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## **Meet the editor**

Ana-María Fernández-Escamilla is a senior researcher at Spanish National Research Council. In 2001, she received her PhD in Biophysical Chemistry at the University of Granada, Spain. She developed her career in prestigious Spanish and international research centers as European Molecular Biology Laboratory (Germany), funded by National and European competing grants.

She participated in the development and testing of the computer algorithm "TANGO", designed to predict protein aggregation propensity. Currently, she develops as PI two R&D projects funded by public bodies and one biotech-company research contract. In 2014, she got positive assessment of research excellence by the Spanish National Evaluation and Foresight Agency. Her scientific research is focused on protein engineering combining theoretical and experimental approaches for biochemical, biophysical, and structural characterization of macromolecules.

## Contents

## **Preface XI**



## Preface

Chapter 8 **Amyloidogenesis and Responses to Stress 203**

Chapter 9 **Developments in the Treatment of Amyloid A Amyloidosis Secondary to Rheumatoid Arthritis 217**

Chapter 10 **The Role of Human IAPP in Stress and Inflammatory Processes**

Chapter 12 **Amyloid Nephropathy: A Practical Diagnostic Approach and**

Chapter 13 **A Nanobody‐Based Approach to Amyloid Diseases, the Gelsolin**

Chapter 11 **Proteasome Inhibitors to Treat AL Amyloidosis 267** James J. Driscoll and Saulius Girnius

**Review on Pathogenesis 281**

Adriaan Verhelle and Jan Gettemans

Pulze and Gianluca Tettamanti

Tadashi Nakamura

**VI** Contents

Paisit Paueksakon

**Case Study 313**

**in Type 2 Diabetes 245** Joel Montane and Anna Novials

Magda de Eguileor, Rossana Girardello, Annalisa Grimaldi, Laura

Amyloid protein aggregates are involved in a number of "protein-misfolding diseases" of enormous social and economic impact for which there are still no effective therapies or strat‐ egies for their prevention or inhibition available. There is a heterogeneous group of amyloi‐ dogenic proteins associated with amyloid pathologies including amyloid-beta (Aβ) peptide linked to the most common type of dementia, Alzheimer's disease (AD); prion protein (PrP) related to spongiform encephalopathies; islet amyloid polypeptide (IAPP) connected to dia‐ betes type II; transthyretin (TTR) linked to familial amyloidotic polyneuropathy (FAP) that often affects liver, nerves, heart, and kidneys; serum amyloid A protein (SAA) associated with AA amyloidosis that mostly affects kidneys; immunoglobulin light-chain (AL) amyloi‐ dosis that affects heart, kidneys, skin, nerves, and liver; and dialysis-related amyloidosis that develops when serum proteins in the blood (β2-microglobulin) are deposited in the joints and tendons.

One of the most recent biophysical definitions describes amyloids as elongated unbranched fibrils comprising a repeating intermolecular β-sheet motifs positioned perpendicular to the fibril axis. The resulting insoluble fibrillar structure is a highly ordered cross-beta structure that in the majority of the cases seems to be helically twisted. Amyloid-related diseases or amyloidosis are characterized by extracellular deposition of misfolded proteins in different organs, forming insoluble amyloid aggregates. Most of these protein aggregates lose their normal function and lead to cell damage, disruption of the tissue architecture, and organ dysfunction and death. Even though amyloid fibers have been extensively studied, current insights implicate prefibrillar intermediates rather than mature amyloid fibers in causing cell death.

Several events may trigger amyloid formation, such as protein mutations, unusual enzymat‐ ic cleavage patterns, and posttranslational modifications. However, not all amyloid fibers play a detrimental role in the host. An increasing number of studies show how amyloids also play an important role as beneficial (nontoxic) amyloid fibers termed "functional amy‐ loids" (FAs). FAs are related to the formation of buds of insects, spider silk, coating fungal hyphae, bacterial host adhesion, bacterial biofilm formation, melanin synthesis, and human hemostatic system, whose molecular and functional mechanisms still remain undiscovered. Evidently, functional amylogenesis mechanisms require a strong regulation to avoid toxici‐ ty. In addition, I would like to highlight two recent studies. The first very interesting one by Camilla Betti (May 2016) describes the potential biotechnological application of induced tar‐ get aggregation, a method based on expression of specific aggregation-prone peptides de‐ rived from the targeted proteins, as a very useful tool to knockdown protein functions in plants to produce favorable features in crops. The second study by Rubén Hervás et al. (Jan 2016) demonstrates that a functional amyloid (RNA-binding protein Orb2 of *Drosophila* ) shares a wide group of features with toxic amyloids, including conversion to a structurally characteristic insoluble fibrous deposits via toxic oligomeric intermediates, which can be se‐ questered in vivo in hetero-oligomers by pathological amyloids. Furthermore, the toxic in‐ termediates of this protein are extremely transient, corroborating the above-mentioned requirement for the strict regulation of the kinetic parameters during the amyloid formation process to differentiate this functional amyloid from pathological amyloids.

This book opens an exciting door to provide the latest information about the function and the mechanisms of the amyloid formation process from the structural, biophysical, biomedi‐ cal, and nanotechnological perspective, combining the new findings on toxic and functional amyloids studies using the new technological advances in electron microscopy (EM), atomic force microscopy (AFM), and positron emission tomography (PET) among others, to fight against amyloid-based diseases.

When I was reviewing all the chapters, I decided to start the book with a clear and general perspective on amyloids. Chapter 1 by Isabel Cardoso, Luis Miguel Santos, and Mobina Ale‐ mi is written in a very accessible, comprehensive, and educational way that fulfills all the requirements for an introductory chapter. This chapter describes Alzheimer's disease (AD) as a form of localized amyloidosis that affects the central nervous system focusing on the neuroprotective role of transthyretin (TTR) and its amyloidogenic role in Familial Amyloid Polyneuropathy (FAP).

The next chapters deal with the progress in technique used to obtain structural and kinetic information on amyloid fibrils. Chapter 2 by Dmitry Kurouski aims to demonstrate the ap‐ plicability of electron microscopy (EM) including transmission electron microscopy (TEM), scanning transmission electron microscopy (STEM), scanning electron microscopy (SEM), and cryo-SEM, and also atomic force microscopy (AFM) and vibrational circular dichroism (VCD) to advance the understanding of supramolecular architecture and the organization of amyloid fibrils and fibril polymorphism. Chapter 3 by Wonseok Lee, Hyungbeen Lee, Gyu‐ do Lee, and Dae Sung Yoon describes the advances on alternative techniques, such as AFM and its extensions (dynamic liquid AFM; PeakForce quantitative nanomechanical mapping (QNM) and Kelvin probe force microscopy (KFPM)), as excellent methodologies to deter‐ mine the biophysical properties of amyloid fibrils and the possible perspectives and future directions for the characterization of amyloid fibrils. Positron emission tomography (PET) imaging is an interesting technique for in vivo experiments. Chapter 4 by Hideo Tsukada describes PET as an excellent technique for the diagnosis of Alzheimer's disease using 18F-BCPP-EF—a novel PET probe for mitochondrial complex I (MC-I) activity—which could discriminate the neuronal-damaged areas with neuroinflammation, in young and aged mon‐ keys demonstrating the normal aging effects on MC-I activity in the brain.

I consider crucial for the current research the combination of theoretical and experimental approaches to develop any scientific area. This methodology is described in Chapter 5 writ‐ ten by Oxana V. Galzitskaya, Nikita V. Dovidchenko, and Olga M. Selivanova who use theo‐ retical and experimental approaches to describe possible kinetic models to elucidate the key features of amyloid nucleation and growth. Concretely, this chapter reviews the common features of the amyloid formation process by several proteins which have their own specific fibrillogenesis route determined by their amino acid sequence protein and strongly depend‐ ent on environmental conditions and seed addition.

As mentioned above, one of the possible triggers to induce amyloid aggregation is the post‐ translational modifications (PTMs) that affect protein structure and function. Chapter 6 by Clara Iannuzzi, Gaetano Irace, and Ivana Sirangelo is focused on one of the more frequent PTMs, protein glycation, and its determinant role on amyloid aggregation process and its implication on cellular cytotoxicity.

shares a wide group of features with toxic amyloids, including conversion to a structurally characteristic insoluble fibrous deposits via toxic oligomeric intermediates, which can be se‐ questered in vivo in hetero-oligomers by pathological amyloids. Furthermore, the toxic in‐ termediates of this protein are extremely transient, corroborating the above-mentioned requirement for the strict regulation of the kinetic parameters during the amyloid formation

This book opens an exciting door to provide the latest information about the function and the mechanisms of the amyloid formation process from the structural, biophysical, biomedi‐ cal, and nanotechnological perspective, combining the new findings on toxic and functional amyloids studies using the new technological advances in electron microscopy (EM), atomic force microscopy (AFM), and positron emission tomography (PET) among others, to fight

When I was reviewing all the chapters, I decided to start the book with a clear and general perspective on amyloids. Chapter 1 by Isabel Cardoso, Luis Miguel Santos, and Mobina Ale‐ mi is written in a very accessible, comprehensive, and educational way that fulfills all the requirements for an introductory chapter. This chapter describes Alzheimer's disease (AD) as a form of localized amyloidosis that affects the central nervous system focusing on the neuroprotective role of transthyretin (TTR) and its amyloidogenic role in Familial Amyloid

The next chapters deal with the progress in technique used to obtain structural and kinetic information on amyloid fibrils. Chapter 2 by Dmitry Kurouski aims to demonstrate the ap‐ plicability of electron microscopy (EM) including transmission electron microscopy (TEM), scanning transmission electron microscopy (STEM), scanning electron microscopy (SEM), and cryo-SEM, and also atomic force microscopy (AFM) and vibrational circular dichroism (VCD) to advance the understanding of supramolecular architecture and the organization of amyloid fibrils and fibril polymorphism. Chapter 3 by Wonseok Lee, Hyungbeen Lee, Gyu‐ do Lee, and Dae Sung Yoon describes the advances on alternative techniques, such as AFM and its extensions (dynamic liquid AFM; PeakForce quantitative nanomechanical mapping (QNM) and Kelvin probe force microscopy (KFPM)), as excellent methodologies to deter‐ mine the biophysical properties of amyloid fibrils and the possible perspectives and future directions for the characterization of amyloid fibrils. Positron emission tomography (PET) imaging is an interesting technique for in vivo experiments. Chapter 4 by Hideo Tsukada describes PET as an excellent technique for the diagnosis of Alzheimer's disease using 18F-BCPP-EF—a novel PET probe for mitochondrial complex I (MC-I) activity—which could discriminate the neuronal-damaged areas with neuroinflammation, in young and aged mon‐

keys demonstrating the normal aging effects on MC-I activity in the brain.

ent on environmental conditions and seed addition.

I consider crucial for the current research the combination of theoretical and experimental approaches to develop any scientific area. This methodology is described in Chapter 5 writ‐ ten by Oxana V. Galzitskaya, Nikita V. Dovidchenko, and Olga M. Selivanova who use theo‐ retical and experimental approaches to describe possible kinetic models to elucidate the key features of amyloid nucleation and growth. Concretely, this chapter reviews the common features of the amyloid formation process by several proteins which have their own specific fibrillogenesis route determined by their amino acid sequence protein and strongly depend‐

process to differentiate this functional amyloid from pathological amyloids.

against amyloid-based diseases.

Polyneuropathy (FAP).

VIII Preface

Currently, there are ample evidence that associate the inflammatory response to many neu‐ rodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and multiple sclerosis. Chapter 7 by Ruth Kandel, Mitchell R. White, and Kevan L. Hartshorn shows how chronic inflammation in the brain is fundamental to AD pathogenesis which is precipitated through accumulation of amyloid-beta (Aβ) peptides. They suggest that the discovery of the neuroinflammation stimulus for Aβ production would allow early intervention to prevent development of AD.

The majority of the studies includes serious health connotations on amyloids due to clear relation with human pathologies but beyond of amyloid toxicity; each day the studies on amyloid functionality are getting force being "functional amyloids" a term well established. Chapter 8 by Magda de Eguileor, Rossana Girardello, Annalisa Grimaldi, Laura Pulze, and Gianluca Tettamanti describes "amyloidogenesis" as a widely distributed process in differ‐ ent cell types of evolutionary distant organisms that it has been integrated into additional physiological functions such as the reparation of body lesion.

The most prevalent amyloid pathologies are related to neurodegenerative diseases as Alz‐ heimer's disease and Parkinson's disease, among others, but there are many amyloidoses which affect other organs: liver, nerves, heart, kidneys, joints, and tendons. Chapter 9 by Tadashi Nakamura is focused on "amyloid A (AA) amyloidosis", a complication of longstanding inflammatory diseases such as rheumatoid arthritis (RA). He also describes the new developments on its treatment, being currently under investigation, the novel therapies oriented to AA fibril formation and immunotherapy. These therapies will lead to improved prognosis in the near future. Another relevant amyloidogenic pathology is the "diabetes type II". Chapter 10 by Joel Montane and Anna Novials describes the capability of human islet amyloid polypeptide (hIAPP) to induce endoplasmic reticulum (ER) stress, resulting in an inflammatory processes and eventually apoptosis. Light-chain (AL) amyloidosis is the most common form of systemic amyloidosis (nonlocalized), affecting a wide range of or‐ gans, most commonly the kidneys. Chapter 11 by James J. Driscoll and Saulius Girnius shows the treatment advances and the novel therapeutic strategies against "AL amyloido‐ sis" mainly focused on the development of proteasome inhibitors. Another frequent system‐ ic amyloidosis or nonlocalized amyloidosis is the "amyloid nephropathy". Chapter 12 by Paisit Paueksakon discusses practical diagnostic approach and pathogenesis of amyloid nephropathy including discussion of treatment and prognosis. It also reviews several inter‐ esting techniques in typing of this amyloidosis as immunofluorescence, immunohistochem‐ istry, and more recently laser microdissection and mass spectrometry.

From the new nanotechnology point of view, the amyloids are very interesting structures for nano-bio-technological applications, being this technology in turn very useful to obtain new insights on amyloidosis. Chapter 13 by Adriaan Verhelle and Jan Gettemans proposes the use of nanobodies (the heavy chain of heavy-chain antibodies) as possible inhibitors on the formation of the pathogenic gelsolin isoforms.

Finally, I would like to thank all authors for their significant contributions to the 13 chapters of this book and I greatly appreciate their work performed to orient this book not only to the experts in the field but also to those scientists, students and readers who, without any previ‐ ous knowledge on the subject and in an easy way can get a general up-to-date overview on amyloids. I also thank InTech editorial team, especially Ms. Iva Simcic, for her efficient and professional help given to me. All of you have made my assignment as book editor enor‐ mously pleasant and educational as well. I am very grateful to Dr. J.C. Jiménez-López for suggesting me to be editor of this book. My warm thanks to my colleagues Joost Schymko‐ witz and Frederic Rousseau, well-known experts on amyloids, for reading this preface.

#### **Ana-María Fernández-Escamilla, PhD**

Environmental Protection Department, Estación Experimental del Zaidín (EEZ), Spanish National Research Council (CSIC), Granada, Spain

## **Modulating Role of TTR in Aβ Toxicity, from Health to Disease**

Isabel Cardoso, Luis Miguel Santos and Mobina Alemi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63194

#### **Abstract**

Finally, I would like to thank all authors for their significant contributions to the 13 chapters of this book and I greatly appreciate their work performed to orient this book not only to the experts in the field but also to those scientists, students and readers who, without any previ‐ ous knowledge on the subject and in an easy way can get a general up-to-date overview on amyloids. I also thank InTech editorial team, especially Ms. Iva Simcic, for her efficient and professional help given to me. All of you have made my assignment as book editor enor‐ mously pleasant and educational as well. I am very grateful to Dr. J.C. Jiménez-López for suggesting me to be editor of this book. My warm thanks to my colleagues Joost Schymko‐ witz and Frederic Rousseau, well-known experts on amyloids, for reading this preface.

X Preface

**Ana-María Fernández-Escamilla, PhD** Environmental Protection Department, Estación Experimental del Zaidín (EEZ), Spanish National Research Council (CSIC),

Granada, Spain

Amyloidosis is a generic term that refers to a wide spectrum of diseases that are characterized by the deposition of proteins in different organs, forming insoluble aggregates. Examples include islet amyloid polypeptide (IAPP) associated with diabetes type 2, prion protein (PrP) related with spongiform encephalopathies, (TTR) associated with familial amyloidotic polyneuropathy (FAP), and amyloid-beta (Aβ) peptide linked to Alzheimer's disease (AD), the most common form of dementia. Aβ peptide, thought to be the causative agent in AD, is generated upon sequential cleavage of the amyloid precursor protein (APP), by beta- and gamma-secretases, and it is believed that an imbalance between Aβ production and clearance results in its accumulation in the brain. TTR is a 55 kDa homotetrameric protein synthesized by the liver and choroid plexus of the brain and is involved in the transport of thyroid hormones and retinol. TTR protects against Aβ toxicity by binding the peptide, thus inhibiting its aggregation. Also, increased Aβ levels are found in both brain and plasma of AD mice with only one copy of the TTR gene, when compared to animals with two copies of the gene, suggesting a role for TTR in Aβ clearance. Growing evidence also suggests a wider role for TTR in central nervous system neuroprotection, including in the cases of ischemia, regeneration, and memory.

**Keywords:** Alzheimer's disease (AD), Aβ peptide, transthyretin (TTR), neuroprotec‐ tion, blood–brain barrier (BBB)

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Amyloidosis**

## **1.1. Amyloidosis definition**

Amyloidosis has long been used as a general term referring to a wide spectrum of proteinmisfolding diseases [1], which are characterized by the extracellular deposition of those proteins in different organs, consequently forming insoluble aggregates called amyloid, a term popularized by Virchow in 1854 [2]. According to the Nomenclature Committee of the International Society of Amyloidosis (ISA), 31 identified proteins form extracellular amyloid fibrils in humans [3]. Amyloid fibrils are characterized by certain tinctorial properties, independently of the precursor protein forming the deposits, that for a long time were the only diagnosis available. These include apple-green birefringence under polarized light after staining with Congo red and yellow-green fluorescence after staining with thioflavin S and thioflavin T; thioflavin T has also been shown to interact with amyloid in suspension produc‐ ing a specific fluorescent signal with a new excitation maximum at 450 nm. Ultrastructural studies by transmission electron microscopy (TEM) revealed that the amyloid material is fibrillary appearing as bundles of straight or coiled fibrils, non-branched, 7–10 nm wide and variable in length; in most cases, they seem to be helically twisted. Amyloid fibrils present a high content in β-pleated sheet as demonstrated by x-ray diffraction analysis and extensive antiparallel β-sheet strands with their axes running perpendicularly to the axis of the growing fibril (cross-β pattern).

Amyloid deposits are not entirely composed of the amyloid precursor protein. Several components have been found associated with all amyloid fibrils. These include serum amyloid P component (SAP), sulphonated glycosaminoglycans (GAGs), apoliproteins E and J, α1 antichymiotrypsin, several basement membrane components such as fibronectin, laminin and collagen type IV, complement proteins, and metal ions.

The extracellular deposition of fibrillary proteins leads to cell damage, organ dysfunction and death, and thus, these proteins are associated with a unique clinical syndrome, as seen in the case of the islet amyloid polypeptide (IAPP) associated with diabetes type 2 [4], prion protein (PrP) associated with the spongiform encephalopathies [5], transthyretin (TTR) associated with familial amyloidotic polyneuropathy [6] (FAP), and amyloid-beta (Aβ) peptide associated with Alzheimer's disease [7] (AD), among others.

Amyloid disorders are usually divided into two categories depending on the distribution of the amyloid deposits: localized and systemic amyloidosis. In localized amyloidosis, amyloid is restricted to a single tissue or organ, usually in the surroundings of the cells responsible for the synthesis of the precursor protein; in systemic amyloidosis, the amyloidogenic proteins are usually derived from circulating precursors that are either in excess, abnormal or both. Amyloidosis can also be hereditary or non-hereditary.

This chapter will focus in AD, a form of localized amyloidosis affecting the central nervous system, and the most common form of dementia. In particularly, we will discuss the neuro‐ protective role of TTR in AD, in addition to its amyloidogenic role in FAP, an example of systemic amyloidosis with a special involvement of the peripheral nerve.

## **2. Overview of AD**

**1. Amyloidosis**

**1.1. Amyloidosis definition**

2 Exploring New Findings on Amyloidosis

fibril (cross-β pattern).

collagen type IV, complement proteins, and metal ions.

Amyloidosis can also be hereditary or non-hereditary.

Alzheimer's disease [7] (AD), among others.

Amyloidosis has long been used as a general term referring to a wide spectrum of proteinmisfolding diseases [1], which are characterized by the extracellular deposition of those proteins in different organs, consequently forming insoluble aggregates called amyloid, a term popularized by Virchow in 1854 [2]. According to the Nomenclature Committee of the International Society of Amyloidosis (ISA), 31 identified proteins form extracellular amyloid fibrils in humans [3]. Amyloid fibrils are characterized by certain tinctorial properties, independently of the precursor protein forming the deposits, that for a long time were the only diagnosis available. These include apple-green birefringence under polarized light after staining with Congo red and yellow-green fluorescence after staining with thioflavin S and thioflavin T; thioflavin T has also been shown to interact with amyloid in suspension produc‐ ing a specific fluorescent signal with a new excitation maximum at 450 nm. Ultrastructural studies by transmission electron microscopy (TEM) revealed that the amyloid material is fibrillary appearing as bundles of straight or coiled fibrils, non-branched, 7–10 nm wide and variable in length; in most cases, they seem to be helically twisted. Amyloid fibrils present a high content in β-pleated sheet as demonstrated by x-ray diffraction analysis and extensive antiparallel β-sheet strands with their axes running perpendicularly to the axis of the growing

Amyloid deposits are not entirely composed of the amyloid precursor protein. Several components have been found associated with all amyloid fibrils. These include serum amyloid P component (SAP), sulphonated glycosaminoglycans (GAGs), apoliproteins E and J, α1 antichymiotrypsin, several basement membrane components such as fibronectin, laminin and

The extracellular deposition of fibrillary proteins leads to cell damage, organ dysfunction and death, and thus, these proteins are associated with a unique clinical syndrome, as seen in the case of the islet amyloid polypeptide (IAPP) associated with diabetes type 2 [4], prion protein (PrP) associated with the spongiform encephalopathies [5], transthyretin (TTR) associated with familial amyloidotic polyneuropathy [6] (FAP), and amyloid-beta (Aβ) peptide associated with

Amyloid disorders are usually divided into two categories depending on the distribution of the amyloid deposits: localized and systemic amyloidosis. In localized amyloidosis, amyloid is restricted to a single tissue or organ, usually in the surroundings of the cells responsible for the synthesis of the precursor protein; in systemic amyloidosis, the amyloidogenic proteins are usually derived from circulating precursors that are either in excess, abnormal or both.

This chapter will focus in AD, a form of localized amyloidosis affecting the central nervous system, and the most common form of dementia. In particularly, we will discuss the neuro‐ protective role of TTR in AD, in addition to its amyloidogenic role in FAP, an example of

systemic amyloidosis with a special involvement of the peripheral nerve.

AD was firstly described by Alois Alzheimer in 1906 and is characterized by progressive loss of cognitive functions, ultimately leading to death [8]. This condition highly affects not only the life of patients but also the life of their caregivers. Pathologically, the disease is character‐ ized by the presence of extraneuronal amyloid plaques consisting of aggregates of the Aβ peptide, and neurofibrillary tangles (NFTs) which are intracellular aggregates of abnormally hyperphosphorylated tau protein [9]. Aβ peptide is generated upon sequential cleavage of the amyloid precursor protein (APP), by beta- and gamma-secretases, and it is believed that an imbalance between Aβ production and clearance results in its accumulation in the brain.

## **2.1. From the first description to the confirmation**

Alzheimer's disease was first described in the 1907's paper entitled "Uber eine eigenartige Erkankung der Hirnrinde," by Alois Alzheimer [10], in which he reported the behavior of a 51-year-old female patient (Auguste Deter) of the insane asylum of Frankfurt am Main. The patient presented several symptoms that caught Alzheimer's attention, apart from the central nervous system anatomical characteristics. Among others, time and space disorientation, rapid loss of memory, and mood swings were the most prominent symptoms [10]. In relation to pathological features, the observation of something that looked like "thick bundles" [10] of fibrils, later known as senile/amyloid plaques and NFTs [11], transformed AD into a unique condition, distinguishing it from the other neurological conditions known to date.

## *2.1.1. Symptoms and Diagnosis*

Since 1907, clinicians have been trying their best to accurately identify AD-related symptoms and to divide and organize these symptoms in the simplest form. Burns et al. came out in 2002 with three different categories: (1) cognitive deficits that affect memory (amnesia and agnosia), speech (aphasia), and motor behavior; (2) psychiatric symptoms and behavioral disturbances, including depression, anxiety, delusions, and misidentification; (3) difficulties with the daily living activities, such as driving, using the telephone, dealing with money and, later in the disease, all the basic needs (feeding, dressing, toileting) [12–14]. As expected in such a complex condition, a huge symptomatic variation is found in AD patients, although a positive corre‐ lation between symptom severity and disease evolution is observed.

Although AD is seen as an elderly disease due to its higher prevalence in the older population (approximately 5.3 million people solely in the US, in 2015) [15], it is also the most frequent form of dementia under the age of 65, with up to 5% of all cases [16]. Of curiosity, every 67 s, one more person is diagnosed with AD, and, by 2050, one new case of AD is expected to develop every 33 s [15]. Due to this disease complexity, diagnosis guidelines had to be established, and for a long time, the main criteria adopted was the one decided at the 1984 consortium, by the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association (NINCDS–ADRDA). These criteria divided AD in three possible diagnosis scenarios, which were possible, probable, or definite AD [17]. More recently, some minor alterations have been proposed in order to comprise also the stages prior to the clinical observation of symptoms [18, 19], thus prompting three renewed stages: (1) preclinical Alzheimer's disease; (2) mild cognitive impairment (MCI) due to Alzheimer's disease; and (3) dementia due to Alzheimer's disease [20].

Despite all the attentions directed to the establishment of proper diagnostic criteria and guidelines available, the diagnosis of AD is still not an easy task. Actually, a recent metaanalysis showed that the sensitivity and specificity of the clinical diagnosis ranged from 53 to 99% and 55 to 99%, respectively [21]. Although alone it is considered a low value, when combined with other characterizing techniques (as neuroimaging and biomarkers—see Biomarkers section), it is possible to predict/diagnose AD with a high confidence.

Genetically, AD is usually divided in two forms: autosomal dominant familial AD (FAD; predominantly of early-onset—under the age of 65) and sporadic AD (also called and usually associated to the late-onset AD—more than 65 years) [22]. Although extensively used, it is important to point out that this classification is far too simplistic.

Despite all of the effort put into research, the primary event triggering AD remains yet a mystery. Nonetheless, for FAD, several mutations capable of triggering the disease have been identified, especially in three distinct genes: the amyloid precursor protein (APP) [23], the presenilin 1 (PSEN1), and the presenilin 2 (PSEN2) genes [24], in chromosomes 21q, 14q, and 1q, respectively. Although these three genes comprise approximately 55% of all mutations, they are only responsible for less than 1% of all cases of AD (http://www.molgen.vib-ua.be/ ADMutations/). Contrary to FAD, sporadic AD does not exhibit autosomal-dominant inheri‐ tance but up to 60–80% of this form of AD is genetically determined [22].

## **2.2. The biochemical basis of AD**

In spite of its multifactorial etiology, AD is characterized by two specific brain lesions, the amyloid plaques, and the NFTs, considered the hallmarks of AD. Also, associated with these abnormalities, it is often observed severe neural loss and reactive gliosis.

NFTs are filamentous inclusions (intracellular lesions), preferentially observed in pyramidal neurons, which are composed of filamentous aggregates of abnormally hyperphosphorylated microtubule-associated protein tau [25]. Even though NFTs are a hallmark of AD, they are also observed for other neurodegenerative disorders termed tauopathies (e.g., sporadic cortico‐ basal degeneration, palsy, and Pick's disease, progressive supranuclear palsy, Seattle family A, parkinsonism–dementia complex of Guam, and some frontotemporal dementias) [26, 27].

As for amyloid plaques, they can be distinguished in different plaques subtypes, depending on their composition, and being the neuritic and diffuse plaques the two major subtypes in AD. Neuritic plaques are constituted by the 40- and 42-amino acids (aa) Aβ peptides [28] (Aβ40 and Aβ42, respectively), surrounded by dystrophic neurites (axons and dendrites), microglia (monocyte- or macrophage-derived cells that reside in the brain), and reactive astrocytes [29]. Diffuse deposits are mainly composed of Aβ42 [28] and lack the neuritic and glial components [29], but evolve over time with formation of discrete niduses that eventually become neuritic amyloid plaques [30].

## **2.3. Amyloid-β precursor protein and Aβ formation**

comprise also the stages prior to the clinical observation of symptoms [18, 19], thus prompting three renewed stages: (1) preclinical Alzheimer's disease; (2) mild cognitive impairment (MCI)

Despite all the attentions directed to the establishment of proper diagnostic criteria and guidelines available, the diagnosis of AD is still not an easy task. Actually, a recent metaanalysis showed that the sensitivity and specificity of the clinical diagnosis ranged from 53 to 99% and 55 to 99%, respectively [21]. Although alone it is considered a low value, when combined with other characterizing techniques (as neuroimaging and biomarkers—see

Genetically, AD is usually divided in two forms: autosomal dominant familial AD (FAD; predominantly of early-onset—under the age of 65) and sporadic AD (also called and usually associated to the late-onset AD—more than 65 years) [22]. Although extensively used, it is

Despite all of the effort put into research, the primary event triggering AD remains yet a mystery. Nonetheless, for FAD, several mutations capable of triggering the disease have been identified, especially in three distinct genes: the amyloid precursor protein (APP) [23], the presenilin 1 (PSEN1), and the presenilin 2 (PSEN2) genes [24], in chromosomes 21q, 14q, and 1q, respectively. Although these three genes comprise approximately 55% of all mutations, they are only responsible for less than 1% of all cases of AD (http://www.molgen.vib-ua.be/ ADMutations/). Contrary to FAD, sporadic AD does not exhibit autosomal-dominant inheri‐

In spite of its multifactorial etiology, AD is characterized by two specific brain lesions, the amyloid plaques, and the NFTs, considered the hallmarks of AD. Also, associated with these

NFTs are filamentous inclusions (intracellular lesions), preferentially observed in pyramidal neurons, which are composed of filamentous aggregates of abnormally hyperphosphorylated microtubule-associated protein tau [25]. Even though NFTs are a hallmark of AD, they are also observed for other neurodegenerative disorders termed tauopathies (e.g., sporadic cortico‐ basal degeneration, palsy, and Pick's disease, progressive supranuclear palsy, Seattle family A, parkinsonism–dementia complex of Guam, and some frontotemporal dementias) [26, 27].

As for amyloid plaques, they can be distinguished in different plaques subtypes, depending on their composition, and being the neuritic and diffuse plaques the two major subtypes in AD. Neuritic plaques are constituted by the 40- and 42-amino acids (aa) Aβ peptides [28] (Aβ40 and Aβ42, respectively), surrounded by dystrophic neurites (axons and dendrites), microglia (monocyte- or macrophage-derived cells that reside in the brain), and reactive astrocytes [29]. Diffuse deposits are mainly composed of Aβ42 [28] and lack the neuritic and glial components [29], but evolve over time with formation of discrete niduses that eventually become neuritic

due to Alzheimer's disease; and (3) dementia due to Alzheimer's disease [20].

Biomarkers section), it is possible to predict/diagnose AD with a high confidence.

important to point out that this classification is far too simplistic.

tance but up to 60–80% of this form of AD is genetically determined [22].

abnormalities, it is often observed severe neural loss and reactive gliosis.

**2.2. The biochemical basis of AD**

4 Exploring New Findings on Amyloidosis

amyloid plaques [30].

The amyloid-β precursor protein (APP) is a transmembrane receptor expressed ubiquitously in both neuronal cells and extra-neuronal tissues [31]. In humans, the APP gene is located in the chromosome 21, explaining partially the increased risk for Down syndrome patients to develop AD, and is composed of 18 exons [32]. Three major isoforms are expressed by alternative splicing: APP770 (full length), APP751 (lacking exon 8), and APP695 (lacking exon 7 and exon 8) [23, 31, 33]. APP belongs to a highly conserved family of type 1 transmembrane glycoproteins that extends also to invertebrate species, including the homologous: APL-1 (*Caenorhabditis elegans*), APPL (*Drosophila*), APLP1, and APLP2 (in mammals, besides APP) [34], and appa and appb (zebrafish) [35]. Following translation, APP is trafficked through the endoplasmic reticulum (ER), Golgi and trans-Golgi network (TGN), where it suffers specific endoproteolytic cleavages [33] that will originate several APP metabolites, among them the Aβ peptide. After reaching the membrane surface, APP can still undergo clathrin-mediated endocytosis and then be recycled to the surface again [36], during which Aβ can also be produced [37].

The 4 kDa Aβ peptide was first isolated and sequenced by Glenner and Wong, in 1984 [7] and can be found in the plasma and cerebrospinal fluid (CSF) of healthy humans and other mammals [38]. It was described as a 24 aa peptide but later, sequencing analysis revealed that the peptide could actually comprise 36–43 aa [39], being the two major species Aβ40 and Aβ42. In healthy individuals, these two forms make up about 90 and 10%, respectively, of the Aβ peptides that are normally produced by brain cells [40]. Despite the small difference in size and sequence of the various isoforms, they differ greatly in properties; for example, Aβ42 is more hydrophobic, thus, more prone to aggregation (compared to the less hydrophobic Aβ40). In fact, it readily aggregates in vitro, being considered the more amyloidogenic and hence pathogenic species [41].

## *2.3.1. Towards amyloid or not?*

APP processing can originate different metabolites that bear very different physiological functions, depending on the proteolysis pathway adopted: the amyloidogenic or nonamyloidogenic pathway (**Figure 1**). In the non-amyloidogenic pathway, APP is firstly cleaved by the α-secretase, a zinc metalloproteinase of the ADAM family [42], followed by the action of γ-secretase. The latter is a high molecular weight complex of four proteins: presenilin 1 or 2 (PSEN1, PSEN2), nicastrin (NCT) [43, 44], anterior pharynx-defective 1 (APH1), and prese‐ nilin enhancer 2 (PEN2) [45]. The cleavage by the α-secretase (at Lys687 of APP770) [46], within the Aβ domain, abrogates the production of Aβ, resulting in the release of a large soluble ectodomain of APP (sAPPα, ~100 kDa), leaving behind a 83-residue carboxi-terminal fragment (CTFα, of ~10 kDa) [47]. Then, γ-secretase cuts the CTFα, liberating the extracellular p3 peptide and the 50 aa APP intracellular domain (AICD, of ~6 kDa) [48].

On the other hand, as suggested by its name, the amyloidogenic pathway gives rise to the amyloidogenic Aβ peptide, and similar to the previous pathway, it consists of two sequential cleavages, first by the β-secretase (beta-site APP–cleaving enzyme 1–BACE-1), and then by γsecretase. The first protease cleaves APP at Met671 [49], releasing the large soluble ectodomain sAPPβ [33]. The remaining 99 aa CTFβ (of ~12 kDa) [50] is then cleaved by the γ-secretase, in the membrane, and originates, as said above, the Aβ peptide and the AICD [48]. This process generates different Aβ species, with variable hydrophobic C-termini (related to the γ-secretase cleaving site), that present different propensity to oligomerize [51] and, consequently, to form the amyloid plaques. Noteworthy, AD-linked mutations in the PSEN1 and PSEN2 proteins, particularly important in the case of FAD, influence γ-secretase-mediated processing of APP, and selectively enhance Aβ42 production compared to Aβ40 [52].

**Figure 1.** The amyloidogenic and non-amyloidogenic pathways of APP. In the non-amyloidogenic pathway, APP is cleaved by the α-secretase releasing the sAPPα neuroprotective N-terminal fragment, which contains part of the Aβ sequence. The 83 aa APP fragment (C83) then suffers the action of the γ-secretase, liberating the p3 peptide and the AICD fragment. The amyloidogenic pathway involves the sequential cleavage of APP by the β-secretase which releas‐ es the soluble ectodomain sAPPβ. The remaining C99 fragment of APP is then cleaved by the γ-secretase, resulting in the formation of the Aβ peptide. Due to its high propensity to aggregate, Aβ peptide oligomerizes, accumulates and forms amyloid senile plaques, in turn leading to the described alterations of AD. Current therapeutic approaches in AD include: (1) inhibition of β- and γ-secretases, (2) improving Aβ clearance, and (3) amelioration of inflammation and synaptic dysregulation.

Although tightly related to AD onset, APP processing is a normal metabolic event and Aβ is a normal product of cellular metabolism throughout life, circulating as a soluble peptide in biological fluids [53]. Plus, Aβ deposition can also be found, together with NFTs, in the brain of non-demented elderly people [54].

#### **2.4. Alzheimer's disease hypotheses**

AD is one of the human diseases with the highest number of hypothesis formulated trying to explain its pathogenesis, with very different and plausible molecular mechanism to back them up. Within the list, the amyloid cascade hypothesis stands out, together with the so-called "tau and tangle" hypothesis, strengthened by the fact that they are based in the two hallmarks of AD.

## *2.4.1. Amyloid cascade hypothesis*

sAPPβ [33]. The remaining 99 aa CTFβ (of ~12 kDa) [50] is then cleaved by the γ-secretase, in the membrane, and originates, as said above, the Aβ peptide and the AICD [48]. This process generates different Aβ species, with variable hydrophobic C-termini (related to the γ-secretase cleaving site), that present different propensity to oligomerize [51] and, consequently, to form the amyloid plaques. Noteworthy, AD-linked mutations in the PSEN1 and PSEN2 proteins, particularly important in the case of FAD, influence γ-secretase-mediated processing of APP,

**Figure 1.** The amyloidogenic and non-amyloidogenic pathways of APP. In the non-amyloidogenic pathway, APP is cleaved by the α-secretase releasing the sAPPα neuroprotective N-terminal fragment, which contains part of the Aβ sequence. The 83 aa APP fragment (C83) then suffers the action of the γ-secretase, liberating the p3 peptide and the AICD fragment. The amyloidogenic pathway involves the sequential cleavage of APP by the β-secretase which releas‐ es the soluble ectodomain sAPPβ. The remaining C99 fragment of APP is then cleaved by the γ-secretase, resulting in the formation of the Aβ peptide. Due to its high propensity to aggregate, Aβ peptide oligomerizes, accumulates and forms amyloid senile plaques, in turn leading to the described alterations of AD. Current therapeutic approaches in AD include: (1) inhibition of β- and γ-secretases, (2) improving Aβ clearance, and (3) amelioration of inflammation and

Although tightly related to AD onset, APP processing is a normal metabolic event and Aβ is a normal product of cellular metabolism throughout life, circulating as a soluble peptide in biological fluids [53]. Plus, Aβ deposition can also be found, together with NFTs, in the brain

AD is one of the human diseases with the highest number of hypothesis formulated trying to explain its pathogenesis, with very different and plausible molecular mechanism to back them up. Within the list, the amyloid cascade hypothesis stands out, together with the so-called "tau

and selectively enhance Aβ42 production compared to Aβ40 [52].

6 Exploring New Findings on Amyloidosis

synaptic dysregulation.

of non-demented elderly people [54].

**2.4. Alzheimer's disease hypotheses**

Since its formalization, in 1992 by Hardy and Higgins, the amyloid cascade hypothesis has had a prominent role in explaining the etiology and pathogenesis of AD. They suggested that amyloid deposition was the primary influence driving AD pathogenesis [55], due to two key observations: the detection of Aβ as the main constituent of amyloid plaques, and the discovery of mutations in the APP, PSEN1 and PSEN2 genes associated with FAD [56]. This hypothesis stated that a dysregulation in APP processing or Aβ clearance would provoke an increase in the Aβ42/Aβ40 ratio, which would promote aggregation, accumulation, and plaque formation. In turn, this would be responsible for the subsequent pathology (including tau aggregation, phosphorylation, neuronal attrition, and clinical dementia) [57]. Due to its inability to entirely explain AD pathogenesis, especially by the lack of correlation between plaque burden and clinical manifestations [58], this hypothesis has been upgraded over the past few years. Scientists started to divert their attention from the effects of the amyloid deposits, to study the other forms (monomers, oligomers, and protofibrils—usually shorter and thinner then mature fibrils) [59] of Aβ peptide-induced neurotoxicity. Some studies suggest that Aβ toxicity functions in a plaque-independent manner, indicating that oligomeric intermediates present higher toxicity to the cells [60] and that activation of signaling pathways due to intraneuronal accumulation of Aβ oligomers is responsible for tau hyperphosphorylation and subsequent deposition [61]. Several explanations have been proposed, with some defending that oligo‐ meric toxicity is related to a greater capacity for diffusion and a larger collective surface area for interacting with neuronal and glial cells [60], while other proposed that it is not related to a specific prefibrillar aggregate (dimer, trimer, and so on) but rather to the propensity that each species has to grow and undergo fibril formation [62].

A more consensual vision about Aβ is that it possesses a dual role: On one hand, it can be a neurotrophic agent or a neuroprotector against excitotoxicity (by activating the phosphatidy‐ linositol-3-kinase (PI-3K) pathway); on the other hand, an inducer of neuronal degeneration (at high concentrations) in mature neurons [63].

Other interesting and amyloid cascade-opposite hypotheses have been proposed, stating that Aβ should not be seen as the initiating factor for neurodegeneration in AD, but instead, its deposition is nothing more than a protective mechanism to neuronal insult, in which Aβ binds and removes harmful substances by blocking them in plaques [64, 65].

## *2.4.2. Tau hypothesis*

"Tauists" defend a collection of ideas that maintain the primacy of NFTs formation as the ADcausing event, which Mudher and Lovestone designated as the "tau and tangle hypothesis" [57]. It started to emerge due to solid evidence that amyloid plaques do not account for the complex pathophysiology of AD [66], opposed to the observed highly positive correlation between NFTs and cognitive deficits [67]. It argues that in AD the normal role of tau (micro‐ tubules stabilization) is impaired and that NFTs accumulate and occupy much of the neuron, resulting in neuronal death. This was supported by the visualization of the extracellular tangles in the shape of neurons, abundant in the late stages of disease [57]. Also, the discovery of mutations within the tau gene that cause fronto-temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), demonstrating that tau dysfunction, in the absence of amyloid pathology, was enough to cause neuronal loss and clinical dementia [68], further strengthened this hypothesis. However, tau mutations do not originate amyloid plaques, whereas APP and presenilin gene mutations give rise to amyloid and tau depositions, strongly evidencing that amyloid pathology is upstream of tau pathology [57]. More recently, a more embracing tau hypothesis was proposed, in which a series of damage signals (Aβ oligomers, oxygen-free radicals, iron overload, cholesterol levels in neuronal rafts, low-density lipoprotein (LDL) species and homocysteine, among other) trigger, by innate immunity, the activation of microglial cells with the consequent release of pro-inflammatory cytokines that modify neuronal behavior through anomalous signaling cascades, which finally, promote tau hyper‐ phosphorylation [66]. In turn, tau hyperphosphorylation will contribute to further activation of microglial cells and stimulation of the deleterious cycle, which will lead to progressive neuronal degeneration [66]. The degree of tau phosphorylation in the AD brain is reasonably well correlated with the severity of AD symptoms; however, fetal tau, a much more phos‐ phorylated form of tau than adult tau, does not induce AD-like pathology [69]. In summary, there is no direct evidence for the neurotoxicity of hyperphosphorylated tau (as in the case of Aβ toxicity).

#### *2.4.3. "Other" hypothesis*

## *2.4.3.1. GSK-3 hypothesis*

Glycogen synthase kinase-3 (GSK-3), a multi-tasking kinase with major roles in brain signaling, has been recently proposed as a central player in AD pathology. This was supported by observing that the deregulation of this protein is responsible for many of the pathological hallmarks of the disease, in both sporadic and familial AD cases [70]. It was suggested that the hyperactivity of GSK-3β, the most abundant of two isoforms (GSK-3α and GSK-3β) expressed in neurons, is intimately involved with cognitive impairment [71], Aβ production [72], tau hyperphosphorylation [73], acetylcholine synthesis [74], neuronal death [75], and neuroin‐ flammation [76] in AD. Furthermore, regarding Aβ interaction, it was observed that Aβ also regulates GSK-3β activity [77, 78] making it difficult to establish which event is located upstream. Thus, this hypothesis is seen as an integration and extension of the amyloid cascade hypothesis, still conferring to Aβ a central role in AD pathology. Although GSK-3 modulation appears to be an excellent therapeutic approach, no effective result has been observed in trials, perhaps due to its activity in multiple targets.

#### *2.4.3.2. Oxidative stress/mitochondrial hypothesis*

The brain is especially vulnerable to free radical damage as a result of its high oxygen con‐ sumption rate, abundant fatty acids content, and the relative low levels of antioxidant enzymes [79]. The most appealing feature of the oxidative stress hypothesis is its slow and cumulative damaging nature that could, over time, account for the late life onset and slowly progressive nature of AD, and neurodegeneration in general [80]. Also supporting this hypothesis is the suggested unbalanced levels of heavy metals in the brain, among others, iron (Fe), copper (Cu), aluminum, and mercury, which function as catalysts for oxygen free radical generation [80]. There has been high controversy in the measurement of these elements, especially Fe, but most studies reveal an apparent unbalance in AD brains compared to controls. In a recent study, Fe was found significantly increased in patients with severe AD [81] (as previously reported [82]). Nonetheless, some consider that this "accepted" elevation, even if significant in some studies of AD pathology, does not account for brain degeneration, and so, presents itself as a mis‐ leading therapeutic target with considerable risks for patients (reviewed in [83]). As for Cu, it has been shown to be decreased in AD brains [81], which at first goes against the oxidation hypothesis. More recently, and also in the presence of some contradictory results [84], copper was found to bind strongly to Aβ aggregates, inhibiting in vitro amyloid fibril formation [85]. In addition, and when bound to the aggregates, copper exhibited a redox role, by degrading hydrogen peroxide [86]. Protein and DNA oxidation (in particular mitochondrial DNA), and lipid peroxidation (which affects the phospholipid-rich membrane) were also found to be increased in AD brains [80].

Another hypothesis intimately related to the brain redox status is the mitochondrial cascade hypothesis [87]. Mitochondria are considered the cell "powerhouses"; however, when in a non-physiological energy production, they can provoke severe damage by increasing the reactive oxygen species (ROS). Curiously, mitochondria are the first target of ROS, suffering DNA oxidation, which may lead to a further increase of ROS production, generating a vicious cycle [88]. The authors of this hypothesis state that sporadic and autosomal dominant AD are not etiologically homogeneous and that mitochondrial dysfunction works as a link for both. Very briefly, in autosomal dominant forms, Aβ-induced mitochondrial dysfunction leads to the other AD-characteristic histopathologies, while in sporadic AD, mitochondrial malfunc‐ tion induces the AD pathologies, including processing of APP to Aβ [89].

#### *2.4.3.3. The cholinergic hypothesis*

tubules stabilization) is impaired and that NFTs accumulate and occupy much of the neuron, resulting in neuronal death. This was supported by the visualization of the extracellular tangles in the shape of neurons, abundant in the late stages of disease [57]. Also, the discovery of mutations within the tau gene that cause fronto-temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), demonstrating that tau dysfunction, in the absence of amyloid pathology, was enough to cause neuronal loss and clinical dementia [68], further strengthened this hypothesis. However, tau mutations do not originate amyloid plaques, whereas APP and presenilin gene mutations give rise to amyloid and tau depositions, strongly evidencing that amyloid pathology is upstream of tau pathology [57]. More recently, a more embracing tau hypothesis was proposed, in which a series of damage signals (Aβ oligomers, oxygen-free radicals, iron overload, cholesterol levels in neuronal rafts, low-density lipoprotein (LDL) species and homocysteine, among other) trigger, by innate immunity, the activation of microglial cells with the consequent release of pro-inflammatory cytokines that modify neuronal behavior through anomalous signaling cascades, which finally, promote tau hyper‐ phosphorylation [66]. In turn, tau hyperphosphorylation will contribute to further activation of microglial cells and stimulation of the deleterious cycle, which will lead to progressive neuronal degeneration [66]. The degree of tau phosphorylation in the AD brain is reasonably well correlated with the severity of AD symptoms; however, fetal tau, a much more phos‐ phorylated form of tau than adult tau, does not induce AD-like pathology [69]. In summary, there is no direct evidence for the neurotoxicity of hyperphosphorylated tau (as in the case of

Glycogen synthase kinase-3 (GSK-3), a multi-tasking kinase with major roles in brain signaling, has been recently proposed as a central player in AD pathology. This was supported by observing that the deregulation of this protein is responsible for many of the pathological hallmarks of the disease, in both sporadic and familial AD cases [70]. It was suggested that the hyperactivity of GSK-3β, the most abundant of two isoforms (GSK-3α and GSK-3β) expressed in neurons, is intimately involved with cognitive impairment [71], Aβ production [72], tau hyperphosphorylation [73], acetylcholine synthesis [74], neuronal death [75], and neuroin‐ flammation [76] in AD. Furthermore, regarding Aβ interaction, it was observed that Aβ also regulates GSK-3β activity [77, 78] making it difficult to establish which event is located upstream. Thus, this hypothesis is seen as an integration and extension of the amyloid cascade hypothesis, still conferring to Aβ a central role in AD pathology. Although GSK-3 modulation appears to be an excellent therapeutic approach, no effective result has been observed in trials,

The brain is especially vulnerable to free radical damage as a result of its high oxygen con‐ sumption rate, abundant fatty acids content, and the relative low levels of antioxidant enzymes

Aβ toxicity).

*2.4.3. "Other" hypothesis*

8 Exploring New Findings on Amyloidosis

*2.4.3.1. GSK-3 hypothesis*

perhaps due to its activity in multiple targets.

*2.4.3.2. Oxidative stress/mitochondrial hypothesis*

The cholinergic hypothesis was proposed after the observation of a decrease of choline acetyltransferase (ChAT) in AD patients [90]. It states that the loss of cholinergic cells in the septal nuclei and basal forebrain (described in patients with advanced AD [91]) compromises the innervation of the cerebral cortex and related structures, which play an important role in cognitive functions, especially memory [92]. More recent studies have suggested a bidirec‐ tional pathway linking Aβ toxicity in cholinergic dysfunction and the interaction of cholinergic regulatory mechanisms in the processing of APP [93]. Discrediting this hypothesis, later studies showed that the cholinergic degeneration [94] and the decrease in ChAT enzyme activity [95] are not observable in the early stages of disease. This was accompanied by the fact that treatment with acetylcholinesterase inhibitors does not offer long term cure, although it has shown consistent, despite modest, benefits in symptoms improvement [96]. Nonetheless, some studies point out that compensatory mechanisms could overcome the cholinergic defects, disguising its effects in early stages of AD or mild cognitive impairment [97].

## *2.4.3.4. Calcium hypothesis*

The calcium hypothesis was first introduced by Khachaturian in 1982, stating that cellular mechanisms which maintain the homeostasis of cytosolic Ca2+ play a key role in brain aging and that sustained changes in Ca2+ homeostasis could provide the final common pathway for age-associated brain changes [98], or in this case, be the proximal cause of neurodegeneration in AD. Out of curiosity, this proposal was purely speculative at the time, only sustained on circumstantial evidence from a handful of studies [98]. It was observed that the persistent elevation of the levels of Ca2+ leads to the constant elimination of newly acquired memories, due to a stimulation of long-term depression mechanisms [99]. In fact, calcium signaling dysfunctions occur during the initial phases of the disease, and even before the development of pronounced symptoms [100]. However, whether it is calcium dyshomeostasis that provokes Aβ production and accumulation [101], or vice-versa [102], remains to be elucidated. Either way, in addition to the Aβ unbalance, increased levels of Ca2+ promoted protein tau hyper‐ phosphorylation [103]. It was also suggested that amyloid oligomers induce membrane permeabilization, leading to increased intracellular Ca2+ concentration [104]. Nevertheless, there is some disagreement as to the mechanism by which amyloid oligomers increase intracellular calcium.

### **2.5. Biomarkers and risk factor**

Despite of the extensive Knowledge on the causative gene mutations responsible for familial AD, the sporadic (non-genetic) form of this disease, which results from the diverse interactions between genetic and environmental factors, is still lacking characterization. Thus, researchers are continuously looking for specific molecules that should be altered exclusively in AD, and preferentially in the asymptomatic period. This, combined with the meticulous description of the patient risk factors, may give the opportunity for an early action and increase the success rate of therapeutics.

#### *2.5.1. Biomarkers*

By definition, and according to the International Programme on Chemical Safety, biomarker is "any substance, structure, or process that can be measured in the body or its products and influence or predict the incidence of outcome or disease" [105]. The search for early AD biomarkers has been highly targeted over the last years, as investigators believe that the generation of an effective treatment for AD is only possible if the disease is detected at very early stages. Thus, the discovery of biomarkers is of extreme importance for the early diagnosis of AD and even for predicting the conversion of MCI into AD patients.

The search for solid AD biomarkers started with those who seem to be altered in this condi‐ tion when compared to normality. Several studies showed that the combination of CSF to‐ tal- and phospho-tau, and CSF and plasma Aβ42 is able to predict with increased sensitivity

and specificity the development of AD in patients with MCI [21, 106, 107], in addition to the already established role of the ApoE-ε4 isoform [108] (major susceptibility gene—see Sec‐ tion 2.5.2. Risk Factors). Studies in FAD-causing mutations carriers showed increased levels of CSF total-tau and plasma Aβ42, although having reduced CSF Aβ42 levels, at least 10 years before the symptoms establishment [106]. Also, the ratio of Aβ42/Aβ40 in CSF and plasma was found decreased, respectively, for non-demented mutation carriers [109] and cognitively normal elders (which evolved to MCI or AD) [110]. Other CSF biomarkers, such as BACE-1 [111] and sAPPα/β [112], are also suggested; however, independent studies were not consistent [106], possibly due to technical difficulties in the biomarkers quantification [113]. Our group has also proposed TTR as a biomarker in plasma, demonstrating a nega‐ tive correlation with AD severity [114], which supported prior observations for CSF levels. In that study, the authors also considered TTR a selective biomarker for AD [115]. Other studies contradict this idea suggesting that TTR potential as biomarker raises some doubt since its levels appear to fluctuate substantially within a single individual over a 2-week interval [116].

In addition to the so-called fluid biomarkers, physicians have at their disposal powerful imaging technology. With the improvement in PET (positron emission tomography) and MRI (magnetic resonance imaging) spectroscopy resolution, neuroimaging has been gaining importance and increasing the confidence in AD diagnosis. Due to the possibility of using specific tracers, such as a derivate of thioflavin T that crosses the blood–brain barrier (BBB) and binds selectively to Aβ (C11-labeled Pittsburgh Compound B–PiB), it is possible to identify amyloid deposition in the brain in vivo [117]. In 2004, Klunk and colleagues performed the first study of brain amyloid imaging (BAI) using the PiB compound, in which they showed a robust relationship between amyloid deposition and PiB retention [118]. The combination of increased BAI signal, low CSF Aβ42, and high CSF p-tau in a subject with dementia is seen as a "definite" diagnosis of AD. Furthermore, BAI and CSF profiles can be used to predict patients with MCI who will progress to frank dementia with high degree of confidence [119]. Despite the value of this compound, the resulting data should be subjected to careful analysis since healthy subjects also present amyloid deposition, hindering the differentiation between symptomatic AD and asymptomatic controls with amyloid plaques [120]. MRI (structural evidence) is also a common technique often used, alone or in combination with CSF tau and Aβ42, to predict development of AD [121]. Curiously, a recent study showed that the MRI together with PiB-PET makes the best combination of biomarkers, thus showing the best AD predictive value [119].

#### *2.5.2. Risk factors*

some studies point out that compensatory mechanisms could overcome the cholinergic defects,

The calcium hypothesis was first introduced by Khachaturian in 1982, stating that cellular mechanisms which maintain the homeostasis of cytosolic Ca2+ play a key role in brain aging and that sustained changes in Ca2+ homeostasis could provide the final common pathway for age-associated brain changes [98], or in this case, be the proximal cause of neurodegeneration in AD. Out of curiosity, this proposal was purely speculative at the time, only sustained on circumstantial evidence from a handful of studies [98]. It was observed that the persistent elevation of the levels of Ca2+ leads to the constant elimination of newly acquired memories, due to a stimulation of long-term depression mechanisms [99]. In fact, calcium signaling dysfunctions occur during the initial phases of the disease, and even before the development of pronounced symptoms [100]. However, whether it is calcium dyshomeostasis that provokes Aβ production and accumulation [101], or vice-versa [102], remains to be elucidated. Either way, in addition to the Aβ unbalance, increased levels of Ca2+ promoted protein tau hyper‐ phosphorylation [103]. It was also suggested that amyloid oligomers induce membrane permeabilization, leading to increased intracellular Ca2+ concentration [104]. Nevertheless, there is some disagreement as to the mechanism by which amyloid oligomers increase

Despite of the extensive Knowledge on the causative gene mutations responsible for familial AD, the sporadic (non-genetic) form of this disease, which results from the diverse interactions between genetic and environmental factors, is still lacking characterization. Thus, researchers are continuously looking for specific molecules that should be altered exclusively in AD, and preferentially in the asymptomatic period. This, combined with the meticulous description of the patient risk factors, may give the opportunity for an early action and increase the success

By definition, and according to the International Programme on Chemical Safety, biomarker is "any substance, structure, or process that can be measured in the body or its products and influence or predict the incidence of outcome or disease" [105]. The search for early AD biomarkers has been highly targeted over the last years, as investigators believe that the generation of an effective treatment for AD is only possible if the disease is detected at very early stages. Thus, the discovery of biomarkers is of extreme importance for the early diagnosis

The search for solid AD biomarkers started with those who seem to be altered in this condi‐ tion when compared to normality. Several studies showed that the combination of CSF to‐ tal- and phospho-tau, and CSF and plasma Aβ42 is able to predict with increased sensitivity

of AD and even for predicting the conversion of MCI into AD patients.

disguising its effects in early stages of AD or mild cognitive impairment [97].

*2.4.3.4. Calcium hypothesis*

10 Exploring New Findings on Amyloidosis

intracellular calcium.

rate of therapeutics.

*2.5.1. Biomarkers*

**2.5. Biomarkers and risk factor**

#### *2.5.2.1. Environmental factors*

As Stephen King wrote in The Gunslinger: "Time's the thief of memory," and so, the most worldwide accepted (and intuitive) risk factor is aging. In every species, age brings a slowing of brain function [122], thus preventing the brain to properly respond/recover from insults. The increasing of life expectancy, in addition to the increasing of population (attributed to the postwar "baby boom"), turned aging in a major risk factor. Also gender appears to play a role in disease development, since data show that women are more prone to this disease than men. Although women present higher life expectancy, also in younger study groups (60–80 years), where differences in death rate are insignificant, women present higher incidence of cases [123]. The specific mechanism is unknown; however, several factors have been proposed to influence, such as: age-related sex hormone reduction, risks of other diseases (diabetes, depression, cardiovascular disease), and differences in brain anatomy and metabolism [124].

