynthesis of Neuroprotectin D-1 (NPD-1)

#### **6. DHA and neurodegenerative diseases**

Several epidemiological, clinical and basic-experimental studies have demonstrated the beneficial effects of n-3 LCPUFA against various diseases, among which; cardiovascular disease (Hamer and Steptoe., 2006), some cancers (Gillet et al., 2011), inflammatory diseases such as rheumatoid arthritis (Kremer et al., 1990) and asthma (Yokoyama et al., 2000), neurological disorders such as schizophrenia (Laugharne et al., 1996), depression (Hibbeln & Salem, 1995), migraine (Wagner & Nootbaar-Wagner, 1997) and neurodegenerative diseases such as Alzheimer's disease (Morley & Banks, 2010), Multiple sclerosis (Mehta et al., 2009) and Parkinson's disease (Calon & Cole, 2007). The evidence of the beneficial effect of DHA has been clearly demonstrated mainly in neurodegenerative diseases.

#### **6.1 DHA and Alzheimer´s disease**

Alzheimer's disease (AD) is a progressive dementia that is early manifested by the loss of synaptic function and memory capacity of the individual. The number of patients who are diagnosed the neuropathological disorder has increased substantially in all countries, mainly in those where has been produced an increase in the life expectancy. In fact, it is estimated that about 5% of the population that borders 65 is affected by AD. The prevalence of the disease doubles every 5 years over 65 years (Cummings, 2004), and many studies suggest that almost half the population up to 85 years show symptoms related to the disease (Nussbaum & Ellis, 2003; Forsyth & Ritzline, 1998).The presence of the β-amyloid peptide, which is associated with neurotoxic effects, is one of the characteristic expression of the molecular damage observed in patients (Hardy & Higgins, 1992; Yankner, 1996). Its origin occurs from the degradation (altered or incomplete) of the β-amyloid peptide precursor (APP) (Selkoe, 1994). A significant reduction in DHA levels, both in erythrocytes and in the brain, is observed in AD, specifically in the frontal lobe, and occipital and temporal cortex (Guan et al., 1994). It is also produced a replacement of DHA in phospholipids by saturated fatty acids (SFA) among which myristic acid (14:0), palmitic (16:0) and stearic acid (18:0) are the most frequent (Skinner et al., 1993). Thus, it is likely that changes in the ratio AGS/n-3 and n-6 LCPUFA could alter the neuronal function, especially at the membrane phospholipids, which in turn could result in neurological deficits. The altered fatty acid composition observed in the brains of AD patients could be caused by a deficiency in the LCPUFA transport from blood to the brain. It is remarkable that in patients with certain types of dementia or cognitive impairment it is observed the same reduction in the levels of n-3 LCPUFA, especially DHA (Kyle et al., 1999; Conquer et al., 2000). Interestingly, a decrease of DHA in plasma does not appear to be unique to AD, it is also common in the general cognitive impairment observed in ageing (Catalán et al., 2002). Many studies have demonstrated that a high intake of DHA is associated with a lower risk of AD, and in individuals diagnosed the disease, consumption of DHA result in a decrease in the progression of the characteristic symptoms, especially in relation to the cognitive impairment (Barberger-Gateau et al., 2002).

#### **6.2 DHA and multiple sclerosis**

132 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

cytokines and oxidative stress may activate the synthesis of NPD-1 (Aksenov & Markesbery,

Several epidemiological, clinical and basic-experimental studies have demonstrated the beneficial effects of n-3 LCPUFA against various diseases, among which; cardiovascular disease (Hamer and Steptoe., 2006), some cancers (Gillet et al., 2011), inflammatory diseases such as rheumatoid arthritis (Kremer et al., 1990) and asthma (Yokoyama et al., 2000), neurological disorders such as schizophrenia (Laugharne et al., 1996), depression (Hibbeln & Salem, 1995), migraine (Wagner & Nootbaar-Wagner, 1997) and neurodegenerative diseases such as Alzheimer's disease (Morley & Banks, 2010), Multiple sclerosis (Mehta et al., 2009) and Parkinson's disease (Calon & Cole, 2007). The evidence of the beneficial effect of DHA has been clearly demonstrated mainly in neurodegenerative

Alzheimer's disease (AD) is a progressive dementia that is early manifested by the loss of synaptic function and memory capacity of the individual. The number of patients who are diagnosed the neuropathological disorder has increased substantially in all countries, mainly in those where has been produced an increase in the life expectancy. In fact, it is

Fig. 3. Biosynthesis of Neuroprotectin D-1 (NPD-1)

**6. DHA and neurodegenerative diseases** 

**6.1 DHA and Alzheimer´s disease** 

2001).

diseases.

In the case of multiple sclerosis (MS), the benefits associated with n-3 LCPUFA, especially DHA, have been shown in both the mental and physical disabilities. Evaluation of patients that has been supplemented with DHA indicates a significant improvement in the symptoms characteristic of the disease (Nussbaum & Ellis, 2003; Shinto et al., 2009). Some of these beneficial effects have been observed even in patients who consume a diet low in fat, but supplemented with n-3 LCPUFA of marine origin (fish oil). However, the evidence regarding a benefic in the progression of MS is not yet fully conclusive (Weinstock-Guttman et al., 2005). Considering the information currently available, it is not yet possible to establish a direct association between the consumption of n-3 LCPUFA and a lower incidence of MS, more studies are required on the issue (Weinstock-Guttman et al., 2005; Marcheselli et al., 2003). A study showed a relationship between reduced risk of this disease and the consumption of fish, but only among women (Nordvik et al., 2000). Currently, most hypotheses about MS suggest that n-3 LCPUFA would provide the molecules needed to rebuild the myelin sheath, which is severely affected in patients with this pathology. Dietary supplementation with n-3 LCPUFA helps to reduce the severity of MS in patients recently diagnosed the pathology and may delay the onset of symptoms. This is especially effective when supplementation is from marine oils along with vitamins and dietary professional counseling (Kelley, 2001). Perhaps the severity of the MS disease can be also reduced by modulating the immune response. Several studies have shown that a reduction in the dietary fat intake and changes in the relationship n-6/n-3 produce changes in the immune

Docosahexaenoic Acid (DHA), in the Prevention and Treatment of Neurodegenerative Diseases 135

Neurodegenerative diseases may significantly alter the functioning of the nervous system, reducing both the number and function of neurons, which seriously affects the quality of life for those suffering these diseases. New strategies aiming to the prevention and/or the treatment of these diseases are of high priority. In this context DHA and its derivative NPD-1, emerged as a new perspective for the prevention and/or therapeutic management of these diseases, especially considering the social and economic devastation that neurological diseases may produce to the individual and the family. Future clinical research and nutritional interventions should be planned directly to establish the necessary doses of DHA needed to achieve significant beneficial effects, as well as to encourage the development and consumption of foods and/or supplements rich in this fatty acid. In this regard, the development of functional foods and/or nutraceuticals containing DHA at different concentrations is an alternative that the pharmaceutical and the food industries should consider very seriously (Valenzuela et al., 2009). To day the increase of the consumption of fish or seafood appear as not entirely feasible, due to the massive depredation of the resource, which has decreased its availability and consequently has increased the price of the products from the sea. Perhaps, in the future the increasing activity of the aquaculture may offer a viable alternative to improve the general consumption of n-3 LCPUFA to the western population and helping to prevent the early onset of neurodegenerative diseases.

The authors are grateful from FONDECYT and INNOVA-Chile the support of their

Aksenov, M.Y. & Markesbery, W.R., (2001). Changes in thiol content and expression of

Aveldano, M.I. & Bazan, N.G., (1983). Molecular species of phosphatidylcholine, -

Barberger-Gateau, P., Letenneur, L., Deschamps, V., et al. (2002). Fish, meat, and risk of

Bazan, N.G., (2009). Neuroprotectin D1-mediated anti-inflammatory and survival signaling

Belayev, L., Khoutorova, L., Atkins, K.D., et al. (2009). Robust docosahexaenoic acid-

Bousquet, M., Saint-Pierre, M., Julien, C., et al. (2008). Beneficial effects of dietary omega-3

model of Parkinson's disease. *FASEB J*, Vol. 22, pp. 1213-1225

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(Suppl), pp. 400-405

*Stroke,* Vol. 40, pp. 3121-3126

of bovine retina. *J. Lipid Res*, Vol. 24, pp. 620–627

dementia: cohort study. *BMJ*, Vol. 325, pp. 932-933

glutathione redox system genes in the hippocampus and cerebellum in Alzheimer's

ethanolamine, -serine, and -inositol in microsomal and photoreceptor membranes

in stroke, retinal degenerations, and Alzheimer's disease. *J Lipid Res*, Vol. 50

mediated neuroprotection in a rat model of transient, focal cerebral ischemia.

polyunsaturated fatty acid on toxin-induced neuronal degeneration in an animal

**8. Conclusion and perspectives** 

**9. Acknowledgment** 

**10. References** 

research.

response (Kew et al., 2003). The use of nutritional supplements rich in n-3 LCPUFA is associated with a reduced activity and plasma levels of circulating immune cells (lymphocytes, polymorph nuclear neutrophils and monocytes), including the reduction in the production of inflammatory mediators (Weinstock-Guttman et al., 2005; Nordvik et al., 2000). Moreover, a reduction in the intake of n-3 LCPUFA improves a number of indexes associated with the immune response, including lymphocyte proliferation, increased macrophage activity and cytokine production (Serhan et al., 2000). These records allow the suggestion of a protective role of n-3 LCPUFA in MS, which would lead to establish the potential of the use of n-3 LCPUFA as anti-inflammatory and neuroprotective in MS, although it remains a topic for further research.

#### **6.3 DHA and Parkinson´s disease**

In contrast to AD, the relationship of fat intake and the risk of developing Parkinson's disease (PD) is very limited. Two studies have only established an association between high consumption of saturated fatty acids, low intake of n-3 LCPUFA and the increased of the risk to develop PD (Chen et al., 2003; de Lau et al., 2005). To date researchers have not been able to establish a direct association between low intake of n-3 LCPUFA and increased risk of developing PD. However, as in patients with AD, in the brains of people with PD it is also observed a significant decrease in the levels of n-3 LCPUFA, especially DHA (Johnson et al., 1999). Research in primates allow to observe a significant reduction in the extent of levodopa-induced dyskinesia (a damage model for the PD) in animals supplemented with DHA, which suggests that these effects would be mediated by the activation of retinoid X receptors (RXR) (Samadi et al., 2006). In addition, data from these investigations show a drastic drop in neural DHA levels (Julien et al., 2006; Breckenridge et al., 1973). Also, the dietary supplementation with DHA of animals reduced the neuronal damage produced by a characteristic PD-inducer agent, the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Bousquet et al., 2008). Currently, the available information is not sufficient to establish a neuroprotective effect of DHA in the development of PD, being necessary to carry out more studies on the subject.

#### **7. Dietary sources and intake of DHA**

The metabolic precursor of n-3 LCPUFA, ALA, is found almost exclusively in land-based plant foods, such as nuts (walnuts 6%), chia seeds (*Salvia hispanica*) and flax seeds (*Linum usitatissimum*) and in some edible oils, such as soy (7 %), canola (11%), evening primrose (27%), chia (58%) and clary sage (60%). While, the already-formed DHA is found exclusively in marine foods, either of animal or vegetal origin, especially in fatty fish such as tuna, mackerel, menhaden, salmon, and some algae and microalgae. Unfortunately, the western consumption of ALA and DHA is very low, which has forced the development of nutritional supplements rich in DHA either from fish oils or microalgae, and also to add this fatty acid to foods such as vegetable oils, milk and derivatives. In this regard, in addition to the capsules containing fish oil or DHA concentrates which are very popular and available DHA can be also added to various foods such as dairy, dairy products, juices, beverages, bakery products, etc. The fatty acid may be provided in the form of triglycerides, phospholipids, and in pure form as ethyl esters (Valenzuela et al., 2006). Today, a wide variety of foods containing DHA are available from the retail and nutraceutical market.

#### **8. Conclusion and perspectives**

134 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

response (Kew et al., 2003). The use of nutritional supplements rich in n-3 LCPUFA is associated with a reduced activity and plasma levels of circulating immune cells (lymphocytes, polymorph nuclear neutrophils and monocytes), including the reduction in the production of inflammatory mediators (Weinstock-Guttman et al., 2005; Nordvik et al., 2000). Moreover, a reduction in the intake of n-3 LCPUFA improves a number of indexes associated with the immune response, including lymphocyte proliferation, increased macrophage activity and cytokine production (Serhan et al., 2000). These records allow the suggestion of a protective role of n-3 LCPUFA in MS, which would lead to establish the potential of the use of n-3 LCPUFA as anti-inflammatory and neuroprotective in MS,

In contrast to AD, the relationship of fat intake and the risk of developing Parkinson's disease (PD) is very limited. Two studies have only established an association between high consumption of saturated fatty acids, low intake of n-3 LCPUFA and the increased of the risk to develop PD (Chen et al., 2003; de Lau et al., 2005). To date researchers have not been able to establish a direct association between low intake of n-3 LCPUFA and increased risk of developing PD. However, as in patients with AD, in the brains of people with PD it is also observed a significant decrease in the levels of n-3 LCPUFA, especially DHA (Johnson et al., 1999). Research in primates allow to observe a significant reduction in the extent of levodopa-induced dyskinesia (a damage model for the PD) in animals supplemented with DHA, which suggests that these effects would be mediated by the activation of retinoid X receptors (RXR) (Samadi et al., 2006). In addition, data from these investigations show a drastic drop in neural DHA levels (Julien et al., 2006; Breckenridge et al., 1973). Also, the dietary supplementation with DHA of animals reduced the neuronal damage produced by a characteristic PD-inducer agent, the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Bousquet et al., 2008). Currently, the available information is not sufficient to establish a neuroprotective effect of DHA in the development of PD, being necessary to

The metabolic precursor of n-3 LCPUFA, ALA, is found almost exclusively in land-based plant foods, such as nuts (walnuts 6%), chia seeds (*Salvia hispanica*) and flax seeds (*Linum usitatissimum*) and in some edible oils, such as soy (7 %), canola (11%), evening primrose (27%), chia (58%) and clary sage (60%). While, the already-formed DHA is found exclusively in marine foods, either of animal or vegetal origin, especially in fatty fish such as tuna, mackerel, menhaden, salmon, and some algae and microalgae. Unfortunately, the western consumption of ALA and DHA is very low, which has forced the development of nutritional supplements rich in DHA either from fish oils or microalgae, and also to add this fatty acid to foods such as vegetable oils, milk and derivatives. In this regard, in addition to the capsules containing fish oil or DHA concentrates which are very popular and available DHA can be also added to various foods such as dairy, dairy products, juices, beverages, bakery products, etc. The fatty acid may be provided in the form of triglycerides, phospholipids, and in pure form as ethyl esters (Valenzuela et al., 2006). Today, a wide variety of foods containing DHA are available from the retail and nutraceutical market.

although it remains a topic for further research.

**6.3 DHA and Parkinson´s disease** 

carry out more studies on the subject.

**7. Dietary sources and intake of DHA** 

Neurodegenerative diseases may significantly alter the functioning of the nervous system, reducing both the number and function of neurons, which seriously affects the quality of life for those suffering these diseases. New strategies aiming to the prevention and/or the treatment of these diseases are of high priority. In this context DHA and its derivative NPD-1, emerged as a new perspective for the prevention and/or therapeutic management of these diseases, especially considering the social and economic devastation that neurological diseases may produce to the individual and the family. Future clinical research and nutritional interventions should be planned directly to establish the necessary doses of DHA needed to achieve significant beneficial effects, as well as to encourage the development and consumption of foods and/or supplements rich in this fatty acid. In this regard, the development of functional foods and/or nutraceuticals containing DHA at different concentrations is an alternative that the pharmaceutical and the food industries should consider very seriously (Valenzuela et al., 2009). To day the increase of the consumption of fish or seafood appear as not entirely feasible, due to the massive depredation of the resource, which has decreased its availability and consequently has increased the price of the products from the sea. Perhaps, in the future the increasing activity of the aquaculture may offer a viable alternative to improve the general consumption of n-3 LCPUFA to the western population and helping to prevent the early onset of neurodegenerative diseases.

#### **9. Acknowledgment**

The authors are grateful from FONDECYT and INNOVA-Chile the support of their research.

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**6** 

*Australia* 

**Effect of Zinc and DHA** 

**on Expression Levels and** 

*1NeuroAllergy Research Laboratory (NARL), 1,2,4School of Life and Environmental Sciences, 3School of Medicine, Deakin University,* 

**Post-Translational Modifications of** 

Andrew Sinclair3, Colin J. Barrow4 and Cenk Suphioglu1

Nadia Sadli1, Nayyar Ahmed1, M. Leigh Ackland2,

**Histones H3 and H4 in Human Neuronal Cells** 

Docosahexaenoic acid (DHA) is an important omega-3 fatty acid required for the development of the human central nervous system and the continuous maintenance of neuronal cell function. The DHA composition of the brain decreases with age possibly as a result of increased oxidative damage to the lipid membranes (Schaefer, Bongard et al. 2006). Epidemiological studies have shown that patients with Alzheimer's disease (AD) have significantly lower levels of omega-3 fatty acids in their plasma phospholipids (Moriguchi,

There is an association between DHA levels in the brain and zinc homeostasis, which is particularly interesting as both are involved in neuroprotection. A reduction of DHA levels in the brain causes over expression of ZnT3, a transmembrane proteins that is associated with sequestration of cytoplasmic Zn2+ into synaptic vesicles (Jayasooriya, Ackland et al. 2005), resulting in zinc toxicity and neuronal cell death in cultured neuronal cells (Suphioglu, De Mel et al. 2010a; Naganska and Matyja 2006). Mice that lacked a zinctransporting gene ZnT3 were shown to develop fewer and smaller plaques than Alzheimer's-prone mice with the gene (Lee, Cole et al. 2002), suggesting that altered zinc homeostasis may contribute to the plaque formation in AD. DHA, on the other hand, has neuroprotective properties against neurodegenerative diseases. Dietary supplement of omega-3 fatty acid may protect against Alzheimer's disease, through inhibiting amyloid plague formation (Calon, Lim et al. 2004; Oksman, Iivonen et al. 2006; Florent-Bechard, Malaplate-Armand et al. 2007). DHA was also observed to significantly increase neuronal survival by preventing cytoskeleton perturbations, caspase activation and apoptosis

Our recent data has shown that histone gene and protein expression were affected by both zinc and DHA. The expression levels of histones H3 and H4, in human neuronal cells, were down-regulated by zinc and up-regulated by DHA (Suphioglu, Sadli et al. 2010b), suggesting a possible interaction between the two nutrients. Further investigations into the

**1. Introduction** 

Greiner et al. 2000; Friedland 2003).

(Florent-Bechard, Malaplate-Armand et al. 2007).

Yokoyama, A., Hamazaki, T., Ohshita, A., et al. (2000). Effects of aerosolized docosahexaenoic acid in mouse model of atopic asthma. *Int Arch Allergy Immunol*, Vol 123, pp. 327-332

## **Effect of Zinc and DHA on Expression Levels and Post-Translational Modifications of Histones H3 and H4 in Human Neuronal Cells**

Nadia Sadli1, Nayyar Ahmed1, M. Leigh Ackland2, Andrew Sinclair3, Colin J. Barrow4 and Cenk Suphioglu1 *1NeuroAllergy Research Laboratory (NARL), 1,2,4School of Life and Environmental Sciences, 3School of Medicine, Deakin University, Australia* 

#### **1. Introduction**

140 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

Yokoyama, A., Hamazaki, T., Ohshita, A., et al. (2000). Effects of aerosolized

Vol 123, pp. 327-332

docosahexaenoic acid in mouse model of atopic asthma. *Int Arch Allergy Immunol*,

Docosahexaenoic acid (DHA) is an important omega-3 fatty acid required for the development of the human central nervous system and the continuous maintenance of neuronal cell function. The DHA composition of the brain decreases with age possibly as a result of increased oxidative damage to the lipid membranes (Schaefer, Bongard et al. 2006). Epidemiological studies have shown that patients with Alzheimer's disease (AD) have significantly lower levels of omega-3 fatty acids in their plasma phospholipids (Moriguchi, Greiner et al. 2000; Friedland 2003).

There is an association between DHA levels in the brain and zinc homeostasis, which is particularly interesting as both are involved in neuroprotection. A reduction of DHA levels in the brain causes over expression of ZnT3, a transmembrane proteins that is associated with sequestration of cytoplasmic Zn2+ into synaptic vesicles (Jayasooriya, Ackland et al. 2005), resulting in zinc toxicity and neuronal cell death in cultured neuronal cells (Suphioglu, De Mel et al. 2010a; Naganska and Matyja 2006). Mice that lacked a zinctransporting gene ZnT3 were shown to develop fewer and smaller plaques than Alzheimer's-prone mice with the gene (Lee, Cole et al. 2002), suggesting that altered zinc homeostasis may contribute to the plaque formation in AD. DHA, on the other hand, has neuroprotective properties against neurodegenerative diseases. Dietary supplement of omega-3 fatty acid may protect against Alzheimer's disease, through inhibiting amyloid plague formation (Calon, Lim et al. 2004; Oksman, Iivonen et al. 2006; Florent-Bechard, Malaplate-Armand et al. 2007). DHA was also observed to significantly increase neuronal survival by preventing cytoskeleton perturbations, caspase activation and apoptosis (Florent-Bechard, Malaplate-Armand et al. 2007).

Our recent data has shown that histone gene and protein expression were affected by both zinc and DHA. The expression levels of histones H3 and H4, in human neuronal cells, were down-regulated by zinc and up-regulated by DHA (Suphioglu, Sadli et al. 2010b), suggesting a possible interaction between the two nutrients. Further investigations into the

Effect of Zinc and DHA on Expression Levels and

Letenneur 2004).

**1.2.1 Zinc in the brain** 

(Paik, Joung et al. 1999).

disease progression.

Post-Translational Modifications of Histones H3 and H4 in Human Neuronal Cells 143

(Breteler, van den Ouweland et al. 1992; Launer, Brayne et al. 1992; McDowell 2001). A family history of dementia, gender (women are more likely to develop dementia than men), a head injury in the past (Plassman, Havlik et al. 2000), atherosclerosis, high cholesterol, hypertension, diabetes and high homocysteine levels, excessive alcohol and tobacco consumption, exposure to environmental substances and non-healthy diets are some of the factors likely to increase risk of dementia (Larrieu, Letenneur et al. 2004;

While there are some risk factors that cannot be controlled, such as genetics or age, many risk factors can be managed through lifestyle changes or appropriate dietary intakes. These dietary and lifestyle interventions cannot stop people from developing dementia but they may reduce the risk (Simopoulos 1999; Simopoulos, Leaf et al. 1999; Crawford, Bazinet et al. 2009). The adequate omega-3 fatty acid and zinc intake are examples of dietary factors associated with a substantially reduced risk of neurodegenerative diseases (Simopoulos

Zinc is the second most prevalent trace element in the body and is present in particularly high concentrations in the mammalian brain (Weiss, Sensi et al. 2000), including synaptic vesicles where it is tightly bound to intracellular proteins and zinc finger-containing transcription factors (Frederickson, Hernandez et al. 1989). The concentration of intracellular free zinc in the brain is thought to be very low under physiological conditions (Frederickson, Hernandez et al. 1989; Outten and O'Halloran 2001). However, it can rise to >300 nM in

Zinc plays an important role in growth and development, the immune response, neurological function and reproduction (Stefanidou, Maravelias et al. 2006). Zinc is also a constituent of many enzymes and is essential for the proper function of various enzymes including carbonic anhydrase (Lukaski 2005), RNA polymerase, and superoxide dismutase

The role of zinc in cognitive function has been studied extensively in both children (Sandstead, Penland et al. 1998) and the elderly (Bertoni-Freddari, Mocchegiani et al. 2006). Zinc deficiency during fetal life is associated with developmental delays and low serum zinc levels in elderly is linked with poor global cognitive function (Golub, Keen et al. 1995), particularly verbal function, and also increases stress (Mocchegiani, Bertoni-Freddari et al. 2005; Mocchegiani, Malavolta et al. 2006). Zinc deficiency most often occurs when zinc intake is inadequate or poorly absorbed (Hambidge, Goodall et al. 1989; Golub, Keen et al. 1995; Paik, Joung et al. 1999), when there are increased losses of zinc from the body or when the body's requirement for zinc increases (Hambidge, Goodall et al. 1989). Nonetheless, despite its importance, recent studies have revealed that excess zinc release in the pathological condition is toxic to the central nervous system. Moreover, disruption of zinc homeostasis has been implicated in several neurodegenerative diseases, such as AD (Huang, Cuajungco et al. 2000; Watt and Hooper 2003) where excess extracellular synaptic zinc was found to induce the formation of amyloid plaques, the characteristic feature of AD brains (Linkous, Adlard et al. 2009; Zatta, Drago et al. 2009). These studies suggest the link between an altered neuronal zinc homeostasis and neurodegenerative

1991; Crawford, Bazinet et al. 2009; Devore, Grodstein et al. 2009).

response to injurious stimuli (Canzoniero, Turetsky et al. 1999).

**1.2 The importance of zinc and DHA in neuronal cells** 

effects of zinc and DHA on histone post-translational modifications (PTMs) were carried out. Zinc was found to reduce acetylation, while increasing histone deacetylase (HDACs) expression levels. This is consistent with dysfunctional acetylation-deacetylation apparatus seen in neurodegenerative diseases (Saha and Pahan 2006). DHA, however, showed an increase in acetylation of histones while it reduced the HDACs to the basal level, indicating that zinc and DHA have distinct epigenetic patterns. Both may be involved in neurodegenerative process possibly mediated by histone post-translational modification. Epigenetic mechanisms, including DNA methylation and histone modifications are critically important in mediating precise neural gene regulation. Studies reported that abnormal epigenetic regulation was associated with mental retardation and neurodegenerative symptoms (Al-Gazali, Padmanabhan et al. 2001).

Although it is still unclear that the change in DHA levels, which result in an alteration in zinc homeostasis, could contribute to the beta amyloid formation (Koh 2001) and neuronal cell death, a growing number of studies do in fact indicate this altered molecular interaction between zinc and DHA as a trigger of the pathology (Jomova, Vondrakova et al., 2010; Tougu, Tiiman et al., 2011).

In this chapter, we will summarize the current findings regarding molecular interactions between zinc and DHA that may provide a potential molecular mechanism to explain the beneficial effects of dietary DHA in neuroprotection.

#### **1.1 Neurodegenerative diseases**

As life expectancies are increasing and populations are ageing, neurodegenerative diseases have become a global issue (Nepal, Brown et al. 2008; Nepal, Ranmuthugala et al. 2008). Neurodegenerative diseases such as Alzheimer's disease is the leading cause of dementia in the elderly, which is characterized by molecular changes in nerve cells that result in nerve cell degeneration and ultimately nerve dysfunction and cell death (Dong, Wang et al. 2009). In 1995, Australia had a population of 18 million and 13,000 people were estimated to have dementia. It is predicted that Australia will have 25 million people in 2041, and 460,000 of these will have dementia (Jorm 2001). In other words, while our total population will increase by 40%, our population with dementia will increase by more than three-fold (Jorm 2001).

The expected human lifespan is now longer than ever due to improved hygiene, the discovery of medicines such as antibiotics, and economic welfare. The consequences for this aging population are the increased incidence of age-related diseases. Therefore, treatments to prevent age related neurodegeneration will have economic benefits as well as major impact on the quality of life of the patients (Karasek 2004). A great deal is already known about the pathology of neuronal diseases, but the molecular mechanisms underlying many of these diseases remain unknown. Thus, more research is needed to find the cause and to improve the treatment methods for these significant mental health problems.

#### **1.1.1 Risk factors associated with neurodegeneration**

The most consistent risk factor for developing neurodegenerative disease is aging (Pardon and Rattray 2008; Yankner, Lu et al. 2008; Fratiglioni and Qiu 2009). While it is possible to develop dementia early in life, the chances of developing it increases dramatically as people get older (Rocca, van Duijn et al. 1991). Although AD can strike people in their 30s, 40s, or 50s, the vast majority of cases of AD are diagnosed in people older than 65 (Breteler, van den Ouweland et al. 1992; Launer, Brayne et al. 1992; McDowell 2001). A family history of dementia, gender (women are more likely to develop dementia than men), a head injury in the past (Plassman, Havlik et al. 2000), atherosclerosis, high cholesterol, hypertension, diabetes and high homocysteine levels, excessive alcohol and tobacco consumption, exposure to environmental substances and non-healthy diets are some of the factors likely to increase risk of dementia (Larrieu, Letenneur et al. 2004; Letenneur 2004).

While there are some risk factors that cannot be controlled, such as genetics or age, many risk factors can be managed through lifestyle changes or appropriate dietary intakes. These dietary and lifestyle interventions cannot stop people from developing dementia but they may reduce the risk (Simopoulos 1999; Simopoulos, Leaf et al. 1999; Crawford, Bazinet et al. 2009). The adequate omega-3 fatty acid and zinc intake are examples of dietary factors associated with a substantially reduced risk of neurodegenerative diseases (Simopoulos 1991; Crawford, Bazinet et al. 2009; Devore, Grodstein et al. 2009).

#### **1.2 The importance of zinc and DHA in neuronal cells**

#### **1.2.1 Zinc in the brain**

142 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

effects of zinc and DHA on histone post-translational modifications (PTMs) were carried out. Zinc was found to reduce acetylation, while increasing histone deacetylase (HDACs) expression levels. This is consistent with dysfunctional acetylation-deacetylation apparatus seen in neurodegenerative diseases (Saha and Pahan 2006). DHA, however, showed an increase in acetylation of histones while it reduced the HDACs to the basal level, indicating that zinc and DHA have distinct epigenetic patterns. Both may be involved in neurodegenerative process possibly mediated by histone post-translational modification. Epigenetic mechanisms, including DNA methylation and histone modifications are critically important in mediating precise neural gene regulation. Studies reported that abnormal epigenetic regulation was associated with mental retardation and neurodegenerative

Although it is still unclear that the change in DHA levels, which result in an alteration in zinc homeostasis, could contribute to the beta amyloid formation (Koh 2001) and neuronal cell death, a growing number of studies do in fact indicate this altered molecular interaction between zinc and DHA as a trigger of the pathology (Jomova, Vondrakova et al., 2010;

In this chapter, we will summarize the current findings regarding molecular interactions between zinc and DHA that may provide a potential molecular mechanism to explain the

As life expectancies are increasing and populations are ageing, neurodegenerative diseases have become a global issue (Nepal, Brown et al. 2008; Nepal, Ranmuthugala et al. 2008). Neurodegenerative diseases such as Alzheimer's disease is the leading cause of dementia in the elderly, which is characterized by molecular changes in nerve cells that result in nerve cell degeneration and ultimately nerve dysfunction and cell death (Dong, Wang et al. 2009). In 1995, Australia had a population of 18 million and 13,000 people were estimated to have dementia. It is predicted that Australia will have 25 million people in 2041, and 460,000 of these will have dementia (Jorm 2001). In other words, while our total population will increase by 40%, our population with dementia will increase by more than three-fold (Jorm

The expected human lifespan is now longer than ever due to improved hygiene, the discovery of medicines such as antibiotics, and economic welfare. The consequences for this aging population are the increased incidence of age-related diseases. Therefore, treatments to prevent age related neurodegeneration will have economic benefits as well as major impact on the quality of life of the patients (Karasek 2004). A great deal is already known about the pathology of neuronal diseases, but the molecular mechanisms underlying many of these diseases remain unknown. Thus, more research is needed to find the cause and to

The most consistent risk factor for developing neurodegenerative disease is aging (Pardon and Rattray 2008; Yankner, Lu et al. 2008; Fratiglioni and Qiu 2009). While it is possible to develop dementia early in life, the chances of developing it increases dramatically as people get older (Rocca, van Duijn et al. 1991). Although AD can strike people in their 30s, 40s, or 50s, the vast majority of cases of AD are diagnosed in people older than 65

improve the treatment methods for these significant mental health problems.

**1.1.1 Risk factors associated with neurodegeneration** 

symptoms (Al-Gazali, Padmanabhan et al. 2001).

beneficial effects of dietary DHA in neuroprotection.

Tougu, Tiiman et al., 2011).

2001).

**1.1 Neurodegenerative diseases** 

Zinc is the second most prevalent trace element in the body and is present in particularly high concentrations in the mammalian brain (Weiss, Sensi et al. 2000), including synaptic vesicles where it is tightly bound to intracellular proteins and zinc finger-containing transcription factors (Frederickson, Hernandez et al. 1989). The concentration of intracellular free zinc in the brain is thought to be very low under physiological conditions (Frederickson, Hernandez et al. 1989; Outten and O'Halloran 2001). However, it can rise to >300 nM in response to injurious stimuli (Canzoniero, Turetsky et al. 1999).

Zinc plays an important role in growth and development, the immune response, neurological function and reproduction (Stefanidou, Maravelias et al. 2006). Zinc is also a constituent of many enzymes and is essential for the proper function of various enzymes including carbonic anhydrase (Lukaski 2005), RNA polymerase, and superoxide dismutase (Paik, Joung et al. 1999).

The role of zinc in cognitive function has been studied extensively in both children (Sandstead, Penland et al. 1998) and the elderly (Bertoni-Freddari, Mocchegiani et al. 2006). Zinc deficiency during fetal life is associated with developmental delays and low serum zinc levels in elderly is linked with poor global cognitive function (Golub, Keen et al. 1995), particularly verbal function, and also increases stress (Mocchegiani, Bertoni-Freddari et al. 2005; Mocchegiani, Malavolta et al. 2006). Zinc deficiency most often occurs when zinc intake is inadequate or poorly absorbed (Hambidge, Goodall et al. 1989; Golub, Keen et al. 1995; Paik, Joung et al. 1999), when there are increased losses of zinc from the body or when the body's requirement for zinc increases (Hambidge, Goodall et al. 1989). Nonetheless, despite its importance, recent studies have revealed that excess zinc release in the pathological condition is toxic to the central nervous system. Moreover, disruption of zinc homeostasis has been implicated in several neurodegenerative diseases, such as AD (Huang, Cuajungco et al. 2000; Watt and Hooper 2003) where excess extracellular synaptic zinc was found to induce the formation of amyloid plaques, the characteristic feature of AD brains (Linkous, Adlard et al. 2009; Zatta, Drago et al. 2009). These studies suggest the link between an altered neuronal zinc homeostasis and neurodegenerative disease progression.

Effect of Zinc and DHA on Expression Levels and

(Zhang, Wang et al. 2008).

and neuronal survival.

Alzheimer's disease.

**1.3 Zinc and DHA affect neuronal histone levels** 

Post-Translational Modifications of Histones H3 and H4 in Human Neuronal Cells 145

Zinc homeostasis in the brain is regulated and tightly controlled by Zn transporters, which are divided into two gene families; the ZnT proteins [solute-linked carrier 30 (SLC30)] and the Zip family [solute-linked carrier 39 (SLC39)](Overbeck, Uciechowski et al. 2008; Lichten and Cousins 2009). ZnT and Zip proteins appear to have opposite roles in cellular zinc homeostasis, where ZnT transporters reduce intracellular cytoplasmic zinc by promoting zinc efflux from cells or into intracellular vesicles, while Zip transporters increase intracellular cytoplasmic zinc by promoting extracellular and, perhaps, vesicular zinc transport into cytoplasm (Murakami and Hirano 2008). In M17 cells, we detected the expression of these two zinc transporter families, including Zip1, Zip2, Zip3, Zip4, Znt1, ZnT2, ZnT3, ZnT4, ZnT5, ZnT6 and ZnT7 (Suphioglu, De Mel et al. 2010a). ZnT3 has been the focus of our studies, as it is associated with brain zinc accumulation, as well as Alzheimer's disease, the condition where the expression was found to be up-regulated

Progressive neuronal cell loss is a pathological hallmark of neurodegenerative diseases. Apoptosis or alternative pathways of neuronal death have been discussed in Alzheimer's disease and other disorders (Culmsee and Landshamer 2006). We propose that the alteration of zinc metabolism may play a significant role in cellular apoptosis, which is a key feature in the pathology of neurodegenerative disorders such as Alzheimer's disease (Mattson and Duan 1999). Using western blot analysis of human neuroblastoma M17 cell line, we observed a link between DHA treatment and inhibition of apoptosis, where more than 66% reduction in active caspase-3 protein levels was detected in cells treated with 20µg/ml DHA, compared with the untreated control (Suphioglu, De Mel et al. 2010a). The suppression of activated caspase-3 might be mediated by phosphatidylinositol 3-kinasedependent pathway resulting in the phosphorylation of Akt and DHA may act through this pathway. Akbar et al. (2005) reported the beneficial effect of DHA in neurosurvival through an increase in phosphatidylserine concentration, which resulted in translocation and phosphorylation of Akt suppressing the activation of caspase-3 (Akbar, Calderon et al. 2005). Zinc on the other hand directly activates Akt by phosphorylation at Ser-473/Thr-308 in H1907 embryonic hippocampal cells, leading to activation of GSK-3beta and cell death (Min, Lee et al. 2007). Therefore, we hypothesize that DHA inhibits apoptosis through decreasing intracellular zinc ion concentration, which leads to an increase in Akt activity

In summary, dietary DHA deficiency is associated with neurodegenerative condition, which has shown to be a factor in zinc toxicity. DHA also inhibits cellular apoptosis in M17 cells through decreasing cellular zinc uptake and reduction of ZnT3 mRNA and protein levels. Therefore, zinc homeostasis plays an important role in neuronal cell survival and altered zinc homeostasis may contribute to the development of neurodegenerative diseases such as

The connection between zinc homeostasis and DHA metabolism contributes significantly towards neuronal survival and neurodegenerative diseases. A greater understanding of the fundamental basis by which dietary DHA plays an important role in regulating zinc homeostasis, may lead to the development of effective strategies for the prevention and treatment of neurodegenerative diseases. In recent years, our research has been focusing on the key proteins that are regulated by both zinc and DHA and we have also studied how

#### **1.2.2 Omega-3 fatty acids in the brain**

Docosahexaenoic acid (DHA) is the predominant omega-3 fatty acid in the brain of mammals which comprises up to 15% of the concentration of fatty acids in the nervous system (Calderon and Kim 2004). It is found in the neuronal phospholipids in high concentrations in synapses (Bazan 2003). Epidemiological studies suggest that dietary DHA, which is commonly found in fish (Kalmijn, Launer et al. 1997), may modify the risk for certain neurodegenerative disorders (Hibbeln and Salem 1995). As evidence, decreased blood levels of omega-3 fatty acids have been associated with several neurodegenerative conditions, including Alzheimer's disease, schizophrenia and depression (Fenton, Hibbeln et al. 2000; Young and Conquer 2005). Communities with regular consumption of fish have shown to possess reduced prevalence of neurodegenerative disease and cognitive decline in general (Fenton, Hibbeln et al. 2000; He, Song et al. 2004; van Gelder, Tijhuis et al. 2007).

DHA can be linked with many aspects of neural function, including neurotransmission, membrane fluidity (Lauritzen, Hansen et al. 2001), ion channel (Lai, Wang et al. 2009), enzyme regulation (Strokin, Sergeeva et al. 2003) and gene expression (Qi, Seo et al. 2006). DHA is found in breast milk, and may be required for early visual (Bazan 2009) and brain development in children (Willatts 2002; Simmer and Patole 2004). Furthermore, studies in animal models have provided support for the protective role of omega-3 fatty acid. For example, mice fed on diets high in omega-3 fatty acids were shown to improve in neurological function, such as better regulation of nerve cell membrane excitability (Xiao and Li 1999), increased levels of neurotransmitters and higher density of neurotransmitter membrane receptors (Innis 2000). Hossain et al (1998) found that administration of DHA led to improvement in memory function and reduction in free radical levels while maintaining high level of antioxidant enzyme, suggesting a role of DHA in antioxidant defense (Hossain, Hashimoto et al. 1998). Study by Calon et al (2004) has reported that dietary DHA protects the cells against apoptosis by decreasing caspase activity (Calon, Lim et al. 2004). While others have supported this finding by showing that the enrichment of dietary DHA prevents apoptosis under damaging conditions (Gomez de Segura, Valderrabano et al. 2004). DHA also increases phosphotidylserine levels (PS) in neuronal membrane, which result in Akt translocation (Akbar, Calderon et al. 2005) and contributes to survival signaling by suppression of caspase-3 (Akbar, Baick et al. 2006).

#### **1.2.3 Molecular link between DHA and zinc in neuronal cells: DHA decreases neuronal cell death in association with altered zinc transport**

The alteration in both DHA and zinc homeostasis are key features of neurodegenerative disorders (Lukiw, Cui et al. 2005) (Cuajungco and Lees 1997). A study by Jayasooriya et al (2005) has demonstrated the link between an altered zinc homeostasis in the brain of rats fed on an omega 3-deficient diet (Jayasooriya, Ackland et al. 2005); this diet lead to a significant decrease in brain DHA levels. Though these data have shown a relationship between DHA and zinc homeostasis, the basis of a molecular mechanism has not been elucidated. We therefore used this fundamental idea to investigate the molecular mechanisms underlying the zinc and DHA interaction. With the use of human neuroblastoma cell line M17, we have shown that DHA reduces cellular zinc uptake, possibly mediated by the zinc transporter ZnT3 followed by a significant reduction in pro-apoptotic marker, caspase-3 (Suphioglu, De Mel et al. 2010a). This indicates the effect of DHA deficiency in the progression of neurodegenerative disease, which is partly mediated by altered zinc fluxes.

Docosahexaenoic acid (DHA) is the predominant omega-3 fatty acid in the brain of mammals which comprises up to 15% of the concentration of fatty acids in the nervous system (Calderon and Kim 2004). It is found in the neuronal phospholipids in high concentrations in synapses (Bazan 2003). Epidemiological studies suggest that dietary DHA, which is commonly found in fish (Kalmijn, Launer et al. 1997), may modify the risk for certain neurodegenerative disorders (Hibbeln and Salem 1995). As evidence, decreased blood levels of omega-3 fatty acids have been associated with several neurodegenerative conditions, including Alzheimer's disease, schizophrenia and depression (Fenton, Hibbeln et al. 2000; Young and Conquer 2005). Communities with regular consumption of fish have shown to possess reduced prevalence of neurodegenerative disease and cognitive decline in general (Fenton, Hibbeln et al. 2000; He, Song et al. 2004; van Gelder, Tijhuis et al. 2007). DHA can be linked with many aspects of neural function, including neurotransmission, membrane fluidity (Lauritzen, Hansen et al. 2001), ion channel (Lai, Wang et al. 2009), enzyme regulation (Strokin, Sergeeva et al. 2003) and gene expression (Qi, Seo et al. 2006). DHA is found in breast milk, and may be required for early visual (Bazan 2009) and brain development in children (Willatts 2002; Simmer and Patole 2004). Furthermore, studies in animal models have provided support for the protective role of omega-3 fatty acid. For example, mice fed on diets high in omega-3 fatty acids were shown to improve in neurological function, such as better regulation of nerve cell membrane excitability (Xiao and Li 1999), increased levels of neurotransmitters and higher density of neurotransmitter membrane receptors (Innis 2000). Hossain et al (1998) found that administration of DHA led to improvement in memory function and reduction in free radical levels while maintaining high level of antioxidant enzyme, suggesting a role of DHA in antioxidant defense (Hossain, Hashimoto et al. 1998). Study by Calon et al (2004) has reported that dietary DHA protects the cells against apoptosis by decreasing caspase activity (Calon, Lim et al. 2004). While others have supported this finding by showing that the enrichment of dietary DHA prevents apoptosis under damaging conditions (Gomez de Segura, Valderrabano et al. 2004). DHA also increases phosphotidylserine levels (PS) in neuronal membrane, which result in Akt translocation (Akbar, Calderon et al. 2005) and contributes to survival signaling by

**1.2.3 Molecular link between DHA and zinc in neuronal cells: DHA decreases neuronal** 

The alteration in both DHA and zinc homeostasis are key features of neurodegenerative disorders (Lukiw, Cui et al. 2005) (Cuajungco and Lees 1997). A study by Jayasooriya et al (2005) has demonstrated the link between an altered zinc homeostasis in the brain of rats fed on an omega 3-deficient diet (Jayasooriya, Ackland et al. 2005); this diet lead to a significant decrease in brain DHA levels. Though these data have shown a relationship between DHA and zinc homeostasis, the basis of a molecular mechanism has not been elucidated. We therefore used this fundamental idea to investigate the molecular mechanisms underlying the zinc and DHA interaction. With the use of human neuroblastoma cell line M17, we have shown that DHA reduces cellular zinc uptake, possibly mediated by the zinc transporter ZnT3 followed by a significant reduction in pro-apoptotic marker, caspase-3 (Suphioglu, De Mel et al. 2010a). This indicates the effect of DHA deficiency in the progression of

neurodegenerative disease, which is partly mediated by altered zinc fluxes.

**1.2.2 Omega-3 fatty acids in the brain** 

suppression of caspase-3 (Akbar, Baick et al. 2006).

**cell death in association with altered zinc transport** 

Zinc homeostasis in the brain is regulated and tightly controlled by Zn transporters, which are divided into two gene families; the ZnT proteins [solute-linked carrier 30 (SLC30)] and the Zip family [solute-linked carrier 39 (SLC39)](Overbeck, Uciechowski et al. 2008; Lichten and Cousins 2009). ZnT and Zip proteins appear to have opposite roles in cellular zinc homeostasis, where ZnT transporters reduce intracellular cytoplasmic zinc by promoting zinc efflux from cells or into intracellular vesicles, while Zip transporters increase intracellular cytoplasmic zinc by promoting extracellular and, perhaps, vesicular zinc transport into cytoplasm (Murakami and Hirano 2008). In M17 cells, we detected the expression of these two zinc transporter families, including Zip1, Zip2, Zip3, Zip4, Znt1, ZnT2, ZnT3, ZnT4, ZnT5, ZnT6 and ZnT7 (Suphioglu, De Mel et al. 2010a). ZnT3 has been the focus of our studies, as it is associated with brain zinc accumulation, as well as Alzheimer's disease, the condition where the expression was found to be up-regulated (Zhang, Wang et al. 2008).

Progressive neuronal cell loss is a pathological hallmark of neurodegenerative diseases. Apoptosis or alternative pathways of neuronal death have been discussed in Alzheimer's disease and other disorders (Culmsee and Landshamer 2006). We propose that the alteration of zinc metabolism may play a significant role in cellular apoptosis, which is a key feature in the pathology of neurodegenerative disorders such as Alzheimer's disease (Mattson and Duan 1999). Using western blot analysis of human neuroblastoma M17 cell line, we observed a link between DHA treatment and inhibition of apoptosis, where more than 66% reduction in active caspase-3 protein levels was detected in cells treated with 20µg/ml DHA, compared with the untreated control (Suphioglu, De Mel et al. 2010a). The suppression of activated caspase-3 might be mediated by phosphatidylinositol 3-kinasedependent pathway resulting in the phosphorylation of Akt and DHA may act through this pathway. Akbar et al. (2005) reported the beneficial effect of DHA in neurosurvival through an increase in phosphatidylserine concentration, which resulted in translocation and phosphorylation of Akt suppressing the activation of caspase-3 (Akbar, Calderon et al. 2005). Zinc on the other hand directly activates Akt by phosphorylation at Ser-473/Thr-308 in H1907 embryonic hippocampal cells, leading to activation of GSK-3beta and cell death (Min, Lee et al. 2007). Therefore, we hypothesize that DHA inhibits apoptosis through decreasing intracellular zinc ion concentration, which leads to an increase in Akt activity and neuronal survival.

In summary, dietary DHA deficiency is associated with neurodegenerative condition, which has shown to be a factor in zinc toxicity. DHA also inhibits cellular apoptosis in M17 cells through decreasing cellular zinc uptake and reduction of ZnT3 mRNA and protein levels. Therefore, zinc homeostasis plays an important role in neuronal cell survival and altered zinc homeostasis may contribute to the development of neurodegenerative diseases such as Alzheimer's disease.

#### **1.3 Zinc and DHA affect neuronal histone levels**

The connection between zinc homeostasis and DHA metabolism contributes significantly towards neuronal survival and neurodegenerative diseases. A greater understanding of the fundamental basis by which dietary DHA plays an important role in regulating zinc homeostasis, may lead to the development of effective strategies for the prevention and treatment of neurodegenerative diseases. In recent years, our research has been focusing on the key proteins that are regulated by both zinc and DHA and we have also studied how

Effect of Zinc and DHA on Expression Levels and

chromatin structure (Davie and Chadee 1998).

the expression of many other genes.

maintenance (Bernstein and Allis 2005).

cause the death of the cells.

Post-Translational Modifications of Histones H3 and H4 in Human Neuronal Cells 147

superhelix into the area surrounding the nucleosome (Luger, Rechsteiner et al. 1997). In contrast to the structural core histone proteins, histone H1 is associated into linker DNA, which connects the nucleosomes together, resulting in the formation of "beads-on-a-string"

**1.3.2 Possible mechanism of the effect of zinc and DHA on H3 and H4 expression**  We observed a significant reduction in both mRNA and protein levels of histones H3 and H4 following zinc treatment suggesting that zinc may inhibit the transcription of histones H3 and H4 in M17 human neuronal cells (Suphioglu, Sadli et al. 2010b). Histones H3 and H4 possess multiple metal response elements upstream of their start codon, which indicates the possible involvement of zinc in their transcription. Previous studies showed that inhibition of DNA synthesis triggers a concerted repression of histone synthesis, indicating that sustained histone synthesis depends on continued DNA synthesis. We proposed that the termination of H3 and H4 synthesis may possibly be caused by the effect of zinc in inhibiting DNA synthesis (Suphioglu, Sadli et al. 2010b). Conversely, DHA was found to upregulate H3 and H4 expression levels and abolished the effect of zinc, suggesting the potential contribution of DHA in increasing DNA synthesis, which result in the increase of histone protein levels. Our results are supported by previous studies, by which zinc regulates a variety of transcription and translation related factors, including the H3 histone family 3A protein (Barcelo-Coblijn, Hogyes et al. 2003). Since there's association between alteration in histone subunit expression and DNA replication, the condition may then alter

From this study, we propose that DHA may contribute positively to minimizing the onset of neurodegenerative disease through maintaining the integrity of DNA and histones H3 and H4 synthesis. The inhibition of DNA synthesis, which subsequently lead to the loss of neurons, however, is a pathological process of neurodegenerative disorders and potentially

In addition to nucleosome assembly, studies have found that histones are potentially important carriers of epigenetic information. They, therefore, play significant role in regulating gene activities, such as DNA damage repair, replication and transcription through post-translational modifications (PTMs) (Hasan and Hottiger 2002). Core histones are characterized by the presence of fold domain (Alva, Ammelburg et al. 2007) and *N*terminal tails which are exposed to nucleosomal surface (Ausio, Dong et al. 1989). These *N*terminal tails of core histones are subjected to extensive post-translational modifications in many cellular processes (Ausio, Dong et al. 1989), which play pivotal roles in the epigenetic control of chromatin structure necessary for DNA accessibility during gene expression (Iizuka and Smith 2003). Some PTMs, including acetylation and phosphorylation, are reversible and are often associated with increase in gene expression. Other PTMs, such as lysine methylation, are often found to be more stable and participate in long term epigenetic

One of the best-studied post-translational modifications is the acetylation of lysine residues, which is a reversible process that is catalyzed by either histone acetyltransferases (HATs) or histone deacetylases (HDACs). The main acetylation sites in histone H3 of most species are at lysine 9, -14, -18 and -23 (Thorne, Kmiciek et al. 1990). Histone acetylation is a hallmark of

**1.4 Histone post-translational modifications (PTMs) and gene activities** 

zinc and DHA, alone and in combination might affect the expression levels of these novel proteins.

Two-dimensional gel electrophoresis and mass spectrometry were applied to identify the major protein changes in the protein lysates of M17 human neuronal cells that had been grown in the presence and absence of zinc and DHA. Four protein spots were selected for mass spectrometry analysis to reveal their identity as human histone variants H3 and H4. In order to investigate the change in the expression levels of the histones, proteomic findings were further investigated using western blot and real–time PCR analyses. Our results have revealed the differential expression of histones, particularly histone H3 and H4 in response to DHA and zinc supplementation (Suphioglu, Sadli et al. 2010b). In this study, we reported for the first time that both H3 and H4 were significantly downregulated by zinc in the absence of DHA (zinc effect) and up-regulated following DHA treatment at the physiological zinc level (DHA effect), suggesting the interrelationship between zinc and DHA in neuroprotection, which is mediated by histones (Suphioglu, Sadli et al. 2010b).

#### **1.3.1 Histones**

Histones are a group of conserved, highly basic proteins that are rich in lysine (K) and arginine (R) (Kinkade and Cole 1966; DeLange and Smith 1971; Elgin and Weintraub 1975; Munishkina, Fink et al. 2004) (Table 1). Histones are the nuclear protein that are involved in the assembly of chromatin through electrostatic interaction between the highly negatively charged polymeric DNA and the positively charged histones, which play a determining role in stabilizing the nucleosomes at physiological conditions (Korolev, Lyubartsev et al. 2004). About 85% of the DNA in chromatin is represented by uniform units, the nucleosomes, which are the complexes of DNA double helix with five histone proteins (H2A, H2B, H3, H4, and H1) (Luger, Rechsteiner et al. 1997). The central part of the nucleosome is called the nucleosome core particle and consists of 147 bp DNA wrapped around the histone octamer, which is formed from one (H3/H4)2 tetramer and two H2A/H2B dimers (Luger, Rechsteiner et al. 1997; Woodcock and Dimitrov 2001). The four core histones have similar isoelectric points (pI) and share a common structural motif called the histone fold, which facilitates the interactions between the individual core histones (Arents and Moudrianakis 1995). Flanking the core domains are the relatively unstructured *N*-terminal tail domains. The histone tails extend out from the face of the nucleosome and through the gyres of DNA


Table 1. Characteristics of histones. Molecular weight (MW) is given in Daltons (Da) and isoelectric points (pI) are shown

zinc and DHA, alone and in combination might affect the expression levels of these novel

Two-dimensional gel electrophoresis and mass spectrometry were applied to identify the major protein changes in the protein lysates of M17 human neuronal cells that had been grown in the presence and absence of zinc and DHA. Four protein spots were selected for mass spectrometry analysis to reveal their identity as human histone variants H3 and H4. In order to investigate the change in the expression levels of the histones, proteomic findings were further investigated using western blot and real–time PCR analyses. Our results have revealed the differential expression of histones, particularly histone H3 and H4 in response to DHA and zinc supplementation (Suphioglu, Sadli et al. 2010b). In this study, we reported for the first time that both H3 and H4 were significantly downregulated by zinc in the absence of DHA (zinc effect) and up-regulated following DHA treatment at the physiological zinc level (DHA effect), suggesting the interrelationship between zinc and DHA in neuroprotection, which is mediated by histones (Suphioglu,

Histones are a group of conserved, highly basic proteins that are rich in lysine (K) and arginine (R) (Kinkade and Cole 1966; DeLange and Smith 1971; Elgin and Weintraub 1975; Munishkina, Fink et al. 2004) (Table 1). Histones are the nuclear protein that are involved in the assembly of chromatin through electrostatic interaction between the highly negatively charged polymeric DNA and the positively charged histones, which play a determining role in stabilizing the nucleosomes at physiological conditions (Korolev, Lyubartsev et al. 2004). About 85% of the DNA in chromatin is represented by uniform units, the nucleosomes, which are the complexes of DNA double helix with five histone proteins (H2A, H2B, H3, H4, and H1) (Luger, Rechsteiner et al. 1997). The central part of the nucleosome is called the nucleosome core particle and consists of 147 bp DNA wrapped around the histone octamer, which is formed from one (H3/H4)2 tetramer and two H2A/H2B dimers (Luger, Rechsteiner et al. 1997; Woodcock and Dimitrov 2001). The four core histones have similar isoelectric points (pI) and share a common structural motif called the histone fold, which facilitates the interactions between the individual core histones (Arents and Moudrianakis 1995). Flanking the core domains are the relatively unstructured *N*-terminal tail domains. The histone tails extend out from the face of the nucleosome and through the gyres of DNA

M.W. (Da) Sequence

length

Isoelectric point (pI)

proteins.