The cardiovascular risk factors, which appear sometimes as a distinct group of factors, include diabetes mellitus [125], overweight [126], hypertension [127], and high cholesterol levels [128]. Individually or in cooperation, these factors increase the predisposition for cognitive decline. The midlife control of the above cardiovascular factors has been associated with a reduction in white matter lesions in late life [127]. As for cholesterol, results appear to be inconsistent; however, lipid-lowering treatments present benefits against white matter lesions [126].

Contrasting with the previous risk factors, wine consumption, coffee consumption, the use of non-steroidal anti-inflammatory drugs (NSAIDs), and physical activity are associated with reduced risks, thus showing some protective effects [129].

## *2.5.2.2. Genetic factors*

ApoE exists as three isoforms ε2, ε3 and ε4, with ε3 having the highest prevalence. As Corder and colleagues stated, in 1993, ApoE plays an important role in AD, with the risk of developing disease increased in carriers of the ApoE-ε4 allele, such that a double dose of this allele was nearly enough to cause AD by the age of 80 [130]. Despite the broad molecular evidence about ApoE role in AD, its genetic variation is also present in other kinds of neurological disorders, including Parkinson's disease and multiple sclerosis [22]. In 2009, three novel AD genes were identified, presenting high degree of association: CLU (clusterin or apolipoprotein J), CR1 (complement component (3b/4b) receptor 1), and PICALM (phosphatidylinositol-binding clathrin assembly protein). Down's syndrome (or trisomy 21) is also considered a genetic risk factor. This condition is the result of a third copy of the chromosome 21, which coincides with the location of the APP gene [131], giving rise to an increased accumulation of Aβ.

## **2.6. Drugs and treatments**

Due to the complexity of AD, a vast number of targets and pathways may be chosen to intervene. Cholinergic degradation inhibitors, immunotherapy, secretase inhibitors, antiinflammatory drugs, and tau- and Aβ-deposition interfering drugs (**Figure 1**) are but a few examples of the many classes of drugs that are being tested at the moment.

The first drugs developed for AD, acetylcholinesterase inhibitors (AchEI), aimed at increasing acetylcholine levels (see "Cholinergic hypothesis"). Currently, FDA has five drugs approved for the "treatment" of AD in the initial stages: 4 AchEI (Donepezil, Rivastigmine, Galantamine, and Tacrine) and 1 NMDA receptor antagonist (Memantine) (http://www.alzforum.org). As referred above, they treat and ameliorate the symptoms, but do not cure.

In 2010, Rinne and colleagues showed for the first time target engagement from a diseasemodifying drug in humans, using the monoclonal anti-Aβ antibody bapineuzumab [132, 133]. This study showed a reduction of fibrillar amyloid in the brain of AD individuals, but did not improve cognition and stopped at phase 3 [133]. Crenezumab, another anti-Aβ, was selected for pre-symptomatic treatment trials of Colombian mutant PSEN1 kindred [133], however, showed extensive cross-reaction with non-Aβ related proteins [134]. Intravenous immunoglobulins (IVIG) have also been proposed as a potential treatment, based on the hypothesis That IVIG contain naturally occurring antibodies that specifically promote clearance of Aβ peptides from the brain [135].

β- and γ-secretase modulators [133], Aβ and tau deposition modulators (e.g., scylloinositol and methylene blue, respectively [39]), and molecules addressing oxidative damage (resver‐ atrol) are also potential drugs under study. Recently, deep brain stimulation (DBS) performed by the group of Dr Lozano in AD patients showed promising results, with improvements and/ or slowing in the rate of cognitive decline [136], increased hippocampus volume and glucose metabolism [137]. The authors propose that DBS is able to influence the structure of the brain and that hippocampal atrophy can potentially be slowed, suggesting restorative properties to DBS [137].

A general recommended therapy is a good diet and a healthy lifestyle, in order to control cardiovascular risk factors, decreasing cerebrovascular events. No effective treatment has been found, thus increasing the interest for the early diagnose of AD, to allow a more effective and early stage intervention.

## **3. Blood–brain barrier and Alzheimer's disease**

postwar "baby boom"), turned aging in a major risk factor. Also gender appears to play a role in disease development, since data show that women are more prone to this disease than men. Although women present higher life expectancy, also in younger study groups (60–80 years), where differences in death rate are insignificant, women present higher incidence of cases [123]. The specific mechanism is unknown; however, several factors have been proposed to influence, such as: age-related sex hormone reduction, risks of other diseases (diabetes, depression, cardiovascular disease), and differences in brain anatomy and metabolism [124].

The cardiovascular risk factors, which appear sometimes as a distinct group of factors, include diabetes mellitus [125], overweight [126], hypertension [127], and high cholesterol levels [128]. Individually or in cooperation, these factors increase the predisposition for cognitive decline. The midlife control of the above cardiovascular factors has been associated with a reduction in white matter lesions in late life [127]. As for cholesterol, results appear to be inconsistent; however, lipid-lowering treatments present benefits against white matter lesions [126].

Contrasting with the previous risk factors, wine consumption, coffee consumption, the use of non-steroidal anti-inflammatory drugs (NSAIDs), and physical activity are associated with

ApoE exists as three isoforms ε2, ε3 and ε4, with ε3 having the highest prevalence. As Corder and colleagues stated, in 1993, ApoE plays an important role in AD, with the risk of developing disease increased in carriers of the ApoE-ε4 allele, such that a double dose of this allele was nearly enough to cause AD by the age of 80 [130]. Despite the broad molecular evidence about ApoE role in AD, its genetic variation is also present in other kinds of neurological disorders, including Parkinson's disease and multiple sclerosis [22]. In 2009, three novel AD genes were identified, presenting high degree of association: CLU (clusterin or apolipoprotein J), CR1 (complement component (3b/4b) receptor 1), and PICALM (phosphatidylinositol-binding clathrin assembly protein). Down's syndrome (or trisomy 21) is also considered a genetic risk factor. This condition is the result of a third copy of the chromosome 21, which coincides with

the location of the APP gene [131], giving rise to an increased accumulation of Aβ.

examples of the many classes of drugs that are being tested at the moment.

referred above, they treat and ameliorate the symptoms, but do not cure.

Due to the complexity of AD, a vast number of targets and pathways may be chosen to intervene. Cholinergic degradation inhibitors, immunotherapy, secretase inhibitors, antiinflammatory drugs, and tau- and Aβ-deposition interfering drugs (**Figure 1**) are but a few

The first drugs developed for AD, acetylcholinesterase inhibitors (AchEI), aimed at increasing acetylcholine levels (see "Cholinergic hypothesis"). Currently, FDA has five drugs approved for the "treatment" of AD in the initial stages: 4 AchEI (Donepezil, Rivastigmine, Galantamine, and Tacrine) and 1 NMDA receptor antagonist (Memantine) (http://www.alzforum.org). As

reduced risks, thus showing some protective effects [129].

*2.5.2.2. Genetic factors*

12 Exploring New Findings on Amyloidosis

**2.6. Drugs and treatments**

The BBB is a profoundly specialized brain endothelial structure of the differentiated neuro‐ vascular system. These specialized endothelial cells interacting with astrocytes, microglia, and pericytes, confine components of the circulating blood from neurons. Furthermore, the BBB controls the chemical composition of the neuronal environment which is required for the functioning of neuronal circuits, synaptic transmission, synaptic remodeling, angiogenesis, and neurogenesis. BBB malfunction, through the disruption of the tight junctions (TJs) and alteration of the transport of molecules between blood and brain, brain hypoperfusion, and inflammatory responses, may begin or contribute to the process of different diseases such as AD, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis. These data support developments of new therapeutic strategies for the neurodegenerative disorders focused at the BBB [138].

## **3.1. The blood–brain barrier: the earliest findings**

In 1885, when scientist Paul Ehrlich injected trypan blue dye into the bloodstream of mice, he noted that the stain colored all of the animal organs, except the brain [139]. In 1913, one of the Ehrlich's students, Edwin Goldmann, performed a follow-up experiment by injecting the same dye into the brain of mice. He observed that injection of trypan blue directly into the CSF stained all cell types in the brain but failed to penetrate into the periphery [140]. Although aiming at finding new compounds that could attack disease-causing microbes, these experi‐ ments suggested a physical barrier between the brain and the blood, becoming the stepping stones on BBB research. Lewandowsky was the first to use the term blood–brain barrier while studying the limited permeation of potassium ferrocyanate into the brain [141]. However, it took until the 1960s for the specific anatomy of the network of brain–blood vessels comprising the BBB to be glimpsed [142]. Using electron microscopy, in 1967, Reese and Karnovsky showed that the BBB is localized at the level of TJs between adjacent brain endothelial cells [143, 144].

It is now known that the brain of mammals is separated from the blood by the BBB, localized to the brain capillaries and pia-subarachnoid membranes, and by the blood-CSF barrier, confined to the choroid plexus [145]. At the junctional complex formed by the TJs and adherens junctions (AJs), brain endothelial cells are connected to each other [142]. Transcellular transport at the BBB occurs through several mechanisms including passive and active transport through different receptors and transporters. Interestingly, endothelial cells, pericytes, and astrocytes present at the BBB also express several enzymes such as cholinesterase, aminopeptidases and endopeptidases that alter endogenous and exogenous molecules, which can negatively influence neuronal function. This produces a metabolic barrier which protects the central nervous system (CNS) [146].

## **3.2. The neurovascular unit at the BBB; endothelial cells, pericytes, and glial cells**

## *3.2.1. Endothelial cells and pericytes*

A cerebral capillary lumen is enclosed by a single endothelial cell. Anatomically, the BBB endothelial cells are distinguished from those at the periphery by enhanced mitochondrial content [147], loss of fenestrations [148], least pinocytotic activity [149], and the presence of TJs [150]. The BBB tightly sealed monolayer of endothelial cells usually prevents the free exchange of solutes between blood and brain [151] except for the lipid-soluble molecules smaller than 400 Da with less than nine hydrogen bonds, which can cross the BBB without any support, via lipid-mediated diffusion [146].

As for pericytes (granular or filamentous), they are connected to the abluminal membrane of the endothelial cells [152]. Pericytes and endothelial cells are ensheathed by the basal lamina, composed of collagen type IV and other extracellular matrix proteins [153]. However, there is not much known about the involvement of pericytes at the BBB, but the addition of pericytes to co-cultures of endothelial cells and astrocytes has been shown to stabilize capillary-like structures [154]. In conditions associated with increased BBB permeability such as hypoxia or traumatic brain injury, pericytes have also been shown to migrate away from brain microves‐ sels [155, 156]. Moreover, pericyte-derived angiopoietin provokes expression of TJs such as occludin in endothelial cells [157], confirming that pericytes are involved in the maintenance of the barrier properties in the cerebral endothelium [142].

## *3.2.2. Glial cells*

stained all cell types in the brain but failed to penetrate into the periphery [140]. Although aiming at finding new compounds that could attack disease-causing microbes, these experi‐ ments suggested a physical barrier between the brain and the blood, becoming the stepping stones on BBB research. Lewandowsky was the first to use the term blood–brain barrier while studying the limited permeation of potassium ferrocyanate into the brain [141]. However, it took until the 1960s for the specific anatomy of the network of brain–blood vessels comprising the BBB to be glimpsed [142]. Using electron microscopy, in 1967, Reese and Karnovsky showed that the BBB is localized at the level of TJs between adjacent brain endothelial cells

It is now known that the brain of mammals is separated from the blood by the BBB, localized to the brain capillaries and pia-subarachnoid membranes, and by the blood-CSF barrier, confined to the choroid plexus [145]. At the junctional complex formed by the TJs and adherens junctions (AJs), brain endothelial cells are connected to each other [142]. Transcellular transport at the BBB occurs through several mechanisms including passive and active transport through different receptors and transporters. Interestingly, endothelial cells, pericytes, and astrocytes present at the BBB also express several enzymes such as cholinesterase, aminopeptidases and endopeptidases that alter endogenous and exogenous molecules, which can negatively influence neuronal function. This produces a metabolic barrier which protects the central

**3.2. The neurovascular unit at the BBB; endothelial cells, pericytes, and glial cells**

A cerebral capillary lumen is enclosed by a single endothelial cell. Anatomically, the BBB endothelial cells are distinguished from those at the periphery by enhanced mitochondrial content [147], loss of fenestrations [148], least pinocytotic activity [149], and the presence of TJs [150]. The BBB tightly sealed monolayer of endothelial cells usually prevents the free exchange of solutes between blood and brain [151] except for the lipid-soluble molecules smaller than 400 Da with less than nine hydrogen bonds, which can cross the BBB without any

As for pericytes (granular or filamentous), they are connected to the abluminal membrane of the endothelial cells [152]. Pericytes and endothelial cells are ensheathed by the basal lamina, composed of collagen type IV and other extracellular matrix proteins [153]. However, there is not much known about the involvement of pericytes at the BBB, but the addition of pericytes to co-cultures of endothelial cells and astrocytes has been shown to stabilize capillary-like structures [154]. In conditions associated with increased BBB permeability such as hypoxia or traumatic brain injury, pericytes have also been shown to migrate away from brain microves‐ sels [155, 156]. Moreover, pericyte-derived angiopoietin provokes expression of TJs such as occludin in endothelial cells [157], confirming that pericytes are involved in the maintenance

[143, 144].

nervous system (CNS) [146].

14 Exploring New Findings on Amyloidosis

*3.2.1. Endothelial cells and pericytes*

support, via lipid-mediated diffusion [146].

of the barrier properties in the cerebral endothelium [142].

Astrocytes are another cell type present in the neurovascular unit. These cells are usually located between neurons, pericytes, and capillary endothelium, and communicate with these cells via their several foot processes [138]. It is long believed that astrocytes are crucial in the development of the BBB properties [158]. It has been shown that co-culture of brain endothelial cells with astrocytes can enhance TJs of the brain endothelium [159]. Furthermore, endothelial cultures incubated with astrocyte-conditioned media have shown to improve BBB character‐ istics in vitro by de novo protein synthesis of γGTP in cerebral microvessel endothelial cells, which indicate that the glial cells may induce the cerebral capillary endothelial cells to express differentiated properties which allow the endothelium to function as the blood–brain barrier [160]. Also, astrocytes have been shown to regulate cerebral microvascular permeability [161], via dynamic calcium signaling between astrocytes and the endothelium [162]. This has been explained by Zonta et al., whereby an increase in neuron-induced astrocyte calcium promotes secretion of vasodilatory substances from perivascular astrocyte endfeet, resulting in im‐ proved local blood flow [163]. This work constituted a breakthrough in the knowledge of both astrocyte function and regulation of the activity-dependent cerebral blood flow [164].

Finally, another glial cell type present in the BBB is microglia which migrates from the yolk sac into the CNS parenchyma during embryogenesis [165]. Microglia performs critical functions in innate and adaptive brain immune responses. Microglia, when activated, trans‐ forms from "ramified" to an "ameboid" and ultimately to a "phagocytic" form. This evolution is correlated with alterations in expression of surface antigens and cytokines release [138]. Studies have shown that perivascular microglial cells are derived from bone marrow [166].

In vivo studies demonstrated that resident microglia cells in the brain parenchyma also communicate with CNS microvessels. This may suggest that microglia plays an important role in regulating BBB features during embryogenesis and diseases [167], besides an indisputable function in CNS development and homeostasis [168]. Furthermore, it has been shown that during embryonic stages of CNS vascularization, stabilization, and fusion of brain endothelial cells are mediated by resident microglial cells [167]. Also, interestingly, it has been shown that specific depletion of microglia results in reduced vessel density in a mouse model of choroidal neo-vascularization [169]. There are studies which suggest that microglial activation may be related to BBB disruption [170] apparently by producing ROS, through NADPH oxidase, which in turn impairs BBB function. Furthermore, TNF-α released from activated microglia has shown to affect BBB integrity, as permeability of endothelial cells co-cultured with microglia, was increased when microglial cells were activated by LPS and it was blocked by a neutralizing antibody against TNF-α, indicating that TNF-α contributes to BBB dysfunction [171]. The post-traumatic inflammatory response is shown to be associated with expression of cytokines such as IL-1β or metalloproteinases particularly MMP2, 3 and 5 produced mainly by microglia at the lesion site. It has also been demonstrated that these proteins can cause disruption of the basal lamina and/or redistribution and degradation of the TJs complexes [172, 173] resulting in BBB breakdown and neurological disorders after traumatic injury [174].

## **3.3. Junctional complexes and cytoskeleton linked proteins at the BBB**

The presence of junctional complexes is one of the main characteristics of inter-endothelial space of the cerebral microvasculature which include TJs, AJs, and possibly gap junctions. Both TJs and AJs restrict permeability across the endothelium, whereas gap junctions (if present) mediate intercellular communication [175].

## *3.3.1. Tight junction proteins*

Occludin, the first integral membrane protein within the TJ family identified [176], is a 60– 65 kDa protein with four transmembrane domains which is highly expressed in the cerebral endothelium [177, 178], whereas it is much less present in non-neural endothelial cells [179]. A construct with a deletion in the N-terminal of occludin showed a considerable effect on the TJ integrity [180]. Deletion of occludin in mice has been shown to cause a complex phenotype and postnatal growth retardation [181]. However, occludin functions are not limited to its role as a TJ protein. For instance, there are studies demonstrating that occludin can regulate epithelial cell differentiation [182] and control cell apoptosis in mouse hepatocytes [183]. Also, it has been shown, in a mouse model of multiple sclerosis, that occludin dephosphorylation leads to noticeable signs of disease which occur just prior to apparent changes in the BBB permeability [184]. In cerebral ischemia, occludin and other TJs were shown to be vulnerable to attack by matrix metalloproteinases [185].

The claudin family, 20–24 kDa proteins, includes more than 20 members that build TJ strands through homophilic interactions [186]. Claudin 3, 5, and 12 play important roles at the BBB [187, 188]. Each claudin regulates the diffusion of a group of molecules of a certain size. For instance, mice neonates with deletion of claudin-5 die due to a size-selective loosening of the BBB for molecules smaller than 800 Da [188]. It is speculated that claudins determine the primary "seal" of the TJ and occludin functions as an additional support structure [142]. It has been demonstrated that overexpression of claudin species can induce development of TJ-like strands, while expression of occludin does not lead to the formation of TJ; rather, occludin only localizes to TJ in cells that have already been transfected with claudins [189].

In addition to the claudin/occluding proteins, junctional adhesion molecules (JAMs) perform an important role in the organization of TJ assembly as it has been reported that these proteins can reduce cell permeability and enhance resistance to macromolecules [190]. JAM-1, a 40-kDa member of the IgG superfamily composed of a single membrane-spanning chain with a large extracellular domain [191], is postulated to mediate the attachment of neighboring cell membranes via homophilic interactions [192]. However, there is not much known about JAMs exact function in the mature BBB, but in the rat cortical cold injury model, a characterized in vivo model of BBB breakdown it has been shown that endothelial JAM-1 is significantly reduced which strengthens the idea that JAM-1 contributes to TJs integrity [193].

#### *3.3.2. Adherens junction proteins*

The first component of AJs is the vascular endothelial (VE)-cadherin, an endothelial-specific integral membrane protein which is linked to the cytoskeleton via catenins [175] and mediates cell-cell adhesion via homophilic interactions between the extracellular domains of proteins expressed in adjacent cells [194]. In vitro and in vivo studies have shown that VE-cadherin is required for the cells to regulate vessel maintenance, that is, for the correct organization of the new vessels and for the endothelial integrity in the quiescent vessels [195]. Various mecha‐ nisms have been suggested for how VE-cadherin regulates endothelial functions; such as direct activation of signaling molecules with a role in survival and organization of the actin cytos‐ keleton and regulation of gene transcription cofactors and formation of complexes with growth factor receptors [138].

Even though AJs are required at the BBB to decrease the endothelial permeability [196], it is principally the TJs that present the low paracellular permeability and high electrical resistance [142, 197].

Another component of AJs is Platelet endothelial cell adhesion molecule 1 (PECAM-1), an integral membrane protein of the Ig superfamily with six extracellular domains, a short transmembrane, and a large cytoplasmic domain which is highly expressed in blood and vascular cells especially endothelial cells [198]. PECAM-1 plays a major role in the migration of leukocytes across endothelium [138] and contributes to the steady-state barrier function of endothelial cells. It also functions as a mechano-sensor and stimulates reconstruction of the barrier integrity following perturbations, including the BBB [199, 200]; all physiologic proc‐ esses that rely on the junctional integrity and signaling [198].

## *3.3.3. Associated proteins*

**3.3. Junctional complexes and cytoskeleton linked proteins at the BBB**

mediate intercellular communication [175].

to attack by matrix metalloproteinases [185].

*3.3.2. Adherens junction proteins*

*3.3.1. Tight junction proteins*

16 Exploring New Findings on Amyloidosis

The presence of junctional complexes is one of the main characteristics of inter-endothelial space of the cerebral microvasculature which include TJs, AJs, and possibly gap junctions. Both TJs and AJs restrict permeability across the endothelium, whereas gap junctions (if present)

Occludin, the first integral membrane protein within the TJ family identified [176], is a 60– 65 kDa protein with four transmembrane domains which is highly expressed in the cerebral endothelium [177, 178], whereas it is much less present in non-neural endothelial cells [179]. A construct with a deletion in the N-terminal of occludin showed a considerable effect on the TJ integrity [180]. Deletion of occludin in mice has been shown to cause a complex phenotype and postnatal growth retardation [181]. However, occludin functions are not limited to its role as a TJ protein. For instance, there are studies demonstrating that occludin can regulate epithelial cell differentiation [182] and control cell apoptosis in mouse hepatocytes [183]. Also, it has been shown, in a mouse model of multiple sclerosis, that occludin dephosphorylation leads to noticeable signs of disease which occur just prior to apparent changes in the BBB permeability [184]. In cerebral ischemia, occludin and other TJs were shown to be vulnerable

The claudin family, 20–24 kDa proteins, includes more than 20 members that build TJ strands through homophilic interactions [186]. Claudin 3, 5, and 12 play important roles at the BBB [187, 188]. Each claudin regulates the diffusion of a group of molecules of a certain size. For instance, mice neonates with deletion of claudin-5 die due to a size-selective loosening of the BBB for molecules smaller than 800 Da [188]. It is speculated that claudins determine the primary "seal" of the TJ and occludin functions as an additional support structure [142]. It has been demonstrated that overexpression of claudin species can induce development of TJ-like strands, while expression of occludin does not lead to the formation of TJ; rather, occludin only

In addition to the claudin/occluding proteins, junctional adhesion molecules (JAMs) perform an important role in the organization of TJ assembly as it has been reported that these proteins can reduce cell permeability and enhance resistance to macromolecules [190]. JAM-1, a 40-kDa member of the IgG superfamily composed of a single membrane-spanning chain with a large extracellular domain [191], is postulated to mediate the attachment of neighboring cell membranes via homophilic interactions [192]. However, there is not much known about JAMs exact function in the mature BBB, but in the rat cortical cold injury model, a characterized in vivo model of BBB breakdown it has been shown that endothelial JAM-1 is significantly

The first component of AJs is the vascular endothelial (VE)-cadherin, an endothelial-specific integral membrane protein which is linked to the cytoskeleton via catenins [175] and mediates

localizes to TJ in cells that have already been transfected with claudins [189].

reduced which strengthens the idea that JAM-1 contributes to TJs integrity [193].

There are various proteins in the cytoplasm that associate with the transmembrane compo‐ nents of the TJ [142]. Multi-domain scaffolding proteins of membrane-associated guanylate kinase-like homolog family, including zonulae occludentes (ZO) proteins, such as ZO-1 and ZO-2, are characterized for their contributing to the cytoskeletal anchorage for the transmem‐ brane TJ proteins, binding to the claudins, occludin, and actin [201, 202] and controlling the correct distribution of claudins [203].

ZO-1, a 220-kDa protein, is one of the first and best-studied proteins in TJs [204]. It is expressed in epithelial and endothelial cells and even other cells which do not have TJs [205]. It has also been observed to be associated with AJs [206] and gap junction proteins [207]. ZO-1 is located just below the TJ membrane contact points and has been found to be important for both function and stability of TJs. ZO-1 dissociation from the junctional complex is shown to be correlated with increased permeability in the BBB in vitro model [208]. ZO-2, a 160-kDa protein with a high sequence homology to ZO-1 [209], interestingly, has been demonstrated to function redundantly with ZO-1, replacing it and promoting the formation of TJ in the cells lacking ZO-1 [210].

Another component present in the junctional complexes is actin. Although actin has been basically considered structural in function, it is now obvious that the anchorage of AJs and TJs to the actin cytoskeleton is critical for both barrier stability and also for the regulation of cell polarity, cellular movement, fluid sensing, and cell-contact inhibition [211]. Studies using mice lacking the actin-binding protein dystrophin have demonstrated increased brain vascular permeability due to disorganized α-actin cytoskeleton in endothelial cells and astrocytes [212]. These findings demonstrate that properly arranged actin filaments and their binding to the TJ and/or AJ proteins are critical for normal barrier function. Studies have also shown that HIV-1 gp120 and alcohol are able to alter the cytoskeleton and induce stress fiber actin formation, causing increased permeability of the human BBB endothelium [213]. It has been suggested that alcohol-mediated changes in the brain endothelial cells (BEC) monolayers may increase diffusion of plasma components and viral penetration across the BBB, and therefore, especially at levels attained in heavy drinkers, accelerate HIV-1 penetration into the brain [138].

## **3.4. Transport at the BBB**

Transcellular transport at the BBB happens through several mechanisms. Small lipophilic molecules can access the brain by passive diffusion. Brain CSF bulk flow mediates transport of molecules with different sizes into the CSF at a slow rate [214]. For potentially toxic molecules and metabolic waste products, the CSF works as a sink. These molecules are then eliminated from the CSF back into the blood by active transport or facilitated diffusion across the choroid plexus epithelium, or by vacuolar transport across the epithelial arachnoid granulations [138]. Efflux pumps return many undesired molecules back to the blood to regulate passive transport into the brain [215]. The flow of the plasma oxygen and carbon dioxide across the BBB is diffusive. Therefore, oxygen supply and carbon dioxide elimination are blood-flow dependent, so the gas transport is sufficient as long as cerebral blood flow is within physiological limits [216].

Small polar molecules, such as glucose, amino acids and nucleosides, can pass the BBB by carrier-mediated transport. These carriers can be multi-ligands or specific to only one mole‐ cule, such as GLUT1 glucose transporter, the L1 large neutral amino acid transporter, and the CNT2 adenosine transporter which have been cloned from BBB-specific cDNA libraries [146]. The direction of the concentration gradient is usually from blood to brain, regulated by brain demands and the concentration of metabolites in plasma [138]. Ion transporters such as the sodium pump localized on the abluminal membrane are important to sustain the highconcentration gradient for sodium at the BBB, so that sodium-dependent transport can happen. Also, sodium–hydrogen exchanger expressed at the luminal membrane and chloride– bicarbonate exchanger expressed at both sides [217] play significant roles in regulating intracellular pH in the endothelium.

Moreover, large solutes, such as proteins and peptides, are transported across the BBB by receptor-mediated or adsorption-mediated endocytic transport [218, 219]. Analysis of the rat BBB transcriptome has shown that 10–15% of all proteins in the neurovascular unit are transporters which emphasize the critical role of these molecules at the BBB [215, 220]. As a consequence of this controlled transport, the concentration of amino acids and proteins can suffer considerable variations, whereas relatively small differences exist in the concentration of ions between blood and CSF [221].

In the BBB, the ABC transporters for efflux are permeability glycoprotein (P-gp, multidrug resistance protein, ABCB1), the multidrug resistance-associated proteins and breast cancer resistance protein [222, 223] whose major role is to operate as active efflux pumps, transporting a diverse range of lipid-soluble compounds out of the brain capillary endothelium and the CNS, eliminating potentially neurotoxic endogenous or xenobiotic molecules [216, 224]. P-gp is expressed at the luminal and abluminal membrane, as well as in pericytes and astrocytes [225], and is distributed along the nuclear envelope, in caveolae, cytoplasmic vesicles, Golgi complex, and rough ER [138]. The endothelial cells at the BBB also express several transporters for hormones, some cytokines, and chemokines [226]. Large proteins, such as transferrin, LDL, leptin, immunoglobulin G (IgG), insulin, and insulin-like growth factor also use receptormediated transport systems to pass through the BBB [146].

Internalization of ligands and receptors from the plasma membrane is cholesterol sensitive [227] and comprises endocytosis of caveolae, vesicles enriched in caveolin-1 [228]. The caveolar membranes carry several receptors including those for insulin, albumin, receptor for advance glycation endproducts (RAGE), LDL, HDL [229] and are also closely associated with P-gp. Moreover, caveolin-1 can affect the levels of TJs in endothelial cells of the BBB [230]. Interest‐ ingly, study of the ultrastructure of the BBB in young and aged mice during ischemia has demonstrated that permeability is associated with a remarkable increase in endothelial caveolae and vacuoles although TJs were generally intact [231].

## **3.5. Aβ clearance at the BBB**

permeability due to disorganized α-actin cytoskeleton in endothelial cells and astrocytes [212]. These findings demonstrate that properly arranged actin filaments and their binding to the TJ and/or AJ proteins are critical for normal barrier function. Studies have also shown that HIV-1 gp120 and alcohol are able to alter the cytoskeleton and induce stress fiber actin formation, causing increased permeability of the human BBB endothelium [213]. It has been suggested that alcohol-mediated changes in the brain endothelial cells (BEC) monolayers may increase diffusion of plasma components and viral penetration across the BBB, and therefore, especially

at levels attained in heavy drinkers, accelerate HIV-1 penetration into the brain [138].

Transcellular transport at the BBB happens through several mechanisms. Small lipophilic molecules can access the brain by passive diffusion. Brain CSF bulk flow mediates transport of molecules with different sizes into the CSF at a slow rate [214]. For potentially toxic molecules and metabolic waste products, the CSF works as a sink. These molecules are then eliminated from the CSF back into the blood by active transport or facilitated diffusion across the choroid plexus epithelium, or by vacuolar transport across the epithelial arachnoid granulations [138]. Efflux pumps return many undesired molecules back to the blood to regulate passive transport into the brain [215]. The flow of the plasma oxygen and carbon dioxide across the BBB is diffusive. Therefore, oxygen supply and carbon dioxide elimination are blood-flow dependent, so the gas transport is sufficient as long as cerebral blood flow is

Small polar molecules, such as glucose, amino acids and nucleosides, can pass the BBB by carrier-mediated transport. These carriers can be multi-ligands or specific to only one mole‐ cule, such as GLUT1 glucose transporter, the L1 large neutral amino acid transporter, and the CNT2 adenosine transporter which have been cloned from BBB-specific cDNA libraries [146]. The direction of the concentration gradient is usually from blood to brain, regulated by brain demands and the concentration of metabolites in plasma [138]. Ion transporters such as the sodium pump localized on the abluminal membrane are important to sustain the highconcentration gradient for sodium at the BBB, so that sodium-dependent transport can happen. Also, sodium–hydrogen exchanger expressed at the luminal membrane and chloride– bicarbonate exchanger expressed at both sides [217] play significant roles in regulating

Moreover, large solutes, such as proteins and peptides, are transported across the BBB by receptor-mediated or adsorption-mediated endocytic transport [218, 219]. Analysis of the rat BBB transcriptome has shown that 10–15% of all proteins in the neurovascular unit are transporters which emphasize the critical role of these molecules at the BBB [215, 220]. As a consequence of this controlled transport, the concentration of amino acids and proteins can suffer considerable variations, whereas relatively small differences exist in the concentration

In the BBB, the ABC transporters for efflux are permeability glycoprotein (P-gp, multidrug resistance protein, ABCB1), the multidrug resistance-associated proteins and breast cancer resistance protein [222, 223] whose major role is to operate as active efflux pumps, transporting

**3.4. Transport at the BBB**

18 Exploring New Findings on Amyloidosis

within physiological limits [216].

intracellular pH in the endothelium.

of ions between blood and CSF [221].

Increase in either total Aβ levels or the relative concentration of both Aβ40 and Aβ42 (where the former is more concentrated in cerebrovascular plaques and the latter in neuritic plaques) have been implicated in the pathogenesis of both familial and sporadic AD.

There are several identified pathways for the removal of the Aβ from the brain. Aβ peptides mainly produced in neurons are degraded by peptidases. Through efflux transporters located in cerebral vessels, Aβ flows out from brain parenchyma into the plasma. Aβ is also removed through perivascular pathways into the cervical lymph nodes as Aβ within ISF diffuses in the extracellular spaces of the brain parenchyma entering basement membranes of capillaries, passing into the tunica media of arteries, and draining out of the brain. Aβ can also be taken up by different cells in the brain [232]. A few AD cases are familial AD, associated with genetic mutations which promote an increase in the production of Aβ [233]. On the other hand, the cause of the sporadic AD, the majority of the AD cases, is considered to be the impaired clearance of Aβ from the brain [234, 235]. In this viewpoint, AD is associated with cerebro‐ vascular disorder, which drives the accumulation of Aβ at the blood vessels (cerebral amyloid angiapothy, CAA) and in the brain parenchyma, extracellularly [138, 236], and intraneuronal lesions—NTFs [237].

In the healthy brain, Aβ concentration is accurately regulated by its rate of production, its enzymatic degradation [238], its rapid clearance across the BBB through LRP1 [239, 240], and influx back into the brain by RAGE [241]. These receptors are multi-ligand cell surface receptors that, in addition to Aβ, mediate the clearance of a large number of proteins. P-gp, belonging to the superfamily of ATP-binding cassette (ABC) transporters, is also involved in effluxing Aβ out of the brain. While LRP1 and P-gp appear to mediate the efflux of Aβ from the brain to the periphery, RAGE has been strongly implicated in Aβ influx back into the CNS. With increasing age and also in AD pathology, the expression of the Aβ efflux transporters is decreased and the Aβ influx transporter expression is increased at the BBB, adding to the amyloid burden in the brain and its gradual neurotoxic oligomerization [242]. Thus, continu‐ ous Aβ elimination by transport across the BBB and/or metabolism is essential to prevent its potentially neurotoxic accumulations in the brain [234]. Studies have demonstrated several transport proteins such as α2-macroglobulin, TTR, apolipoprotein E (apoE), and apolipopro‐ tein J (apoJ), which bind to Aβ and control its clearance, metabolism, and aggregation [243]. It has been shown that apoJ can increase the BBB clearance of Aβ42 [244], while apoE disrupts the clearance of free Aβ across the mouse BBB, in an isoform-specific manner (apoE4>apoE3 or apoE2), by driving Aβ transport from LRP1 to VLDLR which internalizes Aβ-apoE complex at a slower rate than LRP1 [245]. Another transport pathway is the bulk flow of the ISF into the CSF through the perivascular Virchow-Robin arterial spaces, which is followed by drainage into the plasma across the arachnoid villi [243].

## *3.5.1. Aβ transport by LRP1*

LRP1, the major efflux transporter for Aβ across the BBB [239] and a member of the LDL receptor family, acts as both a multifunctional scavenger and a signaling receptor. LRP1 is synthesized as a precursor molecule (600 kDa) in the ER. Then in the Golgi network, a cleavage generates an 85 kDa transmembrane beta-subunit (containing two intracellular NPxY motifs) that remains non-covalently connected to the extracellular 515 kDa alpha-subunit (containing 4 ligand-binding domains for more than 30 ligands) [246].

Transcytosis of Aβ across the BBB starts with its binding to LRP1 at the abluminal side of the cerebral endothelium [239, 240]. However, this has been controversial due to the studies that failed to demonstrate a role for LRP-mediated transcytosis, but rather showed a role for LRP receptors in endocytosis and degradation of Aβ [247]. Anyway, the significant function of LRP1 in AD is not only portrayed by LRP1-mediated endocytosis of Aβ but also by data showing that the cytoplasmic domain of LRP1 has been involved in APP processing [248]. Cleavage of the extracellular domain of LRP1 by beta-secretase (BACE1) releases soluble LRP1 (sLRP1) in plasma [249]. Reduced expression of LRP1 has been described during aging in animal models and in AD individuals [240, 249]. In astrocytes, LRP1 also mediates degradation of amyloid deposits via apoE [250].

## *3.5.2. Aβ transport by RAGE*

RAGE, a multiligand receptor in the immunoglobulin superfamily, which can bind to various ligands including Aβ and advanced glycation end products (AGE proteins) [251], is the most influential influx transporter for Aβ across the BBB [241]. Interestingly, and unlike many receptors (including LRP1), RAGE expression is triggered by the accumulation of RAGE ligands, meaning that the levels of RAGE expression are determined by the levels of its ligands. In the healthy brain, RAGE is expressed at minimal levels at the BBB, except at the endothelium of bigger microvessels of the brain. However, when RAGE ligands increase in the AD brain, RAGE expression rises in the affected cerebral vessels, neurons or microglia [251]. This mechanism worsens the cellular dysfunction due to RAGE-Aβ interactions. Circulating Aβ can enter the brain by a special receptor-mediated transport mechanism that is dependent on RAGE expression on the luminal surface of brain vessels [252]. Following Aβ binding to RAGE at the luminal membrane of the BBB, transcytosis of circulating Aβ across the BBB into the brain parenchyma and its binding to neurons occurs. Moreover, activation of NF-kB in the endothelial cell leads to proinflammatory cytokines secretion and cerebral blood flow sup‐ pression [241]. Aβ-RAGE interaction not only generates oxidative damage to RAGE-express‐ ing neurons, which results in neuronal degeneration, but also activates microglial cells, indirectly leading to inflammation [251]. Therefore, repression of Aβ-RAGE interaction in the BBB can inhibit Aβ influx, oxidant stress, and cytokine production. The inhibitors of Aβ/RAGE interaction have been shown to improve the BBB function and the cerebral blood flow responses to the brain activation, and to reduce neuroinflammation. Some RAGE/Aβ blockers are currently being tested in AD patients [138]. While RAGE is involved in the influx of Aβ into the brain, the soluble isoform of RAGE (sRAGE) has been detected in the plasma. It seems that sRAGE competes with cell-surface RAGE for ligand binding, thus increasing the elimi‐ nation of circulating Aβ [145].

## **3.6. BBB dysfunction in AD**

decreased and the Aβ influx transporter expression is increased at the BBB, adding to the amyloid burden in the brain and its gradual neurotoxic oligomerization [242]. Thus, continu‐ ous Aβ elimination by transport across the BBB and/or metabolism is essential to prevent its potentially neurotoxic accumulations in the brain [234]. Studies have demonstrated several transport proteins such as α2-macroglobulin, TTR, apolipoprotein E (apoE), and apolipopro‐ tein J (apoJ), which bind to Aβ and control its clearance, metabolism, and aggregation [243]. It has been shown that apoJ can increase the BBB clearance of Aβ42 [244], while apoE disrupts the clearance of free Aβ across the mouse BBB, in an isoform-specific manner (apoE4>apoE3 or apoE2), by driving Aβ transport from LRP1 to VLDLR which internalizes Aβ-apoE complex at a slower rate than LRP1 [245]. Another transport pathway is the bulk flow of the ISF into the CSF through the perivascular Virchow-Robin arterial spaces, which is followed by drainage

LRP1, the major efflux transporter for Aβ across the BBB [239] and a member of the LDL receptor family, acts as both a multifunctional scavenger and a signaling receptor. LRP1 is synthesized as a precursor molecule (600 kDa) in the ER. Then in the Golgi network, a cleavage generates an 85 kDa transmembrane beta-subunit (containing two intracellular NPxY motifs) that remains non-covalently connected to the extracellular 515 kDa alpha-subunit (containing

Transcytosis of Aβ across the BBB starts with its binding to LRP1 at the abluminal side of the cerebral endothelium [239, 240]. However, this has been controversial due to the studies that failed to demonstrate a role for LRP-mediated transcytosis, but rather showed a role for LRP receptors in endocytosis and degradation of Aβ [247]. Anyway, the significant function of LRP1 in AD is not only portrayed by LRP1-mediated endocytosis of Aβ but also by data showing that the cytoplasmic domain of LRP1 has been involved in APP processing [248]. Cleavage of the extracellular domain of LRP1 by beta-secretase (BACE1) releases soluble LRP1 (sLRP1) in plasma [249]. Reduced expression of LRP1 has been described during aging in animal models and in AD individuals [240, 249]. In astrocytes, LRP1 also mediates degradation of amyloid

RAGE, a multiligand receptor in the immunoglobulin superfamily, which can bind to various ligands including Aβ and advanced glycation end products (AGE proteins) [251], is the most influential influx transporter for Aβ across the BBB [241]. Interestingly, and unlike many receptors (including LRP1), RAGE expression is triggered by the accumulation of RAGE ligands, meaning that the levels of RAGE expression are determined by the levels of its ligands. In the healthy brain, RAGE is expressed at minimal levels at the BBB, except at the endothelium of bigger microvessels of the brain. However, when RAGE ligands increase in the AD brain, RAGE expression rises in the affected cerebral vessels, neurons or microglia [251]. This mechanism worsens the cellular dysfunction due to RAGE-Aβ interactions. Circulating Aβ can enter the brain by a special receptor-mediated transport mechanism that is dependent on

into the plasma across the arachnoid villi [243].

4 ligand-binding domains for more than 30 ligands) [246].

*3.5.1. Aβ transport by LRP1*

20 Exploring New Findings on Amyloidosis

deposits via apoE [250].

*3.5.2. Aβ transport by RAGE*

Failure in the BBB function is an outstanding event in the development and progression of several CNS diseases including multiple sclerosis [253], ischemia [254], Parkinson's disease, and AD [255]. While in some of the diseases increased BBB permeability is an outcome and consequence of the pathology (such as ischemic stroke), in other cases, BBB failure may be a causative event for the disease (such as multiple sclerosis). Furthermore, BBB dysfunction can be mild with temporary opening of TJs, or it can be a chronic breakdown [256], with changes in transporters and enzymes happening at the same time [216].

Although cerebrovascular abnormalities have been noted in AD, the starting point between BBB failure and AD pathology is not clear yet [142, 257]. Nevertheless, BBB homeostasis is altered in the initial stages of AD leading to the production of proinflammatory cytokines and suppressors of the cerebral blood flow by endothelial cells; then, amyloid deposits are observed in cerebral capillaries and vessels in the later stages of AD [258]. A large number of alterations in the structure and function of the BBB were shown to occur in AD. For instance: decreased LRP1 expression in human brain microvasculature [239], increased LRP1 oxidative damage [259], impaired microvascular P-gp [260], increased expression of RAGE in the cerebral vessels, neurons and microglia [251]. Moreover, it has been shown to occur: decreased glucose consumption by the brain, thus predicting a decay in cognitive function [261], which has been described by the reduction in the GLUT-1 transporters expression (protein levels) in AD hippocampus microvessels [262] but not a decrease in GLUT-1 mRNA levels [263]. Other alterations have been described, such as: decreased endothelial mitochondrial density, increased endothelial vacuolization, accumulation of collagen and perlecans in the basement capillary membrane [264], and increased pinocytotic vesicles [265].Also, it has been reported a reduced number and smaller diameter of capillaries in CNS implying the diminished overall surface for LRP1-mediated transcytosis of Aβ across the BBB, and also a decreased length of brain capillaries [266] which lowers transport of energy substrates and nutrients across the BBB, and reduces the clearance of neurotoxins from the brain [138]; Overexpression and accumulation of occludin in frontal cortex and basal ganglia of AD brains [267] have also been described as well as lower expression of genes such as the homeobox gene MEOX2 (or GAX), a regulator of vascular differentiation [268]. Finally, activated microglia [269] and astrocytes, the resident brain immune cells present in neurovascular unit of the BBB, have been observed surrounding Aβ plaques in AD brains, releasing inflammatory cytokines, such as IL-1 and IL-6, TNF-α, and transforming growth factor-β [174].

## **4. Periphery and Alzheimer's disease**

## **4.1. Aβ levels at the periphery**

Although studies in rodents have shown an increase in plasma Aβ levels, data in human AD patients have been contradictory; while some demonstrate increased levels of circulating Aβ [270], others report decreased [271] or unchanged [272] levels. Nevertheless, the significant role of circulating Aβ in AD pathology cannot be neglected. Interestingly, Aβ is also produced outside the brain in considerable amounts by the platelets, skeletal muscle, vascular walls, kidney, heart, liver, and by other non-neural tissues [273–275]. These pools may also provide a dynamic exchange of Aβ between the brain and periphery. However, Aβ peptides in the periphery cannot form filamentous structures, probably due to the presence of multiple circulating molecules that bind Aβ and thereby change its free-plasma levels [276].

## *4.1.1. Plasma proteins involved in peripheral sink and clearance of Aβ*

Continuous removal of Aβ, not only from the brain but also from blood and from the entire organism, is essential for preventing its accumulation in the brain. Aβ in the plasma is bound to a number of proteins such as albumin [277], apoE and apoJ [278], TTR and a soluble form of LRP1 (sLRP1). In healthy human plasma, sLRP1 is a major endogenous brain Aβ sinker that sequesters 70–90% of plasma Aβ. sLRP1 has been demonstrated to bind the majority of circulating Aβ preventing RAGE-dependent influx of Aβ back to the brain, while improving its systemic clearance [279]. These data confirm the peripheral sink hypothesis, which imply systemic clearance of Aβ via binding proteins in serum and preventing its uptake through RAGE. Accordingly, this explains how some therapeutics, such as peripheral administration of sLRP1 or antibodies against Aβ [280], decrease Aβ levels in the brain. Moreover, AD patients show decreased plasma sLRP1 levels and increased oxidative damage to sLRP1, reducing its binding capacity for Aβ. This, in turn, increases the free Aβ fraction in plasma [279], that is, accessible for RAGE-dependent influx back to the brain [281]**.**

Higher sRAGE levels are linked to reduced risk of developing several disorders such as cardiovascular disease and AD. Levels of sRAGE are decreased in AD and in vascular dementia, which may confer a target for therapeutic purposes [282]. It has been demonstrated that systemic administration of a truncated form of RAGE decreases Aβ load in a transgenic mouse model [241]. Moreover, sRAGE has an inverse relationship with cholesterol, presenting another modulatory impact on Aβ metabolism due to the role of cholesterol as a mediator of inflammation and APP processing [145].

## *4.1.2. Alterations in other plasma proteins in AD*

Few proteins are synthesized solely by the brain or are present in higher concentrations in CSF compared with the blood. In conditions of the BBB failure, these CSF markers can appear or be increased in the plasma [283]. Hence, estimating levels of CSF proteins in the plasma may be a reliable method to control BBB integrity. For instance, S-100 is primarily produced in the brain by astrocytes and when the BBB is disturbed it is quickly released from the brain and appears in the blood [284].

## **4.2. Liver and Aβ elimination**

accumulation of occludin in frontal cortex and basal ganglia of AD brains [267] have also been described as well as lower expression of genes such as the homeobox gene MEOX2 (or GAX), a regulator of vascular differentiation [268]. Finally, activated microglia [269] and astrocytes, the resident brain immune cells present in neurovascular unit of the BBB, have been observed surrounding Aβ plaques in AD brains, releasing inflammatory cytokines, such as IL-1 and

Although studies in rodents have shown an increase in plasma Aβ levels, data in human AD patients have been contradictory; while some demonstrate increased levels of circulating Aβ [270], others report decreased [271] or unchanged [272] levels. Nevertheless, the significant role of circulating Aβ in AD pathology cannot be neglected. Interestingly, Aβ is also produced outside the brain in considerable amounts by the platelets, skeletal muscle, vascular walls, kidney, heart, liver, and by other non-neural tissues [273–275]. These pools may also provide a dynamic exchange of Aβ between the brain and periphery. However, Aβ peptides in the periphery cannot form filamentous structures, probably due to the presence of multiple

Continuous removal of Aβ, not only from the brain but also from blood and from the entire organism, is essential for preventing its accumulation in the brain. Aβ in the plasma is bound to a number of proteins such as albumin [277], apoE and apoJ [278], TTR and a soluble form of LRP1 (sLRP1). In healthy human plasma, sLRP1 is a major endogenous brain Aβ sinker that sequesters 70–90% of plasma Aβ. sLRP1 has been demonstrated to bind the majority of circulating Aβ preventing RAGE-dependent influx of Aβ back to the brain, while improving its systemic clearance [279]. These data confirm the peripheral sink hypothesis, which imply systemic clearance of Aβ via binding proteins in serum and preventing its uptake through RAGE. Accordingly, this explains how some therapeutics, such as peripheral administration of sLRP1 or antibodies against Aβ [280], decrease Aβ levels in the brain. Moreover, AD patients show decreased plasma sLRP1 levels and increased oxidative damage to sLRP1, reducing its binding capacity for Aβ. This, in turn, increases the free Aβ fraction in plasma [279], that is,

Higher sRAGE levels are linked to reduced risk of developing several disorders such as cardiovascular disease and AD. Levels of sRAGE are decreased in AD and in vascular dementia, which may confer a target for therapeutic purposes [282]. It has been demonstrated that systemic administration of a truncated form of RAGE decreases Aβ load in a transgenic mouse model [241]. Moreover, sRAGE has an inverse relationship with cholesterol, presenting another modulatory impact on Aβ metabolism due to the role of cholesterol as a mediator of

circulating molecules that bind Aβ and thereby change its free-plasma levels [276].

*4.1.1. Plasma proteins involved in peripheral sink and clearance of Aβ*

accessible for RAGE-dependent influx back to the brain [281]**.**

inflammation and APP processing [145].

IL-6, TNF-α, and transforming growth factor-β [174].

**4. Periphery and Alzheimer's disease**

**4.1. Aβ levels at the periphery**

22 Exploring New Findings on Amyloidosis

At the periphery, the liver play important roles not only in the storage and in the release of nutrients and proteins but also in the neutralization and elimination of a variety of toxic substances such as Aβ from the plasma.

The receptor for LDL (LDLr), LRP1, and megalin/LRP2 play important roles in endocytosis of lipoproteins and systemic lipid homeostasis. Moreover, LRP1 mediates the clearance of a multitude of extracellular ligands such as Aβ and regulates diverse signaling processes such as growth factor signaling, inflammatory signaling pathways, apoptosis, and phagocytosis in liver.

In the liver, LRP1, which can be blocked by the receptor-associated protein (RAP), has been shown to be the predominant transporter that mediates systemic clearance of Aβ [285]. LRP1 localized to hepatic cells binds to and systemically clears circulating Aβ. Reduced hepatic LRP1 levels are associated with decreased peripheral Aβ clearance in aged rats. In aged squirrel monkeys, systemic clearance of Aβ is also reduced and associated with increased Aβ levels in the brain. In addition to the liver, sLRP1-Aβ complexes and free Aβ are removed through the kidneys [279]. Liver was also demonstrated to be the major source of Aβ and to be able to regulate brain Aβ levels [286].

The half-life of circulating Aβ is short, in the range of minutes [287, 288]. This also suggests that rapid systemic clearance of Aβ prevents reuptake by RAGE after efflux.

It has been shown that some proteins such as insulin [289] and TTR [290] increase LRP1 levels in hepatic plasma membrane, and in turn enhance peripheral Aβ clearance. Insulin-degrading enzyme, a zinc metalloendopeptidase that hydrolyzes numerous peptides, including Aβ [238], insulin [291] and the AICD, has been purified from several mammalian tissues including liver, brain and blood cells [292]. Furthermore, experiments in rats demonstrated that after 3.5 min post-infusion of radiolabelled Aβ into the lateral ventricle, 40% of the injected radioactivity was already in the blood and urine, and internalized by the liver and the kidneys, indicating not only a quick clearance mechanism but also the involvement of systemic organs in the elimination and catabolism of Aβ [293]. Therefore, the capacity of the liver to internalize, catabolize, and eliminate large doses of Aβ, may explain not only the low plasma Aβ levels but also its small variation noted with age and disease stages.

In some cases, both parenchymal and non-parenchymal liver cells take up proteins, whereas in other cases this is done mostly by hepatocytes, as happens for TTR, apoA-I, SAP and Aβ. In vivo and in vitro experiments showed that hepatocytes are the main cells involved in Aβ uptake (about 90%) and in its catabolism [288].

## **5. Transthyretin**

Transthyretin (TTR), formerly called prealbumin due to its electrophoretic characteristics, located just in front of the albumin band, is a plasma protein secreted mainly by the liver and choroid plexus (CP) [294]. The name "transthyretin" discloses its dual physiological role as a carrier for thyroid hormones [295] and retinol, the latter through the binding to retinol-binding protein (RBP) [296]. TTR was first described in the CSF [297] and shortly after in the plasma [298].

Although the involvement of TTR in the transport of thyroid hormones and RBP, as well as in FAP, is very well established, its involvement in neuroprotection is part of a very recent knowledge and constantly evolving.

## **5.1. TTR gene structure and expression**

TTR is codified by a single copy gene localized in the long arm of chromosome 18 [299]. The entire nucleotide sequence including the 5′ (transcription initiating site) and the 3′ (untrans‐ lated region) flanking regions was determined [300, 301] and attributed to the region 18q11.2– q12.1 [302]. The full gene is 7.6 kilobase (kb) long comprising 4 exons and 3 introns [300, 301]. Exon 1 contains 95 basepairs (bp), including 26 bp 5′ untranslated, and codes for a 20 aa residue leader peptide and aa 1–3 of the mature protein; exon 2 (131 bp), 3 (136 bp), and 4 (253 bp) hold the coding sequences for aa residues 4–47, 48–92 and 93–127, respectively. The introns (A, B and C) are 934, 2090 and 3308 bp long, respectively. Introns A and C contain two open reading frames (orf) of unknown significance [301].

The TTR mRNA spans ~0.7 kb and contains a 5′-untranslated region (26–27 nucleotides), a coding region (441 nucleotides), and a 3′-untranslated region (145–148 nucleotides) preceding the poly(A) tail [294, 303]. Human [299], rat [304, 305] and mouse [306] coding regions exhibit a considerable degree of homology (~85%), suggesting a phylogenetically preserved modu‐ lating role in gene expression.

TTR is predominantly synthesized by the liver where more than 90% of the protein is produced. The remaining is produced by the CP and the retina. TTR is detected in the fetal blood very early during development, as soon as eight weeks after conception [307]. TTR plasma concen‐ tration is age dependent, and in healthy newborns, it is about half of that in adults [308, 309]. TTR values vary from 20 to 40 mg/dL. In spite of the low TTR levels in CSF (~2 mg/dL), the CP is presented as the major site of TTR expression, expressed as a ratio of TTR/mass of tissue, corresponding to a ~30-fold higher than that found in plasma [310]. TTR represents 20% of the total CSF proteins [310].

With respect to the regulation of TTR expression, several studies showed that liver and CP TTR expression are increased in response to sex hormones, as demonstrated in mice [311, 312]. In rats, hydrocortisone and psychosocial stress are also inducers of TTR in the CP [313]. Others studies indicate that TTR can be expressed by brain cells, for instance in response to the heat shock factor 1 (HSF1) and to the AICD fragment of APP, as we will discuss further ahead in the context of TTR protection in AD.

## **5.2. TTR protein structure**

vivo and in vitro experiments showed that hepatocytes are the main cells involved in Aβ

Transthyretin (TTR), formerly called prealbumin due to its electrophoretic characteristics, located just in front of the albumin band, is a plasma protein secreted mainly by the liver and choroid plexus (CP) [294]. The name "transthyretin" discloses its dual physiological role as a carrier for thyroid hormones [295] and retinol, the latter through the binding to retinol-binding protein (RBP) [296]. TTR was first described in the CSF [297] and shortly after in the plasma

Although the involvement of TTR in the transport of thyroid hormones and RBP, as well as in FAP, is very well established, its involvement in neuroprotection is part of a very recent

TTR is codified by a single copy gene localized in the long arm of chromosome 18 [299]. The entire nucleotide sequence including the 5′ (transcription initiating site) and the 3′ (untrans‐ lated region) flanking regions was determined [300, 301] and attributed to the region 18q11.2– q12.1 [302]. The full gene is 7.6 kilobase (kb) long comprising 4 exons and 3 introns [300, 301]. Exon 1 contains 95 basepairs (bp), including 26 bp 5′ untranslated, and codes for a 20 aa residue leader peptide and aa 1–3 of the mature protein; exon 2 (131 bp), 3 (136 bp), and 4 (253 bp) hold the coding sequences for aa residues 4–47, 48–92 and 93–127, respectively. The introns (A, B and C) are 934, 2090 and 3308 bp long, respectively. Introns A and C contain two open reading

The TTR mRNA spans ~0.7 kb and contains a 5′-untranslated region (26–27 nucleotides), a coding region (441 nucleotides), and a 3′-untranslated region (145–148 nucleotides) preceding the poly(A) tail [294, 303]. Human [299], rat [304, 305] and mouse [306] coding regions exhibit a considerable degree of homology (~85%), suggesting a phylogenetically preserved modu‐

TTR is predominantly synthesized by the liver where more than 90% of the protein is produced. The remaining is produced by the CP and the retina. TTR is detected in the fetal blood very early during development, as soon as eight weeks after conception [307]. TTR plasma concen‐ tration is age dependent, and in healthy newborns, it is about half of that in adults [308, 309]. TTR values vary from 20 to 40 mg/dL. In spite of the low TTR levels in CSF (~2 mg/dL), the CP is presented as the major site of TTR expression, expressed as a ratio of TTR/mass of tissue, corresponding to a ~30-fold higher than that found in plasma [310]. TTR represents 20% of the

With respect to the regulation of TTR expression, several studies showed that liver and CP TTR expression are increased in response to sex hormones, as demonstrated in mice [311,

uptake (about 90%) and in its catabolism [288].

knowledge and constantly evolving.

**5.1. TTR gene structure and expression**

frames (orf) of unknown significance [301].

lating role in gene expression.

total CSF proteins [310].

**5. Transthyretin**

24 Exploring New Findings on Amyloidosis

[298].

The TTR mRNA codifies for the TTR-monomer; the polypeptide of 147 aa residues whose Nterminal region is a hydrophobic signal peptide of 20 aa residues. The TTR monomer is subjected to a cleaving process, during its migration through the ER, giving rise to the native TTR monomer after breaking of the signal peptide [294]. Assembly of four identical subunits (13,745 Da) occurs yielding the mature tetrameric protein, with a molecular mass of 54,980 Da [314].

**Figure 2.** The human TTR tetrameric structure complexed with two molecules of RBP (light green), which in turn is bound to retinol, and with T4 (binding in the central channel).

The tridimensional structure of TTR was made available at 1.8 Å resolution by X-ray diffraction studies on the crystallized protein [315]. Each monomer contains two β-sheets formed by strands DAGH and CBEF. All, except strands A and G, display an antiparallel orientation, and are arranged in a topology similar to the classic Greek key barrel. The strands are 7–8 residues long, except strand D, which, is only 3 residues in length. Only about 5% of the aa residues are located in one segment of α-helix comprising residues 75–83, at the end of strand E. Two monomers associate forming the dimer by interactions between chains F and H of each monomer. Fixing the chains of one monomer as A–H and the ones of the other monomer as A ′–H′, the arrangement within a dimer is DAGHH′G ′A ′D ′ and CBEFF′E ′B ′C ′. The tetramer consists of two dimers with connecting edges occurring between the AB loop of one dimer with the H strand of the other dimer (**Figure 2**). The quaternary structure of TTR has the shape of a globular protein with an overall size of 70 Å × 55 Å × 50 Å [315]. The two dimers are slightly rotated in relation to each other along the y axis.

## **5.3. Physiology and Metabolism**

## *5.3.1. Transport of thyroxine (T4)*

Thyroid hormones are transported in blood circulation and delivered to the target tissues, with an incredible high amount of the hormones (99%) bound to serum proteins. In human plasma, TTR, thyroxine-binding protein (TBG) and albumin are responsible for the delivery of T4 into the target tissues. Although TBG is much less concentrated in the plasma than TTR, it presents the highest affinity constant for T4 (Ka = 1 × 1010 M−1) and transports about 70% of the plasma T4. TTR has an intermediate affinity for T4 (Ka = 7 × 107 M−1) and transports about 15% of the hormone, and finally, albumin presents the lowest binding affinity (Ka = 7 × 105 M−1) [316].

The four monomers of the TTR tetramer, demarcate an open channel through the molecule where two binding sites for thyroid hormones are located (**Figure 2**). These two binding sites present negative cooperativity [317] implying that when the first thyroid hormone molecule occupies the first site, the affinity for the second molecule is highly reduced.

T4 transport into the brain is of particular interest and has raised much controversy [318]. Although TTR is the major T4-binding protein in the CSF, studies done in *ttr* knock-out mice (TTR−/−) [319] revealed that TTR is not essential for thyroxine to reach the brain and other tissues [320]. In addition, measurement of several parameters of thyroid hormone function indicates that these mice are euthyroid despite strongly reduced total T4 plasma levels [321]. No other protein was found to replace TTR in the transport of T4 in the CSF, and T4 levels were normal in the cortex, cerebellum, and hippocampus while strongly reduced in the CP of the TTR−/− mice [322].

Therefore, it was suggested that TTR might be a reservoir for T4 both in the plasma and in the CP and CSF, which might become important under conditions of increased hormone demand.

## *5.3.2. Transport of vitamin A*

TTR is also responsible for retinol transport through binding to retinol-binding protein (RBP, **Figure 2**). RBP is a 21 kDa monomeric protein comprising 182 aa residues [323]. The confor‐ mational structure of RBP bound to retinol was determined by x-ray crystallography [324]. In most instances, RBP is transported in the bloodstream in the form of a saturated holo-RBP protein equimolarly attached to TTR [325]. Although TTR has four binding sites for RBP [326, 327], under physiological conditions only one RBP molecule is bound to TTR due to the RBP limiting concentration.

#### *5.3.3. TTR metabolism*

The biological half-life of TTR is about 2–3 days in humans [328, 329], 22–23 h in monkeys [330] and 10–13 h in rats [331]. The major sites of TTR degradation in the rat are liver (36–38%), followed by the muscle (12–15%) and skin (8–10%) [332].

The normal physiology of TTR is not completely understood, in particular, its cellular uptake. Nevertheless, several observations suggest that TTR internalization is receptor-mediated, both in human hepatoma cells (HepG2) [333] and in chicken oocytes [334]. Megalin, a receptor implicated in the renal re-uptake of plasma proteins carriers of lipophilic compounds, was shown to play a role in renal uptake of TTR [335]. Furthermore, different TTR mutations presented different levels of cell association and degradation, suggesting that the structure of TTR is important for megalin recognition. TTR internalization was further explored by studying TTR uptake using hepatomas and primary hepatocytes [336]. This work showed direct evidence for TTR internalization by a specific receptor, forming a ~90 kDa complex. TTR internalization was inhibited by RBP (70% decrease) and T4 (20% decrease) and TTR mutants revealed differences in uptake, indicating again that the recognition by receptor is structure dependent. Internalization was also inhibited by lipoproteins and RAP, a ligand for all members of the low-density lipoprotein receptor family (LDLr). All together, these results suggest a common pathway for TTR and lipoprotein metabolism and the existence of a RAPsensitive receptor for TTR internalization. TTR is also internalized by several other cells types in cell culture, such as astrocytoma cells [337], cardiomiocytes [338, 339], neurons [340, 341], endothelial cells and others. More recently, TTR was also shown to be uptaken by sensory neurons, an event also mediated by megalin [340].

#### *5.3.4. Proteolytic activity*

**5.3. Physiology and Metabolism**

26 Exploring New Findings on Amyloidosis

*5.3.1. Transport of thyroxine (T4)*

mice [322].

*5.3.2. Transport of vitamin A*

limiting concentration.

*5.3.3. TTR metabolism*

T4. TTR has an intermediate affinity for T4 (Ka = 7 × 107

Thyroid hormones are transported in blood circulation and delivered to the target tissues, with an incredible high amount of the hormones (99%) bound to serum proteins. In human plasma, TTR, thyroxine-binding protein (TBG) and albumin are responsible for the delivery of T4 into the target tissues. Although TBG is much less concentrated in the plasma than TTR, it presents the highest affinity constant for T4 (Ka = 1 × 1010 M−1) and transports about 70% of the plasma

The four monomers of the TTR tetramer, demarcate an open channel through the molecule where two binding sites for thyroid hormones are located (**Figure 2**). These two binding sites present negative cooperativity [317] implying that when the first thyroid hormone molecule

T4 transport into the brain is of particular interest and has raised much controversy [318]. Although TTR is the major T4-binding protein in the CSF, studies done in *ttr* knock-out mice (TTR−/−) [319] revealed that TTR is not essential for thyroxine to reach the brain and other tissues [320]. In addition, measurement of several parameters of thyroid hormone function indicates that these mice are euthyroid despite strongly reduced total T4 plasma levels [321]. No other protein was found to replace TTR in the transport of T4 in the CSF, and T4 levels were normal in the cortex, cerebellum, and hippocampus while strongly reduced in the CP of the TTR−/−

Therefore, it was suggested that TTR might be a reservoir for T4 both in the plasma and in the CP and CSF, which might become important under conditions of increased hormone demand.

TTR is also responsible for retinol transport through binding to retinol-binding protein (RBP, **Figure 2**). RBP is a 21 kDa monomeric protein comprising 182 aa residues [323]. The confor‐ mational structure of RBP bound to retinol was determined by x-ray crystallography [324]. In most instances, RBP is transported in the bloodstream in the form of a saturated holo-RBP protein equimolarly attached to TTR [325]. Although TTR has four binding sites for RBP [326, 327], under physiological conditions only one RBP molecule is bound to TTR due to the RBP

The biological half-life of TTR is about 2–3 days in humans [328, 329], 22–23 h in monkeys [330] and 10–13 h in rats [331]. The major sites of TTR degradation in the rat are liver (36–38%),

The normal physiology of TTR is not completely understood, in particular, its cellular uptake. Nevertheless, several observations suggest that TTR internalization is receptor-mediated, both

followed by the muscle (12–15%) and skin (8–10%) [332].

hormone, and finally, albumin presents the lowest binding affinity (Ka = 7 × 105

occupies the first site, the affinity for the second molecule is highly reduced.

M−1) and transports about 15% of the

M−1) [316].

TTR was also found to act as a cryptic protease and the first substrate described was apoA-I [342, 343]. TTR is also able to cleave lipidated apoA-I (mainly in the lipid poor pre β-HDL subpopulation) which can be relevant in the lipoprotein metabolism [343]. Moreover, apoA-I cleaved by TTR presents less ability to promote cholesterol efflux [343] and shows increased amyloidogenecity and propensity to form aggregates. Liz et al. also described that TTR was able to cleave amidated neuropeptide Y (NPY) and that its proteolytic activity affects axonal growth, leading to the conclusion that TTR has natural substrates in the nervous system [344]. Newly, the same authors described TTR as a metallopeptidase [345], and this result was supported by another study that showed the involvement of a carboxylate and an ammonium group, possibly from a lysine side chain, in the TTR hydrolytic activity [346]. Costa et al. showed for the first time that TTR can cleave Aβ peptide in vitro [347] and implications in Aβ clearance in AD will be addressed further ahead.

#### **5.4. TTR as a cause of disease**

TTR is associated with the most prevalent type of hereditary systemic amyloidosis. The pathologic conditions include FAP and familial amyloidotic cardiomyopathy (FAC). A nonhereditary condition is also related to TTR, the systemic senile amyloidosis (SSA), and affects about 25% of people over 80 years of age. In SAA, the deposits occur in the heart and are composed of wild-type (WT) TTR.

FAP is related to a peculiar form of hereditary autosomal dominant polyneuropathy. Corino de Andrade first described the disease in 1952 [6] in the Portuguese population mainly from the northern part of the country. Characterized by systemic deposition of amyloid and with a special involvement of the peripheral nerves, the age of onset of the disease is usually between 20 and 35 years of age, with a fast progression to death within 10–15 years.

Clinically, FAP is characterized by early impairment of temperature and pain sensation in the feet, and autonomic dysfunction leading to paresis, malabsorption, and emaciation. Painless injury to the feet complicated by ulcers, cellulitis, osteomyelitis, and Charcots joints may also occur [348]. Motor involvement occurs with disease development causing wasting and weakness, and there is a progressive loss of reflexes. The amyloid deposits can occur in any part of the peripheral nervous system, including the nerve trunks, plexus, and sensory and autonomic ganglia. The other organ frequently involved in FAP, which is the heart. Clinically, the cardiomyopathy may present as an arrhythmia, heart block, or heat failure.

The first report relating immunologically TTR as the main protein in FAP fibrils was in 1978 by Costa and colleagues [349]. In 1984, the Val30Met mutation was identified in the protein isolated from Portuguese FAP patients. This variant was shown to be a biochemical marker for FAP [350] that resulted from a point mutation in the exon 2 of the TTR gene [351]. Since the identification of the Val30Met variant, many others aa substitutions were identified in the TTR protein and now over 100 mutations are described (http://amyloidosismutations.com/ mut-attr.php); these variants are associated with different clinical phenotypes and a consid‐ erable number of non-pathogenic TTR mutations have been identified, including a T119M variant described to be protective.

TTR tetramer dissociation is believed to be on the basis of a series of events leading to TTR amyloid formation. In fact, the amyloidogenic potential of the TTR variants relates inversely with its tetrameric stability, and it is thought that upon dissociation of the tetramer, non-native TTR monomers are formed which can assemble, forming amyloidogenic intermediate species, such as oligomers and aggregates. Similarly to other amyloidogenic proteins, it is now believed that cellular toxicity is derived from the initial intermediate species occurring in the initial stages of FAP [352]. TTR stabilization has been proposed as a key step for the inhibition of TTR fibril formation and has been the basis for FAP therapeutic strategies. Such stabilization can be achieved through the use of small compounds sharing molecular structural similarities with T4 and binding in the T4 central-binding channel. Most of such compounds belong to the class of NSAIDs as it is the case of diflunisal and tafamidis, currently being administrated to FAP patients. For instance, in FAP patients, diflunisal was shown to stabilize TTR, increasing its serum concentration [353], and to reduce the rate of progression of neurological impairment and to preserve quality of life [354].

## **6. TTR and neuroprotection**

Several lines of evidence indicate that TTR possesses neuroprotective properties in multiple contexts. Studies with TTR−/− mice revealed that these animals show reduced signs of de‐ pressive-like behavior, probably due to the modulation of noradrenergic system by the in‐ crease of noradrenaline in the limbic forebrain [355]. Additionally, increased levels of NPY, known as an antidepressant neurotransmitter [356], were reported in dorsal root ganglia (DRG), sciatic nerve, spinal cord, hippocampus, cortex, and CSF of TTR−/− mice [357], sup‐ porting the importance of TTR in the modulation of depressive behavior. Furthermore, Sousa and co-workers also described that TTR−/− mice present memory impairment com‐ pared with wild-type (TTR+/+) animals, indicating that the absence of TTR accelerates cogni‐ tive deficits usually associated with aging [358].

In addition, TTR was associated with nerve regeneration. Fleming et al. revealed, for the first time, that TTR acts as an enhancer of nerve regeneration, following the observation that TTR−/− mice have decreased ability to regenerate from a sciatic crushed nerve [359]. Later, the same authors showed that the absence of TTR leads to impaired retrograde transport and decreased axonal growth, and also that the effect of TTR in neurite outgrowth and nerve regeneration is mediated by megalin-dependent internalization [340].

It was also established a relationship between TTR and ischemia, one of the major causes of brain injuries in world. Santos and co-workers proposed that in a compromised heat-shock response, CSF TTR contributes to control neuronal cell death, edema, and inflammation, influencing the survival endangered neurons [360].

More recently, a new neuroprotective role in the CNS was attributed to TTR, as a transcription inducer of insulin-like growth factor receptor I (IGF-IR), known as a protective receptor against apoptosis [361]. Vieira and colleagues described, for the first time, that TTR induces increased levels of IGF-IR, showing that TTR triggers IGF-IR nuclear translocation in cultured neurons [341].

## **6.1. TTR protection in Alzheimer's Disease**

Clinically, FAP is characterized by early impairment of temperature and pain sensation in the feet, and autonomic dysfunction leading to paresis, malabsorption, and emaciation. Painless injury to the feet complicated by ulcers, cellulitis, osteomyelitis, and Charcots joints may also occur [348]. Motor involvement occurs with disease development causing wasting and weakness, and there is a progressive loss of reflexes. The amyloid deposits can occur in any part of the peripheral nervous system, including the nerve trunks, plexus, and sensory and autonomic ganglia. The other organ frequently involved in FAP, which is the heart. Clinically,

The first report relating immunologically TTR as the main protein in FAP fibrils was in 1978 by Costa and colleagues [349]. In 1984, the Val30Met mutation was identified in the protein isolated from Portuguese FAP patients. This variant was shown to be a biochemical marker for FAP [350] that resulted from a point mutation in the exon 2 of the TTR gene [351]. Since the identification of the Val30Met variant, many others aa substitutions were identified in the TTR protein and now over 100 mutations are described (http://amyloidosismutations.com/ mut-attr.php); these variants are associated with different clinical phenotypes and a consid‐ erable number of non-pathogenic TTR mutations have been identified, including a T119M

TTR tetramer dissociation is believed to be on the basis of a series of events leading to TTR amyloid formation. In fact, the amyloidogenic potential of the TTR variants relates inversely with its tetrameric stability, and it is thought that upon dissociation of the tetramer, non-native TTR monomers are formed which can assemble, forming amyloidogenic intermediate species, such as oligomers and aggregates. Similarly to other amyloidogenic proteins, it is now believed that cellular toxicity is derived from the initial intermediate species occurring in the initial stages of FAP [352]. TTR stabilization has been proposed as a key step for the inhibition of TTR fibril formation and has been the basis for FAP therapeutic strategies. Such stabilization can be achieved through the use of small compounds sharing molecular structural similarities with T4 and binding in the T4 central-binding channel. Most of such compounds belong to the class of NSAIDs as it is the case of diflunisal and tafamidis, currently being administrated to FAP patients. For instance, in FAP patients, diflunisal was shown to stabilize TTR, increasing its serum concentration [353], and to reduce the rate of progression of neurological impairment

Several lines of evidence indicate that TTR possesses neuroprotective properties in multiple contexts. Studies with TTR−/− mice revealed that these animals show reduced signs of de‐ pressive-like behavior, probably due to the modulation of noradrenergic system by the in‐ crease of noradrenaline in the limbic forebrain [355]. Additionally, increased levels of NPY, known as an antidepressant neurotransmitter [356], were reported in dorsal root ganglia (DRG), sciatic nerve, spinal cord, hippocampus, cortex, and CSF of TTR−/− mice [357], sup‐ porting the importance of TTR in the modulation of depressive behavior. Furthermore,

the cardiomyopathy may present as an arrhythmia, heart block, or heat failure.

variant described to be protective.

28 Exploring New Findings on Amyloidosis

and to preserve quality of life [354].

**6. TTR and neuroprotection**

There are several reports suggesting a relevant protective role of TTR in AD. However, the precise mechanism(s) is not entirely understood.

In 1993, Wisniewski described that Aβ40 fibril formation was inhibited upon incubation with human CSF [362], which was explained by the sequestration of Aβ by extracellular proteins circulating in CSF such as apoE and apoJ [362–364]. The earliest description of TTR protection in AD was presented by Schwarzman and colleagues in 1994 when they observed that, in CSF and contrarily to the expectations, apoE was not the major protein binding to Aβ, but TTR [365], proposing the sequestration hypothesis as a possible explanation for the peptide aggregation and consequent progression of AD. This hypothesis suggested that certain extracellular proteins sequester normally produced Aβ, thereby preventing amyloid formation and its toxicity. Amyloid formation would occur when sequestration failed [365, 366], which could be related either with an Aβ overproduction, a reduction in the levels of the sequestering proteins, inability of those proteins to interact with the peptide, deficient clearance mecha‐ nisms, or a combination of all the events above stated.

Supporting a protective role for TTR in AD are its decreased levels observed both in the CSF [367] and in the plasma [114, 368] of AD patients as compared to age-matched subjects. Serot and coworkers suggested that the decrease in TTR levels in the CSF of AD brains was possibly related with an epithelial atrophy in the CP [367]. Interestingly, very recently, studies in an AD transgenic mouse model reported CP dysfunction and revealed a specific increase only of the Aβ42 isoform in epithelial cytosol and in stroma surrounding choroidal capillaries, accompanied by a thickening of the epithelial basal membrane, greater collagen-IV deposition around capillaries in CP that probably restrain solute exchanges, and attenuated expression of epithelial aquaporin-1 and TTR protein compared to non-transgenic mice [369].

Modulation of Aβ aggregation and toxicity was also investigated in brain vascular smooth muscle cells, isolated from dogs and AD patients, containing intracytoplasmatic granules of Aβ produced in the presence of apoE. In this model, TTR was able to rescue the cells from this accumulation and the positive thioflavin S staining, initially observed, was no longer detected [370].

Although not fully consensual, several authors reported the presence of TTR in amyloid plaques both in AD patients [371–373] and in AD transgenic models [372], strengthening a role for TTR in Aβ deposition and in AD. TTR was also identified as a survival gene, and its differential overexpression in mice hippocampus was suggested to be responsible for the lack of neurodegeneration observed in the Tg2576 transgenic mice overexpressing the mutant form of human amyloid precursor protein with the Swedish mutation (APPSw) [374]. Up-regulation of TTR and other survival genes was induced by the sAPPα, a neuroprotective fragment resulting from APP processing by α-secretase [373]. TTR up-regulation was also reported in situations of exposure of AD transgenic mice to an "enriched environment," also resulting in pronounced reductions in cerebral Aβ levels and amyloid deposits, compared to animals raised under "standard housing" conditions [375]. Later on, AD transgenic models with genetic reduction of TTR and/or overexpression of human TTR [372] further showed the ability of TTR to modulate Aβ aggregation and toxicity. While the overexpression of human TTR ameliorated AD features in APP transgenic mice [372], the ablation of the mouse TTR gene resulted in accelerated amyloid deposition and increased Aβ brain levels [372, 376, 377]. In vitro studies further demonstrated that a direct interaction between TTR and Aβ abrogated the noxious properties of Aβ oligomers [378].

Animal models, in particular mice models, also provided evidence for a gender-associated modulation of brain Aβ levels [377] as elevated brain levels of Aβ42 were observed in AD female mice with only one copy of TTR when compared to female with the two copies of the TTR gene, while no significant differences were observed in males. Additionally, this work also indicated that reduced levels of brain testosterone and 17β-estradiol in female mice with TTR genetic reduction might underlie their increased AD-like neuropathology [377]. Interest‐ ingly, estradiol was found to be decreased in female AD patients when compared to healthy age and gender-matched controls [114], which in conjunction with TTR regulation by sex hormones, as already described, can account for TTR lowering in AD and the prevalence of this disease in women. In fact, plasma TTR levels were found decreased in AD women, as compared to healthy age- and gender-matched controls, whereas plasma TTR levels in AD men were not significantly different from their respective controls [114], further confirming the gender modulation by TTR.

#### *6.1.1. TTR and Aβ interaction*

TTR and Aβ binding was initially demonstrated by adding radiolabelled Aβ1-28 synthetic peptide to human CSF and subsequent analysis by SDS-PAGE [365]. Later on, the TTR/Aβ complex was also demonstrated in plasma [379], although the details of the interaction as well as the effects in Aβ fibrillogenesis and toxicity were not known.

around capillaries in CP that probably restrain solute exchanges, and attenuated expression

Modulation of Aβ aggregation and toxicity was also investigated in brain vascular smooth muscle cells, isolated from dogs and AD patients, containing intracytoplasmatic granules of Aβ produced in the presence of apoE. In this model, TTR was able to rescue the cells from this accumulation and the positive thioflavin S staining, initially observed, was no longer detected

Although not fully consensual, several authors reported the presence of TTR in amyloid plaques both in AD patients [371–373] and in AD transgenic models [372], strengthening a role for TTR in Aβ deposition and in AD. TTR was also identified as a survival gene, and its differential overexpression in mice hippocampus was suggested to be responsible for the lack of neurodegeneration observed in the Tg2576 transgenic mice overexpressing the mutant form of human amyloid precursor protein with the Swedish mutation (APPSw) [374]. Up-regulation of TTR and other survival genes was induced by the sAPPα, a neuroprotective fragment resulting from APP processing by α-secretase [373]. TTR up-regulation was also reported in situations of exposure of AD transgenic mice to an "enriched environment," also resulting in pronounced reductions in cerebral Aβ levels and amyloid deposits, compared to animals raised under "standard housing" conditions [375]. Later on, AD transgenic models with genetic reduction of TTR and/or overexpression of human TTR [372] further showed the ability of TTR to modulate Aβ aggregation and toxicity. While the overexpression of human TTR ameliorated AD features in APP transgenic mice [372], the ablation of the mouse TTR gene resulted in accelerated amyloid deposition and increased Aβ brain levels [372, 376, 377]. In vitro studies further demonstrated that a direct interaction between TTR and Aβ abrogated

Animal models, in particular mice models, also provided evidence for a gender-associated modulation of brain Aβ levels [377] as elevated brain levels of Aβ42 were observed in AD female mice with only one copy of TTR when compared to female with the two copies of the TTR gene, while no significant differences were observed in males. Additionally, this work also indicated that reduced levels of brain testosterone and 17β-estradiol in female mice with TTR genetic reduction might underlie their increased AD-like neuropathology [377]. Interest‐ ingly, estradiol was found to be decreased in female AD patients when compared to healthy age and gender-matched controls [114], which in conjunction with TTR regulation by sex hormones, as already described, can account for TTR lowering in AD and the prevalence of this disease in women. In fact, plasma TTR levels were found decreased in AD women, as compared to healthy age- and gender-matched controls, whereas plasma TTR levels in AD men were not significantly different from their respective controls [114], further confirming

TTR and Aβ binding was initially demonstrated by adding radiolabelled Aβ1-28 synthetic peptide to human CSF and subsequent analysis by SDS-PAGE [365]. Later on, the TTR/Aβ

of epithelial aquaporin-1 and TTR protein compared to non-transgenic mice [369].

[370].

30 Exploring New Findings on Amyloidosis

the noxious properties of Aβ oligomers [378].

the gender modulation by TTR.

*6.1.1. TTR and Aβ interaction*

In 2008, Costa and colleagues characterized the TTR/Aβ interaction by competition-binding assays using synthetic Aβ42, which revealed that the WT TTR binds to different Aβ peptide species: soluble (with a Kd of 28 ± 5 nM), oligomers, and fibrils [378]. Other studies showed that TTR drastically decreased the rate of aggregation without affecting the fraction of Aβ in the aggregate pool and an estimated apparent KS of 2300 M−1 was calculated [380]. These data support a hypothesis, wherein TTR preferentially binds to aggregated rather than monomeric Aβ and arrests further growth of the aggregates. Recent work indicates that the intensity of TTR binding to Aβ peptide is highest for partially aggregated materials and decreased for freshly prepared or heavily aggregated Aβ, suggesting that TTR binds selectively to soluble toxic Aβ aggregates [381]. Although Schwarzman and colleagues had shown in 1994 that TTR is capable of inhibiting Aβ1-28 aggregation [365], Costa and co-workers showed by transmis‐ sion electron microscopy (TEM) analysis that TTR is capable of interfering with Aβ fibrilliza‐ tion, both at inhibiting its aggregation and at disrupting pre-formed Aβ fibrils [378]. Thus, new and innovative studies are necessary to clarify the details of this interaction.

Another point of controversy refers to the TTR species involved in Aβ binding. Some studies support that the TTR monomer rather than the tetramer binds more strongly to Aβ [382], and it is even suggested that while the TTR monomer arrests Aβ aggregate growth, the tetramer modestly enhances the peptide aggregation [382]. Another study performed using diverse natural TTR mutants showed that different TTR variants bind differentially to Aβ in the following manner: T119M>WT>V30M<sup>3</sup> Y78F>L55P TTR [378], indicating the lower the amyloi‐ dogenic potential of TTR, the stronger the affinity toward the peptide. Since the amyloidogenic potential correlates inversely with TTR tetrameric stability, authors concluded that the TTR tetramer is the species binding to Aβ peptide. Previous work had already shown that amyloi‐ dogenic TTR mutants such as L55P and E42G, the only ones able to form TTR amyloid fibrils at pH 6.8 amongst the forty-seven variants tested, were unable to bind Aβ [383].

Given the above considerations, it is therefore conceivable that mutations of the TTR gene could alter the TI'R/Aβ sequestration properties. However, the screening study in AD patients found no correlation between TTR variants and AD [384], and therefore other factors, namely conformational changes resulting from aging, should be affecting TTR levels and its binding properties towards Aβ.

Computer-assisted modelling was developed to determine the possible key residues partici‐ pating in the interaction and the data suggested that residues 38–42, Asparagine 62 (E62) and E66 of each TTR monomer had a central role in the interaction [365]. Later studies have confirmed that only the residues 38–42 of TTR were important for the interaction [366]. More recently, Du and Murphy, identified the A strand, in the inner β-sheet of TTR, as well as the EF helix, as regions of TTR that are involved with Aβ [382] association. New data from the same group now indicates the involvement of the G strand of TTR with the particular in‐ volvement of L82A and L110A, suggesting that Aβ binding to TTR is mediated through these bulky hydrophobic leucines [385].

## *6.1.2. Effects of TTR stabilization in AD*

The decrease in TTR levels in the context of AD is found early in disease development as indicated by the lower levels also found in plasma TTR in MCI patients [114]. TTR levels continue to decrease as disease progresses correlating negatively with disease severity, both in CSF [115] and plasma [114], and with senile plaque burden [386]. Similar results were found in AD transgenic mice as TTR was decreased as early as 3 months of age [377], well before amyloid deposition. Mice, however, seem to be able to compensate and female showed restored TTR levels at the age of 10 months [377].

The reason for TTR decrease is not known, but its tetrameric stability seems to play an important role. An unstable TTR can result in accelerated clearance, accounting for the lower levels observed. Further, such instability can also affect the Aβ sequestration properties of TTR. Supporting this stability hypothesis is the observation that plasma TTR from AD patients presents impaired ability to carry T4. This also supports that it is the TTR tetramer that binds Aβ, since T4 binding to TTR implies that the tetramer is assembled. Very interestingly, TTR genetic stabilization, that is, the presence of the T119M allele, was associated to decreased cerebrovascular disease and increased life expectancy [387]. In the context of AD, and arguing in favor of the stability hypothesis is the observation that the TTR/Aβ interaction can be improved in vitro in the presence of small chemical molecules known to bind to the T4-binding channel and to stabilize its tetrameric fold [388]. Importantly, in vivo studies using one of such stabilizer—iododiflunisal, known to be a very potent TTR stabilizer [389] and shown to improve TTR/Aβ interaction [388]—administrated to AD transgenic mice resulted in amelio‐ ration of AD features such as cognitive function and Aβ brain deposition [390]. In addition, plasma levels of Aβ42 were decreased upon iododiflunisal administration. These results opened the possibility for the use of TTR stabilizers in AD therapeutic drug development.

## *6.1.3. Mechanisms of TTR protection in AD*

Aβ sequestration by TTR and other extracellular proteins was the first hypothesis proposed to explain why CSF is able to inhibit Aβ amyloid formation, implying that, when sequestered, Aβ cannot aggregate to form amyloid [362, 365]. However, the precise mechanism leading to final Aβ removal is not yet elucidated.

Following the identification of the TTR proteolytic activity, it was also described that Aβ peptide is cleaved by TTR, in vitro, with consequent generation of non-amyloidogenic frag‐ ments or fragments with amyloidogenic potential inferior to the full-length peptide [347]. Since clearance of Aβ from the brain can occur via proteolytic degradation of the peptide by several enzymes, such as neprilysin (NEP), insulin-degrading enzyme (IDE), Endothelinconverting enzyme (ECE), angiotensin-converting enzyme (ACE), uPA/tPA-plasmin system, cathepsin D, and matrix metalloendopeptidase 9 [391, 392], lower levels of TTR or inhibition of its proteolytic activity would result in less Aβ peptide eliminated by the cells, and there‐ fore in its accumulation and amyloid formation. Interestingly, several sites of Aβ cleavage by TTR are common to several of the proteases mentioned [347]. Further, NEP is known to cleave not only monomeric but also oligomeric forms of Aβ localized intra and extracellu‐ larly, as determined in vitro and in vivo [393, 394]; similarly, TTR was shown to degrade

both monomers and aggregates of Aβ in vitro. Nevertheless, Aβ degradation by TTR was not yet shown in vivo. It is also not known if binding/sequestration of Aβ and its degrada‐ tion by TTR are part of the same mechanism or are independent processes.

*6.1.2. Effects of TTR stabilization in AD*

32 Exploring New Findings on Amyloidosis

restored TTR levels at the age of 10 months [377].

*6.1.3. Mechanisms of TTR protection in AD*

final Aβ removal is not yet elucidated.

The decrease in TTR levels in the context of AD is found early in disease development as indicated by the lower levels also found in plasma TTR in MCI patients [114]. TTR levels continue to decrease as disease progresses correlating negatively with disease severity, both in CSF [115] and plasma [114], and with senile plaque burden [386]. Similar results were found in AD transgenic mice as TTR was decreased as early as 3 months of age [377], well before amyloid deposition. Mice, however, seem to be able to compensate and female showed

The reason for TTR decrease is not known, but its tetrameric stability seems to play an important role. An unstable TTR can result in accelerated clearance, accounting for the lower levels observed. Further, such instability can also affect the Aβ sequestration properties of TTR. Supporting this stability hypothesis is the observation that plasma TTR from AD patients presents impaired ability to carry T4. This also supports that it is the TTR tetramer that binds Aβ, since T4 binding to TTR implies that the tetramer is assembled. Very interestingly, TTR genetic stabilization, that is, the presence of the T119M allele, was associated to decreased cerebrovascular disease and increased life expectancy [387]. In the context of AD, and arguing in favor of the stability hypothesis is the observation that the TTR/Aβ interaction can be improved in vitro in the presence of small chemical molecules known to bind to the T4-binding channel and to stabilize its tetrameric fold [388]. Importantly, in vivo studies using one of such stabilizer—iododiflunisal, known to be a very potent TTR stabilizer [389] and shown to improve TTR/Aβ interaction [388]—administrated to AD transgenic mice resulted in amelio‐ ration of AD features such as cognitive function and Aβ brain deposition [390]. In addition, plasma levels of Aβ42 were decreased upon iododiflunisal administration. These results opened the possibility for the use of TTR stabilizers in AD therapeutic drug development.

Aβ sequestration by TTR and other extracellular proteins was the first hypothesis proposed to explain why CSF is able to inhibit Aβ amyloid formation, implying that, when sequestered, Aβ cannot aggregate to form amyloid [362, 365]. However, the precise mechanism leading to

Following the identification of the TTR proteolytic activity, it was also described that Aβ peptide is cleaved by TTR, in vitro, with consequent generation of non-amyloidogenic frag‐ ments or fragments with amyloidogenic potential inferior to the full-length peptide [347]. Since clearance of Aβ from the brain can occur via proteolytic degradation of the peptide by several enzymes, such as neprilysin (NEP), insulin-degrading enzyme (IDE), Endothelinconverting enzyme (ECE), angiotensin-converting enzyme (ACE), uPA/tPA-plasmin system, cathepsin D, and matrix metalloendopeptidase 9 [391, 392], lower levels of TTR or inhibition of its proteolytic activity would result in less Aβ peptide eliminated by the cells, and there‐ fore in its accumulation and amyloid formation. Interestingly, several sites of Aβ cleavage by TTR are common to several of the proteases mentioned [347]. Further, NEP is known to cleave not only monomeric but also oligomeric forms of Aβ localized intra and extracellu‐ larly, as determined in vitro and in vivo [393, 394]; similarly, TTR was shown to degrade In addition to the peptidolytic removal of Aβ, clearance of the peptide from the brain also occurs via active transport at the BBB and BCSFB, as already discussed. The receptors for Aβ at the BBB bind Aβ directly, or bind to one of its carrier proteins, and transport it across the endothelial cell. The first hint pointing to the involvement of TTR in Aβ transport and clearance came from the analysis of brain and plasma levels of the peptide in mice with different TTR genetic backgrounds. Results showed that AD transgenic mice with just one copy of TTR had lower brain and plasma Aβ levels, as compared to animals with two TTR gene copies, raising the hypothesis that TTR might be involved both in Aβ brain efflux and in its peripheral removal at the liver. Very recently, it has been showed that TTR promotes Aβ internalization and efflux in hCMEC/D3 cells, a BBB cellular model widely used. Importantly, TTR stimulated brain-toblood Aβ permeability in hCMEC/D3 which in turn can be explained because TTR itself can only cross the BBB only in the brain-to-blood direction [290, 332]. Thus, TTR can transport Aβ from, but not into the brain, acting as a neuroprotective molecule.

The presence of TTR in brain areas other than its site of synthesis and secretion—CP and CSF, respectively—has been already shown. In situations of injury, such as ischemia, TTR was detected at the local of infarct and shown to derive from CSF TTR [360]. However, other studies demonstrated TTR synthesis by cortical [395] or hippocampal neurons both in vitro [396], and in vivo [397] showing that TTR expression in the brain can be regulated [396]. For instance, Kerridge and colleagues showed that TTR expressed in SH-SY5Y neuroblastoma cell line is up-regulated by the AICD fragment of amyloid precursor protein (APP), specifically derived from the APP695 isoform [396]. Induced accumulation of functional AICD resulted in TTR upregulation with concomitant Aβ decreased levels. Wang and colleagues reported that TTR expression in SH-SY5Y cells, primary hippocampal neurons, and the hippocampus of APP23 mice is significantly enhanced by HSF1 [397]**.** In any case, TTR is available in the brain and might participate in brain Aβ efflux by promoting BBB permeability to the peptide. It is also possible that TTR contributes to Aβ clearance from the brain through the BCSFB.

TTR was also able to increase Aβ internalization by hepatocytes prompting TTR as an Aβ transporter both in the brain and at periphery. Previous work showed that TTR is internalized by hepatocytes using a RAP-sensitive receptor, which together with the knowledge that as follows: (1) LRP1 is the main Aβ receptor both at the BBB and at the liver, (2) LRP1 is prefer‐ entially expressed at the basolateral membrane of the endothelial cells of the BBB, and (3) TTR can only cross the BBB in the brain-to-blood direction, indicates this receptor is involved in TTR-assisted Aβ transport. So far, it has been shown that mice with TTR genetic ablation present decrease levels of brain and liver LRP1 and that TTR added to hCMEC/D3 cells results in increased LRP1 expression. These findings open new perspectives for TTR/LRP-related therapeutic interventions in AD. However, a direct interaction between LRP1 and TTR is yet to be demonstrated. TTR has also been suggested to act in a chaperone-like manner by binding toxic or pretoxic Aβ aggregates in both the intracellular and extracellular environment [372].

**Figure 3.** Schematic representation of the proposed mechanisms underlying TTR Protection in AD. (A) In a healthy individual, TTR binds Aβ peptide in the brain at the CSF promoting its degradation and elimination through the epi‐ thelial cells of the CP. At the BBB, Aβ effluxes through LRP1 to the blood, a process in which TTR also participates. At the periphery, TTR and other Aβ bindable substances create a peripheral sink avoiding the return of Aβ to the brain, and transport the peptide to the liver, where it will be internalized by liver LRP1 for final degradation. (B) In AD pa‐ tients, TTR decreased tetrameric stability for yet unknown reasons, and decreased expression due to decreased sex hormones, result in lower protein levels in the brain and plasma. In turn, this contributes to failure in Aβ sequestra‐ tion, efflux at the BBB, and peripheral transport to the liver. Impaired Aβ transport at the BBB and internalization/ degradation by the liver is aggravated by LRP1 decreased expression, modulated by TTR.

Therefore, more studies are necessary to unravel the mechanism(s) underlying TTR protection in AD, and to clarify how the hypothesis presented so far fit together (**Figure 3**).

## **Acknowledgements**

This work was funded by national funds through FCT—Fundação para a Ciência e a Tecno‐ logia/MEC—Ministério da Educação e Ciência and when applicable co-funded by FEDER funds within the partnership agreement PT2020 related with the research unit number 4293, and by FEDER funds through the Operational Competitiveness Programme—COMPETE and by National Funds through FCT—Fundação para a Ciência e a Tecnologia under the projects FCOMP-01-0124-FEDER-037277 (PEst-C/SAU/LA0002/2013).

## **Author details**

Isabel Cardoso1,2\*, Luis Miguel Santos1,2 and Mobina Alemi1,2,3

\*Address all correspondence to: icardoso@ibmc.up.pt


## **References**

**Figure 3.** Schematic representation of the proposed mechanisms underlying TTR Protection in AD. (A) In a healthy individual, TTR binds Aβ peptide in the brain at the CSF promoting its degradation and elimination through the epi‐ thelial cells of the CP. At the BBB, Aβ effluxes through LRP1 to the blood, a process in which TTR also participates. At the periphery, TTR and other Aβ bindable substances create a peripheral sink avoiding the return of Aβ to the brain, and transport the peptide to the liver, where it will be internalized by liver LRP1 for final degradation. (B) In AD pa‐ tients, TTR decreased tetrameric stability for yet unknown reasons, and decreased expression due to decreased sex hormones, result in lower protein levels in the brain and plasma. In turn, this contributes to failure in Aβ sequestra‐ tion, efflux at the BBB, and peripheral transport to the liver. Impaired Aβ transport at the BBB and internalization/

Therefore, more studies are necessary to unravel the mechanism(s) underlying TTR protection

This work was funded by national funds through FCT—Fundação para a Ciência e a Tecno‐ logia/MEC—Ministério da Educação e Ciência and when applicable co-funded by FEDER funds within the partnership agreement PT2020 related with the research unit number 4293, and by FEDER funds through the Operational Competitiveness Programme—COMPETE and by National Funds through FCT—Fundação para a Ciência e a Tecnologia under the projects

2 i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal

degradation by the liver is aggravated by LRP1 decreased expression, modulated by TTR.

FCOMP-01-0124-FEDER-037277 (PEst-C/SAU/LA0002/2013).

Isabel Cardoso1,2\*, Luis Miguel Santos1,2 and Mobina Alemi1,2,3

1 IBMC-Instituto de Biologia Molecular e Celular, Porto, Portugal

3 Faculdade de Medicina, Universidade do Porto, Porto, Portugal

\*Address all correspondence to: icardoso@ibmc.up.pt

**Acknowledgements**

34 Exploring New Findings on Amyloidosis

**Author details**

in AD, and to clarify how the hypothesis presented so far fit together (**Figure 3**).


[22] Bertram L, Lill CM, Tanzi RE. The genetics of Alzheimer disease: back to the future. Neuron [Internet]. 2010;68(2):270–81 [cited 2015 Jun 14]. http://www.sciencedir‐ ect.com/science/article/pii/S0896627310008378

[12] Burns A, Byrne EJ, Maurer K. Alzheimer's disease. Lancet (London, England) [Internet] (Elsevier). 2002;360(9327):163–5 [cited 2016 Jan 6]. http://www.thelancet.com/article/

[13] van der Linde RM, Dening T, Matthews FE, Brayne C. Grouping of behavioural and psychological symptoms of dementia. Int J Geriatr Psychiatry [Internet]. 2014;29(6): 562–8 [cited 2015 Oct 20]. http://www.pubmedcentral.nih.gov/articlerender.fcgi?

[14] Petrovic M, Hurt C, Collins D, Burns A, Camus V, Liperoti R, et al. Clustering of behavioural and psychological symptoms in dementia (BPSD): a European Alzheimer's disease consortium (EADC) study. Acta Clin Belg [Internet]. 2016;62(6):426–32 [cited

[15] 2015 Alzheimer's disease facts and figures. Alzheimers Dement [Internet]. 2015;11(3): 332–84 [cited 2015 Jun 2]. http://www.ncbi.nlm.nih.gov/pubmed/25984581

[16] Reitz C, Mayeux R. Alzheimer disease: epidemiology, diagnostic criteria, risk factors and biomarkers. Biochem Pharmacol [Internet] (NIH Public Access) 2014;88(4):640–51

[17] McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group\* under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease. Neurology [Internet]. 1984;34(7):939–939 [cited 2015 Jun 7]. http://

[18] McKhann GM, Knopman DS, Chertkow H, Hyman BT, Jack CR, Kawas CH, et al. The diagnosis of dementia due to Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease. Alzheimers Dement [Internet] (Elsevier). 2011;7(3):263–9 [cited 2014 Jul 9]. http://www.alzheimersanddementia.com/article/S1552526011001014/

[19] Hyman BT, Phelps CH, Beach TG, Bigio EH, Cairns NJ, Carrillo MC, et al. National Institute on Aging-Alzheimer's Association guidelines for the neuropathologic assessment of Alzheimer's disease. Alzheimers Dement [Internet]. 2012;8(1):1–13 [cited 2015 Aug 6]. http://www.sciencedirect.com/science/article/pii/S1552526011029803 [20] 2013 Alzheimer's disease facts and figures. Alzheimers Dement [Internet]. 2013;9(2): 208–45 [cited 2015 Sep 2]. http://www.sciencedirect.com/science/article/pii/

[21] Gaugler JE, Kane RL, Johnston J a, Sarsour K. Sensitivity and specificity of diagnostic accuracy in Alzheimer´s disease: a synthesis of existing evidence. Am J Alzheimer´s

artid=4255309&tool=pmcentrez&rendertype=abstract

www.neurology.org/content/34/7/939

fulltext

S1552526013000769

Dis Other Dement. 2013;28(4):337–47.

2016 Jan 6]. http://www.ncbi.nlm.nih.gov/pubmed/18351187

[cited 2014 Jul 10]. http:/pmc/articles/PMC3992261/?report=abstract

S0140673602094205/fulltext

36 Exploring New Findings on Amyloidosis


[43] Yu G, Nishimura M, Arawaka S, Levitan D, Zhang L, Tandon A, et al. Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction and betaAPP process‐ ing. Nature [Internet]. 2000;407(6800):48–54 [cited 2015 Dec 9]. http:// www.ncbi.nlm.nih.gov/pubmed/10993067

[32] Yoshikai S, Sasaki H, Doh-ura K, Furuya H, Sakaki Y. Genomic organization of the human amyloid beta-protein precursor gene. Gene [Internet]. 1990;87(2):257–63 [cited

[33] Zhang Y, Thompson R, Zhang H, Xu H. APP processing in Alzheimer's disease. Mol Brain [Internet]. 2011;4:3 [cited 2015 Nov 18]. http://www.pubmedcentral.nih.gov/

[34] Zhou Z, Chan CH, Ma Q, Xu X, Xiao Z, Tan E. The roles of amyloid precursor protein (APP) in neurogenesis: implications to pathogenesis and therapy of Alzheimer disease.

[35] Musa A, Lehrach H, Russo VA. Distinct expression patterns of two zebrafish homo‐ logues of the human APP gene during embryonic development. Dev Genes Evol [Internet]. 2001;211(11):563–7 [cited 2015 Nov 18]. http://www.ncbi.nlm.nih.gov/

[36] Xiao Q, Gil S-C, Yan P, Wang Y, Han S, Gonzales E, et al. Role of phosphatidylinositol clathrin assembly lymphoid-myeloid leukemia (PICALM) in intracellular amyloid precursor protein (APP) processing and amyloid plaque pathogenesis. J Biol Chem [Internet]. 2012;287(25):21279–89 [cited 2016 Jan 10]. http://www.pubmedcen‐ tral.nih.gov/articlerender.fcgi?artid=3375549&tool=pmcentrez&rendertype=abstract

[37] Cirrito JR, Kang J-E, Lee J, Stewart FR, Verges DK, Silverio LM, et al. Endocytosis is required for synaptic activity-dependent release of amyloid-beta in vivo. Neuron [Internet]. 2008;58(1):42–51 [cited 2015 Nov 1]. http://www.pubmedcentral.nih.gov/

[38] Seubert P, Vigo-Pelfrey C, Esch F, Lee M, Dovey H, Davis D, et al. Isolation and quantification of soluble Alzheimer's beta-peptide from biological fluids. Nature [Internet]. 1992;359(6393):325–7 [cited 2015 Dec 4]. http://www.ncbi.nlm.nih.gov/

[39] Querfurth HW, LaFerla FM. Alzheimer's disease. N Engl J Med [Internet]. 2010;362(4): 329–44 [cited 2014 Nov 18]. http://www.ncbi.nlm.nih.gov/pubmed/20107219

[40] Selkoe DJ. Alzheimer disease: mechanistic understanding predicts novel therapies. Ann Intern Med [Internet]. 2004;140(8):627–38 [cited 2016 Jan 10]. http://

[41] Jarrett JT, Berger EP, Lansbury PT. The carboxy terminus of the beta amyloid protein is critical for the seeding of amyloid formation: implications for the pathogenesis of Alzheimer's disease. Biochemistry [Internet]. 1993;32(18):4693–7 [cited 2015 Oct 5].

[42] Rossner S. New players in old amyloid precursor protein-processing pathways. Int J Dev Neurosci [Internet]. 2004;22(7):467–74 [cited 2016 Jan 11]. http://www.sciencedir‐

articlerender.fcgi?artid=2390913&tool=pmcentrez&rendertype=abstract

articlerender.fcgi?artid=3022812&tool=pmcentrez&rendertype=abstract

2015 Dec 31]. http://www.ncbi.nlm.nih.gov/pubmed/2110105

Cell Adh Migr. 2016;5(4):280–92.

pubmed/11862463

38 Exploring New Findings on Amyloidosis

pubmed/1406936

www.ncbi.nlm.nih.gov/pubmed/15096334

http://www.ncbi.nlm.nih.gov/pubmed/8490014

ect.com/science/article/pii/S0736574804000899


[66] Maccioni RB, Farías G, Morales I, Navarrete L. The revitalized tau hypothesis on Alzheimer's disease. Arch Med Res [Internet]. 2010;41(3):226–31. http://www.science‐ direct.com/science/article/pii/S0188440910000500

[54] Bennett DA, Schneider JA, Arvanitakis Z, Kelly JF, Aggarwal NT, Shah RC, et al. Neuropathology of older persons without cognitive impairment from two community-

[55] Hardy JA, Higgins GA. Alzheimer's disease: the amyloid cascade hypothesis. Science.

[56] Reitz C. Alzheimer's disease and the amyloid cascade hypothesis: a critical review. Int J Alzheimers Dis [Internet]. 2012;2012:369808. http://www.pubmedcentral.nih.gov/

[57] Mudher A, Lovestone S. Alzheimer's disease - Do tauists and baptists finally shake

[58] Chételat G. Alzheimer disease: aβ-independent processes-rethinking preclinical AD. Nat Rev Neurol [Internet]. 2013;9(3):123–4. http://www.pubmedcentral.nih.gov/

[59] Klein WL, Krafft G a., Finch CE. Targeting small A β oligomers: the solution to an

[60] Gilbert BJ. The role of amyloid β in the pathogenesis of Alzheimer's disease. J Clin Pathol [Internet]. 2013;66(5):362–6 [cited 2016 Jan 16]. http://www.ncbi.nlm.nih.gov/

[61] Jin M, Shepardson N, Yang T, Chen G, Walsh D, Selkoe DJ. Soluble amyloid betaprotein dimers isolated from Alzheimer cortex directly induce Tau hyperphosphory‐ lation and neuritic degeneration. Proc Natl Acad Sci USA [Internet]. 2011;108(14):5819– 24. http://www.pubmedcentral.nih.gov/articlerender.fcgi?ar‐

[62] Jan A, Adolfsson O, Allaman I, Buccarello AL, Magistretti PJ, Pfeifer A, et al. Aβ42 neurotoxicity is mediated by ongoing nucleated polymerization process rather than by

[63] Puzzo D, Arancio O. Amyloid-β peptide: Dr. Jekyll or Mr. Hyde? J Alzheimers Dis [Internet]. 2013;33 Suppl 1:S111–20 [cited 2016 Jan 30]. http://www.pubmedcen‐ tral.nih.gov/articlerender.fcgi?artid=3696497&tool=pmcentrez&rendertype=abstract

[64] Robinson SR, Bishop GM. Aβ as a bioflocculant: implications for the amyloid hypoth‐ esis of Alzheimer's disease. Neurobiol Aging [Internet] (Elsevier). 2002;23(6):1051–72 [cited 2016 Jan 30]. http://www.neurobiologyofaging.org/article/S0197458001003426/

[65] Lee H, Zhu X, Castellani RJ, Nunomura A, Perry G, Smith MA. Amyloid-beta in Alzheimer disease: the null versus the alternate hypotheses. J Pharmacol Exp Ther

[Internet]. 2007;321(3):823–9. http://www.ncbi.nlm.nih.gov/pubmed/17229880

articlerender.fcgi?artid=3313573&tool=pmcentrez&rendertype=abstract

articlerender.fcgi?artid=3935395&tool=pmcentrez&rendertype=abstract

Alzheimer's disease conundrum? Trends Neurosci. 2001;24(4):219–24.

based studies. Neurology. 2006;66(12):1837–44.

hands? Trends Neurosci. 2002;25(1):22–6.

tid=3078381&tool=pmcentrez&rendertype=abstract

discrete Aβ42 species. J Biol Chem. 2011;286(10):8585–96.

1992;256(5054):184–5.

40 Exploring New Findings on Amyloidosis

pubmed/23526599

fulltext


[91] Pinto T, Lanctôt KL, Herrmann N. Revisiting the cholinergic hypothesis of behavioral and psychological symptoms in dementia of the Alzheimer's type. Ageing Res Rev [Internet]. 2011;10(4):404–12. doi:10.1016/j.arr.2011.01.003

[78] Lei P, Ayton S, Bush AI, Adlard P a. GSK-3 in Neurodegenerative Diseases. Int J

[79] Padurariu M, Ciobica A, Lefter R, Serban IL, Stefanescu C, Chirita R. The oxidative stress hypothesis in Alzheimer's disease. Psychiatr Danub [Internet]. 2013;25(4):401–9.

[80] Markesbery WR. Oxidative stress hypothesis in Alzheimer's disease. Free Radic Biol

[81] Graham SF, Nasaruddin M Bin, Carey M, Holscher C, McGuinness B, Kehoe PG, et al. Age-associated changes of brain copper, iron, and zinc in Alzheimer's disease and dementia with Lewy bodies. J Alzheimers Dis [Internet]. 2014;42(4):1407–13. http://

[82] Deibel MA, Ehmann WD, Markesbery WR. Copper, iron, and zinc imbalances in severely degenerated brain regions in Alzheimer's disease: possible relation to

[83] Schrag M, Mueller C, Oyoyo U, Smith M a., Kirsch WM. Iron, zinc and copper in the Alzheimer's disease brain: a quantitative meta-analysis. Some insight on the influence of citation bias on scientific opinion. Prog Neurobiol [Internet]. 2011;94(3):296–306. doi:

[84] Tiiman A, Palumaa P, Tõugu V. The missing link in the amyloid cascade of Alzheimer's disease-Metal ions. Neurochem Int [Internet]. 2013;62(4):367–78. doi:10.1016/j.neuint.

[85] Mold M, Ouro-Gnao L, Wieckowski BM, Exley C. Copper prevents amyloid-β(1-42) from forming amyloid fibrils under near-physiological conditions in vitro. Sci Rep.

[86] Mayes J, Tinker-Mill C, Kolosov O, Zhang H, Tabner BJ, Allsop D. Amyloid fibrils in alzheimer disease are not inert when bound to copper ions but can degrade hydrogen peroxide and generate reactive oxygen species. J Biol Chem. 2014;289(17):12052–62.

[87] Swerdlow RH, Khan SM. A "mitochondrial cascade hypothesis" for sporadic Alzheim‐

[88] Grimm A, Friedland K, Eckert A. Mitochondrial dysfunction: the missing link between aging and sporadic Alzheimer's disease. Biogerontology [Internet]. 2015 [cited 2015 Oct

[89] Russell H. Swerdlow SMK. The Alzheimer's Disease mitochondrial cascade hypothe‐

[90] Perry E, Perry R, Blessed G, Tomlinson B. Necropsy evidence of central cholinergic deficits in senile dementia. Lancet [Internet]. 1977;309(8004):189 [cited 2016 Jan 15].

http://www.sciencedirect.com/science/article/pii/S0140673677917809

content.iospress.com/articles/journal-of-alzheimers-disease/jad140684

Alzheimers Dis. 2011;2011:189246.

Med. 1997;23(1):134–47.

42 Exploring New Findings on Amyloidosis

10.1016/j.pneurobio.2011.05.001

2013.01.023

2013;3(Ii):1256.

http://www.ncbi.nlm.nih.gov/pubmed/24247053

oxidative stress. J Neurol Sci. 1996;143(606):137–42.

er's disease. Med Hypotheses. 2004;63(1):8–20.

sis: an update. Exp Neurol. 2009;218(2):308–15.

16]. http://www.ncbi.nlm.nih.gov/pubmed/26468143


modulation. Curr Alzheimer Res [Internet]. 2012;9(8):881–9. http:// www.ncbi.nlm.nih.gov/pubmed/22698061

[115] Gloeckner SF, Meyne F, Wagner F, Heinemann U, Krasnianski A, Meissner B, et al. Quantitative analysis of transthyretin, tau and amyloid-beta in patients with dementia. J Alzheimers Dis [Internet]. 2008;14(1):17–25 [cited 2016 Jan 5]. http:// www.ncbi.nlm.nih.gov/pubmed/18525124

[103] Pierrot N, Santos SF, Feyt C, Morel M, Brion JP, Octave JN. Calcium-mediated transient phosphorylation of tau and amyloid precursor protein followed by intraneuronal

[104] Glabe CG. Common mechanisms of amyloid oligomer pathogenesis in degenerative

[105] Strimbu K, Tavel JA. What are biomarkers? Curr Opin HIV AIDS [Internet]. 2010;5(6): 463–6. http://www.pubmedcentral.nih.gov/articlerender.fcgi?ar‐

[106] Rosén C, Hansson O, Blennow K, Zetterberg H. Fluid biomarkers in Alzheimer's disease—current concepts. Mol Neurodegener [Internet]. 2013;8(1):20. http://

[107] Hansson O, Zetterberg H, Buchhave P, Londos E, Blennow K, Minthon L. Association between CSF biomarkers and incipient Alzheimer's disease in patients with mild

[108] Andreasson U, Lautner R, Schott JM, Mattsson N, Hansson O, Herukka S-K, et al. CSF biomarkers for Alzheimer's pathology and the effect size of APOE  4. Mol Psychiatry [Internet]. 2014;19(2):148–9 [cited 2016 Jan 17]. http://www.pubmedcentral.nih.gov/

[109] Ringman JM, Younkin SG, Pratico D, Seltzer W, Cole GM, Geschwind DH, et al. Biochemical markers in persons with preclinical familial Alzheimer disease. Neurolo‐

[110] Graff-Radford NR, Crook JE, Lucas J, Boeve BF, Knopman DS, Ivnik RJ, et al. Associ‐ ation of low plasma Abeta42/Abeta40 ratios with increased imminent risk for mild cognitive impairment and Alzheimer disease. Arch Neurol [Internet]. 2007;64(3):354–

[111] Verheijen JH, Huisman LGM, van Lent N, Neumann U, Paganetti P, Hack CE, et al. Detection of a soluble form of BACE-1 in human cerebrospinal fluid by a sensitive activity assay. Clin Chem [Internet]. 2006;52(6):1168–74. http://www.ncbi.nlm.nih.gov/

[112] Perneczky R, Tsolakidou a., Arnold a., Diehl-Schmid J, Grimmer T, Förstl H, et al. CSF soluble amyloid precursor proteins in the diagnosis of incipient Alzheimer disease.

[113] Lopez-Font I, Cuchillo-Ibañez I, Sogorb-Esteve A, García-Ayllón M-S, Sáez-Valero J. Transmembrane amyloid-related proteins in CSF as potential biomarkers for Alzheim‐ er's disease. Front Neurol [Internet]. 2015;6(June):125. http://www.pubmedcen‐ tral.nih.gov/articlerender.fcgi?artid=4451586&tool=pmcentrez&rendertype=abstract

[114] Ribeiro CA, Santana I, Oliveira C, Baldeiras I, Moreira J, Saraiva MJ, et al. Transthyretin decrease in plasma of MCI and AD patients: investigation of mechanisms for disease

cognitive impairment: a follow-up study. Lancet Neurol. 2006;5(3):228–34.

articlerender.fcgi?artid=3903112&tool=pmcentrez&rendertype=abstract

62. http://archneur.jamanetwork.com/article.aspx?articleid=793567

amyloid-β accumulation. J Biol Chem. 2006;281(52):39907–14.

disease. Neurobiol Aging. 2006;27(4):570–5.

gy. 2008;71(2):85–92.

44 Exploring New Findings on Amyloidosis

pubmed/16614000

Neurology. 2011;77(1):35–8.

tid=3078627&tool=pmcentrez&rendertype=abstract

www.molecularneurodegeneration.com/content/8/1/20


Ann Neurol [Internet]. 2010;68(4):521–34 [cited 2016 Jan 18]. http:// www.ncbi.nlm.nih.gov/pubmed/20687206

[137] Laxton AW, Lozano AM. Deep brain stimulation for the treatment of alzheimer disease and dementias. World Neurosurg [Internet]. 2013;80(3–4):S28.e1–S28.e8. doi: 10.1016/ j.wneu.2012.06.028

[126] Vuorinen M, Solomon A, Rovio S, Nieminen L, Kåreholt I, Tuomilehto J, et al. Changes in vascular risk factors from midlife to late life and white matter lesions: a 20-year

[127] Imtiaz B, Tolppanen A-M, Kivipelto M, Soininen H. Future directions in Alzheimer's disease from risk factors to prevention. Biochem Pharmacol [Internet]. 2014;88(4):661–

[128] Shepardson NE, Shankar GM, Selkoe DJ. Cholesterol level and statin use in Alzheimer disease: II. Review of human trials and recommendations. Arch Neurol [Internet]. 2011;68(11):1385–92. http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?

[129] Lindsay J, Laurin D, Verreault R, Hébert R, Helliwell B, Hill GB, et al. Risk factors for Alzheimer's disease: a prospective analysis from the Canadian Study of Health and

[130] Corder EH, Saunders a M, Strittmatter WJ, Schmechel DE, Gaskell PC, Small GW, et al. Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late

[131] Tanzi RE, Bertram L. Twenty years of the Alzheimer's disease amyloid hypothesis: a

[132] Rinne JO, Brooks DJ, Rossor MN, Fox NC, Bullock R, Klunk WE, et al. 11C-PiB PET assessment of change in fibrillar amyloid-beta load in patients with Alzheimer's disease treated with bapineuzumab: a phase 2, double-blind, placebo-controlled, ascendingdose study. Lancet Neurol [Internet]. 2010;9(4):363–72 [cited 2015 Aug 12]. http://

[133] Gandy S, DeKosky ST. Toward the treatment and prevention of Alzheimer's disease: rational strategies and recent progress. Annu Rev Med [Internet]. 2013;64:367–83. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3625402&tool=pmcen‐

[134] Watt AD, Crespi GAN, Down RA, Ascher DB, Gunn A, Perez KA, et al. Do current therapeutic anti-Aβ antibodies for Alzheimer's disease engage the target? Acta

[135] Gong B, Pan Y, Zhao W, Knable L, Vempati P, Begum S, et al. IVIG immunotherapy protects against synaptic dysfunction in Alzheimer's disease through complement anaphylatoxin C5a-mediated AMPA-CREB-C/EBP signaling pathway. Mol Immunol.