Sadli et al. 2010b).

Histone type Class (amino acid

isoelectric points (pI) are shown

distribution)

H1 Very lysine rich ~ 21,500 ~215

H2A Lysine rich 14,004 129 10.9 H2B Lysine rich 13,774 125 10.3 H3 Arginine rich 15,324 135 11.1 H4 Arginine rich 11,282 102 11.4

Table 1. Characteristics of histones. Molecular weight (MW) is given in Daltons (Da) and

**1.3.1 Histones** 

superhelix into the area surrounding the nucleosome (Luger, Rechsteiner et al. 1997). In contrast to the structural core histone proteins, histone H1 is associated into linker DNA, which connects the nucleosomes together, resulting in the formation of "beads-on-a-string" chromatin structure (Davie and Chadee 1998).

#### **1.3.2 Possible mechanism of the effect of zinc and DHA on H3 and H4 expression**

We observed a significant reduction in both mRNA and protein levels of histones H3 and H4 following zinc treatment suggesting that zinc may inhibit the transcription of histones H3 and H4 in M17 human neuronal cells (Suphioglu, Sadli et al. 2010b). Histones H3 and H4 possess multiple metal response elements upstream of their start codon, which indicates the possible involvement of zinc in their transcription. Previous studies showed that inhibition of DNA synthesis triggers a concerted repression of histone synthesis, indicating that sustained histone synthesis depends on continued DNA synthesis. We proposed that the termination of H3 and H4 synthesis may possibly be caused by the effect of zinc in inhibiting DNA synthesis (Suphioglu, Sadli et al. 2010b). Conversely, DHA was found to upregulate H3 and H4 expression levels and abolished the effect of zinc, suggesting the potential contribution of DHA in increasing DNA synthesis, which result in the increase of histone protein levels. Our results are supported by previous studies, by which zinc regulates a variety of transcription and translation related factors, including the H3 histone family 3A protein (Barcelo-Coblijn, Hogyes et al. 2003). Since there's association between alteration in histone subunit expression and DNA replication, the condition may then alter the expression of many other genes.

From this study, we propose that DHA may contribute positively to minimizing the onset of neurodegenerative disease through maintaining the integrity of DNA and histones H3 and H4 synthesis. The inhibition of DNA synthesis, which subsequently lead to the loss of neurons, however, is a pathological process of neurodegenerative disorders and potentially cause the death of the cells.

#### **1.4 Histone post-translational modifications (PTMs) and gene activities**

In addition to nucleosome assembly, studies have found that histones are potentially important carriers of epigenetic information. They, therefore, play significant role in regulating gene activities, such as DNA damage repair, replication and transcription through post-translational modifications (PTMs) (Hasan and Hottiger 2002). Core histones are characterized by the presence of fold domain (Alva, Ammelburg et al. 2007) and *N*terminal tails which are exposed to nucleosomal surface (Ausio, Dong et al. 1989). These *N*terminal tails of core histones are subjected to extensive post-translational modifications in many cellular processes (Ausio, Dong et al. 1989), which play pivotal roles in the epigenetic control of chromatin structure necessary for DNA accessibility during gene expression (Iizuka and Smith 2003). Some PTMs, including acetylation and phosphorylation, are reversible and are often associated with increase in gene expression. Other PTMs, such as lysine methylation, are often found to be more stable and participate in long term epigenetic maintenance (Bernstein and Allis 2005).

One of the best-studied post-translational modifications is the acetylation of lysine residues, which is a reversible process that is catalyzed by either histone acetyltransferases (HATs) or histone deacetylases (HDACs). The main acetylation sites in histone H3 of most species are at lysine 9, -14, -18 and -23 (Thorne, Kmiciek et al. 1990). Histone acetylation is a hallmark of

Effect of Zinc and DHA on Expression Levels and

et al. 2005).

disease.

Sananbenesi et al. 2007).

Post-Translational Modifications of Histones H3 and H4 in Human Neuronal Cells 149

2005). Studies have reported the involvement of H3 phosphorylation in transcriptional activation (Mizzen, Kuo et al. 1998; Clayton, Rose et al. 2000; Nowak and Corces 2000), chromatin fiber decondensation, and chromosomes compaction during cell division (Hendzel, Wei et al. 1997; Hsu, Sun et al. 2000). Histone H3 is phosphorylated during both mitosis and meiosis and initiated at different phase of the cell division in different organisms (Hans and Dimitrov 2001). The phosphorylation of Thr(T)3 of histone H3, which is catalyzed by kinase haspin occurs during mitosis and it plays an essential role in facilitating condensation and resolution of sister chromatids in the late G2 and prophase (Dai, Sultan et al. 2005). To ensure this orderly cell cycle progression, the regulation of chromatin structure and spindle activity must be precisely integrated. The inappropriate H3(T3) phosphorylation causes defects in chromatin structure which might hinder chromosome alignment in mitosis (Enomoto, Koyamazaki et al. 2001), leading to genomic instability (Dai, Sultan et al. 2005). Threonine-3 residue is found in histone H3 of all eukaryotes, suggesting a highly conserved and critical function for this residue (Dai, Sultan

**1.4.1 Histone post-translational modifications (PTMs) and neurodegenerative disease**  As previously discussed, histone post-translational modifications (PTMs) play significant role in regulating gene activities. Therefore, aberrant pattern of epigenetic regulation has been linked to the development of neurodegenerative diseases such as Alzheimer's

During normal neuronal condition, HATs and HDACs remain in a state of balance, which they counteract each other to ensure neurophysiological homeostasis. Such equilibrium (Figure 1A) is responsible for regulating gene expression leading to normal function of neuronal cell activity and memory formation (Saha and Pahan 2006). During neurodegenerative diseases, the acetylation homeostasis is altered when histone acetylation significantly decreases (Rouaux, Jokic et al. 2003), reflecting dysfunctional acetylation-deacetylation apparatus (Figure 1B). General loss of acetylating agent would cause excessive increase in HDAC activity, which is then associated with transcriptional repression (Figure 1B). Studies have reported that reduction in histone acetylation followed by the increase in HDAC activity or DNA methylation is common in many neurodegenerative and neuropsychiatric disorders (Faraco, Pancani et al. 2006; Fischer,

In recent years, the increasing numbers of structurally diverse HDAC inhibitors have been identified with the potential to target specific brain regions and in cell-specific manner to reverse disorder-specific epigenetic dysregulation (Abel and Zukin 2008). The HDAC inhibitors include: short-chain fatty acid (i.e. valproic acid) (Kothari, Joshi et al. 2009), hydroxamic acid (i.e. SAHA, TSA, oxamflatin) (Archin, Espeseth et al. 2009), cyclic tetrapeptides (i.e trapoxin, apicidin) (Masuoka, Shindoh et al. 2008) and benzamide (i.e MS-275) (Gahr, Peter et al. 2008). These HDACs inhibitors are aimed to inhibit its enzymatic activity and to remove the repressive blocks from promoters of essential genes and therefore induce active gene transcription (Saha and Pahan 2006). The X-ray crystallographic studies showed that this type of HDAC inhibitor act as a chelator of zinc ion in the catalytic site of HDACs which therefore block the substrate access to the active zinc ion and subsequently inhibit the deacetylation ativity (Ficner 2009). However, it is still uncertain whether certain

neurodegenerative disorders are mediated by a specific HDAC.

transcriptionally active regions, whereas hypoacetylated histones are associated with tightly compacted nucleosomes, resulting in transcriptional repression due to restricted access of transcriptional factors to their targeted DNA (Oliva, Bazett-Jones et al. 1990). The addition of an acetyl group by a member of HAT family create appropriate 'histone code' for chromatin modification and decrease the interaction between the negatively charged DNA backbone and the positively charged histone tail enhancing DNA accessibility to transcription factors (TFs), which therefore increase gene transcription. Conversely, HDAC removes the acetyl group and potentially leads to general repression of gene transcription (Dangond, Henriksson et al. 2001).

So far, in humans, 18 HDACs enzymes have been identified on the basis of similarity to yeast counterparts and classified based on sequence identity and domain organization as well as cofactor dependency (Heltweg, Dequiedt et al. 2003). The classic HDACs (Class I, II and IV) require Zn2+ for their activity, whereas the sir2-related HDACs (sirtuins) require (nicotinamide adenine dinucleotide) NAD+ as cofactor (Koyama, Adachi et al. 2000). Class I HDACs (HDAC1, 2, 3 and 8), which are homologs of the yeast histone deacetylase RPD3, are found primarily in the nucleus of most cell lines and tissue types (Fischle, Emiliani et al. 1999; Fischle, Kiermer et al. 2001), whereas Class II HDACs (HDAC 4, 5, 6, 7 9 and 10) share a significant degree of homology with the yeast Hda1 and are able to shuttle in and out of the nucleus depending on different signals (Fischle, Emiliani et al. 1999; Fischle, Kiermer et al. 2001), suggesting a potential extranuclear functions by regulating the acetylation status of nonhistone substrates (Grozinger, Hassig et al. 1999; Heltweg, Dequiedt et al. 2003). Class III HDACs are composed of the Sirtuins (SIRT) proteins 1–7 and require NAD+ for deacetylase activity in contrast to the zinc-catalyzed mechanism used by class I and II HDACs (Koyama, Adachi et al. 2000; Lo, Trievel et al. 2000; Blander and Guarente 2004). The most recently described HDACs are Class IV, which is represented by HDAC11. This enzyme is phylogenetically different from class I and II HDACs and is therefore classified separately (Gao, Cueto et al. 2002). So far, very little is known about its function and regulation (Yang and Seto 2008).

In addition to acetylation, important progress has also been made in the studies of other types of covalent modifications including methylation and phosphorylation of histones H3. It has long been known that histone H3 is methylated at a number of lysine (Lys) and arginine (Arg) residues. The major sites of Lys-methylation on histones identified so far are: Lys4, Lys9, Lys27, Lys36, Lys79 and arginine methylation takes place at R2, R17 and R26 (Sims, Nishioka et al. 2003; Lee, Teyssier et al. 2005). The addition of methyl-group on histone tail residues is catalyzed by histone methyltransferases (HMTs). These enzymes can catalyze mono-, di-, or trimethylation on lysine residues and this differential methylation provides further functional diversity to each site of lysine methylation. Similar to histone acetylation, histone methylation can also modulate histone interaction with DNA, which result in an alteration of nucleosome structures and functions and therefore contribute to different cellular process (Rice and Allis 2001). The specific methylation of histone tails such as H3(K4), H3(K36), and H3(K79) have been associated with active transcriptional activity (Strahl, Ohba et al. 1999; Berger 2007), whereas methylation of H3(K9), H3(K27) and H4(K20) have been correlated with gene silencing (Lee, Teyssier et al. 2005).

Histone phosphorylation have also been shown to occur on all histones, and are located within the highly conserved amino acid residues alanine, arginine, lysine and serine (Clayton and Mahadevan 2003). For histone H3, phosphorylation takes place specifically at Thr3, -11 and at Ser10, -28 (Hendzel, Wei et al. 1997; Hsu, Sun et al. 2000; Dai, Sultan et al.

transcriptionally active regions, whereas hypoacetylated histones are associated with tightly compacted nucleosomes, resulting in transcriptional repression due to restricted access of transcriptional factors to their targeted DNA (Oliva, Bazett-Jones et al. 1990). The addition of an acetyl group by a member of HAT family create appropriate 'histone code' for chromatin modification and decrease the interaction between the negatively charged DNA backbone and the positively charged histone tail enhancing DNA accessibility to transcription factors (TFs), which therefore increase gene transcription. Conversely, HDAC removes the acetyl group and potentially leads to general repression of gene transcription (Dangond,

So far, in humans, 18 HDACs enzymes have been identified on the basis of similarity to yeast counterparts and classified based on sequence identity and domain organization as well as cofactor dependency (Heltweg, Dequiedt et al. 2003). The classic HDACs (Class I, II and IV) require Zn2+ for their activity, whereas the sir2-related HDACs (sirtuins) require (nicotinamide adenine dinucleotide) NAD+ as cofactor (Koyama, Adachi et al. 2000). Class I HDACs (HDAC1, 2, 3 and 8), which are homologs of the yeast histone deacetylase RPD3, are found primarily in the nucleus of most cell lines and tissue types (Fischle, Emiliani et al. 1999; Fischle, Kiermer et al. 2001), whereas Class II HDACs (HDAC 4, 5, 6, 7 9 and 10) share a significant degree of homology with the yeast Hda1 and are able to shuttle in and out of the nucleus depending on different signals (Fischle, Emiliani et al. 1999; Fischle, Kiermer et al. 2001), suggesting a potential extranuclear functions by regulating the acetylation status of nonhistone substrates (Grozinger, Hassig et al. 1999; Heltweg, Dequiedt et al. 2003). Class III HDACs are composed of the Sirtuins (SIRT) proteins 1–7 and require NAD+ for deacetylase activity in contrast to the zinc-catalyzed mechanism used by class I and II HDACs (Koyama, Adachi et al. 2000; Lo, Trievel et al. 2000; Blander and Guarente 2004). The most recently described HDACs are Class IV, which is represented by HDAC11. This enzyme is phylogenetically different from class I and II HDACs and is therefore classified separately (Gao, Cueto et al. 2002). So far, very little is known about its function and regulation (Yang

In addition to acetylation, important progress has also been made in the studies of other types of covalent modifications including methylation and phosphorylation of histones H3. It has long been known that histone H3 is methylated at a number of lysine (Lys) and arginine (Arg) residues. The major sites of Lys-methylation on histones identified so far are: Lys4, Lys9, Lys27, Lys36, Lys79 and arginine methylation takes place at R2, R17 and R26 (Sims, Nishioka et al. 2003; Lee, Teyssier et al. 2005). The addition of methyl-group on histone tail residues is catalyzed by histone methyltransferases (HMTs). These enzymes can catalyze mono-, di-, or trimethylation on lysine residues and this differential methylation provides further functional diversity to each site of lysine methylation. Similar to histone acetylation, histone methylation can also modulate histone interaction with DNA, which result in an alteration of nucleosome structures and functions and therefore contribute to different cellular process (Rice and Allis 2001). The specific methylation of histone tails such as H3(K4), H3(K36), and H3(K79) have been associated with active transcriptional activity (Strahl, Ohba et al. 1999; Berger 2007), whereas methylation of H3(K9), H3(K27) and

Histone phosphorylation have also been shown to occur on all histones, and are located within the highly conserved amino acid residues alanine, arginine, lysine and serine (Clayton and Mahadevan 2003). For histone H3, phosphorylation takes place specifically at Thr3, -11 and at Ser10, -28 (Hendzel, Wei et al. 1997; Hsu, Sun et al. 2000; Dai, Sultan et al.

H4(K20) have been correlated with gene silencing (Lee, Teyssier et al. 2005).

Henriksson et al. 2001).

and Seto 2008).

2005). Studies have reported the involvement of H3 phosphorylation in transcriptional activation (Mizzen, Kuo et al. 1998; Clayton, Rose et al. 2000; Nowak and Corces 2000), chromatin fiber decondensation, and chromosomes compaction during cell division (Hendzel, Wei et al. 1997; Hsu, Sun et al. 2000). Histone H3 is phosphorylated during both mitosis and meiosis and initiated at different phase of the cell division in different organisms (Hans and Dimitrov 2001). The phosphorylation of Thr(T)3 of histone H3, which is catalyzed by kinase haspin occurs during mitosis and it plays an essential role in facilitating condensation and resolution of sister chromatids in the late G2 and prophase (Dai, Sultan et al. 2005). To ensure this orderly cell cycle progression, the regulation of chromatin structure and spindle activity must be precisely integrated. The inappropriate H3(T3) phosphorylation causes defects in chromatin structure which might hinder chromosome alignment in mitosis (Enomoto, Koyamazaki et al. 2001), leading to genomic instability (Dai, Sultan et al. 2005). Threonine-3 residue is found in histone H3 of all eukaryotes, suggesting a highly conserved and critical function for this residue (Dai, Sultan et al. 2005).

#### **1.4.1 Histone post-translational modifications (PTMs) and neurodegenerative disease**

As previously discussed, histone post-translational modifications (PTMs) play significant role in regulating gene activities. Therefore, aberrant pattern of epigenetic regulation has been linked to the development of neurodegenerative diseases such as Alzheimer's disease.

During normal neuronal condition, HATs and HDACs remain in a state of balance, which they counteract each other to ensure neurophysiological homeostasis. Such equilibrium (Figure 1A) is responsible for regulating gene expression leading to normal function of neuronal cell activity and memory formation (Saha and Pahan 2006). During neurodegenerative diseases, the acetylation homeostasis is altered when histone acetylation significantly decreases (Rouaux, Jokic et al. 2003), reflecting dysfunctional acetylation-deacetylation apparatus (Figure 1B). General loss of acetylating agent would cause excessive increase in HDAC activity, which is then associated with transcriptional repression (Figure 1B). Studies have reported that reduction in histone acetylation followed by the increase in HDAC activity or DNA methylation is common in many neurodegenerative and neuropsychiatric disorders (Faraco, Pancani et al. 2006; Fischer, Sananbenesi et al. 2007).

In recent years, the increasing numbers of structurally diverse HDAC inhibitors have been identified with the potential to target specific brain regions and in cell-specific manner to reverse disorder-specific epigenetic dysregulation (Abel and Zukin 2008). The HDAC inhibitors include: short-chain fatty acid (i.e. valproic acid) (Kothari, Joshi et al. 2009), hydroxamic acid (i.e. SAHA, TSA, oxamflatin) (Archin, Espeseth et al. 2009), cyclic tetrapeptides (i.e trapoxin, apicidin) (Masuoka, Shindoh et al. 2008) and benzamide (i.e MS-275) (Gahr, Peter et al. 2008). These HDACs inhibitors are aimed to inhibit its enzymatic activity and to remove the repressive blocks from promoters of essential genes and therefore induce active gene transcription (Saha and Pahan 2006). The X-ray crystallographic studies showed that this type of HDAC inhibitor act as a chelator of zinc ion in the catalytic site of HDACs which therefore block the substrate access to the active zinc ion and subsequently inhibit the deacetylation ativity (Ficner 2009). However, it is still uncertain whether certain neurodegenerative disorders are mediated by a specific HDAC.

Effect of Zinc and DHA on Expression Levels and

in particular histone H3 in human neuronal cells.

expression of death-inducing genes (Saha and Pahan 2006).

therefore increasing the activity of histone deacetylation.

(Marks, Richon et al. 2000; Ficner 2009).

**1.5.2 Effect of zinc and DHA on histone deacetylases (HDACs) 1, 2, 3** 

genes (Saha and Pahan 2006).

Post-Translational Modifications of Histones H3 and H4 in Human Neuronal Cells 151

Proper regulation of gene expression in the nervous system is not only controlled by the transcriptional machinery but is also subject to modulation by epigenetic mechanisms such as histone modifications. Following our histone expression study, for the first time, we have also investigated the effect of zinc and DHA on post-translational modifications of histones,

One-dimensional electrophoresis and western immunoblot analysis were used to investigate the change in post-translational modified histones of human neuronal cells that had been grown in the presence and absence of zinc and DHA. Our results showed that zinc decreased acetylation of H3(K9), whereas DHA increase H3(K9) acetylation. This suggests the potential involvement of zinc in the progress of neurodegenerative disease through an altered acetylation homeostasis in neuronal cells. During the acetylation dyshomeostasis, transcriptional regulation may be affected which has been reported to be one of the prime causes of neurodegenerative diseases (Saha and Pahan 2006). This altered gene transcription then caused opposite effects from normal gene regulation pattern in neuronal cells. This attributed to degenerative fate of neurons that subsequently reduced the expression of survival-associated genes by altered acetylation and at the same time, stimulated the

The increase in Histone H3(K9) acetylation in response to DHA, however, indicates the ability of DHA to normalize the histone H3(K9) acetylation to the basal level (control) and abolishes the effect of zinc (Sadli et al., 2011, *unpublished results*). Therefore, DHA may contribute to neuroprotection through reinstating the altered acetylation dyshomeostasis caused by zinc toxicity, which would possibly up-regulate the expression of neuroprotective

We performed western immunoblotting to investigate the change in the expression levels of histone deacetylase (HDACs) 1, 2, 3 using anti-HDAC1, 2 and 3 antibodies, where we found that zinc significantly up-regulated HDAC1, 2 and 3 expression levels compared with the control, while DHA significantly down-regulated HDAC1, 2 and 3 (Sadli et al., 2011, *unpublished results*). It's been reported that the activity of HDACs were increased in dying neurons, due to the loss of counterbalancing effect of HATs activity (Saha and Pahan 2006). From our results, we propose that the increase in zinc can also contribute to the neurodegenerative process through up-regulating HDACs enzyme expression levels, and

The HDACs catalytic domain contains a Zn2+ ion, in the active site, which contributes significantly to its catalytic activity (Vannini, Volpari et al. 2004; Ficner 2009). *In vitro,* the deacetylase activity of the purified HDAC homologue was observed only after incubation with zinc chloride (Finnin, Donigian et al. 1999), which suggests that HDAC activity requires a metal cofactor (Hassig, Tong et al. 1998). X-ray crystallographic studies have shown that HDAC inhibitors could chelate zinc ions in the catalytic sites of HDACs and therefore block substrate access to the active zinc ions and inhibit the deacetylation reaction

It has been established in the scientific literature that the isotopic selective inhibition of HDAC enzyme may be the potential treatment for neurodegenerative diseases. It has also been demonstrated that the transcriptional dysregulation by HDACs may play significant

**1.5 Importance of zinc and DHA in epigenetics of human neuronal cells 1.5.1 Effect of zinc and DHA on acetylation levels of Histone H3(K9)** 

Fig. 1. Neuronal acetylation homeostasis. (i) Weights on the balance represent the protein level of HATs and HDACs. (ii) Enzymatic activity scale represents the activity and dark grey areas are physiologically optimal. (A) Under normal neuronal conditions, the level and activity of both HATs and HDACs are within their point of balance where they counteract each other to maintain internal equilibrium (homeostasis). (B) During neurodegenerative disease condition, acetylation homeostasis is altered resulting in the loss of HATs level and activity which balance towards an excessive production of HDACs and subsequent increase in deacetylation

Aging is also considered as the greatest risk factor for the development of the neurodegenerative diseases, such as Alzheimer's disease where neuronal function declination and gene expression alternation could be detected in the aging human brain (Giovacchini, Chang et al. 2002). Studies have found the altered pattern of histone modification in aging cells, such as, trimethylation of histone H4 at lysine 20, which was increased in kidneys and liver of the old-aged rat (Sarg, Koutzamani et al. 2002), and the level of H4 acetylation, which was decreased in the rat brain cortical neurons with age (Pina, Martinez et al. 1988). Several new methylated sites, such as H3(K24), H3(K128) and H2A(R89) were also detected in the study of aged mouse brain, however, no functional studies on these three sites had been reported (Wang, Tsai et al. 2009). It has been reported that in aging brains, most PTMs sites were found on histone H3 which has the longest N-terminal tails amongst other core histones (Wang, Tsai et al. 2009). These studies suggest the importance of proper epigenetic modification in biological activities and neuronal cell development, while the altered epigenetic regulation leads to neurodegenerative diseases.

(A) Normal condition (B) Neurodegenerative

(i) (i)

**HDAC**

disease condition

**HIGH HIGH HIGH HIGH**

**OPTIMUM**

in deacetylation

diseases.

**HDAC HAT**

**OPTIMUM**

(ii) (ii)

Aging is also considered as the greatest risk factor for the development of the neurodegenerative diseases, such as Alzheimer's disease where neuronal function declination and gene expression alternation could be detected in the aging human brain (Giovacchini, Chang et al. 2002). Studies have found the altered pattern of histone modification in aging cells, such as, trimethylation of histone H4 at lysine 20, which was increased in kidneys and liver of the old-aged rat (Sarg, Koutzamani et al. 2002), and the level of H4 acetylation, which was decreased in the rat brain cortical neurons with age (Pina, Martinez et al. 1988). Several new methylated sites, such as H3(K24), H3(K128) and H2A(R89) were also detected in the study of aged mouse brain, however, no functional studies on these three sites had been reported (Wang, Tsai et al. 2009). It has been reported that in aging brains, most PTMs sites were found on histone H3 which has the longest N-terminal tails amongst other core histones (Wang, Tsai et al. 2009). These studies suggest the importance of proper epigenetic modification in biological activities and neuronal cell development, while the altered epigenetic regulation leads to neurodegenerative

Fig. 1. Neuronal acetylation homeostasis. (i) Weights on the balance represent the protein level of HATs and HDACs. (ii) Enzymatic activity scale represents the activity and dark grey areas are physiologically optimal. (A) Under normal neuronal conditions, the level and activity of both HATs and HDACs are within their point of balance where they counteract each other to maintain internal equilibrium (homeostasis). (B) During neurodegenerative disease condition, acetylation homeostasis is altered resulting in the loss of HATs level and activity which balance towards an excessive production of HDACs and subsequent increase

**LOW LOW LOW LOW**

**Deacetylation Acetylation Deacetylation Acetylation**

**OPTIMUM**

**OPTIMUM**

**H A T**

#### **1.5 Importance of zinc and DHA in epigenetics of human neuronal cells 1.5.1 Effect of zinc and DHA on acetylation levels of Histone H3(K9)**

Proper regulation of gene expression in the nervous system is not only controlled by the transcriptional machinery but is also subject to modulation by epigenetic mechanisms such as histone modifications. Following our histone expression study, for the first time, we have also investigated the effect of zinc and DHA on post-translational modifications of histones, in particular histone H3 in human neuronal cells.

One-dimensional electrophoresis and western immunoblot analysis were used to investigate the change in post-translational modified histones of human neuronal cells that had been grown in the presence and absence of zinc and DHA. Our results showed that zinc decreased acetylation of H3(K9), whereas DHA increase H3(K9) acetylation. This suggests the potential involvement of zinc in the progress of neurodegenerative disease through an altered acetylation homeostasis in neuronal cells. During the acetylation dyshomeostasis, transcriptional regulation may be affected which has been reported to be one of the prime causes of neurodegenerative diseases (Saha and Pahan 2006). This altered gene transcription then caused opposite effects from normal gene regulation pattern in neuronal cells. This attributed to degenerative fate of neurons that subsequently reduced the expression of survival-associated genes by altered acetylation and at the same time, stimulated the expression of death-inducing genes (Saha and Pahan 2006).

The increase in Histone H3(K9) acetylation in response to DHA, however, indicates the ability of DHA to normalize the histone H3(K9) acetylation to the basal level (control) and abolishes the effect of zinc (Sadli et al., 2011, *unpublished results*). Therefore, DHA may contribute to neuroprotection through reinstating the altered acetylation dyshomeostasis caused by zinc toxicity, which would possibly up-regulate the expression of neuroprotective genes (Saha and Pahan 2006).

#### **1.5.2 Effect of zinc and DHA on histone deacetylases (HDACs) 1, 2, 3**

We performed western immunoblotting to investigate the change in the expression levels of histone deacetylase (HDACs) 1, 2, 3 using anti-HDAC1, 2 and 3 antibodies, where we found that zinc significantly up-regulated HDAC1, 2 and 3 expression levels compared with the control, while DHA significantly down-regulated HDAC1, 2 and 3 (Sadli et al., 2011, *unpublished results*). It's been reported that the activity of HDACs were increased in dying neurons, due to the loss of counterbalancing effect of HATs activity (Saha and Pahan 2006). From our results, we propose that the increase in zinc can also contribute to the neurodegenerative process through up-regulating HDACs enzyme expression levels, and therefore increasing the activity of histone deacetylation.

The HDACs catalytic domain contains a Zn2+ ion, in the active site, which contributes significantly to its catalytic activity (Vannini, Volpari et al. 2004; Ficner 2009). *In vitro,* the deacetylase activity of the purified HDAC homologue was observed only after incubation with zinc chloride (Finnin, Donigian et al. 1999), which suggests that HDAC activity requires a metal cofactor (Hassig, Tong et al. 1998). X-ray crystallographic studies have shown that HDAC inhibitors could chelate zinc ions in the catalytic sites of HDACs and therefore block substrate access to the active zinc ions and inhibit the deacetylation reaction (Marks, Richon et al. 2000; Ficner 2009).

It has been established in the scientific literature that the isotopic selective inhibition of HDAC enzyme may be the potential treatment for neurodegenerative diseases. It has also been demonstrated that the transcriptional dysregulation by HDACs may play significant

Effect of Zinc and DHA on Expression Levels and

AD (Fasulo, Ugolini et al. 2000).

analysis.

**apoptotic marker) expression levels** 

death in neurodegenerative diseases.

**1.7 M17 cell line as a model** 

Post-Translational Modifications of Histones H3 and H4 in Human Neuronal Cells 153

to an increase in Aβ production (Tesco, Koh et al. 2007). Studies also indicate that caspases have been implicated in the mechanism of tau-mediated neurodegeneration of AD (Garcia-Sierra, Mondragon-Rodriguez et al. 2008) (Dickson 2004). According to this hypothesis, Aβ peptide was reported to promote neuronal pathological tau filament assembly by triggering caspase activation leading to tau cleavage, which in turn generate more tau pathological filaments (Tesco, Koh et al. 2007) that further contribute to increase of cellular dysfunction in

**1.6.1 Effect of zinc and DHA on Bcl-2 (anti-apoptotic marker) and caspase-3 (pro-**

As mentioned previously, zinc toxicity is one of the important causes of cell death in neurodegenerative disease, including Alzheimer's disease (Naganska and Matyja 2006). It has been reported that intracellular zinc release, as a result of altered zinc metabolism, leads to the activation of caspase-3, which then subsequently trigger neuronal cell apoptosis (Zhang, Wang et al. 2004). In our study, we aimed to determine whether anti-apoptotic Bcl-2 and pro-apoptotic caspase-3 were involved in the cellular pathway affected by zinc and DHA interactions in M17 cells by investigating their expression levels using western blot

Both zinc and DHA have been shown to opposingly modulate the levels of Bcl-2 and caspase-3 in M17 cells (Sadli et al., 2011, *unpublished results*). An increase in zinc levels causes up-regulation of caspase-3 and down-regulation of Bcl-2 expression, suggesting the potential occurrence of apoptosis of zinc-induced M17 cells, which is representative of neurodegenerative conditions such as AD where intracellular zinc ion is elevated while DHA level is reduced. Conversely, DHA treatment of M17 cells increased expression levels of Bcl-2 and reduced caspase-3, which suggest that DHA exclusively activates the extracellular signal regulated kinase/mitogen-activated protein kinases (ERK/MPK) pathway to promote cell survival that lead to the up-regulation of Bcl-2 and inhibition of caspase-3 activation (German, Insua et al. 2006). Our observation was supported by Akbar et al. (2006), which showed the involvement of DHA in neuronal cell survival by driving Akt translocation resulting in activation of Bcl-2 and subsequent suppression of caspase-3

activity leading to inhibition of apoptosis in neuronal cells (Akbar, Baick et al. 2006).

Our findings with Bcl-2 and caspase-3 highlight the importance of DHA in neuroprotection and zinc toxicity in apoptosis. The blockage of caspase-3 activity by DHA might protect against the apoptotic cell death following zinc toxicity, which may offer a useful and alternative therapeutic strategy to delay neuronal loss associated with neurodegenerative diseases. Further investigations on the role of DHA in neuroprotection through inhibition of caspase needs to be done, which will provide additional insights into this cascade activity pathway. This in return will establish the idea whether cascade-induced zinc toxicity is a direct cause of apoptosis or a downstream consequence, which will eventually lead to cell

Neurodegeneration is very difficult to study *in vivo*. Neuronal cells do not regenerate and cannot be observed or manipulated without removing them from the patients. For these reasons, *in vitro* models are very important options. An ideal cell line would possess similar characteristic as the *in vivo* neurons, while having the advantage of immortalization to

role in causing neurodegenerative disease and that HDACs therapy may prevent or slow down the neurodegenerative disease process. So far, the HDAC inhibitors investigated in treating neurodegenerative diseases are very limited and mainly focused on the wellestablish experimental drug trichostatin A (member of hydroxamic acid group) and the clinically used HDAC inhibitors sodium butyrate, valproic acid, phenylbutyrate and vorinostat, which belong to short chain fatty acid group that are known to be able to penetrate the blood-brain barrier (Butler and Bates 2006). From our observation, DHA, being a long chain n-3 fatty acid that is selectively allowed to cross the blood-brain barrier, is likely to have neuroprotective characteristic that mimic the behavior of HDACs inhibitors. This significantly down-regulates the HDACs expression levels and therefore induces histone acetylation, which then allow the transcription and expression of genes, in what had been a too tightly packaged chromatin structure in which certain genes do not get transcribed.

Generally, increase in HDACs during neurodegenerative disease is associated with increase in gene repression and transcriptional dysfunction of certain transcription factors (TFs) such as CREB, which is important in regulating the expression of pro-survival elements such as Bcl-2 (Freeland, Boxer et al. 2001; Saha and Pahan 2006). In our study, we show how zinc contributes to dysfunctional acetylation homesostasis in M17 cells by up-regulating HDACs, which influence the reduction of HATs and consequently histone acetylation levels. DHA, however, is shown to reestablish the imbalance of acetylation homesostasis and therefore capable of correcting the down-regulation of specific genes caused by reduction in histone acetylation (Sadli et al., 2011, *unpublished results*). The mechanism by which DHA inhibits the HDACs expression is unclear, whether DHA directly chelates zinc ion from the catalytic sites of HDACs or hinder the zinc ion binding to the enzymes.

#### **1.6 Link between cellular apoptosis and neurodegenerative diseases**

A characteristic of many neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and stroke - is neuronal cell death (Cavallucci and D'Amelio, 2011; Calissano, Matrone et al. 2009). Central nervous system tissue has very limited regenerative capacity and therefore it is important to limit the damage caused by neuronal cell death (Rossi and Cattaneo 2002) (Kuhn, Palmer et al. 2001). In recent years, the investigation regarding the contribution of caspases and neuronal apoptosis to neurodegenerative diseases has gained increasing attention. This evidence has been generated by using a variety of complementary approaches, including evaluating human tissue and using transgenic mouse and *in vitro* models (Kuhn, Palmer et al. 2001).

Studies have shown the imbalance level of pro-apoptotic (Bax, Bak and Bad) and antiapoptotic Bcl-2 protein (Su, Deng et al. 1997; Kitamura, Shimohama et al. 1998), as well as caspase-3 and -6 in post-mortem brains of AD patients (Stadelmann, Deckwerth et al. 1999). In addition, immunohistochemical and biochemical studies reported the presence of active caspases and caspase-cleaved substrates around senile plague and neurofibrillary tangles in neuron (Gastard, Troncoso et al. 2003). There's also a marked co-localization of hyperphosphorylated tau, caspase-3 and caspase-6 in TUNEL-positive neurons in the brainstem of AD patients (Wai, Liang et al. 2009), suggesting the potential involvement of apoptotic death in the etiology of AD.

Caspase-mediated apoptotic pathways have specifically been linked to the progression of AD, especially toward the formation of amyloid precursor protein (APP) and Aβ peptide production. Caspase-3-mediated APP stabilizes BACE1 (the β-secretase enzyme that is responsible for the cleavage of APP and the associated creation of beta-amyloid), which lead

role in causing neurodegenerative disease and that HDACs therapy may prevent or slow down the neurodegenerative disease process. So far, the HDAC inhibitors investigated in treating neurodegenerative diseases are very limited and mainly focused on the wellestablish experimental drug trichostatin A (member of hydroxamic acid group) and the clinically used HDAC inhibitors sodium butyrate, valproic acid, phenylbutyrate and vorinostat, which belong to short chain fatty acid group that are known to be able to penetrate the blood-brain barrier (Butler and Bates 2006). From our observation, DHA, being a long chain n-3 fatty acid that is selectively allowed to cross the blood-brain barrier, is likely to have neuroprotective characteristic that mimic the behavior of HDACs inhibitors. This significantly down-regulates the HDACs expression levels and therefore induces histone acetylation, which then allow the transcription and expression of genes, in what had been a too tightly packaged chromatin structure in which certain genes do not get transcribed. Generally, increase in HDACs during neurodegenerative disease is associated with increase in gene repression and transcriptional dysfunction of certain transcription factors (TFs) such as CREB, which is important in regulating the expression of pro-survival elements such as Bcl-2 (Freeland, Boxer et al. 2001; Saha and Pahan 2006). In our study, we show how zinc contributes to dysfunctional acetylation homesostasis in M17 cells by up-regulating HDACs, which influence the reduction of HATs and consequently histone acetylation levels. DHA, however, is shown to reestablish the imbalance of acetylation homesostasis and therefore capable of correcting the down-regulation of specific genes caused by reduction in histone acetylation (Sadli et al., 2011, *unpublished results*). The mechanism by which DHA inhibits the HDACs expression is unclear, whether DHA directly chelates zinc ion from the catalytic

sites of HDACs or hinder the zinc ion binding to the enzymes.

transgenic mouse and *in vitro* models (Kuhn, Palmer et al. 2001).

apoptotic death in the etiology of AD.

**1.6 Link between cellular apoptosis and neurodegenerative diseases** 

A characteristic of many neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and stroke - is neuronal cell death (Cavallucci and D'Amelio, 2011; Calissano, Matrone et al. 2009). Central nervous system tissue has very limited regenerative capacity and therefore it is important to limit the damage caused by neuronal cell death (Rossi and Cattaneo 2002) (Kuhn, Palmer et al. 2001). In recent years, the investigation regarding the contribution of caspases and neuronal apoptosis to neurodegenerative diseases has gained increasing attention. This evidence has been generated by using a variety of complementary approaches, including evaluating human tissue and using

Studies have shown the imbalance level of pro-apoptotic (Bax, Bak and Bad) and antiapoptotic Bcl-2 protein (Su, Deng et al. 1997; Kitamura, Shimohama et al. 1998), as well as caspase-3 and -6 in post-mortem brains of AD patients (Stadelmann, Deckwerth et al. 1999). In addition, immunohistochemical and biochemical studies reported the presence of active caspases and caspase-cleaved substrates around senile plague and neurofibrillary tangles in neuron (Gastard, Troncoso et al. 2003). There's also a marked co-localization of hyperphosphorylated tau, caspase-3 and caspase-6 in TUNEL-positive neurons in the brainstem of AD patients (Wai, Liang et al. 2009), suggesting the potential involvement of

Caspase-mediated apoptotic pathways have specifically been linked to the progression of AD, especially toward the formation of amyloid precursor protein (APP) and Aβ peptide production. Caspase-3-mediated APP stabilizes BACE1 (the β-secretase enzyme that is responsible for the cleavage of APP and the associated creation of beta-amyloid), which lead to an increase in Aβ production (Tesco, Koh et al. 2007). Studies also indicate that caspases have been implicated in the mechanism of tau-mediated neurodegeneration of AD (Garcia-Sierra, Mondragon-Rodriguez et al. 2008) (Dickson 2004). According to this hypothesis, Aβ peptide was reported to promote neuronal pathological tau filament assembly by triggering caspase activation leading to tau cleavage, which in turn generate more tau pathological filaments (Tesco, Koh et al. 2007) that further contribute to increase of cellular dysfunction in AD (Fasulo, Ugolini et al. 2000).

#### **1.6.1 Effect of zinc and DHA on Bcl-2 (anti-apoptotic marker) and caspase-3 (proapoptotic marker) expression levels**

As mentioned previously, zinc toxicity is one of the important causes of cell death in neurodegenerative disease, including Alzheimer's disease (Naganska and Matyja 2006). It has been reported that intracellular zinc release, as a result of altered zinc metabolism, leads to the activation of caspase-3, which then subsequently trigger neuronal cell apoptosis (Zhang, Wang et al. 2004). In our study, we aimed to determine whether anti-apoptotic Bcl-2 and pro-apoptotic caspase-3 were involved in the cellular pathway affected by zinc and DHA interactions in M17 cells by investigating their expression levels using western blot analysis.

Both zinc and DHA have been shown to opposingly modulate the levels of Bcl-2 and caspase-3 in M17 cells (Sadli et al., 2011, *unpublished results*). An increase in zinc levels causes up-regulation of caspase-3 and down-regulation of Bcl-2 expression, suggesting the potential occurrence of apoptosis of zinc-induced M17 cells, which is representative of neurodegenerative conditions such as AD where intracellular zinc ion is elevated while DHA level is reduced. Conversely, DHA treatment of M17 cells increased expression levels of Bcl-2 and reduced caspase-3, which suggest that DHA exclusively activates the extracellular signal regulated kinase/mitogen-activated protein kinases (ERK/MPK) pathway to promote cell survival that lead to the up-regulation of Bcl-2 and inhibition of caspase-3 activation (German, Insua et al. 2006). Our observation was supported by Akbar et al. (2006), which showed the involvement of DHA in neuronal cell survival by driving Akt translocation resulting in activation of Bcl-2 and subsequent suppression of caspase-3 activity leading to inhibition of apoptosis in neuronal cells (Akbar, Baick et al. 2006).

Our findings with Bcl-2 and caspase-3 highlight the importance of DHA in neuroprotection and zinc toxicity in apoptosis. The blockage of caspase-3 activity by DHA might protect against the apoptotic cell death following zinc toxicity, which may offer a useful and alternative therapeutic strategy to delay neuronal loss associated with neurodegenerative diseases. Further investigations on the role of DHA in neuroprotection through inhibition of caspase needs to be done, which will provide additional insights into this cascade activity pathway. This in return will establish the idea whether cascade-induced zinc toxicity is a direct cause of apoptosis or a downstream consequence, which will eventually lead to cell death in neurodegenerative diseases.

#### **1.7 M17 cell line as a model**

Neurodegeneration is very difficult to study *in vivo*. Neuronal cells do not regenerate and cannot be observed or manipulated without removing them from the patients. For these reasons, *in vitro* models are very important options. An ideal cell line would possess similar characteristic as the *in vivo* neurons, while having the advantage of immortalization to

Effect of Zinc and DHA on Expression Levels and

understanding, diagnosis and treatment of AD.

**1.9 Conclusions and future perspectives** 

development of neurodegenerative diseases.

acetylation homeostasis.

DNA synthesis and therefore increasing histone protein levels.

Post-Translational Modifications of Histones H3 and H4 in Human Neuronal Cells 155

When considering neurological disorders, one good example for the usability of twodimensional gel electrophoresis (2-DE) in exploring new biomarkers, was the discovery of two unknown protein isoforms p130 and p131 that were suggested to be able to discriminate Creutzfeldt-Jakob disease from other type of dementia (Harrington, Merril et al. 1986). Most efforts in understanding the pathogenesis using 2-DE-based expression proteomics have been made by comparing brain proteomes of AD patients and controls. Some of the first 2- DE studies examined the levels of AD brain proteins where they revealed alterations in the levels of a number of proteins, such as GFAP, tubulin, and creatine kinase (Smirnov, Shevtsov et al. 1991; Burbaeva 1992). Subsequently, the number of 2-DE studies has multiplied and at present, changes in the levels of more than 100 brain protein isoforms have been identified in neurodegenerative disorders (Fountoulakis, Juranville et al. 2002; Butterfield and Castegna 2003; Vlahou and Fountoulakis 2005). Despite the multiplicity of isoform specific protein changes, the findings still remain rather fragmented and novel

Proteomic methods were also successfully applied in the study of tau protein phosphorylation in AD where tau become phosphorylated and accumulated to form neurofibrillary tangles (Ksiezak-Reding, Binder et al. 1990). The increased phosphorylation of elongation factor II has also been demonstrated in AD brain by the 2-D approach (Johnson, Gotlib et al. 1992). As a consequence of rapid demographic aging, AD has become one of the most devastating socioeconomical challenges of the present-day. Now, the new hope of unraveling the secrets of AD is done by the so-called "new technologies" (i.e. proteomics) which have been suggested to represent a breakthrough in improving our

We characterized the effect of zinc and DHA in modulating gene and protein expression in M17 human neuronal cells. This idea was based on the fact that both zinc and DHA play significant roles in neuroprotection and are known to interact biochemically. DHA treatment of M7 cells results in lower zinc transporter ZnT3 protein levels and reduction in pro-apoptotic marker caspase-3 indicates the involvement of zinc in pathways that regulate brain cell survival and that alteration in zinc homeostasis may contribute to the

Both zinc and DHA may possibly be involved in the signaling mechanism that regulates histone expression levels in M17 human neuronal cells. Here we hypothesize that DHA may play a protective role by up-regulating histones H3 and H4, which accounts for the positive effect of DHA in minimizing the onset of neurodegenerative disease through facilitating

Following our previous study, we also investigated the effect of zinc and DHA in regulating gene expression through histones post-translational modifications. Zinc was found to reduce histone acetylation and increase HDACs, which represent a critical step commonly underlying catastrophic neuronal dysfunction, whereas, DHA reinstated the imbalance of acetylation homeostasis indicating its potential neuroprotective ability to ameliorate neurodegenerative diseases. Reduction in acetylation along with parallel gain of HDAC levels represents the crux of the altered situation and we propose that DHA could possibly mimic the action of a HDAC inhibitors and therefore, reverses the zinc-mediated altered

hypothesis related to the pathogenesis of AD still remains to be revealed.

ensure continuous supply of cells. Immortalized cells are also convenient to handle and experiments can be performed during continuous conditions in which biochemical process can easily be studied.

Throughout our studies, M17, a neuronal-derived cell line was used. M17 cells are originally isolated from the bone marrow of a two year old male suffering from disseminated neuroblastoma (Global Bio-resource Center, 2007). Microscopic analysis shows that the cell type indicates a neuronal characteristic; being morphologically small in size and dense with triangular-shaped cell bodies. The advantage of this cell line is that it is of human origin, and by now, M17 cells constitute a well studied and defined cellular system. Our in-house results suggest that M17 cell line is a suitable model for studying the effects of DHA and zinc supplementation on the gene and protein expression profiles of neuronal cells throughout this study.

#### **1.8 Application of proteomic and molecular analysis in neurodegenerative disease research**

The need for protein-level analysis arises because the phenotype of human neuronal cells in relation to neurodegeneration corresponds to the functions of expressed and modified protein networks. Unlike the genome that is relatively static, the proteome is extremely dynamic and constantly adjusted in response to changing internal (e.g aging) and external events (e.g toxic exposure, drugs).

Proteins are composed of a variety of combinations of amino acids, and are subject to co- as well as post-translational modification, such as deletion of amino acid sequences and chemical modification of specific amino acids (e.g oxidation and phosphorylation) (Anderson, Matheson et al. 2001). These modifications will influence the activity state, function and interactions of proteins.

The word "*proteome*" is derived from proteins expressed by a genome, and it refers to all the proteins produced by an organism, first coined by Wilkins et al. in 1996 (Wilkins, Sanchez et al. 1996). In its wider sense, proteomic research assesses protein expression, modification, interaction and localization. By studying global patterns of proteins and their changes dynamically, proteomic research can improve our understanding of system-level cellular behavior. Although proteomics as an entity is relatively new, the methodological and theoretical foundations have been under development for more than three decades (Campostrini, Pascali et al. 2004). Two-dimensional protein electrophoresis, coupled with peptide mass fingerprinting analysis by mass spectrometry (MS), has become the most powerful techniques for proteome analysis (Binz, Muller et al. 1999).

In the future, downstream steps after genomics and proteomics will aim at understanding functional consequences of biomolecule interactions in different biological pathways in a system. Comparative studies to quantitate, identify and characterize the proteins expressed in normal neuronal cells and diseased cells will give insight into the mechanisms of neurodegenerative disease. This will allow the identification of novel diagnostic and treatment reagents for Alzheimer's disease.

Proteomic analysis data has become an important resource in the investigation of neurodegenerative diseases. Proteomic profiling, in particular, has enabled researchers to investigate a vast number of proteins at once. Such principles have been utilized in order to detect specific alterations in the protein expression levels in various regions of the neurodegenerated brain compared to control brain. This may, in turn, facilitate the construction of hypotheses on the mechanisms by which the disease progresses.

ensure continuous supply of cells. Immortalized cells are also convenient to handle and experiments can be performed during continuous conditions in which biochemical process

Throughout our studies, M17, a neuronal-derived cell line was used. M17 cells are originally isolated from the bone marrow of a two year old male suffering from disseminated neuroblastoma (Global Bio-resource Center, 2007). Microscopic analysis shows that the cell type indicates a neuronal characteristic; being morphologically small in size and dense with triangular-shaped cell bodies. The advantage of this cell line is that it is of human origin, and by now, M17 cells constitute a well studied and defined cellular system. Our in-house results suggest that M17 cell line is a suitable model for studying the effects of DHA and zinc supplementation on the gene and protein expression profiles of neuronal cells

**1.8 Application of proteomic and molecular analysis in neurodegenerative disease** 

The need for protein-level analysis arises because the phenotype of human neuronal cells in relation to neurodegeneration corresponds to the functions of expressed and modified protein networks. Unlike the genome that is relatively static, the proteome is extremely dynamic and constantly adjusted in response to changing internal (e.g aging) and external

Proteins are composed of a variety of combinations of amino acids, and are subject to co- as well as post-translational modification, such as deletion of amino acid sequences and chemical modification of specific amino acids (e.g oxidation and phosphorylation) (Anderson, Matheson et al. 2001). These modifications will influence the activity state,

The word "*proteome*" is derived from proteins expressed by a genome, and it refers to all the proteins produced by an organism, first coined by Wilkins et al. in 1996 (Wilkins, Sanchez et al. 1996). In its wider sense, proteomic research assesses protein expression, modification, interaction and localization. By studying global patterns of proteins and their changes dynamically, proteomic research can improve our understanding of system-level cellular behavior. Although proteomics as an entity is relatively new, the methodological and theoretical foundations have been under development for more than three decades (Campostrini, Pascali et al. 2004). Two-dimensional protein electrophoresis, coupled with peptide mass fingerprinting analysis by mass spectrometry (MS), has become the most

In the future, downstream steps after genomics and proteomics will aim at understanding functional consequences of biomolecule interactions in different biological pathways in a system. Comparative studies to quantitate, identify and characterize the proteins expressed in normal neuronal cells and diseased cells will give insight into the mechanisms of neurodegenerative disease. This will allow the identification of novel diagnostic and

Proteomic analysis data has become an important resource in the investigation of neurodegenerative diseases. Proteomic profiling, in particular, has enabled researchers to investigate a vast number of proteins at once. Such principles have been utilized in order to detect specific alterations in the protein expression levels in various regions of the neurodegenerated brain compared to control brain. This may, in turn, facilitate the construction of

powerful techniques for proteome analysis (Binz, Muller et al. 1999).

hypotheses on the mechanisms by which the disease progresses.

can easily be studied.

throughout this study.

events (e.g toxic exposure, drugs).

function and interactions of proteins.

treatment reagents for Alzheimer's disease.

**research** 

When considering neurological disorders, one good example for the usability of twodimensional gel electrophoresis (2-DE) in exploring new biomarkers, was the discovery of two unknown protein isoforms p130 and p131 that were suggested to be able to discriminate Creutzfeldt-Jakob disease from other type of dementia (Harrington, Merril et al. 1986). Most efforts in understanding the pathogenesis using 2-DE-based expression proteomics have been made by comparing brain proteomes of AD patients and controls. Some of the first 2- DE studies examined the levels of AD brain proteins where they revealed alterations in the levels of a number of proteins, such as GFAP, tubulin, and creatine kinase (Smirnov, Shevtsov et al. 1991; Burbaeva 1992). Subsequently, the number of 2-DE studies has multiplied and at present, changes in the levels of more than 100 brain protein isoforms have been identified in neurodegenerative disorders (Fountoulakis, Juranville et al. 2002; Butterfield and Castegna 2003; Vlahou and Fountoulakis 2005). Despite the multiplicity of isoform specific protein changes, the findings still remain rather fragmented and novel hypothesis related to the pathogenesis of AD still remains to be revealed.

Proteomic methods were also successfully applied in the study of tau protein phosphorylation in AD where tau become phosphorylated and accumulated to form neurofibrillary tangles (Ksiezak-Reding, Binder et al. 1990). The increased phosphorylation of elongation factor II has also been demonstrated in AD brain by the 2-D approach (Johnson, Gotlib et al. 1992). As a consequence of rapid demographic aging, AD has become one of the most devastating socioeconomical challenges of the present-day. Now, the new hope of unraveling the secrets of AD is done by the so-called "new technologies" (i.e. proteomics) which have been suggested to represent a breakthrough in improving our understanding, diagnosis and treatment of AD.

#### **1.9 Conclusions and future perspectives**

We characterized the effect of zinc and DHA in modulating gene and protein expression in M17 human neuronal cells. This idea was based on the fact that both zinc and DHA play significant roles in neuroprotection and are known to interact biochemically. DHA treatment of M7 cells results in lower zinc transporter ZnT3 protein levels and reduction in pro-apoptotic marker caspase-3 indicates the involvement of zinc in pathways that regulate brain cell survival and that alteration in zinc homeostasis may contribute to the development of neurodegenerative diseases.

Both zinc and DHA may possibly be involved in the signaling mechanism that regulates histone expression levels in M17 human neuronal cells. Here we hypothesize that DHA may play a protective role by up-regulating histones H3 and H4, which accounts for the positive effect of DHA in minimizing the onset of neurodegenerative disease through facilitating DNA synthesis and therefore increasing histone protein levels.

Following our previous study, we also investigated the effect of zinc and DHA in regulating gene expression through histones post-translational modifications. Zinc was found to reduce histone acetylation and increase HDACs, which represent a critical step commonly underlying catastrophic neuronal dysfunction, whereas, DHA reinstated the imbalance of acetylation homeostasis indicating its potential neuroprotective ability to ameliorate neurodegenerative diseases. Reduction in acetylation along with parallel gain of HDAC levels represents the crux of the altered situation and we propose that DHA could possibly mimic the action of a HDAC inhibitors and therefore, reverses the zinc-mediated altered acetylation homeostasis.

Effect of Zinc and DHA on Expression Levels and

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Fig. 2. Proposed model depicting the role of zinc and DHA in progression of neurodegenerative diseases. Various models representing neurodegenerative diseases are marked by irregular gene expression, altered epigenetic patterns as well as apoptosis. Cellular zinc toxicity contributes to neurodegenerative condition through a number of different pathways, which seem to be reversed by the presence of DHA. Zinc and DHA may share common pathways in the progression of neurodegenerative disease where DHA play a significant role in restoring the condition caused by altered zinc homeostasis

#### **2. References**


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Fig. 2. Proposed model depicting the role of zinc and DHA in progression of

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**7** 

*Colombia* 

**Free Radicals,** 

**Neuronal Death and Neuroprotection** 

The Reactive Oxygen Species (ROS) and the Reactive Nitrogen Species (RNS) are highly reactive molecules participating as mediators in biological processes such as metabolic cellular respiration, neurotransmission, translation and transcription gene, and inflammatory-type reactions among others (D. Almaguer & L.E. Almaguer, 2006; Boots et al., 2008; Uttara, 2009). Additionally, these molecules have the capacity to interact with nucleophilic centers of biomolecules by modulating their activity or by irreversibly modifying them in order to

The main oxygen radicals are the hydroxyl (-OH), the superoxide anion (O2-), and the hydrogen peroxide (H2O2), while the main nitrogen radical is the nitric oxide (•NO), but also, it is known that the ROS can interact with -NO generating new species with high oxidizing power (Martinez et al., 2010). In aerobic systems most of the ROS come from the mitochondrial oxidative metabolism, where 1-2% of the oxygen is converted into free radicals (Uttara et al., 2009). Other less important sources of ROS are the autoxidation of catecholamines and hemoproteins that occurs in the cytoplasm, nuclear membrane, endoplasmic reticulum and peroxisomes (Boots et al., 2008; Martínez et al, 2010). The concentration of ROS or RNS in organisms is determined by the balance between the rate of production of reactive species and the elimination rate of these compounds by the action of enzymes and antioxidants (AO) (Dorado et al., 2003). Thus, under conditions of physiological homeostasis, a balance exists between the cellular processes that contribute to the production of ROS / RNS, and those factors such as superoxide dismutase (SOD), catalase (Cat) and glutathione peroxidase (GPx), which contribute to their elimination (Dorado et al., 2003; Martínez et al., 2010). Thus, alterations in the balance between these systems, pro-oxidants and antioxidants can lead to intracellular accumulation of free radicals (FR), causing oxidative stress states (Dorado et al., 2003; Kelsey, et al , 2010; Uttara,

Specifically, oxidative stress and redox imbalance is the combined result of excessive formation of oxidant species (ROS/RNS) and/or a decreasing in the efficiency of endogenous antioxidant systems. Thus, the combination of these factors converge in damaging to biomolecules such as DNA, RNA, proteins, carbohydrates and lipids. This probably initiate processes of mitochondrial dysfunction and excitotoxicity (Kelsey, et al.,

generate different kind of radicals (D. Almaguer & L.E. Almaguer, 2006).

**1. Introduction** 

2009).

Diana Gallego, Manuel Rojas and Camilo Orozco

*Departamento de Ciencias para La Salud Animal Facultad de Medicina Veterinaria y de Zootecnia Universidad Nacional de Colombia, Sede Bogotá* 


### **Free Radicals, Neuronal Death and Neuroprotection**

Diana Gallego, Manuel Rojas and Camilo Orozco

*Departamento de Ciencias para La Salud Animal Facultad de Medicina Veterinaria y de Zootecnia Universidad Nacional de Colombia, Sede Bogotá Colombia* 

#### **1. Introduction**

164 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

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Xiao, Y. and X. Li (1999). "Polyunsaturated fatty acids modify mouse hippocampal neuronal excitability during excitotoxic or convulsant stimulation." *Brain Res* 846(1): 112-21. Yang, X. J. and E. Seto (2008). "Lysine acetylation: codified crosstalk with other

Young, G. and J. Conquer (2005). "Omega-3 fatty acids and neuropsychiatric disorders."

Zatta, P., D. Drago, et al. (2009). "Alzheimer's disease, metal ions and metal homeostatic

Zhang, L. H., X. Wang, et al. (2008). "Abundant expression of zinc transporters in the amyloid plaques of Alzheimer's disease brain." *Brain Res Bull* 77(1): 55-60. Zhang, Y., H. Wang, et al. (2004). "Peroxynitrite-induced neuronal apoptosis is mediated by

intracellular zinc release and 12-lipoxygenase activation." *J Neurosci* 24(47): 10616-

expressed by a genome should be identified and how to do it." *Biotechnol Genet Eng* 

amyloid-beta peptide. Metal ion binding, contribution to fibrillization and toxicity."

subsequent 5-y cognitive decline in elderly men: the Zutphen Elderly Study." *Am J* 

histone deacetylase, human HDAC8, complexed with a hydroxamic acid inhibitor."

193(3): 701-13.

*Metallomics* 3(3): 250-61.

*Clin Nutr* 85(4): 1142-7.

*Proc Natl Acad Sci U S A* 101(42): 15064-9.

disease." *Trends Pharmacol Sci* 21(10): 395-401.

development." *J Fam Health Care* 12(6 Suppl): 5.

chromosomes." *Curr Opin Genet Dev* 11(2): 130-5.

therapy." *Trends Pharmacol Sci* 30(7): 346-55.

posttranslational modifications." *Mol Cell* 31(4): 449-61. Yankner, B. A., T. Lu, et al. (2008). "The aging brain." *Annu Rev Pathol* 3: 41-66.

*Trends Biochem Sci* 28(8): 406-10.

*Reprod Nutr Dev* 45(1): 1-28.

*Rev* 13: 19-50.

27.

The Reactive Oxygen Species (ROS) and the Reactive Nitrogen Species (RNS) are highly reactive molecules participating as mediators in biological processes such as metabolic cellular respiration, neurotransmission, translation and transcription gene, and inflammatory-type reactions among others (D. Almaguer & L.E. Almaguer, 2006; Boots et al., 2008; Uttara, 2009). Additionally, these molecules have the capacity to interact with nucleophilic centers of biomolecules by modulating their activity or by irreversibly modifying them in order to generate different kind of radicals (D. Almaguer & L.E. Almaguer, 2006).

The main oxygen radicals are the hydroxyl (-OH), the superoxide anion (O2-), and the hydrogen peroxide (H2O2), while the main nitrogen radical is the nitric oxide (•NO), but also, it is known that the ROS can interact with -NO generating new species with high oxidizing power (Martinez et al., 2010). In aerobic systems most of the ROS come from the mitochondrial oxidative metabolism, where 1-2% of the oxygen is converted into free radicals (Uttara et al., 2009). Other less important sources of ROS are the autoxidation of catecholamines and hemoproteins that occurs in the cytoplasm, nuclear membrane, endoplasmic reticulum and peroxisomes (Boots et al., 2008; Martínez et al, 2010). The concentration of ROS or RNS in organisms is determined by the balance between the rate of production of reactive species and the elimination rate of these compounds by the action of enzymes and antioxidants (AO) (Dorado et al., 2003). Thus, under conditions of physiological homeostasis, a balance exists between the cellular processes that contribute to the production of ROS / RNS, and those factors such as superoxide dismutase (SOD), catalase (Cat) and glutathione peroxidase (GPx), which contribute to their elimination (Dorado et al., 2003; Martínez et al., 2010). Thus, alterations in the balance between these systems, pro-oxidants and antioxidants can lead to intracellular accumulation of free radicals (FR), causing oxidative stress states (Dorado et al., 2003; Kelsey, et al , 2010; Uttara, 2009).

Specifically, oxidative stress and redox imbalance is the combined result of excessive formation of oxidant species (ROS/RNS) and/or a decreasing in the efficiency of endogenous antioxidant systems. Thus, the combination of these factors converge in damaging to biomolecules such as DNA, RNA, proteins, carbohydrates and lipids. This probably initiate processes of mitochondrial dysfunction and excitotoxicity (Kelsey, et al.,

Free Radicals, Neuronal Death and Neuroprotection 167

reported that the nervous system is rich in iron (Fe3 +) and unsaturated fatty acids (Halliwell, 2001), this is one of the main features that make this organ particularly sensitive to oxidative stress. Thus, the high lipid content of nervous tissue and its high metabolic activity make it particularly vulnerable to oxidative damage (T.M. Dawson & V.L. Dawson, 1996). It has been reported that iron is essential in the brain, particularly during development, but high amounts can lead to the generation of damage to brain cells, because Fe 3 + can lead to oxidative stress by catalyzing the formation processes of ROS (Butterfield et al, 2002; Gerlach et al, 1994). In addition, the brain consumes a high proportion of the total oxygen, since the energy it uses comes almost exclusively from oxidative metabolism which occurs in the mitochondrial respiratory chain located in the bodies, dendrites, axons and synaptic buttons of cells neural, where the ATPase maintains the ionic gradient across the neuronal membrane (Pavón et al., 1998). Another reason that explains why ROS are particularly active in the brain is that in this tissue, the metabolism of the aminoacids and neurotransmitters acts as an important source of these highly reactive molecules (Uttara et al., 2009). Thus, considering that this organ contains low levels of protective enzymes and other enzymatic antioxidants, it is easy to understand why their susceptibility (Contestabile,

Although oxidative stress is not the direct cause or the etiological factor responsible for the neuropathologies, it is known that it induces toxic effects by oxidizing lipids, proteins, carbohydrates and nucleotides. This causes accumulation of intracellular aggregates, mitochondrial dysfunction, excitotoxicity and apoptosis. Formation of modified lipids by oxidation can cause cellular dysfunction and death in postmitotic cells. Likewise, the peroxidation of polyunsaturated fatty acids in cell membranes, initiates a cumulative deterioration of membrane functions and causes a decrease in flow, reduction in the electrochemical potential and increased permeability of the membrane. Similarly, oxidation causes changes in the structure of some proteins and the formation of protein aggregates. These abnormal proteins induce oxidative damage that has been observed in neurodegenerative diseases as AD and PD. On another hand, ROS also can affect both glial cells and neuronal cells, which exhibit a particular sensitivity to free radicals and therefore are prone to cell degeneration processes (Gilgun-Sherki et al., 2001). For example, there is a biological phenomenon known as "selective neuronal vulnerability", which is involved in the response of different neuronal populations against neurodegenerative conditions. For example, neurons in the entorhinal cortex, the CA1 region of hippocampus, frontal cortex and amygdala, are highly susceptible populations to neurodegeneration associated with oxidative stress in Alzheimer's disease (AD) (Braak et al., 1991; Hyman et al, 1984; Terry et

The relationship between aging and neurodegenerative diseases is more than evident. This is supported by recent investigations that have observed that during the aging process there is an increase in the formation of H2O2 that alters the conditions of the electrons flow in the transport chain, facilitating their 'escape' of normal stream flow. This leads the neurons to suffer the harmful effect of free radicals. Moreover, studies performed by Sohal, et al., 1993, Benzi & Mareti, 1995, and others, have postulated that ROS can inflict damage to the mitochondrial inner membrane, the components of the electron transport chain or the mitochondrial DNA (mtDNA). Oxidative stress further increases ROS production and, consequently, damage to the mitochondria, creating a cycle that perpetuates the effects of ROS (González et al., 1999). Thus, it is obvious because aging itself is a key factor in diseases such as AD or PD; since the increase of oxidative damage, the progressive mitochondrial

2001).

al, 2001).

2010; Dorado et al., 2003), which in turn results in structural and functional alterations of organic macromolecules, leading the affected cells to degenerative processes and cell death by necrosis or apoptosis (D. Almaguer & L.E. Almaguer, 2006; Gonzalez et al. 1999; Martinez et al., 2010). The deleterious effects of ROS on cell integrity, eventually may drive or participate in the development of diseases or pathological processes such as atherosclerosis, cancer, diabetes, rheumatoid arthritis, ischemia-reperfusion syndrome, cardiovascular disease, chronic inflammatory processes, shock Blackwater and other degenerative diseases in humans and animals (Freidovich, 1999; Fang et al., 2002). However, is hard to establish whether these reactive species represent a primary etiologic factor or product from the damaged tissue (Gonzalez et al., 1999).

The biochemical composition of the brain tissue makes it especially susceptible to the action of ROS/RNS, as it contains a pool of unsaturated lipids which are suitable to oxidative modification and lipid peroxidation. The double chains of unsaturated fatty acids are targeted for attack by free radicals, which initiate a cascade or chain reaction that damages these acids (Butterfield et al, 2002). Several researchers consider the brain as an organ highly susceptible to oxidative damage, and several studies have demonstrated how easy occurs the peroxidation of brain cell membranes (Chance, 1979). Also, the brain seems to be more susceptible than other organs to peroxidation, this is probably due to its high oxygen requirements, it uses 20% of the total oxygen intake, while weighing only 2% of the total body weight. Neuronal cells are considered the most susceptible cells to oxidative damage, owing to their low antioxidant activity in comparison to other tissues , as well as the high content of methyl ions in certain brain regions (Floyd, 1992). In this regard, some authors have suggested that the continuous state of oxidative stress may produce specific structural alterations of proteins, leading to abnormal protein aggregate formation, which is responsible for perpetuating oxidative damage. In fact, these abnormal proteins are considered as molecular markers of neurodegenerative diseases such as Alzheimer, Parkinson, and Cognitive Dysfunction Syndrome in geriatric dogs (Gallego et, al., 2010a, 2010b).

There are several risk factors involved in the development of these neuropathologies, one of them is the aging process "per se". In this regard, when the production of ROS / RNS has an exaggerated and prolonged increasing, and the antioxidant response is not enough, the system reach a different point of equilibrium (homeoresis, rather than homeostasis). This equilibrium is accompanied by high concentrations of free amino acids and differences in gene expression patterns, allowing survival but causing irreversible neuronal damage in the long term (Rivas et al., 2001). The original hypothesis of the free radicals in aging was proposed by Harman Gerschman and Harman in the 50´s (Sohal, 1993). The central dogma of this theory is that during the aerobic metabolism there is an incidental and uncontrollable production of oxygen radical species. Then, these species promote reactions damaging the macromolecules. This irreversible damage accumulates over time and results in a gradual loss of functional capacity of the cells (Hayflick, 1993). According to recent studies this might be related to neuronal processes of senescence (Passos, et al, 2010).

#### **2. Oxidative stress (OS) and neurodegenerative diseases (ND)**

There are plenty scientific evidence that considers the brain tissue as a preferential target for the accumulation of ROS and RNS, thereby triggering oxidative stress chronic conditions, which lead to injury and degeneration of neuronal cells (Contestabile, 2001). It has been

2010; Dorado et al., 2003), which in turn results in structural and functional alterations of organic macromolecules, leading the affected cells to degenerative processes and cell death by necrosis or apoptosis (D. Almaguer & L.E. Almaguer, 2006; Gonzalez et al. 1999; Martinez et al., 2010). The deleterious effects of ROS on cell integrity, eventually may drive or participate in the development of diseases or pathological processes such as atherosclerosis, cancer, diabetes, rheumatoid arthritis, ischemia-reperfusion syndrome, cardiovascular disease, chronic inflammatory processes, shock Blackwater and other degenerative diseases in humans and animals (Freidovich, 1999; Fang et al., 2002). However, is hard to establish whether these reactive species represent a primary etiologic factor or

The biochemical composition of the brain tissue makes it especially susceptible to the action of ROS/RNS, as it contains a pool of unsaturated lipids which are suitable to oxidative modification and lipid peroxidation. The double chains of unsaturated fatty acids are targeted for attack by free radicals, which initiate a cascade or chain reaction that damages these acids (Butterfield et al, 2002). Several researchers consider the brain as an organ highly susceptible to oxidative damage, and several studies have demonstrated how easy occurs the peroxidation of brain cell membranes (Chance, 1979). Also, the brain seems to be more susceptible than other organs to peroxidation, this is probably due to its high oxygen requirements, it uses 20% of the total oxygen intake, while weighing only 2% of the total body weight. Neuronal cells are considered the most susceptible cells to oxidative damage, owing to their low antioxidant activity in comparison to other tissues , as well as the high content of methyl ions in certain brain regions (Floyd, 1992). In this regard, some authors have suggested that the continuous state of oxidative stress may produce specific structural alterations of proteins, leading to abnormal protein aggregate formation, which is responsible for perpetuating oxidative damage. In fact, these abnormal proteins are considered as molecular markers of neurodegenerative diseases such as Alzheimer, Parkinson, and Cognitive Dysfunction Syndrome in geriatric dogs (Gallego et, al., 2010a,

There are several risk factors involved in the development of these neuropathologies, one of them is the aging process "per se". In this regard, when the production of ROS / RNS has an exaggerated and prolonged increasing, and the antioxidant response is not enough, the system reach a different point of equilibrium (homeoresis, rather than homeostasis). This equilibrium is accompanied by high concentrations of free amino acids and differences in gene expression patterns, allowing survival but causing irreversible neuronal damage in the long term (Rivas et al., 2001). The original hypothesis of the free radicals in aging was proposed by Harman Gerschman and Harman in the 50´s (Sohal, 1993). The central dogma of this theory is that during the aerobic metabolism there is an incidental and uncontrollable production of oxygen radical species. Then, these species promote reactions damaging the macromolecules. This irreversible damage accumulates over time and results in a gradual loss of functional capacity of the cells (Hayflick, 1993). According to recent studies this

There are plenty scientific evidence that considers the brain tissue as a preferential target for the accumulation of ROS and RNS, thereby triggering oxidative stress chronic conditions, which lead to injury and degeneration of neuronal cells (Contestabile, 2001). It has been

might be related to neuronal processes of senescence (Passos, et al, 2010).

**2. Oxidative stress (OS) and neurodegenerative diseases (ND)** 

product from the damaged tissue (Gonzalez et al., 1999).

2010b).

reported that the nervous system is rich in iron (Fe3 +) and unsaturated fatty acids (Halliwell, 2001), this is one of the main features that make this organ particularly sensitive to oxidative stress. Thus, the high lipid content of nervous tissue and its high metabolic activity make it particularly vulnerable to oxidative damage (T.M. Dawson & V.L. Dawson, 1996). It has been reported that iron is essential in the brain, particularly during development, but high amounts can lead to the generation of damage to brain cells, because Fe 3 + can lead to oxidative stress by catalyzing the formation processes of ROS (Butterfield et al, 2002; Gerlach et al, 1994). In addition, the brain consumes a high proportion of the total oxygen, since the energy it uses comes almost exclusively from oxidative metabolism which occurs in the mitochondrial respiratory chain located in the bodies, dendrites, axons and synaptic buttons of cells neural, where the ATPase maintains the ionic gradient across the neuronal membrane (Pavón et al., 1998). Another reason that explains why ROS are particularly active in the brain is that in this tissue, the metabolism of the aminoacids and neurotransmitters acts as an important source of these highly reactive molecules (Uttara et al., 2009). Thus, considering that this organ contains low levels of protective enzymes and other enzymatic antioxidants, it is easy to understand why their susceptibility (Contestabile, 2001).

Although oxidative stress is not the direct cause or the etiological factor responsible for the neuropathologies, it is known that it induces toxic effects by oxidizing lipids, proteins, carbohydrates and nucleotides. This causes accumulation of intracellular aggregates, mitochondrial dysfunction, excitotoxicity and apoptosis. Formation of modified lipids by oxidation can cause cellular dysfunction and death in postmitotic cells. Likewise, the peroxidation of polyunsaturated fatty acids in cell membranes, initiates a cumulative deterioration of membrane functions and causes a decrease in flow, reduction in the electrochemical potential and increased permeability of the membrane. Similarly, oxidation causes changes in the structure of some proteins and the formation of protein aggregates. These abnormal proteins induce oxidative damage that has been observed in neurodegenerative diseases as AD and PD. On another hand, ROS also can affect both glial cells and neuronal cells, which exhibit a particular sensitivity to free radicals and therefore are prone to cell degeneration processes (Gilgun-Sherki et al., 2001). For example, there is a biological phenomenon known as "selective neuronal vulnerability", which is involved in the response of different neuronal populations against neurodegenerative conditions. For example, neurons in the entorhinal cortex, the CA1 region of hippocampus, frontal cortex and amygdala, are highly susceptible populations to neurodegeneration associated with oxidative stress in Alzheimer's disease (AD) (Braak et al., 1991; Hyman et al, 1984; Terry et al, 2001).

The relationship between aging and neurodegenerative diseases is more than evident. This is supported by recent investigations that have observed that during the aging process there is an increase in the formation of H2O2 that alters the conditions of the electrons flow in the transport chain, facilitating their 'escape' of normal stream flow. This leads the neurons to suffer the harmful effect of free radicals. Moreover, studies performed by Sohal, et al., 1993, Benzi & Mareti, 1995, and others, have postulated that ROS can inflict damage to the mitochondrial inner membrane, the components of the electron transport chain or the mitochondrial DNA (mtDNA). Oxidative stress further increases ROS production and, consequently, damage to the mitochondria, creating a cycle that perpetuates the effects of ROS (González et al., 1999). Thus, it is obvious because aging itself is a key factor in diseases such as AD or PD; since the increase of oxidative damage, the progressive mitochondrial

Free Radicals, Neuronal Death and Neuroprotection 169

factors that may increase the risk of dementia and AD. In contrast, consumption of foods with antioxidants such as fruits, proteins rich in methionine and vitamins have been identified as protective factors against the disease. In this sense, it is possible that the variation in the diets may be associated with the prevalence of AD by geographic area, as several studies have suggested a link between nutrients of each diet and the presence of cognitive changes. However, it is necessary to confirm this experimentally (Ramesh et al.,

Experimental data suggest a relationship between the PD and two mitochondrial-specific conditions, dysfunction and oxidative damage to neuronal mitochondria. This assertion is supported by research suggesting mitochondrial dysfunction and impairment of mitochondrial complex I activity in the neurons of the substantia nigra and frontal cortex in PD patients. In addition, several genes whose mutations or polymorphisms increase the risk of developing PD are related to mitochondrial function. In this regard, dysfunction of mitochondrial complex I, becomes important, since its inhibition creates a biochemical environment that increases the production of FMNH semiquinone flavin, which increases the generation of O2 and in turn, the latter promotes lipid peroxidation, damage oxidative protein and protein nitration mediated by peroxynitrite (ONOO-) and nitrosylation. This is a process leading neurons to apoptosis and α-synuclein aggregation with subsequent death

According to Dorado et al., 2003, the substantia nigra has characteristics that tend to make it more vulnerable to attack by ROS. These features include low levels of glutathione and vitamin E, high levels of free iron (prooxidant), mono amine oxidase (MAO generates ROS), •NO (radical neurotoxic pro-oxidant) and neuromelanin. The neuromelanin is a black pigment found in certain subpopulations of monoaminergic neurons, and is the result of auto-oxidation, condensation and polymerization of dopamine and its oxidation products. Neuromelanin binds to any neutralizing reactive species, but can become a reservoir of toxic under certain conditions (oxidative attack, low levels of glutathione) releases these reactive species. Thus, neurons that contain greater amounts of neuromelanin in the substantia nigra

Glutathione (GSH) which is the most important intracellular molecule for the removal of hydroperoxides in the brain, is decreased in Parkinsonian patients. This could be related to the increased concentration of MDA (a marker of lipid oxidation). One of the facts that suggests the role of ROS in this disease is that they have identified high levels of glycosylation end products (AGE resulting from impaired glucose oxidation and cause irreversible oxidation of protein) in the substantia nigra and cortex. Also, it appears that a factor associated with oxidative damage is that the distribution of transition metals in the brain shows large regional differences, so that regions with large amounts of iron (which is easily oxidized) as the substantia nigra are at risk of suffer a more aggressive oxidative attack (Dorado et al., 2003). However, this is not the only mechanism to explain why dopaminergic neurons are especially sensitive to oxidative stress, because its high metabolic rate and the oxidation of dopamine, either by autoxidation or by the metabolic pathway by means of the MAO (Dorado et al., 2003), they represent a major source of ROS. Likewise, dopamine may act as metal chelator electron donors, and owing to its tendency towards reduction can initiate the Fenton reaction to generate H2O2. Some evidence suggests that mutations in the protein α-synuclein play a crucial role in

of dopaminergic neurons. (Navarro & Boveris, 2008, 2010).

2010).

**2.2 Parkinson disease** 

pars compacta die more easily.

failure accumulated with aging, and coupled with the characteristics of brain, make aging itself a common risk factor in neurodegeneration. Another fact that may explain the relationship between neurodegeneration and aging is that during aging can occurs certain level of iron accumulation, which increases oxidative stress. This occurs mainly through the Fenton reaction, where the production of highly reactive hydroxyl radicals ends up causing damage to DNA, lipids and proteins (Ang et al., 2010; González et al., 1999; Uttara et al., 2009).

In addition to the accumulation of free radicals that can occurs in the brain of individuals with ND, it is also clear that the antioxidant systems decrease during the aging process. Then, the action of ROS turns up in a process even more damaging to neuronal tissue. Scientific data suggest that all this deleterious effect seems to be magnified by pathological proteins such as beta-amyloid in AD and alpha-synuclein in PD, which act as initiators and perpetuators of the intraneuronal oxidative stress, generating injuries and disease-specific symptoms.

#### **2.1 Alzheimer disease**

According to some authors, the ROS in AD induce a prolonged increase pro-oxidant state (Benzi & Mareti, 1995). This statement is supported by clinical and experimental evidence (for example, high concentrations of Fe and Cu identified in the brains of some patients with AD), showing that ROS cause neuronal death and other neuropathological changes associated with this disease (González et al., 1999; Ramesh et al., 2010). However, the role of oxidative processes in AD is still a matter of debate, with conflicting and divergent data in the literature, which could be related to the difficulty of directly measuring the activity of ROS in biological systems due to their short half-life and its high reactivity (Gonzalez et al., 1999).

Several studies have identified the capacity of the protein βA as a chelating agent for transition metals (Cu2, Zn2 and Fe3). Regarding this, it is important to note that the binding of Cu2 and Fe3 provides free radicals OH , and toxic chemical reactions, due to the altered state of oxidation of these two metals, causing the appearance of catalytic H2O2 in the presence transition metal (Uttara et al., 2009). Additionally, the Fe3 in AD neurofibrillary plaque accumulates (NFT) and depots βA, which probably explains the evidence that suggests increased levels of Zn (II), Fe (III) and Cu (II) in the neuropil and senile plaques (Ramesh et al., 2010). Moreover, it appears that the Fe3 directly involved in plaque formation βA, and thus indirectly in the formation of ROS, as it promotes amyloidosis by modulating the ability of the α-secretase to cleave the amyloid precursor protein (APP), or to facilitate the aggregation of Aβ.

In 1994, Behl C. and colleagues showed that toxicity in AD is associated with the Aβ protein, which causes increased production of H2O2. These authors also showed that catalase blocks the toxicity of Aβ, which allows us to understand how the H2O2 and its derivatives such as OH-radical, cause lipid peroxidation leading to neuronal cell death in this disease (González et al., 1999). In addition, hemoxigenasa1 the Cu\ZnSOD and MnSOD, have been identified in neurofibrillary tangles of human brains with AD, suggesting a close interaction between ROS and the products from these enzimes. It has also been suggested that cellular toxicity of βA that is specifically related to damage by ROS or its products, generates insoluble protein aggregates, as is known, is a crucial event in AD (González et al., 1999).

Moreover, there is indirect evidence that ROS can relate to AD. For example, some dietary factors such as saturated fatty acids, high calories and heavy drinking have been reported as factors that may increase the risk of dementia and AD. In contrast, consumption of foods with antioxidants such as fruits, proteins rich in methionine and vitamins have been identified as protective factors against the disease. In this sense, it is possible that the variation in the diets may be associated with the prevalence of AD by geographic area, as several studies have suggested a link between nutrients of each diet and the presence of cognitive changes. However, it is necessary to confirm this experimentally (Ramesh et al., 2010).

#### **2.2 Parkinson disease**

168 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

failure accumulated with aging, and coupled with the characteristics of brain, make aging itself a common risk factor in neurodegeneration. Another fact that may explain the relationship between neurodegeneration and aging is that during aging can occurs certain level of iron accumulation, which increases oxidative stress. This occurs mainly through the Fenton reaction, where the production of highly reactive hydroxyl radicals ends up causing damage to DNA, lipids and proteins (Ang et al., 2010; González et al., 1999; Uttara et al.,

In addition to the accumulation of free radicals that can occurs in the brain of individuals with ND, it is also clear that the antioxidant systems decrease during the aging process. Then, the action of ROS turns up in a process even more damaging to neuronal tissue. Scientific data suggest that all this deleterious effect seems to be magnified by pathological proteins such as beta-amyloid in AD and alpha-synuclein in PD, which act as initiators and perpetuators of the intraneuronal oxidative stress, generating injuries and disease-specific

According to some authors, the ROS in AD induce a prolonged increase pro-oxidant state (Benzi & Mareti, 1995). This statement is supported by clinical and experimental evidence (for example, high concentrations of Fe and Cu identified in the brains of some patients with AD), showing that ROS cause neuronal death and other neuropathological changes associated with this disease (González et al., 1999; Ramesh et al., 2010). However, the role of oxidative processes in AD is still a matter of debate, with conflicting and divergent data in the literature, which could be related to the difficulty of directly measuring the activity of ROS in biological systems due to their short half-life and its high reactivity (Gonzalez et al.,

Several studies have identified the capacity of the protein βA as a chelating agent for transition metals (Cu2, Zn2 and Fe3). Regarding this, it is important to note that the binding of Cu2 and Fe3 provides free radicals OH , and toxic chemical reactions, due to the altered state of oxidation of these two metals, causing the appearance of catalytic H2O2 in the presence transition metal (Uttara et al., 2009). Additionally, the Fe3 in AD neurofibrillary plaque accumulates (NFT) and depots βA, which probably explains the evidence that suggests increased levels of Zn (II), Fe (III) and Cu (II) in the neuropil and senile plaques (Ramesh et al., 2010). Moreover, it appears that the Fe3 directly involved in plaque formation βA, and thus indirectly in the formation of ROS, as it promotes amyloidosis by modulating the ability of the α-secretase to cleave the amyloid precursor protein (APP), or

In 1994, Behl C. and colleagues showed that toxicity in AD is associated with the Aβ protein, which causes increased production of H2O2. These authors also showed that catalase blocks the toxicity of Aβ, which allows us to understand how the H2O2 and its derivatives such as OH-radical, cause lipid peroxidation leading to neuronal cell death in this disease (González et al., 1999). In addition, hemoxigenasa1 the Cu\ZnSOD and MnSOD, have been identified in neurofibrillary tangles of human brains with AD, suggesting a close interaction between ROS and the products from these enzimes. It has also been suggested that cellular toxicity of βA that is specifically related to damage by ROS or its products, generates insoluble protein

Moreover, there is indirect evidence that ROS can relate to AD. For example, some dietary factors such as saturated fatty acids, high calories and heavy drinking have been reported as

aggregates, as is known, is a crucial event in AD (González et al., 1999).

2009).

symptoms.

1999).

**2.1 Alzheimer disease** 

to facilitate the aggregation of Aβ.

Experimental data suggest a relationship between the PD and two mitochondrial-specific conditions, dysfunction and oxidative damage to neuronal mitochondria. This assertion is supported by research suggesting mitochondrial dysfunction and impairment of mitochondrial complex I activity in the neurons of the substantia nigra and frontal cortex in PD patients. In addition, several genes whose mutations or polymorphisms increase the risk of developing PD are related to mitochondrial function. In this regard, dysfunction of mitochondrial complex I, becomes important, since its inhibition creates a biochemical environment that increases the production of FMNH semiquinone flavin, which increases the generation of O2 and in turn, the latter promotes lipid peroxidation, damage oxidative protein and protein nitration mediated by peroxynitrite (ONOO-) and nitrosylation. This is a process leading neurons to apoptosis and α-synuclein aggregation with subsequent death of dopaminergic neurons. (Navarro & Boveris, 2008, 2010).

According to Dorado et al., 2003, the substantia nigra has characteristics that tend to make it more vulnerable to attack by ROS. These features include low levels of glutathione and vitamin E, high levels of free iron (prooxidant), mono amine oxidase (MAO generates ROS), •NO (radical neurotoxic pro-oxidant) and neuromelanin. The neuromelanin is a black pigment found in certain subpopulations of monoaminergic neurons, and is the result of auto-oxidation, condensation and polymerization of dopamine and its oxidation products. Neuromelanin binds to any neutralizing reactive species, but can become a reservoir of toxic under certain conditions (oxidative attack, low levels of glutathione) releases these reactive species. Thus, neurons that contain greater amounts of neuromelanin in the substantia nigra pars compacta die more easily.

Glutathione (GSH) which is the most important intracellular molecule for the removal of hydroperoxides in the brain, is decreased in Parkinsonian patients. This could be related to the increased concentration of MDA (a marker of lipid oxidation). One of the facts that suggests the role of ROS in this disease is that they have identified high levels of glycosylation end products (AGE resulting from impaired glucose oxidation and cause irreversible oxidation of protein) in the substantia nigra and cortex. Also, it appears that a factor associated with oxidative damage is that the distribution of transition metals in the brain shows large regional differences, so that regions with large amounts of iron (which is easily oxidized) as the substantia nigra are at risk of suffer a more aggressive oxidative attack (Dorado et al., 2003). However, this is not the only mechanism to explain why dopaminergic neurons are especially sensitive to oxidative stress, because its high metabolic rate and the oxidation of dopamine, either by autoxidation or by the metabolic pathway by means of the MAO (Dorado et al., 2003), they represent a major source of ROS. Likewise, dopamine may act as metal chelator electron donors, and owing to its tendency towards reduction can initiate the Fenton reaction to generate H2O2. Some evidence suggests that mutations in the protein α-synuclein play a crucial role in

Free Radicals, Neuronal Death and Neuroprotection 171

Due to this variety in the classification of antioxidants, a new classification has been proposed recently, which seeks to involve the full range of antioxidants based on aspects such as chemical nature and mechanism of action (Cui et al., 2004; Pérez, A. et al., 2008).

1. Antioxidant enzymes: They act on specific ROS, in order to change them into less

2. Preventive Antioxidants: These bind to promoters of oxidation and sequester transition metal ions such as iron and copper, which contain unpaired electrons and greatly accelerate the formation of free radicals. Examples of such antioxidants are transferrin and lactoferrin (Pauls & Thompson, 1980). Also, ceruloplasmin Cu hijacking to prevent the formation of free radicals from peroxides, catalyzes the oxidation of ferrous ions to ferric ions due to its ferroxidase activity, and increases the binding of iron to transferrin. In addition, the haptoglobins that bind to hemoglobin, hemopexin that binds heme

3. Antioxidant ROS sequestrant: These inhibit the initiation of chain reactions of free radicals or break the chain of spreading it. The main intracellular sequesters are vitamin E, carotene and coenzyme Q (Murthy, 2001), while extracellular sequesters include protein systems, and water-soluble compounds such as ascorbic acid, uric acid and

4. Nutritional Antioxidants: Diet is the major source of substances with antioxidant properties or elements for the synthesis of antioxidant enzymes. Several metals (copper, zinc, selenium, manganese, iron) are involved as components or cofactors of numerous enzymes antioxidants, and certain vitamins (ascorbic acid, α-tocopherol and β-carotene,

The different antioxidant systems work in a coordinated manner, following a series of metabolic processes where •O2-metabolized by superoxide dismutase SOD generates H2O2, and this in turn is metabolized to H2O and O2 by a catalase or glutathione peroxidase, which act as coupled with glutathione reductase (Dorado et al, 2003). However, it is difficult to think of a single molecular mechanism that acts as a regulator of the generation and the effects of FR, so some authors, for instance, Cadenas, 1997, propouse at least three types of molecular mechanisms that underlie the activities of various antioxidants. Such mechanisms are: (a) a process involving the transfer of the radical nature of ROS, together with the formation of a reactive radical, an antioxidant derived previously, (b) a similar process in which the transfer of the radical, and the formation of a stable or inert radical is carried out through enzymatic activity, and (c) mechanisms of action of small molecules that mimic the activity of enzymes such as SOD and GPx. These mechanisms describe to some extent the action of a variety of molecules with antioxidant properties, for example, enzymes such as SOD, CAT and GPx, which are responsible for initiating the process of neutralization of ROS by the dismutation of O•2 to H2O2 (D. Almaguer & L.E. Almaguer, 2006). Other defense mechanisms used by different antioxidants include, recycling of ROS/RNS or their precursors, inhibition of ROS formation, binding of metal ions required for catalysis of ROS generation and activation of endogenous antioxidant

According to some researchers (Halliwell, B., 1994, 1997; Cadenas, 1997; Dorado et al, 2003), the protective efficiency of this variety of antioxidants, is somewhat dependent on the type

harmful molecules. Examples of such enzymes are SOD, CAT, and GPx.

groups and albumin binds to heme and Cu (Benedetti et al., 1988).

bilirubin (Cui et al, 2004; Frei et al., 1988; Pérez, A. et al., 2008;).

folic acid) act as a sequestrant of ROS (Pérez, A. et al., 2008).

**3.2 Mechanisms of neutralization of ROS** 

defenses (Gilgun-Sherki et al.,2008).

Such a classification sets these compounds into the following groups:

modulating the activity of dopamine, but negatively, initiating neuronal cytoplasmic accumulation and interaction of dopamine with iron to cause the production of ROS (Uttara et al., 2009).

#### **3. Cellular defense systems against oxidative stress (OE)**

ROS and RNS molecules are well known for their deleterious effects on cellular integrity and their relation to different neurodegenerative diseases such as described in the previous section. However, it is important to note that these molecules also play a physiological role, quite distant from the aforementioned pathological role. In homeostatic physiological conditions their responses participate in cell signaling systems such as in the defense response against infectious agents and induction of mitogenic responses (Valko et al., 2007). These cellular responses bring the intra and extracellular environments to a continuous exposure to FR (González et al., 1999). Owing to this condition, aerobic organisms have evolved specific defense mechanisms, programmed to form cell protective barriers, which allow them to restrict the harmful effects of free radicals and neutralize the damage caused by the action of these reactive species (D. Almaguer & L.E. Almaguer, 2006; Cadenas, 1997; Contestabile, 2001; Perez et al., 2008; Valko et al., 2007; Vian et al., 1999). These mechanisms refer to antioxidants. These are molecules of different weights whose function is basically delay or inhibit oxidation. The mechanism is to transfer electrons to the reactive species to saturate its electron affinity, and thus, maintain ROS at a level compatible with metabolic processes and cellular functions (D. Almaguer & L.E. Almaguer, 2006; Contestabile, 2001; Viant, 1999).

#### **3.1 Classification of the main antioxidant systems**

Taking into account specific aspects of antioxidants, such as their chemical nature, mechanism of action or origin, several strategies have been proposed for classification. According to Gilgun-Sherki et al.2008, these compounds can be classified into two major groups: antioxidant enzymes and low molecular weight antioxidants. To the first group belong some antioxidant enzyme systems derived from enzymes as cytochrome oxidase, superoxide dismutase (Cu-ZnSOD and MnSOD), catalases and peroxidases such as glutathione peroxidase and glutathione reductase (Dorado et al., 2003). The group of low molecular weight antioxidants involves a variety of antioxidants, so some authors have subclassified this group into two: indirect antioxidants (eg, chelating agents) and direct antioxidants. The latter group is of great importance in combating oxidative stress and contains hundreds of components, however, only a minority of these molecules such as glutathione and NADPH are synthesized by the cell itself.

Another classification for antioxidant systems, refers to its source. In this sense, we can identify endogenous antioxidants and antioxidant of exogenous origin. The first group are vitamins such as ascorbic acid, tocopherol and retinoic acid also found glutathione in its reduced form, coenzyme Q10, melatonin, uric acid and lipoic acid. Also are included some isoforms of the enzyme superoxide dismutase (copper/zinc SOD, manganese SOD, extracellular SOD), catalase and glutathione peroxidase (Gilgun-Sherki et al.,2008; Chan, 2001; Contestabile, 2001). With respect to the group of antioxidants of exogenous origin, ie those that can only be obtained from external sources, we should mention some substances such as acetyl cysteine and carotenoids, which act as precursors of endogenous antioxidant type (Gilgun-Sherki et al.,2008; Chan, 2001; Contestabille, 2001).

modulating the activity of dopamine, but negatively, initiating neuronal cytoplasmic accumulation and interaction of dopamine with iron to cause the production of ROS

ROS and RNS molecules are well known for their deleterious effects on cellular integrity and their relation to different neurodegenerative diseases such as described in the previous section. However, it is important to note that these molecules also play a physiological role, quite distant from the aforementioned pathological role. In homeostatic physiological conditions their responses participate in cell signaling systems such as in the defense response against infectious agents and induction of mitogenic responses (Valko et al., 2007). These cellular responses bring the intra and extracellular environments to a continuous exposure to FR (González et al., 1999). Owing to this condition, aerobic organisms have evolved specific defense mechanisms, programmed to form cell protective barriers, which allow them to restrict the harmful effects of free radicals and neutralize the damage caused by the action of these reactive species (D. Almaguer & L.E. Almaguer, 2006; Cadenas, 1997; Contestabile, 2001; Perez et al., 2008; Valko et al., 2007; Vian et al., 1999). These mechanisms refer to antioxidants. These are molecules of different weights whose function is basically delay or inhibit oxidation. The mechanism is to transfer electrons to the reactive species to saturate its electron affinity, and thus, maintain ROS at a level compatible with metabolic processes and cellular functions (D. Almaguer & L.E. Almaguer, 2006; Contestabile, 2001;

Taking into account specific aspects of antioxidants, such as their chemical nature, mechanism of action or origin, several strategies have been proposed for classification. According to Gilgun-Sherki et al.2008, these compounds can be classified into two major groups: antioxidant enzymes and low molecular weight antioxidants. To the first group belong some antioxidant enzyme systems derived from enzymes as cytochrome oxidase, superoxide dismutase (Cu-ZnSOD and MnSOD), catalases and peroxidases such as glutathione peroxidase and glutathione reductase (Dorado et al., 2003). The group of low molecular weight antioxidants involves a variety of antioxidants, so some authors have subclassified this group into two: indirect antioxidants (eg, chelating agents) and direct antioxidants. The latter group is of great importance in combating oxidative stress and contains hundreds of components, however, only a minority of these molecules such as

Another classification for antioxidant systems, refers to its source. In this sense, we can identify endogenous antioxidants and antioxidant of exogenous origin. The first group are vitamins such as ascorbic acid, tocopherol and retinoic acid also found glutathione in its reduced form, coenzyme Q10, melatonin, uric acid and lipoic acid. Also are included some isoforms of the enzyme superoxide dismutase (copper/zinc SOD, manganese SOD, extracellular SOD), catalase and glutathione peroxidase (Gilgun-Sherki et al.,2008; Chan, 2001; Contestabile, 2001). With respect to the group of antioxidants of exogenous origin, ie those that can only be obtained from external sources, we should mention some substances such as acetyl cysteine and carotenoids, which act as precursors of endogenous antioxidant

**3. Cellular defense systems against oxidative stress (OE)** 

**3.1 Classification of the main antioxidant systems** 

glutathione and NADPH are synthesized by the cell itself.

type (Gilgun-Sherki et al.,2008; Chan, 2001; Contestabille, 2001).

(Uttara et al., 2009).

Viant, 1999).

Due to this variety in the classification of antioxidants, a new classification has been proposed recently, which seeks to involve the full range of antioxidants based on aspects such as chemical nature and mechanism of action (Cui et al., 2004; Pérez, A. et al., 2008). Such a classification sets these compounds into the following groups:


#### **3.2 Mechanisms of neutralization of ROS**

The different antioxidant systems work in a coordinated manner, following a series of metabolic processes where •O2-metabolized by superoxide dismutase SOD generates H2O2, and this in turn is metabolized to H2O and O2 by a catalase or glutathione peroxidase, which act as coupled with glutathione reductase (Dorado et al, 2003). However, it is difficult to think of a single molecular mechanism that acts as a regulator of the generation and the effects of FR, so some authors, for instance, Cadenas, 1997, propouse at least three types of molecular mechanisms that underlie the activities of various antioxidants. Such mechanisms are: (a) a process involving the transfer of the radical nature of ROS, together with the formation of a reactive radical, an antioxidant derived previously, (b) a similar process in which the transfer of the radical, and the formation of a stable or inert radical is carried out through enzymatic activity, and (c) mechanisms of action of small molecules that mimic the activity of enzymes such as SOD and GPx. These mechanisms describe to some extent the action of a variety of molecules with antioxidant properties, for example, enzymes such as SOD, CAT and GPx, which are responsible for initiating the process of neutralization of ROS by the dismutation of O•2 to H2O2 (D. Almaguer & L.E. Almaguer, 2006). Other defense mechanisms used by different antioxidants include, recycling of ROS/RNS or their precursors, inhibition of ROS formation, binding of metal ions required for catalysis of ROS generation and activation of endogenous antioxidant defenses (Gilgun-Sherki et al.,2008).

According to some researchers (Halliwell, B., 1994, 1997; Cadenas, 1997; Dorado et al, 2003), the protective efficiency of this variety of antioxidants, is somewhat dependent on the type

Free Radicals, Neuronal Death and Neuroprotection 173

and antioxidant compounds, mechanisms to develop or enhance protective responses against a constant state of oxidative stress. In addition, people generally consider that there are several advantages to the use of antioxidants, in fact, there are a consumer culture around these compounds, where people attempt to consume diets rich in antioxidants and/or supplement their diet with one or more of these substances to "improve living conditions for the geriatric stage" (Mullie et al., 2009). Several studies have shown that diet has a long-term effect on general health (Leibson et al., 1997; Uttara et al., 2009; Vermeer, et al., 2003), acting as a principal source of natural antioxidants due to its ability to provide a variety of molecules that activate or enhancer the action of some endogenous antioxidants (Sun et al., 2008). In this sense, it is important to consider the effect of these molecules on processes of neuroprotection, since according to some reports, the diet also has the ability to extend human cognitive longevity (Peter, et al, 2004; Casseta, 2005, Glade, 2010; Ramesh et al., 2010). There are several antioxidant research in the field of ND, such as AD and PD, but despite the variability of results and poor clinical trials, diets rich in vitamins and other natural antioxidants still seem to be publicly recognized for their action as useful supplements in reducing risk of suffering some types of dementia (Kamphuisa &

Evidence of this can be seen in some studies indicating that Indian diets, which contain spices like red chilli, coriander and turmeric (plant widely used as food preservative and medicinal), apparently reduced the prevalence of patients with AD in India, since its incidence is 4.4 times lower compared to countries like the U.S. (James et al., 2009; Ramesh et al., 2010). Additionally, some reports indicate supplementing diets high in fat that usually lead to the development of cardiovascular diseases with a high intake of dietary antioxidants, such as polyphenols, may reduce the risk of disease. Likewise, consumption of nutritional substances such as berries, nuts or fish oil can dramatically impact on the aging brain, possibly leading to improved motor and cognitive skills (James, et al, 2009). In this context, the interest in finding low-cost therapeutic alternatives that improve living conditions and reduce the risk of age-related diseases has led to the identification of a growing list of antioxidant supplements as Vitamins C and E, β-carotene, coenzyme Q, ascorbate and polyphenols, among others (Burton & Ingold, 1989, 1990; Kelsey et al.,

Natural antioxidants can act as a therapeutic tool against excess ROS, because several of them have a high ability to cross the blood brain barrier (Uttara et al., 2009), and can activate various antioxidant mechanisms in the brain in order to create conditions to achieve and maintain the neuronal homeostasis (Gilgun-Sherki et al., 2001). However, an antioxidant substance that has some use in preventing the ND must have some additional capabilities to its ability to sequester free radicals. For example, one of the main AO found in mammalian cells is glutathione (GSH), however, this substance, despite its large capacity anti FR can´t be directly used for supplementation, due to their inability to cross the bloodbrain barrier and reach the brain tissue (Witschi et al., 1992). Despite this, some of its precursors or analogs have been tested in various animal models (Contestabile, 2001). Thus, studies such as Martinez, et al., 2000, suggest that long-term supplementation with GSH precursors such as N-acetylcysteine can partially restore the impaired memory and decreased mitochondrial lipid peroxidation, characteristic of aging process. (Contestabile,

**4.2 Scientific evidence of AO supplementation in the treatment of ND** 

Scheltensb, 2010).

2010).

2001; Prasad et al., 1999).

of ROS generated, the place where they are produced (physical barriers like the blood-brain barrier permeability reduce many antioxidants) and the severity of cell damage.

#### **4. Antioxidants and neurodegeneration**

During the last decades in many populations around the world there has been a notable increase in the number of adults over 60´s (United Nations [ONU], 2009). Consequently, there has been an increase in the incidence and prevalence of various diseases affecting the elders. Among this group of diseases, the neurodegenerative type have become very important, especially in industrialized countries, which are listed as the third leading cause of death after cardiovascular diseases and cancer (Boyd, 2000; Gallego et, al., 2010a; United Nations [ONU], 2009; Segura, 2003; Troenes B, et al 2003). However, the ND related to aging, not only represent a problem for human health because there are substantial data showing that aging also predisposes other species to suffer dementia syndromes. For example, some old dogs can develop neuropathology similar to AD, known as Cognitive Dysfunction Syndrome in Senior Dogs (CDS) or "dog´s Alzheimer". This disease affects dogs over 7 years old and due to its clinical and pathophysiological similarities with AD has been proposed as a model for doing research in the field of neurodegeneration, especially in the study of AD (Adams B, et al., 2000; Gallego, 2010b; Overall, 2001; Ruehl et al., 1995).

As already mentioned, the incidence of ND increases with age, however, we must clarify that the risk of having them can be determined, partially, by factors such as lifestyle, obesity, metabolic syndrome, genetic susceptibility, predisposing medical factors and increased oxidative stress, among others, but the last one is the main factor related to the presentation of these diseases (Contestabile, 2001; James et al., 2009; Kelsey et al., 2010; Uttara et al., 2009;). In this sense, today it seems clear the role of oxidative stress during the onset and course of ND associated with aging (Meydani et al., 1998; Passos et al., 2010). So, recognizing this fact has allowed us to identify therapeutic targets where the activation of cellular mechanisms of antioxidant type appear to be a suitable option for the treatment of neurodegenerative diseases such as AD, PD and CDS (Casetta et al., 2005; James et al., 2009, Contestabile, 2001). In general terms, the main goal of the antioxidant therapy for these diseases is to interrupt or modulate the interaction of pathological neuronal protein (Aβ protein and Tau protein) with redox metals. This is in order to prevent damage or decomposition of metalloenzymes, and innate antioxidant systems by promoting homeostasis of metals and minimizing the OE and its effects (Uttara et al., 2009). Additionally, several studies suggest that increasing cellular protection through the use of antioxidants could be beneficial for maintaining or reducing the rate of neuronal death during the course of certain ND, such as PD (Casseta et al., 2005; Kelsey et al., 2010).

Thus, taking into account the growing interest in antioxidant therapy for the treatment and/or prevention of ND, and considering that diet is a main source of natural Anti-Oxidants (AO), then we present a systematic review of scientific evidence showing the action and effect of some food products on the course or the beginning of the characteristic lesions of this type of pathology.

#### **4.1 The role of diet as a preventive factor against the development of ND**

Given that the lifestyle and the type of diet can act as important risk factors for the emergence of various diseases (Kalaria et al., 2008), the relationship between a particular diet and its effects long-term research center have been some authors who seek through diet

of ROS generated, the place where they are produced (physical barriers like the blood-brain

During the last decades in many populations around the world there has been a notable increase in the number of adults over 60´s (United Nations [ONU], 2009). Consequently, there has been an increase in the incidence and prevalence of various diseases affecting the elders. Among this group of diseases, the neurodegenerative type have become very important, especially in industrialized countries, which are listed as the third leading cause of death after cardiovascular diseases and cancer (Boyd, 2000; Gallego et, al., 2010a; United Nations [ONU], 2009; Segura, 2003; Troenes B, et al 2003). However, the ND related to aging, not only represent a problem for human health because there are substantial data showing that aging also predisposes other species to suffer dementia syndromes. For example, some old dogs can develop neuropathology similar to AD, known as Cognitive Dysfunction Syndrome in Senior Dogs (CDS) or "dog´s Alzheimer". This disease affects dogs over 7 years old and due to its clinical and pathophysiological similarities with AD has been proposed as a model for doing research in the field of neurodegeneration, especially in the

barrier permeability reduce many antioxidants) and the severity of cell damage.

study of AD (Adams B, et al., 2000; Gallego, 2010b; Overall, 2001; Ruehl et al., 1995).

during the course of certain ND, such as PD (Casseta et al., 2005; Kelsey et al., 2010).

**4.1 The role of diet as a preventive factor against the development of ND** 

Thus, taking into account the growing interest in antioxidant therapy for the treatment and/or prevention of ND, and considering that diet is a main source of natural Anti-Oxidants (AO), then we present a systematic review of scientific evidence showing the action and effect of some food products on the course or the beginning of the characteristic

Given that the lifestyle and the type of diet can act as important risk factors for the emergence of various diseases (Kalaria et al., 2008), the relationship between a particular diet and its effects long-term research center have been some authors who seek through diet

As already mentioned, the incidence of ND increases with age, however, we must clarify that the risk of having them can be determined, partially, by factors such as lifestyle, obesity, metabolic syndrome, genetic susceptibility, predisposing medical factors and increased oxidative stress, among others, but the last one is the main factor related to the presentation of these diseases (Contestabile, 2001; James et al., 2009; Kelsey et al., 2010; Uttara et al., 2009;). In this sense, today it seems clear the role of oxidative stress during the onset and course of ND associated with aging (Meydani et al., 1998; Passos et al., 2010). So, recognizing this fact has allowed us to identify therapeutic targets where the activation of cellular mechanisms of antioxidant type appear to be a suitable option for the treatment of neurodegenerative diseases such as AD, PD and CDS (Casetta et al., 2005; James et al., 2009, Contestabile, 2001). In general terms, the main goal of the antioxidant therapy for these diseases is to interrupt or modulate the interaction of pathological neuronal protein (Aβ protein and Tau protein) with redox metals. This is in order to prevent damage or decomposition of metalloenzymes, and innate antioxidant systems by promoting homeostasis of metals and minimizing the OE and its effects (Uttara et al., 2009). Additionally, several studies suggest that increasing cellular protection through the use of antioxidants could be beneficial for maintaining or reducing the rate of neuronal death

**4. Antioxidants and neurodegeneration** 

lesions of this type of pathology.

and antioxidant compounds, mechanisms to develop or enhance protective responses against a constant state of oxidative stress. In addition, people generally consider that there are several advantages to the use of antioxidants, in fact, there are a consumer culture around these compounds, where people attempt to consume diets rich in antioxidants and/or supplement their diet with one or more of these substances to "improve living conditions for the geriatric stage" (Mullie et al., 2009). Several studies have shown that diet has a long-term effect on general health (Leibson et al., 1997; Uttara et al., 2009; Vermeer, et al., 2003), acting as a principal source of natural antioxidants due to its ability to provide a variety of molecules that activate or enhancer the action of some endogenous antioxidants (Sun et al., 2008). In this sense, it is important to consider the effect of these molecules on processes of neuroprotection, since according to some reports, the diet also has the ability to extend human cognitive longevity (Peter, et al, 2004; Casseta, 2005, Glade, 2010; Ramesh et al., 2010). There are several antioxidant research in the field of ND, such as AD and PD, but despite the variability of results and poor clinical trials, diets rich in vitamins and other natural antioxidants still seem to be publicly recognized for their action as useful supplements in reducing risk of suffering some types of dementia (Kamphuisa & Scheltensb, 2010).

Evidence of this can be seen in some studies indicating that Indian diets, which contain spices like red chilli, coriander and turmeric (plant widely used as food preservative and medicinal), apparently reduced the prevalence of patients with AD in India, since its incidence is 4.4 times lower compared to countries like the U.S. (James et al., 2009; Ramesh et al., 2010). Additionally, some reports indicate supplementing diets high in fat that usually lead to the development of cardiovascular diseases with a high intake of dietary antioxidants, such as polyphenols, may reduce the risk of disease. Likewise, consumption of nutritional substances such as berries, nuts or fish oil can dramatically impact on the aging brain, possibly leading to improved motor and cognitive skills (James, et al, 2009). In this context, the interest in finding low-cost therapeutic alternatives that improve living conditions and reduce the risk of age-related diseases has led to the identification of a growing list of antioxidant supplements as Vitamins C and E, β-carotene, coenzyme Q, ascorbate and polyphenols, among others (Burton & Ingold, 1989, 1990; Kelsey et al., 2010).

#### **4.2 Scientific evidence of AO supplementation in the treatment of ND**

Natural antioxidants can act as a therapeutic tool against excess ROS, because several of them have a high ability to cross the blood brain barrier (Uttara et al., 2009), and can activate various antioxidant mechanisms in the brain in order to create conditions to achieve and maintain the neuronal homeostasis (Gilgun-Sherki et al., 2001). However, an antioxidant substance that has some use in preventing the ND must have some additional capabilities to its ability to sequester free radicals. For example, one of the main AO found in mammalian cells is glutathione (GSH), however, this substance, despite its large capacity anti FR can´t be directly used for supplementation, due to their inability to cross the bloodbrain barrier and reach the brain tissue (Witschi et al., 1992). Despite this, some of its precursors or analogs have been tested in various animal models (Contestabile, 2001). Thus, studies such as Martinez, et al., 2000, suggest that long-term supplementation with GSH precursors such as N-acetylcysteine can partially restore the impaired memory and decreased mitochondrial lipid peroxidation, characteristic of aging process. (Contestabile, 2001; Prasad et al., 1999).