[136] Laxton AW, Tang-Wai DF, McAndrews MP, Zumsteg D, Wennberg R, Keren R, et al. A phase I trial of deep brain stimulation of memory circuits in Alzheimer's disease.

dbfrom=pubmed&id=22084122&retmode=ref&cmd=prlinks\npapers3://

follow-up study. Dement Geriatr Cogn Disord. 2011;31(2):119–25.

70. http://www.ncbi.nlm.nih.gov/pubmed/24418410

publication/doi/10.1001/archneurol.2011.242

46 Exploring New Findings on Amyloidosis

Aging. Am J Epidemiol. 2002;156(5):445–53.

onset families. Science. 1993;261(5123):921–3.

genetic perspective. Cell. 2005;120(4):545–55.

www.ncbi.nlm.nih.gov/pubmed/20189881

trez&rendertype=abstract

2013;56(4):619–29.

Neuropathol. 2014;127(6):803–10.


[162] Braet K, Paemeleire K, D'Herde K, Sanderson MJ, Leybaert L. Astrocyte-endothelial cell calcium signals conveyed by two signalling pathways. Eur J Neurosci. 2001;13(1): 79–91.

[150] Kniesel U, Wolburg H. Tight junctions of the blood-brain barrier. Cell Mol Neurobiol.

[151] Ohtsuki S, Terasaki T. Contribution of carrier-mediated transport systems to the bloodbrain barrier as a supporting and protecting interface for the brain; importance for CNS

[152] Tagami M, Nara Y, Kubota A, Fujino H, Yamori Y. Ultrastructural changes in cerebral pericytes and astrocytes of stroke-prone spontaneously hypertensive rats. Stroke.

[153] Farkas E, Luiten PGM. Cerebral microvascular pathology in aging and Alzheimer's

[154] Ramsauer M, Krause D, Dermietzel R. Angiogenesis of the blood-brain barrier in vitro

[155] Gonul E, Duz B, Kahraman S, Kayali H, Kubar A, Timurkaynak E. Early pericyte response to brain hypoxia in cats: an ultrastructural study. Microvasc Res [Internet].

[156] Dore-Duffy P, Owen C, Balabanov R, Murphy S, Beaumont T, Rafols JA. Pericyte migration from the vascular wall in response to traumatic brain injury. Microvasc Res

[157] Hori S, Ohtsuki S, Hosoya K, Nakashima E, Terasaki T. A pericyte-derived angiopoie‐ tin-1 multimeric complex induces occludin gene expression in brain capillary endo‐ thelial cells through Tie-2 activation in vitro. J Neurochem [Internet]. 2004;89(2):503–

[158] Davson H, Oldendorf WH. Symposium on membrane transport. Transport in the central nervous system. Proc R Soc Med [Internet]. 1967;60(4):326–9 [cited 2016 Jan 12]. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1901728&tool=pmcen‐

[159] Tao-Cheng JH, Nagy Z, Brightman MW. Tight junctions of brain endothelium in vitro are enhanced by astroglia. J Neurosci [Internet]. 1987;7(10):3293–9 [cited 2016 Jan 12].

[160] Maxwell K, Berliner JA, Cancilla PA. Induction of gamma-glutamyl transpeptidase in cultured cerebral endothelial cells by a product released by astrocytes. Brain Res [Internet]. 1987;410(2):309–14 [cited 2016 Jan 12]. http://www.ncbi.nlm.nih.gov/

[161] Ballabh P, Braun A, Nedergaard M. The blood-brain barrier: an overview: structure, regulation, and clinical implications. Neurobiol Dis [Internet]. 2004;16(1):1–13 [cited

2014 Jul 11]. http://www.ncbi.nlm.nih.gov/pubmed/15207256

[Internet]. 2000;60(1):55–69. http://www.ncbi.nlm.nih.gov/pubmed/10873515

drug discovery and development. Pharm Res. 2007;24(9):1745–58.

disease. Vol. 64, Progress in Neurobiology. 2001. 575-611 p.

and the function of cerebral pericytes. FASEB J. 2002;16(10):1274–6.

2002;64(1):116–9. http://www.ncbi.nlm.nih.gov/pubmed/12074637

13. http://www.ncbi.nlm.nih.gov/pubmed/15056293

http://www.ncbi.nlm.nih.gov/pubmed/3668629

trez&rendertype=abstract

pubmed/2885071

2000;20(1):57–76.

48 Exploring New Findings on Amyloidosis

1990;21:1064–71.


http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4080800&tool=pmcen‐ trez&rendertype=abstract


[182] Schulzke JD, Gitter a. H, Mankertz J, Spiegel S, Seidler U, Amasheh S, et al. Epithelial transport and barrier function in occludin-deficient mice. Biochim Biophys Acta Biomembr. 2005;1669(1):34–42.

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4080800&tool=pmcen‐

[173] Bolton SJ, Anthony DC, Perry VH. Loss of the tight junction proteins occludin and zonula occludens-1 from cerebral vascular endothelium during neutrophil-induced blood-brain barrier breakdown in vivo. Neuroscience [Internet]. 1998;86(4):1245–57

[174] da Fonseca ACC, Matias D, Garcia C, Amaral R, Geraldo LH, Freitas C, et al. The impact of microglial activation on blood-brain barrier in brain diseases. Front Cell Neurosci [Internet]. 2014;8:362 [cited 2016 Jan 13]. http://www.pubmedcentral.nih.gov/article‐

[175] Bazzoni G, Dejana E. Endothelial cell-to-cell junctions: molecular organization and role in vascular homeostasis. Physiol Rev [Internet]. 2004;84(3):869–901 [cited 2015 Aug 6].

[176] Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, Tsukita S. Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol [Internet]. 1993;123(6 Pt 2):1777–88 [cited 2015 Mar 19]. http://www.pubmedcentral.nih.gov/articleren‐

[177] Lippoldt A, Kniesel U, Liebner S, Kalbacher H, Kirsch T, Wolburg H, et al. Structural alterations of tight junctions are associated with loss of polarity in stroke-prone spontaneously hypertensive rat blood-brain barrier endothelial cells. Brain Res [Internet]. 2000;885(2):251–61 [cited 2016 Jan 13]. http://www.ncbi.nlm.nih.gov/

[178] Hawkins BT, Abbruscato TJ, Egleton RD, Brown RC, Huber JD, Campos CR, et al. Nicotine increases in vivo blood-brain barrier permeability and alters cerebral micro‐ vascular tight junction protein distribution. Brain Res [Internet]. 2004;1027(1–2):48–58

[179] Hirase T, Staddon JM, Saitou M, Ando-Akatsuka Y, Itoh M, Furuse M, et al. Occludin as a possible determinant of tight junction permeability in endothelial cells. J Cell Sci [Internet]. 1997;110(Pt 1):1603–13 [cited 2016 Jan 13]. http://www.ncbi.nlm.nih.gov/

[180] Bamforth SD, Kniesel U, Wolburg H, Engelhardt B, Risau W. A dominant mutant of occludin disrupts tight junction structure and function. J Cell Sci [Internet]. 1999;112(Pt

[181] Saitou M, Furuse M, Sasaki H, Schulzke JD, Fromm M, Takano H, et al. Complex phenotype of mice lacking occludin, a component of tight junction strands. Mol Biol

[cited 2015 Dec 22]. http://www.ncbi.nlm.nih.gov/pubmed/15494156

1):1879–88. http://www.ncbi.nlm.nih.gov/pubmed/10341207

[cited 2016 Jan 13]. http://www.ncbi.nlm.nih.gov/pubmed/9697130

render.fcgi?artid=4217497&tool=pmcentrez&rendertype=abstract

der.fcgi?artid=2290891&tool=pmcentrez&rendertype=abstract

http://www.ncbi.nlm.nih.gov/pubmed/15269339

trez&rendertype=abstract

50 Exploring New Findings on Amyloidosis

pubmed/11102579

pubmed/9247194

Cell. 2000;11(12):4131–42.


barrier leakage and active demyelination. J Pathol [Internet]. 2003;201(2):319–27 [cited 2016 Jan 15]. http://www.ncbi.nlm.nih.gov/pubmed/14517850

[204] Stevenson BR, Siliciano JD, Mooseker MS, Goodenough DA. Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occlu‐ dens) in a variety of epithelia. J Cell Biol [Internet]. 1986;103(3):755–66 [cited 2016 Jan 15]. http://www.pubmedcentral.nih.gov/articlerender.fcgi?ar‐ tid=2114282&tool=pmcentrez&rendertype=abstract

[193] Yeung D, Manias JL, Stewart DJ, Nag S. Decreased junctional adhesion molecule-A expression during blood-brain barrier breakdown. Acta Neuropathol [Internet]. 2008;115(6):635–42 [cited 2016 Jan 14]. http://www.ncbi.nlm.nih.gov/pubmed/18357461

[194] Vincent PA, Xiao K, Buckley KM, Kowalczyk AP. VE-cadherin: adhesion at arm's length. Am J Physiol Cell Physiol [Internet]. 2004;286(5):C987–97 [cited 2016 Jan 14].

[195] Lampugnani MG, Dejana E. Adherens junctions in endothelial cells regulate vessel maintenance and angiogenesis. Thromb Res [Internet]. 2007;120 Suppl :S1–6 [cited 2016

[196] Abbruscato TJ, Davis TP. Protein expression of brain endothelial cell E-cadherin after hypoxia/aglycemia: influence of astrocyte contact. Brain Res [Internet]. 1999;842(2):

[197] Romero IA, Radewicz K, Jubin E, Michel CC, Greenwood J, Couraud P-O, et al. Changes in cytoskeletal and tight junctional proteins correlate with decreased permeability induced by dexamethasone in cultured rat brain endothelial cells. Neurosci Lett [Internet]. 2003;344(2):112–6 [cited 2015 Dec 8]. http://www.ncbi.nlm.nih.gov/pubmed/

[198] Privratsky JR, Newman PJ. PECAM-1: regulator of endothelial junctional integrity. Cell Tissue Res [Internet]. 2014;355(3):607–19 [cited 2016 Jan 14]. http://www.pubmedcen‐ tral.nih.gov/articlerender.fcgi?artid=3975704&tool=pmcentrez&rendertype=abstract

[199] Graesser D, Solowiej A, Bruckner M, Osterweil E, Juedes A, Davis S, et al. Altered vascular permeability and early onset of experimental autoimmune encephalomyelitis in PECAM-1-deficient mice. J Clin Invest [Internet]. 2002;109(3):383–92 [cited 2016 Jan 14]. http://www.pubmedcentral.nih.gov/articlerender.fcgi?ar‐

[200] Tietz S, Engelhardt B. Brain barriers: crosstalk between complex tight junctions and adherens junctions. J Cell Biol [Internet]. 2015;209(4):493–506 [cited 2016 Jan 14]. http://

www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4442813&tool=pmcen‐

[201] Fanning AS, Jameson BJ, Jesaitis LA, Anderson JM. The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoske‐ leton. J Biol Chem [Internet]. 1998;273(45):29745–53 [cited 2016 Jan 15]. http://

[202] Van Itallie CM, Anderson JM. Architecture of tight junctions and principles of molec‐ ular composition. Semin Cell Dev Biol [Internet]. 2014;36:157–65 [cited 2016 Jan 6]. ttp://

www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4254347&tool=pmcen‐

[203] Kirk J, Plumb J, Mirakhur M, McQuaid S. Tight junctional abnormality in multiple sclerosis white matter affects all calibres of vessel and is associated with blood-brain

277–86 [cited 2016 Jan 14]. http://www.ncbi.nlm.nih.gov/pubmed/10526124

http://www.ncbi.nlm.nih.gov/pubmed/15075197

tid=150854&tool=pmcentrez&rendertype=abstract

www.ncbi.nlm.nih.gov/pubmed/9792688

trez&rendertype=abstract

trez&rendertype=abstract

12782340

52 Exploring New Findings on Amyloidosis

Jan 14]. http://www.ncbi.nlm.nih.gov/pubmed/18023702


mice. Glia [Internet]. 2003;42(3):235–51 [cited 2016 Jan 16]. http:// www.ncbi.nlm.nih.gov/pubmed/12673830


[223] Begley DJ. ABC transporters and the blood-brain barrier. Curr Pharm Des [Internet]. 2004;10(12):1295–312 [cited 2016 Jan 17]. http://www.ncbi.nlm.nih.gov/pubmed/ 15134482

mice. Glia [Internet]. 2003;42(3):235–51 [cited 2016 Jan 16]. http://

[213] Shiu C, Barbier E, Di Cello F, Choi HJ, Stins M. HIV-1 gp120 as well as alcohol affect blood-brain barrier permeability and stress fiber formation: involvement of reactive oxygen species. Alcohol Clin Exp Res [Internet]. 2007;31(1):130–7 [cited 2016 Jan 16].

[214] Davson H. Review lecture. The blood-brain barrier. J Physiol [Internet]. 1976;255(1):1– 28 [cited 2016 Jan 17]. http://www.pubmedcentral.nih.gov/articlerender.fcgi?ar‐

[215] Wong AD, Ye M, Levy AF, Rothstein JD, Bergles DE, Searson PC. The blood-brain barrier: an engineering perspective. Front Neuroeng [Internet]. 2013;6:7 [cited 2016 Jan

[216] Abbott NJ, Patabendige AAK, Dolman DEM, Yusof SR, Begley DJ. Structure and function of the blood-brain barrier. Neurobiol Dis [Internet]. 2010;37(1):13–25 [cited

[217] Taylor CJ, Nicola PA, Wang S, Barrand MA, Hladky SB. Transporters involved in regulation of intracellular pH in primary cultured rat brain endothelial cells. J Physiol [Internet]. 2006;576(Pt 3):769–85 [cited 2016 Jan 17]. http://www.pubmedcen‐ tral.nih.gov/articlerender.fcgi?artid=1890423&tool=pmcentrez&rendertype=abstract

[218] Giacomini KM, Huang S-M, Tweedie DJ, Benet LZ, Brouwer KLR, Chu X, et al. Membrane transporters in drug development. Nat Rev Drug Discov [Internet]. 2010;9(3):215–36 [cited 2014 Jul 11]. http://www.pubmedcentral.nih.gov/articleren‐

[219] Ueno M. Mechanisms of the penetration of blood-borne substances into the brain. Curr Neuropharmacol [Internet]. 2009;7(2):142–9 [cited 2016 Jan 16]. http://www.pubmed‐ central.nih.gov/articlerender.fcgi?artid=2730006&tool=pmcentrez&rendertype=ab‐

[220] Enerson BE, Drewes LR. The rat blood-brain barrier transcriptome. J Cereb Blood Flow

[221] Abbott NJ, Rönnbäck L, Hansson E. Astrocyte-endothelial interactions at the bloodbrain barrier. Nat Rev Neurosci [Internet]. 2006;7(1):41–53 [cited 2014 Jul 9]. http://

[222] Dauchy S, Dutheil F, Weaver RJ, Chassoux F, Daumas-Duport C, Couraud P-O, et al. ABC transporters, cytochromes P450 and their main transcription factors: expression at the human blood-brain barrier. J Neurochem [Internet]. 2008;107(6):1518–28 [cited

16]. http://journal.frontiersin.org/article/10.3389/fneng.2013.00007/abstract

2014 Jul 15]. http://www.ncbi.nlm.nih.gov/pubmed/19664713

der.fcgi?artid=3326076&tool=pmcentrez&rendertype=abstract

2016 Jan 17]. http://www.ncbi.nlm.nih.gov/pubmed/19094056

stract

Metab. 2006;26(7):959–73.

www.ncbi.nlm.nih.gov/pubmed/16371949

www.ncbi.nlm.nih.gov/pubmed/12673830

54 Exploring New Findings on Amyloidosis

http://www.ncbi.nlm.nih.gov/pubmed/17207111

tid=1309232&tool=pmcentrez&rendertype=abstract


2007;27(5):909–18 [cited 2015 Oct 22]. http://www.pubmedcentral.nih.gov/articleren‐ der.fcgi?artid=2853021&tool=pmcentrez&rendertype=abstract

[245] Deane R, Sagare A, Hamm K, Parisi M, Lane S, Finn MB, et al. apoE isoform–specific disruption of amyloid β peptide clearance from mouse brain. J Clin Invest [Internet]. 2008;118(12):4002–13 [cited 2015 Oct 17]. http://www.pubmedcentral.nih.gov/article‐ render.fcgi?artid=2582453&tool=pmcentrez&rendertype=abstract

[234] Tanzi RE, Moir RD, Wagner SL. Clearance of Alzheimer's Abeta peptide: the many roads to perdition. Neuron [Internet]. 2004;43(5):605–8 [cited 2016 Jan 17]. http://

[235] Zlokovic B V, Yamada S, Holtzman D, Ghiso J, Frangione B. Clearance of amyloid betapeptide from brain: transport or metabolism? Nat Med [Internet]. 2000;6(7):718–9 [cited

[236] Hardy J. Amyloid double trouble. Nat Genet [Internet]. 2006;38(1):11–2 [cited 2016 Jan

[237] Ballatore C, Lee VM-Y, Trojanowski JQ. Tau-mediated neurodegeneration in Alzheim‐ er's disease and related disorders. Nat Rev Neurosci [Internet]. 2007;8(9):663–72 [cited

[238] Selkoe DJ. Clearing the brain's amyloid cobwebs. Neuron [Internet]. 2001;32(2):177–80

[239] Shibata M, Yamada S, Kumar SR, Calero M, Bading J, Frangione B, et al. Clearance of Alzheimer's amyloid-ss(1-40) peptide from brain by LDL receptor-related protein-1 at the blood-brain barrier. J Clin Invest [Internet]. 2000;106(12):1489–99 [cited 2015 Dec 14]. http://www.pubmedcentral.nih.gov/articlerender.fcgi?ar‐

[240] Deane R, Wu Z, Sagare A, Davis J, Du Yan S, Hamm K, et al. LRP/amyloid beta-peptide interaction mediates differential brain efflux of Abeta isoforms. Neuron [Internet]. 2004;43(3):333–44 [cited 2015 Nov 26]. http://www.ncbi.nlm.nih.gov/pubmed/

[241] Deane R, Du Yan S, Submamaryan RK, LaRue B, Jovanovic S, Hogg E, et al. RAGE mediates amyloid-beta peptide transport across the blood-brain barrier and accumu‐ lation in brain. Nat Med [Internet]. 2003;9(7):907–13 [cited 2015 Sep 21]. http://

[242] Pascale CL, Miller MC, Chiu C, Boylan M, Caralopoulos IN, Gonzalez L, et al. Amyloidbeta transporter expression at the blood-CSF barrier is age-dependent. Fluids Barriers CNS [Internet]. 2011;8:21 [cited 2015 Dec 5]. http://www.pubmedcentral.nih.gov/

[243] Deane R, Bell RD, Sagare A, Zlokovic B V. Clearance of amyloid-beta peptide across the blood-brain barrier: implication for therapies in Alzheimer's disease. CNS Neurol Disord Drug Targets [Internet]. 2009;8(1):16–30 [cited 2015 Oct 5]. http://

www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2872930&tool=pmcen‐

[244] Bell RD, Sagare AP, Friedman AE, Bedi GS, Holtzman DM, Deane R, et al. Transport pathways for clearance of human Alzheimer's amyloid β-peptide and apolipoproteins E and J in the mouse central nervous system. J Cereb Blood Flow Metab [Internet].

articlerender.fcgi?artid=3162580&tool=pmcentrez&rendertype=abstract

[cited 2016 Jan 17]. http://www.ncbi.nlm.nih.gov/pubmed/11683988

2016 Jan 17]. http://www.ncbi.nlm.nih.gov/pubmed/10888892

17]. http://www.ncbi.nlm.nih.gov/pubmed/16380721

tid=387254&tool=pmcentrez&rendertype=abstract

www.ncbi.nlm.nih.gov/pubmed/12808450

trez&rendertype=abstract

www.ncbi.nlm.nih.gov/pubmed/15339642

56 Exploring New Findings on Amyloidosis

2014 Jul 21]. doi: 10.1038/nrn2194

15294142


1;2(10). pii: a011452. doi: 10.1101/cshperspect.a011452. Review. PubMed PMID: 23028132; PubMed Central PMCID: PMC3475405.

[265] Claudio L. Ultrastructural features of the blood-brain barrier in biopsy tissue from Alzheimer's disease patients. Acta Neuropathol [Internet]. 1996;91(1):6–14 [cited 2016 Feb 5]. http://www.ncbi.nlm.nih.gov/pubmed/8773140

[254] Kaur C, Ling EA. Blood brain barrier in hypoxic-ischemic conditions. Curr Neurovasc Res [Internet]. 2008;5(1):71–81 [cited 2016 Jan 18]. http://www.ncbi.nlm.nih.gov/

[255] Desai BS, Monahan AJ, Carvey PM, Hendey B. Blood-brain barrier pathology in Alzheimer's and Parkinson's disease: implications for drug therapy. Cell Transplant [Internet]. 2007;16(3):285–99 [cited 2016 Jan 18]. http://www.ncbi.nlm.nih.gov/

[256] Förster C. Tight junctions and the modulation of barrier function in disease. Histochem Cell Biol [Internet]. 2008;130(1):55–70 [cited 2015 Nov 30]. http://www.pubmedcen‐ tral.nih.gov/articlerender.fcgi?artid=2413111&tool=pmcentrez&rendertype=abstract

[257] Wardlaw JM, Sandercock PAG, Dennis MS, Starr J. Is breakdown of the blood-brain barrier responsible for lacunar stroke, leukoaraiosis, and dementia? Stroke [Internet]. 2003;34(3):806–12 [cited 2015 Dec 5]. http://www.ncbi.nlm.nih.gov/pubmed/12624314

[258] Zlokovic B V. Clearing amyloid through the blood-brain barrier. J Neurochem [Inter‐ net]. 2004;89(4):807–11 [cited 2016 Jan 14]. http://www.ncbi.nlm.nih.gov/pubmed/

[259] Owen JB, Sultana R, Aluise CD, Erickson MA, Price TO, Bu G, et al. Oxidative modifi‐ cation to LDL receptor-related protein 1 in hippocampus from subjects with Alzheimer disease: implications for Aβ accumulation in AD brain. Free Radic Biol Med [Internet]. 2010;49(11):1798–803 [cited 2016 Jan 18]. http://www.pubmedcentral.nih.gov/article‐

[260] Cirrito JR, Deane R, Fagan AM, Spinner ML, Parsadanian M, Finn MB, et al. Pglycoprotein deficiency at the blood-brain barrier increases amyloid-beta deposition in an Alzheimer disease mouse model. J Clin Invest [Internet]. 2005;115(11):3285–90 [cited 2016 Jan 18]. http://www.pubmedcentral.nih.gov/articlerender.fcgi?ar‐

[261] Landau SM, Harvey D, Madison CM, Koeppe RA, Reiman EM, Foster NL, et al. Associations between cognitive, functional, and FDG-PET measures of decline in AD and MCI. Neurobiol Aging [Internet]. 2011;32(7):1207–18 [cited 2016 Jan 18]. http://

www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2891865&tool=pmcen‐

[262] Harik SI. Changes in the glucose transporter of brain capillaries. Can J Physiol Phar‐ macol [Internet]. 1992;70 Suppl:S113–7 [cited 2016 Jan 18]. http://

[263] Mooradian AD, Chung HC, Shah GN. GLUT-1 expression in the cerebra of patients with Alzheimer's disease. Neurobiol Aging [Internet]. 2016;18(5):469–74 [cited 2016 Jan

[264] Sagare AP, Bell RD, Zlokovic BV. Neurovascular dysfunction and faulty amyloid βpeptide clearance in Alzheimer disease. Cold Spring Harb Perspect Med. 2012 Oct

render.fcgi?artid=2970765&tool=pmcentrez&rendertype=abstract

tid=1257538&tool=pmcentrez&rendertype=abstract

trez&rendertype=abstract

www.ncbi.nlm.nih.gov/pubmed/1295661

18]. http://www.ncbi.nlm.nih.gov/pubmed/9390772

pubmed/18289024

58 Exploring New Findings on Amyloidosis

pubmed/17503739

15140180


[284] Kapural M, Krizanac-Bengez L, Barnett G, Perl J, Masaryk T, Apollo D, et al. Serum S-100beta as a possible marker of blood-brain barrier disruption. Brain Res [Internet]. 2002;940(1–2):102–4 [cited 2016 Jan 19]. http://www.ncbi.nlm.nih.gov/pubmed/ 12020881

[274] Li QX, Whyte S, Tanner JE, Evin G, Beyreuther K, Masters CL. Secretion of Alzheimer's disease Abeta amyloid peptide by activated human platelets. Lab Invest [Internet]. 1998;78(4):461–9 [cited 2016 Jan 18]. http://www.ncbi.nlm.nih.gov/pubmed/9564890

[275] Kuo YM, Kokjohn TA, Watson MD, Woods AS, Cotter RJ, Sue LI, et al. Elevated abeta42 in skeletal muscle of Alzheimer disease patients suggests peripheral alterations of AbetaPP metabolism. Am J Pathol [Internet]. 2000;156(3):797–805 [cited 2016 Jan 18]. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1876838&tool=pmcen‐

[276] Kuo YM, Kokjohn TA, Kalback W, Luehrs D, Galasko DR, Chevallier N, et al. Amyloidbeta peptides interact with plasma proteins and erythrocytes: implications for their quantitation in plasma. Biochem Biophys Res Commun [Internet]. 2000;268(3):750–6

[277] Biere AL, Ostaszewski B, Stimson ER, Hyman BT, Maggio JE, Selkoe DJ. Amyloid betapeptide is transported on lipoproteins and albumin in human plasma. J Biol Chem [Internet]. 1996;271(51):32916–22 [cited 2016 Jan 18]. http://www.ncbi.nlm.nih.gov/

[278] Strittmatter WJ, Weisgraber KH, Huang DY, Dong LM, Salvesen GS, Pericak-Vance M, et al. Binding of human apolipoprotein E to synthetic amyloid beta peptide: isoformspecific effects and implications for late-onset Alzheimer disease. Proc Natl Acad Sci U S A [Internet]. 1993;90(17):8098–102 [cited 2016 Jan 18]. http://www.pubmedcen‐ tral.nih.gov/articlerender.fcgi?artid=47295&tool=pmcentrez&rendertype=abstract

[279] Sagare A, Deane R, Bell RD, Johnson B, Hamm K, Pendu R, et al. Clearance of amyloidbeta by circulating lipoprotein receptors. Nat Med [Internet]. 2007;13(9):1029–31 [cited 2015 Oct 1]. http://www.pubmedcentral.nih.gov/articlerender.fcgi?ar‐

[280] Pan W, Solomon B, Maness LM, Kastin AJ. Antibodies to beta-amyloid decrease the blood-to-brain transfer of beta-amyloid peptide. Exp Biol Med (Maywood) [Internet]. 2002;227(8):609–15 [cited 2016 Jan 18]. http://www.ncbi.nlm.nih.gov/pubmed/12192102

[281] Erickson MA, Banks WA. Blood-brain barrier dysfunction as a cause and consequence of Alzheimer's disease. J Cereb Blood Flow Metab [Internet]. 2013;33(10):1500–13 [cited 2016 Jan 16]. http://www.pubmedcentral.nih.gov/articlerender.fcgi?ar‐

[282] Geroldi D, Falcone C, Emanuele E. Soluble receptor for advanced glycation end products: from disease marker to potential therapeutic target. Curr Med Chem [Internet]. 2006;13(17):1971–8 [cited 2016 Jan 18]. http://www.ncbi.nlm.nih.gov/

[283] Marchi N, Fazio V, Cucullo L, Kight K, Masaryk T, Barnett G, et al. Serum transthyretin monomer as a possible marker of blood-to-CSF barrier disruption. J Neurosci [Internet]. 2003;23(5):1949–55 [cited 2016 Jan 18]. http://www.ncbi.nlm.nih.gov/pubmed/12629200

tid=2936449&tool=pmcentrez&rendertype=abstract

tid=3790938&tool=pmcentrez&rendertype=abstract

[cited 2016 Jan 18]. http://www.ncbi.nlm.nih.gov/pubmed/10679277

trez&rendertype=abstract

60 Exploring New Findings on Amyloidosis

pubmed/8955133

pubmed/16842191


[305] Dickson PW, Howlett GJ, Schreiber G. Rat transthyretin (prealbumin). Molecular cloning, nucleotide sequence, and gene expression in liver and brain. J Biol Chem [Internet]. 1985;260(13):8214–9. http://www.ncbi.nlm.nih.gov/pubmed/3839240

[294] Soprano DR, Herbert J, Soprano KJ, Schon EA, Goodman DS. Demonstration of transthyretin mRNA in the brain and other extrahepatic tissues in the rat. J Biol Chem [Internet]. 1985;260(21):11793–8 [cited 2015 Dec 30]. http://www.ncbi.nlm.nih.gov/

[295] Woeber KA, Ingbar SH. The contribution of thyroxine-binding prealbumin to the binding of thyroxine in human serum, as assessed by immunoadsorption. J Clin Invest.

[296] Goodman D. Retinoids and retinol-binding proteins. Harvey Lect. 1987;Series 81:111–

[297] Kabat EA, Moore DH, Landow H. An electrophoretic study of the protein components in cerebrospinal fluid and their relationship to the serum proteins. J Clin Invest [Internet]. 1942;21(5):571–7 [cited 2015 Dec 30]. http://www.pubmedcentral.nih.gov/

[298] Seibert FB NJ. Electrophoretic study of the blood response in tuberculosis. J Biol Chem.

[299] Wallace MR, Naylor SL, Kluve-Beckerman B, Long GL, McDonald L, Shows TB, et al. Localization of the human prealbumin gene to chromosome 18. Biochem Biophys Res Commun [Internet]. 1985;129(3):753–8. http://www.ncbi.nlm.nih.gov/pubmed/

[300] Sasaki H, Yoshioka N, Takagi Y, Sakaki Y. Structure of the chromosomal gene for human serum prealbumin. Gene [Internet]. 1985;37(1–3):191–7 [cited 2015 Dec 30].

[301] Tsuzuki T, Mita S, Maeda S, Araki S, Shimada K. Structure of the human prealbumin

[302] Whitehead AS, Skinner M, Bruns GA, Costello W, Edge MD, Cohen AS, et al. Cloning of human prealbumin complementary DNA. Localization of the gene to chromosome 18 and detection of a variant prealbumin allele in a family with familial amyloid polyneuropathy. Mol Biol Med [Internet]. 1984;2(6):411–23 [cited 2015 Dec 30]. http://

[303] Mita S, Maeda S, Shimada K, Araki S. Cloning and sequence analysis of cDNA for human prealbumin. Biochem Biophys Res Commun [Internet]. 1984;124(2):558–64.

[304] Sundelin J, Melhus H, Das S, Eriksson U, Lind P, Trägårdh L, et al. The primary structure of rabbit and rat prealbumin and a comparison with the tertiary structure of human prealbumin [Internet]. J Biol Chem. 1985;260:6481–7. http:// www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=Ab‐ stractPlus&list\_uids=3922975\nhttp://www.jbc.org/cgi/reprint/260/10/6481

http://www.sciencedirect.com/science/article/pii/0006291X84915900

http://www.ncbi.nlm.nih.gov/pubmed/4054629

gene. J Biol Chem. 1985;260(22):12224–7.

www.ncbi.nlm.nih.gov/pubmed/6100724

articlerender.fcgi?artid=435175&tool=pmcentrez&rendertype=abstract

pubmed/4044580

62 Exploring New Findings on Amyloidosis

1968;47(7):1710–21.

1942;143:29–38.

2990465

32.


albumin. J Chromatogr [Internet]. 1992;579(2):225–35 [cited 2016 Jan 12]. http:// www.ncbi.nlm.nih.gov/pubmed/1429970


Biophys Acta [Internet]. 1976;439(2):449–60 [cited 2016 Jan 13]. http:// www.ncbi.nlm.nih.gov/pubmed/986177

[327] van Jaarsveld PP, Edelhoch H, Goodman DS, Robbins J. The interaction of human plasma retinol-binding protein and prealbumin. J Biol Chem [Internet]. 1973;248(13): 4698–705 [cited 2016 Jan 13]. http://www.ncbi.nlm.nih.gov/pubmed/4718739

albumin. J Chromatogr [Internet]. 1992;579(2):225–35 [cited 2016 Jan 12]. http://

[317] Ferguson RN, Edelhoch H, Saroff HA, Robbins J, Cahnmann HJ. Negative cooperativity in the binding of thyroxine to human serum prealbumin. Preparation of tritium-labeled

[318] Richardson SJ, Wijayagunaratne RC, D'Souza DG, Darras VM, Van Herck SLJ. Trans‐ port of thyroid hormones via the choroid plexus into the brain: The roles of transthyr‐ etin and thyroid hormone transmembrane transporters. Vol. 9, Frontiers in

[319] Manral P, Reixach N. Amyloidogenic and non-amyloidogenic transthyretin variants interact differently with human cardiomyocytes: insights into early events of nonfibrillar tissue damage. Biosci Rep. 2015 Jan 14;35(1). pii: e00172. doi: 10.1042/ BSR20140155. PubMed PMID: 25395306; PubMed Central PMCID: PMC4293901.

[320] Palha JA, Hays MT, Morreale de Escobar G, Episkopou V, Gottesman ME, Saraiva MJ. Transthyretin is not essential for thyroxine to reach the brain and other tissues in transthyretin-null mice. Am J Physiol [Internet]. 1997;272(3 Pt 1):E485–93 [cited 2016

[321] Palha JA, Episkopou V, Maeda S, Shimada K, Gottesman ME, Saraiva MJ. Thyroid hormone metabolism in a transthyretin-null mouse strain. J Biol Chem [Internet]. 1994;269(52):33135–9 [cited 2016 Jan 13]. http://www.ncbi.nlm.nih.gov/pubmed/

[322] Palha JA, Fernandes R, de Escobar GM, Episkopou V, Gottesman M, Saraiva MJ. Transthyretin regulates thyroid hormone levels in the choroid plexus, but not in the brain parenchyma: study in a transthyretin-null mouse model. Endocrinology [Inter‐ net]. 2000;141(9):3267–72 [cited 2016 Jan 13]. http://www.ncbi.nlm.nih.gov/pubmed/

[323] Rask L, Anundi H, Peterson PA. The primary structure of the human retinol-binding protein. FEBS Lett [Internet]. 1979;104(1):55–8 [cited 2016 Jan 13]. http://

[324] Newcomer ME, Jones TA, Aqvist J, Sundelin J, Eriksson U, Rask L, et al. The threedimensional structure of retinol-binding protein. EMBO J [Internet]. 1984;3(7):1451–4 [cited 2016 Jan 13]. http://www.pubmedcentral.nih.gov/articlerender.fcgi?ar‐

[325] Blaner WS. Retinol-binding protein: the serum transport protein for vitamin A. Endocr Rev [Internet]. 1989;10(3):308–16 [cited 2015 Dec 12]. http://www.ncbi.nlm.nih.gov/

[326] Kopelman M, Cogan U, Mokady S, Shinitzky M. The interaction between retinolbinding proteins and prealbumins studied by fluorescence polarization. Biochim

Jan 13]. http://www.ncbi.nlm.nih.gov/pubmed/9124556

www.ncbi.nlm.nih.gov/pubmed/573217

tid=557543&tool=pmcentrez&rendertype=abstract

8-anilino-1-naphthalenesulfonic acid. Biochemistry. 1975;14(2):282–9.

www.ncbi.nlm.nih.gov/pubmed/1429970

Neuroscience. 2015.

64 Exploring New Findings on Amyloidosis

7806543

10965897

pubmed/2550213


[348] Reilly MM, King RH. Familial amyloid polyneuropathy. Brain Pathol [Internet]. 1993;3(2):165–76 [cited 2016 Jan 14]. http://www.ncbi.nlm.nih.gov/pubmed/8293178

[338] Bourgault S, Choi S, Buxbaum JN, Kelly JW, Price JL, Reixach N. Mechanisms of transthyretin cardiomyocyte toxicity inhibition by resveratrol analogs. Biochem Biophys Res Commun [Internet]. 2011;410(4):707–13 [cited 2016 Jan 14]. http://

www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3145458&tool=pmcen‐

[339] Manral P, Reixach N. Amyloidogenic and non-amyloidogenic transthyretin variants interact differently with human cardiomyocytes: insights into early events of nonfibrillar tissue damage. Biosci Rep [Internet]. 2015;35(1) [cited 2016 Jan 14]. http://

www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4293901&tool=pmcen‐

[340] Fleming CE, Mar FM, Franquinho F, Saraiva MJ, Sousa MM. Transthyretin internali‐ zation by sensory neurons is megalin mediated and necessary for its neuritogenic activity. J Neurosci [Internet]. 2009;29(10):3220–32 [cited 2016 Jan 14]. http://

[341] Vieira M, Gomes JR, Saraiva MJ. Transthyretin Induces Insulin-like Growth Factor I Nuclear Translocation Regulating Its Levels in the Hippocampus. Mol Neurobiol [Internet]. 2015;51(3):1468–79. http://link.springer.com/10.1007/s12035-014-8824-4

[342] Liz MA, Faro CJ, Saraiva MJ, Sousa MM. Transthyretin, a new cryptic protease. J Biol Chem [Internet]. 2004;279(20):21431–8 [cited 2016 Jan 13]. http://

[343] Liz MA, Gomes CM, Saraiva MJ, Sousa MM. ApoA-I cleaved by transthyretin has reduced ability to promote cholesterol efflux and increased amyloidogenicity. J Lipid Res [Internet]. 2007;48(11):2385–95 [cited 2016 Jan 13]. http://www.ncbi.nlm.nih.gov/

[344] Liz MA, Fleming CE, Nunes AF, Almeida MR, Mar FM, Choe Y, et al. Substrate specificity of transthyretin: identification of natural substrates in the nervous system. Biochem J [Internet]. 2009;419(2):467–74 [cited 2016 Jan 13]. http://www.pubmedcen‐ tral.nih.gov/articlerender.fcgi?artid=4153561&tool=pmcentrez&rendertype=abstract

[345] Liz MA, Leite SC, Juliano L, Saraiva MJ, Damas AM, Bur D, et al. Transthyretin is a metallopeptidase with an inducible active site. Biochem J [Internet]. 2012;443(3):769–

[346] Gouvea IE, Kondo MY, Assis DM, Alves FM, Liz MA, Juliano MA, et al. Studies on the peptidase activity of transthyretin (TTR). Biochimie [Internet]. 2013;95(2):215–23 [cited

[347] Costa R, Ferreira-da-Silva F, Saraiva MJ, Cardoso I. Transthyretin protects against Abeta peptide toxicity by proteolytic cleavage of the peptide: a mechanism sensitive to the Kunitz protease inhibitor. PLoS One [Internet]. 2008;3(8):e2899 [cited 2015 Dec 9]. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2483353&tool=pmcen‐

78 [cited 2016 Jan 13]. http://www.ncbi.nlm.nih.gov/pubmed/22332999

2016 Jan 13]. http://www.ncbi.nlm.nih.gov/pubmed/23000319

trez&rendertype=abstract

66 Exploring New Findings on Amyloidosis

trez&rendertype=abstract

pubmed/17693625

trez&rendertype=abstract

www.ncbi.nlm.nih.gov/pubmed/19279259

www.ncbi.nlm.nih.gov/pubmed/15033978


Neurosci [Internet]. 2015;9:17. http://www.pubmedcentral.nih.gov/articlerender.fcgi? artid=4319477&tool=pmcentrez&rendertype=abstract

[370] Mazur-Kolecka B, Frackowiak J, Wiśniewski HM. Apolipoproteins E3 and E4 induce, and transthyretin prevents accumulation of the Alzheimer's beta-amyloid peptide in cultured vascular smooth muscle cells. Brain Res [Internet]. 1995;698(1–2):217–22 [cited 2016 Jan 4]. http://www.ncbi.nlm.nih.gov/pubmed/8581485

[359] Fleming CE, Saraiva MJ, Sousa MM. Transthyretin enhances nerve regeneration. J

[360] Santos SD, Lambertsen KL, Clausen BH, Akinc A, Alvarez R, Finsen B, et al. CSF transthyretin neuroprotection in a mouse model of brain ischemia. J Neurochem [Internet]. 2010;115(6):1434–44. http://www.ncbi.nlm.nih.gov/pubmed/21044072 [361] Kooijman R. Regulation of apoptosis by insulin-like growth factor (IGF)-I. Cytokine Growth Factor Rev [Internet]. 2006;17(4):305–23 [cited 2016 Jan 14]. http://

[362] Wisniewski T, Castano E, Ghiso J, Frangione B. Cerebrospinal fluid inhibits Alzheim‐ er ?-amyloid fibril formation in vitro. Ann Neurol [Internet]. 1993;34(4):631–3 [cited

[363] Ghiso J, Matsubara E, Koudinov A, Choi-Miura NH, Tomita M, Wisniewski T, et al. The cerebrospinal-fluid soluble form of Alzheimer's amyloid beta is complexed to SP-40,40 (apolipoprotein J), an inhibitor of the complement membrane-attack complex. Biochem J [Internet]. 1993;293(Pt 1):27–30 [cited 2016 Jan 4]. http://www.pubmedcen‐ tral.nih.gov/articlerender.fcgi?artid=1134315&tool=pmcentrez&rendertype=abstract

[364] Goldgaber D, Schwarzman AI, Bhasin R, Gregori L, Schmechel D, Saunders AM, et al. Sequestration of amyloid beta-peptide. Ann N Y Acad Sci [Internet]. 1993;695:139–43

[365] Schwarzman AL, Gregori L, Vitek MP, Lyubski S, Strittmatter WJ, Enghilde JJ, et al. Transthyretin sequesters amyloid beta protein and prevents amyloid formation. Proc Natl Acad Sci USA [Internet]. 1994;91(18):8368–72 [cited 2015 Dec 6]. http:// www.pubmedcentral.nih.gov/articlerender.fcgi?artid=44607&tool=pmcentrez&ren‐

[366] Schwarzman AL, Goldgaber D. Interaction of transthyretin with amyloid beta-protein: binding and inhibition of amyloid formation. Ciba Found Symp [Internet]. 1996;199:146–60 [cited 2015 Nov 30]; discussion 160–4. http://www.ncbi.nlm.nih.gov/

[367] Serot JM, Christmann D, Dubost T, Couturier M. Cerebrospinal fluid transthyretin: aging and late onset Alzheimer's disease. J Neurol Neurosurg Psychiatry. 1997;63(4):

[368] Han S-H, Jung ES, Sohn J-H, Hong HJS, Hong HJS, Kim JW, et al. Human serum transthyretin levels correlate inversely with Alzheimer's disease. J Alzheimers Dis

[369] González-Marrero I, Giménez-Llort L, Johanson CE, Carmona-Calero EM, Castañeyra-Ruiz L, Brito-Armas JM, et al. Choroid plexus dysfunction impairs beta-amyloid clearance in a triple transgenic mouse model of Alzheimer's disease. Front Cell

[Internet]. 2011;25(1):77–84. http://www.ncbi.nlm.nih.gov/pubmed/21335655

[cited 2016 Jan 4]. http://www.ncbi.nlm.nih.gov/pubmed/8239272

Neurochem. 2007;103(2):831–9.

68 Exploring New Findings on Amyloidosis

dertype=abstract

pubmed/8915609

506–8.

www.ncbi.nlm.nih.gov/pubmed/16621671

2016 Jan 4]. http://www.ncbi.nlm.nih.gov/pubmed/8215255


[389] Cardoso I, Almeida MR, Ferreira N, Arsequell G, Valencia G, Saraiva MJ. Comparative in vitro and ex vivo activities of selected inhibitors of transthyretin aggregation: relevance in drug design. Biochem J. 2007;408(1):131–8.

[379] Carro E, Trejo JL, Gomez-Isla T, LeRoith D, Torres-Aleman I. Serum insulin-like growth factor I regulates brain amyloid-beta levels. Nat Med [Internet]. 2002;8(12):1390–7 [cited

[380] Liu L, Murphy RM. Kinetics of inhibition of beta-amyloid aggregation by transthyretin. Biochemistry [Internet]. 2006;45(51):15702–9 [cited 2016 Jan 4]. http://

[381] Yang DT, Joshi G, Cho PY, Johnson JA, Murphy RM. Transthyretin as both a sensor and a scavenger of β-amyloid oligomers. Biochemistry [Internet]. 2013;52(17):2849–61 [cited 2016 Jan 5]. http://www.pubmedcentral.nih.gov/articlerender.fcgi?ar‐

[382] Du J, Murphy RM. Characterization of the interaction of β-amyloid with transthyretin monomers and tetramers. Biochemistry [Internet]. 2010;49(38):8276–89 [cited 2016 Jan 5]. http://www.pubmedcentral.nih.gov/articlerender.fcgi?ar‐

[383] Schwarzman AL, Tsiper M, Wente H, Wang A, Vitek MP, Vasiliev V, et al. Amyloido‐ genic and anti-amyloidogenic properties of recombinant transthyretin variants. Amyloid [Internet]. 2004;11(1):1–9 [cited 2015 Dec 23]. http://www.ncbi.nlm.nih.gov/

[384] Palha JA, Moreira P, Wisniewski T, Frangione B, Saraiva MJ. Transthyretin gene in Alzheimer's disease patients. Neurosci Lett [Internet]. 1996;204(3):212–4 [cited 2016 Jan

[385] Du J, Cho PY, Yang DT, Murphy RM. Identification of beta-amyloid-binding sites on transthyretin. Protein Eng Des Sel [Internet]. 2012;25(7):337–45 [cited 2016 Feb 1]. http://

www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3530273&tool=pmcen‐

[386] Merched A, Serot JM, Visvikis S, Aguillon D, Faure G, Siest G. Apolipoprotein E, transthyretin and actin in the CSF of Alzheimer's patients: relation with the senile plaques and cytoskeleton biochemistry. FEBS Lett [Internet]. 1998;425(2):225–8 [cited

[387] Hornstrup LS, Frikke-Schmidt R, Nordestgaard BG, Tybjærg-Hansen A. Genetic stabilization of transthyretin, cerebrovascular disease, and life expectancy. Arterioscler Thromb Vasc Biol [Internet]. 2013;33(6):1441–7 [cited 2015 Oct 27]. http://

[388] Ribeiro CA, Saraiva MJ, Cardoso I. Stability of the transthyretin molecule as a key factor in the interaction with a-beta peptide—relevance in Alzheimer's Disease. PLoS One [Internet]. 2012;7(9):e45368. http://www.pubmedcentral.nih.gov/articlerender.fcgi?

2015 Dec 29]. http://www.ncbi.nlm.nih.gov/pubmed/12415260

www.ncbi.nlm.nih.gov/pubmed/17176092

tid=3658121&tool=pmcentrez&rendertype=abstract

tid=2943652&tool=pmcentrez&rendertype=abstract

5]. http://www.ncbi.nlm.nih.gov/pubmed/8938268

2016 Jan 5]. http://www.ncbi.nlm.nih.gov/pubmed/9559653

www.ncbi.nlm.nih.gov/pubmed/23580146

artid=3444465&tool=pmcentrez&rendertype=abstract

pubmed/15185492

70 Exploring New Findings on Amyloidosis

trez&rendertype=abstract


## **Supramolecular Organization of Amyloid Fibrils**

## Dmitry Kurouski

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62672

#### **Abstract**

The current chapter illustrates how both electron and scanning probe microscopy can be utilized to unravel the supramolecular organization of amyloid fibrils, β-sheet-rich protein aggregates, which are strongly associated with various neurodegenerative diseases. It also discusses why morphologically different fibrils can be grown from the same protein under slightly different experimental conditions, which is a phenomen‐ on known as fibril polymorphism. Next, it establishes the high potential of vibrational circular dichroism (VCD) for unraveling the supramolecular organization of amyloid fibrils. Finally, it discusses several hypotheses of amyloid fibril formation that were developed based on numerous microscopic and spectroscopic studies.

**Keywords:** amyloid fibrils, supramolecular chirality, microscopy, VCD, polymor‐ phism or fibril polymorphism

## **1. Introduction**

Under normal physiological conditions, the native structure of partially unfolded proteins is restored by numerous self-sustaining pathways of cell homeostasis. However, under some pathological conditions, such as neurodegenerative diseases, partially unfolded proteins may adopt β-sheet structure [1, 2]. This primarily happens because the β-sheet structure is energet‐ ically more favorable when compared to the native structure of the protein. When two βsheet moieties associate together plane-to-plane, they form a very stable structure, known as a cross-β-sheet.Thecross-β-sheetiscapableoftemplatingtheaggregationofmisfoldedorpartially unfolded proteins. As a result, fibril filament, the simplest supramolecular architecture, is formed. It stretches microns in length in the direction perpendicular the peptide strands in the β-sheets [3–5]. Following electrostatic energy minimization, filaments tend to twist along their longitudinalaxis.Andasaresult,localchargesonthefibrilsurfaceincreasetheirmutualdistance.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Contrastingly, twisted filaments have much higher elastic energy than a flat structure. And consequently,the filaments tendtoadoptbecomingas relaxedandflatgeometricallyaspossible to reach the elastic energy minimum. The net between these two competing energies, electro‐ static and elastic, determines the degree of the filament twist [6, 7].

Twisted filaments also braid and coil with other filaments [8–10], forming higher hierarchical supramolecular structures named proto-fibrils and fibrils [11, 12]. Alternatively, twisted filaments may associate side-by-side, forming cross-β-sheet tape-like fibrils [10, 11]. Different filaments' propagation pathways result in a large diversity of fibril morphologies. This phenomenon is known as fibril polymorphism [3, 13]. *Post-mortem* microscopic examination of fibrils that were detected in the organs and tissue of patients, who were diagnosed with different neurodegenerative diseases, also revealed their morphological heterogeneity [14, 15]. Moreover, *in vitro* studies have demonstrated that morphologically or structurally different fibril polymorphs had different toxicity [16]. And as a result, a clear understanding of fibril supramolecular organization will help to unravel the origin of fibril toxicity.

Methodologically, unraveling the supramolecular organization of amyloid fibrils is a challenging task. This is primarily because fibrils are insoluble and cannot be crystalized, which unfortunately limits X-ray and NMR, classical tools of structural biology, for their studies [17, 18]. Historically, microscopy was the first, and most commonly utilized technique which is capable of unraveling supramolecular organization of amyloid fibrils. There are several microscopes that are effective in resolving amyloid aggregates which include: electron (EM), transmission (TEM), and scanning electron (SEM) microscopes, and lastly probe microscopes. In addition to microscopy, vibrational circular dichroism (VCD) can directly probe supramolecular organization of amyloid fibrils [11, 19]. It was recently discovered that VCD can detect the twist of the fibril filaments, which not always may be visualized using SEM or atomic force microscopy (AFM) [8]. The observed VCD intensities of fully developed fibrils and their filaments are one to two orders of magnitude larger than VCD intensities observed from solutions of individual proteins. This indicates that enhanced VCD arises from the long-range supramolecular chirality of fibrils, making VCD a unique *solution-phase*, stereospecific probe of protein fibril structure and chiral morphology. A growing body of literature indicates that VCD has already become a powerful and broadly used tool for the supramolecular characterization of amyloid aggregates [11, 20, 21].

## **2. Unraveling supramolecular chirality of amyloid fibrils using microscopy**

Conventional light microscopy is not capable of visualizing individual amyloid aggregates due to their small sizes. According to Abbe diffraction limit, a resolution (*d*) directly depends on a wavelength of light (*λ*), where *α* is the half-angle of the maximum cone of light that can enter the microscope objective (1).

#### Supramolecular Organization of Amyloid Fibrils http://dx.doi.org/10.5772/62672 75

$$d = \frac{\lambda}{2\sin(a)}\tag{1}$$

For a microscope objective, the aperture angle is described by the numerical aperture (NA), where NA = n•sinα (n is a media refractive index). Consequently, *d* of any confocal microscope can be expressed as (2).

Contrastingly, twisted filaments have much higher elastic energy than a flat structure. And consequently,the filaments tendtoadoptbecomingas relaxedandflatgeometricallyaspossible to reach the elastic energy minimum. The net between these two competing energies, electro‐

Twisted filaments also braid and coil with other filaments [8–10], forming higher hierarchical supramolecular structures named proto-fibrils and fibrils [11, 12]. Alternatively, twisted filaments may associate side-by-side, forming cross-β-sheet tape-like fibrils [10, 11]. Different filaments' propagation pathways result in a large diversity of fibril morphologies. This phenomenon is known as fibril polymorphism [3, 13]. *Post-mortem* microscopic examination of fibrils that were detected in the organs and tissue of patients, who were diagnosed with different neurodegenerative diseases, also revealed their morphological heterogeneity [14, 15]. Moreover, *in vitro* studies have demonstrated that morphologically or structurally different fibril polymorphs had different toxicity [16]. And as a result, a clear understanding

of fibril supramolecular organization will help to unravel the origin of fibril toxicity.

Methodologically, unraveling the supramolecular organization of amyloid fibrils is a challenging task. This is primarily because fibrils are insoluble and cannot be crystalized, which unfortunately limits X-ray and NMR, classical tools of structural biology, for their studies [17, 18]. Historically, microscopy was the first, and most commonly utilized technique which is capable of unraveling supramolecular organization of amyloid fibrils. There are several microscopes that are effective in resolving amyloid aggregates which include: electron (EM), transmission (TEM), and scanning electron (SEM) microscopes, and lastly probe microscopes. In addition to microscopy, vibrational circular dichroism (VCD) can directly probe supramolecular organization of amyloid fibrils [11, 19]. It was recently discovered that VCD can detect the twist of the fibril filaments, which not always may be visualized using SEM or atomic force microscopy (AFM) [8]. The observed VCD intensities of fully developed fibrils and their filaments are one to two orders of magnitude larger than VCD intensities observed from solutions of individual proteins. This indicates that enhanced VCD arises from the long-range supramolecular chirality of fibrils, making VCD a unique *solution-phase*, stereospecific probe of protein fibril structure and chiral morphology. A growing body of literature indicates that VCD has already become a powerful and broadly used tool for the

**2. Unraveling supramolecular chirality of amyloid fibrils using microscopy**

Conventional light microscopy is not capable of visualizing individual amyloid aggregates due to their small sizes. According to Abbe diffraction limit, a resolution (*d*) directly depends on a wavelength of light (*λ*), where *α* is the half-angle of the maximum cone of light that can

static and elastic, determines the degree of the filament twist [6, 7].

74 Exploring New Findings on Amyloidosis

supramolecular characterization of amyloid aggregates [11, 20, 21].

enter the microscope objective (1).

$$d = \frac{0.61\lambda}{NA} \tag{2}$$

The expression 2 indicates that the resolution increase with the decrease in the wavelength and an increase in the NA of the objective. The best optical systems would be equipped with 100× oil immersed objective (NA=1.4) will give the resolution of ~200 nm. At the same time, amyloid fibrils, as we will discuss below, are only 10–20 nm in diameter. Nevertheless, it should be mentioned that the light microscopy is the most commonly utilized technique in the clinical practice for the confirmatory diagnostic of amyloid plaques. Upon staining with Congo-red, in the linearly polarized light, these amyloid deposits exhibit green birefringence.

Recently invented super-resolution microscopy, such as photoactivated localization micro‐ scopy (PALM) and direct stochastical optical reconstruction microscopy (d-STORM), allowed for overcoming the Abbe diffraction limit and reach 2–10 nm spatial resolution [22–24]. For example, d-STORM was capable of resolving individual amyloid β aggregates [25]. However, very limited information about their supramolecular organization could be obtained. Partially, because sample labeling with fluorophores is required, which obscures tiny details of fibril topology. Also, since there are only a few fluorophores are activated per every light exposure in super-resolution microscopy, long acquisition time is typically required for an image processing. Therefore, thermal drift of current super-resolution microscopes becomes the serious issue which limits their spatial resolution.

#### **2.1. Unraveling the shape and localization of amyloid fibrils by TEM**

The invention of the electron microscope, by Max Knoll and Ernst Ruska at the Berlin Tech‐ nische Hochschule, in 1931, allowed for overcoming the limitation of a visible light in obtaining higher spatial resolution. Using TEM, the first morphological characterization of the individual amyloid fibrils was achieved [26, 30]. This microscopic technique is relatively facile when compared to other microscopic tools which will be discussed below. In most cases, only a drop of the analyzed sample is needed to be placed onto a carbon or formvar-coated Cu film support and dried under ambient conditions. Uranium or osmium salts are often applied to the dried sample to increase the contrast of biological samples. Alternatively, samples for TEM can be prepared by cutting plastic embedded biological samples. For example, TEM imaging of the thin-cut plastic embedded amyloid tissues revealed long unbranched rod-like aggregates that were named amyloid fibrils (**Figure 1A**) [26–29]. Aggregates with the same morphology were later reported by M. Kidd upon TEM imaging of the extract of amyloid-loaded liver tissue (**Figure 1B**) [30].

**Figure 1.** TEM images of amyloid fibrils in (A) thin-cut plastic embedded amyloid tissue (×50,000) and (B) extract of amyloid-loaded liver tissue. Locations of amyloid fibrils and their twisted filaments are marked by "A" and "F" re‐ spectively. Adapted from Tosoni et al. [26] and Kidd [30].

Aggregates of Tau proteins, histologically known as neurofibrillary tangles, are commonly copresent together with amyloid plaques in brains of patients, who have been diagnosed with Alzheimer's disease. Their detailed microscopic study conducted by Wischik et al. (**Figure 2**) [14, 15]. Using TEM, Wischik et al. studied the extracts from the human brains that contained neurofibrillary tangles. Authors observed fibrils with flat and twisted topologies. Using metal

**Figure 2.** TEM images of uncoated (A, B) and metal coated (C, D) Tau fibrils extracted from the human brain. Scale bards are 100 nm (A, C, D) and 1000 nm (B) [14, 15].

shadowing, Wischik et al. determined the twist direction, which appeared to be left-handed. Additional, it was discovered that these left-twisted fibrils were composed of two intertwined left-handed proto-fibrils. The exposure of left-twisted fibrils to an alkali solution led to their untwining, which made Wischik et al. to conclude that the integrity of left-twisted fibrils was held by electrostatic interactions between their proto-fibrils.

later reported by M. Kidd upon TEM imaging of the extract of amyloid-loaded liver tissue

**Figure 1.** TEM images of amyloid fibrils in (A) thin-cut plastic embedded amyloid tissue (×50,000) and (B) extract of amyloid-loaded liver tissue. Locations of amyloid fibrils and their twisted filaments are marked by "A" and "F" re‐

Aggregates of Tau proteins, histologically known as neurofibrillary tangles, are commonly copresent together with amyloid plaques in brains of patients, who have been diagnosed with Alzheimer's disease. Their detailed microscopic study conducted by Wischik et al. (**Figure 2**) [14, 15]. Using TEM, Wischik et al. studied the extracts from the human brains that contained neurofibrillary tangles. Authors observed fibrils with flat and twisted topologies. Using metal

**Figure 2.** TEM images of uncoated (A, B) and metal coated (C, D) Tau fibrils extracted from the human brain. Scale

(**Figure 1B**) [30].

76 Exploring New Findings on Amyloidosis

spectively. Adapted from Tosoni et al. [26] and Kidd [30].

bards are 100 nm (A, C, D) and 1000 nm (B) [14, 15].