Free Radicals, Neuronal Death and Neuroprotection 175

Additionally, some authors suggest that the severity of demyelination and neuronal necrosis is reduced in brain areas such as cortex, hippocampus, cerebellar cortex and optic nerve of rats consuming acetyl-L-carnitine (Ramacci et al., 1998; Glade, 2010). Likewise, the use of acetyl-L-carnitine as a dietary supplement to improve cognitive longevity has been demonstrated by the results of a study by Passeri et al., 1990, in which two parallel groups and assessed homogeneous subjects of both sexes aged 65 years and with mild cognitive functions. One group was supplemented with 2 gr/day of acetyl-L-carnitine for three months, while the other group was treated with a placebo. The group of patients treated with acetyl-L-carnitine showed a significant improvement in learning abilities, and longterm memory skills, suggesting the therapeutic importance of this metabolite for the

Besides the natural AO, there is also a growing list of synthetic AO that have been widely studied, for example, conjugated forms of the enzymes SOD and CAT (Greenwald, 1990) and supplements of selenium (Parnham et al., 1991). Similarly, there is growing evidence that give, some drugs with different therapeutic uses to neuroprotection such as probucol (hypocholesterolemic) and salicylates, a certain capacity as recycler of free radicals under

However, despite the existing literature suggesting beneficial effects of AO, there are several questions regarding the therapeutic value that some natural AO may have compared to pathological processes such as AD and PD. There are conflicting data emerging from research on in vitro and in vivo neurodegeneration models, and some authors argue that the fact that one type of antioxidant molecule is produced physiologically or is taken normally from the diet is not a guarantee that this supplementation can be safe and advantageous therapeutic standpoint, since it must be took into account the physiological regulation of the redox state of the cell (Contestabile, 2001). In conclusion, experimental data are converging in terms of the therapeutic benefits of various natural antioxidants with neuroprotective capabilities, however, according to some authors, there are very few clinical data to demonstrate a clear and lasting effect of this type of treatments. Some possible reasons for such a disadvantage might be specific criteria such as dose, stability, duration of treatment, side effects and ability to cross the blood brain barrier, among others (Contestabile, 2001; James, 2009; Meydani et al., 1998). Because of this, it is essential to expand research on the use of AO diet supplements

experimental conditions (Cui et al., 2004; Juliano et al., 1995; Zhao et al., 1995).

compared to the onset and development of ND as AD and PD.

**4.3 Dietary supplements with antioxidant used in the management of ND** 

prevent the onset of neurodegenerative conditions such as AD or PD.

Considering the cascade of degenerative events of diseases such as AD and PD (degradation and abnormal folding of proteins, inadequate energy production in CNS degeneration by oxidative damage, excitotoxicity and inflammation), there is interest in whether the AO, or changes in eating habits can prevent and/or block one or more of these pathways of neurodegeneration in slowing the progression of the disease (Mazzio, et al., 2011). In this regard, several studies have suggested some properties of antioxidants as protective factors against the risk of diseases such as AD and PD. In addition, it is considered that changes in the concentration of AO in states of neurodegeneration may be a primary event or secondary to the ingestion of a particular type of diet (Kedar, 2003). Therefore, considering the usefulness of certain AO and its possible uses in therapy of ND, the following describes some of the main AO that are consumed in the diet or supplemented in order to treat or

treatment of geriatric patients with mental disfunction.

The use of AO decreases oxidative damage and also reduce the cognitive decline associated with age, this in both human and animal models (Joseph et al., 1998; Milgram et al., 2002). Examples are the studies conducted in geriatric dogs, which indicate that oxidative damage may be related to cognitive dysfunction and that long-term treatment with AO, with a behavioral enrichment program, reduce cognitive decline in dogs CDS (Cotman et al., 2002; Gallego et al, 2010a; Head E, 2002; Landsberg, 2005). Also, some authors argue that the intake of fruits and vegetables may reduce the risk of cognitive decline associated with aging in rodents, dogs and even humans, attributing that property to the capabilities of some antioxidants and anti-inflammatory compounds found in these foods (Araujo et al., 2005; Gallego et al., 2010; Landsberg, 2005; Opii et al., 2008).

With regard to vitamins, their results seem contradictory. Studies conducted with various vitamins have suggested these compounds as potential protective factors against states of neurodegeneration. Thus, recently demonstrated that supplementation of Vitamin E 500UI long term in the rats diet can protect against cognitive decline associated with aging (Morris, et al., 2005; Peterson et al., 2005). However, other studies in this category showed contrasting results, for example, a research conducted by Young KW, et, al, 2005, which included subjects with mild cognitive impairment, who were given a daily dose of 2.000UI of Vitamin E and 10 mg of donepezil (compound with anticholinergic activity) or a placebo for a period of three years. The results of this study showed that the overall rate of progression from mild cognitive impairment to clinical expression of AD was 16% per year, and no difference was found between the subjects who were administered placebo, and subjects who received vitamin E, suggesting a disagreement over the validity of vitamin supplements to patients with AD (Kelsey et al., 2010).

With respect to the PD, some of the AO compounds that have been suggested as protective factors include vitamins A, C (3000 mg/d) and E (3200 mg/d) (Fahn, 1991). Several epidemiological studies have shown that consumption of these vitamins may improve cognition and reduce the risk of developing clinical symptoms characteristic of this disease (Masaki et al., 1994). Likewise, the study by Chen et al, 1997, showed that administration of coenzyme Q10 improved clinical symptoms in patients with mitochondrial encephalopathy. Similarly, Birkmayer, et al, 1990, in a study of 415 patients with PD showed that administration of a dose of 1.4 mg/kg NADH can be an effective therapeutic tool in the treatment of PD. According to some authors, the efficiency of AO such as vitamins E and C, is the most convincing evidence of the involvement of free radicals in PD. However, in order to determine the efficacy of an antioxidant treatment, it is necessary to perform additional studies including high doses of vitamin E (3200 mg/d) in combination with vitamin C (3000 mg/d), before the administration of levodopa in patients with early PD (Fahn, 1991; Rao & Balachandran, 2002).

Acetyl-L-carnitine as a metabolite of vitamins involved in the process of synaptic transmission and has a potent neuroprotective effect can reduce the structural damage caused by states of oxidative stress in neurons. Neuroprotective capacity of this metabolite is evidenced through the increase in resistance to oxidation of cellular components such as mitochondrial RNA, and various proteins (Sharman et al., 2002; Poon et al., 2006). Previous research has shown that the brains of old rats respond positively to the diets supplemented with acetyl-L-carnitine in the long term. This effect is generated by activating the expression of intracellular enzymes such as GSH and SOD, which leads to a reduction in the formation of 4-hydroxynonenal in the mitochondria, and thus decreases the degree of carboxylation and oxidative nitrosylation mitochondrial protein (Calabrese, et al., 2006; Poon et al., 2006).

The use of AO decreases oxidative damage and also reduce the cognitive decline associated with age, this in both human and animal models (Joseph et al., 1998; Milgram et al., 2002). Examples are the studies conducted in geriatric dogs, which indicate that oxidative damage may be related to cognitive dysfunction and that long-term treatment with AO, with a behavioral enrichment program, reduce cognitive decline in dogs CDS (Cotman et al., 2002; Gallego et al, 2010a; Head E, 2002; Landsberg, 2005). Also, some authors argue that the intake of fruits and vegetables may reduce the risk of cognitive decline associated with aging in rodents, dogs and even humans, attributing that property to the capabilities of some antioxidants and anti-inflammatory compounds found in these foods (Araujo et al.,

With regard to vitamins, their results seem contradictory. Studies conducted with various vitamins have suggested these compounds as potential protective factors against states of neurodegeneration. Thus, recently demonstrated that supplementation of Vitamin E 500UI long term in the rats diet can protect against cognitive decline associated with aging (Morris, et al., 2005; Peterson et al., 2005). However, other studies in this category showed contrasting results, for example, a research conducted by Young KW, et, al, 2005, which included subjects with mild cognitive impairment, who were given a daily dose of 2.000UI of Vitamin E and 10 mg of donepezil (compound with anticholinergic activity) or a placebo for a period of three years. The results of this study showed that the overall rate of progression from mild cognitive impairment to clinical expression of AD was 16% per year, and no difference was found between the subjects who were administered placebo, and subjects who received vitamin E, suggesting a disagreement over the validity of vitamin

With respect to the PD, some of the AO compounds that have been suggested as protective factors include vitamins A, C (3000 mg/d) and E (3200 mg/d) (Fahn, 1991). Several epidemiological studies have shown that consumption of these vitamins may improve cognition and reduce the risk of developing clinical symptoms characteristic of this disease (Masaki et al., 1994). Likewise, the study by Chen et al, 1997, showed that administration of coenzyme Q10 improved clinical symptoms in patients with mitochondrial encephalopathy. Similarly, Birkmayer, et al, 1990, in a study of 415 patients with PD showed that administration of a dose of 1.4 mg/kg NADH can be an effective therapeutic tool in the treatment of PD. According to some authors, the efficiency of AO such as vitamins E and C, is the most convincing evidence of the involvement of free radicals in PD. However, in order to determine the efficacy of an antioxidant treatment, it is necessary to perform additional studies including high doses of vitamin E (3200 mg/d) in combination with vitamin C (3000 mg/d), before the administration of levodopa in patients with early PD (Fahn, 1991;

Acetyl-L-carnitine as a metabolite of vitamins involved in the process of synaptic transmission and has a potent neuroprotective effect can reduce the structural damage caused by states of oxidative stress in neurons. Neuroprotective capacity of this metabolite is evidenced through the increase in resistance to oxidation of cellular components such as mitochondrial RNA, and various proteins (Sharman et al., 2002; Poon et al., 2006). Previous research has shown that the brains of old rats respond positively to the diets supplemented with acetyl-L-carnitine in the long term. This effect is generated by activating the expression of intracellular enzymes such as GSH and SOD, which leads to a reduction in the formation of 4-hydroxynonenal in the mitochondria, and thus decreases the degree of carboxylation and oxidative nitrosylation mitochondrial protein (Calabrese, et al., 2006; Poon et al., 2006).

2005; Gallego et al., 2010; Landsberg, 2005; Opii et al., 2008).

supplements to patients with AD (Kelsey et al., 2010).

Rao & Balachandran, 2002).

Additionally, some authors suggest that the severity of demyelination and neuronal necrosis is reduced in brain areas such as cortex, hippocampus, cerebellar cortex and optic nerve of rats consuming acetyl-L-carnitine (Ramacci et al., 1998; Glade, 2010). Likewise, the use of acetyl-L-carnitine as a dietary supplement to improve cognitive longevity has been demonstrated by the results of a study by Passeri et al., 1990, in which two parallel groups and assessed homogeneous subjects of both sexes aged 65 years and with mild cognitive functions. One group was supplemented with 2 gr/day of acetyl-L-carnitine for three months, while the other group was treated with a placebo. The group of patients treated with acetyl-L-carnitine showed a significant improvement in learning abilities, and longterm memory skills, suggesting the therapeutic importance of this metabolite for the treatment of geriatric patients with mental disfunction.

Besides the natural AO, there is also a growing list of synthetic AO that have been widely studied, for example, conjugated forms of the enzymes SOD and CAT (Greenwald, 1990) and supplements of selenium (Parnham et al., 1991). Similarly, there is growing evidence that give, some drugs with different therapeutic uses to neuroprotection such as probucol (hypocholesterolemic) and salicylates, a certain capacity as recycler of free radicals under experimental conditions (Cui et al., 2004; Juliano et al., 1995; Zhao et al., 1995).

However, despite the existing literature suggesting beneficial effects of AO, there are several questions regarding the therapeutic value that some natural AO may have compared to pathological processes such as AD and PD. There are conflicting data emerging from research on in vitro and in vivo neurodegeneration models, and some authors argue that the fact that one type of antioxidant molecule is produced physiologically or is taken normally from the diet is not a guarantee that this supplementation can be safe and advantageous therapeutic standpoint, since it must be took into account the physiological regulation of the redox state of the cell (Contestabile, 2001). In conclusion, experimental data are converging in terms of the therapeutic benefits of various natural antioxidants with neuroprotective capabilities, however, according to some authors, there are very few clinical data to demonstrate a clear and lasting effect of this type of treatments. Some possible reasons for such a disadvantage might be specific criteria such as dose, stability, duration of treatment, side effects and ability to cross the blood brain barrier, among others (Contestabile, 2001; James, 2009; Meydani et al., 1998). Because of this, it is essential to expand research on the use of AO diet supplements compared to the onset and development of ND as AD and PD.

#### **4.3 Dietary supplements with antioxidant used in the management of ND**

Considering the cascade of degenerative events of diseases such as AD and PD (degradation and abnormal folding of proteins, inadequate energy production in CNS degeneration by oxidative damage, excitotoxicity and inflammation), there is interest in whether the AO, or changes in eating habits can prevent and/or block one or more of these pathways of neurodegeneration in slowing the progression of the disease (Mazzio, et al., 2011). In this regard, several studies have suggested some properties of antioxidants as protective factors against the risk of diseases such as AD and PD. In addition, it is considered that changes in the concentration of AO in states of neurodegeneration may be a primary event or secondary to the ingestion of a particular type of diet (Kedar, 2003). Therefore, considering the usefulness of certain AO and its possible uses in therapy of ND, the following describes some of the main AO that are consumed in the diet or supplemented in order to treat or prevent the onset of neurodegenerative conditions such as AD or PD.

Free Radicals, Neuronal Death and Neuroprotection 177

the most active and widely distributed (Elejalde, 2001); it is found naturally in foods such as vegetable oils, fats, vegetables, egg yolks, nuts, seeds, fruits and green vegetables (Berman & Brodaty, 2004). In addition to its antioxidant properties, also has capabilities such as specific enzymes regulating agent, anti-inflammatory and neuroprotective (Martin et al., 1999). Due to its neuroprotective properties, researchers worldwide have taken a particular interest in elucidating their protective mechanisms, as opposed to development of cognitive

The neuroprotective effect of vitamin E was first described in 1992 in an in vitro study using neuronal cells cultures (Behl C, et al, 1992). However, in order to know whether dietary supplementation with antioxidants can increase their brain levels, it is essential to investigate the effect of such molecules in animal models of ND like the EP (Prasad et al., 1999). In this regard, previous studies suggest that long-term feeding of rats (6 to 15 months of age) with a dietary supplement of 500 IU of vitamin E, induces protective effects against cognitive deficit related to age (Morris et al., 2005; Peterson et al., 2005). Similarly, it has been reported that supplementation with vitamin E in combination with vitamin C, increases the concentration of these vitamins in plasma and cerebrospinal fluid, where the resistance of lipoproteins to oxidation in in vitro studies is increased (Kontush et al, 2001; Casetta et al., 2005). It has also been reported that dietary supplementation with αtocopherol (1000 IU/day) for four months increases brain levels of vitamin E in rats (Vatassery et al., 1988), as in the brain and cerebrospinal fluid of dogs treated during two years (SR Pillai, 1993). These results suggest that dietary supplementation with vitamin E

On the other hand, one of the largest studies with antioxidants as a treatment for AD, included 341 patients with moderate dementia who were given 4 different treatments: 10mg/day of selegiline (a selective MAO inhibitor), 2000 IU daily α-tocopherol, a combination of selegiline and-tocopherol, or placebo. The effect of the treatments was assessed through the presentation of clinical signs such as loss of ability to perform basic activities of daily living, severe dementia or death. After the treatments the authors observed a significant delay in the time of presentation of clinical signs in patients treated with selegiline, alpha-tocopherol, or in combination compared with placebo (Sano, 1996, 1997). However, there was no additive effect of selegiline and vitamin E, possibly due to a common mechanism of action in which lower levels of free radicals, and prevents its formation through inhibition of oxidative metabolism of catecholamines (Casetta et al., 2005). Similarly, studies by Behl, C., 2000, suggest a neuroprotective activity of both, the natural tocopherol and the synthetic. In addition, the effect appears to be superior to that described for estradiol (powerful antioxidant), in terms of the ability of neuronal protection against oxidative damage generated by β-amyloid protein in AD. Additionally, there are studies that seek to enhance the antioxidant effect of tocopherol, in combination with other vitamins such as Vitamin C. For example, in a prospective study in the Netherlands (Engelhart et al., 2002), they used a population of 5395 subjects at least 55 years, where the high intake of vitamin C and E was associated with a lower risk of developing AD after a follow up of 6.5 years. The relative risk was 0. for vitamin E, and 0.82 for vitamin C (Casetta

With respect to the source, the vitamin E supplements do not appear to offer better results than those obtained by eating foods rich in this vitamin (Kontush & Schekatolina, 2004). This may be due to the cumulative and synergistic, as the bioavailability of vitamins is

dysfunction related to aging and various ND (Table 1) (Ramesh et al, 2010).

may be valuable in animal models of PD (Prasad et al., 1999).

et al., 2005).

#### **4.3.1 Vitamins**

#### **4.3.1.1 Vitamin E**

Vitamin E or tocopherol is a powerful antioxidant capable of stopping the spread of the chain reaction of free radicals in the lipid portion of cell membranes, inhibiting lipid peroxidation in plasma membrane phospholipids (Mandel et al., 2003; McCay, 1985; Gilgun-Sherki et al., 2001). There are eight tocopherols with vitamin E activity, and α-tocopherol is


Table 1. Vitamin E or Tocopherol starring in several studies evaluating the antioxidant supplementation and its effects on cognitive deficits related with ND

Vitamin E or tocopherol is a powerful antioxidant capable of stopping the spread of the chain reaction of free radicals in the lipid portion of cell membranes, inhibiting lipid peroxidation in plasma membrane phospholipids (Mandel et al., 2003; McCay, 1985; Gilgun-Sherki et al., 2001). There are eight tocopherols with vitamin E activity, and α-tocopherol is

**Use Results Reference** 

two vitamins.

patients.

patients.

Table 1. Vitamin E or Tocopherol starring in several studies evaluating the antioxidant

supplementation and its effects on cognitive deficits related with ND

placebo groups

The different forms of tocopherol that

Morris et al. 2005

Kontush et

Fahns A, 1998

Zandi et al., 2004

Sano et al., 1997

Fahn S, 1991

Jae Hee Kang, 2006

Pappert, E.J

al. [281],

protective effect associated with AD, compared with α-tocopherol alone

Increases in plasma levels of vitamin E does not affect the oxidation resistance of lipoproteins, it is necessary to use combinations of the

Increase the time interval between the onset of the disease and the need for treatment with L-dopa in 75% of

The combined use of vitamin supplements showed a decrease in the incidence and prevalence of AD in a population of 5092 individuals

Treatment delays functional impairment in patients with mild

The combination of these natural antioxidants delayed by 2.5 years, the

administration of L-dopa in PD

No differences were found between groups treated with vitamin E and

No increased levels of vitamin E in the cerebrospinal fluid of patients with PD. However, the subjects had clinical symptoms of the disease when vitamin E was administered.

Alzheimer's disease.

time for the start of the

make vitamin E exert greater

**4.3.1 Vitamins 4.3.1.1 Vitamin E** 

The individual or combined action of tocopherols are protective factors against the incidence of AD

Supplementation with vitamins E and C to reduce lipid peroxidation

Supplementation with 3000 IU / day vitamin E and C can delay the onset of symptoms associated with

Combination of supplements of vitamins C and E affect the prevalence and incidence of AD.

Supplementation with 2000 IU / day vitamin E reduces the EO implicated in the pathogenesis of

High doses of α-tocopherol and ascorbate can delay the time of administration of L-dopa in PD

Long-term supplementation with vitamin E may provide cognitive

The intake of high doses of vitamin E (400-4000 IU / day) can slow the progression of PD by inhibiting nigral cell death

dopamine deficiency in PD

patients

AD.

patients

benefits.

and cognitive dysfunction

in patients with AD.

the most active and widely distributed (Elejalde, 2001); it is found naturally in foods such as vegetable oils, fats, vegetables, egg yolks, nuts, seeds, fruits and green vegetables (Berman & Brodaty, 2004). In addition to its antioxidant properties, also has capabilities such as specific enzymes regulating agent, anti-inflammatory and neuroprotective (Martin et al., 1999). Due to its neuroprotective properties, researchers worldwide have taken a particular interest in elucidating their protective mechanisms, as opposed to development of cognitive dysfunction related to aging and various ND (Table 1) (Ramesh et al, 2010).

The neuroprotective effect of vitamin E was first described in 1992 in an in vitro study using neuronal cells cultures (Behl C, et al, 1992). However, in order to know whether dietary supplementation with antioxidants can increase their brain levels, it is essential to investigate the effect of such molecules in animal models of ND like the EP (Prasad et al., 1999). In this regard, previous studies suggest that long-term feeding of rats (6 to 15 months of age) with a dietary supplement of 500 IU of vitamin E, induces protective effects against cognitive deficit related to age (Morris et al., 2005; Peterson et al., 2005). Similarly, it has been reported that supplementation with vitamin E in combination with vitamin C, increases the concentration of these vitamins in plasma and cerebrospinal fluid, where the resistance of lipoproteins to oxidation in in vitro studies is increased (Kontush et al, 2001; Casetta et al., 2005). It has also been reported that dietary supplementation with αtocopherol (1000 IU/day) for four months increases brain levels of vitamin E in rats (Vatassery et al., 1988), as in the brain and cerebrospinal fluid of dogs treated during two years (SR Pillai, 1993). These results suggest that dietary supplementation with vitamin E may be valuable in animal models of PD (Prasad et al., 1999).

On the other hand, one of the largest studies with antioxidants as a treatment for AD, included 341 patients with moderate dementia who were given 4 different treatments: 10mg/day of selegiline (a selective MAO inhibitor), 2000 IU daily α-tocopherol, a combination of selegiline and-tocopherol, or placebo. The effect of the treatments was assessed through the presentation of clinical signs such as loss of ability to perform basic activities of daily living, severe dementia or death. After the treatments the authors observed a significant delay in the time of presentation of clinical signs in patients treated with selegiline, alpha-tocopherol, or in combination compared with placebo (Sano, 1996, 1997). However, there was no additive effect of selegiline and vitamin E, possibly due to a common mechanism of action in which lower levels of free radicals, and prevents its formation through inhibition of oxidative metabolism of catecholamines (Casetta et al., 2005). Similarly, studies by Behl, C., 2000, suggest a neuroprotective activity of both, the natural tocopherol and the synthetic. In addition, the effect appears to be superior to that described for estradiol (powerful antioxidant), in terms of the ability of neuronal protection against oxidative damage generated by β-amyloid protein in AD. Additionally, there are studies that seek to enhance the antioxidant effect of tocopherol, in combination with other vitamins such as Vitamin C. For example, in a prospective study in the Netherlands (Engelhart et al., 2002), they used a population of 5395 subjects at least 55 years, where the high intake of vitamin C and E was associated with a lower risk of developing AD after a follow up of 6.5 years. The relative risk was 0. for vitamin E, and 0.82 for vitamin C (Casetta et al., 2005).

With respect to the source, the vitamin E supplements do not appear to offer better results than those obtained by eating foods rich in this vitamin (Kontush & Schekatolina, 2004). This may be due to the cumulative and synergistic, as the bioavailability of vitamins is

Free Radicals, Neuronal Death and Neuroprotection 179

complex, and the contribution of gluconeogenesis oxygen to the brain. In addition, due to the critical role that these nutrients play in the metabolism of glucose and mitochondrial respiration justifies the use of B vitamins for patients with PD (Mazzio et al., 2011). Some authors argue that a relationship exists between the PD, vitamin B6, vitamin B12 and folic acid. This relationship refers to the role played by these molecules in the regulation of homocysteine, since they are responsible for their cleavage to methionine and tetrahydrofolate. Such effects may attenuate the neurotoxicity associated with a condition known as hyperhomocysteinemia, which is associated with PD and cytotoxicity related Mitochondrial Transitory Permeability Pore (Mazzio et al., 2011). Additionally, it has been suggested that elevated levels of homocysteine may increase the severity of PD, since this amino acid could mediate neuronal toxicity through NMDA receptors, precipitating oxidative stress, calcium overload and apoptosis (Mazzio et al., 2011). Homocysteine has also been associated with states of oxidative stress related with AD. In fact, there are some reports of an increased intake of vitamin B6 (Tucker et al., 2005; Corrada et al., 2005), vitamin B12 or folate (Wang et al., 2001; Morris et al., 2005), in middle-aged or advanced age people, with the belief of obtaining a beneficial effect on the incidence of AD or cognitive

In this regard, several studies seeking to assess the impact of high doses of vitamin supplements on plasma homocysteine levels in patients with AD (Aisen et al., 2003). For example, a study in patients with AD evaluated the effects of supplementation of these individuals over a period of 18 months. The results indicated that the groups treated with high doses of folic acid, vitamin B6 and B12 reduced by 20-30% peripheral levels of homocysteine, however, the study showed no cognitive differences between individuals

As mentioned above, the use of vitamin supplements may promote the biological integrity of systems, however, the combined use of vitamins and plant-derived polyphenolic compounds, seems to have good recognition as antioxidants (Mazzio et al., 2011). The use of supplements from plants to improve health is an issue that is gaining popularity among most people because it is considered that the use of natural products is safe and produces fewer side effects, compared to synthetic drug, in fact, to date over 50 different species of plants, and more than 8000 phenolic compounds have been identified with beneficial effects

Polyphenols can be divided into different groups depending on the number of rings of phenol and the chemical group attached to these rings. The most representative of this group of substances are the flavonoids, which are subdivided into flavonoids (catechin, epicatechin), flavonols (quercetin, myricetin, kaempferol), flavanones (hesperetin, naringenin), flavones (apigenin, luteolin), isoflavones (genistein, daidzein) and anthocyanins (cyanide, malvidin) (Ramassamy, 2006; Sun, et al., 2008). These molecules are found in a wide variety of food products from plants. One of the most important aspects of the polyphenols current research is their neuroprotective capacity. This section will define and describe in detail the neuroprotective mechanisms of these macromolecules, and also, will discuss recent evidence regarding their potentially antioxidant effect to prevent or to control the development of

treated with antioxidants and individuals treated with placebo (Aisen et al., 2003).

impairment (Coley et al, 2008).

**4.4 Polyphenols** 

on health (Sun et al., 2008).

neuropathology as AD or PD.

concerned. However, not be ruled out that the apparent protection provided by the supplementation of vitamins E and C, is the result of the synergy of these vitamins and other substances in fruits and vegetables such as flavonoids, which have both antiinflammatory properties as antioxidants (Ramesh et al., 2010; Seshadri & Wolf, 2003). It has also been suggested that vitamin E supplements can reduce levels of β-amyloid in a transgenic model of AD. However, this effect was only observed in young mice but not in older animals. These results may suggest that antioxidant therapy may be beneficial only if given at an early stage of the disease (Sung et al., 2004; Casetta et al., 2005).

The inconsistencies found between the results of these and other studies of vitamin E, lead to consider that most supplements used are composed exclusively of α-tocopherol, leaving aside the action of other forms of tocopherol that make this vitamin. Therefore, it has been suggested that the protective effect of vitamin E in the brain is the result of the combined intake of all forms of tocopherol (Farrell & Roberts, 1994).

#### **4.3.1.2 Vitamin C**

Vitamin C or ascorbic acid is a soluble molecule that has a variety of functions, which include the recycling of oxidized forms of vitamin E and the activation of certain enzymes (McCay, 1985; Chan, 1993; Elejalde, 2001). Importantly, in vivo there are interactions between vitamin E and vitamin C, where the role of antioxidant vitamin E has proven to be improved by supplementation of vitamin C. This interaction, however, involves the two vitamins, whose levels are not regulated by metabolism, but depend on consumption in the diet (Kagan, & Tyurina, 1998). Humans and other primates are unable to synthesize this vitamin, while most mammals, including rats and mice produce the endogenous form of this molecule in the liver (Chatterjee et al., 1975).

Vitamin C is found at high levels in a variety of cells, including neurons, where it participates in the biosynthesis of catecholamines and plays an important role as a cofactor of dopamine-hydroxylase. Vitamine C and vitamin E inhibit peroxidation of membrane phospholipids and act as free radicals scavenger (Gilgun-Sherki et al., 2001). Some authors report that ascorbic acid protects low density lipoprotein from oxidation and reduces oxidizing molecules that damage the integrity of the Central Nervous System (Sales et al., 2009). Given these properties, the scientific interest in this vitamin is to know about their effects on neuronal damage induced by agents such as pilocarpine, where has been found that antioxidant treatment significantly reduces the level of lipid peroxidation and nitrite content, and also potentiates the activity of SOD and CAT in the hippocampus of adult rats after pilocarpine-induced seizures. (Sales et al., 2009).

With respect to the ND, Hellenbrand et al., 1996, found that vitamin C has a protective effect against PD, with statistically significant trend. Several studies in populations aged 65 or older focused their interest on the combined effect of vitamin C and other antioxidants such as vitamin E, beta-carotene, or flavonoids, and these could be associated with reduction of dementia / incidence of AD or reduction of cognitive decline (Esposito et al, 2002; Coley et al., 2008). Zandi et al., 2004 reported that using the combination of high doses of vitamins E and C are associated with reduced prevalence and incidence of AD, even, once initiated the disease.

#### **4.3.1.3 Vitamin B**

B complex vitamins such as thiamin (vitamin B1), lipoic acid, biotin, vitamin B6, folic acid, vitamin B12, pantothenate, symbiotically work together to boost the pyruvate dehydrogenase

concerned. However, not be ruled out that the apparent protection provided by the supplementation of vitamins E and C, is the result of the synergy of these vitamins and other substances in fruits and vegetables such as flavonoids, which have both antiinflammatory properties as antioxidants (Ramesh et al., 2010; Seshadri & Wolf, 2003). It has also been suggested that vitamin E supplements can reduce levels of β-amyloid in a transgenic model of AD. However, this effect was only observed in young mice but not in older animals. These results may suggest that antioxidant therapy may be beneficial only if

The inconsistencies found between the results of these and other studies of vitamin E, lead to consider that most supplements used are composed exclusively of α-tocopherol, leaving aside the action of other forms of tocopherol that make this vitamin. Therefore, it has been suggested that the protective effect of vitamin E in the brain is the result of the combined

Vitamin C or ascorbic acid is a soluble molecule that has a variety of functions, which include the recycling of oxidized forms of vitamin E and the activation of certain enzymes (McCay, 1985; Chan, 1993; Elejalde, 2001). Importantly, in vivo there are interactions between vitamin E and vitamin C, where the role of antioxidant vitamin E has proven to be improved by supplementation of vitamin C. This interaction, however, involves the two vitamins, whose levels are not regulated by metabolism, but depend on consumption in the diet (Kagan, & Tyurina, 1998). Humans and other primates are unable to synthesize this vitamin, while most mammals, including rats and mice produce the endogenous form of

Vitamin C is found at high levels in a variety of cells, including neurons, where it participates in the biosynthesis of catecholamines and plays an important role as a cofactor of dopamine-hydroxylase. Vitamine C and vitamin E inhibit peroxidation of membrane phospholipids and act as free radicals scavenger (Gilgun-Sherki et al., 2001). Some authors report that ascorbic acid protects low density lipoprotein from oxidation and reduces oxidizing molecules that damage the integrity of the Central Nervous System (Sales et al., 2009). Given these properties, the scientific interest in this vitamin is to know about their effects on neuronal damage induced by agents such as pilocarpine, where has been found that antioxidant treatment significantly reduces the level of lipid peroxidation and nitrite content, and also potentiates the activity of SOD and CAT in the hippocampus of adult rats

With respect to the ND, Hellenbrand et al., 1996, found that vitamin C has a protective effect against PD, with statistically significant trend. Several studies in populations aged 65 or older focused their interest on the combined effect of vitamin C and other antioxidants such as vitamin E, beta-carotene, or flavonoids, and these could be associated with reduction of dementia / incidence of AD or reduction of cognitive decline (Esposito et al, 2002; Coley et al., 2008). Zandi et al., 2004 reported that using the combination of high doses of vitamins E and C are associated with reduced prevalence and incidence of AD, even, once initiated the

B complex vitamins such as thiamin (vitamin B1), lipoic acid, biotin, vitamin B6, folic acid, vitamin B12, pantothenate, symbiotically work together to boost the pyruvate dehydrogenase

given at an early stage of the disease (Sung et al., 2004; Casetta et al., 2005).

intake of all forms of tocopherol (Farrell & Roberts, 1994).

this molecule in the liver (Chatterjee et al., 1975).

after pilocarpine-induced seizures. (Sales et al., 2009).

**4.3.1.2 Vitamin C** 

disease.

**4.3.1.3 Vitamin B** 

complex, and the contribution of gluconeogenesis oxygen to the brain. In addition, due to the critical role that these nutrients play in the metabolism of glucose and mitochondrial respiration justifies the use of B vitamins for patients with PD (Mazzio et al., 2011). Some authors argue that a relationship exists between the PD, vitamin B6, vitamin B12 and folic acid. This relationship refers to the role played by these molecules in the regulation of homocysteine, since they are responsible for their cleavage to methionine and tetrahydrofolate. Such effects may attenuate the neurotoxicity associated with a condition known as hyperhomocysteinemia, which is associated with PD and cytotoxicity related Mitochondrial Transitory Permeability Pore (Mazzio et al., 2011). Additionally, it has been suggested that elevated levels of homocysteine may increase the severity of PD, since this amino acid could mediate neuronal toxicity through NMDA receptors, precipitating oxidative stress, calcium overload and apoptosis (Mazzio et al., 2011). Homocysteine has also been associated with states of oxidative stress related with AD. In fact, there are some reports of an increased intake of vitamin B6 (Tucker et al., 2005; Corrada et al., 2005), vitamin B12 or folate (Wang et al., 2001; Morris et al., 2005), in middle-aged or advanced age people, with the belief of obtaining a beneficial effect on the incidence of AD or cognitive impairment (Coley et al, 2008).

In this regard, several studies seeking to assess the impact of high doses of vitamin supplements on plasma homocysteine levels in patients with AD (Aisen et al., 2003). For example, a study in patients with AD evaluated the effects of supplementation of these individuals over a period of 18 months. The results indicated that the groups treated with high doses of folic acid, vitamin B6 and B12 reduced by 20-30% peripheral levels of homocysteine, however, the study showed no cognitive differences between individuals treated with antioxidants and individuals treated with placebo (Aisen et al., 2003).

#### **4.4 Polyphenols**

As mentioned above, the use of vitamin supplements may promote the biological integrity of systems, however, the combined use of vitamins and plant-derived polyphenolic compounds, seems to have good recognition as antioxidants (Mazzio et al., 2011). The use of supplements from plants to improve health is an issue that is gaining popularity among most people because it is considered that the use of natural products is safe and produces fewer side effects, compared to synthetic drug, in fact, to date over 50 different species of plants, and more than 8000 phenolic compounds have been identified with beneficial effects on health (Sun et al., 2008).

Polyphenols can be divided into different groups depending on the number of rings of phenol and the chemical group attached to these rings. The most representative of this group of substances are the flavonoids, which are subdivided into flavonoids (catechin, epicatechin), flavonols (quercetin, myricetin, kaempferol), flavanones (hesperetin, naringenin), flavones (apigenin, luteolin), isoflavones (genistein, daidzein) and anthocyanins (cyanide, malvidin) (Ramassamy, 2006; Sun, et al., 2008). These molecules are found in a wide variety of food products from plants. One of the most important aspects of the polyphenols current research is their neuroprotective capacity. This section will define and describe in detail the neuroprotective mechanisms of these macromolecules, and also, will discuss recent evidence regarding their potentially antioxidant effect to prevent or to control the development of neuropathology as AD or PD.

Free Radicals, Neuronal Death and Neuroprotection 181

lipoprotein. These features have drawn public attention, since several of these substances have therapeutic potential against diseases such as cancer, ischemia, heart, liver, and neurodegenerative diseases (Mandel et al., 2003). Thus, numerous studies in different models of neurodegeneration *in vitro* and *in vivo* have shown that polyphenols can prevent and/or reduce oxidative damage by free radicals generated (Mandel & Youdim, 2004; Scalbert et al., 2005). Numerous epidemiological estudies have shown neuroprotective effects of polyphenols and have established a clear relationship between these effects and decreased risk of neurological dysfunction associated with aging (Mandel et al., 2003). However, the nature of these protective effects, is not limited to the antioxidant properties, since recent evidence derived from *in vitro* cellular models, suggest that polyphenols such as resveratrol and EGCG, besides having the ability to recycle free radicals directly, also may regulate the cytotoxic effects of oligomers of βA via phosphorylation of phosphokinase C. In addition, polyphenols such as EGCG and resveratrol possess the ability to activate the enzyme transmembrane α-secretase, which catalyzes the formation of a soluble and amyloidogenic (no plaque-forming) from the amyloid precursor protein (APP). Through this pathway, APP is formed and therefore do not allow the formation of neuritic plaques, a hallmark of AD. This information indicates that polyphenols may be used in therapies to exert control over the APP related molecules, and may suggest avenues for the development of new treatments that reduce the risk of developing AD according to the aging process.

Additionally, studies such as James et al., 1999, showed the extracts of blueberry or strawberry (high in polyphenols) as substances that can significantly attenuate cognitive and motor deficits related to aging in rodents. In this study, rodents of all treatments showed improvement in short-term memory according to the Morris water test. However, while these diets were supplemented based on an equal antioxidant capacity (determined by absorbance capacity test the oxygen radical, ORAC) was not found equal effectiveness in the prevention or reversal of the changes associated with aging. Additionally, the antioxidant capacity alone was not predictive in evaluating the potential of these compounds against certain age-related disorders. In fact, markers of oxidative stress (DCF fluorescence, and glutathione peroxidase level in the brain) were slightly reduced by the diets, suggesting that the polyphenols from berries can have multiple actions in addition to

Other possible mechanisms for the beneficial effects of these foods are: direct effects on signaling to enhance neuronal communication, the ability to act as a buffer against excess calcium, enhancement of neuroprotective proteins and reduction of signs of stress such as NF -κβ. (Calabrese et al., 2010). According to studies in cell cultures and animal models, there is a cascade of signaling between the molecules and effects of eating berries. For example, treatment with berries to COS-7 cells exposed to dopamine or to hippocampal primary neurons, significantly increased the expression of MAPK mitogen. Additionally, mice supplemented with berries APP/PS1, exhibited high levels of hippocampal extracellular signaling regulated by ERK, such as protein kinase C (PKC) α, compared with transgenic mice maintained on control diets. In addition, Brannon & Trygve, 2010, suggests that treatment with berries is effective protection against the toxic effect of βA and against the decline in the induction of dopamine in the regulation of intracellular calcium in COS-7 cells transfected hippocampal neurons. This protection suggests an increase in

(Brannon & Trygve, 2010).

the antioxidant.

phosphorylated MAPK and decreased PKCγ.

#### **4.4.1 Plant polyphenols**

Polyphenols are a class of phytoalexins found in a wide range of plants, fruits and vegetables (Bastianetto & Quirion, 2002; Brannon et al., 2010; Ramassamy, 2006; Ramesh et al., 2010). When ingested they are transported from the circulatory system to various body organs including the brain (Sun et al., 2008). It has also been found to be potent recyclers of superoxide radicals, hydrogen peroxide and oxygen (Morel et al., 1993; Nanjo et al., 1996; Ramassamy, 2006), mechanisms that together with the anti-inflammatory activity have been extensively studied in order to know their beneficial effects against aging-related processes (Table 2) (Brannon & Trygve, 2010; James et al., 2009).

The ability of polyphenols to act as antioxidants is given by its ability to chelate metal ions, which is achieved by suppressing reactive species that contribute to oxidative damage (Brannon & Trygve, 2010). This antioxidant capacity depends on the molecular structure of each polyphenol, the position of hydroxyl groups, and other substitutions in their chemical structure (Sun et al., 2008). In addition to the antioxidant capacities, several polyphenols exhibit multiple biological properties among which are the anti-inflammatory, anticancer, antiviral, antimicrobial, vasodilator and anti-coagulant (Rahman et al., 2007). Additionally, *in vitro* studies demonstrate that polyphenols may possess the ability to activate or inhibit several signaling pathways such as NF-κβ, SIRT1, MAPK's, heat shock proteins and other regulatory molecules, which may play an important role in basic functions such as senescence, apoptosis and the activation or production of transcription factors (Brannon & Trygve, 2010).

Furthermore, polyphenols are natural antioxidants that after consumption tend to produce an increase in plasma antioxidant capacity, and also can inhibit the oxidation of low density


Table 2. Attenuation of diseases associated to aging by action de polyphenols (Brannon & Trygve, 2010)

Polyphenols are a class of phytoalexins found in a wide range of plants, fruits and vegetables (Bastianetto & Quirion, 2002; Brannon et al., 2010; Ramassamy, 2006; Ramesh et al., 2010). When ingested they are transported from the circulatory system to various body organs including the brain (Sun et al., 2008). It has also been found to be potent recyclers of superoxide radicals, hydrogen peroxide and oxygen (Morel et al., 1993; Nanjo et al., 1996; Ramassamy, 2006), mechanisms that together with the anti-inflammatory activity have been extensively studied in order to know their beneficial effects against aging-related processes

The ability of polyphenols to act as antioxidants is given by its ability to chelate metal ions, which is achieved by suppressing reactive species that contribute to oxidative damage (Brannon & Trygve, 2010). This antioxidant capacity depends on the molecular structure of each polyphenol, the position of hydroxyl groups, and other substitutions in their chemical structure (Sun et al., 2008). In addition to the antioxidant capacities, several polyphenols exhibit multiple biological properties among which are the anti-inflammatory, anticancer, antiviral, antimicrobial, vasodilator and anti-coagulant (Rahman et al., 2007). Additionally, *in vitro* studies demonstrate that polyphenols may possess the ability to activate or inhibit several signaling pathways such as NF-κβ, SIRT1, MAPK's, heat shock proteins and other regulatory molecules, which may play an important role in basic functions such as senescence, apoptosis and the activation or production of transcription factors (Brannon &

Furthermore, polyphenols are natural antioxidants that after consumption tend to produce an increase in plasma antioxidant capacity, and also can inhibit the oxidation of low density

**Quercetine** Regulates production of TNF-α Anti-inflammatory properties that

Table 2. Attenuation of diseases associated to aging by action de polyphenols (Brannon &

Influence on senescence,

to stress and metabolism Increment of half life

with atherosclerosis

Prevention of metastasis Prevention of tumor growing

reduce development of

atherosclerosis.

inflammation, apoptosis, resistance

TTR sequesters beta-amyloid fibrils

Prevents inflammation associated

**4.4.1 Plant polyphenols** 

Trygve, 2010).

Trygve, 2010)

(Table 2) (Brannon & Trygve, 2010; James et al., 2009).

**Polyphenol Effect Result** 

deacetylation of p53, NF-κβ, HSF-1,

It binds to receptors in the brain to stimulate the production of

Regulates NO production in

Blocks EGF receptor in cervical

**Resveratrol** Activation of SIRT1 driving to

caloric restriction

transthyretin (TTR)

**EGCG** Regulates NO production in endothelial cells

carcinoma cells

cancer cells

FOXO1/3/4 y PGC-1α. Activation of SIRT1 can mimic lipoprotein. These features have drawn public attention, since several of these substances have therapeutic potential against diseases such as cancer, ischemia, heart, liver, and neurodegenerative diseases (Mandel et al., 2003). Thus, numerous studies in different models of neurodegeneration *in vitro* and *in vivo* have shown that polyphenols can prevent and/or reduce oxidative damage by free radicals generated (Mandel & Youdim, 2004; Scalbert et al., 2005). Numerous epidemiological estudies have shown neuroprotective effects of polyphenols and have established a clear relationship between these effects and decreased risk of neurological dysfunction associated with aging (Mandel et al., 2003). However, the nature of these protective effects, is not limited to the antioxidant properties, since recent evidence derived from *in vitro* cellular models, suggest that polyphenols such as resveratrol and EGCG, besides having the ability to recycle free radicals directly, also may regulate the cytotoxic effects of oligomers of βA via phosphorylation of phosphokinase C. In addition, polyphenols such as EGCG and resveratrol possess the ability to activate the enzyme transmembrane α-secretase, which catalyzes the formation of a soluble and amyloidogenic (no plaque-forming) from the amyloid precursor protein (APP). Through this pathway, APP is formed and therefore do not allow the formation of neuritic plaques, a hallmark of AD. This information indicates that polyphenols may be used in therapies to exert control over the APP related molecules, and may suggest avenues for the development of new treatments that reduce the risk of developing AD according to the aging process. (Brannon & Trygve, 2010).

Additionally, studies such as James et al., 1999, showed the extracts of blueberry or strawberry (high in polyphenols) as substances that can significantly attenuate cognitive and motor deficits related to aging in rodents. In this study, rodents of all treatments showed improvement in short-term memory according to the Morris water test. However, while these diets were supplemented based on an equal antioxidant capacity (determined by absorbance capacity test the oxygen radical, ORAC) was not found equal effectiveness in the prevention or reversal of the changes associated with aging. Additionally, the antioxidant capacity alone was not predictive in evaluating the potential of these compounds against certain age-related disorders. In fact, markers of oxidative stress (DCF fluorescence, and glutathione peroxidase level in the brain) were slightly reduced by the diets, suggesting that the polyphenols from berries can have multiple actions in addition to the antioxidant.

Other possible mechanisms for the beneficial effects of these foods are: direct effects on signaling to enhance neuronal communication, the ability to act as a buffer against excess calcium, enhancement of neuroprotective proteins and reduction of signs of stress such as NF -κβ. (Calabrese et al., 2010). According to studies in cell cultures and animal models, there is a cascade of signaling between the molecules and effects of eating berries. For example, treatment with berries to COS-7 cells exposed to dopamine or to hippocampal primary neurons, significantly increased the expression of MAPK mitogen. Additionally, mice supplemented with berries APP/PS1, exhibited high levels of hippocampal extracellular signaling regulated by ERK, such as protein kinase C (PKC) α, compared with transgenic mice maintained on control diets. In addition, Brannon & Trygve, 2010, suggests that treatment with berries is effective protection against the toxic effect of βA and against the decline in the induction of dopamine in the regulation of intracellular calcium in COS-7 cells transfected hippocampal neurons. This protection suggests an increase in phosphorylated MAPK and decreased PKCγ.

Free Radicals, Neuronal Death and Neuroprotection 183

Quercetin is a flavonoid found in different types of food such as apples, capers, onions, broccoli, tea and wine (Boots et al., 2008; Esposito et al., 2002; Kelsey et al, 2010). As EGCG, quercetin has been widely studied in *in vitro* and *in vivo* assays in neural models (Ossola et al., 2009). Thus, PC12 cell studies showed that quercetin enhances cell survival in the presence of hydrogen peroxide (Dajas et al., 2003; Heo & Lee, 2004), linoleic acid (Sasaki, et al., 2003), and tert-butyl (Silva et al., 2008). Furthermore, in human neuroblastoma cells SH-SY5Y used as experimental models for PD, this substance has shown to have protective ability against toxicity by 6-OHDA (Kelsey et al., 2010). Also, other study about neurodegeneration in animal models have suggested that the neuroprotective capacity of quercetin could be related to increased blood-brain barrier permeability, thus facilitating the

Taken together, these studies indicate that quercetin has the potential, such as EGCG, to block the starting of the enzymatic oxidation of dopamine (Tamura et al., 1994), and this could means a new therapy against neurodegenerative diseases such as PD (Kelsey et al., 2010). However, Ossola, et al., 2009 states that despite the fact that quercetin has not shown significant toxicity in several animal studies, the risk of neurotoxicity is not negligible due to its narrow therapeutic dose range in *in vitro* experiments, also the effectiveness of quercetin

Resveratrol (trans-3, 4 ',5-trihydroxystilbene) is a polyphenol found abundantly in grapes and red wine, it is known for its antioxidant and neuroprotective properties in several experiments, therefore, consumption of wine has been proposed as a possible benefit in neurodegenerative processes (Calabrese et al., 2010; Esposito et al., 2002; Kiziltepe et al., 2004; Ramasamy, 2006). The main biological activities attributed to resveratrol include: inhibition of lipid peroxidation and free radicals in cell cultures and rat brains (Virgili & Contestabile, 2000, Casetta et al., 2005), vasodilator, anti-inflammatory and anticancer. Also, It has been shown that mice fed with daily dose of resveratrol for 45 days, had resveratrol or its metabolites in the brain, indicating its bioavailability to neuronal cells (Casseta et al.,

In the field of ND has been suggested that resveratrol not only attenuated the cytotoxicity induced by β-amyloid, but also blocks the accumulation of intracellular reactive oxygen species typical of apoptosis (Casetta et al., 2005; Jang & Surh, 2003). In addition, partial neuroprotection was demonstrated in rats with chronic supplementation of resveratrol in *in vivo* studies of excitotoxicity related to the administration of agonists for glutamate and kainic acid receptors, (Contestabille, 2001; Virgili & Contestabile, 2000). The consumption of about 8 mg/kg/day of resveratrol for 45 days decreased excitotoxic damage measured on the basis of the reduction of certain neuromarcador of GABAergic neurons, from 38% to 14% in the olfactory cortex and 27 % to 12% in the hippocampus. This was the first report of neuroprotection by long-term administration of resveratrol in an *in vivo* model of neurodegeneration (Contestabille, 2001). Similarly, Han, 2003, showed that cell death induced by administration of β-amyloid peptide (20μM), decreased significantly, and protein concentration-dependent by treatment with resveratrol administered 2 hours later. Also, in two different transfected cell lines (HEK293 and N2A), Marambaud et al., 2005, showed that resveratrol may reduce the secretion of β-amyloid peptide, perhaps through the activation of proteosomal degradation of the peptide. This effect of resveratrol occurred

2005; Contestabile, 2001; Karuppagounder et al., 2008; Ramesh et al., 2010).

penetration of the substance in the brain (Ossola et al., 2009).

**4.4.1.2 Quercetin** 

in ND is quite low. **4.4.1.3 Resveratrol** 

#### **4.4.1.1 Epigallocatechin 3-Gallate (EGCG)**

EGCG is a polyphenol flavonoid type found in large quantities in green tea. According to some authors, this compound has exerts significant neuroprotective effects against a wide range of oxidative insults in a multitude of neuronal cell models (Calabrese et al., 2010; Kelsey et al., 2010). In one study, CGNs incubated cells with an inhibitor of Bcl-2 known as HA14-1, which generates oxidation and mitochondrial intrinsic apoptosis (Zimmermann et al., 2007), applying the co-treatment with EGCG, was found that the microtubule network of CGNs exposed to HA14-1 was significantly preserved, and so, was prevented the apoptotic nuclear morphology (Kelsey et al., 2010). In fact, studies like that of Weinreb et al., 2004, showed that treatment of neuronal cells with EGCG affects the expression levels of various proteins, including proteins related to components of the cytoskeleton, metabolism, and binding proteins (Calabrese et al., 2010; Izumi et al., 2005). EGCG similarly protects human neuroblastoma cells (SH-SY5Y) against the cytotoxicity associated with the amyloid precursor protein (APP), and the 6-hydroxydopamine (6-OHDA) (Avramovich et al., 2007), thus, rescues PC12 cells from serum deprivation-induced apoptosis or paraquat (Hou et al., 2008; Kelsey et al., 2010; Mandel et al., 2003). Also, supplementation of transgenic mice over expressing APP (APPsw) substantially reduced amyloid plaque burden and reduced cognitive impairment (Rezai-Zadeh et al., 2005, Kelsey et al., 2010). Similarly, in murine N2A cells transfected with a mutant form of human APP (Rezai-Zadeh et al., 2005) was found that EGCG reduced the generation of β-amyloid (Ramassamy, 2006).

In addition to the neuroprotective effects of EGCG observed in *in vitro* studies, this antioxidant also preserved neuronal survival and function in several *in vivo* models of neurodegeneration. For example, supplementation to mice with EGCG protected dopaminergic neurons in the substantia nigra pars compacta from toxicity induced MPTP, therefore it could preserve the levels of dopamine in the striatum (Levites et al., 2001). Similarly, the acute and chronic administration of EGCG has been evaluated in various cell and animal models of AD, where it has been suggested that EGCG significantly reduced the toxicity induced by β-amyloid (Kelsey et al., 2010). With respect to PD, Sung et al, 2010, used models *in vitro* and *in vivo* to investigate the modulation of the effects of EGCG on L-dopa and induced neuronal damage. The results indicated that oral supplementation with this antioxidant initiated potential beneficial effects in patients with PD treated with L-dopa as moderately inhibits methylation of this molecule. Similarly, Levitas et al., 2001, using mice as animal model of PD which were given a pre-treatment with green tea extract (0.5 *and* 1 mg / kg) or EGCG (2 and 10 mg / kg) prevented the damage generated by the neurotoxin (MPTP) on dopaminergic neurons in the nigrostriatal pathway.

Another type of molecular mechanism involved in neuroprotection by EGCG is mediated gene activation in apoptosis. Evidence of this assertion is found in studies such as that by Levites et al., 2002, which results in neuroblastoma cells SH-SY5Y showed that EGCG decreased the gene expression of pro-apoptotic such as Bax, Bad, Fas ligand and TRAIL (tumor necrosis factor-related apoptosis-inducingligand), however, the expression of Bcl-2 and Bcl-x was not affected (Levites et al., 2002). These results suggest that the neuroprotective effects of EGCG may involve the inactivation of proapoptotic genes, rather than the action of anti-apoptotic mitochondrial proteins (Ramassamy, 2006). Taken together, these findings indicate that EGCG may be a therapeutic candidate for chronic neurodegenerative diseases like AD and PD (Weinreb et al.,2004; Frank & Gupta, 2005), and may be beneficial in acute episodes of neuronal damage, such as spinal cord trauma.

#### **4.4.1.2 Quercetin**

182 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

EGCG is a polyphenol flavonoid type found in large quantities in green tea. According to some authors, this compound has exerts significant neuroprotective effects against a wide range of oxidative insults in a multitude of neuronal cell models (Calabrese et al., 2010; Kelsey et al., 2010). In one study, CGNs incubated cells with an inhibitor of Bcl-2 known as HA14-1, which generates oxidation and mitochondrial intrinsic apoptosis (Zimmermann et al., 2007), applying the co-treatment with EGCG, was found that the microtubule network of CGNs exposed to HA14-1 was significantly preserved, and so, was prevented the apoptotic nuclear morphology (Kelsey et al., 2010). In fact, studies like that of Weinreb et al., 2004, showed that treatment of neuronal cells with EGCG affects the expression levels of various proteins, including proteins related to components of the cytoskeleton, metabolism, and binding proteins (Calabrese et al., 2010; Izumi et al., 2005). EGCG similarly protects human neuroblastoma cells (SH-SY5Y) against the cytotoxicity associated with the amyloid precursor protein (APP), and the 6-hydroxydopamine (6-OHDA) (Avramovich et al., 2007), thus, rescues PC12 cells from serum deprivation-induced apoptosis or paraquat (Hou et al., 2008; Kelsey et al., 2010; Mandel et al., 2003). Also, supplementation of transgenic mice over expressing APP (APPsw) substantially reduced amyloid plaque burden and reduced cognitive impairment (Rezai-Zadeh et al., 2005, Kelsey et al., 2010). Similarly, in murine N2A cells transfected with a mutant form of human APP (Rezai-Zadeh et al., 2005) was

found that EGCG reduced the generation of β-amyloid (Ramassamy, 2006).

(MPTP) on dopaminergic neurons in the nigrostriatal pathway.

In addition to the neuroprotective effects of EGCG observed in *in vitro* studies, this antioxidant also preserved neuronal survival and function in several *in vivo* models of neurodegeneration. For example, supplementation to mice with EGCG protected dopaminergic neurons in the substantia nigra pars compacta from toxicity induced MPTP, therefore it could preserve the levels of dopamine in the striatum (Levites et al., 2001). Similarly, the acute and chronic administration of EGCG has been evaluated in various cell and animal models of AD, where it has been suggested that EGCG significantly reduced the toxicity induced by β-amyloid (Kelsey et al., 2010). With respect to PD, Sung et al, 2010, used models *in vitro* and *in vivo* to investigate the modulation of the effects of EGCG on L-dopa and induced neuronal damage. The results indicated that oral supplementation with this antioxidant initiated potential beneficial effects in patients with PD treated with L-dopa as moderately inhibits methylation of this molecule. Similarly, Levitas et al., 2001, using mice as animal model of PD which were given a pre-treatment with green tea extract (0.5 *and* 1 mg / kg) or EGCG (2 and 10 mg / kg) prevented the damage generated by the neurotoxin

Another type of molecular mechanism involved in neuroprotection by EGCG is mediated gene activation in apoptosis. Evidence of this assertion is found in studies such as that by Levites et al., 2002, which results in neuroblastoma cells SH-SY5Y showed that EGCG decreased the gene expression of pro-apoptotic such as Bax, Bad, Fas ligand and TRAIL (tumor necrosis factor-related apoptosis-inducingligand), however, the expression of Bcl-2 and Bcl-x was not affected (Levites et al., 2002). These results suggest that the neuroprotective effects of EGCG may involve the inactivation of proapoptotic genes, rather than the action of anti-apoptotic mitochondrial proteins (Ramassamy, 2006). Taken together, these findings indicate that EGCG may be a therapeutic candidate for chronic neurodegenerative diseases like AD and PD (Weinreb et al.,2004; Frank & Gupta, 2005), and

may be beneficial in acute episodes of neuronal damage, such as spinal cord trauma.

**4.4.1.1 Epigallocatechin 3-Gallate (EGCG)** 

Quercetin is a flavonoid found in different types of food such as apples, capers, onions, broccoli, tea and wine (Boots et al., 2008; Esposito et al., 2002; Kelsey et al, 2010). As EGCG, quercetin has been widely studied in *in vitro* and *in vivo* assays in neural models (Ossola et al., 2009). Thus, PC12 cell studies showed that quercetin enhances cell survival in the presence of hydrogen peroxide (Dajas et al., 2003; Heo & Lee, 2004), linoleic acid (Sasaki, et al., 2003), and tert-butyl (Silva et al., 2008). Furthermore, in human neuroblastoma cells SH-SY5Y used as experimental models for PD, this substance has shown to have protective ability against toxicity by 6-OHDA (Kelsey et al., 2010). Also, other study about neurodegeneration in animal models have suggested that the neuroprotective capacity of quercetin could be related to increased blood-brain barrier permeability, thus facilitating the penetration of the substance in the brain (Ossola et al., 2009).

Taken together, these studies indicate that quercetin has the potential, such as EGCG, to block the starting of the enzymatic oxidation of dopamine (Tamura et al., 1994), and this could means a new therapy against neurodegenerative diseases such as PD (Kelsey et al., 2010). However, Ossola, et al., 2009 states that despite the fact that quercetin has not shown significant toxicity in several animal studies, the risk of neurotoxicity is not negligible due to its narrow therapeutic dose range in *in vitro* experiments, also the effectiveness of quercetin in ND is quite low.

#### **4.4.1.3 Resveratrol**

Resveratrol (trans-3, 4 ',5-trihydroxystilbene) is a polyphenol found abundantly in grapes and red wine, it is known for its antioxidant and neuroprotective properties in several experiments, therefore, consumption of wine has been proposed as a possible benefit in neurodegenerative processes (Calabrese et al., 2010; Esposito et al., 2002; Kiziltepe et al., 2004; Ramasamy, 2006). The main biological activities attributed to resveratrol include: inhibition of lipid peroxidation and free radicals in cell cultures and rat brains (Virgili & Contestabile, 2000, Casetta et al., 2005), vasodilator, anti-inflammatory and anticancer. Also, It has been shown that mice fed with daily dose of resveratrol for 45 days, had resveratrol or its metabolites in the brain, indicating its bioavailability to neuronal cells (Casseta et al., 2005; Contestabile, 2001; Karuppagounder et al., 2008; Ramesh et al., 2010).

In the field of ND has been suggested that resveratrol not only attenuated the cytotoxicity induced by β-amyloid, but also blocks the accumulation of intracellular reactive oxygen species typical of apoptosis (Casetta et al., 2005; Jang & Surh, 2003). In addition, partial neuroprotection was demonstrated in rats with chronic supplementation of resveratrol in *in vivo* studies of excitotoxicity related to the administration of agonists for glutamate and kainic acid receptors, (Contestabille, 2001; Virgili & Contestabile, 2000). The consumption of about 8 mg/kg/day of resveratrol for 45 days decreased excitotoxic damage measured on the basis of the reduction of certain neuromarcador of GABAergic neurons, from 38% to 14% in the olfactory cortex and 27 % to 12% in the hippocampus. This was the first report of neuroprotection by long-term administration of resveratrol in an *in vivo* model of neurodegeneration (Contestabille, 2001). Similarly, Han, 2003, showed that cell death induced by administration of β-amyloid peptide (20μM), decreased significantly, and protein concentration-dependent by treatment with resveratrol administered 2 hours later. Also, in two different transfected cell lines (HEK293 and N2A), Marambaud et al., 2005, showed that resveratrol may reduce the secretion of β-amyloid peptide, perhaps through the activation of proteosomal degradation of the peptide. This effect of resveratrol occurred

Free Radicals, Neuronal Death and Neuroprotection 185

with the progression of cognitive impairment, in fact, several studies argue that extracts of Ginkgo biloba may be effective in delaying the clinical deterioration of patients with dementia (Ernst & Pittler, 1999; Casseta et al., 2005). The effects of EGb 761 may be explained, at least in part, based on their protective actions in animal models of hypoxia and ischemia (Droy-Lefaix et al., 1995) and *in vitro* models of toxicity (Ni et al., 1996; Oyama et al., 1996; Xin et al., 2000), also a prospective placebo-controlled study demonstrated the therapeutic efficacy of oral administration of EGb 761 in dementia patients and healthy adults showed improved memory and attention (Bastianetto & Quirion, 2002; Maurer et al., 1997; Mix et al., 2000). A case-control study that used a cohort of 1,462 women aged 75 years was conducted to test the effectiveness of the prevention of AD using EGb761, the conclusion is that the small number of women who developed dementia were prescribed with the supplement at least for two years. These results suggest that EGb 761 treatment may reduce the risk of developing Alzheimer's dementia in older women (Andrieu et al,

Additionally, Bastianetto & Quirion, in 2002, conducted a study *in vitro* with embryonic mouse hippocampal cells on which were used two different protocols of citotoxididad, one with β-amyloid (A\_25-35 (25\_M) A\_1-40 (5\_M) A\_1-42 or (25\_M)), and the other with sodium nitroprusside (SNP) to assess whether the components of red wine were able to reduce cell death caused by β-amyloid and oxidative stress. We found that EGb 761, possibly through the antioxidant properties of its flavonoids was able to protect

The mechanisms of action underlying the protective effects of EGb 761, have been evaluated in different studies. For example Bastianetto& Quirion, in 2002, noted in his study that the treatment with EGb 761 were able to inhibit the injury induced by βA 25-35, and NO. In addition, the extract showed protective effect against the harmful effects of H2O2, a supposed mediator of the toxicity caused by β-amyloid (Behl et al., 1994). These data suggest that the scavenging properties of hydroxyl radical of the EGb 761, may be part of the protection against β-amyloid toxicity (Oyama et al., 1996; Bastianetto & Quirion, 2002). Finally, it has been reported that EGb 761 inhibits a number of apoptotic events induced by βA 25-35, a process that may be relevant to the neuro-degeneration that occurs in AD (Johnson, 1994). In addition, these anti-apoptotic effects of EGb 761 are apparently related to the ability to inhibit the toxicity induced by H2O2, and that this natural extract has been reported as an effective tool to the apoptosis hydroxyl radical-induced in cultured neurons

The curcuminoids are the active component of turmeric, which have been attributed to have capacity as an inhibitor of lipid peroxidation, and free radical scavenger, it is a potent antiinflammatory and anticancer, and is also traditionally used in Asia (Aggarwal et al., 2007; James, et al., 2009; Ramesh et al., 2010). In light of its antioxidant, anti-inflammatory and anti-amyloid actions, curcumin is being investigated as a candidate compound for the prevention or treatment of diseases such as multiple myeloma, pancreatic cancer, myelodysplastic syndromes, colon cancer, psoriasis and AD (Calabrese et al., 2010; Goel, et

The curcumin reduces pro-inflammatory cytokines, oxidative damage, the Aβ42 and cognitive deficits in models of AD (Frautschy et al., 2001). Also, has been told that it is a direct inhibitor of βA aggregation and fibril formation (Cole et al., 2003; James et al., 2009).

hippocampal cells against the toxic effects previously described.

(Bastianetto & Quirion, 2002; Ni et al., 1996, Xin et al., 2000).

2003; Casseta, et al., 2005).

**4.4.1.5 Curcuminoides** 

al., 2008).

without the direct involvement of the β and γ-secretase, but, Brannon & Trygve, 2010, argues that resveratrol can activate transmembrane protein α-secretase. However, it is still unclear the effect of resveratrol on the mechanism of degradation of ß-amyloid levels in neurons, although it is suggested that may have a key effect on the route of clearance of beta-amyloid (Marambaud et al., 2005; Ramesh et al., 2010).

With respect to cell signaling pathways related to the effects of resveratrol has been shown that its protective activity is related to the PCK phosphorylation, leading to its activation, and the activation of no amyloidogenic cleavage pathways of APP, decreasing the release of β-amyloid (Han, 2003; Ramesh et al., 2010). Similarly, studies in neuroblastoma cells SH-SY5Y by Miloso et al.,1999, argue that resveratrol can induce activation of MAP kinases ERK1 and ERK2. In addition to these signaling pathways, resveratrol can also induce the expression of the response of the transcription early growth factor (Egr1) (Della Ragione et al., 2002), which could regulate some aspects of synaptic plasticity related to learning and memory (Li et al., 2005). Additionally, resveratrol may interact with other proteins, including members of the sirtuins family. Deacetylases sirtuins are related to mechanisms of cellular longevity (Guarente, 2001), resveratrol acts as a potent activator of these molecules, thus related to neuroprotective pathways (Araki et al., 2004; Ramesh et al., 2010). Other intracellular signaling mechanisms that may be implicated with the neuroprotective effect of resveratrol against β-amyloid peptide include the modulation of NF-kB pathways or NFκB/SIRT1, where resveratrol can inhibit the activity of NF-kB induced β-amyloid peptide through the activation of SIRT1 (Ramassamy, 2006).

With respect to the high amounts of resveratrol contained in red wine, investigations that assess moderate alcohol consumption show that this practice was significantly associated with lower risk of acquiring dementia and AD, compared to non-consumption (Casseta et al., 2005). Similarly, several epidemiological studies indicate that moderate wine consumption may be associated with a lower incidence of AD (Lindsay et al., 2002; Orgogozo et al., 1997; Truelsen et al., 2002), and additionally, different studies *in vitro* and *in vivo* have investigated the basis for this association. For example, doses from 10 microM of resveratrol in PC12 cells have demonstrated protective ability against the cytotoxicity induced by amyloid β (Jang & Surh, 2003). It has also been reported that the combination of resveratrol with other flavonoids such as catechin, may exert synergistic protection against the toxicity of amyloid β peptide in PC12 cells (Conte et al., 2003). However, there has not been demonstrated the relevance of these findings *in vivo* models (Ramassamy et al., 2010). Moreover, it is difficult to reconcile the therapeutic potential of resveratrol with the well known toxic effects of ethanol (Calabrese et al., 2010), because it does not seem reasonable to recommend alcohol consumption to those with tendency to addiction (Ramesh et al, 2010; Resnick & Junlapeeya, 2004).

#### **4.4.1.4 Ginkgo biloba**

The extract of Ginkgo biloba EGb 761 is a substance from the green leaves of Ginkgo biloba (Drieu, 1986). This extract was patented in 1990 and has a wide range of biochemical and pharmacological activities, including the antioxidant activity (Marcocci et al., 1994), the neurotrophic capacity in the hippocampal formation (Barkats et al., 1995; Bastianetto & Quirion, 2002), and the neuroprotective ability against neurotoxicity induced by β-amyloid peptide (Casseta, et al., 2005; Luo et al., 2002; Yao et al., 2001;).

The therapeutic use of Ginkgo biloba has been proposed due to its high content of flavonoids and terpenoids, and is widely used in Europe to alleviate symptoms associated

without the direct involvement of the β and γ-secretase, but, Brannon & Trygve, 2010, argues that resveratrol can activate transmembrane protein α-secretase. However, it is still unclear the effect of resveratrol on the mechanism of degradation of ß-amyloid levels in neurons, although it is suggested that may have a key effect on the route of clearance of

With respect to cell signaling pathways related to the effects of resveratrol has been shown that its protective activity is related to the PCK phosphorylation, leading to its activation, and the activation of no amyloidogenic cleavage pathways of APP, decreasing the release of β-amyloid (Han, 2003; Ramesh et al., 2010). Similarly, studies in neuroblastoma cells SH-SY5Y by Miloso et al.,1999, argue that resveratrol can induce activation of MAP kinases ERK1 and ERK2. In addition to these signaling pathways, resveratrol can also induce the expression of the response of the transcription early growth factor (Egr1) (Della Ragione et al., 2002), which could regulate some aspects of synaptic plasticity related to learning and memory (Li et al., 2005). Additionally, resveratrol may interact with other proteins, including members of the sirtuins family. Deacetylases sirtuins are related to mechanisms of cellular longevity (Guarente, 2001), resveratrol acts as a potent activator of these molecules, thus related to neuroprotective pathways (Araki et al., 2004; Ramesh et al., 2010). Other intracellular signaling mechanisms that may be implicated with the neuroprotective effect of resveratrol against β-amyloid peptide include the modulation of NF-kB pathways or NFκB/SIRT1, where resveratrol can inhibit the activity of NF-kB induced β-amyloid peptide

With respect to the high amounts of resveratrol contained in red wine, investigations that assess moderate alcohol consumption show that this practice was significantly associated with lower risk of acquiring dementia and AD, compared to non-consumption (Casseta et al., 2005). Similarly, several epidemiological studies indicate that moderate wine consumption may be associated with a lower incidence of AD (Lindsay et al., 2002; Orgogozo et al., 1997; Truelsen et al., 2002), and additionally, different studies *in vitro* and *in vivo* have investigated the basis for this association. For example, doses from 10 microM of resveratrol in PC12 cells have demonstrated protective ability against the cytotoxicity induced by amyloid β (Jang & Surh, 2003). It has also been reported that the combination of resveratrol with other flavonoids such as catechin, may exert synergistic protection against the toxicity of amyloid β peptide in PC12 cells (Conte et al., 2003). However, there has not been demonstrated the relevance of these findings *in vivo* models (Ramassamy et al., 2010). Moreover, it is difficult to reconcile the therapeutic potential of resveratrol with the well known toxic effects of ethanol (Calabrese et al., 2010), because it does not seem reasonable to recommend alcohol consumption to those with tendency to addiction (Ramesh et al, 2010;

The extract of Ginkgo biloba EGb 761 is a substance from the green leaves of Ginkgo biloba (Drieu, 1986). This extract was patented in 1990 and has a wide range of biochemical and pharmacological activities, including the antioxidant activity (Marcocci et al., 1994), the neurotrophic capacity in the hippocampal formation (Barkats et al., 1995; Bastianetto & Quirion, 2002), and the neuroprotective ability against neurotoxicity induced by β-amyloid

The therapeutic use of Ginkgo biloba has been proposed due to its high content of flavonoids and terpenoids, and is widely used in Europe to alleviate symptoms associated

peptide (Casseta, et al., 2005; Luo et al., 2002; Yao et al., 2001;).

beta-amyloid (Marambaud et al., 2005; Ramesh et al., 2010).

through the activation of SIRT1 (Ramassamy, 2006).

Resnick & Junlapeeya, 2004).

**4.4.1.4 Ginkgo biloba** 

with the progression of cognitive impairment, in fact, several studies argue that extracts of Ginkgo biloba may be effective in delaying the clinical deterioration of patients with dementia (Ernst & Pittler, 1999; Casseta et al., 2005). The effects of EGb 761 may be explained, at least in part, based on their protective actions in animal models of hypoxia and ischemia (Droy-Lefaix et al., 1995) and *in vitro* models of toxicity (Ni et al., 1996; Oyama et al., 1996; Xin et al., 2000), also a prospective placebo-controlled study demonstrated the therapeutic efficacy of oral administration of EGb 761 in dementia patients and healthy adults showed improved memory and attention (Bastianetto & Quirion, 2002; Maurer et al., 1997; Mix et al., 2000). A case-control study that used a cohort of 1,462 women aged 75 years was conducted to test the effectiveness of the prevention of AD using EGb761, the conclusion is that the small number of women who developed dementia were prescribed with the supplement at least for two years. These results suggest that EGb 761 treatment may reduce the risk of developing Alzheimer's dementia in older women (Andrieu et al, 2003; Casseta, et al., 2005).

Additionally, Bastianetto & Quirion, in 2002, conducted a study *in vitro* with embryonic mouse hippocampal cells on which were used two different protocols of citotoxididad, one with β-amyloid (A\_25-35 (25\_M) A\_1-40 (5\_M) A\_1-42 or (25\_M)), and the other with sodium nitroprusside (SNP) to assess whether the components of red wine were able to reduce cell death caused by β-amyloid and oxidative stress. We found that EGb 761, possibly through the antioxidant properties of its flavonoids was able to protect hippocampal cells against the toxic effects previously described.

The mechanisms of action underlying the protective effects of EGb 761, have been evaluated in different studies. For example Bastianetto& Quirion, in 2002, noted in his study that the treatment with EGb 761 were able to inhibit the injury induced by βA 25-35, and NO. In addition, the extract showed protective effect against the harmful effects of H2O2, a supposed mediator of the toxicity caused by β-amyloid (Behl et al., 1994). These data suggest that the scavenging properties of hydroxyl radical of the EGb 761, may be part of the protection against β-amyloid toxicity (Oyama et al., 1996; Bastianetto & Quirion, 2002). Finally, it has been reported that EGb 761 inhibits a number of apoptotic events induced by βA 25-35, a process that may be relevant to the neuro-degeneration that occurs in AD (Johnson, 1994). In addition, these anti-apoptotic effects of EGb 761 are apparently related to the ability to inhibit the toxicity induced by H2O2, and that this natural extract has been reported as an effective tool to the apoptosis hydroxyl radical-induced in cultured neurons (Bastianetto & Quirion, 2002; Ni et al., 1996, Xin et al., 2000).

#### **4.4.1.5 Curcuminoides**

The curcuminoids are the active component of turmeric, which have been attributed to have capacity as an inhibitor of lipid peroxidation, and free radical scavenger, it is a potent antiinflammatory and anticancer, and is also traditionally used in Asia (Aggarwal et al., 2007; James, et al., 2009; Ramesh et al., 2010). In light of its antioxidant, anti-inflammatory and anti-amyloid actions, curcumin is being investigated as a candidate compound for the prevention or treatment of diseases such as multiple myeloma, pancreatic cancer, myelodysplastic syndromes, colon cancer, psoriasis and AD (Calabrese et al., 2010; Goel, et al., 2008).

The curcumin reduces pro-inflammatory cytokines, oxidative damage, the Aβ42 and cognitive deficits in models of AD (Frautschy et al., 2001). Also, has been told that it is a direct inhibitor of βA aggregation and fibril formation (Cole et al., 2003; James et al., 2009).

Free Radicals, Neuronal Death and Neuroprotection 187

be used in conjunction with the approach of Neurogerontology (James et al, 2009), so, it is necessary to continue investigating the potential benefits of various AO against the

As described earlier in this chapter, a variety of scientific evidence that describes the importance of different oxidative mechanisms which are part of the dynamic biological relevance of the ND as AD, PD or the CDS. Such approaches have led to a series of theories on the therapeutic use of antioxidants to slow down the chain reaction of oxidative events and thus to reduce its cytotoxic effects. Thus, consumption of antioxidants such as vitamin E and C, polyphenols and other antioxidants has became very important because they provide a series of defense mechanisms that promote longevity, reduce the risk of developing certain

However, there are some data that are incompatible with these theories, leading to various disputes regarding antioxidant supplementation and its possible beneficial effects on the body. In this sense, it is important to consider factors that may limit somewhat the research in this field. Among these factors: choosing an appropriate dose, long-term monitoring of a large cohort study, the inclusion or exclusion of different environmental factors, and *in vivo*

Occasionally, the start of antioxidant therapy for ND, is given when there may be a significant number of injured neurons, giving rise to specific clinical symptoms. In this case, antioxidants act on viable neurons, but do not recover the population of dead neurons. Therefore, in several studies of neuroprotection, supplying long-term supplements at the onset of the disease or even earlier is recommended; these results provide a valid evidence

Aisen P.S., Egelko S., Andrews H., Diaz-Arrastia R., Weiner, M. DeCarli, C., Jagust, W.;

Aggarwal BB, Sundaram C, Malani N, Ichikawa H (2007). Curcumin: the Indian solid gold.

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Avramovich-Tirosh, Y.; Reznichenko, L.; Mit, T.; Zheng, H.; Fridkin, M.; Weinreb, O.;

Miller, J.W.; Green, R K. Bell & Sano, M. (2003). A pilot study of vitamins to lower plasma homocysteine levels in Alzheimer disease, *Am J Geriatr Psychiatry,* Vol*.*11,

EPIDOS study. Association of Alzheimer's disease onset with ginkgo biloba and other symptomatic cognitive treatments in a population of women aged 75 years and older from the EPIDOS study. *J Gerontol A Biol Sci Med Sci*, Vol.58, pp. 372-7. Araki T, Sasaki Y, Milbrandt J. (2004). Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration. *Science*, Vol.13, pp.1010–1013. Araujo, J.A.; Studzinski, C.M. & Milgram, N.W. (2005). Further evidence for the cholinergic

hypothesis of aging and dementia from the canine model of aging. *Prog.* 

Mandel, S. & Youdim, M.B. (2007). Neurorescue activity, APP regulation and amyloid-beta peptide reduction by novel multi-functional brain permeable iron-

of the therapeutic effects of these substances and are a guarantee of further trials.

prevention and/or treatment of ND as AD and PD.

neuropathologies, and also can be consumed in a daily diet.

application of the results obtained in *in vitro* studies.

*Adv Exp Med Biol*, Vol.595, pp.1–75.

*Neuropsychopharm. Biol. Psychiat*, Vol.29, pp.411-422.

**6. Conclusions** 

**7. References** 

pp.246–249.

Similarly, curcumin has anti-AD activities, including, limitation of the kinase JNK (c-Jun Nterminal protein kinase) and stimulation of neurogenesis (Cole et al., 2007; James et al., 2009). Moreover, previous research has shown that turmeric reduces inflammation and oxidative damage in Tg2576 transgenic mouse brain AβPPSw (Kumar & Singh, 2008; Lim et al., 2001,), and that curcumin reduces the level of soluble and insoluble βA in several brain regions. Therefore, it has been suggested that this substance could prevent the onset of AD, not only by scavenging reactive oxygen species, but also by inhibiting the aggregation of βA in the brain (Ramesh et al., 2010). Similarly, Lim et al., 2001, studied the effects of the curcumin in transgenic mice carrying a human mutation of the amyloid precursor protein (APPsw) that causes AD (Lim et al., 2001) and found a reduction in brain level of oxidized proteins, and a decrease in both the level of soluble and insoluble βA, and the plaque burden (Lim et al., 2001). However, more studies are needed to test the potential therapeutic use of curcumin for the treatment or prevention of AD in humans (Calabrese et al., 2010).

The benefits of curcumin derive from its complex chemical structure and its ability to influence multiple signaling pathways, for instance, survival pathways such as those regulated by NF-kB, the Nrf2-dependent cytoprotective pathways, and routes of metastasis and angiogenesis (Calabrese et al., 2010; Goel et al.,2008; Ramsewak et al., 2000).

Because curcumin is highly toxic and has a limited bioavailability, the assessment of their impact on clinical practice has not been easy, however, using lipid formulations, this obstacle has been largely mitigated (Begum et al., 2008; James et al., 2009; Calabrese et al., 2010).

#### **5. Dietary recommendations for patients with ND as AD and PD**

Some evidence suggest that high intake of dietary antioxidants or fruits and vegetables provides nutritional compounds with antioxidant properties that may contribute to improving the quality of life, due to the decreased risk of degenerative diseases associated with aging and the accumulation of free radicals (Meydani et al.,1998). Therefore, although it is important to consider that dietary supplementation with AO may enhance cognitive longevity, and to some extent, reduces the risk of developing these diseases, it is also important to do some additional considerations. For example, the use of a single antioxidant, is not the best option, since the oxidation of individual antioxidants may promote oxidative stress, therefore, we recommend using combinations of antioxidants at the appropriate doses. Also, as mentioned previously, it has been found that diets rich in vitamin E may reduce the risk of EP, and has also been suggested that moderate wine consumption may reduce the risk to suffer from AD. However, there is still controversy about the epidemiological data related to these hypotheses, which could be due partially to the intrinsic difficulties of epidemiological surveys on the eating habits in large populations (Esposito, et al., 2002).

However, nutritional factors remain a very relevant topic when setting up a comprehensive treatment in patients with dementia. In addition, the formulation of a specific diet for people with AD or PD, requires a prior careful review of the patient, in order to identify their nutritional deficiencies, and thus design a healthy diet to ensure good physical health. For example, certain vitamins such as B12 and folic acid must be replaced to ensure that AD dementia is not due in part to this deficiency (Ramesh et al, 2010).

Finally, some authors argue that there is a lack of knowledge on these issues among medical professionals and clinicians, so they overlooked some dietary recommendations that could be used in conjunction with the approach of Neurogerontology (James et al, 2009), so, it is necessary to continue investigating the potential benefits of various AO against the prevention and/or treatment of ND as AD and PD.

#### **6. Conclusions**

186 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

Similarly, curcumin has anti-AD activities, including, limitation of the kinase JNK (c-Jun Nterminal protein kinase) and stimulation of neurogenesis (Cole et al., 2007; James et al., 2009). Moreover, previous research has shown that turmeric reduces inflammation and oxidative damage in Tg2576 transgenic mouse brain AβPPSw (Kumar & Singh, 2008; Lim et al., 2001,), and that curcumin reduces the level of soluble and insoluble βA in several brain regions. Therefore, it has been suggested that this substance could prevent the onset of AD, not only by scavenging reactive oxygen species, but also by inhibiting the aggregation of βA in the brain (Ramesh et al., 2010). Similarly, Lim et al., 2001, studied the effects of the curcumin in transgenic mice carrying a human mutation of the amyloid precursor protein (APPsw) that causes AD (Lim et al., 2001) and found a reduction in brain level of oxidized proteins, and a decrease in both the level of soluble and insoluble βA, and the plaque burden (Lim et al., 2001). However, more studies are needed to test the potential therapeutic use of curcumin for the treatment or prevention of AD in humans (Calabrese et al., 2010). The benefits of curcumin derive from its complex chemical structure and its ability to influence multiple signaling pathways, for instance, survival pathways such as those regulated by NF-kB, the Nrf2-dependent cytoprotective pathways, and routes of metastasis

and angiogenesis (Calabrese et al., 2010; Goel et al.,2008; Ramsewak et al., 2000).

**5. Dietary recommendations for patients with ND as AD and PD** 

2010).

(Esposito, et al., 2002).

Because curcumin is highly toxic and has a limited bioavailability, the assessment of their impact on clinical practice has not been easy, however, using lipid formulations, this obstacle has been largely mitigated (Begum et al., 2008; James et al., 2009; Calabrese et al.,

Some evidence suggest that high intake of dietary antioxidants or fruits and vegetables provides nutritional compounds with antioxidant properties that may contribute to improving the quality of life, due to the decreased risk of degenerative diseases associated with aging and the accumulation of free radicals (Meydani et al.,1998). Therefore, although it is important to consider that dietary supplementation with AO may enhance cognitive longevity, and to some extent, reduces the risk of developing these diseases, it is also important to do some additional considerations. For example, the use of a single antioxidant, is not the best option, since the oxidation of individual antioxidants may promote oxidative stress, therefore, we recommend using combinations of antioxidants at the appropriate doses. Also, as mentioned previously, it has been found that diets rich in vitamin E may reduce the risk of EP, and has also been suggested that moderate wine consumption may reduce the risk to suffer from AD. However, there is still controversy about the epidemiological data related to these hypotheses, which could be due partially to the intrinsic difficulties of epidemiological surveys on the eating habits in large populations

However, nutritional factors remain a very relevant topic when setting up a comprehensive treatment in patients with dementia. In addition, the formulation of a specific diet for people with AD or PD, requires a prior careful review of the patient, in order to identify their nutritional deficiencies, and thus design a healthy diet to ensure good physical health. For example, certain vitamins such as B12 and folic acid must be replaced to ensure that AD

Finally, some authors argue that there is a lack of knowledge on these issues among medical professionals and clinicians, so they overlooked some dietary recommendations that could

dementia is not due in part to this deficiency (Ramesh et al, 2010).

As described earlier in this chapter, a variety of scientific evidence that describes the importance of different oxidative mechanisms which are part of the dynamic biological relevance of the ND as AD, PD or the CDS. Such approaches have led to a series of theories on the therapeutic use of antioxidants to slow down the chain reaction of oxidative events and thus to reduce its cytotoxic effects. Thus, consumption of antioxidants such as vitamin E and C, polyphenols and other antioxidants has became very important because they provide a series of defense mechanisms that promote longevity, reduce the risk of developing certain neuropathologies, and also can be consumed in a daily diet.

However, there are some data that are incompatible with these theories, leading to various disputes regarding antioxidant supplementation and its possible beneficial effects on the body. In this sense, it is important to consider factors that may limit somewhat the research in this field. Among these factors: choosing an appropriate dose, long-term monitoring of a large cohort study, the inclusion or exclusion of different environmental factors, and *in vivo* application of the results obtained in *in vitro* studies.

Occasionally, the start of antioxidant therapy for ND, is given when there may be a significant number of injured neurons, giving rise to specific clinical symptoms. In this case, antioxidants act on viable neurons, but do not recover the population of dead neurons. Therefore, in several studies of neuroprotection, supplying long-term supplements at the onset of the disease or even earlier is recommended; these results provide a valid evidence of the therapeutic effects of these substances and are a guarantee of further trials.

#### **7. References**


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**Part 3** 

**Intracellular Signaling of Neurodegeneration** 


## **Part 3**

## **Intracellular Signaling of Neurodegeneration**

198 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

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Witschi. A.; Reddy, S.; Stofer, B. & Lauterburg, B.H. (1992). The systemic availability of oral glutathione. European Journal of Clinical Pharmacology, Vol.43, pp.667–669. Xin W, Wei T, Chen C, Ni Y, Zhao B, Hou J. (2000). Mechanisms of apoptosis in rat

Yao Z, Drieu K & Papadopoulos V. (2001). The Ginkgo biloba extract EGb 761 rescues the

Zandi PP, Anthony JC, Khachaturian AS, Stone SV, Gustafson D, Tschanz JT, et al. (2004).

Zhao, W., Richardson, J.S., Mombourquette, M.J. & Weil, J.A. (1995). An in vitro EPR study

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cerebellar granule cells induced by hydroxyl radicals and the effects of EGb 761 and

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Vol.282, pp.29296-29304.

**8** 

**Neuropathological Disorders and** 

**Phospholipase A2 Activities in the Brain** 

Phospholipases A2 (PLA2s) constitute a large and diverse group of enzymes with broad biological functions, ranging from membrane synthesis and turnover to the generation of signaling molecules. So far, more than 20 isoforms of PLA2 presenting diverse characteristics, including calcium requirement and subcellular localization, have been documented. Based on their nucleotide sequence and other properties, PLA2 enzymes have been categorized into 15 groups (I-XV) − according to the classification of Dennis (Burke & Dennis, 2009a, 2009b). Released by cells, several groups of PLA2s are relatively small proteins (~14 kDa) that require millimolar amounts of calcium for their optimal activation. These groups of enzymes have historically been called the secreted forms of PLA2 (or sPLA2). The remaining groups are larger proteins, localized in intracellular compartments,

The first intracellular PLA2 to be cloned was a 85-kD protein, classified as a group IV PLA2 (Dennis, 1997; Leslie, 1997). This enzyme, now designated as cytosolic PLA2α (cPLA2α), is known to be under the influence of extracellular signals likely to induce calcium mobilization and phosphorylation (Leslie, 1997). Another group of PLA2 (group VI), which does not require calcium variations for its activity, has been cloned (Balboa et al., 1997; Ma et al., 1997; Tang et al., 1997). This PLA2 isoform has been designated as calciumindependent PLA2 (iPLA2) (Balsinde & Dennis, 1997; Dennis, 1997) and, according to numerous lines of biochemical evidence, may account for most of the PLA2 activity detected in resting cells. From a pharmacological perspective, iPLA2 activity is markedly reduced by bromoenol lactone (BEL) suicide substrate, which is not an effective inhibitor of sPLA2 or cPLA2 enzymes at comparable concentrations (Balboa et al., 1997; Kudo & Murakami, 2002). Several interesting reviews have considered the functional and pathological implications of PLA2 enzymes (Balsinde & Balboa, 2005; Bazan et al., 1993; Brown et al., 2003; Farooqui & Horrocks, 2004; Farooqui et al., 2004; Hooks & Cummings, 2008; Kolko et al., 2007; Kudo & Murakami, 2002; Leslie, 2004; Phillis & O'Regan, 2004; Sun et al., 2004; Sun et al., 2005). In this report, we will describe new and unique functional roles of iPLA2 in the regulation of brain glutamate receptor functions, neuronal plasticity

**1. Introduction** 

which are either dependent or not on calcium ions.

and neurodegenerative processes.

**Calcium Independent Forms of** 

Julie Allyson and Guy Massicotte *Université du Québec à Trois-Rivières* 

*Trois-Rivières, Québec,* 

*Canada* 

### **Neuropathological Disorders and Calcium Independent Forms of Phospholipase A2 Activities in the Brain**

Julie Allyson and Guy Massicotte *Université du Québec à Trois-Rivières Trois-Rivières, Québec, Canada* 

#### **1. Introduction**

Phospholipases A2 (PLA2s) constitute a large and diverse group of enzymes with broad biological functions, ranging from membrane synthesis and turnover to the generation of signaling molecules. So far, more than 20 isoforms of PLA2 presenting diverse characteristics, including calcium requirement and subcellular localization, have been documented. Based on their nucleotide sequence and other properties, PLA2 enzymes have been categorized into 15 groups (I-XV) − according to the classification of Dennis (Burke & Dennis, 2009a, 2009b). Released by cells, several groups of PLA2s are relatively small proteins (~14 kDa) that require millimolar amounts of calcium for their optimal activation. These groups of enzymes have historically been called the secreted forms of PLA2 (or sPLA2). The remaining groups are larger proteins, localized in intracellular compartments, which are either dependent or not on calcium ions.

The first intracellular PLA2 to be cloned was a 85-kD protein, classified as a group IV PLA2 (Dennis, 1997; Leslie, 1997). This enzyme, now designated as cytosolic PLA2α (cPLA2α), is known to be under the influence of extracellular signals likely to induce calcium mobilization and phosphorylation (Leslie, 1997). Another group of PLA2 (group VI), which does not require calcium variations for its activity, has been cloned (Balboa et al., 1997; Ma et al., 1997; Tang et al., 1997). This PLA2 isoform has been designated as calciumindependent PLA2 (iPLA2) (Balsinde & Dennis, 1997; Dennis, 1997) and, according to numerous lines of biochemical evidence, may account for most of the PLA2 activity detected in resting cells. From a pharmacological perspective, iPLA2 activity is markedly reduced by bromoenol lactone (BEL) suicide substrate, which is not an effective inhibitor of sPLA2 or cPLA2 enzymes at comparable concentrations (Balboa et al., 1997; Kudo & Murakami, 2002). Several interesting reviews have considered the functional and pathological implications of PLA2 enzymes (Balsinde & Balboa, 2005; Bazan et al., 1993; Brown et al., 2003; Farooqui & Horrocks, 2004; Farooqui et al., 2004; Hooks & Cummings, 2008; Kolko et al., 2007; Kudo & Murakami, 2002; Leslie, 2004; Phillis & O'Regan, 2004; Sun et al., 2004; Sun et al., 2005). In this report, we will describe new and unique functional roles of iPLA2 in the regulation of brain glutamate receptor functions, neuronal plasticity and neurodegenerative processes.

Neuropathological Disorders and

enantiomer blocks the iPLA2γ isoform more efficiently.

Calcium Independent Forms of Phospholipase A2 Activities in the Brain 203

Group VIB iPLA2γ is a membrane-bound iPLA2 enzyme with unique features, such as utilization of distinct translation initiation sites producing different sizes of enzymes with distinct subcellular localizations (Kinsey,McHowat,Beckett et al., 2007; Mancuso et al., 2000; Mancuso et al., 2004; Murakami et al., 2005; Tanaka et al., 2000; J. Yang et al., 2003) and phospholipid selectivity in terms of sn-1/sn-2 positional specificity that differs among substrates (Yan et al., 2005) iPLA2γ has a mitochondrial localization signal in the N-terminal region and a peroxisomal localization signal near the C-terminus, and the 88-kDa full-length and 63-kDa translation products of iPLA2γ are preferentially distributed in mitochondria and peroxisomes, respectively (Kinsey,McHowat,Beckett et al., 2007; Mancuso et al., 2004; Murakami et al., 2005). In the brain, iPLA2 represents predominant phospholipase activity in cells under resting conditions (Wolf et al., 1995; H. C. Yang et al., 1999). Reverse transcription-polymerase chain reaction experiments have revealed that rat brains constitutively express messenger RNAs for at least 3 calcium-independent PLA2 isoforms, iPLA2β, iPLA2γ and cPLA2γ (Kinsey et al., 2005; Tang et al., 1997; Underwood et al., 1998). These isoforms are characterized by differential sensitivity to PLA2 inhibitors and, by isolating each enantiomer of the iPLA2 inhibitor BEL, Jenkins et al. (Jenkins et al., 2002) established that the (S)-enantiomer of BEL selectively reduces iPLA2β activity, while its (R)-

Although little is known about iPLA2 functions in neurons, a growing body of evidence suggests their involvement in hippocampal long-term potentiation (LTP) of excitatory synaptic transmission (Fujita et al., 2001; Wolf et al., 1995). Hippocampal LTP, first described by Bliss and Lomo in 1973, is commonly regarded as a functional model of synaptic adaptation (i.e. plasticity) that likely participates in learning and memory (Bliss & Collingridge, 1993). PLA2 activities are increased in membranes of slices prepared from the dentate gyrus after LTP induction in anaesthetized rats (Clements et al., 1991) and could be involved in hippocampal LTP expression by elevating the production of arachidonic acid (AA) that retrogradely increases transmitter release at glutamatergic synapses (Drapeau et al., 1990; J. H. Williams et al., 1989). Facilitation of transmitter release by PLA2s during LTP is also reinforced by the fact that iPLA2 activity plays an important role in membrane fusion

The notion that iPLA2 activity may facilitate LTP expression by increasing glutamate release is complicated, however, by an abundant number of reports demonstrating that synaptic potentiation, at least in area CA1 of the hippocampus, is not dependent on changes in transmitter release, but is rather mediated by mechanisms involving the up-regulation of postsynaptic responses mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors at glutamatergic synapses (Hayashi et al., 2000). Several alterations have been reported at postsynaptic sites during LTP, including faster kinetics of receptor-associated ion channels (Ambros-Ingerson & Lynch, 1993; Ambros-Ingerson et al., 1993), redistribution of existing receptors within the postsynaptic density (Xie et al., 1997) and insertion of new receptors at synapses (Lu et al., 2001; Pickard et al., 2001). Consistent with these observations, we recently demonstrated that pretreatment of hippocampal slices with the iPLA2 inhibitor BEL completely abolishes AMPA receptor translocation in synaptic membranes and expression of CA1 hippocampal LTP (Martel et al., 2006). Interestingly, both LTP and AMPA receptor translocation display enantio-selective impairment by the iPLA2γ blocker (R)-BEL but not by the iPLA2β inhibitor (S)-BEL, suggesting that iPLA2γ

processes required for exocytosis (Brown et al., 2003; Takuma & Ichida, 1997).

represents the crucial isoform governing hippocampal synaptic strengthening.

### **2. iPLA2 isoforms and functions**

Among PLA2 enzymes, group IV (cPLA2) and group VI (iPLA2) families represent intracellular enzymes with a catalytic serine in their lipase consensus motif. Various studies, including gene targeting, have indicated that group IVA cPLA2 (cPLA2α), which is regulated by calcium-dependent membrane translocation and mitogen-activated protein kinase (MAPK)-dependent phosphorylation, is central in stimulus-dependent eicosanoid biosynthesis (Bonventre et al., 1997; Uozumi et al., 1997). On the other hand, group VIA iPLA2 (iPLA2β) and group VIB iPLA2 (iPLA2γ) isoforms mainly exhibit PLA2 activity, whereas other iPLA2 isoforms δ, ε, ξ and η display triglyceride lipase and transacylase activities (Table 1) in marked preference to PLA2 activity (Jenkins et al., 2004; Quistad et al., 2003). Group VIA iPLA2β, the most extensively studied iPLA2 isoform, has been implicated in various cellular events, such as phospholipid remodelling (Balsinde et al., 1997; Balsinde & Dennis, 1997), eicosanoid formation (Tay & Melendez, 2004), cell proliferation (Herbert & Walker, 2006), apoptosis (Atsumi et al., 1998), and activation of store-operated channels and capacitative calcium influx (Smani et al., 2004). Disruption of the iPLA2β gene causes impaired sperm motility (Bao et al., 2004), mitigated insulin secretion (Bao, Bohrer et al., 2006; Bao, Song et al., 2006) and neuronal disorders presenting iron dyshomeostasis (Morgan et al., 2006).


Table 1. Calcium-independent group VI phospholipase A2 (iPLA2) (Adapted from (Schaloske & Dennis, 2006))

Among PLA2 enzymes, group IV (cPLA2) and group VI (iPLA2) families represent intracellular enzymes with a catalytic serine in their lipase consensus motif. Various studies, including gene targeting, have indicated that group IVA cPLA2 (cPLA2α), which is regulated by calcium-dependent membrane translocation and mitogen-activated protein kinase (MAPK)-dependent phosphorylation, is central in stimulus-dependent eicosanoid biosynthesis (Bonventre et al., 1997; Uozumi et al., 1997). On the other hand, group VIA iPLA2 (iPLA2β) and group VIB iPLA2 (iPLA2γ) isoforms mainly exhibit PLA2 activity, whereas other iPLA2 isoforms δ, ε, ξ and η display triglyceride lipase and transacylase activities (Table 1) in marked preference to PLA2 activity (Jenkins et al., 2004; Quistad et al., 2003). Group VIA iPLA2β, the most extensively studied iPLA2 isoform, has been implicated in various cellular events, such as phospholipid remodelling (Balsinde et al., 1997; Balsinde & Dennis, 1997), eicosanoid formation (Tay & Melendez, 2004), cell proliferation (Herbert & Walker, 2006), apoptosis (Atsumi et al., 1998), and activation of store-operated channels and capacitative calcium influx (Smani et al., 2004). Disruption of the iPLA2β gene causes impaired sperm motility (Bao et al., 2004), mitigated insulin secretion (Bao, Bohrer et al., 2006; Bao, Song et al., 2006) and neuronal disorders presenting iron dyshomeostasis

VIA-1 Human/Murine 84-85 8 ankyrin repeats iPLA2 VIA-2 Human/Murine 88-90 7 ankyrin repeats iPLA2<sup>β</sup> VIB Human/Murine 88-91 Membrane-bound iPLA2<sup>γ</sup>

protein

lipase

lipase

lipase

VIC Human/Murine 146 Integral membrane

**Group Source Feature**

VID Human 53 Acylglycerol

VIE Human 57 Acylglycerol

VIF Human 28 Acylglycerol

Table 1. Calcium-independent group VI phospholipase A2 (iPLA2) (Adapted from

**Molecular Alternate Mass (kDa) names**

transacylase,triglycerol

transacylase,triglycerol

transacylase,triglycerol

iPLA2

δ

iPLA2ε

iPLA2ζ

iPLA2η

**2. iPLA2 isoforms and functions** 

(Morgan et al., 2006).

(Schaloske & Dennis, 2006))

Group VIB iPLA2γ is a membrane-bound iPLA2 enzyme with unique features, such as utilization of distinct translation initiation sites producing different sizes of enzymes with distinct subcellular localizations (Kinsey,McHowat,Beckett et al., 2007; Mancuso et al., 2000; Mancuso et al., 2004; Murakami et al., 2005; Tanaka et al., 2000; J. Yang et al., 2003) and phospholipid selectivity in terms of sn-1/sn-2 positional specificity that differs among substrates (Yan et al., 2005) iPLA2γ has a mitochondrial localization signal in the N-terminal region and a peroxisomal localization signal near the C-terminus, and the 88-kDa full-length and 63-kDa translation products of iPLA2γ are preferentially distributed in mitochondria and peroxisomes, respectively (Kinsey,McHowat,Beckett et al., 2007; Mancuso et al., 2004; Murakami et al., 2005). In the brain, iPLA2 represents predominant phospholipase activity in cells under resting conditions (Wolf et al., 1995; H. C. Yang et al., 1999). Reverse transcription-polymerase chain reaction experiments have revealed that rat brains constitutively express messenger RNAs for at least 3 calcium-independent PLA2 isoforms, iPLA2β, iPLA2γ and cPLA2γ (Kinsey et al., 2005; Tang et al., 1997; Underwood et al., 1998). These isoforms are characterized by differential sensitivity to PLA2 inhibitors and, by isolating each enantiomer of the iPLA2 inhibitor BEL, Jenkins et al. (Jenkins et al., 2002) established that the (S)-enantiomer of BEL selectively reduces iPLA2β activity, while its (R) enantiomer blocks the iPLA2γ isoform more efficiently.

Although little is known about iPLA2 functions in neurons, a growing body of evidence suggests their involvement in hippocampal long-term potentiation (LTP) of excitatory synaptic transmission (Fujita et al., 2001; Wolf et al., 1995). Hippocampal LTP, first described by Bliss and Lomo in 1973, is commonly regarded as a functional model of synaptic adaptation (i.e. plasticity) that likely participates in learning and memory (Bliss & Collingridge, 1993). PLA2 activities are increased in membranes of slices prepared from the dentate gyrus after LTP induction in anaesthetized rats (Clements et al., 1991) and could be involved in hippocampal LTP expression by elevating the production of arachidonic acid (AA) that retrogradely increases transmitter release at glutamatergic synapses (Drapeau et al., 1990; J. H. Williams et al., 1989). Facilitation of transmitter release by PLA2s during LTP is also reinforced by the fact that iPLA2 activity plays an important role in membrane fusion processes required for exocytosis (Brown et al., 2003; Takuma & Ichida, 1997).

The notion that iPLA2 activity may facilitate LTP expression by increasing glutamate release is complicated, however, by an abundant number of reports demonstrating that synaptic potentiation, at least in area CA1 of the hippocampus, is not dependent on changes in transmitter release, but is rather mediated by mechanisms involving the up-regulation of postsynaptic responses mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors at glutamatergic synapses (Hayashi et al., 2000). Several alterations have been reported at postsynaptic sites during LTP, including faster kinetics of receptor-associated ion channels (Ambros-Ingerson & Lynch, 1993; Ambros-Ingerson et al., 1993), redistribution of existing receptors within the postsynaptic density (Xie et al., 1997) and insertion of new receptors at synapses (Lu et al., 2001; Pickard et al., 2001). Consistent with these observations, we recently demonstrated that pretreatment of hippocampal slices with the iPLA2 inhibitor BEL completely abolishes AMPA receptor translocation in synaptic membranes and expression of CA1 hippocampal LTP (Martel et al., 2006). Interestingly, both LTP and AMPA receptor translocation display enantio-selective impairment by the iPLA2γ blocker (R)-BEL but not by the iPLA2β inhibitor (S)-BEL, suggesting that iPLA2γ represents the crucial isoform governing hippocampal synaptic strengthening.

Neuropathological Disorders and

(AD) (Kwak & Weiss, 2006; Villmann & Becker, 2007).

Calcium Independent Forms of Phospholipase A2 Activities in the Brain 205

deleterious or beneficial effects on neurons. For instance, acute inhibition of iPLA2 activity by racemic BEL has been found to be neuroprotective in organotypic hippocampal slices exposed to oxygen-glucose deprivation (Strokin et al., 2006). In contrast, immature cultures of primary cortical neurons exposed for several days to BEL show decreased neuritogenesis and cellular viability (Forlenza et al., 2007; Mendes et al., 2005). Moreover, iPLA2β knockout mice exhibit abnormal motor behaviors accompanied by the appearance of vacuoles and ubiquitin-positive axonal swelling (spheroids) in many brain regions (Malik et al., 2008; Shinzawa et al., 2008), suggesting that iPLA2β dysfunction leads to neuroaxonal dystrophy. While the reported impact of iPLA2 on cell viablility is mostly attributable to iPLA2β, involvement of the iPLA2γ isoform is much less understood. A previous report demonstrated that iPLA2γ localized in mitochondria catalyzes AA liberation that mediates mitochondrial permeability transition, a key control point for apoptosis (Kinsey,McHowat,Patrick et al., 2007). On the other hand, iPLA2γ expression may exert cytoprotective effects during complement-mediated glomerular epithelial cell injury (Cohen et al., 2008). In addition, recent findings from our laboratory have revealed that constitutive iPLA2γ activity might represent an important neuroprotective system capable of limiting brain excitotoxic damage. We have shown that inhibition of iPLA2γ by the enantio-specific inhibitor (R)-BEL makes hippocampal slice cultures more vulnerable to AMPA-mediated excitotoxicity (Menard et al., 2007). Overactivation of N-methyl-D-aspartic acid (NMDA) or AMPA glutamatergic receptors, allowing the entry of high cation levels into cells, activates a number of enzymes, including ATPases, lipases, proteases and endonucleases that, in turn, deplete energy stores or damage cell membranes, cytoarchitecture or nucleus, respectively. Excitotoxicity has been reported to contribute to a variety of neuropathological disorders, including ischemic stroke, epilepsy, amyotrophic lateral sclerosis and Alzheimer's disease

Interestingly, the harmful effect of iPLA2γ inhibition on AMPA-mediated toxicity is associated with selective up-regulation of AMPA receptor GluR1 subunit (but not GluR2) phosphorylation with a subsequently increased level in synaptic membrane fractions (Menard et al., 2007; Menard et al., 2005; Villmann & Becker, 2007). In the hippocampus, AMPA receptors generally form heterodimers containing 2 copies of each of the GluR1 and GluR2 subunits. It is now well-recognized that GluR2 subunits render AMPA receptors impermeable to calcium. Consequently, its presence or absence plays a critical role in cellular calcium homeostasis and in determining susceptibility to excitotoxicity (Geiger et al., 1995; Sommer et al., 1991). Hence, the reduction of iPLA2γ activity, by promoting surface expression of the GluR1 subunit over the GluR2 subunit (which is reflected by a rise in the GluR1/GluR2 ratio in the membrane fraction), could exacerbate excitotoxic cell death through the formation of GluR2-lacking AMPA receptors that would allow adverse Ca2+ influx upon prolonged AMPA receptor activation. Consistent with this possibility, the greater cell death observed under iPLA2γ inhibition is prevented by GluR2-lacking AMPA receptor antagonists (Menard et al., 2007). How inhibition of iPLA2γ influences the expression of AMPA receptor subtypes in synaptic membranes remains an open question. As mentioned earlier, this may occur by the sorting of protein transport through intracellular secretory pathways (Pechoux et al., 2005). There are other circumstances in which GluR1 subunits are selectively up-regulated in hippocampal neurons, such as after activity deprivation elicited by prolonged blockade of AMPA receptors (Thiagarajan et al., 2005) or tumor necrosis factor-alpha receptor activation (Stellwagen et al., 2005). In the latter

iPLA2γ mRNAs and proteins are enriched in the endoplasmic reticulum (ER)-Golgi apparatus in several cell types (Kinsey et al., 2005), where they may be essential for diverse intracellular trafficking pathways, such as retrograde movement from the Golgi complex to the ER, transport of material from the trans-Golgi network to the plasma membrane or recycling of membrane and receptors through endocytic pathways (Brown et al., 2003). In this matter, Pechoux et al. (Pechoux et al., 2005) reported that iPLA2 inhibition slowed down the transport of caseins from the ER to the Golgi apparatus and from the trans-Golgi network to the plasma membrane, indicating that iPLA2 could participate in membrane trafficking events leading to the secretion of milk proteins. Interestingly, translocation of AMPA receptors originating from the ER-Golgi complex to postsynaptic membranes might be critically involved in LTP (Broutman & Baudry, 2001). Thus, the iPLA2γ isoform may be well-suited to favour AMPA receptor translocation from intracellular pools to synaptic membranes during LTP.

Interestingly, impairment in synaptic plasticity by PLA2 inhibition is correlated with loss of animal abilities to perform on memory tasks. For instance, intracerebral injection of widespectrum PLA2 inhibitors into the chick intermediate medial hyperstriatum ventrale curbs the learning of a passive avoidance task (Holscher & Rose, 1994), while intraperitoneal injections in rats impede spatial learning tested in the Morris water maze (Holscher et al., 1995). Additionally, intracerebroventricular injection of specific iPLA2 inhibitors 30 min before a learning session impairs spatial working memory in rodents (Fujita et al., 2000). Acquisition of 1-trial step-down inhibitory avoidance in rats correlates with iPLA2 activity in the hippocampus, and bilateral injection of iPLA2 inhibitors in region CA1 of the dorsal hippocampus prior to training hinders both short-term and long-term memory (Schaeffer & Gattaz, 2005). Hence, intact iPLA2 activity seems important for proper acquisition of new memories. In a modified protocol developed to test memory retrieval, the same group recently showed that injection of the dual cPLA2 and iPLA2 inhibitor palmitoyl trifluoromethylketone in region CA1 of the rat dorsal hippocampus before performance testing impaired trained behavior in the step-down inhibitory avoidance task (Schaeffer & Gattaz, 2007). Importantly, memory retrieval was re-established after recovery of PLA2 activity, indicating that these PLA2s are indeed necessary for memory retrieval. However, identification of iPLA2 isoforms in memory acquisition and retrieval remains to be addressed.