These pioneering works were followed by numerous studies that report TEM microphoto‐ graphs of various protein aggregates and amyloid fibrils [31, 32]. It has been demonstrated that TEM was capable of detecting twists in ribbon-like fibrils, curvature of fibrils, and the roughness of their surfaces. TEM images could also be used to obtain quantitative data, including dimensions of fibrils and their precursors, such as oligomers and filaments. In addition, TEM was able to reveal a number of filaments in the mature fibril, as well as the periodicity of the fibril twists. However, in most cases, TEM imaging was simply used to confirm a presence of fibril species rather than to provide their morphological characterization. This is in parts because such microscopic examination can be performed relatively quickly, allowing researchers to verify whether fibril formation has occurred or not.

In parallel, TEM was used to monitor changes in the structure of mature fibrils which were caused by various chemical and physical factors [33, 34]. For example, it has been demonstrated that integrity of insulin fibrils could be perturbed through alteration of fibril electrostatic interactions [33]. Shammas et al. exposed insulin fibrils which were grown at pH 2 to solutions with pH 4, 6, 8, 10, and 12. Using TEM, Shammas et al. found that at pH above 4 insulin fibrils drastically changed their morphology (**Figure 3**). Moreover, full disintegration of fibrils was observed if the solution pH was higher than pH 10. It has been concluded that these changes were caused by the strong electrostatic repulsion of the protein amino acid sequence in the fibril cross-β-sheet.

**Figure 3.** TEM images of insulin fibrils (pH 2.0) after prolong exposition in solvents with higher pHs. Scale bars are 200 nm [34].

Separately, using VCD, Kurouski et al. demonstrated that a small change in pH drastically changes morphology and supramolecular chirality of insulin fibrils [35]. This process was irreversible and occurred only when the pH was raised from 1.5 to 2.5. No effect of solution ionic strength was found. An addition of sodium chloride up to 1 M concentration to pH 1.5 fibrils did not change the kinetics of the polymorphs' inter-conversion.

## **2.2. Mass-per-length measurements of amyloid fibrils using scanning transmission electron microscopy**

As the analog of TEM, scanning transmission electron microscopy (STEM) is often used to explore the morphological organization of amyloid aggregates. Similar to TEM, the back‐ ground of STEM images has lower intensity than protein specimens. This is due to stronger electron scattering from protein aggregates comparing to a thin carbon film on which they are adsorbed on. As a result, STEM image intensities appear to be proportional to mass densities (per unit area). This can be used to determine mass-per-length (MPL) values of the analyzed specimens [17, 36, 37]. MPL can be determined directly by measuring the incident electron beam flux if the electron scattering cross-sections, as well as the detector geometry and sensitivity are known. Alternatively, MPL can be evaluated by a comparison of the intensities of the analyzed specimens with the intensities of objects with known mass densities. Tobacco mosaic virus (TMV) is the most commonly used reference for such a comparison-based MPL imaging. MPL values can be utilized to determine secondary structure of amyloid fibrils. For

**Figure 4.** TEM microphotograph (A) of amyloid fibrils grown from Rnq1 prion and their MPL (B) histogram. STEM microphotographs (C and D) or the same fibrils and TMV rods (indicated by double-headed arrows). MPL values (kDa/nm) are shown for fibril segments enclosed in rectangles [39].

example, using MPL, it has been found that 40-residue amyloid β peptide (Aβ1–40) formed two distinct fibril polymorphs with 2- and 3-fold symmetry [36]. MPL measurements con‐ firmed a β-helix-like structure of HET-s prion (HET-s218–289) protein fibrils, where each peptide molecule spanned two turns of the β-helix [38]. It has been also shown that MPL measurements could be used to investigate structural organization of fibrils formed by the yeast prion protein Rnq1 (**Figure 4**) [39]. Chen et al. found that the prion sequence was folded into in-register parallel β-sheet structure, with one Rnq1 molecule per 0.47-nm β-sheet repeat spacing.

## **2.3. Probing topology and supramolecular chirality of amyloids by SEM**

irreversible and occurred only when the pH was raised from 1.5 to 2.5. No effect of solution ionic strength was found. An addition of sodium chloride up to 1 M concentration to pH 1.5

**2.2. Mass-per-length measurements of amyloid fibrils using scanning transmission electron**

As the analog of TEM, scanning transmission electron microscopy (STEM) is often used to explore the morphological organization of amyloid aggregates. Similar to TEM, the back‐ ground of STEM images has lower intensity than protein specimens. This is due to stronger electron scattering from protein aggregates comparing to a thin carbon film on which they are adsorbed on. As a result, STEM image intensities appear to be proportional to mass densities (per unit area). This can be used to determine mass-per-length (MPL) values of the analyzed specimens [17, 36, 37]. MPL can be determined directly by measuring the incident electron beam flux if the electron scattering cross-sections, as well as the detector geometry and sensitivity are known. Alternatively, MPL can be evaluated by a comparison of the intensities of the analyzed specimens with the intensities of objects with known mass densities. Tobacco mosaic virus (TMV) is the most commonly used reference for such a comparison-based MPL imaging. MPL values can be utilized to determine secondary structure of amyloid fibrils. For

**Figure 4.** TEM microphotograph (A) of amyloid fibrils grown from Rnq1 prion and their MPL (B) histogram. STEM microphotographs (C and D) or the same fibrils and TMV rods (indicated by double-headed arrows). MPL values

(kDa/nm) are shown for fibril segments enclosed in rectangles [39].

fibrils did not change the kinetics of the polymorphs' inter-conversion.

**microscopy**

78 Exploring New Findings on Amyloidosis

In SEM, both detector and the electron source are located on the same side of the sample. Consequently, only scattered electrons (rather than electrons that go through the sample, as in the case of TEM) are used to obtain the image of the analyzed specimen. SEM also requires much lower energy of the electron beam (0.2–40 keV vs 100–120 keV, as used for TEM) to obtain the contrast image. Similar to TEM, protein samples, such as amyloid fibrils, are commonly stained with uranium or osmium salts prior to microscopic examination to gain a better contrast. While SEM is not capable of resolving amyloid fibrils in the fixed tissues, it provides excessive morphological information about the *in vitro* prepared or *ex vivo* extracted fibrils. One of the most valuable sides of SEM morphological examination is its capacity to determine the twist handedness of amyloid fibrils. For example, using SEM Rubin et al. evaluated supramolecular chirality of fibrils grown from short amino acid fragments of serum amyloid A (SAA) protein [40, 41]. SAA are a group of apolipoproteins associated with high-density

**Figure 5.** SEM image of simultaneously grown amyloid fibrils of serum amyloid A SAA2–6 peptide that exhibit left- and right-twisted fibrils [40].

lipoprotein (HDL) in blood plasma that are often expressed in response to inflammatory stimuli in liver.

Rubin et al. found that SAA fragment with the sequence SFFSFLG (SAA2–6) simultaneously formed both left- and right-twisted fibrils (**Figure 5**). It was also found that fibrils with the same twist direction had different twist periodicity. Some of the right-twisted fibrils were highly twisted (**Figure 3**, fibril marked R at the top), while others had much more of a loose twist (**Figure 3**, fibrils marked R on the right and bottom). This indicated that several righttwisted fibril polymorphs have simultaneously grown. Rubin et al. also discovered that SAA2–12 with the sequence SFFSFLGEAFD exclusively formed right-twisted fibrils, while its analog with only two different amino acids, SAA1–11RSFFSFLGEAF aggregated in 100% lefttwisted fibrils [40]. This discovery suggested that amino acid sequence may control the su‐ pramolecular chirality of amyloid aggregates.

At the same time, Kurouski et al. demonstrated that fibril morphology and supramolecular chirality can be directly controlled by pH [8, 42]. Insulin and lysozyme aggregation at pH below 2 (25°C) led to a formation of tape-like flat fibrils [11]. At the same time, at pH above this point, both of these proteins formed left-twisted fibrils (**Figure 6**) [8]. A fragment of transthyretin (105–115) and HET-s prion (HET-s218–289) protein, on the opposite, formed left twisted fibrils at low pH (25°C), while their aggregation at pH above 2.5 resulted in flat tape-like fibrils [11]. HET-s is a prion protein of the fungus *Podospora anserine*, which C-terminus fragment is capable of forming amyloid fibrils at low pH. Authors suggested that protonation/deprotonation of aspartic and glutamic amino acid residues, as well as peptide C-termini, which was taking place around this pH, changed the charge on the surface of the filaments. As a result, filaments adopted either left-handed or right handed twist. Left twisted filaments tended to braid and coil forming left-handed fibrils. At the same time, right twisted filaments associated side-byside forming tape-like fibrils [11].

**Figure 6.** SEM images of insulin (A, B) and lysozyme (C and D) tape-like (top panel) and twisted (bottom panel) fibrils [8]. Twists on the fibril surface are marked by red arrows. Scale bars are 100 nm.

## **2.4. Application of cryo-SEM for determination of amyloid fibril topology**

lipoprotein (HDL) in blood plasma that are often expressed in response to inflammatory

Rubin et al. found that SAA fragment with the sequence SFFSFLG (SAA2–6) simultaneously formed both left- and right-twisted fibrils (**Figure 5**). It was also found that fibrils with the same twist direction had different twist periodicity. Some of the right-twisted fibrils were highly twisted (**Figure 3**, fibril marked R at the top), while others had much more of a loose twist (**Figure 3**, fibrils marked R on the right and bottom). This indicated that several righttwisted fibril polymorphs have simultaneously grown. Rubin et al. also discovered that SAA2–12 with the sequence SFFSFLGEAFD exclusively formed right-twisted fibrils, while its analog with only two different amino acids, SAA1–11RSFFSFLGEAF aggregated in 100% lefttwisted fibrils [40]. This discovery suggested that amino acid sequence may control the su‐

At the same time, Kurouski et al. demonstrated that fibril morphology and supramolecular chirality can be directly controlled by pH [8, 42]. Insulin and lysozyme aggregation at pH below 2 (25°C) led to a formation of tape-like flat fibrils [11]. At the same time, at pH above this point, both of these proteins formed left-twisted fibrils (**Figure 6**) [8]. A fragment of transthyretin (105–115) and HET-s prion (HET-s218–289) protein, on the opposite, formed left twisted fibrils at low pH (25°C), while their aggregation at pH above 2.5 resulted in flat tape-like fibrils [11]. HET-s is a prion protein of the fungus *Podospora anserine*, which C-terminus fragment is capable of forming amyloid fibrils at low pH. Authors suggested that protonation/deprotonation of aspartic and glutamic amino acid residues, as well as peptide C-termini, which was taking place around this pH, changed the charge on the surface of the filaments. As a result, filaments adopted either left-handed or right handed twist. Left twisted filaments tended to braid and coil forming left-handed fibrils. At the same time, right twisted filaments associated side-by-

**Figure 6.** SEM images of insulin (A, B) and lysozyme (C and D) tape-like (top panel) and twisted (bottom panel) fibrils

[8]. Twists on the fibril surface are marked by red arrows. Scale bars are 100 nm.

stimuli in liver.

80 Exploring New Findings on Amyloidosis

pramolecular chirality of amyloid aggregates.

side forming tape-like fibrils [11].

Typically, both TEM and SEM require sample dehydration. Protein specimens in general and amyloid fibrils in particular are very sensitive to dehydration, which may cause drastic changes in their morphology. To overcome this limitation, cryo-procedure of the sample preparation is often used. In cryo-SEM, protein specimens are imaged at temperature below ambient (typically between −100 and −175°C). This allows for the sample to be preserved and recorded in the fully hydrated and chemically unmodified state.

Cryo-SEM has been extensively used to investigate supramolecular organization of amyloid aggregates that strongly associated with Alzheimer's and Parkinson's diseases. For example, using cryo-EM, Meinhardt et al. found that Aβ(1–40) peptide could form amyloid fibrils with a range of different morphologies [43]. Authors showed that proto-fibrils could associate either side-by-side, forming tape-like fibrils, or coil (intertwine), forming twisted cables. It has been also shown that despite the width of the coiled fibrils varied from 10 to 21 nm, all of them exhibited the same left-handed twist with a turnover from 65 to 163 nm. It was concluded that such difference in the fibril thickness could be due to a different number of proto-filaments that were taking place in their formation [44].

Antzutkin et al. investigated an aggregation of Arctic mutant of Aβ(1–40). It has been shown that this peptide formed seven fibril polymorphs (three non-coiled and four coiled) if aggre‐ gated at pH 7.4, 22°C [45]. Moreover, Antzutkin et al. demonstrated that these fibril poly‐ morphs with different morphologies had dramatically different growth rates. It has been found that Aβ(1–40) fibril morphologies could have significant variations in both width and twist tightness, depending on the ionic strength (and the nature of ions) in the aggregation solution [46]. Finally, Petkova et al. investigated the sample agitation impact on the morphology of Aβ(1–40) fibrils. Using microscopy and solid-state NMR (ss-NMR), significant structural differences between fibrils that were grown with and without agitation were found [47].

Supramolecular organization and polymorphism of Aβ(1–42) fibrils are much less investigated comparing to Aβ(1–40) fibrils. Using cryo-SEM, Lührs et al. investigated structural organiza‐ tion of Aβ(1–42) fibrils and found that they were composed of several filaments that had ~2.5 nm in height and around 4.5 nm in width [48]. These filaments braided and coiled together forming thick left-handed cables. Later, Zhang et al. confirmed that Aβ(1–42) formed exclu‐ sively left-handed fibrils [49]. It has been also suggested that Aβ(1–42) filaments intertwined around each other, forming a hollow core.

In 2008, Vilar et al. reported results of a very detailed investigation of supramolecular organ‐ ization of α-synuclein fibrils [50]. Using cryo-SEM and ss-NMR, Vilar et al. found that αsynuclein aggregated formed straight filaments that were around 2 nm in width. These filaments tended to dimerize into thicker proto-fibrils via side-to-side association, forming straight tape-like fibrils. At the same time, they it has been noticed that these filaments could braid together forming left-handed proto-fibrils, with a diameter of ~6.5 nm, and fibrils with a diameter of 9.8 nm (**Figure 7**) [50]. In fact, both straight and twisted α-synuclein fibril polymorphs were detected upon the *post mortem* examination of brains of people who were diagnosed with Parkinson's disease [51, 52].

**Figure 7.** Cryo-SEM images of tape-like (top row) and twisted (bottom row) α-synuclein fibrils. Scale bars are 20 nm [50].

Recently, using cryo-SEM, Rubin et al. examined how the absolute chiral configuration of the amino acids in a peptide sequence impacts on the supramolecular organization of the peptide aggregates [41].

**Figure 8.** Cryo-SEM images of fibrils grown from all-L amino acid (A) and all-R (B) amino acid fragment of serum amyloid A (SAA1–12) protein [41].

It has been found that all L amino acid peptide with the sequence RSFFSFLGEAFD (SAA1–12) formed right-handed fibrils (**Figure 8**). At the same time, aggregation of the same amino acid sequence with all D amino acids led to a formation of exclusively left-twisted fibrils. Separately, Harper et al. demonstrated the same for Aβ(1–40) peptide. It has been found that all L amino acid Aβ(1–40) peptide formed left-handed fibrils twist. At the same time, aggregation of all D amino acid peptide Aβ(1–40) resulted in right-handed fibrils. These experimental pieces of evidence demonstrate that absolute chiral configuration of the peptide sequence may determine whether one or another chiral polymorph will be grown.

## **2.5. AFM is capable of unraveling mechanisms of fibril formation and structural organization of amyloids**

**Figure 7.** Cryo-SEM images of tape-like (top row) and twisted (bottom row) α-synuclein fibrils. Scale bars are 20 nm

Recently, using cryo-SEM, Rubin et al. examined how the absolute chiral configuration of the amino acids in a peptide sequence impacts on the supramolecular organization of the peptide

**Figure 8.** Cryo-SEM images of fibrils grown from all-L amino acid (A) and all-R (B) amino acid fragment of serum

[50].

aggregates [41].

82 Exploring New Findings on Amyloidosis

amyloid A (SAA1–12) protein [41].

In AFM, silicon or silicon nitride cantilever is used to image the sample. Changes in the frequency oftip oscillation, as it is moved across the surface (tapping mode AFM), are recorded by the laser that is reflected from the back side of the cantilever. As higher the object on the surface, as smaller becomes the amplitude of the tip oscillation. In another modification of AFM, known as contact-mode AFM (CM-AFM), the tip is not oscillating above the surface, but rather steadily kept at it. When CM-AFM tip is moved across the surface, it simply reflects its roughness, similar to a finger of a blind person who reads the Braille font. One can imagine that the CM-AFM cantilever can easily damage fine protein samples upon such microscopic examination. This much rarely happens in AC-AFM because the tip is being repulsed from the surface as soon as van der Waals forces appear between the two. Therefore, CM-AFM is much less frequently used for imaging biological samples, including amyloid fibrils.

One advantage of AFM over EM is its ability to image the specimen without any dehydra‐ tion or fixation. This is extremely important for microscopic characterization of protein samples, such as amyloid fibrils. It should be noted that AFM provides an accurate height of the imaged specimen, while width often appear slightly larger than the real width of the imaged object. This error is known as the tip convolution effect. It arises from the tip that is used for the sample imaging: as larger the tip diameter and smaller the object on the surface, as larger the tip convolution error. Therefore, AFM and SEM are commonly used complimen‐ tary to each other for a determination of accurate height (AFM) and width (SEM) of the analyzed specimens [8, 11].

Using AFM, Mezenga group recently investigated aggregation of bovine serum albumin (BSA) [53]. Usov et al. found that BSA aggregated forming flexible filaments with left-handed twisted morphology. On later stages of fibril formation, they observed rigid fibrils that either had no twist or were right-twisted. Usov et al. proposed that tube-like structures could be formed if the left-twisted filaments would continue twisting along their longitudinal axis. Such supertwisting would finally result in a formation of a hollow tube. Usov et al. also proposed that observed right-handed twisted ribbons could be formed if left-handed filaments would continue to twist passing through the tube stage. This paradigm is very easy to visualize with a piece of rope. If a degree of left-handed twist will be increased, the left-twitted rope would suddenly start making right-handed twists. Therefore, Usov et al. concluded that observed right-twisted fibrils had the left-handed twist of their internal filaments [53].

Adamcik et al. recently demonstrated that the degree of the fibril twist could be changed by the solvent ionic strength. It has been shown that if β-lactoglobulin was aggregated in 100 mM NaCl, it formed tape-like fibrils. However, at 0 mM NaCl, grown fibrils tended to adopt a lefthanded twist. It has been concluded that as lower the ionic strength, as higher the twist degree of the formed β-lactoglobulin fibrils [54].

Mezzenga group also explored how pH controlled supramolecular organization of amyloid fibril growth at water-air interface (AWI) [55, 56]. It was discovered that small change in pH caused significant differences in interfacial properties of β-lactoglobulin fibrils, such as their alignment, entanglement, multilayer formation, and fibril fracture. For example, at pH 2, βlactoglobulin fibrils did not change their aggregation state after 5-hour exposition at AWI (**Figure 9**). However, at pH 3, these fibrils tended to associate into bundles rather than stay in nematic domains. It has been concluded that such drastic changes were caused by the change of the charge on the fibril surface [56].

**Figure 9.** β-Lactoglobulin fibrils on water-air interface at pH 2 (top) and 3 (bottom) after (A) 1, (B) 2, and 5 hours (C). Modified from Jordens et al. [56].

Nearly a decade ago, it was demonstrated that vortexing of insulin solutions during protein aggregation at 60°C caused formation of similar bundled fibril superstructures [57, 58]. In addition to AFM, Dzwolak group explored chiroptical properties of these insulin bundles using induced circular dichroism (ICD) [59]. In ICD, an achiral chromophore is used to probe the structure of a chiral molecule that has very low intensity of circular dichroism signal. It has been found that these fibril superstructures are capable of binding thioflavin T (ThT), which results in a negative induced circular dichroism (−ICD). However, if the protein aggregation was carried out at steady-state conditions, insulin fibrils did not form bundles and conse‐ quently no enhanced ICD signal was observed.

Adamcik et al. recently demonstrated that the degree of the fibril twist could be changed by the solvent ionic strength. It has been shown that if β-lactoglobulin was aggregated in 100 mM NaCl, it formed tape-like fibrils. However, at 0 mM NaCl, grown fibrils tended to adopt a lefthanded twist. It has been concluded that as lower the ionic strength, as higher the twist degree

Mezzenga group also explored how pH controlled supramolecular organization of amyloid fibril growth at water-air interface (AWI) [55, 56]. It was discovered that small change in pH caused significant differences in interfacial properties of β-lactoglobulin fibrils, such as their alignment, entanglement, multilayer formation, and fibril fracture. For example, at pH 2, βlactoglobulin fibrils did not change their aggregation state after 5-hour exposition at AWI (**Figure 9**). However, at pH 3, these fibrils tended to associate into bundles rather than stay in nematic domains. It has been concluded that such drastic changes were caused by the change

**Figure 9.** β-Lactoglobulin fibrils on water-air interface at pH 2 (top) and 3 (bottom) after (A) 1, (B) 2, and 5 hours (C).

Nearly a decade ago, it was demonstrated that vortexing of insulin solutions during protein aggregation at 60°C caused formation of similar bundled fibril superstructures [57, 58]. In addition to AFM, Dzwolak group explored chiroptical properties of these insulin bundles using induced circular dichroism (ICD) [59]. In ICD, an achiral chromophore is used to probe the structure of a chiral molecule that has very low intensity of circular dichroism signal. It has been found that these fibril superstructures are capable of binding thioflavin T (ThT), which results in a negative induced circular dichroism (−ICD). However, if the protein aggregation

of the formed β-lactoglobulin fibrils [54].

84 Exploring New Findings on Amyloidosis

of the charge on the fibril surface [56].

Modified from Jordens et al. [56].

Mezzenga group also explored aggregation of α-synuclein at AWI and solid-liquid interfa‐ ces using AFM [60]. They found that at AWI, α-synuclein rapidly aggregated into amyloid fibrils that remained adsorbed to the AWI. Instead, when the protein aggregation was taken place at solid-liquid interface, α-synuclein aggregation is greatly reduced. This finding demonstrated that protein aggregation is a very complex process that can be altered by varying solution conditions and presence of interfaces that can either accelerate or decelerate fibril formation.

Separately, Qin et al. aggregated α-synuclein in similar conditions (10 mM phosphate buffer, pH 7.4) and found that mature fibrils have a left-handed twist [61]. They also elucidated the role of two terminal fragments of α-synuclein. It was found that the protein without the first 29 amino acids from the N terminus (syn30–140) formed fibrils similar to the intact α-synu‐ clein. These fibrils had a right-twisted twist and were composed of two filaments and were around 107 nm in height (AFM), **Figure 10**.

**Figure 10.** Morphology of the fibrils derived from three truncated α-synucleins. The inserted panels in each AFM im‐ age are a single fibril (upper) and its second derivative image (lower). Syn30–140 fibrils show long, straight morpholo‐ gy, and the double-filament twisted structure is observed in the enlarged fibril and its second derivative images (attributed to a pair of twisted protofibrils). Syn1–103 gives much thinner fibrils, but double-filament twisted structure is also observed (attributed to a protofibril consisting of two protofilaments). Syn30–103, however, shows very thin fibrils with a single filament and untwisted structure (attributed to a protofilament) [61].

Intriguingly, according to their AFM images, full-length α-synuclein fibrils were almost 140 nm in height. It has also been found that C-terminal truncated α-synuclein (syn1–103) was able to aggregate. However, this 103 amino acid peptide formed fibrils with smaller height (~57 nm). These fibrils, like syn30–140, also exhibited a right-handed twist. According to the Qin et al., syn1–103 fibrils looked rather like proto-fibrils than mature fibrils [61]. Based on this observation, authors made a conclusion that C-terminus of α-synuclein was highly impor‐ tant for the assembly of two proto-fibrils into a mature fibril. Finally, the central sequence fragment of α-synuclein that lacked both N- and C-termini, syn30–103, was found to be able to aggregate forming long un-branched fibrils. However, these fibrils were only 28.8 nm in height with no clear twist evident from the obtained AFM images.

Using AFM, Jansen et al. performed a detailed investigation of insulin aggregates that were grown at pH 1.6 [62]. It was determined that insulin proto-fibrils, which had around 1.2 nm in diameter, intertwined, forming left-handed fibrils with 3–7 nm in diameter. In addition, right-handed fibrils were simultaneously observed. Intriguingly, that it has been proposed that these right-handed fibrils were formed not as a result of proto-fibril coiling, but rather as a result of a lateral aggregation or small fibril blocks (10 × 60 nm). This aggregation mecha‐ nism will be further discussed in the last section of this chapter. This type (side-by-side) of aggregation was also proposed as the origin of binary, tape-like ribbons that were also observed upon insulin aggregation. Finally, authors observed that many insulin fibrils formed from small (~150 nm in length) subunits that are linearly extend one other. Based on this observation, Jansen et al. proposed that insulin fibrils can assemble via chainlike quenching of these subunits.

Human amylin is a 3.9 kDa protein secreted by islet β-cells of the pancreas. Together with insulin, it is involved in glucose metabolism. Amylin fibril aggregates are toxic and strongly associated with diabetes type II. Upon *invitro* aggregation at neutral pH (7.4), amylin forms thin proto-fibrils, which are around 5 nm in width and tend to aggregate further, forming two morphologically distinct polymorphs. In one case, proto-fibrils intertwine (coil), and form cable-like structures 8–10 nm in width with a distinct left-handed twist with a crossover of 25 nm. These cable-like structures could coil further, forming thick left-handed cables with larger widths. In addition, the height of these twisted fibrils has been determined. It has been shown that it varies from 3 (proto-fibrils) to 7 nm mature fibril cables [63]. Alternatively, three, four, or more proto-fibrils could aggregate side-by-side, forming flat, single-layer ribbons. Intriguing that the ribbons also twist at moderate regular intervals in a left-handed fashion [31]. Based on these observations, Goldsbury et al. proposed that amylin fibril polymor‐ phism originated from different ways of proto-fibril associations, while all observed fibril polymorphs have the same structure [64]. There was no clear understanding about the nature of a force that determined the formation of either type of those polymorphs. Three years later, Goldsbury et al. investigated aggregation of the full-length amylin (37 amino acids) and two fragments of amylin: 8–37 and 20–29. It was found that 20–29 fragment formed exclusively flat-ribbons, which were around 40 nm in width. However, 8–37 fragment, similar to the fulllength amylin, formed fibril polymorphs with variable morphologies. Most of them were lefthanded cables with 25 or 50 nm cross-over and flat-like ribbons [32]. Thus, based on this observation, authors proposed that peptide sequence was directly responsible for the determination of the fibril morphology.

#### *2.5.1. Changes in supramolecular chirality upon fibril disintegration*

Recently, Kurouski et al. explored how supramolecular architecture of insulin fibrils could be changed by bacterial chaperonins. The interactions of heat shock proteins with amyloid fibrils have drawn significant attention in recently years. However, most of these studies focused on one group of heat shock proteins with small molecular mass, so called small heat shock proteins [65–67]. Many small heat shock proteins have been reported to disassemble fibrils or prevent the fibrillation process *in vitro* [68].

Kurouski et al. investigated the effect on the mutant chaperonin complex from *Pyrococcusfur‐ iosus*(Pf) on insulin fibrils [69]. This chaperonin (Cpn) was composed of identical subunits and commonly found in most hyperthermophiles [70]. Cpn was mutated to reach its optimum activity below 50°C. Using AFM, Kurouski et al. found that after 5 min of incubation of insulin fibrils with Cpn, fibrils were found fragmented (**Figure 11**) [69].

Using AFM, Jansen et al. performed a detailed investigation of insulin aggregates that were grown at pH 1.6 [62]. It was determined that insulin proto-fibrils, which had around 1.2 nm in diameter, intertwined, forming left-handed fibrils with 3–7 nm in diameter. In addition, right-handed fibrils were simultaneously observed. Intriguingly, that it has been proposed that these right-handed fibrils were formed not as a result of proto-fibril coiling, but rather as a result of a lateral aggregation or small fibril blocks (10 × 60 nm). This aggregation mecha‐ nism will be further discussed in the last section of this chapter. This type (side-by-side) of aggregation was also proposed as the origin of binary, tape-like ribbons that were also observed upon insulin aggregation. Finally, authors observed that many insulin fibrils formed from small (~150 nm in length) subunits that are linearly extend one other. Based on this observation, Jansen et al. proposed that insulin fibrils can assemble via chainlike quenching

Human amylin is a 3.9 kDa protein secreted by islet β-cells of the pancreas. Together with insulin, it is involved in glucose metabolism. Amylin fibril aggregates are toxic and strongly associated with diabetes type II. Upon *invitro* aggregation at neutral pH (7.4), amylin forms thin proto-fibrils, which are around 5 nm in width and tend to aggregate further, forming two morphologically distinct polymorphs. In one case, proto-fibrils intertwine (coil), and form cable-like structures 8–10 nm in width with a distinct left-handed twist with a crossover of 25 nm. These cable-like structures could coil further, forming thick left-handed cables with larger widths. In addition, the height of these twisted fibrils has been determined. It has been shown that it varies from 3 (proto-fibrils) to 7 nm mature fibril cables [63]. Alternatively, three, four, or more proto-fibrils could aggregate side-by-side, forming flat, single-layer ribbons. Intriguing that the ribbons also twist at moderate regular intervals in a left-handed fashion [31]. Based on these observations, Goldsbury et al. proposed that amylin fibril polymor‐ phism originated from different ways of proto-fibril associations, while all observed fibril polymorphs have the same structure [64]. There was no clear understanding about the nature of a force that determined the formation of either type of those polymorphs. Three years later, Goldsbury et al. investigated aggregation of the full-length amylin (37 amino acids) and two fragments of amylin: 8–37 and 20–29. It was found that 20–29 fragment formed exclusively flat-ribbons, which were around 40 nm in width. However, 8–37 fragment, similar to the fulllength amylin, formed fibril polymorphs with variable morphologies. Most of them were lefthanded cables with 25 or 50 nm cross-over and flat-like ribbons [32]. Thus, based on this observation, authors proposed that peptide sequence was directly responsible for the

Recently, Kurouski et al. explored how supramolecular architecture of insulin fibrils could be changed by bacterial chaperonins. The interactions of heat shock proteins with amyloid fibrils have drawn significant attention in recently years. However, most of these studies focused on one group of heat shock proteins with small molecular mass, so called small heat shock proteins [65–67]. Many small heat shock proteins have been reported to disassemble fibrils or

of these subunits.

86 Exploring New Findings on Amyloidosis

determination of the fibril morphology.

prevent the fibrillation process *in vitro* [68].

*2.5.1. Changes in supramolecular chirality upon fibril disintegration*

**Figure 11.** AFM kinetic examination of the insulin fibrils (Fibrils) mixed with Cpn and deposited right away (Fibrils + Cpn), five (5 min later) and half an hour (30 min later) later on mica surface. Immediately upon mixing, Cpn absorb on the fibril surfaces (shown with red arrow). As a result of these Cpn-Fibril interactions the last swell and break apart (5 min later). Even some fibril fragments that are evident on the early microscopic examination (5 min later) disappear after 30 min (30 min later). The scale bar is 1 μm [69].

The foreshortened fibrils looked like swollen clamps with significantly lower height (~6 nm) and width up to 200–400 nm. Interestingly, white image features on the edges of these clamps mimicked the outline of the original fibrils. Most likely they were fibrillar regions that were not melted by Cpn because the reaction was quickly terminated. These observations con‐ firmed that Cpn was able to change the fibril architecture. Kurouski et al. concluded that Cpn melted the fibril core and formed an amorphous protein mass from regular β-sheet structure. Microscopic observations of this phenomenon also indicated a fibril swelling. Authors also found that longer exposure of insulin fibrils to Cpn resulted in theirfurtherfragmentation into smaller and smaller species with irregular shapes. These species coagulated during late stages, forming large amorphous aggregates. Most of the fibril-shaped species disappeared and predominantly amorphous objects form after 30 min of Cpn-fibril co-incubation were observed (**Figure 11**) [69].

In this section, supramolecular organization of amyloid fibrils that were formed by most of known amyloid-associated proteins was discussed. It was shown that an aggregation of these proteins may result in either flat, left- or right-twisted fibrils. Very often several different fibril polymorphs can be grown simultaneously. Numerous research findings indicate that fibril polymorphism can be controlled by pH. At the same time, there are several studies which showed that amino acid sequence can determine supramolecular organization of amyloid fibrils. This section also demonstrated that microscopy can be utilized to monitor changes in the fibril morphology during fibril formation. Moreover, microscopy can be used to monitor changes in the structure of mature fibrils, which can be initiated by chemical or physicalfactors, or triggered by biological molecules, such as chaperonins. Based on these finding one can envision that amyloid fibrils are dynamic rather than static thermodynamic systems and that small change in pH or salinity may change fibril morphology or aggregation state of fibrils at the interfaces.

## **3. Vibrational circular dichroism a unique tool for the determination of fibril supramolecular organization**

VCD is a unique spectroscopic technique that is capable of probing supramolecular chirality of amyloid fibrils [19, 42, 71]. In VCD, left (L)- and right (R)-circularly polarized infra-red (IR) light pass through the sample. Itis well known that solutions oftwo enantiomers have different absorption of left- vs right-circularly polarized light. This physical principle is used to determine the absolute configuration or small chiral molecules, as well as to unravel supra‐ molecular organization of macromolecules. VCD probes deeper levels of fibril supramolecu‐ lar chiral organization that may not be apparent to existing forms of microscopy [8]. It was also shown that enhanced VCD sensitivity arose directly from the long-range supramolecu‐ lar chirality of fibril structures at all hierarchical levels [11]. Measey and Schweitzer-Stenner modeled VCD spectra of a fibril filament that had only two peptide units long, run perpen‐ dicular to the filament axis direction [72]. The model was based on exciton coupling among amide I transition dipoles arrayed as dual, stacked β-sheet ribbons. They introduced a 2° lefthand twist between the strands yielding a long-range gradual helical twist of the filament with a one full-turn distance of 180 strands. It was found that such a structure yields enhanced VCD with a negative VCD band near 1620 cm–1 and a positive VCD band to higher wavenumber frequencies. A corresponding 2° intrastrand helical twist in the opposite direction, corre‐ sponding to a right-hand helical filament, reversed the sign of the enhanced VCD couplet and corresponds to the reversed VCD. This theoretical work demonstrated that VCD directly was capable of a determination of the supramolecular chiral organization of fibril filaments.

Recently, it has been found that insulin aggregation at pH 2.1 and higher results in the formation of fibrils that show a strong VCD spectrum with peaks near 1554, 1593, 1627, 1647, 1670 cm–1 that have (+ + – + +) sign pattern [42]. The fibril VCD spectrum with this sign patters was called "normal VCD." Microscopic examination of these fibrils indicated that a majority of them had a left-handed helical twist [8]. However, if the pH of the aggregation solution is lower than 2.4, the distribution of fibrils shifts to increasingly flat, tape-like, or binary fibrils as the incubation pH continues to be lowered that under microscopic examination show no noticeable chirality ortwist on their surface. Nevertheless, these fibrils show a strong, but often somewhat smaller, VCD with a sign pattern (− – + – −) that is nearly the mirror-image of "normal" VCD fibril spectrum and is referred to as the "reversed" polymorph. The fact that flat tape-like fibrils show a strong reversed VCD signal indicates that they must be com‐ posed of right-handed filaments, the chirality of which lies below the limit of AFM or SEM detection. The combined VCD-microscopic studies showed that pH determines not only the net handedness of the filaments, precursors of mature fibrils, but also controls their associa‐ tion pathways. Left-handed filaments intertwine, forming left-handed proto-fibrils and mature fibrils that have normal VCD. On the other hand, right-handed filaments associate side-by-side, forming flat, tape-like, or binary fibrils. Thus, pH most likely alters proteinsolvent interactions or causes protonation of some amino acid side chains, which are lying on the surface of the filaments. These changes cause variations in the way these filaments bind together to form mature fibrils that either twist together to form braids or align side-by-side without braiding. In addition, it was discovered that pH not only determines insulin fibril morphology and net chirality at the stage of the protein aggregation, but also may dramati‐ cally change the morphology of mature fibrils and overturn their initial chirality.

Most likely, the sensitivity of amino-acid side chains to the aqueous solvent is responsible for this pH sensitivity. Moreover, different ways of constituent side-chains, exposed or not to the solvent, result in different pH sensitivity (high versus low) for the sense of filament chirality observed. This is a long-range fibril property that likely cannot be predicted, even qualitative‐ ly, without a realistic model of protein side chain influence on the sense of helical chirality as a function of pH. Moreover, the chirality of individual fibril filaments lies below the sensitiv‐ ity of AFM or SEM imaging, but can be observed with VCD at the initial and subsequent stages of fibril formation [11].

A growing body of literature indicates that VCD has become a useful tool for the chiral characterization of amyloid aggregates. For example, Measey and Schweitzer-Stennerrecently reported a large enhancement of VCD upon aggregation of short polypeptides [73]. They also demonstrated that mature fibrils formed from the N-terminal peptide fragment of the yeast prion protein, Sup35, and the amyloidogenic alanine-rich peptide AKY8 have opposite signed VCD. It has been also demonstrated that opposite signed VCD spectra could be obtained for mature fibrils formed from poly-L or -D glutamic acid [74]. Polyglutamic acid formed spiral‐ ly twisted aggregates with handedness determined by the amino acid chirality (left-handed for Land right-handed for D).

## **4. Models of amyloid fibril formation**

small change in pH or salinity may change fibril morphology or aggregation state of fibrils at

**3. Vibrational circular dichroism a unique tool for the determination of**

VCD is a unique spectroscopic technique that is capable of probing supramolecular chirality of amyloid fibrils [19, 42, 71]. In VCD, left (L)- and right (R)-circularly polarized infra-red (IR) light pass through the sample. Itis well known that solutions oftwo enantiomers have different absorption of left- vs right-circularly polarized light. This physical principle is used to determine the absolute configuration or small chiral molecules, as well as to unravel supra‐ molecular organization of macromolecules. VCD probes deeper levels of fibril supramolecu‐ lar chiral organization that may not be apparent to existing forms of microscopy [8]. It was also shown that enhanced VCD sensitivity arose directly from the long-range supramolecu‐ lar chirality of fibril structures at all hierarchical levels [11]. Measey and Schweitzer-Stenner modeled VCD spectra of a fibril filament that had only two peptide units long, run perpen‐ dicular to the filament axis direction [72]. The model was based on exciton coupling among amide I transition dipoles arrayed as dual, stacked β-sheet ribbons. They introduced a 2° lefthand twist between the strands yielding a long-range gradual helical twist of the filament with a one full-turn distance of 180 strands. It was found that such a structure yields enhanced VCD with a negative VCD band near 1620 cm–1 and a positive VCD band to higher wavenumber frequencies. A corresponding 2° intrastrand helical twist in the opposite direction, corre‐ sponding to a right-hand helical filament, reversed the sign of the enhanced VCD couplet and corresponds to the reversed VCD. This theoretical work demonstrated that VCD directly was capable of a determination of the supramolecular chiral organization of fibril filaments.

Recently, it has been found that insulin aggregation at pH 2.1 and higher results in the formation of fibrils that show a strong VCD spectrum with peaks near 1554, 1593, 1627, 1647, 1670 cm–1 that have (+ + – + +) sign pattern [42]. The fibril VCD spectrum with this sign patters was called "normal VCD." Microscopic examination of these fibrils indicated that a majority of them had a left-handed helical twist [8]. However, if the pH of the aggregation solution is lower than 2.4, the distribution of fibrils shifts to increasingly flat, tape-like, or binary fibrils as the incubation pH continues to be lowered that under microscopic examination show no noticeable chirality ortwist on their surface. Nevertheless, these fibrils show a strong, but often somewhat smaller, VCD with a sign pattern (− – + – −) that is nearly the mirror-image of "normal" VCD fibril spectrum and is referred to as the "reversed" polymorph. The fact that flat tape-like fibrils show a strong reversed VCD signal indicates that they must be com‐ posed of right-handed filaments, the chirality of which lies below the limit of AFM or SEM detection. The combined VCD-microscopic studies showed that pH determines not only the net handedness of the filaments, precursors of mature fibrils, but also controls their associa‐ tion pathways. Left-handed filaments intertwine, forming left-handed proto-fibrils and mature fibrils that have normal VCD. On the other hand, right-handed filaments associate side-by-side, forming flat, tape-like, or binary fibrils. Thus, pH most likely alters protein-

the interfaces.

88 Exploring New Findings on Amyloidosis

**fibril supramolecular organization**

Accumulated experimental evidence suggested three protein aggregation pathways that lead to the discussed above morphological heterogeneity of amyloid fibrils. According to the first one, fibril polymorphism could be caused by deviations at the stage of monomer-monomer associations, while protein oligomers are formed [75]. Consequently, these structurally different oligomers will lead to structurally and morphologically different fibrils. For exam‐ ple, Srinivasan et al. recently investigated morphologies and structural organization of prefibrilar oligomers and mature fibrils formed from two murine serum amyloid A (SAA) isoforms, named SAA1.1 and SAA2.2 [75]. These two isoforms only have a difference in six amino acids [76]. SAA1.1 had an oligomer-rich fibrillation lag phase of a few days, while SAA2.2 formed small fibrils within a few hours, exhibiting virtually no lag phase [77]. Structural differences of SAA 1.1 and SAA 2.2 resulted in morphological differences of their filaments and consequently fibrils. Specifically, SAA 2.2 formed worm-like filaments that were able to coil and braid. At the same time, the filaments and proto-fibrils of SAA 1.1 had a rodlike supramolecular organization. They were unable to braid and formed thick straight fibrils. Using CD and Raman spectroscopy Srinivasan et al. confirmed structural differences be‐ tween SAA 1.1 and SAA 2.2 oligomers and fibrils.

**Scheme 1.** Models of amyloid fibril formation. Differences in the morphology of mature fibrils can be caused by differ‐ ent ways of protein-protein aggregation on the stage of fibril oligomers' formation (A). Alternatively, protein aggrega‐ tion leads to the formation of a filament that is able to intertwine forming twisted fibrils, or associate side-by-side (B). In the late case, tape-like fibrils are formed. Adapted from Srinivasan et al. [77] and Kurouski et al. [11].

Alternatively, the heterogeneity of fibril morphologies could be due to different ways of filament associations. Specifically, fibril filaments can either braid and coil or associate sideby-side, forming twisted or tape-like fibrils, respectively (**Scheme 1B**). These morphological‐ ly different fibrils will consequently have the same structure [8, 11]. Recently, Kurouski et al. investigated polymorphism of insulin, lysozyme, apo-α-lactalbumin, HET-s (218–289) prion, and a short polypeptide fragment of transtherytin, TTR (105–115). Authors demonstrated that all these proteins form two distinct fibril polymorphs: tape-like and twisted. They have also found that pH directly controls which polymorph will be formed. Using IR and Raman spectroscopy it has been demonstrated that both tape-like and twisted fibril polymorphs have the same secondary structure [11].

Besides braiding and side-by-side association, filament propagation can occur via several alternative pathways. According to Jansen et al. filament fragmentation may result in short filament fragments that serve as templates forlateralratherthan axial protein aggregation [62]. As a result, short fibril blocks are formed (**Scheme 2A**). These blocks tend to associate side-byside forming the fibril that has a right-handed twist upon the microscopic examination.

**Scheme 2.** Models of amyloid filament propagation. Mechanism of side-driven templation (A) and super-coiling of left-twisted fibril filament that leads to the appearance of a right-handed twist on the mature fibril (B). Adapted from R. Jansen et al. [62] and Usov et al. [53].

**Scheme 1.** Models of amyloid fibril formation. Differences in the morphology of mature fibrils can be caused by differ‐ ent ways of protein-protein aggregation on the stage of fibril oligomers' formation (A). Alternatively, protein aggrega‐ tion leads to the formation of a filament that is able to intertwine forming twisted fibrils, or associate side-by-side (B).

Alternatively, the heterogeneity of fibril morphologies could be due to different ways of filament associations. Specifically, fibril filaments can either braid and coil or associate sideby-side, forming twisted or tape-like fibrils, respectively (**Scheme 1B**). These morphological‐ ly different fibrils will consequently have the same structure [8, 11]. Recently, Kurouski et al. investigated polymorphism of insulin, lysozyme, apo-α-lactalbumin, HET-s (218–289) prion, and a short polypeptide fragment of transtherytin, TTR (105–115). Authors demonstrated that all these proteins form two distinct fibril polymorphs: tape-like and twisted. They have also found that pH directly controls which polymorph will be formed. Using IR and Raman spectroscopy it has been demonstrated that both tape-like and twisted fibril polymorphs have

Besides braiding and side-by-side association, filament propagation can occur via several alternative pathways. According to Jansen et al. filament fragmentation may result in short filament fragments that serve as templates forlateralratherthan axial protein aggregation [62]. As a result, short fibril blocks are formed (**Scheme 2A**). These blocks tend to associate side-byside forming the fibril that has a right-handed twist upon the microscopic examination.

In the late case, tape-like fibrils are formed. Adapted from Srinivasan et al. [77] and Kurouski et al. [11].

the same secondary structure [11].

90 Exploring New Findings on Amyloidosis

Separately, Usov et al. proposed two additional mechanisms that resulted in formation of morphologically different fibril polymorphs [53]. According to one, the left-twisted filament may continue twisting along the longitude axis. As a result, a hollow tube-like protein aggregate will be formed. Alternatively, it has been proposed that the left-twisted fibril filament may flip the handedness of its twist, as was discussed in the Section 2.5.

Summarizing, this chapter aimed to demonstrate how microscopy, including electron and probe microscopy, can be utilized to investigate supramolecular organization of amyloid aggregates. It demonstrated that microscopy was capable of elucidating mechanisms of fibril formation and unraveling the origin offibril polymorphism. It was also shown that using AFM, SEM, and cryo-SEM, chiral nature of fibril supramolecular organization can be determined. The author also wanted to attract reader's attention to VCD. This powerful spectroscopic tool is capable of probing supramolecular chirality of fibril filaments that may to always be accessible for currently available microscopic techniques.

This chapter reviewed supramolecular organization of amyloid fibrils formed by almost all known amyloidogenic proteins, including amyloid β, α-synuclein, tau, insulin, and lyso‐ zyme proteins. It demonstrated how physical and chemical factors could change morpholo‐ gy of fibril aggregates at the stage of their formation. It was also shown how these factors, as well as chaperonins, could change the supramolecular organization of already grown fibrils.

One should note that microscopy itself often may not be sufficient for the determination of fibril structure. Therefore, microscopy is often used with numerous spectroscopic techni‐ ques, such as ss-NMR, Raman, and IR spectroscopy. These techniques allow for the determi‐ nation of ψ and φ angles of the peptide backbone, which is necessary to fully elucidate the structure of the fibril cross-β core.

## **Author details**

Dmitry Kurouski\*

Address all correspondence to: dkurouski@northwestern.edu

Chemistry Department, Northwestern University, Evanston, IL, USA

## **References**


[8] Kurouski D, Dukor RK, Lu X, Nafie LA, Lednev IK. Normal and reversed supramo‐ lecular chirality of insulin fibrils probed by vibrational circular dichroism at the protofilament level of fibril structure. Biophys. J. 2012;103:522–531.

is capable of probing supramolecular chirality of fibril filaments that may to always be

This chapter reviewed supramolecular organization of amyloid fibrils formed by almost all known amyloidogenic proteins, including amyloid β, α-synuclein, tau, insulin, and lyso‐ zyme proteins. It demonstrated how physical and chemical factors could change morpholo‐ gy of fibril aggregates at the stage of their formation. It was also shown how these factors, as well as chaperonins, could change the supramolecular organization of already grown fibrils. One should note that microscopy itself often may not be sufficient for the determination of fibril structure. Therefore, microscopy is often used with numerous spectroscopic techni‐ ques, such as ss-NMR, Raman, and IR spectroscopy. These techniques allow for the determi‐ nation of ψ and φ angles of the peptide backbone, which is necessary to fully elucidate the

accessible for currently available microscopic techniques.

Address all correspondence to: dkurouski@northwestern.edu

aggregates. Cell. Mol. Life Sci. 2007;64:2066–2078.

Chemistry Department, Northwestern University, Evanston, IL, USA

[1] Dobson CM. Protein folding and misfolding. Nature 2003;426:884–890.

[2] Sipe JD, Cohen AS. Review: history of the amyloid fibril. J. Struct. Biol. 2000;130:88–98.

[3] Kurouski D, Van Duyne RP, Lednev IK. Exploring the structure and formation mechanism of amyloidfibrils byRaman spectroscopy: a review. Analyst 2015;140:4967–

[4] Fandrich M. On the structural definition of amyloid fibrils and other polypeptide

[5] Dobson CM. Protein aggregation and its consequences for human disease. Prot. Pept.

[6] Usov I, Nystrom G, Adamcik J, Handschin S, Schutz C, Fall A*, et al.* Understanding nanocellulose chirality and structure-properties relationship at the single fibril level.

[7] Assenza S, Adamcik J, Mezzenga R, De Los Rios P. Universal behavior in the meso‐

scale properties of amyloid fibrils. Phys. Rev. Lett. 2014;113:268103.

structure of the fibril cross-β core.

92 Exploring New Findings on Amyloidosis

**Author details**

Dmitry Kurouski\*

**References**

4980.

Lett. 2006;13:219–227.

Nat. Commun. 2015;6:7564.


by a strongly enhanced vibrational circular dichroism signal. J. Am. Chem. Soc. 2009;131:18218–18219.


[33] Shammas SL, Knowles TP, Baldwin AJ, Macphee CE, Welland ME, Dobson CM, Devlin GL. Perturbation of the stability of amyloid fibrils through alteration of electrostatic interactions. Biophys. J. 2011;100:2783–2791.

by a strongly enhanced vibrational circular dichroism signal. J. Am. Chem. Soc.

[21] Lednev LK. Amyloid fibrils: the Eighth Wonder of the world in protein folding and

[22] Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science

[23] Hell SW, Wichmann J. Breaking the diffraction resolution limit by stimulated emis‐ sion: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 1994;19:780–

[24] Heilemann M, van de Linde S, Schuttpelz M, Kasper R, Seefeldt B, Mukherjee A, et al. Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes.

[25] Kaminski Schierle GS, van de Linde S, Erdelyi M, Esbjorner EK, Klein T, Rees E, et al. In situ measurements of the formation and morphology of intracellular beta-amyloid fibrils by super-resolution fluorescence imaging. J. Am. Chem. Soc. 2011;133:12902–

[26] Tosoni A., Barbiano di Belgiojoso G. and Nebuloni M. (2011). Electron Microscopy in the Diagnosis of Amyloidosis, Amyloidosis - Mechanisms and Prospects for Therapy, Dr. Svetlana Sarantseva (Ed.), ISBN: 978- 953-307-253-1, InTech, Available from: http:// www.intechopen.com/books/amyloidosis-mechanisms-andprospects-for-therapy/ electron-microscopy-in-the-diagnosis-of-amyloidosis Locatoin: University Campus

[27] Inoue S, Kuroiwa M, KisilevskyR. Basement membranes, microfibrils and beta amyloid fibrillogenesis in Alzheimer's disease: high resolution ultrastructural findings. Brain

[28] Inoue S, Kuroiwa M, Tan R, Kisilevsky R. A high resolution ultrastructural compari‐ son of isolated and in situ murine AA amyloid fibrils. Amyloid 1998;5:99–110.

[29] Inoue S, Kuroiwa M, Saraiva MJ, Guimaraes A, Kisilevsky R. Ultrastructure of familial amyloid polyneuropathy amyloid fibrils: examination with high-resolution electron

[30] Kidd M. Paired helical filaments in electron microscopy of Alzheimer's disease.

[31] Goldsbury CS, Cooper GJ, Goldie KN, Muller SA, Saafi EL, Gruijters WT, et al. Polymorphic fibrillar assembly of human amylin. J. Struct. Biol. 1997;119:17–27.

[32] Goldsbury C, Goldie K, Pellaud J, Seelig J, Frey P, Muller SA, et al. Amyloid fibril formation from full-length and fragments of amylin. J. Struct. Biol. 2000;130:352–362.

2009;131:18218–18219.

94 Exploring New Findings on Amyloidosis

2006;313:1642–1645.

782.

12905.

aggregation. Biophys. J. 2014;106:1433–1435.

Angew. Chem. Int. Ed. Engl. 2008;47:6172–6176.

STeP Ri Slavka Krautzeka 83/A 51000 Rijeka, Croatia

Res. Rev. 1999;29:218–231.

Nature 1963;197:192–193.

microscopy. J. Struct. Biol. 1998;124:1–12.


[60] Campioni S, Carret G, Jordens S, Nicoud L, Mezzenga R, Riek R. The presence of an air-water interface affects formation and elongation of alpha-Synuclein fibrils. J. Am. Chem. Soc. 2014;136:2866–2875.

[46] Klement K, Wieligmann K, Meinhardt J, Hortschansky P, Richter W, Fandrich M. Effect of different salt ions on the propensity of aggregation and on the structure of

[47] Petkova AT, Leapman RD, Guo Z, Yau WM, Mattson MP, Tycko R. Self-propagating, molecular-level polymorphism in Alzheimer's beta-amyloid fibrils. Science

[48] Luhrs T, Ritter C, Adrian M, Riek-Loher D, Bohrmann B, Dobeli H, et al. 3D structure of Alzheimer's amyloid-beta(1–42) fibrils. Proc. Natl. Acad. Sci. U. S. A. 2005,102:17342–

[49] Zhang R, Hu X, Khant H, Ludtke SJ, Chiu W, Schmid MF, et al. Interprotofilament interactions between Alzheimer's Abeta1–42 peptides in amyloid fibrils revealed by

[50] Vilar M, Chou HT, Luhrs T, Maji SK, Riek-Loher D, Verel R, et al. The fold of alpha-

[51] Crowther RA, Daniel SE, Goedert M. Characterisation of isolated alpha-synuclein filaments from substantia nigra of Parkinson's disease brain. Neurosci. Lett.

[52] Spillantini MG, Crowther RA, Jakes R, Cairns NJ, Lantos PL, Goedert M. Filamen‐ tous alpha-synuclein inclusions link multiple system atrophy with Parkinson's disease

[53] Usov I, Adamcik J, Mezzenga R. Polymorphism complexity and handedness inver‐

[54] Adamcik J, Mezzenga R. Adjustable twisting periodic pitch of amyloid fibrils. Soft

[55] Jordens S, Isa L, Usov I, Mezzenga R. Non-equilibrium nature of two-dimensional isotropic and nematic coexistence in amyloid fibrils at liquid interfaces. Nat. Com‐

[56] Jordens S, Ruhs PA, Sieber C, Isa L, Fischer P, Mezzenga R. Bridging the gap be‐ tween the nanostructural organization and macroscopic interfacialrheology of amyloid

[57] Loksztejn A, Dzwolak W. Chiral bifurcation in aggregating insulin: an induced circular

[58] Dzwolak W, Pecul M. Chiral bias of amyloid fibrils revealed by the twisted conforma‐ tion of Thioflavin T: an induced circular dichroism/DFT study. FEBS Lett.

[59] Dzwolak W. Vortex-induced chiral bifurcation in aggregating insulin. Chirality

cryoEM. Proc. Natl. Acad. Sci. U. S. A. 2009;106:4653–4658.

synuclein fibrils. Proc. Natl. Acad. Sci. U. S. A. 2008;105:8637–8642.

and dementia with Lewy bodies. Neurosci. Lett. 1998;251:205–208.

fibrils at liquid interfaces. Langmuir 2014;30:10090–10097.

dichroism study. J. Mol. Biol. 2008;379:9–16.

sion in serum albumin amyloid fibrils. ACS Nano 2013;7:10465–10474.

Alzheimer's abeta(1–40) amyloid fibrils. J. Mol. Biol. 2007;373:1321–1333.

2005;307:262–265.

96 Exploring New Findings on Amyloidosis

2000;292:128–130.

Matter 2011;7:5437–5443.

mun. 2013;4:1917.

2005,579:6601–6603.

2010;22(Suppl. 1):E154–E160.

17347.


## **Advances in AFM Imaging Applications for Characterizing the Biophysical Properties of Amyloid Fibrils**

Wonseok Lee, Hyungbeen Lee, Gyudo Lee and Dae Sung Yoon

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63316

#### **Abstract**

[74] Yu J, Zhu H, Guo JT, de Beer FC, Kindy MS. Expression of mouse apolipoprotein SAA1.1 in CE/J mice: isoform-specific effects on amyloidogenesis. Lab. Invest.

[75] Malle E, Sodin-Semrl S, Kovacevic A. Serum amyloid A: an acute-phase protein

[76] Srinivasan S, Patke S, Wang Y, Ye Z, Litt J, Srivastava SK, et al. Pathogenic serum amyloid A 1.1 shows a long oligomer-rich fibrillation lag phase contrary to the highly

[77] Kurouski D, Handen JD, Dukor RK, Nafie LA, Lednev IK. Supramolecular chirality in

amyloidogenic non-pathogenic SAA2.2. J. Biol. Chem. 2013;288:2744–2755.

involved in tumour pathogenesis. Cell. Mol. Life Sci. 2009;66:9–26.

peptide microcrystals. Chem. Commun. 2015;51:89–92.

2000;80:1797–1806.

98 Exploring New Findings on Amyloidosis

Although the formation mechanism of amyloid fibrils in bodies is still debated, it has recently been reported how amyloid fibrils can be formed in vitro. Accordingly, we have gained a better understanding of the self-assembly mechanism and intrinsic proper‐ ties of amyloid fibrils. Because the structure of amyloid fibrils consists of nanoscaled insoluble strands (a few nanometers in diameter and micrometers long), a special tool is needed to study amyloid fibrils atlength. Atomic force microscopy (AFM)is supposed to be a versatile toolkit to probe such a tiny biomolecule. The physical/chemical properties of amyloid fibrils have been explored by AFM. In particular, AFM enables the visualization of amyloid fibrillation with different incubation times as well as the concentrations of the formed amyloid fibrils as affected by fibril diameters and lengths. Very recently, the minute structural changes and/or electrical properties of amyloid fibrils have been made by using advanced AFM techniques including dynamic liquid AFM, PeakForce QNM (quantitative nanomechanical mapping), and Kelvin probe force microscopy (KPFM). Herein, we summarize the biophysical properties of amyloid fibrils that are newly discovered with the help of those advanced AFM techniques and suggest our perspectives and future directions for the study of amyloid fibrils.