#### **3. iPLA2 and neuronal cell death mechanisms**

Recently, evidence from non-neuronal cells has suggested that iPLA2 enzymes may have diverse effects on cell death. First, constitutive iPLA2 activity may contribute to cell death since iPLA2β overexpression amplifies thapsigargin-induced apoptosis in INS-1 insulinoma cells (Ramanadham et al., 2004) and accelerates U937 cell death after long-term exposure to hydrogen peroxide (Perez et al., 2004). iPLA2 has been shown to play a pivotal role in oxidant damage of astrocytes (Xu et al., 2003), and its blockade by BEL dampens oligomeric amyloid-beta (Aβ1-42-induced mitochondrial membrane potential loss and reactive oxygen species production in these cells (Zhu et al., 2006). Moreover, iPLA2 inhibition reduces the size of infarcts produced by global ischemia (S. D. Williams & Gottlieb, 2002). On the other hand, iPLA2 activity has also been shown to protect against cell death, as inhibition of iPLA2 accentuates oxidant-induced cell death in renal proximal tubule cells and astrocytes (Cummings et al., 2002; Peterson et al., 2007). Likewise, iPLA2 activity may also have

iPLA2γ mRNAs and proteins are enriched in the endoplasmic reticulum (ER)-Golgi apparatus in several cell types (Kinsey et al., 2005), where they may be essential for diverse intracellular trafficking pathways, such as retrograde movement from the Golgi complex to the ER, transport of material from the trans-Golgi network to the plasma membrane or recycling of membrane and receptors through endocytic pathways (Brown et al., 2003). In this matter, Pechoux et al. (Pechoux et al., 2005) reported that iPLA2 inhibition slowed down the transport of caseins from the ER to the Golgi apparatus and from the trans-Golgi network to the plasma membrane, indicating that iPLA2 could participate in membrane trafficking events leading to the secretion of milk proteins. Interestingly, translocation of AMPA receptors originating from the ER-Golgi complex to postsynaptic membranes might be critically involved in LTP (Broutman & Baudry, 2001). Thus, the iPLA2γ isoform may be well-suited to favour AMPA receptor translocation from intracellular pools to synaptic

Interestingly, impairment in synaptic plasticity by PLA2 inhibition is correlated with loss of animal abilities to perform on memory tasks. For instance, intracerebral injection of widespectrum PLA2 inhibitors into the chick intermediate medial hyperstriatum ventrale curbs the learning of a passive avoidance task (Holscher & Rose, 1994), while intraperitoneal injections in rats impede spatial learning tested in the Morris water maze (Holscher et al., 1995). Additionally, intracerebroventricular injection of specific iPLA2 inhibitors 30 min before a learning session impairs spatial working memory in rodents (Fujita et al., 2000). Acquisition of 1-trial step-down inhibitory avoidance in rats correlates with iPLA2 activity in the hippocampus, and bilateral injection of iPLA2 inhibitors in region CA1 of the dorsal hippocampus prior to training hinders both short-term and long-term memory (Schaeffer & Gattaz, 2005). Hence, intact iPLA2 activity seems important for proper acquisition of new memories. In a modified protocol developed to test memory retrieval, the same group recently showed that injection of the dual cPLA2 and iPLA2 inhibitor palmitoyl trifluoromethylketone in region CA1 of the rat dorsal hippocampus before performance testing impaired trained behavior in the step-down inhibitory avoidance task (Schaeffer & Gattaz, 2007). Importantly, memory retrieval was re-established after recovery of PLA2 activity, indicating that these PLA2s are indeed necessary for memory retrieval. However, identification of iPLA2 isoforms in memory acquisition and retrieval remains to be

Recently, evidence from non-neuronal cells has suggested that iPLA2 enzymes may have diverse effects on cell death. First, constitutive iPLA2 activity may contribute to cell death since iPLA2β overexpression amplifies thapsigargin-induced apoptosis in INS-1 insulinoma cells (Ramanadham et al., 2004) and accelerates U937 cell death after long-term exposure to hydrogen peroxide (Perez et al., 2004). iPLA2 has been shown to play a pivotal role in oxidant damage of astrocytes (Xu et al., 2003), and its blockade by BEL dampens oligomeric amyloid-beta (Aβ1-42-induced mitochondrial membrane potential loss and reactive oxygen species production in these cells (Zhu et al., 2006). Moreover, iPLA2 inhibition reduces the size of infarcts produced by global ischemia (S. D. Williams & Gottlieb, 2002). On the other hand, iPLA2 activity has also been shown to protect against cell death, as inhibition of iPLA2 accentuates oxidant-induced cell death in renal proximal tubule cells and astrocytes (Cummings et al., 2002; Peterson et al., 2007). Likewise, iPLA2 activity may also have

membranes during LTP.

addressed.

**3. iPLA2 and neuronal cell death mechanisms** 

deleterious or beneficial effects on neurons. For instance, acute inhibition of iPLA2 activity by racemic BEL has been found to be neuroprotective in organotypic hippocampal slices exposed to oxygen-glucose deprivation (Strokin et al., 2006). In contrast, immature cultures of primary cortical neurons exposed for several days to BEL show decreased neuritogenesis and cellular viability (Forlenza et al., 2007; Mendes et al., 2005). Moreover, iPLA2β knockout mice exhibit abnormal motor behaviors accompanied by the appearance of vacuoles and ubiquitin-positive axonal swelling (spheroids) in many brain regions (Malik et al., 2008; Shinzawa et al., 2008), suggesting that iPLA2β dysfunction leads to neuroaxonal dystrophy. While the reported impact of iPLA2 on cell viablility is mostly attributable to iPLA2β, involvement of the iPLA2γ isoform is much less understood. A previous report demonstrated that iPLA2γ localized in mitochondria catalyzes AA liberation that mediates mitochondrial permeability transition, a key control point for apoptosis (Kinsey,McHowat,Patrick et al., 2007). On the other hand, iPLA2γ expression may exert cytoprotective effects during complement-mediated glomerular epithelial cell injury (Cohen et al., 2008). In addition, recent findings from our laboratory have revealed that constitutive iPLA2γ activity might represent an important neuroprotective system capable of limiting brain excitotoxic damage. We have shown that inhibition of iPLA2γ by the enantio-specific inhibitor (R)-BEL makes hippocampal slice cultures more vulnerable to AMPA-mediated excitotoxicity (Menard et al., 2007). Overactivation of N-methyl-D-aspartic acid (NMDA) or AMPA glutamatergic receptors, allowing the entry of high cation levels into cells, activates a number of enzymes, including ATPases, lipases, proteases and endonucleases that, in turn, deplete energy stores or damage cell membranes, cytoarchitecture or nucleus, respectively. Excitotoxicity has been reported to contribute to a variety of neuropathological disorders, including ischemic stroke, epilepsy, amyotrophic lateral sclerosis and Alzheimer's disease (AD) (Kwak & Weiss, 2006; Villmann & Becker, 2007).

Interestingly, the harmful effect of iPLA2γ inhibition on AMPA-mediated toxicity is associated with selective up-regulation of AMPA receptor GluR1 subunit (but not GluR2) phosphorylation with a subsequently increased level in synaptic membrane fractions (Menard et al., 2007; Menard et al., 2005; Villmann & Becker, 2007). In the hippocampus, AMPA receptors generally form heterodimers containing 2 copies of each of the GluR1 and GluR2 subunits. It is now well-recognized that GluR2 subunits render AMPA receptors impermeable to calcium. Consequently, its presence or absence plays a critical role in cellular calcium homeostasis and in determining susceptibility to excitotoxicity (Geiger et al., 1995; Sommer et al., 1991). Hence, the reduction of iPLA2γ activity, by promoting surface expression of the GluR1 subunit over the GluR2 subunit (which is reflected by a rise in the GluR1/GluR2 ratio in the membrane fraction), could exacerbate excitotoxic cell death through the formation of GluR2-lacking AMPA receptors that would allow adverse Ca2+ influx upon prolonged AMPA receptor activation. Consistent with this possibility, the greater cell death observed under iPLA2γ inhibition is prevented by GluR2-lacking AMPA receptor antagonists (Menard et al., 2007). How inhibition of iPLA2γ influences the expression of AMPA receptor subtypes in synaptic membranes remains an open question. As mentioned earlier, this may occur by the sorting of protein transport through intracellular secretory pathways (Pechoux et al., 2005). There are other circumstances in which GluR1 subunits are selectively up-regulated in hippocampal neurons, such as after activity deprivation elicited by prolonged blockade of AMPA receptors (Thiagarajan et al., 2005) or tumor necrosis factor-alpha receptor activation (Stellwagen et al., 2005). In the latter

Neuropathological Disorders and

pathology is represented in Figure 2

medium to label nuclei. Scale bar = 25 µm

 **Control**

**(R)-BEL**

Calcium Independent Forms of Phospholipase A2 Activities in the Brain 207

These changes appear to be associated with up-regulation of P25, an activator of cyclindependent kinase 5, and phosphorylation/activation of MAPK. These data provide strong evidence that constitutive iPLA2γ activity is important in the regulation of tau hyperphosphorylation in hippocampal pyramidal neurons, raising the possibility that iPLA2 dysfunctions might contribute to the development of tauopathies in AD. In this line, a putative biochemical model that accounts for the potential influence of iPLA2γ on Tau

AT231 DAPI Merge

Fig. 1. Inhibition of iPLA2γ induces Tau phosphorylation in area CA1 of the hippocampus. Cultured hippocampal slices from P301L tau transgenic mice were pre-exposed to the iPLA2γ inhibitor R-BEL. Slices were then processed for confocal immunofluorescence microscopy with an antibody known to recognize the Thr-231 Tau epitope (AT231, in green). When compared to controls (upper panel), immunostaining revealed increased phosphorylation in the CA1 region of cultured hippocampal slices pre-exposed to 3 µM (R)- BEL for a period of 12 h (lower panel). DAPI (in blue) was included in the mounting

One of the central hypotheses underlying the pathophysiology of AD is the production of cytotoxic Aβ peptides that impairs neuronal activity and leads to a decline in memory and cognition (Palop et al., 2006). The exact mechanisms by which Aβ peptides contribute to AD pathogenesis remain uncertain. PLA2 enzymes may be involved in this condition, as Aβ peptides accentuate cPLA2α activity in neuronal cultures (Zhu et al., 2006) and primary cortical astrocytes (Sanchez-Mejia et al., 2008), while Aβ-induced learning and memory deficits in a transgenic mouse model of AD are prevented after genetic ablation of cPLA2α activity in the brain (Sanchez-Mejia et al., 2008). Regarding the iPLA2 system, it appears that its activity is essential for maintaining membrane phospholipid integrity by reducing peroxidative damage, especially injuries originating in the mitochondria. In this

case, it has been proposed that up-regulation of GluR1 homomeric receptors could derive from a reserve pool of non-GluR2-containing AMPA receptors existing near the membrane.

### **4. iPLA2 dysfunction and neuropathological disorders**

Whereas cPLA2 and sPLA2 are commonly believed to be preferentially involved in AA release, emerging evidence indicates that iPLA2 activity can contribute to docosahexaenoic acid (DHA) release from brain phospholipids (J. T. Green et al., 2008). To our knowledge, the first suggestion that brain iPLA2 activity may be crucial for DHA release came from a study by Strokin et al. (Strokin et al., 2003) who showed that racemic BEL inhibited DHA release from astrocytes. Later, using siRNA silencing procedures, the same group demonstrated that DHA release from phospholipids of astrocytes was mainly dependent on iPLA2γ activity (Strokin et al., 2007). DHA is one of the most abundant omega-3 polyunsaturated fatty acids (PUFA) present in phospholipids of the mammalian brain (Glomset, 2006), where it is recognized to be important for the maintenance of neural membranes and brain function integrity (Youdim et al., 2000). Deficient dietary intake of DHA has been associated with lower performance of learning abilities in rodents (Catalan et al., 2002; Fedorova & Salem, 2006; Takeuchi et al., 2002). On the other hand, DHA dietary supplementation could decrease the risk of developing AD (Calon & Cole, 2007; Calon et al., 2005; Calon et al., 2004) or exert neuroprotective actions in a mouse model presenting numerous aspects of Parkinson's disease (Bousquet et al., 2008), while high-fat consumption combined with low omega-3 PUFA intake promotes AD-like neuropathology (Julien et al., 2008).

Both iPLA2 activity and DHA levels have been reported to be decreased in the plasma of AD patients (Conquer et al., 2000; Gattaz et al., 2004). iPLA2 activity is also lower in AD brains (Ross et al., 1999; Talbot et al., 2000). Whether or not decreased iPLA2γ activity, through its capacity to alter DHA release from brain astrocytes, is a factor that contributes to AD pathology remains to be established. Numerous neurobiological studies have demonstrated that DHA may be acting at different fundamental levels to counteract the cellular manifestations of AD. There are, for instance, strong indications that DHA release in the brain may diminish oxidative stress (Wu et al., 2004; Yavin et al., 2002) and glutamateinduced toxicity (Wang et al., 2003). In this line, DHA-induced reduction of excitotoxic damage in the hippocampus might, in fact, be dependent on internalization of AMPA receptors (Menard et al., 2009). The potential ability of DHA to reduce caspase activation (Calon et al., 2005; Calon et al., 2004), Aβ peptide accumulation and tau hyperphosphorylation (K. N. Green et al., 2007; Oksman et al., 2006) also strongly supports the notion that DHA deficiency, through iPLA2 down-regulation, could represent a precursor event that likely initiates the cellular manifestations of AD pathology.

This has been the premise of our recent investigation on the influence of iPLA2 inhibition on microtubule-associated protein tau phosphorylation. We determined whether iPLA2 blockade could contribute to the development of tau hyperphosphorylation in cultured hippocampal slices from transgenic P301L mice expressing human tau. In this experimental model, treatment for up to 12 h with the specific iPLA2γ inhibitor (R)-BEL resulted in significantly increased tau phosphorylation at Thr231, Ser199/202 and Ser404 sites, and in total tau levels. High-resolution imaging studies have demonstrated that hyperphosphorylation is primarily localized in cell bodies and dendrites of hippocampal pyramidal neurons (Fig. 1).

case, it has been proposed that up-regulation of GluR1 homomeric receptors could derive from a reserve pool of non-GluR2-containing AMPA receptors existing near the membrane.

Whereas cPLA2 and sPLA2 are commonly believed to be preferentially involved in AA release, emerging evidence indicates that iPLA2 activity can contribute to docosahexaenoic acid (DHA) release from brain phospholipids (J. T. Green et al., 2008). To our knowledge, the first suggestion that brain iPLA2 activity may be crucial for DHA release came from a study by Strokin et al. (Strokin et al., 2003) who showed that racemic BEL inhibited DHA release from astrocytes. Later, using siRNA silencing procedures, the same group demonstrated that DHA release from phospholipids of astrocytes was mainly dependent on iPLA2γ activity (Strokin et al., 2007). DHA is one of the most abundant omega-3 polyunsaturated fatty acids (PUFA) present in phospholipids of the mammalian brain (Glomset, 2006), where it is recognized to be important for the maintenance of neural membranes and brain function integrity (Youdim et al., 2000). Deficient dietary intake of DHA has been associated with lower performance of learning abilities in rodents (Catalan et al., 2002; Fedorova & Salem, 2006; Takeuchi et al., 2002). On the other hand, DHA dietary supplementation could decrease the risk of developing AD (Calon & Cole, 2007; Calon et al., 2005; Calon et al., 2004) or exert neuroprotective actions in a mouse model presenting numerous aspects of Parkinson's disease (Bousquet et al., 2008), while high-fat consumption combined with low omega-3 PUFA intake promotes AD-like neuropathology (Julien et al.,

Both iPLA2 activity and DHA levels have been reported to be decreased in the plasma of AD patients (Conquer et al., 2000; Gattaz et al., 2004). iPLA2 activity is also lower in AD brains (Ross et al., 1999; Talbot et al., 2000). Whether or not decreased iPLA2γ activity, through its capacity to alter DHA release from brain astrocytes, is a factor that contributes to AD pathology remains to be established. Numerous neurobiological studies have demonstrated that DHA may be acting at different fundamental levels to counteract the cellular manifestations of AD. There are, for instance, strong indications that DHA release in the brain may diminish oxidative stress (Wu et al., 2004; Yavin et al., 2002) and glutamateinduced toxicity (Wang et al., 2003). In this line, DHA-induced reduction of excitotoxic damage in the hippocampus might, in fact, be dependent on internalization of AMPA receptors (Menard et al., 2009). The potential ability of DHA to reduce caspase activation (Calon et al., 2005; Calon et al., 2004), Aβ peptide accumulation and tau hyperphosphorylation (K. N. Green et al., 2007; Oksman et al., 2006) also strongly supports the notion that DHA deficiency, through iPLA2 down-regulation, could represent a

precursor event that likely initiates the cellular manifestations of AD pathology.

This has been the premise of our recent investigation on the influence of iPLA2 inhibition on microtubule-associated protein tau phosphorylation. We determined whether iPLA2 blockade could contribute to the development of tau hyperphosphorylation in cultured hippocampal slices from transgenic P301L mice expressing human tau. In this experimental model, treatment for up to 12 h with the specific iPLA2γ inhibitor (R)-BEL resulted in significantly increased tau phosphorylation at Thr231, Ser199/202 and Ser404 sites, and in total tau levels. High-resolution imaging studies have demonstrated that hyperphosphorylation is primarily localized in cell bodies and dendrites of hippocampal

**4. iPLA2 dysfunction and neuropathological disorders** 

2008).

pyramidal neurons (Fig. 1).

These changes appear to be associated with up-regulation of P25, an activator of cyclindependent kinase 5, and phosphorylation/activation of MAPK. These data provide strong evidence that constitutive iPLA2γ activity is important in the regulation of tau hyperphosphorylation in hippocampal pyramidal neurons, raising the possibility that iPLA2 dysfunctions might contribute to the development of tauopathies in AD. In this line, a putative biochemical model that accounts for the potential influence of iPLA2γ on Tau pathology is represented in Figure 2

Fig. 1. Inhibition of iPLA2γ induces Tau phosphorylation in area CA1 of the hippocampus. Cultured hippocampal slices from P301L tau transgenic mice were pre-exposed to the iPLA2γ inhibitor R-BEL. Slices were then processed for confocal immunofluorescence microscopy with an antibody known to recognize the Thr-231 Tau epitope (AT231, in green). When compared to controls (upper panel), immunostaining revealed increased phosphorylation in the CA1 region of cultured hippocampal slices pre-exposed to 3 µM (R)- BEL for a period of 12 h (lower panel). DAPI (in blue) was included in the mounting medium to label nuclei. Scale bar = 25 µm

One of the central hypotheses underlying the pathophysiology of AD is the production of cytotoxic Aβ peptides that impairs neuronal activity and leads to a decline in memory and cognition (Palop et al., 2006). The exact mechanisms by which Aβ peptides contribute to AD pathogenesis remain uncertain. PLA2 enzymes may be involved in this condition, as Aβ peptides accentuate cPLA2α activity in neuronal cultures (Zhu et al., 2006) and primary cortical astrocytes (Sanchez-Mejia et al., 2008), while Aβ-induced learning and memory deficits in a transgenic mouse model of AD are prevented after genetic ablation of cPLA2α activity in the brain (Sanchez-Mejia et al., 2008). Regarding the iPLA2 system, it appears that its activity is essential for maintaining membrane phospholipid integrity by reducing peroxidative damage, especially injuries originating in the mitochondria. In this

Neuropathological Disorders and

**6. References** 

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hyperdopaminergia (Wiedholz et al., 2008), a well-established biological defect involved in schizophrenia-related behaviours. Interestingly, the relationship between iPLA2s and the dopaminergic system is reinforced by the fact that iPLA2 inhibition or knockdown in the rat striatum, motor cortex and thalamus results in the apparition of Parkinson-related behaviours (Lee et al., 2007), which are also known to depend on dopamine dysfunction. Thus, given the growing evidence relating the importance of iPLA2s in physiological and pathological conditions, targeting iPLA2 activity may represent a potentially new

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Fig. 2. A putative model illustrating the potential implication of iPLA2γ in Alzheimer's disease. In this simplified model, iPLA2 dysfunction leads to delivery of new GluR1 containing receptors on neuronal membranes. These receptors are then inclined to induce calcium influx and, eventually, Tau phosphorylation by calcium-dependent protein kinases such as Cdk5 and GSK-3β

#### **5. Conclusion**

Besides AD, aberrant function of iPLA2s has also been observed in several other neurological disorders. For instance, increased iPLA2 activity might be an important factor that contributes to phospholipid abnormalities in schizophrenia or bipolar patients with a history of psychosis (Ross et al., 2006; Ross et al., 1999). However, the relationship between iPLA2 up-regulation and cellular manifestations of schizophrenia requires further investigation. As mentioned earlier, because iPLA2γ regulates glutamate receptor subunit expression on cell membranes and functions, it will be interesting to examine whether the increase in iPLA2γ activity can lead to down-regulation of the AMPA receptor GluR1 subunit. This is of particular importance, since GluR1 down-regulation may evoke striatal hyperdopaminergia (Wiedholz et al., 2008), a well-established biological defect involved in schizophrenia-related behaviours. Interestingly, the relationship between iPLA2s and the dopaminergic system is reinforced by the fact that iPLA2 inhibition or knockdown in the rat striatum, motor cortex and thalamus results in the apparition of Parkinson-related behaviours (Lee et al., 2007), which are also known to depend on dopamine dysfunction. Thus, given the growing evidence relating the importance of iPLA2s in physiological and pathological conditions, targeting iPLA2 activity may represent a potentially new therapeutic strategy against several neurological disorders.

#### **6. References**

208 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

regard, iPLA2 expression prevents the loss of mitochondrial membrane potential and attenuates the release of cytochrome c as well as apoptotic proteins, and ultimately diminishes apoptosis in INS-1 cells exposed to staurosporine (Seleznev et al., 2006). Furthermore, Kinsey et al. (Kinsey et al., 2008; Kinsey,McHowat,Patrick et al., 2007) reported that prominent PLA2 activity in the mitochondria of rabbit renal proximal tubular cells comes from iPLA2γ and is of capital importance for the prevention and repair of basal lipid peroxidation and the maintenance of mitochondrial viability. Based on recent studies, it has been proposed that Aβ-induced neurotoxicity might derive from mitochondrial defects. Indeed, in vitro experiments have shown that Aβ peptides can be internalized by cells, imported into mitochondria and ultimately elicit mitochondrial dysfunctions (Hansson Petersen et al., 2008). Given its localization, it is thus tempting to propose that iPLA2γ might represent an important cellular component that prevents mitochondrial dysfunctions. Experiments are required to determine whether iPLA2γ overexpression activity might exert

protective effects against Aβ peptide-induced mitochondrial dysfunctions.

Fig. 2. A putative model illustrating the potential implication of iPLA2γ in Alzheimer's disease. In this simplified model, iPLA2 dysfunction leads to delivery of new GluR1 containing receptors on neuronal membranes. These receptors are then inclined to induce calcium influx and, eventually, Tau phosphorylation by calcium-dependent protein kinases

Besides AD, aberrant function of iPLA2s has also been observed in several other neurological disorders. For instance, increased iPLA2 activity might be an important factor that contributes to phospholipid abnormalities in schizophrenia or bipolar patients with a history of psychosis (Ross et al., 2006; Ross et al., 1999). However, the relationship between iPLA2 up-regulation and cellular manifestations of schizophrenia requires further investigation. As mentioned earlier, because iPLA2γ regulates glutamate receptor subunit expression on cell membranes and functions, it will be interesting to examine whether the increase in iPLA2γ activity can lead to down-regulation of the AMPA receptor GluR1 subunit. This is of particular importance, since GluR1 down-regulation may evoke striatal

such as Cdk5 and GSK-3β

**5. Conclusion** 


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**9** 

*USA* 

**ASK1 and Its Role in** 

**Neurodegenerative Diseases** 

*Translational Research Institute Scripps Florida Jupiter, FL* 

The apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed serine/threonine protein kinase and one of more than 20 members that make up the triple MAP kinase (MAP3K) family of enzymes. Over the past decade, genetic studies have revealed that ASK1 plays a pivitol role in the cellular response to a wide variety of environmental and biological stressors including; reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), endoplasmic reticulum (ER) stress caused by protein aggregation, influx of calcium ions, and receptor-mediated signals transduced via lipopolysaccharides (LPS), Fas ligand, cytokines (TNFα) and certain G protein-coupled receptor (GPCR) agonists [1-5]. In addition, exogenous expression of ASK1 in cells has shown that ASK1 signaling engages the intrinsic apoptosis pathway promoting cytochrome *c* release from mitochodria and subsequent activation of caspase 3 and 9 [1, 6, 7]. Conversely, ASK1 deficient cells are resistant to cell death induced by oxidative and ER stress, indicating

Once activated, ASK1 relays cellular stress signals via the classical three tierd mitogen activated protein kinase (MAPK) signaling cascade, whereby a MAP3K phosphorylates and activates a MAP2K, that in turn phosphorylates and activates a MAPK [9] (Figure 1). More specifically, the ASK1 signaling axis activates the p38 and the c-jun NH2-terminal kinases (JNK) family of MAPKs, via activation of MKK3/MKK6 and MKK4/MKK7 respectively [1, 2, 4]. In addition to its role in the cellular stress response, ASK1 also regulates physiological processes including neuronal differentiation, synaptic plasticity and the innate immune response [10-13]. Thus, ASK1 acts as an important regulator of several important biological

Regulation of ASK1 activity is accomplished via a number of mechanisms including; protein-protein interactions as well as both spatial and temporal control. Firstly, more than 30 ASK1 interacting partners have been shown to regulate ASK1 activity (either positively or negatively) by posttranslational modifications and/or by inducing conformational changes through protein-protein interactions. Secondly, ASK1 signaling complexes are located in both the cytoplasm and mitochondria [14], with nuclear translocation observed upon stress induction indicating that ASK1 localization might also dictate the biological outcome [15,16] and thirdly, duration of ASK1 signaling can influence the nature of the

that ASK1 acts as the lynch pin in certain forms of stress-induced cell death [8].

processess and not surprisingly, ASK1 activation is under tight regulatory control.

**1. Introduction** 

Emmanuel Sturchler, Daniel Feurstein, Patricia McDonald and Derek Duckett *Department of Molecular Therapeutics and* 


### **ASK1 and Its Role in Neurodegenerative Diseases**

Emmanuel Sturchler, Daniel Feurstein, Patricia McDonald and Derek Duckett *Department of Molecular Therapeutics and Translational Research Institute Scripps Florida Jupiter, FL USA* 

#### **1. Introduction**

216 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

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The apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed serine/threonine protein kinase and one of more than 20 members that make up the triple MAP kinase (MAP3K) family of enzymes. Over the past decade, genetic studies have revealed that ASK1 plays a pivitol role in the cellular response to a wide variety of environmental and biological stressors including; reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), endoplasmic reticulum (ER) stress caused by protein aggregation, influx of calcium ions, and receptor-mediated signals transduced via lipopolysaccharides (LPS), Fas ligand, cytokines (TNFα) and certain G protein-coupled receptor (GPCR) agonists [1-5]. In addition, exogenous expression of ASK1 in cells has shown that ASK1 signaling engages the intrinsic apoptosis pathway promoting cytochrome *c* release from mitochodria and subsequent activation of caspase 3 and 9 [1, 6, 7]. Conversely, ASK1 deficient cells are resistant to cell death induced by oxidative and ER stress, indicating that ASK1 acts as the lynch pin in certain forms of stress-induced cell death [8].

Once activated, ASK1 relays cellular stress signals via the classical three tierd mitogen activated protein kinase (MAPK) signaling cascade, whereby a MAP3K phosphorylates and activates a MAP2K, that in turn phosphorylates and activates a MAPK [9] (Figure 1). More specifically, the ASK1 signaling axis activates the p38 and the c-jun NH2-terminal kinases (JNK) family of MAPKs, via activation of MKK3/MKK6 and MKK4/MKK7 respectively [1, 2, 4]. In addition to its role in the cellular stress response, ASK1 also regulates physiological processes including neuronal differentiation, synaptic plasticity and the innate immune response [10-13]. Thus, ASK1 acts as an important regulator of several important biological processess and not surprisingly, ASK1 activation is under tight regulatory control.

Regulation of ASK1 activity is accomplished via a number of mechanisms including; protein-protein interactions as well as both spatial and temporal control. Firstly, more than 30 ASK1 interacting partners have been shown to regulate ASK1 activity (either positively or negatively) by posttranslational modifications and/or by inducing conformational changes through protein-protein interactions. Secondly, ASK1 signaling complexes are located in both the cytoplasm and mitochondria [14], with nuclear translocation observed upon stress induction indicating that ASK1 localization might also dictate the biological outcome [15,16] and thirdly, duration of ASK1 signaling can influence the nature of the

ASK1 and Its Role in Neurodegenerative Diseases 219

Based on sequence homology, three human ASK1 related genes have been identified, namely ASK1 (MAP3K5), ASK2 (MAP3K6) and ASK3 (MAP3K15). In this review we will focus specifically on the biology of ASK1, commenting only on ASK2 and 3 where appropriate. ASK1 is a 170 kDa protein composed of an inhibitiory N-terminal domain, an internal kinase domain and a C-terminal regulatory domain. Although the primary stuctures of the N- and C regulatory domains within the ASK family exhibit a fair degree of divergence, those within the kinase domains are highly conseved. High resolution crystal structure studies revealed that the ASK1 catalytic domain structure displays a typical protein kinase fold, comprised of five β sheets and helix αC constituting the small lobe and a larger mainly α helical C-terminal lobe. The hinge region connecting the two domains lines the catalytic ATP binding site and although catalytically active, the recombinant enzyme

It is well established that both the subcellular localization as well as the magnitude and duration of MAPK activation are important for determining cellular fate. Thus, specific mechanisms may enable ASK1 modules to be rapidly activated or inactivated in a spatial and temporal manner. Increasing evidence indicates that depending on the stimulus and the cellular context, ASK1 activity is tightly regulated by multiple and distinct molecular events including phosphorylation/dephosphorylation of key residues, protein-protein interactions, and ubiquitination, resulting in ASK1 degradation and feedback regulation. Defects in these mechanisms may lead to aberrant ASK1 activity and to certain pathologies in humans.

In resting cells, ASK1 forms homo-oligomers through protein interactions via the C-terminal coiled-coiled domain (CCC). Cell-based and biochemical studies have demonstrated that ASK1 is associated with a number of interacting proteins which can lead to the formation of high molecular weight complexes (1000-3000 kDa), designated the ASK1 signalosome [18, 19]. To date, over 30 proteins have been shown to interact with ASK1 and regulate its activity. While the presice nature of the ASK1 signalosome remains to be fully characterised, it is postulated to be highly dynamic, serving as a foundation for the assembly of specific signaling modules and the subsets of regulatory proteins that it recruits depends upon the

Through genetic screening for ASK1-binding proteins, the redox protein Thioredoxin (Trx1) was one of the first ASK1 interacting proteins identified and has been shown to play a key role in the regulation of ASK1 in response to oxidative stress induced by H2O2 or tumor necrosis factor-α (TNFα) (Figure 2) [20, 21]. The thiol reductase activity of Trx1 is provided by cysteines 32 (Cys32) and 35 (Cys35) which forms a redox cataylitic CXPC motif [22]. Several studies demonstrated that tight association of a reduced form of Trx1 with the Nterminus of ASK1 suppresses ASK1 kinase activity by inhibiting N-terminal interactions between ASK1 monomers. The interaction has been shown to involve the catalytic cysteines Cys32 and Cys35 of Trx1, as mutation of either is sufficient to inhibit the dissociation of Trx1 from ASK1 [21]. Moreover, antioxidants such as catalase and N-acetylycysteine (NAC) block the release of Trx1 from ASK1 after challenge with H2O2, preventing ASK1 activation and associated cell death [23, 24]. These studies led to a model whereby upon H2O2 stimulation, Trx-1 is oxidized on Cys32 and Cys35 promoting formation of an intramolecular disulfide

**3. Modulation of ASK1 activity by postranslational modification** 

**2. Regulation of ASK1 activity** 

context of the initial stress.

adopts a non-active conformation in the crystal state [17].

cellular response. In this context, defects in the fine tuning of ASK1 activity can contribute to a number of pathological conditions including inflammatory, cardiac and several neurodegenerative diseases. In this chapter, we will discuss in detail the molecular mechanisms that regulate ASK1 activity, and focus on the contribution of ASK1 to neurodegenerative diseases such as Alzheimer's Disease, Parkinson's Disease, Polyglutamine (polyQ) Diseases, amyotrophic lateral sclerosis and stroke, together with the potential of ASK1 as a therapeutic target for the treatment of such disorders.

Fig. 1. ASK1, along with at least 20 other kinases belongs to the MAP3K family of enzymes. MAP3K selectively activate MAP2Ks with in turn activate members of the MAPK family. There are 7 MAP2K and 11 MAPK family members with the global MAPK family constituting 8% of the kinome. Specifically, ASK1 responds to multiple extracellular and intracellular stimuli by selectively phosphorylating MKK3/6 and/or MKK4/7 leading to the specific activation of the p38 and JNK MAPK pathways

#### **2. Regulation of ASK1 activity**

218 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

cellular response. In this context, defects in the fine tuning of ASK1 activity can contribute to a number of pathological conditions including inflammatory, cardiac and several neurodegenerative diseases. In this chapter, we will discuss in detail the molecular mechanisms that regulate ASK1 activity, and focus on the contribution of ASK1 to neurodegenerative diseases such as Alzheimer's Disease, Parkinson's Disease, Polyglutamine (polyQ) Diseases, amyotrophic lateral sclerosis and stroke, together with the potential of

**H2O2**

**ASK1**

**UV TNF**

**Angiotensin**

**ER stress**

**Calcium overload**

**MKK3/6 MKK4/7**

**p38 ,β,γ,δ JNK1,2,3**

α

**Cell survival/death, inflammation**

Fig. 1. ASK1, along with at least 20 other kinases belongs to the MAP3K family of enzymes. MAP3K selectively activate MAP2Ks with in turn activate members of the MAPK family. There are 7 MAP2K and 11 MAPK family members with the global MAPK family constituting 8% of the kinome. Specifically, ASK1 responds to multiple extracellular and intracellular stimuli by selectively phosphorylating MKK3/6 and/or MKK4/7 leading to the

ASK1 as a therapeutic target for the treatment of such disorders.

**MAP3K**

**Cell membrane**

**MAP2K**

**MAPK**

specific activation of the p38 and JNK MAPK pathways

Based on sequence homology, three human ASK1 related genes have been identified, namely ASK1 (MAP3K5), ASK2 (MAP3K6) and ASK3 (MAP3K15). In this review we will focus specifically on the biology of ASK1, commenting only on ASK2 and 3 where appropriate. ASK1 is a 170 kDa protein composed of an inhibitiory N-terminal domain, an internal kinase domain and a C-terminal regulatory domain. Although the primary stuctures of the N- and C regulatory domains within the ASK family exhibit a fair degree of divergence, those within the kinase domains are highly conseved. High resolution crystal structure studies revealed that the ASK1 catalytic domain structure displays a typical protein kinase fold, comprised of five β sheets and helix αC constituting the small lobe and a larger mainly α helical C-terminal lobe. The hinge region connecting the two domains lines the catalytic ATP binding site and although catalytically active, the recombinant enzyme adopts a non-active conformation in the crystal state [17].

It is well established that both the subcellular localization as well as the magnitude and duration of MAPK activation are important for determining cellular fate. Thus, specific mechanisms may enable ASK1 modules to be rapidly activated or inactivated in a spatial and temporal manner. Increasing evidence indicates that depending on the stimulus and the cellular context, ASK1 activity is tightly regulated by multiple and distinct molecular events including phosphorylation/dephosphorylation of key residues, protein-protein interactions, and ubiquitination, resulting in ASK1 degradation and feedback regulation. Defects in these mechanisms may lead to aberrant ASK1 activity and to certain pathologies in humans.

#### **3. Modulation of ASK1 activity by postranslational modification**

In resting cells, ASK1 forms homo-oligomers through protein interactions via the C-terminal coiled-coiled domain (CCC). Cell-based and biochemical studies have demonstrated that ASK1 is associated with a number of interacting proteins which can lead to the formation of high molecular weight complexes (1000-3000 kDa), designated the ASK1 signalosome [18, 19]. To date, over 30 proteins have been shown to interact with ASK1 and regulate its activity. While the presice nature of the ASK1 signalosome remains to be fully characterised, it is postulated to be highly dynamic, serving as a foundation for the assembly of specific signaling modules and the subsets of regulatory proteins that it recruits depends upon the context of the initial stress.

Through genetic screening for ASK1-binding proteins, the redox protein Thioredoxin (Trx1) was one of the first ASK1 interacting proteins identified and has been shown to play a key role in the regulation of ASK1 in response to oxidative stress induced by H2O2 or tumor necrosis factor-α (TNFα) (Figure 2) [20, 21]. The thiol reductase activity of Trx1 is provided by cysteines 32 (Cys32) and 35 (Cys35) which forms a redox cataylitic CXPC motif [22]. Several studies demonstrated that tight association of a reduced form of Trx1 with the Nterminus of ASK1 suppresses ASK1 kinase activity by inhibiting N-terminal interactions between ASK1 monomers. The interaction has been shown to involve the catalytic cysteines Cys32 and Cys35 of Trx1, as mutation of either is sufficient to inhibit the dissociation of Trx1 from ASK1 [21]. Moreover, antioxidants such as catalase and N-acetylycysteine (NAC) block the release of Trx1 from ASK1 after challenge with H2O2, preventing ASK1 activation and associated cell death [23, 24]. These studies led to a model whereby upon H2O2 stimulation, Trx-1 is oxidized on Cys32 and Cys35 promoting formation of an intramolecular disulfide

ASK1 and Its Role in Neurodegenerative Diseases 221

ASK1 activity, it was demonstrated that mutation of ASK1 Cys250 to alanine (Cys250A) blocks the binding of Trx1. In this instance, ASK1 is still able to form disulfide bond-linked multimers but it is not constitutively phosphorylated on Thr838, nor can it activate downstream MAPK pathway members. These results suggest that simple dissociation of Trx1 from ASK1, while a trigger is not sufficient to activate ASK1. Further studies are required to define the regulatory role that Cys250 plays in the activation of ASK1 in

Additional studies have demonstrated that in the absence of Trx1, tumor necrosis factor-α receptor-associated factors (TRAF) 2 and 6 are recruited to the ASK1 signalosome [14, 19, 30]. Recruitment of TRAF2 and 6 induces ASK1 phosphorylation and activation and stabilizes a higher molecular weight ASK1 signalosome complex. Consistent with this, H2O2-induced activation of ASK1 was strongly inhibited in mouse embyonic fibroblats (MEF's) deficient in TRAF 2 and 6 [19]. In addition, the residue Thr838 (Thr845 in mouse) located in the activation loop of the kinase is trans/auto-phosphorylated [18] and the affinity of ASK1 for its substrate MKK6, was observed to be significantly increased [31]. ASK1 is phosphorylated at multiple sites and to date, seven phosphorylation sites have been identified *in vitro*. As mentioned above phosphorylation of Thr838 located in the kinase activation loop is required for ASK1 kinase activity. In addition to ASK1 autophosphorylation, positive regulators of ASK1 activity including the family member ASK2 [32], and the murine protein serine/threonine kinase 38 (MPK38), a member of the AMP-activated protein kinase-related serine/threonine kinase family [33], were found to complex with ASK1 and to stimulate its activity by phosphorylating the Thr838 residue in response to H2O2 or TNF treatment. Conversely, several mechanisms negatively regulate ASK1 activity by modulating the phosphorylation status of this critical residue (Figure 3). For example, the protein phosphatase 5 (PP5), a member of the serine/threonine protein phosphatase family is reported to be recruited into the ASK1 signalosome and to dephosphorylate Thr838 subsequent to H2O2 treatment [34]. By dephosphorylating this critical residue, PP5 was shown to inhibit H2O2-induced ASK1 catalytic activity as well as ASK1-mediated apoptosis. Dephosphorylation of Thr838 inactivates ASK1 in a negative feedback manner and thereby modulates the activation of JNK/p38 and apoptosis. Such a negative feedback system most likely interplays with other cellular signal transduction pathways and is critical for determining cell fate (survival or cell death) in response to cellular stressors. Interestingly, overexpression of PP5 was shown to prevent amyloid-β-

The calcium and intergrin binding protein 1 (CIB1) was also found to inhibit autophosphorylation of ASK1 at Thr838, by directly interacting with ASK1. This interaction was observed to compete with and inhibit TRAF2 recruitment to the ASK1 complex [36], repressing ASK1 activation in response to both tunicomycin, an ER stressor and 6-hydroxydopamine (6-OHDA) in dopaminergic cells. Furthermore, the block to ASK1 activation occured in a Ca2+-dependent manner indicating that CIB1 functions as a Ca2+ sensitive negative regulator of ASK1 activity. Thus, in an aging brain where calcium homeostasis is dysregulated [37, 38], the function of this calcium sensitive ASK1 repressor may be altered, leading to abnormal ASK1 activity and to the development of age-related

ASK1 activity is further regulated by additional phosphorylation/dephosphorylation events that occur at serine-83 (Ser83), serine-967 (Ser967), serine-1034 (Ser1034), and tyrosine-718 (Tyr718). The N-terminal domain of ASK1, surrounding the Ser83 residue was

response to H2O2 signaling.

induced MAPK activation and neurotoxicity [35].

neurodegenerative disorders.

bond beetween these two cysteines. This allows dissociation of Trx1 from ASK1 promoting multimerization and activation of ASK1 [20, 23].

Studies conducted by Nadeau *et al*., revealed an alternative mechanism for the function of Trx1 in the regulation of ASK1 in response to H2O2. These studies demonstrated that H2O2 induces ASK1 oxidation leading to the formation of interchain disulfide bond-linked ASK1 multimers [25, 26]. These authors demonstrated that changing all oxidation sensitive cysteine residues responsible for disulfide bond-linked multimers, prevented H2O2-induced ASK1 proapototic activity. Specifically, the cysteine residue at position 250 (Cys250) in ASK1 was identified as an essential residue for JNK activation in response to H2O2-induced stress. Recently, redox sensitive molecules such as the Parkinson's associated protein DJ-1 (a.k.a PARK7) and Peroxiredoxin-2 have also been shown to attenuate ASK1 activity in response to toxic stress in dopaminergic neurons [27-29]. Interestingly, mutation analyses demonstrated, that the ER stress-inducing agent thapsigargin, while inducing ASK1 activation (as determined by Thr838 phosphorylation) was observed to be independent of Cys250 [25]. In attempting to address the role of Cys250 and the role of Trx1 in regulating

Fig. 2. ASK1-MKKs-JNK/p38 signaling cascade. Under basal conditions, ASK1 forms a high molecular mass complex with multiple interacting proteins including thioredoxin (Trx). In response to oxidative stress, ER stress, or calcium overload, firstly Trx is oxidized and released from the signalosome, secondly the TNF receptor associated factors 2 and 6 (TRAF2/6) are recruited to the complex leading to ASK1 autophosphorylation/transphosphorylation and activation. Subsequently, the affinity of the signalosome for its substrates, (MKKs) is increased, favoring ASK1-MKK6/4 interaction and MKK6/4 phosphoryation which in turn activate JNK/p38. Depending upon the cell type and initial stressor, a complex ASK1 regulatory mechanism is invoked, balancing the processes of phosphorylation/dephosphorylation together with ubiquitination/deubiquitination which fine tunes the specificity and duration of the ASK1 signaling cascade and ultimately the cellular response

bond beetween these two cysteines. This allows dissociation of Trx1 from ASK1 promoting

Studies conducted by Nadeau *et al*., revealed an alternative mechanism for the function of Trx1 in the regulation of ASK1 in response to H2O2. These studies demonstrated that H2O2 induces ASK1 oxidation leading to the formation of interchain disulfide bond-linked ASK1 multimers [25, 26]. These authors demonstrated that changing all oxidation sensitive cysteine residues responsible for disulfide bond-linked multimers, prevented H2O2-induced ASK1 proapototic activity. Specifically, the cysteine residue at position 250 (Cys250) in ASK1 was identified as an essential residue for JNK activation in response to H2O2-induced stress. Recently, redox sensitive molecules such as the Parkinson's associated protein DJ-1 (a.k.a PARK7) and Peroxiredoxin-2 have also been shown to attenuate ASK1 activity in response to toxic stress in dopaminergic neurons [27-29]. Interestingly, mutation analyses demonstrated, that the ER stress-inducing agent thapsigargin, while inducing ASK1 activation (as determined by Thr838 phosphorylation) was observed to be independent of Cys250 [25]. In attempting to address the role of Cys250 and the role of Trx1 in regulating

Fig. 2. ASK1-MKKs-JNK/p38 signaling cascade. Under basal conditions, ASK1 forms a high molecular mass complex with multiple interacting proteins including thioredoxin (Trx). In response to oxidative stress, ER stress, or calcium overload, firstly Trx is oxidized and released from the signalosome, secondly the TNF receptor associated factors 2 and 6 (TRAF2/6) are recruited to the complex leading to ASK1 autophosphorylation/transphosphorylation and activation. Subsequently, the affinity of the signalosome for its substrates, (MKKs) is increased, favoring ASK1-MKK6/4 interaction and MKK6/4 phosphoryation which in turn activate JNK/p38. Depending upon the cell type and initial stressor, a complex ASK1 regulatory mechanism is invoked, balancing the processes of phosphorylation/dephosphorylation together with ubiquitination/deubiquitination which fine tunes the specificity and duration

of the ASK1 signaling cascade and ultimately the cellular response

multimerization and activation of ASK1 [20, 23].

ASK1 activity, it was demonstrated that mutation of ASK1 Cys250 to alanine (Cys250A) blocks the binding of Trx1. In this instance, ASK1 is still able to form disulfide bond-linked multimers but it is not constitutively phosphorylated on Thr838, nor can it activate downstream MAPK pathway members. These results suggest that simple dissociation of Trx1 from ASK1, while a trigger is not sufficient to activate ASK1. Further studies are required to define the regulatory role that Cys250 plays in the activation of ASK1 in response to H2O2 signaling.

Additional studies have demonstrated that in the absence of Trx1, tumor necrosis factor-α receptor-associated factors (TRAF) 2 and 6 are recruited to the ASK1 signalosome [14, 19, 30]. Recruitment of TRAF2 and 6 induces ASK1 phosphorylation and activation and stabilizes a higher molecular weight ASK1 signalosome complex. Consistent with this, H2O2-induced activation of ASK1 was strongly inhibited in mouse embyonic fibroblats (MEF's) deficient in TRAF 2 and 6 [19]. In addition, the residue Thr838 (Thr845 in mouse) located in the activation loop of the kinase is trans/auto-phosphorylated [18] and the affinity of ASK1 for its substrate MKK6, was observed to be significantly increased [31].

ASK1 is phosphorylated at multiple sites and to date, seven phosphorylation sites have been identified *in vitro*. As mentioned above phosphorylation of Thr838 located in the kinase activation loop is required for ASK1 kinase activity. In addition to ASK1 autophosphorylation, positive regulators of ASK1 activity including the family member ASK2 [32], and the murine protein serine/threonine kinase 38 (MPK38), a member of the AMP-activated protein kinase-related serine/threonine kinase family [33], were found to complex with ASK1 and to stimulate its activity by phosphorylating the Thr838 residue in response to H2O2 or TNF treatment. Conversely, several mechanisms negatively regulate ASK1 activity by modulating the phosphorylation status of this critical residue (Figure 3). For example, the protein phosphatase 5 (PP5), a member of the serine/threonine protein phosphatase family is reported to be recruited into the ASK1 signalosome and to dephosphorylate Thr838 subsequent to H2O2 treatment [34]. By dephosphorylating this critical residue, PP5 was shown to inhibit H2O2-induced ASK1 catalytic activity as well as ASK1-mediated apoptosis. Dephosphorylation of Thr838 inactivates ASK1 in a negative feedback manner and thereby modulates the activation of JNK/p38 and apoptosis. Such a negative feedback system most likely interplays with other cellular signal transduction pathways and is critical for determining cell fate (survival or cell death) in response to cellular stressors. Interestingly, overexpression of PP5 was shown to prevent amyloid-βinduced MAPK activation and neurotoxicity [35].

The calcium and intergrin binding protein 1 (CIB1) was also found to inhibit autophosphorylation of ASK1 at Thr838, by directly interacting with ASK1. This interaction was observed to compete with and inhibit TRAF2 recruitment to the ASK1 complex [36], repressing ASK1 activation in response to both tunicomycin, an ER stressor and 6-hydroxydopamine (6-OHDA) in dopaminergic cells. Furthermore, the block to ASK1 activation occured in a Ca2+-dependent manner indicating that CIB1 functions as a Ca2+ sensitive negative regulator of ASK1 activity. Thus, in an aging brain where calcium homeostasis is dysregulated [37, 38], the function of this calcium sensitive ASK1 repressor may be altered, leading to abnormal ASK1 activity and to the development of age-related neurodegenerative disorders.

ASK1 activity is further regulated by additional phosphorylation/dephosphorylation events that occur at serine-83 (Ser83), serine-967 (Ser967), serine-1034 (Ser1034), and tyrosine-718 (Tyr718). The N-terminal domain of ASK1, surrounding the Ser83 residue was

ASK1 and Its Role in Neurodegenerative Diseases 223

A study conducted by Zhang *et al*., revealed that the association of ASK1 with 14-3-3 protein suppresses ASK1-mediated apoptosis [44]. These authors demonstrated that phosphorylation of Ser967, a residue located C-terminal to the ASK1 kinase domain, is critical for ASK1/14-3- 3 complex formation. Interestingly, the 14-3-3 binding motif in ASK1 is conserved among its homologues from human, mouse and Drosophila, suggesting the evolutionary importance of this interaction. Importantly, exogenous expression of ASK1S967A, a 14-3-3 defective mutant, dramatically enhanced cell death, suggesting that 14-3-3 association inhibits the death promoting activity of ASK1. More recently, Seong *et al*., demonstrated that Ser967 was phosphorylated by the 3-phosphoinositide-dependent protein kinase 1 (PDK1), a member of the protein kinase A,G, and C subfamily of protein kinases [45] and that binding of PDK1 to ASK1 was mediated through the pleckstrin homology domain of PDK1 and the C-terminal regulatory domain of ASK1. This interaction was shown to suppress H2O2-induced ASK1- JNK-p38 signaling as well as ASK1-mediated apoptosis. In addition, ASK1 was also observed to phosphorylate and inhibit PDK1 acitivity, suggesting a novel mechanism whereby ASK1 and PDK1 negatively regulate their respective kinase activity in a reciprocal

Oxidative stress such as H2O2, was found to increase ASK1 catalytic activity by inducing dephosphorylation of ASK1 at Ser967 leading to ASK1/14-3-3 complex dissociation [46]. Two phosphatases that dephosphorylate ASK1 at Ser967 have been identified so far; Calcineurin B (protein phosphatase 2B) and protein phosphatse 2A (PP2A). Calcineurin B was found to directly interact with the ASK1 C-terminus and to dephosphorylate ASK1 at Ser967 leading to the disassociation of ASK1 from 14-3-3 proteins, ASK1 activation and enhanced cardiomyocyte apoptosis [47]. A study conducted in vascular endothelial cells demonstrated that in resting cells PP2A forms a complex with the ASK1-interacting protein (AIP1), a ras GTPase-activating protein [48]. Upon TNFα treatment the AIP1/PP2A complex was found to interact with and dephophorylate ASK1 at Ser967, leading to the dissociation of its inhibitor 14-3-3 and ASK1 activation [49]. Furthermore, Aβ was found to induce ASK1 Ser967 dephosphorylation and its dissociation from the 14-3-3 protein leading to p38 activation, and induction of the pro-apoptopic BCl-2 family member, Bax [50]. Selective inhibition of PP2A prevented the activation of this signaling cascade linking ASK1 Ser967

Similar to phosphorylation at Ser967, phosphorylation at Ser1034, a residue contained with in the C-terminal regulatory domain of ASK1 was also found to negatively regulate its kinase and proapoptotic activity [51]. While distinct from the Akt and 14-3-3 mechanisms, candidate kinases/phosphatases implicated in the modulation of Ser1034 phosphorylation

As outlined above, ASK1 cellular activity is tightly controlled in both a spatial and temporal fashion by distinct and multiple mechanisms (Figure 3). In addition to phosphorylation, other posttranslational modifications such as ubiquitination have been observed to play an important role in regulating ASK1 activity. Ubiquitination, is a reversible posttranslational modification that is reciprocally regulated by E3 ubiquitin ligases and deubiquitinating enzymes (DUBs). A study conducted by Liu *et al.*, demonstrated that the association of Trx with ASK1 suppresses ASK1 kinase activity not only by inhibiting N-terminal interactions between ASK1 molecules, but also by controling

manner [45].

phosphorylation status to Aβ-induced toxicity.

**4. Regulation of ASK1 protein levels** 

status remain to be identified.

found to contain a consensus Akt phorphorylation site and biochemical and cell-based studies confirmed this site as a substrate for Akt [39]. In addition, Hsp90 was found to form a complex with Akt and ASK1 in unstimulated cells, and to stabilize the Akt-ASK1 interaction under oxidative stress conditions in order to suppress apoptosis [40]. Importantly, Akt-induced inhibition of ASK1 was observed to promote cell survival and to mediate selenite-induced neuroprotection after cerebral ischemia in rat hippocampus [41]. In a study conducted by Nakagami *et al*., activation of Akt was also observed to inhibit the toxic action of amyloid-β and to protect neurons from apoptosis [42]. These authors hypothesized that the suppression of cell death was mediated at least in part, by the ability of Akt to repress ASK1 activity. More recently, the proto-oncogene serine/threonine kinase, PIM1 was also shown to interact with and to phosphorylate ASK1 on Ser83 [43]. PIM1 phosphorylation of ASK1 decreased ASK1 activity and attenuated H2O2-induced ASK1 mediated activation of JNK/p38 and caspase-3. Thus, phosphorylation of ASK1 on Ser83 by Akt or PIM1 maintains ASK1 in an inactive state and suppresses ASK1-mediated p38/JNK downstream signaling.

Fig. 3. Schematic representation of the multiple mechanisms regulating the catalytic activity and the stablility of ASK1 signalosome. ASK1 catalytic activity is modulated by the phosphorylation/dephosphorylation of critical residues as well as by the interaction with redox sensitive molecules. In addition, ASK1 signaling can be regulated by proteins that modulate ubiquitination of the complex and thus its stability. Green and purple represent the proteins involved in mechanisms that enhance ASK1 activity whereas red and blue represent proteins that inhibit ASK1 signaling. Together, it's the complex regulation of these mechanisms that are thought to modulate the specificity and strength of the ASK1-MKKs-JNK/p38 signaling cascade

found to contain a consensus Akt phorphorylation site and biochemical and cell-based studies confirmed this site as a substrate for Akt [39]. In addition, Hsp90 was found to form a complex with Akt and ASK1 in unstimulated cells, and to stabilize the Akt-ASK1 interaction under oxidative stress conditions in order to suppress apoptosis [40]. Importantly, Akt-induced inhibition of ASK1 was observed to promote cell survival and to mediate selenite-induced neuroprotection after cerebral ischemia in rat hippocampus [41]. In a study conducted by Nakagami *et al*., activation of Akt was also observed to inhibit the toxic action of amyloid-β and to protect neurons from apoptosis [42]. These authors hypothesized that the suppression of cell death was mediated at least in part, by the ability of Akt to repress ASK1 activity. More recently, the proto-oncogene serine/threonine kinase, PIM1 was also shown to interact with and to phosphorylate ASK1 on Ser83 [43]. PIM1 phosphorylation of ASK1 decreased ASK1 activity and attenuated H2O2-induced ASK1 mediated activation of JNK/p38 and caspase-3. Thus, phosphorylation of ASK1 on Ser83 by Akt or PIM1 maintains ASK1 in an inactive state and suppresses ASK1-mediated

Fig. 3. Schematic representation of the multiple mechanisms regulating the catalytic activity

and the stablility of ASK1 signalosome. ASK1 catalytic activity is modulated by the phosphorylation/dephosphorylation of critical residues as well as by the interaction with redox sensitive molecules. In addition, ASK1 signaling can be regulated by proteins that modulate ubiquitination of the complex and thus its stability. Green and purple represent the proteins involved in mechanisms that enhance ASK1 activity whereas red and blue represent proteins that inhibit ASK1 signaling. Together, it's the complex regulation of these mechanisms that are thought to modulate the specificity and strength of the ASK1-MKKs-

p38/JNK downstream signaling.

JNK/p38 signaling cascade

A study conducted by Zhang *et al*., revealed that the association of ASK1 with 14-3-3 protein suppresses ASK1-mediated apoptosis [44]. These authors demonstrated that phosphorylation of Ser967, a residue located C-terminal to the ASK1 kinase domain, is critical for ASK1/14-3- 3 complex formation. Interestingly, the 14-3-3 binding motif in ASK1 is conserved among its homologues from human, mouse and Drosophila, suggesting the evolutionary importance of this interaction. Importantly, exogenous expression of ASK1S967A, a 14-3-3 defective mutant, dramatically enhanced cell death, suggesting that 14-3-3 association inhibits the death promoting activity of ASK1. More recently, Seong *et al*., demonstrated that Ser967 was phosphorylated by the 3-phosphoinositide-dependent protein kinase 1 (PDK1), a member of the protein kinase A,G, and C subfamily of protein kinases [45] and that binding of PDK1 to ASK1 was mediated through the pleckstrin homology domain of PDK1 and the C-terminal regulatory domain of ASK1. This interaction was shown to suppress H2O2-induced ASK1- JNK-p38 signaling as well as ASK1-mediated apoptosis. In addition, ASK1 was also observed to phosphorylate and inhibit PDK1 acitivity, suggesting a novel mechanism whereby ASK1 and PDK1 negatively regulate their respective kinase activity in a reciprocal manner [45].

Oxidative stress such as H2O2, was found to increase ASK1 catalytic activity by inducing dephosphorylation of ASK1 at Ser967 leading to ASK1/14-3-3 complex dissociation [46]. Two phosphatases that dephosphorylate ASK1 at Ser967 have been identified so far; Calcineurin B (protein phosphatase 2B) and protein phosphatse 2A (PP2A). Calcineurin B was found to directly interact with the ASK1 C-terminus and to dephosphorylate ASK1 at Ser967 leading to the disassociation of ASK1 from 14-3-3 proteins, ASK1 activation and enhanced cardiomyocyte apoptosis [47]. A study conducted in vascular endothelial cells demonstrated that in resting cells PP2A forms a complex with the ASK1-interacting protein (AIP1), a ras GTPase-activating protein [48]. Upon TNFα treatment the AIP1/PP2A complex was found to interact with and dephophorylate ASK1 at Ser967, leading to the dissociation of its inhibitor 14-3-3 and ASK1 activation [49]. Furthermore, Aβ was found to induce ASK1 Ser967 dephosphorylation and its dissociation from the 14-3-3 protein leading to p38 activation, and induction of the pro-apoptopic BCl-2 family member, Bax [50]. Selective inhibition of PP2A prevented the activation of this signaling cascade linking ASK1 Ser967 phosphorylation status to Aβ-induced toxicity.

Similar to phosphorylation at Ser967, phosphorylation at Ser1034, a residue contained with in the C-terminal regulatory domain of ASK1 was also found to negatively regulate its kinase and proapoptotic activity [51]. While distinct from the Akt and 14-3-3 mechanisms, candidate kinases/phosphatases implicated in the modulation of Ser1034 phosphorylation status remain to be identified.

#### **4. Regulation of ASK1 protein levels**

As outlined above, ASK1 cellular activity is tightly controlled in both a spatial and temporal fashion by distinct and multiple mechanisms (Figure 3). In addition to phosphorylation, other posttranslational modifications such as ubiquitination have been observed to play an important role in regulating ASK1 activity. Ubiquitination, is a reversible posttranslational modification that is reciprocally regulated by E3 ubiquitin ligases and deubiquitinating enzymes (DUBs). A study conducted by Liu *et al.*, demonstrated that the association of Trx with ASK1 suppresses ASK1 kinase activity not only by inhibiting N-terminal interactions between ASK1 molecules, but also by controling

ASK1 and Its Role in Neurodegenerative Diseases 225

was demonstrated to be critical for complex formation between ASK1 and SOCS1. In unstimulated cells this residue is phophorylated, allowing SOCS1 to interact with ASK1 and repress its activity by decreasing its stability. Conversely, stimulation with TNF induced SHP2-mediated dephosphorylation of Tyr718 and activation of ASK1 signaling leading to cell death. More recently, the glycogen synthase kinase-3 (GSK-3β) was found to mediate

In addition to posttranslational modifications, ASK1 protein levels may also be transcriptionally regulated via upregulation of *ASK1* gene expression. Studies have demonstrated that the E2F family of transcription factors (E2 promoter-binding factors) regulate the expression of ASK1 [60]. Suzuki *et al.,* recently found that onset of spinal and bulbar muscular atrophy (SBMA), a neurodegenerative disorder caused by a polyglutamine repeat (polyQ) expansion within the human androgen receptor correlates with aberrant E2F activation [61]. Thus, multiple posttranslational modifications and other regulatory events work in concert to govern the activity of ASK1 under both physiological and pathological conditions. The aberrant activation of ASK1 observed in neurodegenerative diseases may be triggered by multiple stimuli (e.g. oxidative stress, ER stress) by acting directly on ASK1 molecules or by impairing the activity of other proteins implicated in regulating its activity.

Alzheimer's Disease (AD), the most common form of dementia, was first described by Alois Alzheimer 100 years ago. It is a neurodegenerative disorder characterized clinically by the progressive loss of memory and cognitive impairment. AD is pathologically characterized by the accumulation of cerebral neuritic plaques of amyloid-β (Aβ), neurofibrillary tangle (NFT) formation as well as by neuronal cell death. Accumulation of misfolded proteins together with increased oxidative stress and mitochondrial dysfunction are mechanisms that correlate with the pathogenesis of the disease [62, 63]. Although age is the greatest risk factor for AD, the molecular mechanisms underlying the cause of the disease remain mainly elusive. As ASK1 is activated by ROS and ER-stress (UPR), several studies have implicated ASK1 in cell death processes associated with AD. Cell-based studies including, studies employing primary hybrid neuron cells (F11, hybrid cells of rat embryonic day 13 primary cultured neurons and a mouse neuroblastoma) demonstrated that ASK1 can form a complex with the amyloid precursor protein (APP) via JIP-1b (JNK-interacting protein), phospho-MKK6 and JNK1 [65-67] resulting in caspase dependent neuronal cell death [65]. In addition, the resulting neurotoxicity was significantly blocked by exogenous expression of a dominant negative mutant form of ASK1 as well as by the JNK inhibitor SP600125 [65]

The ASK1/APP complex formation was also confirmed by three-dimensional reconstruction of confocal microscopic Z-stacks obtained from fixed brains of APP transgenic mice that revealed an up-regulation of ASK1 expression in these mice compared to non-transgenic controls [66]. Beside ROS-induced ASK1 activation, Aβ, a toxic cleavage product of APP, was also demonstrated to activate ASK1 and subsequently JNK [68]. Whereas primary neuronal cultures derived from E14.5 ASK+/+ mice demonstrated an 80% reduction in cell viability after exposure to Aβ25-35, the survival in ASK-/- derived neurons treated with Aβ25-35 was significantly elevated (only 30% reduction in viability). Furthermore, postmortem analysis of AD patient brains compared with age-matched controls revealed strong expression of the downstream ASK1 substrate MKK6 [69]. The activated phospho-MKK6

TLR4-induced ASK1 activation by increasing ASK1 stability [59].

strongly suggesting an ASK1/JNK-mediated death pathway in AD.

**5. ASK1 in Alzheimer's Disease** 

ASK1 protein levels through ubiquitination and degradation via the 26S proteosome [23]. This work provided the first evidence that ASK1 protein expression is regulated by ubiquitination. Recently, our laboratory demonstrated that the duration of the ASK1 signal in response to oxidative stress is regulated by mechanisms modulating the degradation of the ASK1 signalosome [31]. LC-MS/MS analysis of the ASK1 signalosome, purified from cells treated with H2O2, revealed the presence of ubiquitinated ASK1 together with several proteins associated with the process of protein degradation such as the 26S proteasome regulatory subunit, ubiquitin-like modifier-activating enzyme1 and ubiquitin specific protease 9 X-linked (USP9X). USP9X belongs to the USP subfamiliy of deubiquitinating enzymes and is thought to regulate multiple cellular functions. In an earlier study conducted by Nagai *et al.*, a ubiquitin-like sequence in ASK1 responsible for USP9X recognition was identified [52]. These authors demonstrated that in response to oxidative stress, ubiquitination of ASK1 C-termini mediates the proteosomal degredation of ASK1. In addition, it was demonstrated that in complex with ASK1, USP9X cleaves ubiquitin from the C-terminus of stress induced ASK1, preventing degradation and stabilizing the activated form of ASK1. In keeping with this observation, knockdown of USP9X mediated by siRNA in HeLa cells, reduced H2O2 induced JNK and p38 activation equivalent to that observed in ASK1 deficient cells. H2O2-induced ASK1 activity is therefore regulated by a complex mechanism involving a balance between phosphorylation/dephosphorylation and ubiquitination levels, whereby ubiquitin dependent regulation of ASK1 is closely coupled to its activity. In this regard these authors postulated that USP9X may be a key regulator that fine-tunes the ASK1-dependent signaling cascade. Recently, Zhang *et al.* characterized region specific protein level changes in the brains of mice treated with the neurotoxin MPTP [53]. In comparison to normal brain, USP9X was significantly upregulated within the striatum, cerebellum and cortex of the MPTP treated mice, raising the possibility of a role for USP9X in neurodegeneration.

Several additional reports have emerged describing the ubiquitin dependent regulation of ASK1. It is well established that activation of the TNF receptor 2 (TNFR2) leads to activation of ASK1 and duration of TNFR2 mediated ASK1 signaling is proposed to be controled by ubiquitin-dependent proteosomal degradation of ASK1 [54]. This mechanism was shown to involve the ubiquitin protein ligase activity of the cellular inhibitor of apoptosis protein 1 (cIAP1) and genetic knockdown experiments confirmed that cIAP1 was critical for limiting TNFR2 mediated p38 and JNK activation. Moreover, in a model of glaucoma, a neurodegenerative disease leading to impaired visual function, Kisiswa *et al.* observed an age dependent down-regulation of cIAP1 accompanied by accumulation of TRAF2 in the retinal ganglion cell layer [55]. Interestingly, dysregulated ASK1 activity was recently reported to be involved in glaucoma, indicating that interrupting ASK1 dependent pathways may be beneficial in the treatment of this pathology [56]. In addition to identifying USP9X in H2O2 induced ASK1 signalosome complexes, we also confirmed the presence of Hsp70 in these complexes. Previous studies demonstrate that Hsp70 mediates ASK1 degradation by recruiting the chaperone and ubiquitin ligase CHIP (C-terminus of Hsp70-interacting protein) [57]. Hsp70 together with CHIP have been implicated in protecting cells against cellular stress that cause neurodegenerative diseases, including Alzheimer's Disease (AD), Parkinson's Disease (PD), Huntington Disease (HD), and Amyotrophic Lateral Sclerosis (ALS). The suppressor of cytokine signaling 1 (SOCS1), another protein regulating proteosomal degradation, was also reported to interact with ASK1 and to mediate its degradation [58]. Phosphorylation of ASK1 Tyr718 residue by JAK2

ASK1 protein levels through ubiquitination and degradation via the 26S proteosome [23]. This work provided the first evidence that ASK1 protein expression is regulated by ubiquitination. Recently, our laboratory demonstrated that the duration of the ASK1 signal in response to oxidative stress is regulated by mechanisms modulating the degradation of the ASK1 signalosome [31]. LC-MS/MS analysis of the ASK1 signalosome, purified from cells treated with H2O2, revealed the presence of ubiquitinated ASK1 together with several proteins associated with the process of protein degradation such as the 26S proteasome regulatory subunit, ubiquitin-like modifier-activating enzyme1 and ubiquitin specific protease 9 X-linked (USP9X). USP9X belongs to the USP subfamiliy of deubiquitinating enzymes and is thought to regulate multiple cellular functions. In an earlier study conducted by Nagai *et al.*, a ubiquitin-like sequence in ASK1 responsible for USP9X recognition was identified [52]. These authors demonstrated that in response to oxidative stress, ubiquitination of ASK1 C-termini mediates the proteosomal degredation of ASK1. In addition, it was demonstrated that in complex with ASK1, USP9X cleaves ubiquitin from the C-terminus of stress induced ASK1, preventing degradation and stabilizing the activated form of ASK1. In keeping with this observation, knockdown of USP9X mediated by siRNA in HeLa cells, reduced H2O2 induced JNK and p38 activation equivalent to that observed in ASK1 deficient cells. H2O2-induced ASK1 activity is therefore regulated by a complex mechanism involving a balance between phosphorylation/dephosphorylation and ubiquitination levels, whereby ubiquitin dependent regulation of ASK1 is closely coupled to its activity. In this regard these authors postulated that USP9X may be a key regulator that fine-tunes the ASK1-dependent signaling cascade. Recently, Zhang *et al.* characterized region specific protein level changes in the brains of mice treated with the neurotoxin MPTP [53]. In comparison to normal brain, USP9X was significantly upregulated within the striatum, cerebellum and cortex of the MPTP treated mice, raising

Several additional reports have emerged describing the ubiquitin dependent regulation of ASK1. It is well established that activation of the TNF receptor 2 (TNFR2) leads to activation of ASK1 and duration of TNFR2 mediated ASK1 signaling is proposed to be controled by ubiquitin-dependent proteosomal degradation of ASK1 [54]. This mechanism was shown to involve the ubiquitin protein ligase activity of the cellular inhibitor of apoptosis protein 1 (cIAP1) and genetic knockdown experiments confirmed that cIAP1 was critical for limiting TNFR2 mediated p38 and JNK activation. Moreover, in a model of glaucoma, a neurodegenerative disease leading to impaired visual function, Kisiswa *et al.* observed an age dependent down-regulation of cIAP1 accompanied by accumulation of TRAF2 in the retinal ganglion cell layer [55]. Interestingly, dysregulated ASK1 activity was recently reported to be involved in glaucoma, indicating that interrupting ASK1 dependent pathways may be beneficial in the treatment of this pathology [56]. In addition to identifying USP9X in H2O2 induced ASK1 signalosome complexes, we also confirmed the presence of Hsp70 in these complexes. Previous studies demonstrate that Hsp70 mediates ASK1 degradation by recruiting the chaperone and ubiquitin ligase CHIP (C-terminus of Hsp70-interacting protein) [57]. Hsp70 together with CHIP have been implicated in protecting cells against cellular stress that cause neurodegenerative diseases, including Alzheimer's Disease (AD), Parkinson's Disease (PD), Huntington Disease (HD), and Amyotrophic Lateral Sclerosis (ALS). The suppressor of cytokine signaling 1 (SOCS1), another protein regulating proteosomal degradation, was also reported to interact with ASK1 and to mediate its degradation [58]. Phosphorylation of ASK1 Tyr718 residue by JAK2

the possibility of a role for USP9X in neurodegeneration.

was demonstrated to be critical for complex formation between ASK1 and SOCS1. In unstimulated cells this residue is phophorylated, allowing SOCS1 to interact with ASK1 and repress its activity by decreasing its stability. Conversely, stimulation with TNF induced SHP2-mediated dephosphorylation of Tyr718 and activation of ASK1 signaling leading to cell death. More recently, the glycogen synthase kinase-3 (GSK-3β) was found to mediate TLR4-induced ASK1 activation by increasing ASK1 stability [59].

In addition to posttranslational modifications, ASK1 protein levels may also be transcriptionally regulated via upregulation of *ASK1* gene expression. Studies have demonstrated that the E2F family of transcription factors (E2 promoter-binding factors) regulate the expression of ASK1 [60]. Suzuki *et al.,* recently found that onset of spinal and bulbar muscular atrophy (SBMA), a neurodegenerative disorder caused by a polyglutamine repeat (polyQ) expansion within the human androgen receptor correlates with aberrant E2F activation [61]. Thus, multiple posttranslational modifications and other regulatory events work in concert to govern the activity of ASK1 under both physiological and pathological conditions. The aberrant activation of ASK1 observed in neurodegenerative diseases may be triggered by multiple stimuli (e.g. oxidative stress, ER stress) by acting directly on ASK1 molecules or by impairing the activity of other proteins implicated in regulating its activity.

#### **5. ASK1 in Alzheimer's Disease**

Alzheimer's Disease (AD), the most common form of dementia, was first described by Alois Alzheimer 100 years ago. It is a neurodegenerative disorder characterized clinically by the progressive loss of memory and cognitive impairment. AD is pathologically characterized by the accumulation of cerebral neuritic plaques of amyloid-β (Aβ), neurofibrillary tangle (NFT) formation as well as by neuronal cell death. Accumulation of misfolded proteins together with increased oxidative stress and mitochondrial dysfunction are mechanisms that correlate with the pathogenesis of the disease [62, 63]. Although age is the greatest risk factor for AD, the molecular mechanisms underlying the cause of the disease remain mainly elusive. As ASK1 is activated by ROS and ER-stress (UPR), several studies have implicated ASK1 in cell death processes associated with AD. Cell-based studies including, studies employing primary hybrid neuron cells (F11, hybrid cells of rat embryonic day 13 primary cultured neurons and a mouse neuroblastoma) demonstrated that ASK1 can form a complex with the amyloid precursor protein (APP) via JIP-1b (JNK-interacting protein), phospho-MKK6 and JNK1 [65-67] resulting in caspase dependent neuronal cell death [65]. In addition, the resulting neurotoxicity was significantly blocked by exogenous expression of a dominant negative mutant form of ASK1 as well as by the JNK inhibitor SP600125 [65] strongly suggesting an ASK1/JNK-mediated death pathway in AD.

The ASK1/APP complex formation was also confirmed by three-dimensional reconstruction of confocal microscopic Z-stacks obtained from fixed brains of APP transgenic mice that revealed an up-regulation of ASK1 expression in these mice compared to non-transgenic controls [66]. Beside ROS-induced ASK1 activation, Aβ, a toxic cleavage product of APP, was also demonstrated to activate ASK1 and subsequently JNK [68]. Whereas primary neuronal cultures derived from E14.5 ASK+/+ mice demonstrated an 80% reduction in cell viability after exposure to Aβ25-35, the survival in ASK-/- derived neurons treated with Aβ25-35 was significantly elevated (only 30% reduction in viability). Furthermore, postmortem analysis of AD patient brains compared with age-matched controls revealed strong expression of the downstream ASK1 substrate MKK6 [69]. The activated phospho-MKK6

ASK1 and Its Role in Neurodegenerative Diseases 227

mice was evaluated. Inhibition of ASK1 was observed to significantly attenuate the 6-OHDA induced ASK1/JNK signaling axis. Moreover, knockdown of ASK1 significantly protected against 6-OHDA induced death of dopaminergic neurons, improved motor function and significantly elevated dopamine levels in the striatum. Interestingly, immunological analysis of postmortem PD brain sections clearly indicate that active-ASK1 is frequently observed in SNpc neurons and co-localized in 33% of the cases with Lewy bodies and more than 60% of phospho-ASK1 neurons also revealed abnormal α-synuclein staining [27]. Taken together, the data demonstrate that redox sensitive molecules (e.g. PRX2) are able to modulate apoptotic pathways by influencing ASK1 activity and suggest that targeting either PRX2 or

The expansion of CAG trinucleotide repeat units which encode for uninterrupted glutamine residues or (polyQ) is the underlying cause of at least nine inherited human neurodegenerative disorders including Huntington's Disease (HD), Spinobulbar Muscular Atrophy (SBMA) and several forms of spinocereballar ataxia (SCA) [81, 82]. HD is clinically characterized by abnormal involuntary movements, including chorea and dystonia, and cognitive impairment through a selective loss of neurons mainly in the basal ganglia and cerebral cortex [83]. HD is caused by a mutation in the *huntingtin* gene (abnormal CAG repeats in the open reading frame) which encodes a large protein (350 kDa) with an expanded polyglutamine (polyQ) tract. Interestingly, the number of polyglutamine repeats is correlated with the severity of symptoms and once expanded over a repeat of 40, HD occurs [84]. It has been demonstrated that the expanded polyQ repeats form intracellular cytoplasmic and/or nuclear aggregates with subsequent neurotoxic effects *in vitro*, in transgenic animals (overexpressing polyQ proteins) and in postmortem brains of polyQ disease patients. The neurotoxic insult is mediated by dysfunction of the ubiquitin

As described above, the accumulation of misfolded proteins and induction of ER stress is a process that is known to activate ASK1. Thus, it was hypothesized that ASK1 could play a significant role in HD by modifying huntingtin [87], acting as a signal transducer at the protein level as well as a cell death modulator at the post-translational level [88]. Indeed, by comparison to wild type cells, neuronal cell viability derived from ASK1 knockout mice was significantly protected against cell death mediated by expression of polyQ79 (i.e. 79 glutamine repeats) [8]. In addition, neurons derived from ASK1 knockout mice, were also observed to be defective in proteasome inhibitor and ER stress-induced JNK activation. Recently, it was shown that inhibition of ASK1 through administration of ASK1 antibodies using a micro-osmotic pump reduces ER stress and toxicity in a HD mouse model. In addition, nuclear translocation of huntington fragments was observed in cells harboring active ASK1 enzyme, whereas inactivated ASK1-bound huntingtin prevented its nuclear translocation and improved motor dysfunction in mice [89]. Similarly, ASK1 protein levels are also increased in the striatum following injection of 3-nitropropionic acid (3-NP), a mitochondrial toxin producing age-dependent oxidative stress and a model of Huntington's Disease [90], whereas reduction of ASK1 expression using siRNA was accompanied by a reduction in cell death. Therefore, regulating the activity of ASK1 by small molecule inhibitors and/or antibodies could also reveal promising treatment

ASK1 may be a promising approach for neuroprotective intervention in PD.

**7. ASK1 in Huntington's Disease** 

proteosome function resulting in ER stress [85, 86].

strategies for HD.

also co-immunoprecipitated with the paired helical filament Tau from human AD hippocampal supernatants [67] and overlapped with active p38. Both are found to be exclusively localized in classic pathological AD structures like NFT and senile plaques [69]. These data strongly suggest that ASK1 could play a significant role in the pathogenesis of AD by mediating ROS and/or Aβ induced neuronal cell death via the MKK6/JNK/p38 pathway. In addition, Aβ was not only shown to cause neuron-specific toxicity, but also demonstrated to cause vascular degeneration in cerebral amyloid angiopathy; Hsu and coworkers employed primary murine cerebral endothelial cells (CEC) to investigate the mode of cell death mediated by Aβ in ASK1 transfected CECs [50]. Aβ exposure was observed to result in an induction of the ASK1-MKK3/6-p38-p53 signaling machinery and increased levels of the pro-apoptotic protein Bax resulting in CEC programmed cell death. ASK1 activity is also known to be modulated by oxidized thioredoxin-1 (TRX1) and glutaredoxin-1 (GRX1). Postmortem brain samples of AD patients revealed that whereas TRX1 was decreased in neurons (frontal cortex and hippocampal CA1 regions) GRX1 expression was increased [70]. In addition, the same authors demonstrated that in SH-SY5Y cells, Aβ1-41 exposure resulted in TRX1/GRX1 oxidation with subsequent induction of apoptosis. Current studies therefore suggest that ER-stress and ROS-mediated ASK1 activation represents an important signal transduction mechanism in AD.

#### **6. ASK1 in Parkinson Disease**

Parkinson's Disease (PD) is the second most common neurodegenerative disease after AD. The main pathology of PD is characterized by the severe loss of dopaminergic neurons from the substantia nigra pars compacta (SNpc) that project into the striatum. PD is also characterized by the misfolding of α-synuclein which generates protein aggregates called Lewy bodies [71]. Clinical signs of PD, which include rest tremor, rigidity and bradykinesia become evident when approximately 80% of striatal dopamine and 50% of nigral neurons are lost [72]. Like AD, age is the greatest known risk factor for PD. While the mechanism underlying the 'area-specific' neuronal loss in PD remains unclear, both oxidative and ERstress are strongly implicated as contributing factors of the disease state [73-76].

The neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes the selective loss of dopaminergic neurons in SNpc in mice in a manner similar to that seen in PD and is often used in mouse models to study the molecular mechanisms in PD pathogenesis. Activation of JNK and p38 appear to be critical in mediating MPTP induced toxicity, as dopaminergic neurons in JNK3 knockout mice were significantly protected from the toxic effects of MPTP [77, 78]. The activation of JNK and p38 in dopaminergic neurons is thought to be mediated by ASK1 [79]. Karunakaran and co-workers demonstrated that administration of the neurotoxin MPTP, induced ASK1 activation and the subsequent activation of its downstream targets MKK4 and JNK [80]. This pathway triggered the nucleus to cytoplasmic translocalization of the death-associated protein Daxx, specifically in neurons located in the SNpc. Co-administration of MPTP with α-lipoic acid, a thiol antioxidant inhibited the activation of ASK1 and subsequent activation of its downstream targets [80]. Recently, peroxiredoxin2 (PRX2), an antioxidant enzyme, was demonstrated to protect against 6-OHDA induced neurotoxicity both *in vitro* and *in vivo* [27]. The proposed mechanism of inhibition was through blockade of ASK1 activation and its downstream JNK/p38 signaling pathway. Infusion of a lentiviral vector expressing a short hairpin RNA (shRNA) to specifically knock down ASK1 protein expression in the left SNpc of C57BL/6

also co-immunoprecipitated with the paired helical filament Tau from human AD hippocampal supernatants [67] and overlapped with active p38. Both are found to be exclusively localized in classic pathological AD structures like NFT and senile plaques [69]. These data strongly suggest that ASK1 could play a significant role in the pathogenesis of AD by mediating ROS and/or Aβ induced neuronal cell death via the MKK6/JNK/p38 pathway. In addition, Aβ was not only shown to cause neuron-specific toxicity, but also demonstrated to cause vascular degeneration in cerebral amyloid angiopathy; Hsu and coworkers employed primary murine cerebral endothelial cells (CEC) to investigate the mode of cell death mediated by Aβ in ASK1 transfected CECs [50]. Aβ exposure was observed to result in an induction of the ASK1-MKK3/6-p38-p53 signaling machinery and increased levels of the pro-apoptotic protein Bax resulting in CEC programmed cell death. ASK1 activity is also known to be modulated by oxidized thioredoxin-1 (TRX1) and glutaredoxin-1 (GRX1). Postmortem brain samples of AD patients revealed that whereas TRX1 was decreased in neurons (frontal cortex and hippocampal CA1 regions) GRX1 expression was increased [70]. In addition, the same authors demonstrated that in SH-SY5Y cells, Aβ1-41 exposure resulted in TRX1/GRX1 oxidation with subsequent induction of apoptosis. Current studies therefore suggest that ER-stress and ROS-mediated ASK1 activation

Parkinson's Disease (PD) is the second most common neurodegenerative disease after AD. The main pathology of PD is characterized by the severe loss of dopaminergic neurons from the substantia nigra pars compacta (SNpc) that project into the striatum. PD is also characterized by the misfolding of α-synuclein which generates protein aggregates called Lewy bodies [71]. Clinical signs of PD, which include rest tremor, rigidity and bradykinesia become evident when approximately 80% of striatal dopamine and 50% of nigral neurons are lost [72]. Like AD, age is the greatest known risk factor for PD. While the mechanism underlying the 'area-specific' neuronal loss in PD remains unclear, both oxidative and ER-

The neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes the selective loss of dopaminergic neurons in SNpc in mice in a manner similar to that seen in PD and is often used in mouse models to study the molecular mechanisms in PD pathogenesis. Activation of JNK and p38 appear to be critical in mediating MPTP induced toxicity, as dopaminergic neurons in JNK3 knockout mice were significantly protected from the toxic effects of MPTP [77, 78]. The activation of JNK and p38 in dopaminergic neurons is thought to be mediated by ASK1 [79]. Karunakaran and co-workers demonstrated that administration of the neurotoxin MPTP, induced ASK1 activation and the subsequent activation of its downstream targets MKK4 and JNK [80]. This pathway triggered the nucleus to cytoplasmic translocalization of the death-associated protein Daxx, specifically in neurons located in the SNpc. Co-administration of MPTP with α-lipoic acid, a thiol antioxidant inhibited the activation of ASK1 and subsequent activation of its downstream targets [80]. Recently, peroxiredoxin2 (PRX2), an antioxidant enzyme, was demonstrated to protect against 6-OHDA induced neurotoxicity both *in vitro* and *in vivo* [27]. The proposed mechanism of inhibition was through blockade of ASK1 activation and its downstream JNK/p38 signaling pathway. Infusion of a lentiviral vector expressing a short hairpin RNA (shRNA) to specifically knock down ASK1 protein expression in the left SNpc of C57BL/6

stress are strongly implicated as contributing factors of the disease state [73-76].

represents an important signal transduction mechanism in AD.

**6. ASK1 in Parkinson Disease** 

mice was evaluated. Inhibition of ASK1 was observed to significantly attenuate the 6-OHDA induced ASK1/JNK signaling axis. Moreover, knockdown of ASK1 significantly protected against 6-OHDA induced death of dopaminergic neurons, improved motor function and significantly elevated dopamine levels in the striatum. Interestingly, immunological analysis of postmortem PD brain sections clearly indicate that active-ASK1 is frequently observed in SNpc neurons and co-localized in 33% of the cases with Lewy bodies and more than 60% of phospho-ASK1 neurons also revealed abnormal α-synuclein staining [27]. Taken together, the data demonstrate that redox sensitive molecules (e.g. PRX2) are able to modulate apoptotic pathways by influencing ASK1 activity and suggest that targeting either PRX2 or ASK1 may be a promising approach for neuroprotective intervention in PD.

#### **7. ASK1 in Huntington's Disease**

The expansion of CAG trinucleotide repeat units which encode for uninterrupted glutamine residues or (polyQ) is the underlying cause of at least nine inherited human neurodegenerative disorders including Huntington's Disease (HD), Spinobulbar Muscular Atrophy (SBMA) and several forms of spinocereballar ataxia (SCA) [81, 82]. HD is clinically characterized by abnormal involuntary movements, including chorea and dystonia, and cognitive impairment through a selective loss of neurons mainly in the basal ganglia and cerebral cortex [83]. HD is caused by a mutation in the *huntingtin* gene (abnormal CAG repeats in the open reading frame) which encodes a large protein (350 kDa) with an expanded polyglutamine (polyQ) tract. Interestingly, the number of polyglutamine repeats is correlated with the severity of symptoms and once expanded over a repeat of 40, HD occurs [84]. It has been demonstrated that the expanded polyQ repeats form intracellular cytoplasmic and/or nuclear aggregates with subsequent neurotoxic effects *in vitro*, in transgenic animals (overexpressing polyQ proteins) and in postmortem brains of polyQ disease patients. The neurotoxic insult is mediated by dysfunction of the ubiquitin proteosome function resulting in ER stress [85, 86].

As described above, the accumulation of misfolded proteins and induction of ER stress is a process that is known to activate ASK1. Thus, it was hypothesized that ASK1 could play a significant role in HD by modifying huntingtin [87], acting as a signal transducer at the protein level as well as a cell death modulator at the post-translational level [88]. Indeed, by comparison to wild type cells, neuronal cell viability derived from ASK1 knockout mice was significantly protected against cell death mediated by expression of polyQ79 (i.e. 79 glutamine repeats) [8]. In addition, neurons derived from ASK1 knockout mice, were also observed to be defective in proteasome inhibitor and ER stress-induced JNK activation. Recently, it was shown that inhibition of ASK1 through administration of ASK1 antibodies using a micro-osmotic pump reduces ER stress and toxicity in a HD mouse model. In addition, nuclear translocation of huntington fragments was observed in cells harboring active ASK1 enzyme, whereas inactivated ASK1-bound huntingtin prevented its nuclear translocation and improved motor dysfunction in mice [89]. Similarly, ASK1 protein levels are also increased in the striatum following injection of 3-nitropropionic acid (3-NP), a mitochondrial toxin producing age-dependent oxidative stress and a model of Huntington's Disease [90], whereas reduction of ASK1 expression using siRNA was accompanied by a reduction in cell death. Therefore, regulating the activity of ASK1 by small molecule inhibitors and/or antibodies could also reveal promising treatment strategies for HD.

ASK1 and Its Role in Neurodegenerative Diseases 229

long-term neuroprotection against cerebral ischemia, as measured by infarct volume and sensory motor function. In addition, improvement in postischemic neurobehavioral recovery was also observed up to three weeks following cerebral ischemia [104]. At the molecular level, Hsp-27 was demonstrated to physically interact with ASK1 resulting in inhibition of ASK1 activity. Subsequent genetic knockdown of ASK1 or inhibition of the ASK1/MKK4 cascade also effectively abolished neuronal ischemia. Hsp-27 mediated inhibition of the pro-apoptotic ASK1 pathway may be a promising novel neuroprotective

Aberrant regulation of ASK1 activity is observed in a variety of neurodegenerative stress associated diseases and genetic knockout studies have delivered a strong case for ASK1 as a candidate therapeutic target in the treatment of such disorders. While ASK1 inhibitors have been claimed in the patent literature, no small molecule ASK1 inhibitors have obtained sufficient optimization characteristics for candidate selection and approval for first time in human studies. As such, there is little data available in the peer-reviewed literature concerning these inhibitors. In addition to their potential as future therapeutics, there is little doubt that small molecule inhibitors targeting ASK1 would be highly useful assets to facilitate understanding of the complex biology of ASK1. To rely on a molecular probe to make firm mechanistic conclusions about ASK1's role in cellular signaling, the selectivity of the final compound must be devoid of off-target activity. Recent characterization of the structure of the ASK1 kinase domain may facilitate development of ASK1-specific inhibitors [17]. Interestingly, Bunkoczi *et al.* observed that apart from its closely related isoform (ASK2), the nearest phylogenic neighbor to ASK1 shares sequence identity of only 50% within the kinase domain. In this regard, ASK1 may form a chemically diverse catalytic domain, which may allow a high level of kinase selectivity even with an inhibitor with an ATP competitive mode of action. Our laboratory has recently developed a biochemical assay using the full length ASK1 signalosome complex and full length substrate to identify inhibitors that are not only ATP competitive, but also substrate competitive and noncompetitive with respect to ATP or substrate [31]. Since several ASK1 interacting proteins have been shown to modulate the activity of ASK1 within the signalosome complex, identification of cell permeable peptide inhibitors or development of ATP-noncompetative small molecule inhibitors that alter conformation or block ASK1 regultory protein interactions could serve as highly specific probes for ASK1. Regardless of the approach taken, careful analysis of the first generation of ASK1 inhibitors will be needed to define both the benefits and potential liabilities of mechanistically inhibiting ASK1 in cellular and animal systems.

[1] Ichijo, H., et al., Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. *Science*, 1997. 275(5296): p. 90-4. [2] Chen, Z., et al., ASK1 mediates apoptotic cell death induced by genotoxic stress.

[3] Matsukawa, J., et al., The ASK1-MAP kinase cascades in mammalian stress response.

[4] McDonald, P.H., et al., Beta-arrestin 2: a receptor-regulated MAPK scaffold for the

activation of JNK3. *Science*, 2000. 290(5496): p. 1574-7.

strategy for stroke.

**10. Conclusion** 

**11. References** 

*Oncogene*, 1999. 18(1): p. 173-80.

*J Biochem*, 2004. 136(3): p. 261-5.

#### **8. ASK1 in Amyotrophic Lateral Sclerosis**

Amyotrophic Lateral Sclerosis (ALS) is a late onset neurodegenerative disorder, characterized by a selective loss of motor neurons in the spinal cord, brain stem and cerebral cortex [89]. Although the precise mechanism(s) for the pathogenesis of ALS remains unclear, studies highlight oxidative stress, excitotoxicity, protein aggregation and ER-stress as culprits in motor neuron demise. About 10% of ALS cases are familial (FALS) and about 1-2% are caused by mutations in the Cu/Zn-superoxide dismutase (SOD1) gene. SOD1-/ mice show no ALS phenotypes, indicating that the gain of function mutations in SOD1 (and over 120 have been identified in FALS) is critical for the death of motor neurons and FALS. At the cellular level mutant SOD1 was suggested to play several roles in the pathogenesis of ALS, such as triggering abnormal protein interactions and activating caspases [92] [93]. Studies in transgenic mice expressing the ALS-linked SOD1 mutants showed an increase in the number of motor neurons with activated ASK1 and p38, strongly suggesting that ASK1 p38 pathway may be involved in neuronal cell death in FALS [94, 95]. Furthermore, several groups have demonstrated both *in vitro* and *in vivo* that the activation of ASK1 and/or its downstream pathways are associated with a selective motor neuron loss induced by the mutant SOD1 [96, 97]. Taken together these studies suggest a functional link between ER-stress and ASK1/p38 signaling axis in FALS. In a recent study by Nishitoh *et al.*, a specific interaction of SOD-1 and Derlin-1, a protein of the ER-associated degradation (ERAD) complex was observed to trigger ER-stress through dysfunction of ERAD [98]. The resulting ERAD dysfunction promoted ASK1 activation and subsequent apoptosis. These authors demonstrated that motor neuron death could be significantly reduced by the forced dissociation of mutant SOD1 from Derlin-1. Additional *in vivo* experiments revealed that deletion of ASK1 reduced the motor neuron loss and prolonged the lifespan of mutant SOD1 transgenic mice. These results suggest that Derlin-1/SOD-1 interaction promotes ER-stress, ASK1/p38 activation resulting in motor neuron cell death, a mechanism that is a key component of the disease progression of familial ALS [98].

#### **9. ASK1 in ischemic brain injury**

Ischemic brain injury is an acute or chronic disorder induced by insufficient blood flow into the brain. Hypoxic, or in the case of no oxygen supply, anoxic conditions trigger the induction of complex and overlapping signaling pathways, leading ultimately to neuronal cell death. Experimental models have demonstrated the involvement of pathways involving excitotoxicity, ionic imbalance, oxidative and nitrosactive stress resulting in neuronal cell death [99]. The activation of JNK was demonstrated in a murine model of transient focal ischemia to be a crucial signaling component mediating neuronal cell death [100] and strong evidence indicating a potential role for ASK1 in the pathogenesis of ischemic brain injury has emerged [101]. Using a cerebral ischemia rat model as well as in an *in vitro* kinase assay, ASK1 exhibited increased autophosphorylation and activity at various time points after the induction of cerebral ischemia. Furthermore, ASK1 autophosphorylation and activity were inhibited by the pre-administration of the antioxidant N-acetylcystein. Thus, activation of ASK1 may play a significant role in the apoptotic pathway following cerebral ischemia.

The heat shock protein-27 (Hsp27) was observed to be upregulated in cells surviving ischemic insults [102] and in ischemic preconditioning models [103], suggesting that Hsp-27 is associated with pro-survival cascades. Recently, studies overexpressing human Hsp-27 by viral mediated transfer and in Hsp27 transgenic mice demonstrated that Hsp-27 promoted long-term neuroprotection against cerebral ischemia, as measured by infarct volume and sensory motor function. In addition, improvement in postischemic neurobehavioral recovery was also observed up to three weeks following cerebral ischemia [104]. At the molecular level, Hsp-27 was demonstrated to physically interact with ASK1 resulting in inhibition of ASK1 activity. Subsequent genetic knockdown of ASK1 or inhibition of the ASK1/MKK4 cascade also effectively abolished neuronal ischemia. Hsp-27 mediated inhibition of the pro-apoptotic ASK1 pathway may be a promising novel neuroprotective strategy for stroke.

#### **10. Conclusion**

228 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

Amyotrophic Lateral Sclerosis (ALS) is a late onset neurodegenerative disorder, characterized by a selective loss of motor neurons in the spinal cord, brain stem and cerebral cortex [89]. Although the precise mechanism(s) for the pathogenesis of ALS remains unclear, studies highlight oxidative stress, excitotoxicity, protein aggregation and ER-stress as culprits in motor neuron demise. About 10% of ALS cases are familial (FALS) and about 1-2% are caused by mutations in the Cu/Zn-superoxide dismutase (SOD1) gene. SOD1-/ mice show no ALS phenotypes, indicating that the gain of function mutations in SOD1 (and over 120 have been identified in FALS) is critical for the death of motor neurons and FALS. At the cellular level mutant SOD1 was suggested to play several roles in the pathogenesis of ALS, such as triggering abnormal protein interactions and activating caspases [92] [93]. Studies in transgenic mice expressing the ALS-linked SOD1 mutants showed an increase in the number of motor neurons with activated ASK1 and p38, strongly suggesting that ASK1 p38 pathway may be involved in neuronal cell death in FALS [94, 95]. Furthermore, several groups have demonstrated both *in vitro* and *in vivo* that the activation of ASK1 and/or its downstream pathways are associated with a selective motor neuron loss induced by the mutant SOD1 [96, 97]. Taken together these studies suggest a functional link between ER-stress and ASK1/p38 signaling axis in FALS. In a recent study by Nishitoh *et al.*, a specific interaction of SOD-1 and Derlin-1, a protein of the ER-associated degradation (ERAD) complex was observed to trigger ER-stress through dysfunction of ERAD [98]. The resulting ERAD dysfunction promoted ASK1 activation and subsequent apoptosis. These authors demonstrated that motor neuron death could be significantly reduced by the forced dissociation of mutant SOD1 from Derlin-1. Additional *in vivo* experiments revealed that deletion of ASK1 reduced the motor neuron loss and prolonged the lifespan of mutant SOD1 transgenic mice. These results suggest that Derlin-1/SOD-1 interaction promotes ER-stress, ASK1/p38 activation resulting in motor neuron cell death, a mechanism that is a

Ischemic brain injury is an acute or chronic disorder induced by insufficient blood flow into the brain. Hypoxic, or in the case of no oxygen supply, anoxic conditions trigger the induction of complex and overlapping signaling pathways, leading ultimately to neuronal cell death. Experimental models have demonstrated the involvement of pathways involving excitotoxicity, ionic imbalance, oxidative and nitrosactive stress resulting in neuronal cell death [99]. The activation of JNK was demonstrated in a murine model of transient focal ischemia to be a crucial signaling component mediating neuronal cell death [100] and strong evidence indicating a potential role for ASK1 in the pathogenesis of ischemic brain injury has emerged [101]. Using a cerebral ischemia rat model as well as in an *in vitro* kinase assay, ASK1 exhibited increased autophosphorylation and activity at various time points after the induction of cerebral ischemia. Furthermore, ASK1 autophosphorylation and activity were inhibited by the pre-administration of the antioxidant N-acetylcystein. Thus, activation of ASK1 may play a significant role in the apoptotic pathway following cerebral ischemia. The heat shock protein-27 (Hsp27) was observed to be upregulated in cells surviving ischemic insults [102] and in ischemic preconditioning models [103], suggesting that Hsp-27 is associated with pro-survival cascades. Recently, studies overexpressing human Hsp-27 by viral mediated transfer and in Hsp27 transgenic mice demonstrated that Hsp-27 promoted

**8. ASK1 in Amyotrophic Lateral Sclerosis** 

key component of the disease progression of familial ALS [98].

**9. ASK1 in ischemic brain injury** 

Aberrant regulation of ASK1 activity is observed in a variety of neurodegenerative stress associated diseases and genetic knockout studies have delivered a strong case for ASK1 as a candidate therapeutic target in the treatment of such disorders. While ASK1 inhibitors have been claimed in the patent literature, no small molecule ASK1 inhibitors have obtained sufficient optimization characteristics for candidate selection and approval for first time in human studies. As such, there is little data available in the peer-reviewed literature concerning these inhibitors. In addition to their potential as future therapeutics, there is little doubt that small molecule inhibitors targeting ASK1 would be highly useful assets to facilitate understanding of the complex biology of ASK1. To rely on a molecular probe to make firm mechanistic conclusions about ASK1's role in cellular signaling, the selectivity of the final compound must be devoid of off-target activity. Recent characterization of the structure of the ASK1 kinase domain may facilitate development of ASK1-specific inhibitors [17]. Interestingly, Bunkoczi *et al.* observed that apart from its closely related isoform (ASK2), the nearest phylogenic neighbor to ASK1 shares sequence identity of only 50% within the kinase domain. In this regard, ASK1 may form a chemically diverse catalytic domain, which may allow a high level of kinase selectivity even with an inhibitor with an ATP competitive mode of action. Our laboratory has recently developed a biochemical assay using the full length ASK1 signalosome complex and full length substrate to identify inhibitors that are not only ATP competitive, but also substrate competitive and noncompetitive with respect to ATP or substrate [31]. Since several ASK1 interacting proteins have been shown to modulate the activity of ASK1 within the signalosome complex, identification of cell permeable peptide inhibitors or development of ATP-noncompetative small molecule inhibitors that alter conformation or block ASK1 regultory protein interactions could serve as highly specific probes for ASK1. Regardless of the approach taken, careful analysis of the first generation of ASK1 inhibitors will be needed to define both the benefits and potential liabilities of mechanistically inhibiting ASK1 in cellular and animal systems.

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**10** 

*1,2,4Chile 3France* 

**Role of Connexin** 

**Hemichannels in Neurodegeneration** 

Juan A. Orellana1,2, Christian Giaume3 and Juan C. Sáez1,4

*3CIRB; UMR CNRS 7241/ INSERM U1050, Collège de France, Paris* 

*4Instituto milenio, Centro Interdiciplinario de Neurociencias de Valparaiso, Valparaiso,* 

Progressive loss of neuronal structure and function occur in several neurodegenerative diseases. Cellular responses to brain injury depend on properties of the cells (e.g., hormonal nutritional status) and insult (e.g., duration, intensity, and quality), whereas, tissue responses depend on interactions between their constituent cells, including chemical and electrical transmission as well as paracrine and autocrine signaling. In vertebrate cells, autocrine and paracrine communication occur in part via release of chemical signals through connexin hemichannels (Sáez et al. 2010), the precursors of gap junction channels that are formed by two hemichannels provided by one of each apposed cells (Fig. 1). Each hemichannel is composed of six protein subunits termed connexins, which are highly conserved proteins encoded by 21 genes in human and 20 in mouse with orthologs in other vertebrate species (Cruciani and Mikalsen 2005). Connexins are abundantly expressed in cells of the central nervous system (CNS) (Orellana et al. 2009) (Fig. 2), and they are named after their predicted molecular mass expressed in kDa, so that connexin43 (Cx43) has a

For a long time the main function attributed to connexin hemichannels was the formation of gap junction channels. Nevertheless, in the last decade, the presence of functional connexin hemichannels in nonjunctional membranes has been demonstrated by several experimental approaches (Sáez et al. 2010). These channels allow cellular release of relevant quantities of autocrine/paracrine signaling molecules (e.g., ATP, glutamate, NAD+ and PGE2) to the extracellular milieu (Bruzzone et al. 2001; Cherian et al. 2005; Stout et al. 2002), as well as uptake of small molecules (e.g., glucose) (Retamal et al. 2007a). Recently, another gene family encoding a set of three membrane proteins, named pannexins (Panxs 1-3), has been identified (Bruzzone et al. 2003). Up to now, only Panx3 has been shown to form gap junctions in osteoblasts (Ishikawa et al. 2011) and further studies will be required to identify pannexin gap junctions in other cell types. Connexins and pannexins present similar membrane topology, with four α-helical transmembrane domains connected by two

**1. Introduction** 

molecular mass of ~43 kDa.

*1Departamento de Fisiología; Facultad de Ciencias Biológicas*

*2Departamento de Neurología; Facultad de Medicina Pontificia Universidad Católica de Chile, Santiago,* 


### **Role of Connexin Hemichannels in Neurodegeneration**

Juan A. Orellana1,2, Christian Giaume3 and Juan C. Sáez1,4 *1Departamento de Fisiología; Facultad de Ciencias Biológicas 2Departamento de Neurología; Facultad de Medicina Pontificia Universidad Católica de Chile, Santiago, 3CIRB; UMR CNRS 7241/ INSERM U1050, Collège de France, Paris 4Instituto milenio, Centro Interdiciplinario de Neurociencias de Valparaiso, Valparaiso, 1,2,4Chile* 

*3France* 

#### **1. Introduction**

234 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

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in a transgenic mouse model. *Brain Res*, 2004. 1027(1-2): p. 73-86.

sclerosis in transgenic mice. *Brain Res*, 2005. 1045(1-2): p. 185-98.

of familial ALS. *Mol Cell Neurosci*, 2006. 31(2): p. 218-31.

in stroke. *Nat Rev Neurosci*, 2003. 4(5): p. 399-415.

*Yao Za Zhi*, 2005. 30(12): p. 930-2.

*Neurosci Lett*, 2002. 329(2): p. 232-6.

*J Neurosci*, 2008. 28(49): p. 13038-55.

1-7.

disease. *J Mol Med* (Berl), 2008. 86(4): p. 485-90.

disease. *Neuroscience*, 2009. 163(4): p. 1128-34.

striatum. *Neurosci Lett*, 2008. 430(2): p. 142-6.

model. *Science*, 2000. 288(5464): p. 335-9.

Progressive loss of neuronal structure and function occur in several neurodegenerative diseases. Cellular responses to brain injury depend on properties of the cells (e.g., hormonal nutritional status) and insult (e.g., duration, intensity, and quality), whereas, tissue responses depend on interactions between their constituent cells, including chemical and electrical transmission as well as paracrine and autocrine signaling. In vertebrate cells, autocrine and paracrine communication occur in part via release of chemical signals through connexin hemichannels (Sáez et al. 2010), the precursors of gap junction channels that are formed by two hemichannels provided by one of each apposed cells (Fig. 1). Each hemichannel is composed of six protein subunits termed connexins, which are highly conserved proteins encoded by 21 genes in human and 20 in mouse with orthologs in other vertebrate species (Cruciani and Mikalsen 2005). Connexins are abundantly expressed in cells of the central nervous system (CNS) (Orellana et al. 2009) (Fig. 2), and they are named after their predicted molecular mass expressed in kDa, so that connexin43 (Cx43) has a molecular mass of ~43 kDa.

For a long time the main function attributed to connexin hemichannels was the formation of gap junction channels. Nevertheless, in the last decade, the presence of functional connexin hemichannels in nonjunctional membranes has been demonstrated by several experimental approaches (Sáez et al. 2010). These channels allow cellular release of relevant quantities of autocrine/paracrine signaling molecules (e.g., ATP, glutamate, NAD+ and PGE2) to the extracellular milieu (Bruzzone et al. 2001; Cherian et al. 2005; Stout et al. 2002), as well as uptake of small molecules (e.g., glucose) (Retamal et al. 2007a). Recently, another gene family encoding a set of three membrane proteins, named pannexins (Panxs 1-3), has been identified (Bruzzone et al. 2003). Up to now, only Panx3 has been shown to form gap junctions in osteoblasts (Ishikawa et al. 2011) and further studies will be required to identify pannexin gap junctions in other cell types. Connexins and pannexins present similar membrane topology, with four α-helical transmembrane domains connected by two

Role of Connexin Hemichannels in Neurodegeneration 237

Fig. 2. Cellular distribution of pannexin and connexin hemichannels and connexin gap junctions in brain cells. This figure includes only those cases in which the available information has been obtained *in vivo* and/or *in vitro* with more than one experimental approach. Homocellular connexin gap junction channels and connexin and pannexin hemichannels are indicated within the encircled regions. Neuron (N), astrocyte (A),

Despite the advances in the understanding of infectious diseases, including bacterial meningitis, pathogen-host interactions and the widespread use of chemotherapeutic agents, infections are still an important cause of mortality, morbidity and social burden worldwide. Bacterial meningitis promotes inflammation of the pia, arachnoid, and subarachnoid space. Inflammation may also affect the brain parenchyma leading to encephalitis. Bacteria present in the bloodstream induce an innate immune response which produces systemic release of cytokines, mainly TNF-α (Dietzman et al. 1974; Ring et al. 1998). Then, bacteria colonize and cross the inflamed blood-brain barrier (BBB) and components of their wall, such as lipopolysaccharide (LPS) (Bannerman et al. 1998), peptidoglycan (PGN) (Bannerman et al. 1998), or streptococcal hemolysin/cytolysin (Doran et al. 2003), induce BBB activation and permeabilization (Freyer et al. 1999) (Fig. 3). BBB activation is characterized by numerous

microglia (M) and oligodendrocyte (O). [Modified from Orellana et al. 2009]

**2. What is known about hemichannels in neurological disorders?** 

**2.1 Bacterial meningitis** 

extracellular loops, where both N- and C-termini are intracellular. However, there is only 16% overall identity when their full-length amino acid sequences are compared.

Fig. 1. Diagram illustrating basic structures of connexins and undocked hemichannels present at the cell surface. The membrane topology of a connexin consists of 4 membranespanning domains (M1-M4), 2 extracellular loops (E1 and E2) and 1 cytoplasmic loop (CL). The amino (-NH2) and carboxy (-COOH) terminal tail are intracellular. A hemichannel is formed by six connexins that oligomerize laterally leaving a central pore. In cultured cells under resting conditions hemichannels remain preferentially closed, but they can be activated by diverse physiological and pathological conditions, offering a diffusional transmembrane route between the intra and extracellular milieu. [Modified from Orellana et al. 2011a]

A main role in cellular proliferation and tissue remodelling has been attributed to hemichannels (Burra and Jiang 2009; Schalper et al. 2008), while in the CNS they have been proposed to mediate ischemic tolerance (Lin et al. 2008; Schock et al. 2008) and establish adhesive interactions (Cotrina et al. 2008). To date, most studies suggest that under normal brain conditions hemichannels release physiological molecules relevant for intercellular signalling, including propagation of intercellular Ca2+ waves (Orellana et al. 2011a). However, an increasing body of evidence has situated the hemichannels as potential regulators of the beginning and maintenance of homeostatic imbalances present in diverse brain diseases (Orellana et al. 2009). Pioneering findings by Contreras and co-workers (Contreras et al. 2002) showed that astroglial death induced by ischemia-like conditions is accelerated by the opening of Cx43 hemichannels. In this sense, a constant increase of [Ca2+]i mediated by augmented Ca2+ entry through hemichannels, which are permeable to Ca2+ (Schalper et al. 2010), and deficient or insufficient Ca2+ handling by injured cells could lead to cell death. Here, we review and discuss the current evidence about the role of hemichannels in three major neurodegenerative diseases, namely, bacterial meningitis, stroke and Alzheimer's disease.

extracellular loops, where both N- and C-termini are intracellular. However, there is only

16% overall identity when their full-length amino acid sequences are compared.

Fig. 1. Diagram illustrating basic structures of connexins and undocked hemichannels present at the cell surface. The membrane topology of a connexin consists of 4 membranespanning domains (M1-M4), 2 extracellular loops (E1 and E2) and 1 cytoplasmic loop (CL). The amino (-NH2) and carboxy (-COOH) terminal tail are intracellular. A hemichannel is formed by six connexins that oligomerize laterally leaving a central pore. In cultured cells under resting conditions hemichannels remain preferentially closed, but they can be activated by diverse physiological and pathological conditions, offering a diffusional

transmembrane route between the intra and extracellular milieu. [Modified from Orellana et

A main role in cellular proliferation and tissue remodelling has been attributed to hemichannels (Burra and Jiang 2009; Schalper et al. 2008), while in the CNS they have been proposed to mediate ischemic tolerance (Lin et al. 2008; Schock et al. 2008) and establish adhesive interactions (Cotrina et al. 2008). To date, most studies suggest that under normal brain conditions hemichannels release physiological molecules relevant for intercellular signalling, including propagation of intercellular Ca2+ waves (Orellana et al. 2011a). However, an increasing body of evidence has situated the hemichannels as potential regulators of the beginning and maintenance of homeostatic imbalances present in diverse brain diseases (Orellana et al. 2009). Pioneering findings by Contreras and co-workers (Contreras et al. 2002) showed that astroglial death induced by ischemia-like conditions is accelerated by the opening of Cx43 hemichannels. In this sense, a constant increase of [Ca2+]i mediated by augmented Ca2+ entry through hemichannels, which are permeable to Ca2+ (Schalper et al. 2010), and deficient or insufficient Ca2+ handling by injured cells could lead to cell death. Here, we review and discuss the current evidence about the role of hemichannels in three major neurodegenerative diseases, namely, bacterial meningitis, stroke and Alzheimer's

al. 2011a]

disease.