**Keywords:** amyloid fibril, biophysical property, atomic force microscopy (AFM), Kel‐ vin probe force microscopy (KPFM), PeakForce QNM (quantitative nanomechanical mapping)

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

Proteins and peptides can be transformed under some physiological conditions from their soluble forms into highly ordered fibrous aggregates, which are called "amyloid fibrils" [1–3]. Amyloid fibrils can be deposited in the tissues and organs in the human body, resulting in the generation of degenerative diseases such asAlzheimer's, Parkinson's, Creutzfeldt-Jakob's, and Huntington's diseases depending on specific sequences of proteins or peptides [4, 5]. On the other hand, the amyloid fibrils have often been exploited for functional roles (e.g., extracellu‐ lar matrix) by living creatures [6–8]. These amyloid fibrils, commonly known as functional amyloid,arefoundinbacteria,fungi,insects,invertebrates,andhumans.Becauseoftheirunique mechanical and biological properties, amyloid fibrils had been used in a variety of organisms as a bacterial coatings, scaffolds, biofilm, information storage and transfer, natural adhesives, and structures for the storage of peptide hormones [7–11]. Although the precise formation mechanism of amyloid fibrils and their pathological/functional behaviors in bodies are still controversial, it has been reported how amyloid fibrils can be formed in vitro, allowing us to understand the fibrillation mechanism and/orthe intrinsic properties ofthe fibrils [12]. Because thesefibrilshaveadistinctivestructurethat consistsofβ-sheets thatareperpendicularlystacked in thousands of units, aggregating to a few nanometers in diameter and several micrometers in length, a special tool is required to study the conformational (structural) changes of amyloid fibrils in various microenvironments. To investigate the structural changes of amyloid fibrils, many technical tools such as fluorescence microscopy with Congo red and thioflavin-T, transmission electron microscopy (TEM), X-ray diffraction, and nuclear magnetic resonance (NMR) have been used [13, 14]. Recently, atomic force microscopy (AFM) is a good candidate for characterizing such a thin fibrous material *via* high-resolution imaging of the molecular structure and interactions. A sharp stylus mounted at the end of a flexible cantilever is used to probe the amyloid fibrils placed on a piezoelectric scanner which controls the cantilever on the sample in the horizontal (*x* and *y*) and vertical (*z*) axis (**Figure 1**). The deflection of the cantile‐ verasaresultoftheinteractions(suchasvanderWaalsforces,capillaryforces,chemicalbonding, and electrostatic forces) between the tip and the sample on substrate is converted into a laser beam reflected on the back of the cantilever. In this system, laser light from diode reflects off the back of the cantilever, and it is recorded as the amplified signal by a position-sensitive detec‐ tor (PSD), which reconstructs the molecular structure or interactions of amyloid fibrils. AFM has its advantages, for example, the convenience to prepare a sample, working at room temperature and atmospheric pressure, and high-resolution imaging at the single-molecule level, allowing for measuring the physical/chemical properties of amyloid fibrils. In particu‐ lar, AFM enables scanning to follow the morphological changes of amyloid fibrils in liquid environments, which is powerful as it permits us to visualize amyloid fibrillation under physiological conditions [15].Recentinvitrostudiesof amyloidfibrilshavedemonstratedtimelapse AFM imaging of amyloid fibrillation with different incubation times and concentrations under physiological conditions [16]. Much more recently, AFM application techniques that lie beyond its primary function (i.e., high-resolution imaging) have shown that abundant infor‐ mation of amyloid fibrils can now be accessible. For instance, PeakForce QNM (quantitative nanomechanical mapping) and KPFM (Kelvin probe force microscopy) are, respectively, beginningtobeusedtomapmechanicalpropertiesandtomeasurethesurfacechargeofamyloid fibrils. Consequently, someunprecedentedcharacteristics (e.g.,minute structural changes and/ or electrical properties) of amyloid fibrils have been investigated, providing a better under‐ standing of amyloid fibrils [17–23]. In this chapter, we introduce the advanced AFM applica‐ tion methods (i.e., dynamic liquid AFM, PeakForce QNM, and KPFM) for measuring amyloid fibril's features that could not be accessed by conventional AFM technique and summarize the newly discovered biophysical properties of amyloid fibrils with the advanced AFM techni‐ ques and suggest our perspectives and future directions for characterizing amyloid fibrils.

**Figure 1.** Schematic diagram of the principle of AFM.

**1. Introduction**

1002 Exploring New Findings on Amyloidosis

Proteins and peptides can be transformed under some physiological conditions from their soluble forms into highly ordered fibrous aggregates, which are called "amyloid fibrils" [1–3]. Amyloid fibrils can be deposited in the tissues and organs in the human body, resulting in the generation of degenerative diseases such asAlzheimer's, Parkinson's, Creutzfeldt-Jakob's, and Huntington's diseases depending on specific sequences of proteins or peptides [4, 5]. On the other hand, the amyloid fibrils have often been exploited for functional roles (e.g., extracellu‐ lar matrix) by living creatures [6–8]. These amyloid fibrils, commonly known as functional amyloid,arefoundinbacteria,fungi,insects,invertebrates,andhumans.Becauseoftheirunique mechanical and biological properties, amyloid fibrils had been used in a variety of organisms as a bacterial coatings, scaffolds, biofilm, information storage and transfer, natural adhesives, and structures for the storage of peptide hormones [7–11]. Although the precise formation mechanism of amyloid fibrils and their pathological/functional behaviors in bodies are still controversial, it has been reported how amyloid fibrils can be formed in vitro, allowing us to understand the fibrillation mechanism and/orthe intrinsic properties ofthe fibrils [12]. Because thesefibrilshaveadistinctivestructurethat consistsofβ-sheets thatareperpendicularlystacked in thousands of units, aggregating to a few nanometers in diameter and several micrometers in length, a special tool is required to study the conformational (structural) changes of amyloid fibrils in various microenvironments. To investigate the structural changes of amyloid fibrils, many technical tools such as fluorescence microscopy with Congo red and thioflavin-T, transmission electron microscopy (TEM), X-ray diffraction, and nuclear magnetic resonance (NMR) have been used [13, 14]. Recently, atomic force microscopy (AFM) is a good candidate for characterizing such a thin fibrous material *via* high-resolution imaging of the molecular structure and interactions. A sharp stylus mounted at the end of a flexible cantilever is used to probe the amyloid fibrils placed on a piezoelectric scanner which controls the cantilever on the sample in the horizontal (*x* and *y*) and vertical (*z*) axis (**Figure 1**). The deflection of the cantile‐ verasaresultoftheinteractions(suchasvanderWaalsforces,capillaryforces,chemicalbonding, and electrostatic forces) between the tip and the sample on substrate is converted into a laser beam reflected on the back of the cantilever. In this system, laser light from diode reflects off the back of the cantilever, and it is recorded as the amplified signal by a position-sensitive detec‐ tor (PSD), which reconstructs the molecular structure or interactions of amyloid fibrils. AFM has its advantages, for example, the convenience to prepare a sample, working at room temperature and atmospheric pressure, and high-resolution imaging at the single-molecule level, allowing for measuring the physical/chemical properties of amyloid fibrils. In particu‐ lar, AFM enables scanning to follow the morphological changes of amyloid fibrils in liquid environments, which is powerful as it permits us to visualize amyloid fibrillation under physiological conditions [15].Recentinvitrostudiesof amyloidfibrilshavedemonstratedtimelapse AFM imaging of amyloid fibrillation with different incubation times and concentrations under physiological conditions [16]. Much more recently, AFM application techniques that lie beyond its primary function (i.e., high-resolution imaging) have shown that abundant infor‐ mation of amyloid fibrils can now be accessible. For instance, PeakForce QNM (quantitative nanomechanical mapping) and KPFM (Kelvin probe force microscopy) are, respectively,

## **2. Imaging of self-assembled amyloid fibrils in liquid environments**

Amyloid fibrils are protein aggregates arising from misfolded proteins, transformed to βsheet-rich and insoluble fibrils, which can be tangled and deposited in tissues and organs [2]. Characterizing the structure of amyloid fibrils can provide some critical clues about the mechanism of fibrogenesis and accordingly the pathological/functional roles of the fibrils. So far a variety of instruments (e.g., light, X-ray scattering, and NMR) [24, 25] and microscopic methods (e.g., fluorescence microscopy with Congo red and thioflavin-T [26, 27], TEM, and scanning electron microscopy (SEM) [14]) had been used to explore the structure of amyloid fibrils. Although many research results about the structure of amyloid fibrils had been reported by using those tools and/or methods, a fully comprehensive investigation of the self-assembly behavior and structure of various amyloid fibrils has not yet been conducted. AFM provides additional capabilities and advantages relative to other microscopic methods. For example, it can directly show and measure the structure of an individual fibril. In addition, it is easy to prepare a sample and to get high-resolution imaging at the single-molecule level, allowing for measuring the physical/chemical properties of amyloid fibrils. Furthermore, AFM can scan the conformational changes of amyloid fibrils in a liquid environment, which is powerful to visualize and measure amyloid fibrillation under physiological conditions.

## **2.1. Conventional AFM imaging techniques for amyloid fibrils**

Since AFM was invented in the 1980s, it has widely been used as a versatile tool for direct imaging of nano/biomolecules and measuring molecular interactions at the single-molecule level [28–33]. In amyloid research, it has been reported that AFM is used to elucidate the fibril formation mechanism and to monitor kinetics and morphology of amyloid fibrils such as αsynuclein, insulin, and the B1 domain of protein G, a base model for immunoglobulin lightchain variable domains related with fibrils [34]. For AFM imaging, the fibrils were deposited on a freshly cleaved mica substrate. AFM images were collected in air under ambient condi‐ tions at a scan frequency of 1–2 Hz. From the analysis of AFM images, all fibrils (α-synuclein, insulin, and the B1 domain of protein G) are in correspondence with the general hierarchical assembly model, like amyloid-β (Aβ; 1–40 and 1–42), with which mature amyloid fibrils are formed by the intertwining of protofibrils. When first aggregated into protofibrils, the measured heights (i.e., fibril diameters) range from 1.2 to 3.5 nm. The structural information of the fibrils such as the fibril diameter and contour length depends on the protein species. Several protofibrils can pack together to form a mature fibril (5.2 ± 0.3 nm in height).

It has been reported that different stages of amyloid aggregation (i.e., hierarchy) can be examined by conducting a statistical analysis of acquired high-resolution AFM images [35]. β-Lactoglobulin (β-lg) fibrils are synthesized in vitro by heat denaturation at pH 2. It was shown that the β-lg amyloid-like fibrils are considered to be a semiflexible fibril structure (**Figure 2**). This means that the globular proteins (β-lg monomer) can be transformed into semiflexible fibrillary structures. The average height and contour length of β-lg fibrils were precisely measured by performing a statistical analysis on the AFM images. Each fibril has contour lengths in the range of 50 nm to 10 μm with a persistence length ranging from 16 nm to 1.6 μm. These distinct structural information of fibrils make them ideal model systems for the study of semiflexible polymers [36]. In that study, valuable structural information con‐ cerning individual fibrils in AFM images was produced, such as splitting or thinning of fibrils and even periodic height fluctuations of the fibrils. The periodic fluctuations in height provide a highly important clue about the packing of the protofibrils to form mature fibrils. **Figure 2** presents AFM images of fibrils with four different periods ranging from 35 to 135 nm, corresponding to average heights of fibrils ranging from 4 to 10 nm, respectively. Thus, the periodicity is found to increase linearly with the height of the fibrils. These hierarchical structures of β-lg fibrils can be represented from single to five-stranded packed twisted ribbon, helix-like structures, which can be compared with dynamic simulations using a coarse-grain molecular model.

additional capabilities and advantages relative to other microscopic methods. For example, it can directly show and measure the structure of an individual fibril. In addition, it is easy to prepare a sample and to get high-resolution imaging at the single-molecule level, allowing for measuring the physical/chemical properties of amyloid fibrils. Furthermore, AFM can scan the conformational changes of amyloid fibrils in a liquid environment, which is powerful to

Since AFM was invented in the 1980s, it has widely been used as a versatile tool for direct imaging of nano/biomolecules and measuring molecular interactions at the single-molecule level [28–33]. In amyloid research, it has been reported that AFM is used to elucidate the fibril formation mechanism and to monitor kinetics and morphology of amyloid fibrils such as αsynuclein, insulin, and the B1 domain of protein G, a base model for immunoglobulin lightchain variable domains related with fibrils [34]. For AFM imaging, the fibrils were deposited on a freshly cleaved mica substrate. AFM images were collected in air under ambient condi‐ tions at a scan frequency of 1–2 Hz. From the analysis of AFM images, all fibrils (α-synuclein, insulin, and the B1 domain of protein G) are in correspondence with the general hierarchical assembly model, like amyloid-β (Aβ; 1–40 and 1–42), with which mature amyloid fibrils are formed by the intertwining of protofibrils. When first aggregated into protofibrils, the measured heights (i.e., fibril diameters) range from 1.2 to 3.5 nm. The structural information of the fibrils such as the fibril diameter and contour length depends on the protein species.

Several protofibrils can pack together to form a mature fibril (5.2 ± 0.3 nm in height).

It has been reported that different stages of amyloid aggregation (i.e., hierarchy) can be examined by conducting a statistical analysis of acquired high-resolution AFM images [35]. β-Lactoglobulin (β-lg) fibrils are synthesized in vitro by heat denaturation at pH 2. It was shown that the β-lg amyloid-like fibrils are considered to be a semiflexible fibril structure (**Figure 2**). This means that the globular proteins (β-lg monomer) can be transformed into semiflexible fibrillary structures. The average height and contour length of β-lg fibrils were precisely measured by performing a statistical analysis on the AFM images. Each fibril has contour lengths in the range of 50 nm to 10 μm with a persistence length ranging from 16 nm to 1.6 μm. These distinct structural information of fibrils make them ideal model systems for the study of semiflexible polymers [36]. In that study, valuable structural information con‐ cerning individual fibrils in AFM images was produced, such as splitting or thinning of fibrils and even periodic height fluctuations of the fibrils. The periodic fluctuations in height provide a highly important clue about the packing of the protofibrils to form mature fibrils. **Figure 2** presents AFM images of fibrils with four different periods ranging from 35 to 135 nm, corresponding to average heights of fibrils ranging from 4 to 10 nm, respectively. Thus, the periodicity is found to increase linearly with the height of the fibrils. These hierarchical structures of β-lg fibrils can be represented from single to five-stranded packed twisted ribbon, helix-like structures, which can be compared with dynamic simulations using a coarse-grain

visualize and measure amyloid fibrillation under physiological conditions.

**2.1. Conventional AFM imaging techniques for amyloid fibrils**

1024 Exploring New Findings on Amyloidosis

molecular model.

**Figure 2.** Different periods of β-lactoglobulin fibrils. (a) AFM height image of fibrils with a period of 35 nm (for fibrils with a maximum height of 4 nm), 75 nm (maximum height, 6 nm), and 100 nm (maximum height, 8 nm). (b) Example of a fibril with a period of 135 nm and a maximum height of 10 nm. (c) AFM images and corresponding coarse-grain molecular dynamics reconstructions of left-handed helical fibril formation from the twisting of multistranded ribbons, with the number of filaments ranging between 1 and 5. Figures reproduced with permission from Ref. [35], © 2010 NPG.

It is highly important to study initial fibrillation of amyloid proteins for developing an inhibition method for amyloid fibrils and plaques. Although attempts with many conventional methods such as fluorescent assays with dye and optical spectroscopy had been reported, insight into the mechanisms of formation of initial fibrils at the molecular level has yet to provide clarity. To address that issue, AFM techniques can investigate growing fibrils in greater detail. **Figure 3** shows topological images of the β-lg fibrils acquired after 30 and 45 min incubation [37]. As the incubation time was increased, the contour length of fibrils increased in the range from 520 to 860 nm. The two-filament packed structure was only observed after a heating time of 30 min. But after a heating time of 45 min, fibrils of more than two filaments were observed. From those results, AFM analysis can provide us with some significant structural information not only topological images but also details such as height, contour length, and periodicity from fluctuations along the fibril length. In addition, it can show the formation characteristics of amyloid fibrils, including the critical steps driven by nucleation and growth of individual fibrils. These findings from the use of AFM imaging may help researchers gain understanding of the origin of fibrils and may assist with the development of therapeutic treatments for degenerative diseases that are generated by many different amyloidogenic proteins.

**Figure 3.** AFM height images of heated β-lactoglobulin at pH 2 and 90°C for the heating time of (a and b) 30 min and (c and d) 45 min. Figures reproduced with permission from Ref. [37], © 2011 The Royal Society of Chemistry.

#### **2.2. Advantages of AFM imaging of amyloid fibrils in liquid environments**

As we mentioned above, AFM imaging of amyloid fibrils is strong advantageous not only in the characterization of the structure of amyloid fibrils but also for the observation of initial fibrillation of amyloidogenic proteins. These investigations of amyloid fibrils in ambient atmospheric conditions by AFM imaging yield the basic understandings of the characteristics of amyloid fibrils, but all of these processes (such as fibrillation and conformation changes) actually take place in a liquid environment (i.e., in human body fluid). In addition, the formation of amyloid fibrils continuously occurs in the human body, not discretely, which is why liquid AFM is particularly important to more precisely and continuously observe to fibrils in comparing with conventional AFM.

greater detail. **Figure 3** shows topological images of the β-lg fibrils acquired after 30 and 45 min incubation [37]. As the incubation time was increased, the contour length of fibrils increased in the range from 520 to 860 nm. The two-filament packed structure was only observed after a heating time of 30 min. But after a heating time of 45 min, fibrils of more than two filaments were observed. From those results, AFM analysis can provide us with some significant structural information not only topological images but also details such as height, contour length, and periodicity from fluctuations along the fibril length. In addition, it can show the formation characteristics of amyloid fibrils, including the critical steps driven by nucleation and growth of individual fibrils. These findings from the use of AFM imaging may help researchers gain understanding of the origin of fibrils and may assist with the development of therapeutic treatments for degenerative diseases that are generated by many different

**Figure 3.** AFM height images of heated β-lactoglobulin at pH 2 and 90°C for the heating time of (a and b) 30 min and (c

As we mentioned above, AFM imaging of amyloid fibrils is strong advantageous not only in the characterization of the structure of amyloid fibrils but also for the observation of initial fibrillation of amyloidogenic proteins. These investigations of amyloid fibrils in ambient atmospheric conditions by AFM imaging yield the basic understandings of the characteristics of amyloid fibrils, but all of these processes (such as fibrillation and conformation changes) actually take place in a liquid environment (i.e., in human body fluid). In addition, the

and d) 45 min. Figures reproduced with permission from Ref. [37], © 2011 The Royal Society of Chemistry.

**2.2. Advantages of AFM imaging of amyloid fibrils in liquid environments**

amyloidogenic proteins.

1046 Exploring New Findings on Amyloidosis

In the research field of amyloid fibril, real-time monitoring of fibril growth is necessary to investigate the mechanism of amyloid fibril formation. It has been reported that the direct observation of fibrillation of Aβ peptide on different substrates is achieved by using in situ AFM analysis in a liquid environment [15]. They used two types of substrate: hydrophilic mica and hydrophobic graphite. Hydrophilic mica can be considered a model of the hydrophilic components of cell membranes such as anionic phospholipids. The hydrophobic graphite may be used as a model of hydrophobic elements of cell membrane (e.g., lipid bilayers and lipoprotein particles), in which elements Aβ peptide can localize in both plasma and cerebro‐ spinal fluid in vivo. An in situ AFM study of Aβ fibril aggregation was followed by continuous imaging in phosphate-buffered saline (pH 7.4, protein concentration 10–500 mM). Aβ peptides initially were formed with pseudomicellar aggregates and then tend to form linear structures, such as protofibrils, as the Aβ concentration was increased on the hydrophilic mica substrate. However, Aβ peptides were formed with elongated sheets structures on the hydrophobic graphite substrate. This difference by substrates is the distinguishable characteristics between β-sheets and α-helices. These studies show that an in situ AFM study is able to directly observe not only the amyloid aggregation in physiological fluids but also fibrillation driven by interactions at the interface of hydrophilic and hydrophobic substrates. It may be helpful to understand the mechanism of formation of Aβ fibrils in membranes and lipoprotein particles in vivo.

AFM has been employed not only to study the formation of amyloid fibrils in vitro but also to investigate ex vivo amyloid material isolated from organs, specifically hearts, of patients. The AFM analysis of natural amyloid fibrils produced by the Leu174Ser apolipoprotein A-I variant (ApoA-I-LS) has been reported. ApoA-I-LS amyloid fibrils were obtained from two different patients, and conformational changes of the fibrils in both air and liquid environments were measured [38]. The fibrils from both patients were identified by small-angle X-ray diffraction patterns, indicative of residual α-helical structure. In a liquid environment, it was observed that amyloid fibrils show poor adhesion properties on a mica substrate. But, from acquired AFM images, it was found that globular aggregates were easily adsorbed on mica. These results revealed the existence of fibrils as well as pre-fibrillar aggregates, with common morphologies and compatible sizes of fibrils and globules for both patients.

In addition, AFM imaging in a liquid environment is able to reveal unique and intrinsic properties of amyloid fibrils, which cannot be seen by any other method. It has been reported that the conformations of C-terminal tails outside of the α-synuclein amyloid fibrils are variable depending on environmental conditions by using structural information from liquid AFM imaging, as in **Figure 4** [39]. They suggested that the exposed C-terminus is unstructured and looks like a polymer bush and that the strongly negatively charged C-terminus has various functions and conformations with different ion concentrations (i.e., in the range of 0–5 M NaCl). It was also observed that the height of α-synuclein fibrils imaged in air appears to be about 17% lower than those of the same fibrils imaged in a liquid environment. This phenomenon is attributed to the collapse of C-terminus depending on the drying of the fibrils. Measuring the conformational changes of the C-terminus in amyloid fibrils by AFM imaging sheds light on the estimation of the transformation of the exterior structures of amyloid fibrils.

**Figure 4.** (a) Schematic representation of a fibril with stacked β-sheet folded monomers perpendicular to the fibril axis and an unstructured C-terminus outside the fibril. (b) Different conformations of C-terminal tails outside the fibril core in liquid conditions with different ion concentrations and in ambient air. Figure reproduced with permission Ref. [39], © 2012 American Chemical Society.

## **3. PeakForce QNM (quantitative nanomechanical mapping) of amyloid fibrils**

Amyloid fibrils have been known as β-sheet-rich structures, which are consisted with dense hydrogen-bonding networks [40]. The self-assembly aggregates can be formed with stack‐ ings of several hundreds to thousands of cross β-sheet units, a few nanometers in diameter, and several micrometers in length. Such hierarchical structure of fibrils is highly related to pathological meanings as well as to functional features due to their marvelous mechanical properties, which are comparable to those of silk and/or steel [13, 41]. Characterization of biophysical properties of amyloid fibrils has significant implications ranging from applica‐ tion to medicines and engineering for designing amyloid-based nanomaterials. To measure biophysical properties of amyloid fibrils, although in recent decades many research results had been reported that had been obtained by using various techniques such as optical, mag‐ netic tweezer and dynamic mechanical analysis [13], AFM techniques are commonly used for both manipulation at the single-molecule level and force spectroscopy of nanomaterials (i.e., amyloid fibrils). Moreover, AFM nanoindentation has been used to study nanomechan‐ ics of amyloid fibrils [42].

AFM nanoindentation is performed by mapping force-distance (F-D) curve on the region of interest in the sample [43, 44]. F-D curve measurement is based on the deflection theory of a cantilever beam and the contact mechanics. The degree of nanomechanical deflection of AFM cantilever originated from the physical interactions between the AFM tip and the sample can be converted to the applied forces to the cantilever. When F-D curve records, the cantilever approaches and indents the sample until a certain predefined force is reached, and then the cantilever is retracted. During the approach and retract cycle, the force is continuously measured, which is a F-D curve for one point of the sample (**Figure 5**). The mechanical properties such as Young's modulus, adhesion force, and deformation energies of amyloid fibrils can be determined directly from the recorded data (F-D curve) by AFM nanoindentation. In particular, to extract the accurate Young's modulus of the sample from F-D curves, contact mechanics theory is used. There are three different types of representative contact mechanics models (Hertz, DMT, and JKR) depending on either the type of sample or the structure of AFM tip [18]. The Hertz model presumes a nonadhesive contact between the tip and the sample, while the Derjaguin-Muller-Toporov (DMT) and Johnson-Kendall-Roberts (JKR) models are considered to be adhesive materials. DMT model further includes the extra constant adhesion term, which indicates the adhesive force outside the contact area. The JKR model demonstrates the adhesion forces inside the contact area between the tip and the sample. Those model equations are as follows:

attributed to the collapse of C-terminus depending on the drying of the fibrils. Measuring the conformational changes of the C-terminus in amyloid fibrils by AFM imaging sheds light on

**Figure 4.** (a) Schematic representation of a fibril with stacked β-sheet folded monomers perpendicular to the fibril axis and an unstructured C-terminus outside the fibril. (b) Different conformations of C-terminal tails outside the fibril core in liquid conditions with different ion concentrations and in ambient air. Figure reproduced with permission Ref. [39],

**3. PeakForce QNM (quantitative nanomechanical mapping) of amyloid**

Amyloid fibrils have been known as β-sheet-rich structures, which are consisted with dense hydrogen-bonding networks [40]. The self-assembly aggregates can be formed with stack‐ ings of several hundreds to thousands of cross β-sheet units, a few nanometers in diameter, and several micrometers in length. Such hierarchical structure of fibrils is highly related to pathological meanings as well as to functional features due to their marvelous mechanical properties, which are comparable to those of silk and/or steel [13, 41]. Characterization of biophysical properties of amyloid fibrils has significant implications ranging from applica‐ tion to medicines and engineering for designing amyloid-based nanomaterials. To measure biophysical properties of amyloid fibrils, although in recent decades many research results had been reported that had been obtained by using various techniques such as optical, mag‐ netic tweezer and dynamic mechanical analysis [13], AFM techniques are commonly used for both manipulation at the single-molecule level and force spectroscopy of nanomaterials (i.e., amyloid fibrils). Moreover, AFM nanoindentation has been used to study nanomechan‐

© 2012 American Chemical Society.

1068 Exploring New Findings on Amyloidosis

ics of amyloid fibrils [42].

**fibrils**

the estimation of the transformation of the exterior structures of amyloid fibrils.

$$Hert \mathbf{z} \colon F = \frac{4}{3} E^\* \cdot \overline{R} \,\delta^{\frac{3}{2}}$$

$$\frac{\mathbf{1}}{E^\*} = \frac{\mathbf{1} - \boldsymbol{\nu}\_m{}^2}{E\_m} + \frac{\mathbf{1} - \boldsymbol{\nu}\_\ell{}^2}{E\_\ell}$$

where *F* is the force, *δ* is the AFM indentation depth (distance), *R* is the radius of cantilever tip, *E*\* is the reduced elastic modulus, and *γ* is the work of adhesion. *E*\* is calculated by the relationship between the elastic modulus and Poisson ratio of AFM cantilever (*E***<sup>i</sup>** , *ν***<sup>i</sup>** ) and sample (*E***m**, *ν***m**).

**Figure 5.** The schematic illustration of F-D curve acquisition process during AFM nanoindentation. Approach (blue) and retraction (red) F-D curves. Illustration depicts the cantilever approaching to and retracting from the sample as follows: (1) approaching, (2) initial contact, and (3) repulsive contact. (4) Adhesion and (5) noncontact regimes record‐ ed upon retracting the cantilever and sample.

In recent decades, to measure the physical properties of amyloid fibrils more precisely and quantitatively, PeakForce QNM technique has been developed. PeakForce QNM is a robust F-D curve mapping technique, which is beneficial for soft delicate biological samples. It is able to map and distinguish between nanomechanical features, including the DMT modulus (1 kPa to 100 GPa), adhesion, energy dissipation, and deformation by utilizing PeakForce tapping technology [45, 46]. It can acquire high-resolution topographic images and simultaneously collect a quantitative mapping of the intrinsic mechanical properties of amyloid fibril.

## **3.1. Principle of PeakForce QNM**

PeakForce tapping is one of the scanning modes, which is especially suited to applications to the measurement of mechanical properties of biological samples [18]. In this mode, the probe is oscillated at a low frequency (1–2 kHz), while capturing a force-distance curve (F-D curve) plots each time the AFM probe taps on the sample by directly controlling the probe-sample interaction. To describe the details, one cycle of the PeakForce tapping trajectory is shown in **Figure 6a** [17]. During the approach to the sample, the probe begins (A) with a noncontact force (i.e., attractive force) until contact on the sample. The Z piezo presses it downward until the probe contacts the sample (B); the probe continuously presses until the probe reaches the setpoint (i.e., maximum force) (C). The setpoint, which is one of the parameters of the probesample interaction, is used during AFM topography. After reaching the peak force at point C, the tip is pulled off the surface at point D, which means maximum adhesion. Finally, the curve levels and the probe tip returns to its original position (E).

Accurate controlling of the probe position and feedback permits calculation of several properties from the F-D curve produced by the direct measurements simultaneously at each pixel in **Figure 6b**. From this curve, the peak force is measured at point C, which is used as the imaging feedback signal. As the probe is scanned over the surface, this signal produces a topological image. Adhesion force is measured at D, which is calculated as the force difference between the baseline and the minimum force value of the curve. Simultaneously, Young's modulus is obtained by fitting the DMT contact mechanics model to the retraction curve (at points C to D). The dissipation information is obtained by integrating the area between the extension and retraction curves and the deformation of the sample is calculated as the distance between the contact point on the adhesion curve and the maximum indentation. This unique technique is called PeakForce QNM. Compared to a typical AFM imaging method, PeakForce QNM can simultaneously yield high-resolution AFM imaging with quantitative mechanical property mapping. PeakForce QNM has recently emerged as a means of measuring the intrinsic and biophysical properties of nanomaterials [47], including amyloid fibrils [17, 19]. The following section will discuss the mapping the mechanical properties of amyloid fibrils by PeakForce QNM.

**Figure 6.** Principle of PeakForce QNM operation. (a) One cycle of the PeakForce tapping curve. (b) Calculation of the different peak force channels. Figure reproduced with permission Ref. [17], © 2011 AIP.

## **3.2. Mapping the mechanical properties of amyloid fibrils by PeakForce QNM**

**Figure 5.** The schematic illustration of F-D curve acquisition process during AFM nanoindentation. Approach (blue) and retraction (red) F-D curves. Illustration depicts the cantilever approaching to and retracting from the sample as follows: (1) approaching, (2) initial contact, and (3) repulsive contact. (4) Adhesion and (5) noncontact regimes record‐

In recent decades, to measure the physical properties of amyloid fibrils more precisely and quantitatively, PeakForce QNM technique has been developed. PeakForce QNM is a robust F-D curve mapping technique, which is beneficial for soft delicate biological samples. It is able to map and distinguish between nanomechanical features, including the DMT modulus (1 kPa to 100 GPa), adhesion, energy dissipation, and deformation by utilizing PeakForce tapping technology [45, 46]. It can acquire high-resolution topographic images and simultaneously collect a quantitative mapping of the intrinsic mechanical properties of amyloid fibril.

PeakForce tapping is one of the scanning modes, which is especially suited to applications to the measurement of mechanical properties of biological samples [18]. In this mode, the probe is oscillated at a low frequency (1–2 kHz), while capturing a force-distance curve (F-D curve) plots each time the AFM probe taps on the sample by directly controlling the probe-sample interaction. To describe the details, one cycle of the PeakForce tapping trajectory is shown in **Figure 6a** [17]. During the approach to the sample, the probe begins (A) with a noncontact force (i.e., attractive force) until contact on the sample. The Z piezo presses it downward until the probe contacts the sample (B); the probe continuously presses until the probe reaches the setpoint (i.e., maximum force) (C). The setpoint, which is one of the parameters of the probesample interaction, is used during AFM topography. After reaching the peak force at point C, the tip is pulled off the surface at point D, which means maximum adhesion. Finally, the curve

Accurate controlling of the probe position and feedback permits calculation of several properties from the F-D curve produced by the direct measurements simultaneously at each pixel in **Figure 6b**. From this curve, the peak force is measured at point C, which is used as the imaging feedback signal. As the probe is scanned over the surface, this signal produces a topological image. Adhesion force is measured at D, which is calculated as the force difference

ed upon retracting the cantilever and sample.

10810 Exploring New Findings on Amyloidosis

**3.1. Principle of PeakForce QNM**

levels and the probe tip returns to its original position (E).

In the folding process, synthesized peptide chains can be transformed into three-dimensional protein structure, which has diverse functions in biological organism, through intermolecular interactions such as hydrogen and hydrophobic bonding [3, 48]. The proteins have intrinsic mechanical properties depending on the specific amino acid sequence, lengths, and complex environments. Meanwhile, misfolded proteins, commonly known as "amyloid protein," can be transformed into β-sheet-rich fibrillary structure (i.e., amyloid fibril) during fibrillation process. Amyloid fibrils have tremendous mechanical properties (a few GPa), implying a comparatively high stiffness. Moreover, it displays structural polymorphism, including twisted ribbons, helical ribbons, and nanotube structure [19], which is supposed to show a difference in mechanical properties. Amyloid species (amyloid proteins and fibrils) can be accumulated and deposited in the human body, which is associated with proteoglycans, in particular, heparan sulfate proteoglycans [48, 49]. These findings raise the question about the underpinning nature of the correlation between the mechanical properties of amyloid fibrils and its pathological roles in living systems as well as the use of fibrils as nanoscale materials.

PeakForce QNM has emerged as mechanical property mapping variant of AFM nanoinden‐ tation that had been used for several decades. PeakForce QNM has been used to measure the Young's modulus of β-lg fibrils deposited on mica [17]. It is of importance to measure the mechanical properties of β-lg fibrils because they have been used to make new nanomaterials by mixing with nanoparticles and nanowires for applications in various research fields. Using PeakForce QNM, the average height of the fibrils was measured to be 2.5 ± 0.5 nm and DMT modulus images were concurrently acquired. The Young's modulus of β-lg fibrils was estimated to be 3.7 ± 1.1 GPa. This result from PeakForce QNM analysis is similar to that of a previously reported Young's modulus of insulin fibrils (~3.3 GPa).

Amyloid fibrils can be generally formed with many different proteins such as Aβ (1–42), αsynuclein, albumin, etc., which have pathological and functional meanings. These fibrils often exhibit structural polymorphism due to interweaving of the several protofibrils. It has been demonstrated that amyloid fibrils appear in various structures such as ribbon-like and nanotube-like packed fibrils. PeakForce can precisely measure the Young's moduli of several amyloid fibrils, assembled from α-synuclein, Aβ (1–42), tau protein, insulin, β-lg, lysozyme, ovalbumin, and bovine serum albumin fibrils, as in **Figure 7** [19]. In addition, it demonstrated that PeakForce QNM can provide us some clues regarding structural polymorphism of amyloid fibrils by the exact estimation of Young's modulus. The measured Young's modulus values are twisted ribbon structure, 2.3 ± 0.6 GPa, and helical ribbon and nanotube structures,

**Figure 7.** AFM height image of (a) twisted ribbon, (d) helical ribbon, and (g) nanotube-like structures of the end-cap‐ ped heptapeptide CH3CONH-βAβAKLVFF-CONH2. AFM DMT modulus image of (b) twisted ribbon, (e) helical rib‐ bon, and (h) nanotube-like structures. The dashed lines mark the positions at which the DMT modulus was analyzed. Profiles of DMT moduli along (c) twisted ribbon, (f) helical ribbon, and (i) nanotube-like structures. The data were ob‐ tained by the analysis of 20 fibrils for each type. Figure reproduced with permission Ref. [19], © 2012 The Royal Society of Chemistry.

2.3 ± 0.7 GPa and 2.4 ± 0.5 GPa, respectively. They also measured the value of Young's modulus for insulin amyloid fibrils (3.2 ± 0.6 GPa), which is very similar to that of a previously study of insulin (3.3 ± 0.4 GPa).

mechanical properties of β-lg fibrils because they have been used to make new nanomaterials by mixing with nanoparticles and nanowires for applications in various research fields. Using PeakForce QNM, the average height of the fibrils was measured to be 2.5 ± 0.5 nm and DMT modulus images were concurrently acquired. The Young's modulus of β-lg fibrils was estimated to be 3.7 ± 1.1 GPa. This result from PeakForce QNM analysis is similar to that of a

Amyloid fibrils can be generally formed with many different proteins such as Aβ (1–42), αsynuclein, albumin, etc., which have pathological and functional meanings. These fibrils often exhibit structural polymorphism due to interweaving of the several protofibrils. It has been demonstrated that amyloid fibrils appear in various structures such as ribbon-like and nanotube-like packed fibrils. PeakForce can precisely measure the Young's moduli of several amyloid fibrils, assembled from α-synuclein, Aβ (1–42), tau protein, insulin, β-lg, lysozyme, ovalbumin, and bovine serum albumin fibrils, as in **Figure 7** [19]. In addition, it demonstrated that PeakForce QNM can provide us some clues regarding structural polymorphism of amyloid fibrils by the exact estimation of Young's modulus. The measured Young's modulus values are twisted ribbon structure, 2.3 ± 0.6 GPa, and helical ribbon and nanotube structures,

**Figure 7.** AFM height image of (a) twisted ribbon, (d) helical ribbon, and (g) nanotube-like structures of the end-cap‐ ped heptapeptide CH3CONH-βAβAKLVFF-CONH2. AFM DMT modulus image of (b) twisted ribbon, (e) helical rib‐ bon, and (h) nanotube-like structures. The dashed lines mark the positions at which the DMT modulus was analyzed. Profiles of DMT moduli along (c) twisted ribbon, (f) helical ribbon, and (i) nanotube-like structures. The data were ob‐ tained by the analysis of 20 fibrils for each type. Figure reproduced with permission Ref. [19], © 2012 The Royal Society

of Chemistry.

previously reported Young's modulus of insulin fibrils (~3.3 GPa).

11012 Exploring New Findings on Amyloidosis

Recently, amyloid fibrils have attracted great attention because of outstanding features based on their mechanical properties as nanomaterials. To investigate the potential of amyloid fibrils as biomaterials, it is important to understand the mechanism of the fibrillation process and to quantify the mechanical properties. By performing the PeakForce QNM technique, the mechanical properties of the Aβ-42 fibrils during the fibrillation process have been measured, as in **Figure 8** [21]. The Young's modulus of the oligomers, protofibrils, and mature Aβ-42 fibrils was evaluated. During fibrillation, in which oligomers grow into protofibrils and then finally formed mature fibrils, the mechanical property (i.e., Young's modulus) increased. Hydrogen bonds between β-sheets may need to be reckoned with when one tries to determine the mechanical properties of amyloid fibrils. The changes in β-sheets in oligomers, protofibrils, and mature fibrils may have effects on the mechanical properties during the fibrillation process. These results could be of great significance for the elucidation of the mechanical properties of amyloid fibrils for the planning of new material strategies.

**Figure 8.** Evolution of the Young's modulus of Aβ-42 during the fibrillation process. DMT modulus AFM images of oligomers at 0 h (a), protofibrils at 48 h (b), and fibrils at 72 h (c). Distribution of the measured Young's modulus for oligomers (d), protofibrils (e), and fibrils (f). Figure reproduced with permission Ref. [21], © 2015 Wiley-VCH Verlag GmbH & Co. KGaA.

## **4. Kelvin probe force microscopy (KPFM) of amyloid fibrils**

The amyloid fibril has been implicated in the various neurodegenerative diseases such as Alzheimer's, Parkinson's, Creutzfeldt-Jakob's, and Huntington's diseases [1–3], but the molecular mechanism of an amyloid toxicity is still debated [49–51]. To address this issue, scientists have investigated different possibilities for molecular behavior of amyloid-forming proteins that are correlated with the amyloid toxicity mechanism [52–54]. The leading proposed mechanism of the amyloid toxicity refers to the electrostatic interactions between the lipid membrane of cells and amyloid oligomers/fibrils (i.e., cytotoxicity) [55–57]. In addition, the electrostatic forces play important roles in the accumulation of proteinaceous aggregates as amyloid plaques or Lewy bodies. Therefore, the measurement of electrical properties of amyloid fibrils offers a new insight into the mechanism of amyloid fibril forma‐ tion and toxicity.

KPFM has been considered as a powerful instrument for detecting the electrical properties of nano/biomaterials including molecules [58–62] by mapping the surface potential (the work function) of them. KPFM is carried out by a conducting AFM probe, which ascends to lift scan height. While the probe scans a sample surface of interest, a potential offset between the tip and the surface is recorded at each point of the scanned area. Furthermore, a high spatial resolution of KPFM is attributed to highly sharp conducting AFM tip by measuring the local contact potential difference (CPD) between the tip and the sample (**Figure 9**). Accordingly, KPFM has recently been used for characterizing the surface potential of amyloid fibrils [20, 23, 63] to unveil the molecular mechanism governing amyloid fibril formation and toxicities.

**Figure 9.** A schematic illustration of Kelvin probe force microscopy (KPFM) method for the measurement of electrical properties of material.

#### **4.1. Principle of KPFM**

In KPFM studies of mapping the surface potential, the surface potential of the materials is measured by the CPD between the material surface and the KPFM conducting cantilever tip, where the CPD is the variation in work function between different surfaces (the material surface and the tip) [58]. The CPD of two materials is determined, depending on their elec‐ tronic/electrical characteristics. The characterization of the CPD is able to use not only for measuring the work function of metallic material and semiconductor surfaces but also for mapping the surface potential of biomolecules such as various proteins [64], DNA molecules [59, 60], and amyloids [20, 23, 63].

molecular mechanism of an amyloid toxicity is still debated [49–51]. To address this issue, scientists have investigated different possibilities for molecular behavior of amyloid-forming proteins that are correlated with the amyloid toxicity mechanism [52–54]. The leading proposed mechanism of the amyloid toxicity refers to the electrostatic interactions between the lipid membrane of cells and amyloid oligomers/fibrils (i.e., cytotoxicity) [55–57]. In addition, the electrostatic forces play important roles in the accumulation of proteinaceous aggregates as amyloid plaques or Lewy bodies. Therefore, the measurement of electrical properties of amyloid fibrils offers a new insight into the mechanism of amyloid fibril forma‐

KPFM has been considered as a powerful instrument for detecting the electrical properties of nano/biomaterials including molecules [58–62] by mapping the surface potential (the work function) of them. KPFM is carried out by a conducting AFM probe, which ascends to lift scan height. While the probe scans a sample surface of interest, a potential offset between the tip and the surface is recorded at each point of the scanned area. Furthermore, a high spatial resolution of KPFM is attributed to highly sharp conducting AFM tip by measuring the local contact potential difference (CPD) between the tip and the sample (**Figure 9**). Accordingly, KPFM has recently been used for characterizing the surface potential of amyloid fibrils [20, 23, 63] to unveil the molecular mechanism governing amyloid fibril formation and toxicities.

**Figure 9.** A schematic illustration of Kelvin probe force microscopy (KPFM) method for the measurement of electrical

In KPFM studies of mapping the surface potential, the surface potential of the materials is measured by the CPD between the material surface and the KPFM conducting cantilever tip, where the CPD is the variation in work function between different surfaces (the material surface and the tip) [58]. The CPD of two materials is determined, depending on their elec‐ tronic/electrical characteristics. The characterization of the CPD is able to use not only for

tion and toxicity.

11214 Exploring New Findings on Amyloidosis

properties of material.

**4.1. Principle of KPFM**

The hardware system of KPFM is founded upon the principle of the Kelvin method, which is a standard measurement method of the determination the CPD [65] (this is a reason why KPFM applies the term "Kelvin"). In the Kelvin probe method, the CPD (*V*CPD) between two different materials is plainly defined as

$$V\_{CPD} = \frac{\varphi\_1 - \varphi\_2}{-e} \tag{1}$$

where *φ*1 and *φ*2 are the work functions of two different materials (in KPFM, *φ*1 = *φ*tip and *φ*2 = *φ*sample) and *e* is the charge of an electron. The probe, which consists of material with a known work function, is aligned to the sample surface for forming a capacitor. The reference electrode probe is vibrated, causing a variation in the capacitance between two plates, resulting in an electric current *i*(*t*) and the generated current is given by

$$i(t) = V\_{CPD} 
a \Delta C \cos \alpha t \tag{2}$$

where *ω* is the frequency of vibration, Δ*C* is the change of capacitance, and *V*CPD is the contact potential between the probe and the sample surface. *V*CPD is measured by applying an con‐ trasting external DC (direct current) bias voltage (*V*DC), which is varied until the *i*(*t*) is mini‐ mized.

The fundamental KPFM system is similar to the Kelvin method [65]. Mapping the surface potential of a material using KPFM, an electrostatic force (*FES*) *via* the *V*CPD between a KPFM conducting tip and a material, is caused by an AC (alternating current) voltage *V*AC with a DC offset bias *V*DC which is applied between a tip and a material [66]. The *FES* between the tip and sample is defined as

$$F\_{ES}\left(\mathbf{x},t\right) = -\frac{1}{2}\frac{\partial C\left(\mathbf{x}\right)}{\partial \mathbf{x}} \left[ \left( V\_{DC} \pm V\_{CPD} \right) + V\_{AC} \sin \alpha t \right]^2 \tag{3}$$

where *x* is the distance between the KFPM conducting tip and a material. The electrical force component with frequency *ω* (related to differential value of "(*V*DC ± *V*CPD) *V*AC sin ω*t*") is obtained from the PSD) signals through a lock-in amplifier. A feedback controller applies the *V*DC to the KPFM system until the electrical force component with frequency *ω* is minimized. Accordingly, *V*CPD between a KPFM conducting tip and a material is determined based on altering the DC offset bias *V*DC.

#### **4.2. Mapping the surface potential of amyloid fibrils**

As mentioned above, the evaluation of electrical properties of amyloid fibrils would offer a better understanding of the mechanism of amyloid fibril formation and toxicity. To date, some of researchers have reported KPFM analysis for quantitative estimation of electrical properties of amyloid fibrils. It has been reported that synthesis of the amyloid-β-(25–35) peptide (Aβ25–35) (with sequence GSNKGAIIGLM) fibrils and KPFM analysis of them [67]. Although the major components of neurotic plaque found in AD are 10-to-42-residue-long Aβ peptides (Aβ1–40/Aβ1–42), shorter fragments of peptides are also involved, such as Aβ25–35. It has been reported that Aβ25–35 can form readily β-sheet aggregates and is toxic to neurons. The surface potential analysis of Aβ25–35 fibrils was performed by KPFM. The samples were prepared for KPFM imaging by depositing the solutions on chemically cleaned silicon (Si) substrates, and KPFM records were made for both the sample topography and the phase shift by using a two-pass method, namely, lift mode KPFM. It was found that the five fibrils have almost the same CPD values, which are about −10~ to −15 mV relative to the Si substrate. By calibrating the work function of cantilever tip, the work function of the Aβ25–35 fibril was determined to be about 4.62 eV. However, the main thrust of this study is the surface potential analysis in regards to the interaction of gold nanoparticles (AuNPs) with Aβ25–35 peptides, which we shall mention further on.

**Figure 10.** AFM and KPFM images of β-lactoglobulin amyloid fibrils. In each image of AFM topography (panel a) and surface potential (panel b), the white arrow shows a single amyloid fibril. A blue arrow indicates the location of amy‐ loid fibril, at which the transverse profile of surface potential and topography were obtained. In the profiles of both topography and surface potential at longitudinal direction. Figures reproduced with permission from Ref. [20], © 2012 AIP.

Recently, β-lg has attracted much interest as an alternative amyloid precursor to be used to investigate the fibrillation mechanism and to fabricate various composites because of its inexpensive price and easy fabrication. The electrical properties of the β-lg fibrils using KPFM have been studied, which was the first attempt to visualize mapping the surface potential distribution of a single amyloid fibril [20]. As shown in **Figure 10**, the surface potential of the fibril is positive (e.g., ~12 mV) since the pH of a buffer solution is lower than the isoelectric point (pI) of β-lg (p*I* ~ 5.0). Interestingly, it is found that the surface potential profile of the fibril is critically dependent on the fibril diameter and periodicity, which implies that the surface charge distribution of amyloid fibril is closely related to its conformation. This report demonstrated that KPFM is able to accurately obtain the surface potential of the amyloid fibrils with their conformational structures. Moreover, KPFM was shown to have the possibility of being usable as a detection platform for peptide identification of amyloid fibrils because KPFM measurement has proven to be powerful enough to detect a delicate difference of the surface potential.

**4.2. Mapping the surface potential of amyloid fibrils**

11416 Exploring New Findings on Amyloidosis

which we shall mention further on.

AIP.

As mentioned above, the evaluation of electrical properties of amyloid fibrils would offer a better understanding of the mechanism of amyloid fibril formation and toxicity. To date, some of researchers have reported KPFM analysis for quantitative estimation of electrical properties of amyloid fibrils. It has been reported that synthesis of the amyloid-β-(25–35) peptide (Aβ25–35) (with sequence GSNKGAIIGLM) fibrils and KPFM analysis of them [67]. Although the major components of neurotic plaque found in AD are 10-to-42-residue-long Aβ peptides (Aβ1–40/Aβ1–42), shorter fragments of peptides are also involved, such as Aβ25–35. It has been reported that Aβ25–35 can form readily β-sheet aggregates and is toxic to neurons. The surface potential analysis of Aβ25–35 fibrils was performed by KPFM. The samples were prepared for KPFM imaging by depositing the solutions on chemically cleaned silicon (Si) substrates, and KPFM records were made for both the sample topography and the phase shift by using a two-pass method, namely, lift mode KPFM. It was found that the five fibrils have almost the same CPD values, which are about −10~ to −15 mV relative to the Si substrate. By calibrating the work function of cantilever tip, the work function of the Aβ25–35 fibril was determined to be about 4.62 eV. However, the main thrust of this study is the surface potential analysis in regards to the interaction of gold nanoparticles (AuNPs) with Aβ25–35 peptides,

**Figure 10.** AFM and KPFM images of β-lactoglobulin amyloid fibrils. In each image of AFM topography (panel a) and surface potential (panel b), the white arrow shows a single amyloid fibril. A blue arrow indicates the location of amy‐ loid fibril, at which the transverse profile of surface potential and topography were obtained. In the profiles of both topography and surface potential at longitudinal direction. Figures reproduced with permission from Ref. [20], © 2012

Recently, β-lg has attracted much interest as an alternative amyloid precursor to be used to investigate the fibrillation mechanism and to fabricate various composites because of its

#### **4.3. Quantifying the electrical properties of amyloid fibrils in different environments**

For understanding electrostatic interactions between various nano/biomaterials and amyloid oligomers/fibrils, or the accumulation of proteinaceous aggregates as amyloid fibrils/plaques, many researchers have reported quantifying the electrical properties of amyloid fibrils in different environments. In recent decades, nanomaterials, especially nanoparticles, have been increasingly applied in biomedicine for therapeutic and diagnostic purposes [68–70]. In particular, AuNPs have been widely used in the field of chemistry and biomedical science [71– 73] due to their electronic [74] and molecular-recognition properties [75]. Due to the properties of AuNP, it has been found that the AuNPs can either promote or inhibit the aggregation of Aβ proteins, depending on the size and functionality of AuNPs or the pH value of the solution. As mentioned above, the surface potential analysis on the interaction of AuNPs with Aβ25–35 peptides has been studied [67]. To investigate the effect of AuNPs on fibrillation, Aβ25–35 peptides were incubated with AuNPs at room temperature for 5–12 days. KPFM was used to record both the sample topography and the surface potential of AuNPs with Aβ25–35 fibrils. From the KPFM images, it was found that the CPD values of the AuNPs are about −0.7 to −1.1 V, whereas the value for pure Aβ25–35 peptide fibrils was about 4.62 eV. *Via* calibration of the work function of cantilever tip, the work function of the AuNPs is obtained as about 4.95– 5.20 eV. The work functions of fibrils connected by AuNPs are about 4.78 eV, slightly different from the pure fibrils, which difference may be induced by the local electric fields modified by the presence of AuNPs. From the results, it can be suggested that the presence of AuNPs in Aβ25–35 solution has a significant influence on protein aggregation and on their electrical properties. This report demonstrated that it is plausible to suggest that KPFM will be quite useful for monitoring the conformational change and the electrical properties of the amyloid fibrils during self-assembly with nanomaterials.

To precisely investigate the mechanism of amyloid fibril formation, it is necessary to quantify the electrical properties of amyloid fibrils synthesized at different chemical environments. In the fibrillation of amyloid fibrils, the pH condition is one of the most important regulating chemical factors. It has been reported that the surface potential distribution of β-lg fibril is variable as a function of the pH of a buffer solution [20]. As mentioned above, the surface potential of amyloid fibril is critically dependent on the conformation of amyloid fibril. Therefore, the authors have defined the point charge density as related to the surface potential of β-lg fibril per unit fibril thickness: α(x) ≡ σmax(x)/*d*max(x), where *σ*(x) is the measured surface potential and *d*(x) is the diameter of an amyloid fibril [20]. It is shown that a parameter *α*(x) is varied with respect to pH and that *α*(x) becomes zero at pH ~ 4.7, indicating that the p*I* value of β-lg fibril is given by p*I* = 4.7 (**Figure 11a**). The surface potential of amyloid fibrils ranged from 12 mV (pH ~ 2) to −12 mV (pH ~ 7), depending on the pH of the buffer solution. For precise observation of the β-lg fibril's structure, the authors presented high-resolution KPFM images of single amyloid fibril for pH 3 < p*I* (~5.0) and pH 6 > p*I* (**Figure 11b**). With pH 3, they have found pristine β-lg protofilaments; with pH 6, the β-lg fibril had a complex structure of β-lg oligomer-binding protofilaments that had irregular topological height. It was remarkably observed that the pH-dependent electrostatic property of amyloid fibril was responsible for binding affinity between the amyloid small aggregates and the amyloid fibrils.

**Figure 11.** (a) The surface charge distribution of amyloid fibril as a function of the pH of buffer solution in which amy‐ loid fibril is prepared. The isoelectric point (p*I*) value of amyloid fibril is determined in such a way that the pH value, at which surface charge distribution becomes zero, is identical to p*I* value. (b) AFM topography and surface potential images of β-lg amyloid fibrils when they are prepared in buffer solutions, whose pH values are pH 3 and pH 6, respec‐ tively. All scale bars are 250 nm. Figures reproduced with permission from Ref. [20], © 2012 AIP.

Moreover, in recent studies of amyloid fibril with AFM, some of scientists have reported about various structural features of amyloid fibrils such as steric zipper pattern [76, 77], helical pattern [48], and length [78], which is a key design parameter that determines the material properties of amyloid fibrils. Nevertheless, it is not quite understood yet how the molecular structures and conformational diversity (heterogeneous conformations) of amyloid fibrils are regulated.

To figure out how amyloid fibrils with heterogeneous conformations are formed, the synthesis of the β-lg fibrils with microwave-assisted chemistry method is considered, which enables the acceleration of biochemical reactions, and performed KPFM analysis of them [63]. It has been reported already that the microwave-assisted chemistry for the synthesis of amyloid fibrils makes the acceleration of fibrillation possible. The mechanism of amyloid fibrillation under microwave irradiation is still debated, but it seems to be related to the enhanced collision frequency due to the microwave-driven temperature changes of the protein solution. The temperature of the β-lg protein solution is rapidly increased by rotating water molecules through microwave irradiation. As such, microwave-assisted chemistry accelerates speed of chemical reactions regarding fibrillations of the β-lg proteins within a few hours (even less than an hour) [79]. It is a remarkable result compared to the classical (conventional) heating method which generally requires long incubation time (several hours) and a large volume of protein solution. However the results of this study show that microwave-assisted chemistry not only allows the acceleration of fibrillation but also enables tuning of the β-lg amyloid fibrils by controlling the microwave irradiation time *τ*, the time interval for irradiation *λ*, or the number of irradiations *N*. It was remarkably observed that a stable protein aggregation is produced in the fibril structure that depends on *λ*.

potential of amyloid fibril is critically dependent on the conformation of amyloid fibril. Therefore, the authors have defined the point charge density as related to the surface potential of β-lg fibril per unit fibril thickness: α(x) ≡ σmax(x)/*d*max(x), where *σ*(x) is the measured surface potential and *d*(x) is the diameter of an amyloid fibril [20]. It is shown that a parameter *α*(x) is varied with respect to pH and that *α*(x) becomes zero at pH ~ 4.7, indicating that the p*I* value of β-lg fibril is given by p*I* = 4.7 (**Figure 11a**). The surface potential of amyloid fibrils ranged from 12 mV (pH ~ 2) to −12 mV (pH ~ 7), depending on the pH of the buffer solution. For precise observation of the β-lg fibril's structure, the authors presented high-resolution KPFM images of single amyloid fibril for pH 3 < p*I* (~5.0) and pH 6 > p*I* (**Figure 11b**). With pH 3, they have found pristine β-lg protofilaments; with pH 6, the β-lg fibril had a complex structure of β-lg oligomer-binding protofilaments that had irregular topological height. It was remarkably observed that the pH-dependent electrostatic property of amyloid fibril was responsible for

binding affinity between the amyloid small aggregates and the amyloid fibrils.

**Figure 11.** (a) The surface charge distribution of amyloid fibril as a function of the pH of buffer solution in which amy‐ loid fibril is prepared. The isoelectric point (p*I*) value of amyloid fibril is determined in such a way that the pH value, at which surface charge distribution becomes zero, is identical to p*I* value. (b) AFM topography and surface potential images of β-lg amyloid fibrils when they are prepared in buffer solutions, whose pH values are pH 3 and pH 6, respec‐

Moreover, in recent studies of amyloid fibril with AFM, some of scientists have reported about various structural features of amyloid fibrils such as steric zipper pattern [76, 77], helical pattern [48], and length [78], which is a key design parameter that determines the material properties of amyloid fibrils. Nevertheless, it is not quite understood yet how the molecular structures and conformational diversity (heterogeneous conformations) of amyloid fibrils are

To figure out how amyloid fibrils with heterogeneous conformations are formed, the synthesis of the β-lg fibrils with microwave-assisted chemistry method is considered, which enables the acceleration of biochemical reactions, and performed KPFM analysis of them [63]. It has been reported already that the microwave-assisted chemistry for the synthesis of amyloid fibrils makes the acceleration of fibrillation possible. The mechanism of amyloid fibrillation under microwave irradiation is still debated, but it seems to be related to the enhanced collision

tively. All scale bars are 250 nm. Figures reproduced with permission from Ref. [20], © 2012 AIP.

regulated.

11618 Exploring New Findings on Amyloidosis

**Figure 12** represents the topographic and surface potential images of amyloid fibrils that were synthesized based on microwave-assisted chemistry and their conformational heterogeneities. From these results, it is obvious that except for *λ* = 60 s, the surface potential and the fibril diameter of β-lg amyloid fibrils increase in monotone fashion as *λ* increases. This result revealed that heterogeneous conformations of β-lg amyloid fibrils are due to the wave-driven change of electrostatic interaction, which has a critical impact on the radial growth mechanisms of amyloid fibrils. As described above, KPFM indeed was a good analytical tool for monitoring the conformational change and the electrical properties of amyloid fibrils with respect to microwave-assisted chemistry.

**Figure 12.** (a) AFM images of amyloid fibrils that are synthesized based on microwave-assisted chemistry using differ‐ ent *λ* values. (b) KPFM images of the fibrils formed using different *λ* values. (c) Distribution of surface charges for amyloid fibril as a function of its diameter. The surface potential of an amyloid fibril is almost linearly proportional to its diameter. Figures reprinted with permission from Ref. [63], © 2015 NPG.

## **5. Conclusion and perspectives**

As we surveyed above, because of the marvelous capabilities of the conventional AFM device, it and its extensions as liquid AFM, PeakForce QNM, and KPFM have been widely used to perform accurate measurements of mechanical, electrical, and biophysical properties of various amyloid fibrils. AFM applications can provide researchers with the strong advantage of being able to measure the surface potentials of nanomaterials at the single-molecule level. Moreover, it has been demonstrated that AFM applications enable monitoring the fibrillation behavior of various proteins by measuring morphological properties of amyloid fibrils in air or liquid environments. The changes in morphology of the fibrils indicated that amyloid fibrils can be changed morphologically in liquid environments (e.g., human body) and that implies amyloid fibrils would interact differently with other biomolecules, cells, and tissues in human body. Liquid AFM enables measurement of the characteristics of amyloid fibrils in situ by monitoring the fibrillation and conformation changes of amyloid fibrils. Furthermore, AFM application techniques usually require no labeling with chemical dyes and no chemical reagents for improving the image quality or contrast. Accordingly, AFM is able to get highresolution images of amyloid fibrils for the detection of conformational changes, by topological imaging in air and liquid environments. We dealt with PeakForce QNM that can simultane‐ ously measure several mechanical properties of amyloid fibrils such as Young's modulus, deformation, and adhesion force by using a precisely operating oscillating probe. With a Kelvin probe, KPFM is highly important for imaging and characterizing the electrical properties (i.e., surface potential and surface potential density of amyloid fibrils) while getting high-resolution topological imaging. Because of these advantages, AFM application is expected to attract much attention as an analytical tool for amyloid research in the future. These AFM-related results shed light on understanding the fibrillation of amyloidogenic proteins and will be the cornerstone for the development of not only the effective prevention methods for amyloido‐ genic diseases but also therapeutic agents and drugs. These approaches could also contribute to the development of amyloid fibril-based functional nanomaterials such as biocompatible adhesives [80, 81], solar-cell devices [82], and gene delivery systems [83].

## **Author details**

Wonseok Lee1 , Hyungbeen Lee1 , Gyudo Lee2 and Dae Sung Yoon3\*

\*Address all correspondence to: dsyoon@korea.ac.kr


## **References**

**5. Conclusion and perspectives**

11820 Exploring New Findings on Amyloidosis

As we surveyed above, because of the marvelous capabilities of the conventional AFM device, it and its extensions as liquid AFM, PeakForce QNM, and KPFM have been widely used to perform accurate measurements of mechanical, electrical, and biophysical properties of various amyloid fibrils. AFM applications can provide researchers with the strong advantage of being able to measure the surface potentials of nanomaterials at the single-molecule level. Moreover, it has been demonstrated that AFM applications enable monitoring the fibrillation behavior of various proteins by measuring morphological properties of amyloid fibrils in air or liquid environments. The changes in morphology of the fibrils indicated that amyloid fibrils can be changed morphologically in liquid environments (e.g., human body) and that implies amyloid fibrils would interact differently with other biomolecules, cells, and tissues in human body. Liquid AFM enables measurement of the characteristics of amyloid fibrils in situ by monitoring the fibrillation and conformation changes of amyloid fibrils. Furthermore, AFM application techniques usually require no labeling with chemical dyes and no chemical reagents for improving the image quality or contrast. Accordingly, AFM is able to get highresolution images of amyloid fibrils for the detection of conformational changes, by topological imaging in air and liquid environments. We dealt with PeakForce QNM that can simultane‐ ously measure several mechanical properties of amyloid fibrils such as Young's modulus, deformation, and adhesion force by using a precisely operating oscillating probe. With a Kelvin probe, KPFM is highly important for imaging and characterizing the electrical properties (i.e., surface potential and surface potential density of amyloid fibrils) while getting high-resolution topological imaging. Because of these advantages, AFM application is expected to attract much attention as an analytical tool for amyloid research in the future. These AFM-related results shed light on understanding the fibrillation of amyloidogenic proteins and will be the cornerstone for the development of not only the effective prevention methods for amyloido‐ genic diseases but also therapeutic agents and drugs. These approaches could also contribute to the development of amyloid fibril-based functional nanomaterials such as biocompatible

adhesives [80, 81], solar-cell devices [82], and gene delivery systems [83].

, Gyudo Lee2

1 Department of Biomedical Engineering, Yonsei University, Wonju, Korea

2 School of Public Health, Harvard University, Boston, Massachusetts, USA

3 Department of Bio-convergence Engineering, Korea University, Seoul, Korea

and Dae Sung Yoon3\*

**Author details**

, Hyungbeen Lee1

\*Address all correspondence to: dsyoon@korea.ac.kr

Wonseok Lee1


[30] Muller DJ, Dufrene YF. Atomic force microscopy as a multifunctional molecular toolbox in nanobiotechnology. Nat Nanotechnol. 2008;3(5):261–9.

[16] Jeong JS, Ansaloni A, Mezzenga R, Lashuel HA, Dietler G. Novel mechanistic insight into the molecular basis of amyloid polymorphism and secondary nucleation during

[17] Adamcik J, Berquand A, Mezzenga R. Single-step direct measurement of amyloid fibrils stiffness by peak force quantitative nanomechanical atomic force microscopy. Appl

[18] Sweers K, van der Werf K, Bennink M, Subramaniam V. Nanomechanical properties of α-synuclein amyloid fibrils: a comparative study by nanoindentation, harmonic

[19] Adamcik J, Lara C, Usov I, Jeong JS, Ruggeri FS, Dietler G, et al. Measurement of intrinsic properties of amyloid fibrils by the peak force QNM method. Nanoscale.

[20] Lee G, Lee W, Lee H, Lee SW, Yoon DS, Eom K, et al. Mapping the surface charge

[21] Ruggeri FS, Adamcik J, Jeong JS, Lashuel HA, Mezzenga R, Dietler G. Influence of the β-sheet content on the mechanical properties of aggregates during amyloid fibrilliza‐

[22] Lamour G, Yip CK, Li H, Gsponer J. High intrinsic mechanical flexibility of mouse prion nanofibrils revealed by measurements of axial and radial Young's moduli. ACS Nano.

[23] Lee W, Jung H, Son M, Lee H, Kwak TJ, Lee G, et al. Characterization of the regrowth behavior of amyloid-like fragmented fibrils decomposed by ultrasonic treatment. RSC

[24] Jaroniec CP, MacPhee CE, Bajaj VS, McMahon MT, Dobson CM, Griffin RG. Highresolution molecular structure of a peptide in an amyloid fibril determined by magic angle spinning NMR spectroscopy. Proc Natl Acad Sci U S A. 2004;101(3):711–6. [25] Meersman F, Cabrera RQ, McMillan PF, Dmitriev V. Compressibility of insulin amyloid fibrils determined by X-ray diffraction in a diamond anvil cell. High Pressure Res.

[26] Westermark P, Benson MD, Buxbaum JN, Cohen AS, Frangione B, Ikeda S, et al.

[27] Khurana R, Coleman C, Ionescu-Zanetti C, Carter SA, Krishna V, Grover RK, et al. Mechanism of thioflavin T binding to amyloid fibrils. J Struct Biol. 2005;151(3):229–38.

[29] Binnig G, Quate CF, Gerber C. Atomic force microscope. Phys Rev Lett. 1986;56(9):930.

[28] Giessibl FJ. Advances in atomic force microscopy. Rev Mod Phys. 2003;75(3):949.

Amyloid fibril protein nomenclature—2002. Amyloid. 2002;9(3):197.

distribution of amyloid fibril. Appl Phys Lett. 2012;101(4):043703–4.

tion. Angew Chem Int Ed. 2015;54(8):2462–6.

force microscopy, and Peakforce QNM. Nanoscale Res Lett. 2011;6(1):1–10.

amyloid formation. J Mol Biol. 2013;425(10):1765–81.

Phys Lett. 2011;98(19):193701.

2012;4(15):4426–9.

12022 Exploring New Findings on Amyloidosis

2014;8(4):3851–61.

2009;29(4):665–70.

Adv 2014;4(100):56561–6.


[56] Bokvist M, Lindström F, Watts A, Gröbner G. Two types of Alzheimer's β-amyloid (1– 40) peptide membrane interactions: aggregation preventing transmembrane anchoring versus accelerated surface fibril formation. J Mol Biol. 2004;335(4):1039–49.

[43] Pfreundschuh M, Martinez-Martin D, Mulvihill E, Wegmann S, Muller DJ. Multipara‐ metric high-resolution imaging of native proteins by force-distance curve-based AFM.

[44] Ferencz R, Sanchez J, Blümich B, Herrmann W. AFM nanoindentation to determine Young's modulus for different EPDM elastomers. Polym Test. 2012;31(3):425–32. [45] Foster B. New atomic force microscopy (AFM) approaches life sciences gently, quan‐

[46] Trtik P, Kaufmann J, Volz U. On the use of peak-force tapping atomic force microscopy for quantification of the local elastic modulus in hardened cement paste. Cem Concr

[47] Lee G, Lee H, Nam K, Han J-H, Yang J, Lee SW, et al. Nanomechanical characterization of chemical interaction between gold nanoparticles and chemical functional groups.

[48] Knowles TP, Fitzpatrick AW, Meehan S, Mott HR, Vendruscolo M, Dobson CM, et al. Role of intermolecular forces in defining material properties of protein nanofibrils.

[49] Strohman R. Maneuvering in the complex path from genotype to phenotype. Science.

[50] Verdile G, Fuller S, Atwood CS, Laws SM, Gandy SE, Martins RN. The role of beta amyloid in Alzheimer's disease: still a cause of everything or the only one who got

[51] Drolle E, Hane F, Lee B, Leonenko Z. Atomic force microscopy to study molecular mechanisms of amyloid fibril formation and toxicity in Alzheimer's disease. Drug

[52] Bucciantini M, Giannoni E, Chiti F, Baroni F, Formigli L, Zurdo J, et al. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature.

[53] Kayed R, Head E, Thompson JL, McIntire TM, Milton SC, Cotman CW, et al. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis.

[54] Glabe CG, Kayed R. Common structure and toxic function of amyloid oligomers implies a common mechanism of pathogenesis. Neurology. 2006;66(1 suppl 1):S74–S8.

[55] Hertel C, Terzi E, Hauser N, Jakob-Røtne R, Seelig J, Kemp J. Inhibition of the electro‐ static interaction between β-amyloid peptide and membranes prevents β-amyloid-

induced toxicity. Proc Natl Acad Sci. 1997;94(17):9412–6.

Nat Protoc. 2014;9(5):1113–30.

12224 Exploring New Findings on Amyloidosis

Res. 2012;42(1):215–21.

Nanoscale Res Lett. 2012;7(1):1–11.

Science. 2007;318(5858):1900–3.

Metab Rev. 2014;46(2):207–23.

Science. 2003;300(5618):486–9.

2002;416(6880):507–11.

caught? Pharmacol Res. 2004;50(4):397–409.

2002;296(5568):701–3.

titatively, and correlatively. Am Lab 2012(4):24–8.


## **Effects of Amyloid-β Deposition on Mitochondrial Complex I Activity in Brain: A PET Study in Monkeys**

Hideo Tsukada

[71] Lin Y-C, Yu B-Y, Lin W-C, Lee S-H, Kuo C-H, Shyue J-J. Tailoring the surface potential of gold nanoparticles with self-assembled monolayers with mixed functional groups.

[72] Conklin D, Nanayakkara S, Park T-H, Lagadec MF, Stecher JT, Therien MJ, et al. Electronic transport in porphyrin supermolecule-gold nanoparticle assemblies. Nano

[73] Stehlik S, Petit T, Girard HA, Kromka A, Arnault J-C, Rezek B. Surface potential of diamond and gold nanoparticles can be locally switched by surrounding materials or

[74] Gutiérrez-Sánchez C, Pita M, Vaz-Domínguez C, Shleev S, De Lacey AL. Gold nano‐ particles as electronic bridges for laccase-based biocathodes. J Am Chem Soc.

[75] Raschke G, Kowarik S, Franzl T, Sönnichsen C, Klar T, Feldmann J, et al. Biomolecular recognition based on single gold nanoparticle light scattering. Nano Lett. 2003;3(7):935–

[76] Yoon G, Lee M, Kim JI, Na S, Eom K. Role of sequence and structural polymorphism on the mechanical properties of amyloid fibrils. PLoS one. 2014;9(2):e88502.

[77] Sawaya MR, Sambashivan S, Nelson R, Ivanova MI, Sievers SA, Apostol MI, et al. Atomic structures of amyloid cross-β spines reveal varied steric zippers. Nature.

[78] Choi B, Yoon G, Lee SW, Eom K. Mechanical deformation mechanisms and properties

[79] Hettiarachchi CA, Melton LD, Gerrard JA, Loveday SM. Formation of β-lactoglobulin nanofibrils by microwave heating gives a peptide composition different from conven‐

[80] Mankar S, Anoop A, Sen S, Maji SK. Nanomaterials: amyloids reflect their brighter side.

[81] Mostaert AS, Higgins MJ, Fukuma T, Rindi F, Jarvis SP. Nanoscale mechanical characterisation of amyloid fibrils discovered in a natural adhesive. J Biol Phys.

[82] Bolisetty S, Adamcik J, Heier J, Mezzenga R. Amyloid directed synthesis of titanium dioxide nanowires and their applications in hybrid photovoltaic devices. Adv Funct

[83] Yolamanova M, Meier C, Shaytan AK, Vas V, Bertoncini CW, Arnold F, et al. Peptide nanofibrils boost retroviral gene transfer and provide a rapid means for concentrating

of amyloid fibrils. Phys Chem Chem Phys. 2015;17(2):1379–89.

tional heating. Biomacromolecules. 2012;13(9):2868–80.

J Colloid Interface Sci. 2009;340(1):126–30.

applied voltage. J Nanopart Res. 2014;16(4):1–11.

Lett. 2012;12(5):2414–9.

12426 Exploring New Findings on Amyloidosis

2012;134(41):17212–20.

2007;447(7143):453–7.

Nano Rev 2011;2.

2006;32(5):393–401.

Mater. 2012;22(16):3424–28.

viruses. Nat Nanotechnol. 2013;8(2):130–6.

8.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62901

## **Abstract**

This chapter discusses the capabilities of positron emission tomography (PET) imaging for the diagnosis of Alzheimer's disease (AD) with deposition of amyloid-β (Aβ). We conducted a PET scan using 18F-2-tert-butyl-4-chloro-5-{6-[2-(2-fluoroethoxy)-ethoxy] pyridin-3-ylmethoxy}-2H-pyridazin-3-one (18F-BCPP-EF), a novel PET probe for mitochondrial complex I (MC-I) activity, in young and aged monkeys to demonstrate the normal aging effects on MC-I activity in the brain. The results revealed an age-related impairment of MC-I activity in the brain. Then, we conducted PET scan using 11C-PIB to detect the Aβ deposition in the some parts, not all, of the brains of some part of aged monkeys. For further assessments, PET scans using <sup>11</sup>C-PIB for Aβ, 11C-DPA-713 for inflammation, 18F-fluoro-2-deoxy-D-glucose (18F-FDG) for regional cerebral metabolic rate of glucose (rCMRglc), and 18F-BCPP-EF for MC-I were performed in aged animals. When 18F-BCPP-EF uptake is plotted against 11C-PIB uptake in the cerebral cortical regions, it showed a significant negative correlation between them. Plotting of 11C-DPA-713 uptake against 11C-PIB resulted in a significant positive correlation. In contrast, plotting of rCMRglc against 11C-PIB did not reach a statistically significant level. Taken together, these results strongly suggested that 18F-BCPP-EF could discriminate the neuronally damaged areas with neuroinflammation where 18F-FDG could not, owing to its high uptake into the activated microglia.

**Keywords:** Alzheimer's disease, aging, brain, mitochondrial complex I, PET

## **1. Introduction**

Alzheimer's disease (AD) is neuropathologically characterized by the presence of neurofibril‐ lary tangles with the deposition of hyperphosphorylated tau protein inside nerve cells and

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

senile plaques with extracellular aggregation of amyloid-β (Aβ) protein, and the imaging of tau and Aβ is expected to provide quantitative information noninvasively for the diagnosis of AD. Pathological aging processes are thought to be a degenerative process caused by accumulated damages, which leads to cellular dysfunction, tissue failure, and death, result‐ ing in detectable changes in brain structure and function. Modern *in vivo* imaging techni‐ ques, such as X-ray computed tomography (X-CT), magnetic resonance imaging (MRI), and positron emission tomography (PET), provide useful ways to examine these alterations and to separate normal age-related changes from pathological states. Because functional distur‐ bances precede structural changes determined by X-CT or MRI, the *in vivo* imaging obtained with PET may be abnormal, whereas the brain anatomy appears normal.

One trend is to apply target-specific PET probes for quantitative imaging of the specific neurological target molecules related to diseases, such as the dopaminergic system for schizophrenia [1] and Parkinson's disease (PD) [2], the serotonergic system for depression [3], and the cholinergic system forAD-type dementia [4]. Anothertrend is to measure more general indices for the pathophysiological abnormalities in diseases. To diagnose these diseases and to assess the treatment efficacy of developing drug candidates noninvasively with PET, we have anticipatedPETprobes that canprovide general indices of brain function, such as regional cerebral blood flow (rCBF) and regional cerebral metabolic rate of oxygen (rCMRO2), both of which have been recognized to be the gold standard indices for cerebral functions [5]. Other useful indicators are the regional cerebral metabolic rate of glucose (rCMRglc) assessed with <sup>18</sup>F-fluoro-2-deoxy-D-glucose (<sup>18</sup>F-FDG; **Figure 1C**) [6]. PET using <sup>18</sup>F-FDG is a well-establish‐ ed technique for the quantitative imaging of brain function in the living brain. Some re‐ search demonstrated that <sup>18</sup>F-FDG could predict an onset of AD. However, as demonstrated in the brains of rat and monkey ischemic models [7, 8], the unexpectedly high uptake of 18F-FDG in damaged areas suggested that <sup>18</sup>F-FDG was taken up into not only normal tissues but also inflammatory regions with microglial activation, which hampers the accurate diagnosis of brain function using <sup>18</sup>F-FDG.

**Figure 1.** PET probes used: (A) <sup>11</sup>C-PIB for Aβ, (B) <sup>11</sup>C-DPA-713 for TSPO, (C) <sup>18</sup>F-FDG for rCMRglc, (D) <sup>11</sup>C-(+)-3-MPB for mAChR, (E) <sup>11</sup>C-(*R*)-MeQAA for α7-nAChR, and (F) <sup>18</sup>F-BCPP-EF for MC-I.

Several hypotheses of aging at the molecular level, such as shortening of telomerase, DNA methylation, reactive oxygen species (ROS) generation, and mitochondrial abnormalities, have been proposed. Among these, the "mitochondrial free radical theory of aging" has been highlighted [9]. In mammalian cells, the electron transport chain in mitochondria consists of five complexes from I to V, and the main role of mitochondria was recognized to be energy transduction of ATP. Their dysfunction was thought to be limited to ATP deficiency result‐ ing in necrotic cell death; however, the mechanisms of cell death have been found to include mitochondrial contribution to oxidative stress and apoptosis [10]. Previous studies demon‐ strated an age-related increase in ROS production using rat brain homogenates [11], cortical slices [12], or synaptosomes [13]. Furthermore, mitochondrial dysfunction contributes to the pathophysiology of acute and chronic neurodegenerative disorders [14].

In the present chapter, the capability of assessment of mitochondrial complex I (MC-I) activity is introduced for detection of neurodegenerative damage associated with Aβ deposition. We recently developed and evaluated 18F-2-tert-butyl-4-chloro-5-{6-[2-(2-fluoroethoxy)-ethoxy] pyridin-3-ylmethoxy}-2H-pyridazin-3-one (18F-BCPP-EF), a novel PET probe for MC-I activity (**Figure 1E**) [15]. The translational research has been conducted using an animal PET to assess the aging as well as Aβ deposition effects on MC-I activity in the living brains of young (3–5 years old, corresponding to high-teens in humans) and aged (20–24 years old, corresponding to 75 years old and more) male monkeys [16, 17].

## **2. Aging and cholinergic neuronal system**

senile plaques with extracellular aggregation of amyloid-β (Aβ) protein, and the imaging of tau and Aβ is expected to provide quantitative information noninvasively for the diagnosis of AD. Pathological aging processes are thought to be a degenerative process caused by accumulated damages, which leads to cellular dysfunction, tissue failure, and death, result‐ ing in detectable changes in brain structure and function. Modern *in vivo* imaging techni‐ ques, such as X-ray computed tomography (X-CT), magnetic resonance imaging (MRI), and positron emission tomography (PET), provide useful ways to examine these alterations and to separate normal age-related changes from pathological states. Because functional distur‐ bances precede structural changes determined by X-CT or MRI, the *in vivo* imaging obtained

One trend is to apply target-specific PET probes for quantitative imaging of the specific neurological target molecules related to diseases, such as the dopaminergic system for schizophrenia [1] and Parkinson's disease (PD) [2], the serotonergic system for depression [3], and the cholinergic system forAD-type dementia [4]. Anothertrend is to measure more general indices for the pathophysiological abnormalities in diseases. To diagnose these diseases and to assess the treatment efficacy of developing drug candidates noninvasively with PET, we have anticipatedPETprobes that canprovide general indices of brain function, such as regional cerebral blood flow (rCBF) and regional cerebral metabolic rate of oxygen (rCMRO2), both of which have been recognized to be the gold standard indices for cerebral functions [5]. Other useful indicators are the regional cerebral metabolic rate of glucose (rCMRglc) assessed with <sup>18</sup>F-fluoro-2-deoxy-D-glucose (<sup>18</sup>F-FDG; **Figure 1C**) [6]. PET using <sup>18</sup>F-FDG is a well-establish‐ ed technique for the quantitative imaging of brain function in the living brain. Some re‐ search demonstrated that <sup>18</sup>F-FDG could predict an onset of AD. However, as demonstrated in the brains of rat and monkey ischemic models [7, 8], the unexpectedly high uptake of 18F-FDG in damaged areas suggested that <sup>18</sup>F-FDG was taken up into not only normal tissues but also inflammatory regions with microglial activation, which hampers the accurate diagnosis

**Figure 1.** PET probes used: (A) <sup>11</sup>C-PIB for Aβ, (B) <sup>11</sup>C-DPA-713 for TSPO, (C) <sup>18</sup>F-FDG for rCMRglc, (D) <sup>11</sup>C-(+)-3-MPB

for mAChR, (E) <sup>11</sup>C-(*R*)-MeQAA for α7-nAChR, and (F) <sup>18</sup>F-BCPP-EF for MC-I.

with PET may be abnormal, whereas the brain anatomy appears normal.

of brain function using <sup>18</sup>F-FDG.

126 128Exploring New Findings on Amyloidosis

There have been a number of reports on age-related alterations in neurochemical and neurophysiological functions in the brain, associated with changes in the neurotransmitter synthesis in presynaptic neurons, release into synaptic cleft, reuptake availability, binding to receptors, and signal transduction, all of which are related to the declines of specific motor, cognitive and emotional functions in primates. Among the variety of neurotransmitter receptors, we focused on the effects of aging process on cholinergic receptors, which had previously assessed in the postmortem primate brain tissues [18].

## **2.1. Aging effects on muscarinic cholinergic receptor function**

The cholinergic receptor (AChR) population is divided into muscarinic (mAChR) and nicotinic (nAChR) subclasses in the central nervous system (CNS), and the CNS mAChR system plays an important role in memory and cognitive functions. AD is neuropathological‐ ly characterized by the presence of neurofibrillary tangles with the deposition of Hyperphos‐ phorylated tau protein inside nerve cells and senile plaques with extracellular aggregation of Aβ protein [19, 20]. Accompanied with tau and Aβ depositions, loss of cholinergic neurons in the forebrain, reduced cholinergic activity in the hippocampus and cortical loss of choline acetyltransferase, and reduced central mAChR binding have been observed in the brain of AD patients [21]. The severity of these cholinergic abnormalities is closely correlated with the degree of memory impairment in aged monkeys [22] and dementia patients [21].

Several antagonist-based 11C-labeled PET probes for imaging mAChR have been developed and attempted to determine quantitatively the age-related alterations of mAChR in the living brain using 11C-benztropine [23], *N*-11C-methyl-4-piperidyl benzilate (11C-4-MPB) [24], and 11Ctropanyl benzilate (TBZ) [25]. These PET probes for mAChR, however, showed relatively low uptake to the brain and also slow dissociation rates from mAChR, which may limit the estimation of the density of binding sites *in vivo* [26]. To solve these problems, we proposed a novel mAChR probe, *N*-11C-methyl-3-piperidyl benzilate (11C-3-MPB) (**Figure 1D**) [27, 28], and assessed the aging effects on mAChR binding in comparison to young and aged monkeys [29].

11C-(+)3-MPB was labeled by *N*-methylation of respective nor-compound with 11C-methyl iodide converted from 11C-CO2 by LiAlH4 reduction followed by reaction with HI [27, 28]. A monkey (*Macaca mulatta*) was seated on a monkey chair under conscious condition and fixed with stereotactic coordinates under conscious state [30]. For the kinetic analysis of 11C-(+)3- MPB binding, arterial blood sampling was conducted to determine the input function using metabolic profile in plasma. The PET data obtained were reconstructed by the filtered backprojection (FBP) method. Volumes of interest (VOIs) in brain regions were drawn manually on the MRI, and VOIs of MRI were superimposed on the coregistered PET images to meas‐ ure the time activity curves (TACs) of each PET probe for kinetic analyses using Logan graphical analysis with metabolite-corrected plasma input in the living brain [31].

The TACs of 11C-(+)3-MPB in the frontal, temporal, and occipital cortices reached their peaks 40 min after injection, whereas the striatal and hippocampal regions reached peak values 60

**Figure 2.** MRI and PET images of 11C-(+)3-MPB in the young and aged monkey brains (*M. mulatta*). PET data were col‐ lected in the conscious state with a high-resolution PET scanner. Each PET image was generated by summation of im‐ age data from 60 to 91 min post-injection. The stereotactic coordinates of PET and MRI were adjusted based on the orbitomeatal (OM) line.

min after injection [28]. In aged monkeys, the TACs of 11C-(+)3-MPB in regions rich in mAChR peaked at earlier time points, with faster elimination rates than those in young monkeys. As a result of Logan graphical analysis using metabolite-corrected plasma input, significant agerelated alterations of the *in vivo* binding of 11C-(+)3-MPB were observed in the temporal and frontal cortices and the striatum (**Figure 2**). Aged animals showed the age-related reduction of the maximum number of binding sites (*B*max) of mAChR, whereas there were no age-related alterations of the affinity (1/Kd) of mAChR [29].

## **2.2. Aging effects on nicotinic cholinergic receptor function**

Several antagonist-based 11C-labeled PET probes for imaging mAChR have been developed and attempted to determine quantitatively the age-related alterations of mAChR in the living

tropanyl benzilate (TBZ) [25]. These PET probes for mAChR, however, showed relatively low uptake to the brain and also slow dissociation rates from mAChR, which may limit the estimation of the density of binding sites *in vivo* [26]. To solve these problems, we proposed a

assessed the aging effects on mAChR binding in comparison to young and aged monkeys [29]. 11C-(+)3-MPB was labeled by *N*-methylation of respective nor-compound with 11C-methyl iodide converted from 11C-CO2 by LiAlH4 reduction followed by reaction with HI [27, 28]. A monkey (*Macaca mulatta*) was seated on a monkey chair under conscious condition and fixed with stereotactic coordinates under conscious state [30]. For the kinetic analysis of 11C-(+)3- MPB binding, arterial blood sampling was conducted to determine the input function using metabolic profile in plasma. The PET data obtained were reconstructed by the filtered backprojection (FBP) method. Volumes of interest (VOIs) in brain regions were drawn manually on the MRI, and VOIs of MRI were superimposed on the coregistered PET images to meas‐ ure the time activity curves (TACs) of each PET probe for kinetic analyses using Logan

graphical analysis with metabolite-corrected plasma input in the living brain [31].

The TACs of 11C-(+)3-MPB in the frontal, temporal, and occipital cortices reached their peaks 40 min after injection, whereas the striatal and hippocampal regions reached peak values 60

**Figure 2.** MRI and PET images of 11C-(+)3-MPB in the young and aged monkey brains (*M. mulatta*). PET data were col‐ lected in the conscious state with a high-resolution PET scanner. Each PET image was generated by summation of im‐ age data from 60 to 91 min post-injection. The stereotactic coordinates of PET and MRI were adjusted based on the

11C-methyl-4-piperidyl benzilate (11C-4-MPB) [24], and 11C-

11C-methyl-3-piperidyl benzilate (11C-3-MPB) (**Figure 1D**) [27, 28], and

brain using 11C-benztropine [23], *N*-

novel mAChR probe, *N*-

128 130Exploring New Findings on Amyloidosis

orbitomeatal (OM) line.

The CNSAChRsystems, classified into mAChRand nAChR, play an importantrole in memory and cognitive functions. In the last two decades, 17 different nAChR subunits (α1–α10, β1– β4,γ, δ and ε) have been cloned, and the prominent nAChRs are the α4β2 heteromeric and α7 homomeric subtypes in the brain [32]. Among them, because α7 has high permeability for Ca2+, it can be assumed that, in addition to the ionotropic function induced by membrane depola‐ rization, α7-nAChR is associated with metabotropic activity coupled to Ca2+-regulated secondmessenger signaling required for the modulation of neuron excitability, neurotransmitter release, induction of long-term potentiation (LTP), and cognitive-associated processing of learning and memory. In addition, α7-nAChR may contribute to neuroprotection by modu‐ lating the neurotrophic system crucial for the maintenance of cholinergic neuron integrity and also by stimulating signal transduction pathways that support neuron survival [32].

A PET study indicated that decreases in 11C-nicotine brain uptake were significantly correlat‐ ed with cognitive deficits in AD patients [33]; however, nAChR deficits in the different types of dementia are assumed to be reflected by subtype and region specificity. For the noninva‐ sive imaging of α7-nAChR with more subtype specificity, several 11C-labeled PET probes have been developed and evaluated, including 11C-CHIBA-1001, 11C-A-582941, and <sup>11</sup>C-A-844606 [34]. However, because of low brain uptake, high nonspecific binding, and/or low selectivity to α7-nAChR against 5-HT3R, almost none of these PET probes have demonstrated clinically useful specific binding to α7-nAChR in nonhuman primate brain. Because we recently developed novel PET probes for α7-nAChR, (*R*)-2-[11C]methylamino-benzoic acid 1-azabicycle[2.2.2]oct-3-yl ester [11C-(*R*)-MeQAA; **Figure 1E**] [35], the aging effects on α7-nAChR binding was assessed using 11C-(*R*)-MeQAA in comparison to young and aged monkeys [36].

11C-(*R*)-MeQAA was labeled by *N*-methylation of respective nor-compounds [(*R*)-BH3QAA] with 11C-methyl triflate prepared from 11C-methyl iodide through a glass column containing silver triflate [35]. PET measurements of conscious monkeys were conducted as described in Section 2.2 with arterial blood sampling for plasma metabolic analysis followed by PET image reconstruction and TAC acquisition in the VOIs for kinetic analyses. The values of nondis‐ placeable binding potential (BPND) were evaluated by two-compartment models (2-TC) analysis using the metabolite-corrected plasma input [37] and simplified reference tissue model (SRTM) analysis using the TAC in the cerebellum as an indirect input function [38].

11C-(*R*)-MeQAA images in the brain of young normal monkeys, showing high and heteroge‐ neous uptake of [11C](*R*)-MeQAA into the brain, were determined between 60 and 90 min after the bolus injection. The uptake of radioactivity was high in the thalamus and striatum,

intermediate in the hippocampus, frontal, temporal, and occipital cortices, and low in the cerebellum [35, 36]. The uptake of 11C-(*R*)-MeQAA was significantly higherthan that of 11C-(*S*)- MeQAA in the thalamus, hippocampus, and cortical regions, and the specific binding of 11C- (*R*)-MeQAA was inhibited by the preadministration of SSR180711, an α7-nAChR partial agonist [35]. In contrast, the uptake of 11C-(*R*)-MeQAA into the cerebellum of monkeys was not affected by SSR180711 [35], suggesting that the cerebellum could be applicable for the reference region for the quantitative analysis of 11C-(*R*)-MeQAA binding in the living brain. The clearance rate of 11C-(*R*)-MeQAA in plasma was rapid and relatively stable in plasma. To determine the practicality of the simplified analytical method of 11C-(*R*)-MeQAA with SRTM to calculate the BPND using the TAC in the cerebellum as an indirect input function, we verified it by correlation analyses with 2-TC-BPND values calculated using the metabolite-corrected plasma input. Because of a good correlation between 2-TC and SRTM analyses, 11C-(*R*)- MeQAA binding to α7-nAChRin young and aged monkey brain was determined as the SRTM-BPND values without arterial blood sampling to avoid excessive stress on aged animals. When determining the aging effects on 11C-(*R*)-MeQAA binding, all regions, except the occipital cortex, revealed no significant differences in the BPND values of 11C-(*R*)-MeQAA in the brains of aged and young animals [36]. Although human aging is known to preferentially affect the rCMRglc in the frontal and temporal lobes, our previous studies reported that the occipital cortex showed the most profound reductions of CBF [39], rCMRglc [39], and MC-I [16] in aged monkeys. Because the differences in cortical activity between monkey and human reflect the evolutionary significance of their frontal cortex, the most marked age-related alterations of α7-nAChR activity would be determined in regions such as the frontal cortex, not in the occipital cortex, in humans. These results apparently revealed that aging effects were much less on α7-nAChR compared to mAChR in the living brain.

## **3. Aging and MC-I activity**

Mitochondria are called "cellular power plants" because they generate most of the ATP used as a source of chemical energy. In mammalian cells, the electron transport chain in mitochon‐ dria consists of five complexes from I to V, and complex I (MC-I; NADH-ubiquinone oxidor‐ eductase, EC 1.6.5.3) is the first and rate-limiting step of the overall respiratory activity and oxidative phosphorylation under physiological conditions. Glucose is converted to pyruvate followed by transformation into acetyl-CoA by pyruvate dehydrogenase (PDH) in the mitochondria, which is subsequently fed into the tricarboxylic acid (TCA) cycle, ultimately producing ATP via the electron transport system and oxidative phosphorylation (which is indispensable for cell survival). Mitochondrial dysfunction contributes to the pathophysiolo‐ gy of neurodegenerative diseases [40], some part of which has been considered to relate to the fact that mitochondria are the main intracellular source of ROS in cells and also the main target of ROS-mediated damage. Noninvasive assessment of living brain could be useful for the diagnostic, prognostic, and treatment monitoring of neurodegenerative diseases related to impaired MC-I function; however, no proper PET probes for MC-I imaging in the brain have been developed prior to those that we have recently described [15].

## **3.1. Development of PET probe for MC-I imaging**

intermediate in the hippocampus, frontal, temporal, and occipital cortices, and low in the cerebellum [35, 36]. The uptake of 11C-(*R*)-MeQAA was significantly higherthan that of 11C-(*S*)- MeQAA in the thalamus, hippocampus, and cortical regions, and the specific binding of 11C- (*R*)-MeQAA was inhibited by the preadministration of SSR180711, an α7-nAChR partial agonist [35]. In contrast, the uptake of 11C-(*R*)-MeQAA into the cerebellum of monkeys was not affected by SSR180711 [35], suggesting that the cerebellum could be applicable for the reference region for the quantitative analysis of 11C-(*R*)-MeQAA binding in the living brain. The clearance rate of 11C-(*R*)-MeQAA in plasma was rapid and relatively stable in plasma. To determine the practicality of the simplified analytical method of 11C-(*R*)-MeQAA with SRTM to calculate the BPND using the TAC in the cerebellum as an indirect input function, we verified it by correlation analyses with 2-TC-BPND values calculated using the metabolite-corrected plasma input. Because of a good correlation between 2-TC and SRTM analyses, 11C-(*R*)- MeQAA binding to α7-nAChRin young and aged monkey brain was determined as the SRTM-BPND values without arterial blood sampling to avoid excessive stress on aged animals. When determining the aging effects on 11C-(*R*)-MeQAA binding, all regions, except the occipital cortex, revealed no significant differences in the BPND values of 11C-(*R*)-MeQAA in the brains of aged and young animals [36]. Although human aging is known to preferentially affect the rCMRglc in the frontal and temporal lobes, our previous studies reported that the occipital cortex showed the most profound reductions of CBF [39], rCMRglc [39], and MC-I [16] in aged monkeys. Because the differences in cortical activity between monkey and human reflect the evolutionary significance of their frontal cortex, the most marked age-related alterations of α7-nAChR activity would be determined in regions such as the frontal cortex, not in the occipital cortex, in humans. These results apparently revealed that aging effects were much

Mitochondria are called "cellular power plants" because they generate most of the ATP used as a source of chemical energy. In mammalian cells, the electron transport chain in mitochon‐ dria consists of five complexes from I to V, and complex I (MC-I; NADH-ubiquinone oxidor‐ eductase, EC 1.6.5.3) is the first and rate-limiting step of the overall respiratory activity and oxidative phosphorylation under physiological conditions. Glucose is converted to pyruvate followed by transformation into acetyl-CoA by pyruvate dehydrogenase (PDH) in the mitochondria, which is subsequently fed into the tricarboxylic acid (TCA) cycle, ultimately producing ATP via the electron transport system and oxidative phosphorylation (which is indispensable for cell survival). Mitochondrial dysfunction contributes to the pathophysiolo‐ gy of neurodegenerative diseases [40], some part of which has been considered to relate to the fact that mitochondria are the main intracellular source of ROS in cells and also the main target of ROS-mediated damage. Noninvasive assessment of living brain could be useful for the diagnostic, prognostic, and treatment monitoring of neurodegenerative diseases related to impaired MC-I function; however, no proper PET probes for MC-I imaging in the brain have

less on α7-nAChR compared to mAChR in the living brain.

been developed prior to those that we have recently described [15].

**3. Aging and MC-I activity**

130 132Exploring New Findings on Amyloidosis

As PET probe for MC-I imaging, BMS-747158-01 showed inhibitory activity on MC-I func‐ tion by binding to MC-I, and its F-18 derivative 18F-BMS-747158-01 was originally developed as a myocardial perfusion imaging agent [41]. However, we also realized that 18F-BMS-747158-01 revealed relatively high nonspecific binding in the brain based on the lower degree of inhibition with rotenone, a specific MC-I inhibitor, in both *in vitro* and *in vivo* assessments [7, 8]. To solve the problem, we redesigned to modify the chemical structure of 18F-BMS-747158-01 to induce lower lipophilicity and lower affinity [15, 42]. We recently developed a novel PET probe, 18F-BCPP-EF, and evaluated its properties in the *in vitro* and *in vivo* assessments [7, 8, 15, 16].

For the analysis of the affinity of 18F-BCPP-EF, an *in vitro* binding assay was conducted using 3 H-dihydrorotenone and bovine cardiomyocyte submitochondrial particles (SMP) to deter‐ mine the 50% inhibition (IC50) values, which were converted to the inhibition constant (Ki). 18F-BCPP-EF was radiolabeled by the nucleophilic 18F-fluorination of the corresponding tosylate precursor, toluene-4-sulfonic acid 2-{2-[5-(1-tert-butyl-5-chloro-6-oxo-1,6-dihydro-pyrida‐ zin-4-yloxymethyl)-pyridin-2-yloxy]-ethoxy}-ethyl ester [15]. For the assessment of binding specificity of the PET probe to MC-I, vehicle or rotenone, a specific MC-I inhibitor, at a dose of 0.1 mg/kg was infused to anesthetised rats or conscious monkeys through a vein for 1 h, and then 18F-BCPP-EF was injected as a bolus for PET measurement. PET measurements of young monkeys (3–5 years old) were conducted under conscious state as described in Section 2.2 with arterial blood sampling for plasma metabolic analysis followed by PET image reconstruction and TAC acquisition in the VOIs aided by MRI of individual animals for kinetic analyses [16, 17]. The total distribution volume (DV) values were evaluated by Logan plot graphical analysis using the metabolite-corrected plasma input [33].

18F-BCPP-EF showed the lower affinity (Ki=2.31 nM) with lower lipophilicity (logD7.4=3.03) for MC-I of bovine cardiomyocytes than that of BMS-747158-01 (Ki=0.95 nM, logD7.4=3.69). In PET study in rats, the radioactivity level of 18F-BCPP-EF in the brain showed rapid uptake and gradual decrease with time just after the injection under vehicle condition. In the heart, 18F-BCPP-EF exhibited slow accumulation up to 30 min after the injection followed by a slight washout. With preadministration of rotenone, although a dose escalation study of rotenone was impossible because of its lethal effects on cardiac function, a significant reduction of 18F-BCPP-EF uptake was observed in the brain and heart, even at a relatively low dose of 0.1 mg/ kg/h. 18F-BMS-747158-01 showed a tendency of decreased brain uptake with rotenone infusion but was not fully inhibited [7].

In the conscious monkey brain, TACs of 18F-BCPP-EF peaked between 10 and 20 min after the injection, except in the occipital cortex (40 min), followed by the gradual elimination with time. With preadministration of rotenone, a specific MC-I inhibitor, at a dose of 0.1 mg/kg/h, the uptake of 18F-BCPP-EF into the brain, especially in the frontal and temporal cortices and striatum, was significantly facilitated just afterthe injection followed by faster elimination than normal from the brain regions. The washout and metabolic rates of 18F-BCPP-EF in plasma were rapid; only 10% of nonmetabolized 18F-BCPP-EF was detected 60 min after the injection, which was were nearly identical with that of rotenone-treated animals. The DV values calculated by Logan plot graphical analysis were the highest in the occipital cortex, higher in the striatum, intermediate in the frontal and temporal cortices and cerebellum, and lowest in the hippocampus of young monkey. Rotenone administration resulted in a significant and marked reduction of the binding of 18F-BCPP-EF to MC-Iin the living monkey brain [16]. Taken together, these results clearly suggested that 18F-BCPP-EF was a useful PET probe for the quantitative imaging of MC-I activity in the living brain.

## **3.2. Aging effects on MC-I activity**

Several hypotheses of aging have been proposed, and mitochondrialrespiratory chain failures have been implicated as factors in the aging process, which was called the "mitochondrial free radical theory of aging" [9–13]. This theory is based on the results that (1) mitochondrial ROS production increases with age, (2) the activity of ROS-scavenging enzymes declines with age, (3) mutations of mitochondrial DNA (mtDNA) accumulate during aging, and (4) somatic mtDNA mutations impair respiratory chain function, which results in a further increase in ROS production [9–13]. Mitochondria are the main intracellular source of ROS and also the main target of oxyradical-mediated damage, and cumulative free radical damage leads to significant changes in brain mitochondrial function.

As described in Section 3.1, because we could confirm the capability of 18F-BCPP-EF as PET probe for the noninvasive assessment of MC-I activity in the living brain, we further ex‐ plored to see the aging effects on MC-I activity in comparison to young and aged monkeys under conscious condition [16]. All study procedures were identical as described in Section 3.1, except that the subjects were aged male monkeys (20–24 years old).

Aided by MRI of individual monkeys, the VOIs were set on PET images of 18F-BCPP-EF to obtain TACs. The peak of 18F-BCPP-EF slightly shifted to a later time period between 20 and 30 min after the injection and also provided significantly lower levels than those in young animals shown in Section 3.1. The washout and metabolic patterns of 18F-BCPP-EF in plasma of aged monkeys were nearly identical to those in young animals. When determining the effects of aging on 18F-BCPP-EF binding to MC-I assessed by Logan plot graphical analysis, the data revealed significantly lower DV values of 18F-BCPP-EF in every brain region ana‐ lyzed in aged animals compared to those in young ones [16].

The remarkable finding of the present study was that 18F-BCPP-EF detected the age-related reduction of MC-I activity in the living brain of monkey under conscious conditions with PET (**Figure 3**). It was of interest that the activity of complexes I and IV decreased with age in the brain of humans [43], whereas that of complexes II, III, and V remained mostly un‐ changed [44]. Because all these findings described above were obtained by the *in vitro* assessments of dissected organ samples, the present data are the first to demonstrate the alterations of MC-I activity in the living brain of a nonhuman primate by a noninvasive method using 18F-BCPP-EF and PET [16].

**Figure 3.** MRI and PET images of 18F-BCPP-EF in young and aged monkeys (*M. mulatta*). PET scans were acquired for 91 min after 18F-BCPP-EF injection with sequential arterial blood sampling. The binding of 18F-BCPP-EF to MC-I was calculated using Logan graphical analysis with metabolite-corrected plasma input.

## **4. Effects of Aβ deposition on AChR and MC-I**

calculated by Logan plot graphical analysis were the highest in the occipital cortex, higher in the striatum, intermediate in the frontal and temporal cortices and cerebellum, and lowest in the hippocampus of young monkey. Rotenone administration resulted in a significant and marked reduction of the binding of 18F-BCPP-EF to MC-Iin the living monkey brain [16]. Taken together, these results clearly suggested that 18F-BCPP-EF was a useful PET probe for the

Several hypotheses of aging have been proposed, and mitochondrialrespiratory chain failures have been implicated as factors in the aging process, which was called the "mitochondrial free radical theory of aging" [9–13]. This theory is based on the results that (1) mitochondrial ROS production increases with age, (2) the activity of ROS-scavenging enzymes declines with age, (3) mutations of mitochondrial DNA (mtDNA) accumulate during aging, and (4) somatic mtDNA mutations impair respiratory chain function, which results in a further increase in ROS production [9–13]. Mitochondria are the main intracellular source of ROS and also the main target of oxyradical-mediated damage, and cumulative free radical damage leads to

As described in Section 3.1, because we could confirm the capability of 18F-BCPP-EF as PET probe for the noninvasive assessment of MC-I activity in the living brain, we further ex‐ plored to see the aging effects on MC-I activity in comparison to young and aged monkeys under conscious condition [16]. All study procedures were identical as described in Section

Aided by MRI of individual monkeys, the VOIs were set on PET images of 18F-BCPP-EF to obtain TACs. The peak of 18F-BCPP-EF slightly shifted to a later time period between 20 and 30 min after the injection and also provided significantly lower levels than those in young animals shown in Section 3.1. The washout and metabolic patterns of 18F-BCPP-EF in plasma of aged monkeys were nearly identical to those in young animals. When determining the effects of aging on 18F-BCPP-EF binding to MC-I assessed by Logan plot graphical analysis, the data revealed significantly lower DV values of 18F-BCPP-EF in every brain region ana‐

The remarkable finding of the present study was that 18F-BCPP-EF detected the age-related reduction of MC-I activity in the living brain of monkey under conscious conditions with PET (**Figure 3**). It was of interest that the activity of complexes I and IV decreased with age in the brain of humans [43], whereas that of complexes II, III, and V remained mostly un‐ changed [44]. Because all these findings described above were obtained by the *in vitro* assessments of dissected organ samples, the present data are the first to demonstrate the alterations of MC-I activity in the living brain of a nonhuman primate by a noninvasive method

quantitative imaging of MC-I activity in the living brain.

significant changes in brain mitochondrial function.

3.1, except that the subjects were aged male monkeys (20–24 years old).

lyzed in aged animals compared to those in young ones [16].

using 18F-BCPP-EF and PET [16].

**3.2. Aging effects on MC-I activity**

132 134Exploring New Findings on Amyloidosis

Hallmark pathologies of AD have been assumed to the formation of extracellular aggrega‐ tion of Aβ protein (senile plaques) [19] and intraneuronal aggregation of phosphorylated tau protein (neurofibrillary tangle) [20], pathogenic microglial activation [45], and oxidative stress reactions [13]. Furthermore, recent reports suggested that nondeposited and nonfibrillar assemblies of Aβ peptides are considered to play a primary role in AD, which might be precursors in fibrillogenesis to mediate the neurotoxicity, including oxidative stress in AD [46]. This session discusses the effects of Aβ-related pathological changes on nAChR binding [36] and also on MC-I activity [17] assessed in aged monkey brain using animal PET.

## **4.1. Aging effects on Aβ deposition and neuroinflammation**

For the quantitative measurements of Aβ deposition in the living brain, 11C-PIB was synthe‐ sized by *N*-methylation of nor-compound *N*-desmethyl-PIB with 11C-methyl triflate (**Figure 1A**) [47]. For the assessment of neuroinflammation, 11C-DPA-713 was synthesized by *N*methylation of nor-compound*N*-desmethyl-DPA with 11C-methyltriflate (**Figure 1B**) [48]. PET measurements with these two PET probes were conducted under conscious state as descri‐ bed in Section 2.2. For the analysis of 11C-PIB uptake, standard uptake value (SUV) images were created, and the VOIs were set on each SUV images with the aid of MRI. For the analysis of 11C-DPA-713 uptake, SUV images were created, and VOIs were set on each SUV images.

We had previously found that some, but not all, of aged monkeys exhibited higher 11C-PIB uptake than young ones, suggesting Aβ deposition even in the brain of monkey [49]. Although the SUVs of 11C-PIB were slightly high in the striatal regions, hippocampus, parietal cortex, and thalamus in aged monkeys, it was reported that aged monkeys did not reveal as high Aβ deposition as determined in AD patients [50] and also that no species besides humans has yet shown drastic neuron loss or cognitive decline approaching clinical-grade AD in humans. The SUVs of 11C-DPA-713 were relatively high in the striatal regions, hippocampus, temporal cortex, and thalamus. The plot of SUV of 11C-DPA-713 against SUV of 11C-PIB revealed significant positive correlation both in the cortical regions. When rCMRglc ratio is plotted against SUV of 11C-DPA-713, the results provided the significant positive correlation in the cortical regions. There results strongly suggest that the Aβ deposition as measured with 11C-PIB induced neuroinflammation with microglial activation as determined with 11C-DPA-713 as well as 18F-FDG (**Figure 4**).

**Figure 4.** PET/MRI fusion images of 11C-PIB, 11C-DPA-713, and 18F-BCPP-EF uptake in the brains of aged monkeys (*M. mulatta*). 18F-BCPP-EF brain imaging was performed in monkey 1 with the highest binding of all 20 animals in the conscious state and in monkey 2 with the lowest binding.

## **4.2. Effects of Aβ deposition on nAChR binding**

Postmortem studies of human brains have suggested that a deficit of α7-nAChR is related to AD, dementia with Lewy bodies, and schizophrenia [32]. Aβ has a high affinity to α7-nAChR upon being enriched in basal forebrain areas, and initial Aβ deposition in early AD overlaps with α7-nAChR expression in these regions [51]. α7-nAChR facilitates binding, internaliza‐ tion, and accumulation of Aβ1–42 and may result in the selective vulnerability of specific cells expressing α7-nAChR [52]. Oligomers of Aβ disrupt synaptic plasticity and cognitive function when administered at nanomolar range concentration through the modulation of NMDA receptor function to depress NMDA-evoked currents [53, 54].

To assess the effect of Aβ deposition on α7-nAChR function, the correlation between 11C-PIB uptake and 11C-(*R*)-MeQAA was analyzed [36]. As described in Section 2.2, all regions, except

the occipital cortex, revealed no significant differences in the BPND values of 11C-(*R*)-MeQAA in the brains of aged and young animals. However, of interest, when the BPND values of 11C-(*R*)- MeQAA are plotted against the SUVR of 11C-PIB in the corresponding VOIs of aged animals, the results indicated a significant positive correlation between them (**Figure 5**). The α7-nAChR had been assumed to decrease with the aging process; however, the data seem to be contro‐ versial because of the dose-dependent interaction aspects between of Aβ and α7-nAChR. In young and normal conditions, there is an equilibrium between production and elimination of Aβ, which maintains Aβ in a steady state to perform physiologic roles by the interaction with α7-nAChR; thus, low (on the order of picomolar) concentrations of Aβ1–42 may play a role in modulating synaptic plasticity and enhancing cognitive function in mice via interaction with α7-nAChR, probably in presynaptic neurons [55]. In aging with pathologic conditions, the balance between Aβ formation and clearance is impaired, which leads to Aβ accumula‐ tion and α7-nAChR deficit as observed in postmortem brains of AD patients [32]. These results suggest that α7-nAChR-Aβ interaction has dual effects on brain function following aging or injury as well as preserving physiologic roles, causing the controversial results in the aging effects on the α7-nAChR level. One previous study clearly demonstrated the significant reduction of α7-nAChR level in neurons and also Aβ-induced up-regulation of α7-nAChR on astrocytes in postmortem AD brain compared to that in age-matched control [56]. α7-nAChR may be chronically inactivated in an antagonistic fashion through prolonged interaction with increased levels (on the order of nanomolar) ofAβ, resulting in the up-regulation of α7-nAChR.

and thalamus in aged monkeys, it was reported that aged monkeys did not reveal as high Aβ deposition as determined in AD patients [50] and also that no species besides humans has yet shown drastic neuron loss or cognitive decline approaching clinical-grade AD in humans. The SUVs of 11C-DPA-713 were relatively high in the striatal regions, hippocampus, temporal cortex, and thalamus. The plot of SUV of 11C-DPA-713 against SUV of 11C-PIB revealed significant positive correlation both in the cortical regions. When rCMRglc ratio is plotted against SUV of 11C-DPA-713, the results provided the significant positive correlation in the cortical regions. There results strongly suggest that the Aβ deposition as measured with 11C-PIB induced neuroinflammation with microglial activation as determined with 11C-DPA-713

**Figure 4.** PET/MRI fusion images of 11C-PIB, 11C-DPA-713, and 18F-BCPP-EF uptake in the brains of aged monkeys (*M. mulatta*). 18F-BCPP-EF brain imaging was performed in monkey 1 with the highest binding of all 20 animals in the

Postmortem studies of human brains have suggested that a deficit of α7-nAChR is related to AD, dementia with Lewy bodies, and schizophrenia [32]. Aβ has a high affinity to α7-nAChR upon being enriched in basal forebrain areas, and initial Aβ deposition in early AD overlaps with α7-nAChR expression in these regions [51]. α7-nAChR facilitates binding, internaliza‐ tion, and accumulation of Aβ1–42 and may result in the selective vulnerability of specific cells expressing α7-nAChR [52]. Oligomers of Aβ disrupt synaptic plasticity and cognitive function when administered at nanomolar range concentration through the modulation of NMDA

To assess the effect of Aβ deposition on α7-nAChR function, the correlation between 11C-PIB uptake and 11C-(*R*)-MeQAA was analyzed [36]. As described in Section 2.2, all regions, except

as well as 18F-FDG (**Figure 4**).

134 136Exploring New Findings on Amyloidosis

conscious state and in monkey 2 with the lowest binding.

**4.2. Effects of Aβ deposition on nAChR binding**

receptor function to depress NMDA-evoked currents [53, 54].

**Figure 5.** PET/MRI fusion images of 11C-PIB, 18F-BCPP-EF, and 11C-(*R*)-MeQAA in the brains of aged monkeys (*M. mu‐ latta*). PET images of an aged monkey with the lowest and highest 11C-PIB uptake are shown along with 18F-BCPP-EF and 11C-(*R*)-MeQAA.

## **4.3. Effects of Aβ deposition on MC-I activity**

As shown in Section 3.2, 18F-BCPP-EF binding to MC-I in each brain region indicated much larger variation (CV=25.1%) in aged animals than in young ones (7.4%) [16]. Since we had previously found that some of the aged monkeys exhibited high 11C-PIB uptake, suggesting Aβ deposition in the brain [49], the effects of Aβ deposition level assessed in Section 4.1 on MC-I activity evaluated in Section 3.2 were determined. When the 18F-BCPP-EF binding is plotted against the 11C-PIB uptake in the corresponding VOIs of all animals, the results indicated a significant reversal correlation (**Figure 4**). In contrast, no significant relationships were observed in the plot of rCMRglc measured using 18F-FDG against 11C-PIB uptake in the cerebral corticalregions [17]. There results strongly suggestthattheAβ deposition as measured with 11C-PIB induced neuroinflammation with microglial activation as determined with 11C-DPA-713 as well as 18F-FDG. Apart from developed cells such as neurons, activated inflam‐ matory cells produce lactate from glucose, known as the Warburg effect [57] or aerobic glycolysis, accounting for only approximately 5% glucose utilization in oxidative phosphor‐ ylation. Because activated inflammatory cells exclusively produce ATP through enhanced glycolysis with a low contribution of the electron transport system, these cells need more glucose to survive than normal neuronal and glial tissues, resulting in the higher 18F-FDG uptake in the neurodegenerative damaged regions. The present results demonstrated that 18F-BCPP-EF could be used to image MC-I activity specifically as well as to detect impaired MC-I activity correlated to Aβ deposition in the living brain of monkeys.

## **5. Conclusions**

In this chapter, the effects of Aβ deposition on α7-nAChR binding, TSPO activity, an estab‐ lished marker of microglial activation, and rCMRglc were assessed simultaneously with MC-I activity. PET using 18F-FDG is a well-established technique for the quantitative imaging of brain function as rCMRglc in the living brain. However, the unexpectedly high uptake of 18F-FDG in damaged brain regions suggested that 18F-FDG was taken up into not only normal tissues but also inflammatory regions with microglial activation, which hampers the accu‐ rate diagnosis of brain function using 18F-FDG. Neuroinflammation has recently emerged in several neurodegenerative diseases, including schizophrenia, depression, autism, PD, andAD. PET measurements of MC-I activity using 18F-BCPP-EF will be a superior diagnostic, prog‐ nostic, and treatment monitoring tool for AD-type dementia without being affected by inflammation.

## **Author details**

Hideo Tsukada\*

Address all correspondence to: tsukada@crl.hpk.co.jp

Central Research Laboratory, Hamamatsu Photonics K.K., Hamamatsu, Japan

## **References**

**4.3. Effects of Aβ deposition on MC-I activity**

136 138Exploring New Findings on Amyloidosis

I activity correlated to Aβ deposition in the living brain of monkeys.

**5. Conclusions**

inflammation.

**Author details**

Hideo Tsukada\*

Address all correspondence to: tsukada@crl.hpk.co.jp

As shown in Section 3.2, 18F-BCPP-EF binding to MC-I in each brain region indicated much larger variation (CV=25.1%) in aged animals than in young ones (7.4%) [16]. Since we had previously found that some of the aged monkeys exhibited high 11C-PIB uptake, suggesting Aβ deposition in the brain [49], the effects of Aβ deposition level assessed in Section 4.1 on MC-I activity evaluated in Section 3.2 were determined. When the 18F-BCPP-EF binding is plotted against the 11C-PIB uptake in the corresponding VOIs of all animals, the results indicated a significant reversal correlation (**Figure 4**). In contrast, no significant relationships were observed in the plot of rCMRglc measured using 18F-FDG against 11C-PIB uptake in the cerebral corticalregions [17]. There results strongly suggestthattheAβ deposition as measured with 11C-PIB induced neuroinflammation with microglial activation as determined with 11C-DPA-713 as well as 18F-FDG. Apart from developed cells such as neurons, activated inflam‐ matory cells produce lactate from glucose, known as the Warburg effect [57] or aerobic glycolysis, accounting for only approximately 5% glucose utilization in oxidative phosphor‐ ylation. Because activated inflammatory cells exclusively produce ATP through enhanced glycolysis with a low contribution of the electron transport system, these cells need more glucose to survive than normal neuronal and glial tissues, resulting in the higher 18F-FDG uptake in the neurodegenerative damaged regions. The present results demonstrated that 18F-BCPP-EF could be used to image MC-I activity specifically as well as to detect impaired MC-

In this chapter, the effects of Aβ deposition on α7-nAChR binding, TSPO activity, an estab‐ lished marker of microglial activation, and rCMRglc were assessed simultaneously with MC-I activity. PET using 18F-FDG is a well-established technique for the quantitative imaging of brain function as rCMRglc in the living brain. However, the unexpectedly high uptake of 18F-FDG in damaged brain regions suggested that 18F-FDG was taken up into not only normal tissues but also inflammatory regions with microglial activation, which hampers the accu‐ rate diagnosis of brain function using 18F-FDG. Neuroinflammation has recently emerged in several neurodegenerative diseases, including schizophrenia, depression, autism, PD, andAD. PET measurements of MC-I activity using 18F-BCPP-EF will be a superior diagnostic, prog‐ nostic, and treatment monitoring tool for AD-type dementia without being affected by


rat brain and their dietary modulation. Arch Pharm Res. 1999;22:361–366. DOI: 10.1007/ BF02979058.


measured with positron emission tomography (PET). J Neurosci Res. 1990;27:569–575. DOI: 10.1002/jnr.490270418.