Fig. 2. Cellular distribution of pannexin and connexin hemichannels and connexin gap junctions in brain cells. This figure includes only those cases in which the available information has been obtained *in vivo* and/or *in vitro* with more than one experimental approach. Homocellular connexin gap junction channels and connexin and pannexin hemichannels are indicated within the encircled regions. Neuron (N), astrocyte (A), microglia (M) and oligodendrocyte (O). [Modified from Orellana et al. 2009]

#### **2. What is known about hemichannels in neurological disorders?**

#### **2.1 Bacterial meningitis**

Despite the advances in the understanding of infectious diseases, including bacterial meningitis, pathogen-host interactions and the widespread use of chemotherapeutic agents, infections are still an important cause of mortality, morbidity and social burden worldwide. Bacterial meningitis promotes inflammation of the pia, arachnoid, and subarachnoid space. Inflammation may also affect the brain parenchyma leading to encephalitis. Bacteria present in the bloodstream induce an innate immune response which produces systemic release of cytokines, mainly TNF-α (Dietzman et al. 1974; Ring et al. 1998). Then, bacteria colonize and cross the inflamed blood-brain barrier (BBB) and components of their wall, such as lipopolysaccharide (LPS) (Bannerman et al. 1998), peptidoglycan (PGN) (Bannerman et al. 1998), or streptococcal hemolysin/cytolysin (Doran et al. 2003), induce BBB activation and permeabilization (Freyer et al. 1999) (Fig. 3). BBB activation is characterized by numerous

Role of Connexin Hemichannels in Neurodegeneration 239

changes including cytokine production, overexpression of cell adhesion molecules and NO synthesis (Freyer et al. 1999). Recently, hemichannels have been implicated in the pathogenesis of bacterial meningitis. Using a model of bacterial brain abscess, Karpuk and co-workers (Karpuk et al. 2011) showed that a transient hemichannel activity is induced in astrocytes within close proximity to the abscess border, which dissipates with increasing distance from the inflammatory site. Moreover, this transient hemichannel opening was blocked with the Cx43 mimetic peptide Gap26, carbenoxolone, the Panx1 mimetic peptide 10panx1, and probenecid (Karpuk et al. 2011), indicating the involvement of both Cx43 and Panx1 hemichannels in this response. In addition, astroglial gap junction coupling was significantly reduced in areas immediately surrounding the abscess margins, while regions far from abscess presented normal coupling (Karpuk et al. 2011). These data are consistent with previous studies showing opposite regulation of gap junction channels versus hemichannels in astrocytes subjected to pro-inflammatory conditions (Froger et al. 2010; Froger et al. 2009; Orellana et al. 2011b; Orellana et al. 2010;

Pioneering findings by Retamal and co-workers (Retamal et al. 2007a) showed that TNF-α and IL-1β released from LPS-treated microglia induce an increase and decrease in astroglial

The consequence of this opposite regulation on the homeostasis of the infected and uninfected brain parenchyma and how they may influence CNS function remain to be elucidated. A possible consequence of increased astroglial hemichannel opening could be an enhanced glucose uptake, which might explain the observed changes in the metabolic status of astrocytes under inflammatory conditions (Rtamal et al. 2007a). Moreover, hemichannel-mediated astroglial release of neurotoxic and/or inflammatory compounds such as glutamate and ATP could promote paracrine neuronal damage (Iglesias et al. 2009; Jiang et al. 2011; Kang et al. 2008; Orellana et al. 2011b; Orellana et al. 2011c; Ye et al.

Since the BBB critically regulates the passage of molecules into the CNS, the possibility of defective hemichannels in cells of the BBB during bacterial meningitis may be relevant. In this regard, TNF-α blocks the ATP release induced by photoliberation of InsP3 or zero [Ca2+]o, but increases the basal ATP release and hemichannel-mediated dye uptake in brain cortical endothelium derived cell lines, RBE4 and GP8 (Vandamme et al. 2004). Since the increase in basal ATP release induced by TNF-α is not affected by the mimetic peptide Gap26, a prominent blocker of InsP3- and zero Ca2+-triggered connexin-dependent ATP release (Braet et al. 2003), it was concluded that the InsP3- and zero Ca2+-induced ATP release would involve a mechanism distinct from the one involved in the TNF-α induced elevation of basal ATP release (Vandamme et al. 2004). However, the authors did not rule out the involvement of other type of hemichannels (connexin/pannexins), including hemichannels formed by Cx40, which is highly expressed in brain endothelial cells (Nagasawa et al. 2006). Thus, the rise in basal activity induced by TNF-α could be related to Cx40 and/or pannexin hemichannels. At least in peripheral endothelial cells, the ATP release induced by brief exposure to PGN depends exclusively on Cx43 hemichannels

Enhanced endothelial hemichannel activity could elevate the ATP release, which would recruit microglia to the injury site (Davalos et al. 2005). In agreement with a role of hemichannels in ATP release during inflammatory conditions triggered by bacterial

hemichannel and gap junction channel activity, respectively (Retamal et al. 2007a).

Retamal et al. 2007a).

(Robertson et al. 2010).

2003).

Fig. 3. Connexin based channels in brain cells during bacterial meningitis. During bacterial infection blood levels of cytokine (e.g., TNF-α and IL-1β) are elevated. Both, TNF-α and IL-1β enhance the hemichannel activity (1) of brain endothelial cells. Furthermore, these cytokines induce BBB discontinuity favoring bacterial extravasation (2). Once in the interstitium, bacteria and their extracellular wall components such as LPS and PGN are recognized by microglia (3), which are activated and release cytokines that further activate them (reciprocal arrows). ATP released via hemichannels from microglia (4) promotes microglial migration from less affected regions. Activated microglia can also release glutamate through hemichannels and oxygen and nitrogen derived free radicals that are neurotoxic (5). The enhanced hemichannel activity of activated astrocytes induces neuronal damage through the release of neurotoxic and/or inflammatory compounds such glutamate and PGE2 (6). These compounds may also increase the activity of neuronal connexin/pannexin hemichannels (7) causing electrochemical imbalance and Ca2+ overload in neurons. In contrast to increased opening of hemichannels, astroglial gap junction communication is reduced (8), impairing glutamate and K+ spatial buffering that enhances neuronal susceptibility to insults. Bacterial meningitis can also induce demyelination (9), possibly via microglial cytokine release. Severe inflammation induces recruitment of leukocytes (10) to the infected loci. Gap junction communication between leukocytes and endothelial cells (11) may contribute to strength heterocellular adhesion and allow transfer of signals that regulate leukocyte diapedesis across the endothelium (12). Activated microglia may perform antigen cross-presentation interaction with infiltrating leukocytes in which gap junctions between them may play an important role (13). Direct microglial interaction with LPS or PGN induces gap junction communication between microglia, which can coordinate microglial function (14). [Modified from Orellana et al. 2009]

Fig. 3. Connexin based channels in brain cells during bacterial meningitis. During bacterial infection blood levels of cytokine (e.g., TNF-α and IL-1β) are elevated. Both, TNF-α and IL-1β enhance the hemichannel activity (1) of brain endothelial cells. Furthermore, these cytokines induce BBB discontinuity favoring bacterial extravasation (2). Once in the interstitium, bacteria and their extracellular wall components such as LPS and PGN are recognized by microglia (3), which are activated and release cytokines that further activate them (reciprocal arrows). ATP released via hemichannels from microglia (4) promotes microglial migration from less affected regions. Activated microglia can also release glutamate through hemichannels and oxygen and nitrogen derived free radicals that are neurotoxic (5). The enhanced hemichannel activity of activated astrocytes induces neuronal damage through the release of neurotoxic and/or inflammatory compounds such glutamate

connexin/pannexin hemichannels (7) causing electrochemical imbalance and Ca2+ overload in neurons. In contrast to increased opening of hemichannels, astroglial gap junction communication is reduced (8), impairing glutamate and K+ spatial buffering that enhances neuronal susceptibility to insults. Bacterial meningitis can also induce demyelination (9), possibly via microglial cytokine release. Severe inflammation induces recruitment of leukocytes (10) to the infected loci. Gap junction communication between leukocytes and endothelial cells (11) may contribute to strength heterocellular adhesion and allow transfer of signals that regulate leukocyte diapedesis across the endothelium (12). Activated

microglia may perform antigen cross-presentation interaction with infiltrating leukocytes in which gap junctions between them may play an important role (13). Direct microglial interaction with LPS or PGN induces gap junction communication between microglia, which can coordinate microglial function (14). [Modified from Orellana et al. 2009]

and PGE2 (6). These compounds may also increase the activity of neuronal

changes including cytokine production, overexpression of cell adhesion molecules and NO synthesis (Freyer et al. 1999). Recently, hemichannels have been implicated in the pathogenesis of bacterial meningitis. Using a model of bacterial brain abscess, Karpuk and co-workers (Karpuk et al. 2011) showed that a transient hemichannel activity is induced in astrocytes within close proximity to the abscess border, which dissipates with increasing distance from the inflammatory site. Moreover, this transient hemichannel opening was blocked with the Cx43 mimetic peptide Gap26, carbenoxolone, the Panx1 mimetic peptide 10panx1, and probenecid (Karpuk et al. 2011), indicating the involvement of both Cx43 and Panx1 hemichannels in this response. In addition, astroglial gap junction coupling was significantly reduced in areas immediately surrounding the abscess margins, while regions far from abscess presented normal coupling (Karpuk et al. 2011). These data are consistent with previous studies showing opposite regulation of gap junction channels versus hemichannels in astrocytes subjected to pro-inflammatory conditions (Froger et al. 2010; Froger et al. 2009; Orellana et al. 2011b; Orellana et al. 2010; Retamal et al. 2007a).

Pioneering findings by Retamal and co-workers (Retamal et al. 2007a) showed that TNF-α and IL-1β released from LPS-treated microglia induce an increase and decrease in astroglial hemichannel and gap junction channel activity, respectively (Retamal et al. 2007a).

The consequence of this opposite regulation on the homeostasis of the infected and uninfected brain parenchyma and how they may influence CNS function remain to be elucidated. A possible consequence of increased astroglial hemichannel opening could be an enhanced glucose uptake, which might explain the observed changes in the metabolic status of astrocytes under inflammatory conditions (Rtamal et al. 2007a). Moreover, hemichannel-mediated astroglial release of neurotoxic and/or inflammatory compounds such as glutamate and ATP could promote paracrine neuronal damage (Iglesias et al. 2009; Jiang et al. 2011; Kang et al. 2008; Orellana et al. 2011b; Orellana et al. 2011c; Ye et al. 2003).

Since the BBB critically regulates the passage of molecules into the CNS, the possibility of defective hemichannels in cells of the BBB during bacterial meningitis may be relevant. In this regard, TNF-α blocks the ATP release induced by photoliberation of InsP3 or zero [Ca2+]o, but increases the basal ATP release and hemichannel-mediated dye uptake in brain cortical endothelium derived cell lines, RBE4 and GP8 (Vandamme et al. 2004). Since the increase in basal ATP release induced by TNF-α is not affected by the mimetic peptide Gap26, a prominent blocker of InsP3- and zero Ca2+-triggered connexin-dependent ATP release (Braet et al. 2003), it was concluded that the InsP3- and zero Ca2+-induced ATP release would involve a mechanism distinct from the one involved in the TNF-α induced elevation of basal ATP release (Vandamme et al. 2004). However, the authors did not rule out the involvement of other type of hemichannels (connexin/pannexins), including hemichannels formed by Cx40, which is highly expressed in brain endothelial cells (Nagasawa et al. 2006). Thus, the rise in basal activity induced by TNF-α could be related to Cx40 and/or pannexin hemichannels. At least in peripheral endothelial cells, the ATP release induced by brief exposure to PGN depends exclusively on Cx43 hemichannels (Robertson et al. 2010).

Enhanced endothelial hemichannel activity could elevate the ATP release, which would recruit microglia to the injury site (Davalos et al. 2005). In agreement with a role of hemichannels in ATP release during inflammatory conditions triggered by bacterial

Role of Connexin Hemichannels in Neurodegeneration 241

initiator cell, and the Ca2+ waves extend as far as the ATP diffuses; (iv) the waves are blocked by extracellular apyrase; (v) are blocked by suramin (P2 receptor blocker); and (vi) jump cell-free gaps and are deflected by flow of medium (Orellana et al. 2011a). Probably, *in vivo* these two mechanisms coexist and are subjected to regulation by neuro- and gliotransmitters, playing a key role in the functional synchronization of neurovascular

As mentioned before, in the brain parenchyma, bacteria may undergo lysis and thus, they release pro-inflammatory and toxic factors such as PGN and LPS (Stuertz et al. 1998; Stuertz et al. 1999), while microglia interact directly with intact bacteria (Kim 2003). Bacterial derived pro-inflammatory factors such as LPS induce neurodegeneration (Qin et al. 2007). PGN and LPS stimulate microglial Toll-like receptors (TLRs), induce translocation of nuclear factor (NF) κB (Schwandner et al. 1999), and activation of MAPK signaling and transcription of genes encoding inflammatory cytokines (Laflamme and Rivest 2001; Nau and Bruck 2002). Moreover, PGN increases microglial Cx43 mRNA and protein expression, which correlates with development of gap junction communication *in vitro* (Garg et al. 2005). Similarly, treatment with LPS plus IFN-γ increases Cx43 expression in rat microglia and induces gap junction communication (Eugenín et al. 2001). In addition, brain stab wounds induce recruitment of Cx43 immunoreactive microglia to the injured foci, suggesting that Cx43 is important for coordinating microglial responses (Eugenín et al. 2001). However, up to now, the functional state of hemichannels in microglia has been not examined in model of

Bacterial meningitis also causes axonal damage and demyelination (Nau et al. 2004). These effects may be related to microglial cytokine release, which could promote opening of oligodendrocyte hemichannels, possibly composed of Panx1, Cx32 or Cx29 [Cx29 does not form gap junctions and faces the periaxonal space (Li et al. 2002)] and, thus, promoting ion

Adhesion between leukocytes and endothelial cells could result in part from leukoendothelial gap junction formation (Veliz et al. 2008). In fact, treatment with diverse gap junction channel blockers reduces leukocyte adhesion to venular endothelium *in vivo* (Veliz et al. 2008) as well as transmigration across a BBB model (Eugenín et al. 2003). Gap junctions between lymphoma and endothelial cells have also been demonstrated, and α-GA attenuates the transmigration of lymphoma cells across an endothelial barrier (Haddad et al. 2008). However, it is important to keep in mind that most gap junction blockers will also block hemichannels and that connexin knockout animals will also have impaired transmembrane diffusional transport mediated by hemichannels. Indeed, the autocrine release of ATP through Cx37 hemichannels in monocyte/macrophages limits their adhesion to the endothelial wall and recruitment to the subendothelial compartment (Wong et al. 2006). Recent findings also suggest that connexin and pannexin hemichannels participate in acute and chronic inflammatory responses mediated by macrophagic cells. In fact, LPS or TNF-α promote microglial release of neurotoxic glutamate concentrations via Cx32 hemichannels (Takeuchi et al. 2006). Panx1 hemichannel opening occur in primary macrophages, macrophage cell lines and microglia exposed to pro-inflammatory conditions (Orellana et al. 2011c; Pelegrin and Surprenant 2006; Pelegrin and Surprenant 2007). In addition, caspase-1/inflammasome-mediated release of members of the IL-1 family including IL-1β from mouse peritoneal macrophages requires hemichannel activation

through P2X7 purinergic receptors (Pelegrin and Surprenant 2006).

coupling.

bacterial brain infection.

gradient imbalance and Ca2+ overload.

infections, Shigella infection of epithelial cells promote ATP release through Cx26 hemichannels, resulting in the activation of purinergic receptors on neighboring cells and bacterial dissemination (Tran Van Nhieu et al. 2003). In the same way, normal calcium signaling between astrocytes could be affected under pro-inflammatory conditions, eliciting one of the two calcium waves reported (Orellana et al. 2011a) (Fig. 4). In one of them, Ca2+ waves propagate by diffusion of cytoplasmic inositol (1,4,5)-trisphosphate (IP3) through gap junctions between astrocytes, after phospholipase C (PLC) activation (Fig. 4). Evidence for this mechanism includes: (i) the waves are dependent on gap junctional communication; (ii) the waves are not blocked by extracellular apyrase, which is an ATPase; (iii) are not blocked by purine-receptor antagonists such as suramin; and (iv) do not jump a gap between cells (Orellana et al. 2011a). Other possible mechanism for astroglial Ca2+ waves is through the ATP released by Cx43 and/or Panx1 hemichannels after ATP-mediated P2 receptor activation (Fig. 4). Evidence for this mechanism include: (i) the waves require Cx43 and/or Panx1 expression (ii) hemichannel blockers prevent the waves; (iii) ATP is released by the

Fig. 4. Two models for conduction of Ca2+ waves in astrocytes. (Top panel) Upstream receptor stimulation leads to activation of phospholipase C (PLC) and formation of cytoplasmic inositol (1,4,5)-trisphosphate (IP3), which promote the release of Ca2+ stored in the endoplasmic reticulum. Both IP3 and Ca2+ diffuse to neighboring cells through gap junction channels generating waves of rises in intracellular Ca2+ concentration [Ca2+]i. (Bottom panel) ATP released from vesicles and/or ion channels diffuses through the extracellular space and activates membrane purinergic (P2) receptors. Stimulation of metabotropic P2Y receptors leads to activation of phospholipase C (PLC) and formation of IP3. Whereas, activation of ionotropic P2X receptors leads to Ca2+ influx. The increase in free [Ca2+]i induced by IP3 and P2X receptor opening could promote ATP release through Cx43 and Px1 hemichannels, extending the Ca2+ wave to neighboring cells. [Modified from Orellana et al. 2011a]

infections, Shigella infection of epithelial cells promote ATP release through Cx26 hemichannels, resulting in the activation of purinergic receptors on neighboring cells and bacterial dissemination (Tran Van Nhieu et al. 2003). In the same way, normal calcium signaling between astrocytes could be affected under pro-inflammatory conditions, eliciting one of the two calcium waves reported (Orellana et al. 2011a) (Fig. 4). In one of them, Ca2+ waves propagate by diffusion of cytoplasmic inositol (1,4,5)-trisphosphate (IP3) through gap junctions between astrocytes, after phospholipase C (PLC) activation (Fig. 4). Evidence for this mechanism includes: (i) the waves are dependent on gap junctional communication; (ii) the waves are not blocked by extracellular apyrase, which is an ATPase; (iii) are not blocked by purine-receptor antagonists such as suramin; and (iv) do not jump a gap between cells (Orellana et al. 2011a). Other possible mechanism for astroglial Ca2+ waves is through the ATP released by Cx43 and/or Panx1 hemichannels after ATP-mediated P2 receptor activation (Fig. 4). Evidence for this mechanism include: (i) the waves require Cx43 and/or Panx1 expression (ii) hemichannel blockers prevent the waves; (iii) ATP is released by the

Fig. 4. Two models for conduction of Ca2+ waves in astrocytes. (Top panel) Upstream receptor stimulation leads to activation of phospholipase C (PLC) and formation of

Orellana et al. 2011a]

cytoplasmic inositol (1,4,5)-trisphosphate (IP3), which promote the release of Ca2+ stored in the endoplasmic reticulum. Both IP3 and Ca2+ diffuse to neighboring cells through gap junction channels generating waves of rises in intracellular Ca2+ concentration [Ca2+]i. (Bottom panel) ATP released from vesicles and/or ion channels diffuses through the extracellular space and activates membrane purinergic (P2) receptors. Stimulation of metabotropic P2Y receptors leads to activation of phospholipase C (PLC) and formation of IP3. Whereas, activation of ionotropic P2X receptors leads to Ca2+ influx. The increase in free [Ca2+]i induced by IP3 and P2X receptor opening could promote ATP release through Cx43 and Px1 hemichannels, extending the Ca2+ wave to neighboring cells. [Modified from

initiator cell, and the Ca2+ waves extend as far as the ATP diffuses; (iv) the waves are blocked by extracellular apyrase; (v) are blocked by suramin (P2 receptor blocker); and (vi) jump cell-free gaps and are deflected by flow of medium (Orellana et al. 2011a). Probably, *in vivo* these two mechanisms coexist and are subjected to regulation by neuro- and gliotransmitters, playing a key role in the functional synchronization of neurovascular coupling.

As mentioned before, in the brain parenchyma, bacteria may undergo lysis and thus, they release pro-inflammatory and toxic factors such as PGN and LPS (Stuertz et al. 1998; Stuertz et al. 1999), while microglia interact directly with intact bacteria (Kim 2003). Bacterial derived pro-inflammatory factors such as LPS induce neurodegeneration (Qin et al. 2007). PGN and LPS stimulate microglial Toll-like receptors (TLRs), induce translocation of nuclear factor (NF) κB (Schwandner et al. 1999), and activation of MAPK signaling and transcription of genes encoding inflammatory cytokines (Laflamme and Rivest 2001; Nau and Bruck 2002). Moreover, PGN increases microglial Cx43 mRNA and protein expression, which correlates with development of gap junction communication *in vitro* (Garg et al. 2005). Similarly, treatment with LPS plus IFN-γ increases Cx43 expression in rat microglia and induces gap junction communication (Eugenín et al. 2001). In addition, brain stab wounds induce recruitment of Cx43 immunoreactive microglia to the injured foci, suggesting that Cx43 is important for coordinating microglial responses (Eugenín et al. 2001). However, up to now, the functional state of hemichannels in microglia has been not examined in model of bacterial brain infection.

Bacterial meningitis also causes axonal damage and demyelination (Nau et al. 2004). These effects may be related to microglial cytokine release, which could promote opening of oligodendrocyte hemichannels, possibly composed of Panx1, Cx32 or Cx29 [Cx29 does not form gap junctions and faces the periaxonal space (Li et al. 2002)] and, thus, promoting ion gradient imbalance and Ca2+ overload.

Adhesion between leukocytes and endothelial cells could result in part from leukoendothelial gap junction formation (Veliz et al. 2008). In fact, treatment with diverse gap junction channel blockers reduces leukocyte adhesion to venular endothelium *in vivo* (Veliz et al. 2008) as well as transmigration across a BBB model (Eugenín et al. 2003). Gap junctions between lymphoma and endothelial cells have also been demonstrated, and α-GA attenuates the transmigration of lymphoma cells across an endothelial barrier (Haddad et al. 2008). However, it is important to keep in mind that most gap junction blockers will also block hemichannels and that connexin knockout animals will also have impaired transmembrane diffusional transport mediated by hemichannels. Indeed, the autocrine release of ATP through Cx37 hemichannels in monocyte/macrophages limits their adhesion to the endothelial wall and recruitment to the subendothelial compartment (Wong et al. 2006). Recent findings also suggest that connexin and pannexin hemichannels participate in acute and chronic inflammatory responses mediated by macrophagic cells. In fact, LPS or TNF-α promote microglial release of neurotoxic glutamate concentrations via Cx32 hemichannels (Takeuchi et al. 2006). Panx1 hemichannel opening occur in primary macrophages, macrophage cell lines and microglia exposed to pro-inflammatory conditions (Orellana et al. 2011c; Pelegrin and Surprenant 2006; Pelegrin and Surprenant 2007). In addition, caspase-1/inflammasome-mediated release of members of the IL-1 family including IL-1β from mouse peritoneal macrophages requires hemichannel activation through P2X7 purinergic receptors (Pelegrin and Surprenant 2006).

Role of Connexin Hemichannels in Neurodegeneration 243

breakdown as observed in astrocytes exposed to ischemic conditions (Kimelberg 2005) (Fig. 5). Calcium overload induced in part by hemichannel opening may also activate cyclooxygenase/lipoxygenase pathways leading to increased free radicals, lipid peroxidation and further plasma membrane damage. It is noteworthy that arachidonic acid and decrease in the intracellular redox potential also activates Cx43 hemichannels, which

The first *in vivo* evidence for the involvement of connexin-based channels in the spread of death signals came from experiments in which octanol, a non-selective gap junction and hemichannel blocker, reduced the infarct size after focal cerebral ischemia (Rawanduzy et al. 1997). However, these observations disagree with the findings obtained in heterozygous Cx43 knockout mice or mouse astrocytes lacking Cx43 expression, in which focal ischemia causes larger lesions (Nakase et al. 2003; Nakase et al. 2004; Siushansian et al. 2001). In a rat model of transient global ischemia, pretreatment with compounds that block both hemichannels and gap junction channels (i.e. CBX, α-GA and endothelin) reduces the number of apoptotic neurons as compared to the contralateral hippocampus treated with saline (Perez Velazquez et al. 2006). In addition, hemichannels present in surrounding cells or in the stromal component of diverse organs may also participate in the ischemic responses. For example, the CA1 region of Cx32- deficient mice show increased sensitivity to global ischemia (Oguro et al. 2001). Since Cx32 forms hemichannels in activated microglia (Takeuchi et al. 2006), it is possible that Cx32 knockout animals may show attenuated microglial release of regulatory paracrine signals. When acute diseases like stroke have the presence of other pro-inflammatory components (e.g. high glucose produced by diabetes mellitus), brain damage and cognitive functions in patients is worse (Pasquier et al. 2006). Indeed, it has long been known that hyperglycemia worsens the outcome of acute brain ischemia by increasing the extent of tissue injury in animals and humans (Kagansky et al. 2001). Recently, we showed in astrocytes that high levels of extracellular glucose increase hemichannel activity and decrease gap junction permeability induced by hypoxia (Orellana et al. 2010). These changes are transient after 3 hours of hypoxia in high glucose. However, they are more prominent after 6 hours of hypoxia and last for over 3 hours followed by death of numerous astrocytes (Orellana et al. 2010). Because high glucose worsens the effect on ischemia-induced cell damage in endothelial cells, neurons, and microglia (Kagansky et al. 2001; Lin et al. 1998a; Tsuruta et al. ; Wang et al. 2001), it would be of interest to study if elevated hemichannel activity plays a relevant role as in astrocytes. Importantly, we also demonstrated that microglia treated with amyloid β peptide (Aβ) potentiate the increase in astroglial hemichannel activity and reduction in gap junctional communication induced by hypoxia in high glucose, suggesting that these changes are a common denominator of

may exacerbate cell damage (De Vuyst et al. 2009; Retamal et al. 2007b).

inflamed or activated astrocytes (Orellana et al. 2011b) (Fig. 6).

Panx1, respectively.

In addition, the extracellular media of activated astrocytes was neurotoxic due to its glutamate and ATP content that activate neuronal Panx1 hemichannels via NMDA/P2X receptors leading to neuronal death (Orellana et al. 2011b). Therefore, in a more integrated system (e.g., brain or brain slices) neurons could be efficiently protected from ischemia and neurotoxicity by blocking NMDA and P2X receptors as already proposed (Dirnagl et al. 1999), but also by targeting either glial or neuronal hemichannels composed by Cx43 and

Hemichannels may also be involved in tissue response to stroke through their participation in a phenomenon known as ischemic preconditioning, in which a sublethal ischemic insult induces resistance to a subsequent more severe insult (Gidday 2006). It was recently shown

#### **2.2 Stroke**

Stroke is a major cause of death in industrialized countries and results from a transient or permanent reduction in cerebral blood flow produced, in most cases, by cerebral artery occlusion by an embolus or local thrombosis, i.e., focal ischemia (Dirnagl et al. 1999). Severe and/or prolonged reduction in cerebral blood flow leads to deprivation of oxygen and glucose as well as build-up of potentially toxic substances. During stroke, decreased cellular oxygen levels, loss of oxidative phosphorylation and reduced ATP synthesis are the initial steps leading to cell death (Dirnagl et al. 1999). The ATP depletion may induce a rapid decrease in ATPase activity, leading to imbalanced electrochemical gradients across the plasma membrane. Notably, increased pannexin and/or connexin hemichannel activity occur in cortical astrocytes (Contreras et al. 2002; Orellana et al. 2011b; Orellana et al. 2010; Retamal et al. 2006), olygodendrocytes (Domercq et al. 2010) and hippocampal neurons (Lin et al. 2008; Schock et al. 2008; Thompson et al. 2006) subjected to ischemic like conditions. The enhanced hemichannel activity induced by ischemic like conditions accelerates cell death, at least in cultured rat astrocytes (Contreras et al. 2002; Orellana et al. 2010) (Fig. 5). Possibly, sustained hemichannel opening contribute to increased [Ca2+]i, which in turn may favour even more the connexin hemichannel activity (De Vuyst et al. 2007; Schalper et al. 2008), inducing Ca2+ and Na+ intracellular overload (Fig. 5). The ionic (or electrolyte) imbalance leads to an osmotic imbalance that results in cell swelling and plasma membrane phospholipase A2, with the subsequent generation of arachidonic acid and activation of

Fig. 5. Three mechanisms of death amplification. (A) Initially, a brain injury produced by ischemia, infection or necrosis affecting astrocytes (green), neurons (orange) or resting microglia (blue), could start a wave of death propagated (yellow arrows) and amplified through diffusible toxins and molecules (e.g., Ca2+, NO, superoxide ion, peroxinitrite, glutamate, and NAD+) present in high concentration in injured cells (depicted in the figure as dark colored cells). These molecules could be transferred through connexin gap junctions and connexin and pannexin hemichannels from injured cells (less and more affected cells in gray and black, respectively) to healthier cells. (B) Later, a second wave of death (yellow arrows) may be mediated by microglial cells overactivated by ATP and cytokines released by injured cells. (C) Still later inflammation-induced edema that reduces tissue perfusion could worsen the inflammatory response, recruiting leucocytes and increasing the extent of the lesion. [Modified from Orellana et al. 2009]

Stroke is a major cause of death in industrialized countries and results from a transient or permanent reduction in cerebral blood flow produced, in most cases, by cerebral artery occlusion by an embolus or local thrombosis, i.e., focal ischemia (Dirnagl et al. 1999). Severe and/or prolonged reduction in cerebral blood flow leads to deprivation of oxygen and glucose as well as build-up of potentially toxic substances. During stroke, decreased cellular oxygen levels, loss of oxidative phosphorylation and reduced ATP synthesis are the initial steps leading to cell death (Dirnagl et al. 1999). The ATP depletion may induce a rapid decrease in ATPase activity, leading to imbalanced electrochemical gradients across the plasma membrane. Notably, increased pannexin and/or connexin hemichannel activity occur in cortical astrocytes (Contreras et al. 2002; Orellana et al. 2011b; Orellana et al. 2010; Retamal et al. 2006), olygodendrocytes (Domercq et al. 2010) and hippocampal neurons (Lin et al. 2008; Schock et al. 2008; Thompson et al. 2006) subjected to ischemic like conditions. The enhanced hemichannel activity induced by ischemic like conditions accelerates cell death, at least in cultured rat astrocytes (Contreras et al. 2002; Orellana et al. 2010) (Fig. 5). Possibly, sustained hemichannel opening contribute to increased [Ca2+]i, which in turn may favour even more the connexin hemichannel activity (De Vuyst et al. 2007; Schalper et al. 2008), inducing Ca2+ and Na+ intracellular overload (Fig. 5). The ionic (or electrolyte) imbalance leads to an osmotic imbalance that results in cell swelling and plasma membrane phospholipase A2, with the subsequent generation of arachidonic acid and activation of

Fig. 5. Three mechanisms of death amplification. (A) Initially, a brain injury produced by ischemia, infection or necrosis affecting astrocytes (green), neurons (orange) or resting microglia (blue), could start a wave of death propagated (yellow arrows) and amplified through diffusible toxins and molecules (e.g., Ca2+, NO, superoxide ion, peroxinitrite, glutamate, and NAD+) present in high concentration in injured cells (depicted in the figure as dark colored cells). These molecules could be transferred through connexin gap junctions and connexin and pannexin hemichannels from injured cells (less and more affected cells in gray and black, respectively) to healthier cells. (B) Later, a second wave of death (yellow arrows) may be mediated by microglial cells overactivated by ATP and cytokines released by injured cells. (C) Still later inflammation-induced edema that reduces tissue perfusion could worsen the inflammatory response, recruiting leucocytes and increasing the extent of

the lesion. [Modified from Orellana et al. 2009]

**2.2 Stroke** 

breakdown as observed in astrocytes exposed to ischemic conditions (Kimelberg 2005) (Fig. 5). Calcium overload induced in part by hemichannel opening may also activate cyclooxygenase/lipoxygenase pathways leading to increased free radicals, lipid peroxidation and further plasma membrane damage. It is noteworthy that arachidonic acid and decrease in the intracellular redox potential also activates Cx43 hemichannels, which may exacerbate cell damage (De Vuyst et al. 2009; Retamal et al. 2007b).

The first *in vivo* evidence for the involvement of connexin-based channels in the spread of death signals came from experiments in which octanol, a non-selective gap junction and hemichannel blocker, reduced the infarct size after focal cerebral ischemia (Rawanduzy et al. 1997). However, these observations disagree with the findings obtained in heterozygous Cx43 knockout mice or mouse astrocytes lacking Cx43 expression, in which focal ischemia causes larger lesions (Nakase et al. 2003; Nakase et al. 2004; Siushansian et al. 2001). In a rat model of transient global ischemia, pretreatment with compounds that block both hemichannels and gap junction channels (i.e. CBX, α-GA and endothelin) reduces the number of apoptotic neurons as compared to the contralateral hippocampus treated with saline (Perez Velazquez et al. 2006). In addition, hemichannels present in surrounding cells or in the stromal component of diverse organs may also participate in the ischemic responses. For example, the CA1 region of Cx32- deficient mice show increased sensitivity to global ischemia (Oguro et al. 2001). Since Cx32 forms hemichannels in activated microglia (Takeuchi et al. 2006), it is possible that Cx32 knockout animals may show attenuated microglial release of regulatory paracrine signals. When acute diseases like stroke have the presence of other pro-inflammatory components (e.g. high glucose produced by diabetes mellitus), brain damage and cognitive functions in patients is worse (Pasquier et al. 2006). Indeed, it has long been known that hyperglycemia worsens the outcome of acute brain ischemia by increasing the extent of tissue injury in animals and humans (Kagansky et al. 2001). Recently, we showed in astrocytes that high levels of extracellular glucose increase hemichannel activity and decrease gap junction permeability induced by hypoxia (Orellana et al. 2010). These changes are transient after 3 hours of hypoxia in high glucose. However, they are more prominent after 6 hours of hypoxia and last for over 3 hours followed by death of numerous astrocytes (Orellana et al. 2010). Because high glucose worsens the effect on ischemia-induced cell damage in endothelial cells, neurons, and microglia (Kagansky et al. 2001; Lin et al. 1998a; Tsuruta et al. ; Wang et al. 2001), it would be of interest to study if elevated hemichannel activity plays a relevant role as in astrocytes. Importantly, we also demonstrated that microglia treated with amyloid β peptide (Aβ) potentiate the increase in astroglial hemichannel activity and reduction in gap junctional communication induced by hypoxia in high glucose, suggesting that these changes are a common denominator of inflamed or activated astrocytes (Orellana et al. 2011b) (Fig. 6).

In addition, the extracellular media of activated astrocytes was neurotoxic due to its glutamate and ATP content that activate neuronal Panx1 hemichannels via NMDA/P2X receptors leading to neuronal death (Orellana et al. 2011b). Therefore, in a more integrated system (e.g., brain or brain slices) neurons could be efficiently protected from ischemia and neurotoxicity by blocking NMDA and P2X receptors as already proposed (Dirnagl et al. 1999), but also by targeting either glial or neuronal hemichannels composed by Cx43 and Panx1, respectively.

Hemichannels may also be involved in tissue response to stroke through their participation in a phenomenon known as ischemic preconditioning, in which a sublethal ischemic insult induces resistance to a subsequent more severe insult (Gidday 2006). It was recently shown

Role of Connexin Hemichannels in Neurodegeneration 245

unknown. Final demonstration of the relative importance of enhanced hemichannel activity on cell viability during ischemia *in vivo* will require new approaches including better controlled experimental models and molecules that selectively block connexin- or pannexin-

Alzheimer's Disease (AD) is an age-related neurodegenerative disease that results in memory loss, behaviour and personality changes, among other symptoms. This disorder is characterized by the accumulation of the Aβ into amyloid plaques in the extracellular brain parenchyma, formation of tangles inside neurons as a result of abnormal phosphorylation of the microtubule associated protein tau, dendritic atrophy, and changes in neurotransmission in specific brain regions (Parihar and Hemnani 2004). Aβ is generated by proteolytic cleavage of the amyloid precursor protein (APP), which plays a role in neuronal adhesion, synaptogenesis, and axonal growth (Parihar and Hemnani 2004). High concentrations of Aβ are toxic to several neuronal types (Loo et al. 1993; Parihar and Hemnani 2004; Pike et al. 1995). The mechanisms underlying Aβ-neurotoxicity are complex but involve activation of NMDA receptors, sustained elevations of [Ca2+]i, and oxidative stress (Ekinci et al. 2000; Forloni et al. 1993), which are effects common to those induced by ischemia-reperfusion but

In addition to the above, the cerebral cortex of individuals with AD present activated microglia and astroglia closely associated with amyloid plaques (Kalaria 1999; Wisniewski and Wegiel 1991). Notably, increased Cx43 immunoreactivity is detected in about 80% of Aβ plaques in postmortem human samples from AD patients (Nagy et al. 1996). Accordingly, a recent study performed in a murine model of AD showed that the immunoreactivity for Cxs 30 and 43 is increased at the proximity of most Aβ plaques (Mei et al. 2010). Imbalance in brain homeostasis may explain the increase expression of Cxs 30 and 43 close to amyloid plaques as compensatory mechanism to ensure normal brain function (Mei et al. 2010)(Nagy et al. 1996). Alternatively, increased astroglial gap junctions may serve as a pathway for the propagation of neuronal damage, transferring death signals generated in the microenvironment of amyloid plaques to distant neurons, as death signals can propagate from C6 glioma cells injured with calcium ionophore (Lin et al. 1998b). In agreement with this interpretation, inhibition of gap junctions with octanol abolishes the ability of Aβ to enhance the velocity and extent of propagation of astroglial calcium waves (Haughey and Mattson 2003). However, octanol also blocks P2X7 receptors expressed by spinal astrocytes that also show calcium waves (Suadicani et al. 2006). In support of P2 receptor mediation of the Aβ-induced increase of calcium wave velocity in cortical astrocytes is the fact that suramin, a P2Y and P2X receptors blocker, reduces this response (Haughey and Mattson

Recently, it was shown that the treatment with the neurotoxic fragment of Aβ, 25-35 (Aβ25-35) increases hemichannel opening in microglia, astrocytes and neurons monitored by singlechannel recordings and by time-lapse ethidium uptake (Orellana et al. 2011c). The hemichannel forming proteins responsible of this activity were Cx43 and Panx1 in the case of microglia, Cx43 in the case of astrocytes and Panx1 and possibly Cx36 in neurons (Orellana et al. 2011c). Moreover, Aβ25-35 increased the surface level of Cx43 in microglia and astrocytes and for the first time it was detected an increase in surface Panx1 in Aβ25-35 treated microglia (Orellana et al. 2011c). Panx1 was also detected in astrocytes, but not at

based hemichannels.

**2.3 Alzheimer's Disease** 

on a different time scale.

2003).

Fig. 6. Involvement of extracellular signals released by inflamed glial cells in neuronal death. Activated microglia (by for example Aβ) release pro-inflammatory cytokines (e.g., TNF-α /IL-1β), which increase astroglial hemichannel activity when another pro-inflammatory agent is involved (e.g., hypoxia). Then, astrocytes release glutamate and ATP via Cx43 hemichannels, which can activate more microglia and could promote activation of neuronal NMDA and P2X receptors and further opening of Panx1 hemichannels in neurons. ATP released as a result of Panx1 hemichannel opening could contribute to the progression of neuronal death by a vicious cycle since it will activate more P2X receptors leading to more Ca2+ entry and activation of intracellular neurotoxic cascades. Moreover, dead neurons can activate more microglia and thus, can either restart or potentiate the toxic circuit. [Modified from Orellana et al. 2011b]

that preconditioning reduces degradation of Cx43 in astrocytes, leading to a marked increase in the amount of surface Cx43 hemichannels (Lin et al. 2008). In agreement with the possible involvement of hemichannels in preconditioning responses, Cx43 null mice are insensitive to hypoxic preconditioning whereas wild-type littermates mice exhibit a prominent reduction in infarct volume after induction of preconditioning through occlusion of the middle cerebral artery (Lin et al. 2008). The mechanism of neuroprotection in this model involves the release of ATP through Cx43 hemichannels to the extracellular milieu, where it becomes hydrolyzed to adenosine, a potent neuroprotective molecule. The involvement of Cx36 hemichannels in the preconditioning response of cultured cerebellar granule neurons has been also recently demonstrated (Schock et al. 2008). The possible involvement of pannexin based hemichannels in brain preconditioning responses remains unknown. Final demonstration of the relative importance of enhanced hemichannel activity on cell viability during ischemia *in vivo* will require new approaches including better controlled experimental models and molecules that selectively block connexin- or pannexinbased hemichannels.

#### **2.3 Alzheimer's Disease**

244 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

Fig. 6. Involvement of extracellular signals released by inflamed glial cells in neuronal death. Activated microglia (by for example Aβ) release pro-inflammatory cytokines (e.g., TNF-α /IL-1β), which increase astroglial hemichannel activity when another pro-inflammatory agent is involved (e.g., hypoxia). Then, astrocytes release glutamate and ATP via Cx43 hemichannels, which can activate more microglia and could promote activation of neuronal NMDA and P2X receptors and further opening of Panx1 hemichannels in neurons. ATP released as a result of Panx1 hemichannel opening could contribute to the progression of neuronal death by a vicious cycle since it will activate more P2X receptors leading to more Ca2+ entry and activation of intracellular neurotoxic cascades. Moreover, dead neurons can activate more microglia and thus, can either restart or potentiate the toxic circuit. [Modified

that preconditioning reduces degradation of Cx43 in astrocytes, leading to a marked increase in the amount of surface Cx43 hemichannels (Lin et al. 2008). In agreement with the possible involvement of hemichannels in preconditioning responses, Cx43 null mice are insensitive to hypoxic preconditioning whereas wild-type littermates mice exhibit a prominent reduction in infarct volume after induction of preconditioning through occlusion of the middle cerebral artery (Lin et al. 2008). The mechanism of neuroprotection in this model involves the release of ATP through Cx43 hemichannels to the extracellular milieu, where it becomes hydrolyzed to adenosine, a potent neuroprotective molecule. The involvement of Cx36 hemichannels in the preconditioning response of cultured cerebellar granule neurons has been also recently demonstrated (Schock et al. 2008). The possible involvement of pannexin based hemichannels in brain preconditioning responses remains

from Orellana et al. 2011b]

Alzheimer's Disease (AD) is an age-related neurodegenerative disease that results in memory loss, behaviour and personality changes, among other symptoms. This disorder is characterized by the accumulation of the Aβ into amyloid plaques in the extracellular brain parenchyma, formation of tangles inside neurons as a result of abnormal phosphorylation of the microtubule associated protein tau, dendritic atrophy, and changes in neurotransmission in specific brain regions (Parihar and Hemnani 2004). Aβ is generated by proteolytic cleavage of the amyloid precursor protein (APP), which plays a role in neuronal adhesion, synaptogenesis, and axonal growth (Parihar and Hemnani 2004). High concentrations of Aβ are toxic to several neuronal types (Loo et al. 1993; Parihar and Hemnani 2004; Pike et al. 1995). The mechanisms underlying Aβ-neurotoxicity are complex but involve activation of NMDA receptors, sustained elevations of [Ca2+]i, and oxidative stress (Ekinci et al. 2000; Forloni et al. 1993), which are effects common to those induced by ischemia-reperfusion but on a different time scale.

In addition to the above, the cerebral cortex of individuals with AD present activated microglia and astroglia closely associated with amyloid plaques (Kalaria 1999; Wisniewski and Wegiel 1991). Notably, increased Cx43 immunoreactivity is detected in about 80% of Aβ plaques in postmortem human samples from AD patients (Nagy et al. 1996). Accordingly, a recent study performed in a murine model of AD showed that the immunoreactivity for Cxs 30 and 43 is increased at the proximity of most Aβ plaques (Mei et al. 2010). Imbalance in brain homeostasis may explain the increase expression of Cxs 30 and 43 close to amyloid plaques as compensatory mechanism to ensure normal brain function (Mei et al. 2010)(Nagy et al. 1996). Alternatively, increased astroglial gap junctions may serve as a pathway for the propagation of neuronal damage, transferring death signals generated in the microenvironment of amyloid plaques to distant neurons, as death signals can propagate from C6 glioma cells injured with calcium ionophore (Lin et al. 1998b). In agreement with this interpretation, inhibition of gap junctions with octanol abolishes the ability of Aβ to enhance the velocity and extent of propagation of astroglial calcium waves (Haughey and Mattson 2003). However, octanol also blocks P2X7 receptors expressed by spinal astrocytes that also show calcium waves (Suadicani et al. 2006). In support of P2 receptor mediation of the Aβ-induced increase of calcium wave velocity in cortical astrocytes is the fact that suramin, a P2Y and P2X receptors blocker, reduces this response (Haughey and Mattson 2003).

Recently, it was shown that the treatment with the neurotoxic fragment of Aβ, 25-35 (Aβ25-35) increases hemichannel opening in microglia, astrocytes and neurons monitored by singlechannel recordings and by time-lapse ethidium uptake (Orellana et al. 2011c). The hemichannel forming proteins responsible of this activity were Cx43 and Panx1 in the case of microglia, Cx43 in the case of astrocytes and Panx1 and possibly Cx36 in neurons (Orellana et al. 2011c). Moreover, Aβ25-35 increased the surface level of Cx43 in microglia and astrocytes and for the first time it was detected an increase in surface Panx1 in Aβ25-35 treated microglia (Orellana et al. 2011c). Panx1 was also detected in astrocytes, but not at

Role of Connexin Hemichannels in Neurodegeneration 247

Moreover, these two mechanisms could also be linked since transient activation of NMDA receptors induced a nonselective cationic current that develops slowly and mediates Ca2+ influx directly linked to neuronal death. Interestingly, this secondary current was reported recently to be mediated by neuronal Panx1 hemichannels (Thompson et al. 2008). Moreover, activation of Panx1 hemichannels might be triggered by protein–protein interaction with activated P2 receptors (Iglesias et al. 2008). Alternatively, Panx1 hemichannels could be activated by the rise in [Ca2+]i caused by opening of NMDA and P2X receptors (Locovei et al. 2006). In this mechanism, neuronal ATP released as a result of Panx1 hemichannel opening is also likely to contribute in the progression of neuronal death by a vicious cycle since it will activate more ionotropic P2 receptors enhancing the Ca2+ entry and activation of intracellular neurotoxic cascades (Fig. 7). Studies in AD models will help to confirm or reject

In the last decade, connexin hemichannels have been implicated in paracrine and autocrine cellular communication in several normal and pathologic conditions (Sáez et al. 2010). Currently, most of the available data regarding hemichannel involvement in brain pathologic events are from cultured cells and animal models of diseases. However, the current knowledge of human brain disease processes and the documented presence of hemichannels forming proteins in most studied human CNS tissues allows to speculate about the involvement of hemichannels in neurodegenerative processes. The role of glial cells in mediating nervous tissue inflammation has been recognized previously as leading to neuronal death; these cells can be bad neighbours for neurons (Block et al. 2007). Dysfunction of astroglial and microglial hemichannels, as well as gap junction channels, are likely mechanisms commonly elicited in all brain diseases associated with inflammatory responses. Therefore, normalization of connexin- and pannexin-based channel dysfunctions should confer tissue protection, improve quality of life, and extend survival of patients suffering acute or chronic brain inflammatory responses. Thus, it is proposed that chronic or acute processes of neurodegeneration might be prevented by blocking glial and neuronal hemichannels. Prevention might also be accomplished by reducing the effects of soluble factors (i.e., glutamate, ATP, prostaglandins, and cytokines) accumulated in the

This work was partially supported by the CONICYT 24080055 (to JAO); CONICYT 79090028 (to JAO); CRPCEN (to CG); INSERM (to CG); FONDECYT 1070591 (to JCS); FONDEF DO7I1086 (to JCS); ANILLO ACT-71 (to JCS) and ECOS/CONICYT C10S01 (to CG and JCS)

Bannerman DD, Fitzpatrick MJ, Anderson DY, Bhattacharjee AK, Novitsky TJ, Hasday JD,

Cross AS, Goldblum SE. 1998. Endotoxin-neutralizing protein protects against endotoxin-induced endothelial barrier dysfunction. Infect Immun 66(4):1400-7.

the above interpretations.

microenvironment of the inflamed CNS.

**4. Acknowledgment** 

grants.

**5. References** 

**3. Conclusions** 

their surface, either in the absence or presence of Aβ25-35, which is in disagreement with a recent study in which astroglial Panx1 hemichannels were activated by extracellular ATP (Iglesias et al. 2009). Importantly, conditioned media harvested either from Aβ25-35-treated microglia or astrocytes, increased neuronal ethidium uptake and mortality, an effect prevented by inhibitors of P2X/NMDA receptors and Panx1 hemichannels, indicating that ATP and glutamate contribute to these changes (Orellana et al. 2011c). The contribution of these two molecules in neurotoxicity is well known (Lipton and Rosenberg 1994) and the involvement of hemichannels in glutamate and ATP release has also been documented

Fig. 7. Model of Aβ-induced cascade resulting in glial and neuronal hemichannel activation that leads to neuronal death. Microglia exposed to the amyloid β (Aβ) peptide become first activated (1), enhancing the opening of Cx43 and Panx1 hemichannels. Under these conditions, they release pro-inflammatory cytokines (2) that contribute to the Aβ-induced Cx43 hemichannel opening in astrocytes (3). Activated microglia might release glutamate and ATP through hemichannels (4), while astrocytes could release the same molecules through Cx43 hemichannels (5). This gliotransmission activates neuronal purinergic and NMDA receptors, resulting in an elevation of the intracellular free Ca2+ concentration that might trigger massive Cx36 and Panx1 hemichannel opening and further neuronal death (6). [Modified from Orellana et al. 2011c]

(Iglesias et al. 2009; Jiang et al.; Kang et al. 2008; Orellana et al. 2011b; Orellana et al. 2011c; Takeuchi et al. 2006; Ye et al. 2003). Moreover, these two molecules were shown to induce neuronal death via activation of Panx1 hemichannels in neurons (Orellana et al. 2011b). Their effect on neuronal death may proceed according to at least two mechanisms: 1) through the stimulation of NMDA and/or P2X receptors or 2) through Cx36 and Panx1 hemichannels themselves that could evoke large Ca2+ influxes resulting in neuronal death (Orellana et al. 2009) (Fig. 7).

Moreover, these two mechanisms could also be linked since transient activation of NMDA receptors induced a nonselective cationic current that develops slowly and mediates Ca2+ influx directly linked to neuronal death. Interestingly, this secondary current was reported recently to be mediated by neuronal Panx1 hemichannels (Thompson et al. 2008). Moreover, activation of Panx1 hemichannels might be triggered by protein–protein interaction with activated P2 receptors (Iglesias et al. 2008). Alternatively, Panx1 hemichannels could be activated by the rise in [Ca2+]i caused by opening of NMDA and P2X receptors (Locovei et al. 2006). In this mechanism, neuronal ATP released as a result of Panx1 hemichannel opening is also likely to contribute in the progression of neuronal death by a vicious cycle since it will activate more ionotropic P2 receptors enhancing the Ca2+ entry and activation of intracellular neurotoxic cascades (Fig. 7). Studies in AD models will help to confirm or reject the above interpretations.

#### **3. Conclusions**

246 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

their surface, either in the absence or presence of Aβ25-35, which is in disagreement with a recent study in which astroglial Panx1 hemichannels were activated by extracellular ATP (Iglesias et al. 2009). Importantly, conditioned media harvested either from Aβ25-35-treated microglia or astrocytes, increased neuronal ethidium uptake and mortality, an effect prevented by inhibitors of P2X/NMDA receptors and Panx1 hemichannels, indicating that ATP and glutamate contribute to these changes (Orellana et al. 2011c). The contribution of these two molecules in neurotoxicity is well known (Lipton and Rosenberg 1994) and the involvement of hemichannels in glutamate and ATP release has also been documented

Fig. 7. Model of Aβ-induced cascade resulting in glial and neuronal hemichannel activation that leads to neuronal death. Microglia exposed to the amyloid β (Aβ) peptide become first activated (1), enhancing the opening of Cx43 and Panx1 hemichannels. Under these conditions, they release pro-inflammatory cytokines (2) that contribute to the Aβ-induced Cx43 hemichannel opening in astrocytes (3). Activated microglia might release glutamate and ATP through hemichannels (4), while astrocytes could release the same molecules through Cx43 hemichannels (5). This gliotransmission activates neuronal purinergic and NMDA receptors, resulting in an elevation of the intracellular free Ca2+ concentration that might trigger massive Cx36 and Panx1 hemichannel opening and further neuronal death (6).

(Iglesias et al. 2009; Jiang et al.; Kang et al. 2008; Orellana et al. 2011b; Orellana et al. 2011c; Takeuchi et al. 2006; Ye et al. 2003). Moreover, these two molecules were shown to induce neuronal death via activation of Panx1 hemichannels in neurons (Orellana et al. 2011b). Their effect on neuronal death may proceed according to at least two mechanisms: 1) through the stimulation of NMDA and/or P2X receptors or 2) through Cx36 and Panx1 hemichannels themselves that could evoke large Ca2+ influxes resulting in neuronal death

[Modified from Orellana et al. 2011c]

(Orellana et al. 2009) (Fig. 7).

In the last decade, connexin hemichannels have been implicated in paracrine and autocrine cellular communication in several normal and pathologic conditions (Sáez et al. 2010). Currently, most of the available data regarding hemichannel involvement in brain pathologic events are from cultured cells and animal models of diseases. However, the current knowledge of human brain disease processes and the documented presence of hemichannels forming proteins in most studied human CNS tissues allows to speculate about the involvement of hemichannels in neurodegenerative processes. The role of glial cells in mediating nervous tissue inflammation has been recognized previously as leading to neuronal death; these cells can be bad neighbours for neurons (Block et al. 2007). Dysfunction of astroglial and microglial hemichannels, as well as gap junction channels, are likely mechanisms commonly elicited in all brain diseases associated with inflammatory responses. Therefore, normalization of connexin- and pannexin-based channel dysfunctions should confer tissue protection, improve quality of life, and extend survival of patients suffering acute or chronic brain inflammatory responses. Thus, it is proposed that chronic or acute processes of neurodegeneration might be prevented by blocking glial and neuronal hemichannels. Prevention might also be accomplished by reducing the effects of soluble factors (i.e., glutamate, ATP, prostaglandins, and cytokines) accumulated in the microenvironment of the inflamed CNS.

#### **4. Acknowledgment**

This work was partially supported by the CONICYT 24080055 (to JAO); CONICYT 79090028 (to JAO); CRPCEN (to CG); INSERM (to CG); FONDECYT 1070591 (to JCS); FONDEF DO7I1086 (to JCS); ANILLO ACT-71 (to JCS) and ECOS/CONICYT C10S01 (to CG and JCS) grants.

#### **5. References**

Bannerman DD, Fitzpatrick MJ, Anderson DY, Bhattacharjee AK, Novitsky TJ, Hasday JD, Cross AS, Goldblum SE. 1998. Endotoxin-neutralizing protein protects against endotoxin-induced endothelial barrier dysfunction. Infect Immun 66(4):1400-7.

Role of Connexin Hemichannels in Neurodegeneration 249

Ekinci FJ, Linsley MD, Shea TB. 2000. Beta-amyloid-induced calcium influx induces

Eugenín EA, Branes MC, Berman JW, Sáez JC. 2003. TNF-alpha plus IFN-gamma induce

Eugenín EA, Eckardt D, Theis M, Willecke K, Bennett MV, Sáez JC. 2001. Microglia at brain

Forloni G, Chiesa R, Smiroldo S, Verga L, Salmona M, Tagliavini F, Angeretti N. 1993.

Freyer D, Manz R, Ziegenhorn A, Weih M, Angstwurm K, Docke WD, Meisel A, Schumann

Froger N, Orellana JA, Calvo CF, Amigou E, Kozoriz MG, Naus CC, Saez JC, Giaume C.

Froger N, Orellana JA, Cohen-Salmon M, Ezan P, Amigou E, Saez JC, Giaume C. 2009.

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**Roles of Glial Cells in Neurodegeneration** 

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