[24] Zubieta JK, Koeppe RA, Frey KA, Kilbourn MR, Mangner TJ, Foster NL, Kuhl DE. Assessment of muscarinic receptor concentrations in aging and Alzheimer disease with 11C-NMPB and PET. Synapse. 2001;39:275–287. DOI: 10.1002/1098-2396(20010315)39:4<275::AID-SYN1010>3.0.CO;2–3.

rat brain and their dietary modulation. Arch Pharm Res. 1999;22:361–366. DOI: 10.1007/

[12] Kannurpatti SS, Sanganahalli BG, Mishra S, Joshi PG, Joshi NB. Glutamate-induced differential mitochondrial response in young and adult rats. Neurochem Int.

[13] Choi BH. Oxidative stress and Alzheimer's disease. Neurobiol Aging. 1995;16:675–678.

[14] Schon EA, Przedborski S. Mitochondria: The next (neurode)generation. Neuron.

[15] Harada N, Nishiyama S, Kanazawa M, Tsukada H. Development of novel PET probes, [18F]BCPP-EF, [18F]BCPP-BF, and [11C]BCPP-EM for mitochondrial complex I imaging. J Labelled Compd Radiopharm. 2013;56:553–561. DOI: 10.1002/jlcr.3056. [16] Tsukada H, Ohba H, Kanazawa M, Kakiuchi T, Harada N. Evaluation of 18F-BCPP-EF for mitochondrial complex 1 imaging in conscious monkey brain using PET. EurJ Nucl

[17] Tsukada H, Nishiyama S, Ohba H, Kanazawa M, Kakiuchi T, Harada N. Comparing amyloid-β deposition, neuroinflammation, glucose metabolism, and mitochondrial complex I activity in brain: A PET study in aged monkeys. Eur J Nucl Med Mol

[18] Court JA, Lloyd S, Johnson M, Griffiths M, Birdsall NJM, Piggott MA, Oakley AE, Ince PG, Perry EK, Perry RH. Nicotinic and muscarinic cholinergic receptor binding in the human hippocampal formation during development and aging. Dev Brain Res.

[19] Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer's disease: Progress and problems on the road to therapeutics. Science. 2002;297:353–356. DOI: 10.1126/science.

[20] Grundke-Iqbal I, Iqbal K, Tung YC, Quinlan M, Wisniewski HM, Binder LI. Abnor‐ mal phosphorylation of the microtubule-associated protein tau (τ) in Alzheimer

[21] Terry AV Jr, Buccafusco JJ. The cholinergic hypothesis of age and Alzheimer's diseaserelated cognitive deficits: Recent challenges and their implications for novel drug development. J Pharmacol Exp Ther. 2003;306:821–827. DOI: 10.1124/jpet.102.041616.

[22] Tsukada H, Nishiyama S, Fukumoto D, Ohba H, Sato K, Kakiuchi T. Effects of acute acetylcholinesterase inhibition on the cerebral cholinergic neuronal system and cognitive function: Functional imaging of the conscious monkey brain using animal PET in combination with microdialysis. Synapse. 2004;52:1–10. DOI: 10.1002/syn.10310.

[23] Dewey SL, Volkow ND, Logan J, MacGregor RR, Fowler JS, Schlyer DJ, Bendriem B. Age-related decrease in muscarinic cholinergic receptor binding in the human brain

cytoskeletal pathology. Proc Natl Acad Sci U S A. 1986;83:4913–4917.

Med Mol Imaging. 2014;41:755–763. DOI: 10.1007/s00259-014-2821-8.

Imaging. 2014;41:2127–2136. DOI: 10.1007/s00259-013-2628-z.

1997;101:93–105. DOI: 10.1016/S0165-3806(97)00052-7.

BF02979058.

138 140Exploring New Findings on Amyloidosis

1072994.

2004;44:361–369. DOI: 10.1007/BF02979058.

2011;70:1033–1053. DOI: 10.1016/j.neuron.2011.06.003.

DOI: 10.1016/0197-4580(95)00065-M.


[42] Waterhouse RN. Determination of lipophilicity and its use as a predictor of blood-brain barrier penetration of molecular imaging agents. Mol Imaging Biol. 2003;5:376–389. DOI: 10.1016/j.mibio.2003.09.014.

[32] Albuquerque EX, Pereira EF, Alkondon M, Rogers SW. Mammalian nicotinic acetyl‐ choline receptors: From structure to function. Physiol Rev. 2009;89:73–120. DOI:

[33] Kadir A, Almkvist O, Wall A, Långström B, Nordberg A. PET imaging of cortical 11Cnicotine binding correlates with the cognitive function of attention in Alzheimer's disease. Psychopharmacology. 2006;188:509–520. DOI: 10.1007/s00213-006-0447-7. [34] Wu J, Ishikawa M, Zhang J, Hashimoto K. Brain imaging of nicotinic receptors in Alzheimer's disease. Int J Alzheimer's Dis. 2010;2010:548913. DOI:

[35] Ogawa M, Nishiyama S, Tsukada H, Hatano K, Fuchigami T, Yamaguchi H, Matsushima Y, Ito K, Magata Y. Synthesis and evaluation of new imaging agent for central nicotinic acetylcholine receptor α7 subtype. Nucl Med Biol. 2010;37:347–355.

[36] Nishiyama S, Ohba H, Kanazawa M, Kakiuchi T, Tsukada H. Comparing α7 nicotinic acetylcholine receptor binding, amyloid-β deposition, and mitochondria complex-I function in living brain: A PET study in aged monkeys. Synapse. 2015;69:475–483.

[37] Mintun MA, Raichle ME, Kilbourn MR, Wooten GF, Welch MJ. A quantitative model for the *in vivo* assessment of drug binding sites with positron emission tomography.

[38] Innis RB, Cunningham VJ, Delforge J, Fujita M, Gjedde A, Gunn RN, Holden J, Houle S, Huang SC, Ichise M, Iida H, Kimura Y, Koeppe RA, Knudsen GM, Knuuti J, Lammertsma AA, Laruelle M, Logan J, Maguire RP, Mintun MA, Morris ED, Parsey R, Price JC, Slifstein M, Sossi V, Suhara T, Votaw JR, Wong DF, Carson RE. Consensus nomenclature for *in vivo* imaging of reversibly binding radioligands. J Cereb Blood

[39] Noda A, Takamatsu H, Minoshima S, Tsukada H, Nishimura S. Determination of kinetic rate constants for 2-[18F]fluoro-2-deoxy-D-glucose and partition coefficient of water in conscious macaques and alterations in aging or anesthesia examined on parametric images with an anatomic standardization technique. J Cereb Blood Flow

[40] Schild L, Huppelsberg J, Kahlert S, Keilhoff G, Reiser G. Brain mitochondria are primed by moderate Ca2+ rise upon hypoxia/reoxygenation for functional breakdown and morphological disintegration. J Biol Chem. 2003;278:25454–25460. DOI: 10.1074/

[41] Yalamanchili P, Wexler E, Hayes M, Yu M, Bozek J, Kagan M, Radeke HS, Azure M, Purohit A, Casebier DS, Robinson SP. Mechanism of uptake and retention of F-18 BMS-747158-02 in cardiomyocytes: A novel PET myocardial imaging agent. J Nucl

Ann Neurol. 1984;15:217–227. DOI: 10.1002/ana.410150302.

Flow Metab. 2007;27:1533–1539. DOI: 10.1038/sj.jcbfm.9600493.

Cardiol. 2007;14:782–788. DOI: 10.1016/j.nuclcard.2007.07.009.

10.1152/physrev.00015.2008.

140 142Exploring New Findings on Amyloidosis

10.4061/2010/548913.

DOI: 10.1002/syn.21842.

Metab. 2003;23:1441–1447.

jbc.M302743200.

DOI: 10.1016/j.nucmedbio.2009.11.007.


**Chapter 5**

## **Kinetics of Amyloid Formation by Different Proteins and Peptides: Polymorphism and Sizes of Folding Nuclei of Fibrils**

Oxana V. Galzitskaya, Nikita V. Dovidchenko and Olga M. Selivanova

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63359

#### **Abstract**

[53] Cleary JP, Walsh DM, Hofmeister JJ, Shankar GM, Kuskowski MA, Selkoe DJ, Ashe KH. Natural oligomers of the amyloid-β protein specifically disrupt cognitive func‐

[54] Lesne S, Koh MT, Kotilinek L, Kayed R, Glabe CG, Yang A, Gallagher M, Ashe KH. A specific amyloid-β protein assembly in the brain impairs memory. Nature.

[55] Puzzo D, Privitera L, Leznik E, Fa M, Staniszewski A, Palmeri A, Arancio O. Picomo‐ lar amyloid-β positively modulates synaptic plasticity and memory in hippocampus.

[56] Yu WF, Guan ZZ, Bogdanovic N, Nordberg A. High selective expression of α7 nicotinic receptors on astrocytes in the brains of patients with sporadic Alzheimer's disease and patients carrying Swedish APP 670/671 mutation: A possible association with neurit‐ ic plaques. Exp Neurol. 2005;192:215–225. DOI: 10.1016/j.expneurol.2004.12.015.

[57] Warburg O. On respiratory impairment in cancer cells. Science. 1956;124:267–272.

J Neurosci. 2008;28:14537–14545. DOI: 10.1523/JNEUROSCI.2692-08.2008.

tion. Nat Neurosci. 2005;8:79–84. DOI: 10.1038/nn1372.

2006;440:352–357. DOI: 10.1038/nature04533.

142 144Exploring New Findings on Amyloidosis

DOI: 10.1126/science.124.3215.267.

Aggregation of peptides and proteins into amyloid structure is one of the most intensively studied biological phenomena at the moment. To date, there is no devel‐ oped theory that would allow one to determine what kind of mechanism presents in the given experiment on the basis of aggregation kinetic data. Debates concerning the mechanism of the amyloid fibrils formation and, in particular, the size of the amyloi‐ dogenic nucleus are still going on. We created such a theory on the basis of the kinetics of amyloid aggregates formation. In the presented chapter, theoretical and experimen‐ tal approaches were employed for studding the process of amyloid formation by different proteins and peptides. The current kinetic models described in this chapter adequately describe the key features of amyloid nucleation and growth.

**Keywords:** amyloidogenic regions, amyloid prediction methods, linear and exponen‐ tial mechanisms of amyloid growth, polymorphism, prion

## **1. Introduction**

Many studies show that the generation of an amyloid fibril is a fundamental property of all protein molecules, rather than being limited to a narrow range of the so-called amyloidforming proteins associated with amyloid diseases, as was believed until recently [1, 2]. Normal proteins become toxic upon fibril formation [3]. At the same time, greater cytotoxici‐ ty is characteristic of immature water-soluble fibrils compared to mature insoluble amyloid

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

fibrils. It seems that the mechanism of toxicity should be similar to that for fibrils formed by different proteins. The structure of toxic immature fibrils is certainly rich in β-strands and should be universal, because specific antibodies against the precursors of amyloid peptide Aβ, associated with Alzheimer's disease, recognize the precursors of amyloid fibrils formed by other proteins with different amino acid sequences [4]. It was demonstrated that samples of blood serum from patients with Parkinson's disease show an autoimmune response to insulin oligomers and fibrils, which should reveal the presence of insulin aggregates in this disease [5]. Fibril formation depends on the experimental conditions and is expedited by denatur‐ ants: to aggregate, proteins should be unfolded, at least partly [6]. Preliminary unfolding is not needed for the aggregation of peptides and proteins associated with amyloidosis such as type II diabetes or Alzheimer's and Parkinson's disease, because these proteins are already unfolded under physiological conditions [7]. However, the majority of natively unfolded proteins do not aggregate in vivo [8] suggesting that unfolding is necessary, but not suffi‐ cient for the formation of amyloid fibrils. Most likely, there are specific amino acid motifs that are exposed to the solvent and are more prone to aggregation as compared to other regions of the amino acid sequence. Experimental findings support the hypothesis that small protein regions are responsible for the amyloidogenic behavior [9–11].

## **2. Identification of aggregation sites in proteins responsible for amyloid formation**

We have done comparison of the prediction results for 30 amyloidogenic proteins [12] using seven methods with experimental data: PASTA2 [13], AmylPred2 [12], Tango [14], MetAmyl [15], Waltz [16], FoldAmyloid [17], ArchCandy [18]. More detail comparison with full list of proteins but without new method Archcandy was presented in our recent paper [19]. One can see that FoldAmyloid (which is a relatively old and simple approach) works better than Waltz and Archcandy. PASTA2 is a new more sophisticated approach that performs best among the non-meta-servers. As evident from **Table 1**, the accuracy and sensitivity of PASTA2 predic‐


**Table 1.** Averaged results of amyloid predictions for 30 proteins by various algorithms.

tions are only smaller better than those of FoldAmyloid. At the same time, comparison of the overall prediction quality shows only modest improvement, which is due to low sensitivity partly caused by overprediction and partly by the coarseness of the methods.

## **3. Possible kinetics schemes for amyloid and prion growth**

fibrils. It seems that the mechanism of toxicity should be similar to that for fibrils formed by different proteins. The structure of toxic immature fibrils is certainly rich in β-strands and should be universal, because specific antibodies against the precursors of amyloid peptide Aβ, associated with Alzheimer's disease, recognize the precursors of amyloid fibrils formed by other proteins with different amino acid sequences [4]. It was demonstrated that samples of blood serum from patients with Parkinson's disease show an autoimmune response to insulin oligomers and fibrils, which should reveal the presence of insulin aggregates in this disease [5]. Fibril formation depends on the experimental conditions and is expedited by denatur‐ ants: to aggregate, proteins should be unfolded, at least partly [6]. Preliminary unfolding is not needed for the aggregation of peptides and proteins associated with amyloidosis such as type II diabetes or Alzheimer's and Parkinson's disease, because these proteins are already unfolded under physiological conditions [7]. However, the majority of natively unfolded proteins do not aggregate in vivo [8] suggesting that unfolding is necessary, but not suffi‐ cient for the formation of amyloid fibrils. Most likely, there are specific amino acid motifs that are exposed to the solvent and are more prone to aggregation as compared to other regions of the amino acid sequence. Experimental findings support the hypothesis that small protein

**2. Identification of aggregation sites in proteins responsible for amyloid**

We have done comparison of the prediction results for 30 amyloidogenic proteins [12] using seven methods with experimental data: PASTA2 [13], AmylPred2 [12], Tango [14], MetAmyl [15], Waltz [16], FoldAmyloid [17], ArchCandy [18]. More detail comparison with full list of proteins but without new method Archcandy was presented in our recent paper [19]. One can see that FoldAmyloid (which is a relatively old and simple approach) works better than Waltz and Archcandy. PASTA2 is a new more sophisticated approach that performs best among the non-meta-servers. As evident from **Table 1**, the accuracy and sensitivity of PASTA2 predic‐

**AmylPred2 Tango MetAmyl**

Sensitivity 0.36 0.41 0.19 0.38 0.19 0.28 0.16 Specificity 0.91 0.86 0.95 0.86 0.94 0.92 0.92

**Table 1.** Averaged results of amyloid predictions for 30 proteins by various algorithms.

**high specificity** 

38 121 37 88 37 31 15

33/**46** 42/**46** 17/**46** 33/**46** 22/**46** 29/**46** 8/**46**

**Waltz best perfo‐ mance** 

**FoldAmy‐ loid** 

**Archcandy default set‐ tings** 

regions are responsible for the amyloidogenic behavior [9–11].

**formation**

1442 Exploring New Findings on Amyloidosis

False regions

gions/**total**

ic

predicted as amyloidogen‐

No. correctly predicted re‐

**Scoring type PASTA2**

**90% specificity** 

To explain the kinetic features of the aggregation process of protein molecules, a large number of schemes were proposed. There are many approaches for describing the kinetics and mechanism of protein aggregation: kinetic, thermodynamic, and other empirical approaches. The purpose of the first two was the best approximation of the experimental data and obtain‐ ment of quantitative values of the rate constants describing such processes as nucleation, growth, and all other parts of the aggregation process. At present, there is no consensus whether a protofilament is formed by joining oligomers or by adding monomers. The following questions can be posed: How many different mechanisms exist in the literature for describing the protein aggregation? What is the essence of each mechanism? What mechanisms or models are used in the literature to describe the experimental kinetic curves? What are the similarities between various mechanisms? The large volume of publications describing the formation of amyloid fibrils allows us to reveal two general mechanisms. There are a mechanism of sequential addition of monomers (linear growth) and a mechanism for describing the aggre‐ gation of prions (exponentional growth). Most of these approaches somehow go back to the late fifties [20]. In this study, a kinetic scheme for polymerization of actin was proposed. The experimental data indicated that the polymerization process is very similar to the condensation reaction: The reaction occurs only when the monomer concentration exceeds the critical threshold. The assumption was justified. The cooperative polymerization reaction was confirmed by two facts: (1) The reaction rate in the early stages of the reaction increased upon increasing the concentration of actin; (2) an addition of seed with an already formed unit led to an immediate transition of all free actin to an aggregate. In 1974, Hofrichter et al. [21] used the mechanism of sequential monomer addition to the aggregate to explain the formation of hemoglobin fibrils in sickle cell anemia. To explain the observed effect of "extreme autocatal‐ ysis" and strong concentration dependence, Ferrone with the co-authors developed a model of "heterogeneous nucleation" [22]. This model assumes the following sequence of events: First, in the normal course of nucleation, fibrils, and then, subsequent fibrils may form on its surface. This model is included as an equation for the first stage—the homogeneous nucleation, and for the second step—heterogeneous nucleation; the equations were solved numerically. The notion of heterogeneous nucleation was a new and important contribution to the devel‐ opment of the theory of protein aggregation. In 1983, Frieden and Goddette [23] used a model of sequential monomer addition to the unit to describe the process of protein polymerization, with the proviso that each step of monomer addition had its own reaction rate constant. In 1986, Goldstein and Stryer [24] modified the model of sequential addition of monomers to the unit to describe the process of protein polymerization. They identified the nucleus as a "seed" of size s, after the formation of which the kinetic constants are changed. Using protein amylin, it was shown that nucleation can occur in two ways: fibril-independent (or primary nucleation) and fibril-dependent (secondary nucleation) paths. The contribution of each of these processes depends on the external interfaces (or surfaces). In the presence of such a surface, the primary nucleation mechanism is dominating; in the absence of such a surface, the secondary nucleation mechanism is dominating [25]. Heterogeneous nucleation can be seen from another point of view, as an autocatalytic growth in mature seeds. It was not shown experimentally whether seeds are formed on the existing surface of the unit, or the growth simply continues from the surface of the same aggregate. In 1967, Griffith [26] suggested three possible theoretical mechanisms that could explain the self-replication agent causing "scrapie" sheep (scrapie). In 1982, Prusiner [27] was able to provide enough experimental evidence that the "scrapie" infectious agent is the protein called a prion. In 1991, Prusiner suggested the scheme of transfer and development of prion infection [28]. Based on the Prusiner's work, in 1993, Lansbury his work published [29], in which the mechanism of sequential monomer addition to the seed was applied for analysis of kinetic data.

**Figure 1.** Alternative scenarios for amyloid growth and corresponding kinetic parameters. T2 is the time of inclusion of all monomers into the aggregate, Lrel is the ratio between the duration of the lag phase and the time of inclusion of all monomers into the growing polymer, [M∑] is the total monomer concentration, n\* is the size of the primary nucleus, and n2 is the size of the secondary nucleus. nS is the smallest number of monomers in the stable polymer. Modified from [33].

and fibril-dependent (secondary nucleation) paths. The contribution of each of these processes depends on the external interfaces (or surfaces). In the presence of such a surface, the primary nucleation mechanism is dominating; in the absence of such a surface, the secondary nucleation mechanism is dominating [25]. Heterogeneous nucleation can be seen from another point of view, as an autocatalytic growth in mature seeds. It was not shown experimentally whether seeds are formed on the existing surface of the unit, or the growth simply continues from the surface of the same aggregate. In 1967, Griffith [26] suggested three possible theoretical mechanisms that could explain the self-replication agent causing "scrapie" sheep (scrapie). In 1982, Prusiner [27] was able to provide enough experimental evidence that the "scrapie" infectious agent is the protein called a prion. In 1991, Prusiner suggested the scheme of transfer and development of prion infection [28]. Based on the Prusiner's work, in 1993, Lansbury his work published [29], in which the mechanism of sequential monomer addition to the seed was

**Figure 1.** Alternative scenarios for amyloid growth and corresponding kinetic parameters. T2 is the time of inclusion of all monomers into the aggregate, Lrel is the ratio between the duration of the lag phase and the time of inclusion of all monomers into the growing polymer, [M∑] is the total monomer concentration, n\* is the size of the primary nucleus, and n2 is the size of the secondary nucleus. nS is the smallest number of monomers in the stable polymer. Modified

applied for analysis of kinetic data.

1464 Exploring New Findings on Amyloidosis

from [33].

In 2004 in the paper by Collins et al. [30], the formation kinetics of sup35p prion was studied. It was shown that amyloid aggregate growth occurs upon monomer addition to the fiber ends. Also the authors performed experiments with agitation and observed catalysis due to fiber fragmentation, so their kinetic model consisted of three steps: at the first step, oligomers are formed; at the second step, amyloidal seeds are formed through nucleation-conversion, after that the autocatalytic growth occurs where autocatalysis takes place because of fragmentation. Thus, major schemes of amyloid aggregates formation were drawn. In general, schemes can be summed up into the following reaction: out of a pool of monomer nuclei (the most unstable species on the reaction path with the largest free energy) are formed, which then are somehow converted into seed (the smallest stable amyloid aggregate) which is able to attach monomers in a way that monomer free energy only decreases, the result of which is that after some time, all the monomers become associated into fibrils. In some cases, catalysis of reaction occurs through secondary nucleation or fragmentation. In the presence of a catalyst, the amyloidal growth regime can be named "exponential," whereas in the absence of catalysis, the amyloidal growth can be named linear. It should be mentioned that in this general scheme, it is not important what one means by the words "monomer protein"—the monomer itself or some protein aggregate. Despite overall success in the understanding of possible mechanisms hidden beneath amyloidal aggregation, the relation of the kinetic models to the experimentally measurable values was still unclear. Thus, the authors of the paper [31] have shown that despite possible different mechanisms of aggregation, all of the kinetic experimental data could be successfully approximated by a simple sigmoid curve. Fortunately in the same year, the careful introspection of the behavior of the logarithm of lag time versus logarithm of concentration for several amyloidogenic agents by Knowles et al. [32] revealed that different amyloidogenic agents possess a similar linear dependence in those coordinates and such dependence is Tlag/Tlag0 <sup>≈</sup>(C/C0)<sup>−</sup>1/2, where Tlag0 and C0 are values corresponding to the smallest concentration. In the paper Dovidchenko et al. [33], the authors studied the variance in the mechanisms within scheme monomer ->nucleus->fibril growth with/without catalysis. In the work, the authors were lucky to find bridges between values which can be calculated directly from experimental data—characteristic times such as lag-time Tlag, time of inclusion of all monomers into aggregate T2 and relation Lrel = Tlag/T2 and mechanism of amyloidogenesis. It turned out that through the study of the concentration dependence of amyloid formation, one can estimate nuclei sizes (the size of the primary nucleus and size of the secondary nucleus if secondary nucleation takes place) and possible mechanisms. For example, linear growth (when no catalysis occurs) has a very narrow range of conditions: Lrel should be independent of the logarithm of concentration and its value should not exceed 0.2. In the case of exponential mechanism, one should plot a graph of two dependencies: lnT2 versus ln[MΣ] and Lrel versus ln[MΣ], where M<sup>Σ</sup> is the initial concentration of monomers. As one can see from **Figure 1**, the dependencies should be linear in those coordinates and thus the calculation of the tangent coefficient of a line drawn through experimental points will point out what mechanism takes place in the given reaction. However, several tangent coefficients (for example, −1/2 which can be either the result of a fragmentation mechanism or a bifurcation mechanism without secondary nucleation) are special cases and can be obtained in different scenarios; for those cases, additional experiments are needed to exclude excess mechanism variants.

## **4. Polymorphism of amyloid fibrils**

The fully matured fibers are not the most toxic species. The importance of intermediate species that define the pathway, toxicity, and even the type of fibers formed at the end should be emphasized. Usually electron microscopy (EM) is used for comprehensive characterization of the amyloid formation and for control of the amyloid morphology. The probes are collected for analyzing periodically over duration of all experiments. The main results of EM experi‐ ments at each of the time intervals are the properties of time-dependent structures and their variability (polymorphism). These analyses usually are performed using both commercial and recombinant samples. The use of EM in studying the process of amyloid formation is due to the importance of polymorphism for determining the protein folding pathways. On the other hand, protein folding pathway depends strongly on its amino acid sequence. Further, the resulting protein conformation affects the formation of amyloid fibrils, and the same poly‐ peptide sequence may correspond to the fibrils of different morphologies [34]. The fibril polymorphism is potentially important for the human disease progression, as it may be the reason for natural variability of amyloid-related pathologies such as amyloidosis of light chains of immunoglobulins (AL) and the phenomenon of strain variability of prion infection [34, 35]. The EM images of amyloid fibrils show insignificant morphological differences, and the fibrils are often represented as twisted or parallel clusters of thin protein filaments. One should note also that polymorphism is a serious obstacle in studying fibril structures. A mixture of fibrils of different morphology is always present in solution, and several different types of fibrils can be seen under the same conditions. This creates a problem for image analyses because it requires separation of fibrils by their morphology and performing separate analyses of each type of insulin, Aβ peptide, β2-microglobulin, glucagon, amylin, calcitonin, etc. It is not completely excluded that different fibril morphologies are due to similar but not identical fibril formation pathways. The differences may be formed at the initial steps of fibril formation including the step of nucleation. The size of the nucleus of a protofibril is still unknown for almost all proteins and peptides. Nucleation is the limiting step of the amyloid fibril formation which can start only when the concentration of amyloid proteins exceeds the critical one. After the nucleation, the fibrils grow rapidly. Proteins may have different sizes of the protofibril nuclei (see the next sections). Also, the conditions at which the fibrils are formed may affect its size. This may result in the formation of the fibrils of different morphologies. In other words, we assume that the fibril formation depends on the nucleation step and goes through different pathways, thus determining the final fibril morphology. Note also that both the morphology and molecular structure are sensitive to small differences under conditions when the fibrils are growing in *de novo* preparations (under fixed pH, temperature, buffer composition, and peptide concentration).

## **4.1. Polymorphism of Aβ fibrils**

It was shown previously that peptide Aβ40 forms fibrils of different morphologies even under the same conditions. For the fibrils formed without mixing, the dominating morphologies are twisted fibrils, while the activated fibrils will be less twisted yet inclined to form lateral associations. The inactivated twisted fibrils are the most toxic for the cells [36]. Therefore, it is important to study all possible formations including oligomers and fibrils, which determine the natural polymorphism. Moreover, it was shown previously that the fibrils associated with diseases have the same nucleation center structure called a β-arcade. The β-strands in these structures are parallel to each other. It is interesting to note that this kind of a nucleus structure is not found in normal proteins and the β-strands are antiparallel to each other [37].

#### *4.1.1. Morphology of recombinant Aβ40 peptides*

**4. Polymorphism of amyloid fibrils**

1486 Exploring New Findings on Amyloidosis

peptide concentration).

**4.1. Polymorphism of Aβ fibrils**

The fully matured fibers are not the most toxic species. The importance of intermediate species that define the pathway, toxicity, and even the type of fibers formed at the end should be emphasized. Usually electron microscopy (EM) is used for comprehensive characterization of the amyloid formation and for control of the amyloid morphology. The probes are collected for analyzing periodically over duration of all experiments. The main results of EM experi‐ ments at each of the time intervals are the properties of time-dependent structures and their variability (polymorphism). These analyses usually are performed using both commercial and recombinant samples. The use of EM in studying the process of amyloid formation is due to the importance of polymorphism for determining the protein folding pathways. On the other hand, protein folding pathway depends strongly on its amino acid sequence. Further, the resulting protein conformation affects the formation of amyloid fibrils, and the same poly‐ peptide sequence may correspond to the fibrils of different morphologies [34]. The fibril polymorphism is potentially important for the human disease progression, as it may be the reason for natural variability of amyloid-related pathologies such as amyloidosis of light chains of immunoglobulins (AL) and the phenomenon of strain variability of prion infection [34, 35]. The EM images of amyloid fibrils show insignificant morphological differences, and the fibrils are often represented as twisted or parallel clusters of thin protein filaments. One should note also that polymorphism is a serious obstacle in studying fibril structures. A mixture of fibrils of different morphology is always present in solution, and several different types of fibrils can be seen under the same conditions. This creates a problem for image analyses because it requires separation of fibrils by their morphology and performing separate analyses of each type of insulin, Aβ peptide, β2-microglobulin, glucagon, amylin, calcitonin, etc. It is not completely excluded that different fibril morphologies are due to similar but not identical fibril formation pathways. The differences may be formed at the initial steps of fibril formation including the step of nucleation. The size of the nucleus of a protofibril is still unknown for almost all proteins and peptides. Nucleation is the limiting step of the amyloid fibril formation which can start only when the concentration of amyloid proteins exceeds the critical one. After the nucleation, the fibrils grow rapidly. Proteins may have different sizes of the protofibril nuclei (see the next sections). Also, the conditions at which the fibrils are formed may affect its size. This may result in the formation of the fibrils of different morphologies. In other words, we assume that the fibril formation depends on the nucleation step and goes through different pathways, thus determining the final fibril morphology. Note also that both the morphology and molecular structure are sensitive to small differences under conditions when the fibrils are growing in *de novo* preparations (under fixed pH, temperature, buffer composition, and

It was shown previously that peptide Aβ40 forms fibrils of different morphologies even under the same conditions. For the fibrils formed without mixing, the dominating morphologies are twisted fibrils, while the activated fibrils will be less twisted yet inclined to form lateral associations. The inactivated twisted fibrils are the most toxic for the cells [36]. Therefore, it is **Figure 2** shows the preparation of recombinant Aβ40 peptide after 27 h incubation at 25°C at 50 mM Tris–HCl (pH 7, 5) and C = 0.2 mg/ml. Polymorphism of generated fibrils is seen. In

**Figure 2.** Electron microscopy images of recombinant Aβ40 peptide [0.2 mg/mL, 27 h incubation at 25°C, 50 mM Tris-HCl (pH 7, 5)]. (a) General view (field) of recombinant Aβ40 peptide after 27 h of incubation. (b–d) Gallery of types of Aβ40 peptide fibril images at higher magnification: (b) single fibrils; (c) ribbon-like type of fibrils; (d) bundle-like type of fibrils; (e, f) single fibrils: (e) at horizontal packing; (f) bending sites of the single fibrils. (g) Gallery of ring oligomers.

addition to single fibrils of several microns in length with a diameter of about 8–9 nm, there are also fibrils, forming bands of different widths, and bundles. It seems that single fibrils consist of two filaments. However, under high magnification, it is seen that the fibrils are formed of rounded ring structures. The matter is that upon staining the preparations, the staining agent dyes not only their contours, but also flows into different cavities and openings. This should be taken into account when interpreting EM images; otherwise, a tobacco mosaic virus fibril would consist of two parallel packed short filaments of 300 nm long. In the case of the Aβ40 peptide preparation, under high magnification, it is also seen that the staining agent flows into the openings of ring structures. These ring oligomer structures have a diameter of about 8–9 nm (the width of a single fibril) and a diameter of the internal opening of about 3 nm. Ring structures or Aβ40 oligomers form single fibrils in such a way, that ring oligomers are layered on each other. If a fibril is absorbed on the formvar film sideways, we see a thinner fibril with a diameter of about 4 nm. This size demonstrates an approximate height of a ringstructured oligomer. As a matter of fact, the height is smaller, because the degree of overlap‐ ping of ring-structured oligomers should be taken into account. The greater is the overlapping, the larger is the height of such packing of fibrils.

## *4.1.2. Morphology of synthetic Aβ42 peptide (Invitrogen, lot #73269340A)*

The EM image of Aβ42 peptide (Invitrogen, lot #73269340A) (**Figure 3**) shows that after 97 h of incubation at 37°C (water), fibrils of several microns long are formed. Polymorphism of

**Figure 3.** Electron microscopy images of Aβ42 peptide from Invitrogen (lot #73269340A). The sample was incubated at 37°C (water). (a) General view of the sample at 97-h incubation. (b, c) Gallery of types of Aβ42 peptide fibrils at higher magnification: (b) single thin and (c) thick fibrils. (d) Gallery of ring oligomers.

fibrils is observed. Thin fibrils with a diameter of about 7–8 nm and more are seen, and more pronounced polymorphism of the generated fibrils is observed (**Figure 3**). At the same time, fibrils of different diameters are present: thin ones of about 7–8 nm and wider fibrils up to 15 nm. Moreover, rounded ring-structured oligomers of about 8 nm in diameter can be seen; the internal diameter of the ring (the diameter of the hole) is about 3 nm (**Figure 3d**). At magnification, it can be seen that an oligomer consists of 4–6 smaller rounded particles (**Figure 3d**). It is notable that both thin and thick fibrils are formed of such ring-structured oligomers. Thin fibrils are characterized by more ordered interaction of separate oligomers, while thick fibrils are generated due to random sticking of additional oligomers along the fibril axis. Because of this way of generation, fibrils look uneven in EM images with a tendency to branching [38].

Thus, using the EM method, we have demonstrated that fibrils of these peptides are formed by association of rounded ring structures. Negatively stained TEM images of fibrils extracted from AD brain tissues [39] are very similar to our EM results. Moreover, similar structures are observed elsewhere [40].

## **4.2. Polymorphism of insulin fibrils**

addition to single fibrils of several microns in length with a diameter of about 8–9 nm, there are also fibrils, forming bands of different widths, and bundles. It seems that single fibrils consist of two filaments. However, under high magnification, it is seen that the fibrils are formed of rounded ring structures. The matter is that upon staining the preparations, the staining agent dyes not only their contours, but also flows into different cavities and openings. This should be taken into account when interpreting EM images; otherwise, a tobacco mosaic virus fibril would consist of two parallel packed short filaments of 300 nm long. In the case of the Aβ40 peptide preparation, under high magnification, it is also seen that the staining agent flows into the openings of ring structures. These ring oligomer structures have a diameter of about 8–9 nm (the width of a single fibril) and a diameter of the internal opening of about 3 nm. Ring structures or Aβ40 oligomers form single fibrils in such a way, that ring oligomers are layered on each other. If a fibril is absorbed on the formvar film sideways, we see a thinner fibril with a diameter of about 4 nm. This size demonstrates an approximate height of a ringstructured oligomer. As a matter of fact, the height is smaller, because the degree of overlap‐ ping of ring-structured oligomers should be taken into account. The greater is the overlapping,

The EM image of Aβ42 peptide (Invitrogen, lot #73269340A) (**Figure 3**) shows that after 97 h of incubation at 37°C (water), fibrils of several microns long are formed. Polymorphism of

**Figure 3.** Electron microscopy images of Aβ42 peptide from Invitrogen (lot #73269340A). The sample was incubated at 37°C (water). (a) General view of the sample at 97-h incubation. (b, c) Gallery of types of Aβ42 peptide fibrils at higher

magnification: (b) single thin and (c) thick fibrils. (d) Gallery of ring oligomers.

the larger is the height of such packing of fibrils.

1508 Exploring New Findings on Amyloidosis

*4.1.2. Morphology of synthetic Aβ42 peptide (Invitrogen, lot #73269340A)*

Insulin has a high tendency to aggregation and forms fibrils. Insulin fibrils were first described in 1940 by Waugh [41]. At present, the conditions leading to the formation of active insulin fibrils are known [42]. Insulin fibrillation occurs upon various environmental changes (pH, ionic conditions, temperature, organic solvents, and agitation); it also depends on the methods for producing insulin, its delivery, and storage. It should be noted that the human insulin fibrillation is slower than that of the best studied bovine insulin [42–47].

Currently, all insulin preparations for therapeutic purposes are prepared in the form of a Zncontaining hexamer, since in this condition it is more stable and resistant to the formation of fibrils. When treating diabetes, insulin penetrates into muscles by injection, where it typically forms deposits. The large size of the insulin hexamer slows its adsorption, because the hexamer must go through the dimers in the monomeric state and only in this way penetrate into the bloodstream. This leads to slowing down the interaction with insulin receptors and the need to take insulin 1–1.5 h before mealtime.

Despite intensive studies of fibrillogenesis of insulin, there is no generally accepted scheme for the formation of mature fibrils. The main difficulties arise in interpreting the beginning of the fibrillation process. Polymerization of insulin begins from the transition of the monomer protein from the native state to the partially unfolded conformation which can be amyloido‐ genic. The strong dependence of the reaction of polymerization on various external parameters leads to the formation of insulin fibrils with different morphology, which complicates the analysis of the general scheme of fibril formation.

For electron microscopy (EM) investigations, insulin samples were used at a concentration of 0.2 mg/ml. Zinc-free recombinant human insulin was obtained from "Gerofarm-Bio" (Obo‐ lensk, Russia) and was checked by the tandem mass spectrometry technique (LCQ Deca XP, Thermo Finnigan, USA). According to the EM data on polymerization of human insulin, the mature protein fibrils appeared only after 8 h of insulin incubation. Active polymerization of insulin occurs up to 10–11 h of incubation. This is accompanied by the elongation of the fibrils for both samples up to 10–12 μm. The fibrils interact laterally with each other, resulting in the formation of big clusters composed from ribbons, bundles, and twisting bundles (**Figure 4**). Protein aggregation is the result of increasing the incubation time up to 24 h. According to the EM data, the number of large fibril clusters increases in the samples after 24 h, but the length of the fibrils does not change (remains about 10–12 μm) [48].

**Figure 4.** Electron microscopy images of human zinc-free recombinant insulin (0.2 mg/ml, 24 h incubation at 37°C, 20% acetic acid (pH 2.0), 140 mM NaCl). (a) General view (field) of human insulin after 24 h of incubation. (b–d) Gallery of images of types of insulin fibrils at higher magnification: (b) single fibrils; (c) ribbon-like type of fibrils; (d) bundle-type of fibrils. (e, f) The thinnest fibrils: (e) at horizontal packing; (f) at vertical packing of ring oligomers.

## **5. Example of amyloid growth by the linear nucleation-elongation mechanism (actin, apolipoprotein C-II)**

Most quantitative models for linear polymerization stem from the work performed more than half a century ago [20] proposing a kinetic model for actin polymerization. Only two of the proteins (apolipoprotein C-II [49]: Lrel < 0.1 with the primary nucleus size *n*\* ≈ 4–5 and actin with *n*\* ≈ 3), studied in our analysis, exhibit linear growth. Formation of actin filaments occurs by linear growth—fibrils increase only due to attachment of monomers to the aggregate ends. This is supported by literature data [50] and by analysis of kinetic data (**Figure 1**). According to the theory advanced in [33] for linear growth (observed in actin), the characteristic time of the relative lag-period Lrel does not exceed 0.2 and is independent of ln[MΣ] as in the case of actin (**Figure 5**) [50]. The size of the primary nucleus was calculated from the slope to be *n* = 3.46 ± 0.28.

mature protein fibrils appeared only after 8 h of insulin incubation. Active polymerization of insulin occurs up to 10–11 h of incubation. This is accompanied by the elongation of the fibrils for both samples up to 10–12 μm. The fibrils interact laterally with each other, resulting in the formation of big clusters composed from ribbons, bundles, and twisting bundles (**Figure 4**). Protein aggregation is the result of increasing the incubation time up to 24 h. According to the EM data, the number of large fibril clusters increases in the samples after 24 h, but the length

**Figure 4.** Electron microscopy images of human zinc-free recombinant insulin (0.2 mg/ml, 24 h incubation at 37°C, 20% acetic acid (pH 2.0), 140 mM NaCl). (a) General view (field) of human insulin after 24 h of incubation. (b–d) Gallery of images of types of insulin fibrils at higher magnification: (b) single fibrils; (c) ribbon-like type of fibrils; (d) bundle-type

Most quantitative models for linear polymerization stem from the work performed more than half a century ago [20] proposing a kinetic model for actin polymerization. Only two of the proteins (apolipoprotein C-II [49]: Lrel < 0.1 with the primary nucleus size *n*\* ≈ 4–5 and actin

of fibrils. (e, f) The thinnest fibrils: (e) at horizontal packing; (f) at vertical packing of ring oligomers.

**mechanism (actin, apolipoprotein C-II)**

**5. Example of amyloid growth by the linear nucleation-elongation**

of the fibrils does not change (remains about 10–12 μm) [48].

15210 Exploring New Findings on Amyloidosis

**Figure 5.** (a) Dependence of normalized ThT fluorescence on time for actin [50]. (b) Dependence of Lrel on lnT2. (c) De‐ pendence of Lrel on ln[MΣ]. The slope angle of the fitted line is about −0.01, that is, Lrel is independent of ln[MΣ]. All Lrel values are also <0.2 which, according to the models of formation of fibrillar aggregates, means that the formation of actin fibrils proceeds by the linear mechanism (see [33]). The errors are computed with the Student's coefficients corre‐ sponding to 0.95 confidence level.

## **6. Examples of amyloid growth by the exponential mechanism and sizes of folding nuclei of fibrils formed by different agents**

In order to estimate the size of fibril nucleus (the most unstable state on the monomer to the fibril pathway) and a possible scenario for the formation of aggregates, it is necessary to make a number of kinetic experiments, where the only variable parameter is the monomer concen‐ tration. Characteristic times Tlag (the lag-period duration), T2 (the time of transition of all monomers into an aggregate), and Lrel (the Tlag/T2 ratio) are calculated for each experimental curve. It was demonstrated that the dependences of lnT2 and Lrel on ln[MΣ] (the logarithm of the initial concentration of monomers) are linear, with gradients that can be used for the computation of fibril nucleus sizes (including those of non-amyloid type) and for elucidation of the mechanism of aggregate formation.

If Lrel > 0.2, the linear growth model is excluded, and, independently of lnT2 and Lrel dependence on ln[MΣ], only the exponential growth scenario remains. The dependences of lnT2 on ln[MΣ] and Lrel on ln[MΣ] are formally different for different exponential scenarios (i.e., "growth from the surface," "fragmentation," and "bifurcation"). However, it is impossible, for example, to distinguish the bifurcation from the fragmentation scenario from the kinetics *alone* if the size

*n*<sup>2</sup> of the secondary nucleus of bifurcation is zero; this implies the need for direct observations of fibril shapes to distinguish these cases (see Refs. [33, 48] for more details).

#### **6.1. Insulin**

Now we can present three general schemes of insulin amyloid formation, which differ by the size of folding nuclei of fibrils. For the first type, the size of nucleus is one monomer [44, 51– 53]. For the second scheme of fibril formation, the size of nucleus is two monomers and the dimers are precursors of fibrils [5, 54, 55]. For the third scheme of polymerization, oligomers are precursors of fibrils, and the size of oligomer can vary from three to six monomers [42, 43, 56–58].

The basis for choosing between different mechanisms of formation of fibrils is to detect various soluble oligomers. However, at low concentrations and at early times, it is difficult to accom‐ plish accurate detection of the initial aggregates. For this purpose, a variety of optical methods, as well as Cryo-transmission-EM, atom force microscopy, and small-angle X-ray scattering are used [55, 57–59]. However, all these methods are not quantitative, have no reliable standards, do not provide the full size distribution, may yield abnormal results (with microscopic approaches—interaction with the surface during adsorption of samples), and require the analysis of the results of certain programs to prove the statistical significance [52]. Although all these methods provide valuable information, none of them can convincingly demonstrate how an unstable nucleus grows to a stable seed for further growth of protofibrils, and many experimental and theoretical models are based on this assumption.

**Figure 6.** (a) Dependence of normalized ThT fluorescence on time for insulin [48]. The approximation line calculated for insulin amyloid formation with the experimental data from (a) by the least-squares method plotted in coordinates (b): {lnT2, ln[MΣ]} and (c): {Lrel, ln[MΣ]}; [MΣ] is in mg/ml, T2 in min. The tangent coefficients of the approximation lines are −0.51 ± 0.13 for d(lnT2)/d(ln[MΣ]) and 0.15 ± 0.31 for d(Lrel)/d(ln[MΣ]); the errors are computed with the Student's coefficients corresponding to 0.95 confidence level.

The growth of amyloid fibrils was studied by thioflavin T (ThT) fluorescence, which drastically increases when ThT is bound to fibrils (**Figure 6a**). The renormalized curves, presented in **Figure 6a**, are very similar to one another. They have large lag periods, thus excluding the linear growth scenario. The obtained analytical solution and computer modeling allowed us to determine the size of the nucleus from the experimentally obtained concentration depend‐ ences of the relationship between the lag-time duration and the time of growth of amyloid fibrils [33]. **Figure 6b, c** illustrates determination of the fibrillation scenario and the nuclei sizes (*n\*, n2*) for human insulin (see **Table 2**) [48]. The value *d*(lnT2)/*d*(ln[MΣ]) ≈ −0.5 is inconsistent with the "growth from the surface" model but consistent with "linear growth" (which, though, should have Lrel < 0.2, which is inconsistent with Lrel), "fragmentation" (which, though, is inconsistent with EM images shown in **Figure 4**), and "bifurcation" (consistent with the EM images), with the secondary nucleus size *n*2 = 0 calculated as −1 − *d*(lnT2)/*d*(ln[MΣ]); the primary nucleus size *n*\* = 1 + *n*2 − *d*(Lrel)/*d*(ln[MΣ]) = 1 (see **Table 2**). According to the elaborated theory, it means that the size of the primary nucleus corresponds to one monomer.


**Table 2.** The range of Lrel and lnT2 values, the sizes of the primary (*n*\*) and secondary (*n*2) nuclei and the amyloid growth scenarios obtained from experimental kinetic data alone.

#### **6.2. LysPro human analog insulin**

*n*<sup>2</sup> of the secondary nucleus of bifurcation is zero; this implies the need for direct observations

Now we can present three general schemes of insulin amyloid formation, which differ by the size of folding nuclei of fibrils. For the first type, the size of nucleus is one monomer [44, 51– 53]. For the second scheme of fibril formation, the size of nucleus is two monomers and the dimers are precursors of fibrils [5, 54, 55]. For the third scheme of polymerization, oligomers are precursors of fibrils, and the size of oligomer can vary from three to six monomers [42, 43,

The basis for choosing between different mechanisms of formation of fibrils is to detect various soluble oligomers. However, at low concentrations and at early times, it is difficult to accom‐ plish accurate detection of the initial aggregates. For this purpose, a variety of optical methods, as well as Cryo-transmission-EM, atom force microscopy, and small-angle X-ray scattering are used [55, 57–59]. However, all these methods are not quantitative, have no reliable standards, do not provide the full size distribution, may yield abnormal results (with microscopic approaches—interaction with the surface during adsorption of samples), and require the analysis of the results of certain programs to prove the statistical significance [52]. Although all these methods provide valuable information, none of them can convincingly demonstrate how an unstable nucleus grows to a stable seed for further growth of protofibrils, and many

**Figure 6.** (a) Dependence of normalized ThT fluorescence on time for insulin [48]. The approximation line calculated for insulin amyloid formation with the experimental data from (a) by the least-squares method plotted in coordinates (b): {lnT2, ln[MΣ]} and (c): {Lrel, ln[MΣ]}; [MΣ] is in mg/ml, T2 in min. The tangent coefficients of the approximation lines are −0.51 ± 0.13 for d(lnT2)/d(ln[MΣ]) and 0.15 ± 0.31 for d(Lrel)/d(ln[MΣ]); the errors are computed with the Student's

The growth of amyloid fibrils was studied by thioflavin T (ThT) fluorescence, which drastically increases when ThT is bound to fibrils (**Figure 6a**). The renormalized curves, presented in **Figure 6a**, are very similar to one another. They have large lag periods, thus excluding the linear growth scenario. The obtained analytical solution and computer modeling allowed us to determine the size of the nucleus from the experimentally obtained concentration depend‐

of fibril shapes to distinguish these cases (see Refs. [33, 48] for more details).

experimental and theoretical models are based on this assumption.

coefficients corresponding to 0.95 confidence level.

**6.1. Insulin**

15412 Exploring New Findings on Amyloidosis

56–58].

We have investigated the kinetics of formation of fibrils by monomeric rapid-acting insulin LysPro [48]. According to our data, fibril formation of the insulin analog at pH 2 proceeds by

**Figure 7.** (a) Dependence of normalized ThT fluorescence on time for LysPro insulin [48] The approximation line calcu‐ lated for LysPro insulin amyloid formation with experimental data from (a) by the least-squares method plotted in co‐ ordinates (b): {lnT2, ln[MΣ]} and (c): {Lrel, ln[MΣ]}; [MΣ] is in mg/ml, T2 in min. The tangent coefficients of the approximation lines are −0.35 ± 0.25 for d(lnT2)/d(ln[MΣ]) and 0.07 ± 0. 80 for d(Lrel)/d(ln[MΣ]); the errors are computed with the Student's coefficients corresponding to 0.95 confidence level.

5 h longer than that of the recombinant human insulin. These data seem to us important because the rate of aggregation of insulin preparations is an important health problem not only in the production and storage of insulin, but also for its frequent injections. In this regard, the process of fibrillation should be analyzed for all developed analogs, already prepared for clinical trials and for mass production. Examination of EM images of growing insulin LysPro fibrils shows that they occurred with a kind of the "bifurcation plus lateral growth" scenario [48]. The growth of amyloid fibrils was studied (**Figure 7**) by thioflavin T (ThT) fluorescence, which drastically increases when ThT is bound to fibrils. The dependence of Lrel and lnT2 on ln[MΣ] (**Figure 7**) was used to estimate sizes of the primary and secondary nuclei. Similar to insulin fibrils, the size of the first nucleus is one monomer, and the size of the secondary nucleus is zero (see **Table 2**). It should be noted that "one monomer" may correspond to the oligomer structure consisting of 2–6 monomers, if we did not start from the pure monomers in solution.

#### **6.3. Aβ40**

The experimental kinetics data on the formation of amyloid aggregates, which we used to calculate the sizes of nuclei of amyloid protofibrils formed by Aβ40, were taken from paper [60]. For all the experimental data, we obtained Lrel >> 0.2, which means that the process goes by the exponential mechanism of fibril formation (**Figure 8**). For Aβ40, we obtained that the size of the primary nucleus is two monomers, and the secondary nucleus size is one monomer.

**Figure 8.** (a) Dependence of normalized ThT fluorescence on time for Aβ40 [60]. The approximation [Insert figure cap‐ tion here] line calculated for Aβ40 amyloid formation with experimental data from (a) by the least-squares method plotted in coordinates (b): {lnT2, ln[MΣ]} and (c): {Lrel, ln[MΣ]}; [MΣ] is in μM, T2 in hours. The tangent coefficients of the approximation lines are −0.79 ± 0.18 for d(lnT2)/d(ln[MΣ]) and −0.03 ± 0.32 for d(Lrel)/d(ln[MΣ]); the errors are computed with the Student's coefficients corresponding to 0.95 confidence level.

#### **6.4. Aβ42**

The exponential growth with branching takes place for both Aβ40 and Aβ42 peptides (see **Figures 8** and **9**). Interestingly, according to our analysis, for Aβ42, the size of the primary nucleus of the amyloid protofibril is larger than that of Aβ40, although the mechanism of amyloid fibril formation must be similar. This is consistent with the recent data, which compares the behavior of the two peptides during the aggregation process [60]. The primary nucleus size for Aβ42 is three monomers, and the secondary nucleus size is two monomers.

**Figure 9.** (a) Dependence of normalized ThT fluorescence on time for Aβ42 [61]. The approximation line calculated for Aβ42 amyloid formation with experimental data from (a) by the least-squares method plotted in coordinates (b): {lnT2, ln[MΣ]} and (c): {Lrel, ln[MΣ]}; [MΣ] is in μM, T2 in hours. The tangent coefficients of the approximation lines are −1.48 ± 0.14 for d(lnT2)/d(ln[MΣ]) and 0.22 ± 0.13 for d(Lrel)/d(ln[MΣ]); the errors are computed with the Student's coeffi‐ cients corresponding to 0.95 confidence level.

#### **6.5. Human cardiac titin immunoglobulin domain I27 (TI27)**

5 h longer than that of the recombinant human insulin. These data seem to us important because the rate of aggregation of insulin preparations is an important health problem not only in the production and storage of insulin, but also for its frequent injections. In this regard, the process of fibrillation should be analyzed for all developed analogs, already prepared for clinical trials and for mass production. Examination of EM images of growing insulin LysPro fibrils shows that they occurred with a kind of the "bifurcation plus lateral growth" scenario [48]. The growth of amyloid fibrils was studied (**Figure 7**) by thioflavin T (ThT) fluorescence, which drastically increases when ThT is bound to fibrils. The dependence of Lrel and lnT2 on ln[MΣ] (**Figure 7**) was used to estimate sizes of the primary and secondary nuclei. Similar to insulin fibrils, the size of the first nucleus is one monomer, and the size of the secondary nucleus is zero (see **Table 2**). It should be noted that "one monomer" may correspond to the oligomer structure consisting of 2–6 monomers, if we did not start from the pure monomers in solution.

The experimental kinetics data on the formation of amyloid aggregates, which we used to calculate the sizes of nuclei of amyloid protofibrils formed by Aβ40, were taken from paper [60]. For all the experimental data, we obtained Lrel >> 0.2, which means that the process goes by the exponential mechanism of fibril formation (**Figure 8**). For Aβ40, we obtained that the size of the primary nucleus is two monomers, and the secondary nucleus size is one monomer.

**Figure 8.** (a) Dependence of normalized ThT fluorescence on time for Aβ40 [60]. The approximation [Insert figure cap‐ tion here] line calculated for Aβ40 amyloid formation with experimental data from (a) by the least-squares method plotted in coordinates (b): {lnT2, ln[MΣ]} and (c): {Lrel, ln[MΣ]}; [MΣ] is in μM, T2 in hours. The tangent coefficients of the approximation lines are −0.79 ± 0.18 for d(lnT2)/d(ln[MΣ]) and −0.03 ± 0.32 for d(Lrel)/d(ln[MΣ]); the errors are computed

The exponential growth with branching takes place for both Aβ40 and Aβ42 peptides (see **Figures 8** and **9**). Interestingly, according to our analysis, for Aβ42, the size of the primary nucleus of the amyloid protofibril is larger than that of Aβ40, although the mechanism of amyloid fibril formation must be similar. This is consistent with the recent data, which

with the Student's coefficients corresponding to 0.95 confidence level.

**6.3. Aβ40**

15614 Exploring New Findings on Amyloidosis

**6.4. Aβ42**

To determine the sizes of folding nuclei for amyloid formation, calculations were done using the data of aggregation kinetics for the I27 immunoglobulin domain from human cardiac titin (TI27) [62]. The set of data was digitized, and in the case of TI27, part of the curve was excluded from the analysis. It was found that the growth of amyloids formed by TI27 is exponential, that is, in addition to the primary core nucleation secondary nucleation by a branching mechanism also takes place, accelerating the formation of new fibrils.

**Figure 10.** (a) Dependence of normalized ThT fluorescence on time for TI27 [62]. (b) Dependence of lnT2 on ln[MΣ] for TI27. This was used to calculate the size of the secondary nucleus. The slope of the fitted line is about −1.26 which, according to formulas from [33], indicates that the size of the secondary nucleus is n2 = 1.52 ± 0.36. (c) Dependence of Lrel on ln[MΣ] for TI27. The slope of the fitted line is −0.1, that is, Lrel is dependent on ln[MΣ]. Some Lrel values exceed 0.2 and, according to [33], cannot be explained by the linear model. The size of the primary nucleus was calculated based on the size of the secondary nucleus, and the slope of the straight line is 2.42 ± 0.46. The errors are computed with the Student's coefficients corresponding to 0.95 confidence level.

It is seen from **Figure 10c** that though the values of Lrel for TI27 do not exceed 0.2, Lrel depends on ln[MΣ] and so, as mentioned above, cannot occur by the linear mechanism. This effect can be explained by the presence of second nucleation cores with n2 = 1.52 ± 0.36 (**Figure 10b**). It is interesting that in spite of the difference in the aggregation mechanism, the size of the primary nucleation core for TI27 was 2.42 ± 0.46 (**Figure 10c**) similar (within error) to the primary nucleation core, 3.46 ± 0.28 (**Figure 5**), calculated for actin [50].

## **7. Concluding remarks**

Amyloid fibril polymorphism suggests various ways of their formation from monomers through different oligomers (intermediates of various sizes and shapes) to mature fibrils of different morphology. It is very likely that the surface of the formed fibrils can serve as a matrix for lateral polymerization of monomers/oligomers on it, as reported in the literature [47, 63, 64]. The above complicates the explanation of the mechanism of formation of fibrils and the proposal of a single scheme of polymerization for many proteins. It should be noted that a combination of several mechanisms is quite possible and can be observed sometimes in experiments.

Thus, despite some common features of formation of fibrils of different proteins, each of them has its own specific route for fibrillogenesis, which is originally determined by the amino acid sequence of the protein and is strongly dependent on environmental conditions and seed addition. It is clear that there is need for a serious analysis of data on various proteins to explain various routes for fibril formation that will help identify conditions that affect the initial formation paths (lag time) and find ways to block the entire process, which is an important task in the prevention and treatment of diseases associated with amyloid deposits.

A detailed study of molecular and cellular processes underlying the development of protein‐ opathy, search for targets for drug development and the use of the obtained data in the development of methods for early diagnosis are essential for the successful treatment and prevention of common diseases. Due to the new generation of neuroprotective drugs acting directly on the pathogenesis of the disease, all components of the cascade of pathological key protein aggregation proteinopathy can be regarded as potential targets. We believe that the combination of theoretical and experimental approaches employed throughout our research is particularly useful and productive for obtaining new important results.

## **Acknowledgements**

We thank T.B. Kuvshinkina, E.I. Grigorashvili, M. Yu. Suvorina, A.V. Finkelstein, and A.K. Surin for assistance in the manuscript preparation. This study was supported by the Russian Science Foundation No. 14-14-00536.

## **Author details**

It is seen from **Figure 10c** that though the values of Lrel for TI27 do not exceed 0.2, Lrel depends on ln[MΣ] and so, as mentioned above, cannot occur by the linear mechanism. This effect can be explained by the presence of second nucleation cores with n2 = 1.52 ± 0.36 (**Figure 10b**). It is interesting that in spite of the difference in the aggregation mechanism, the size of the primary nucleation core for TI27 was 2.42 ± 0.46 (**Figure 10c**) similar (within error) to the primary

Amyloid fibril polymorphism suggests various ways of their formation from monomers through different oligomers (intermediates of various sizes and shapes) to mature fibrils of different morphology. It is very likely that the surface of the formed fibrils can serve as a matrix for lateral polymerization of monomers/oligomers on it, as reported in the literature [47, 63, 64]. The above complicates the explanation of the mechanism of formation of fibrils and the proposal of a single scheme of polymerization for many proteins. It should be noted that a combination of several mechanisms is quite possible and can be observed sometimes in

Thus, despite some common features of formation of fibrils of different proteins, each of them has its own specific route for fibrillogenesis, which is originally determined by the amino acid sequence of the protein and is strongly dependent on environmental conditions and seed addition. It is clear that there is need for a serious analysis of data on various proteins to explain various routes for fibril formation that will help identify conditions that affect the initial formation paths (lag time) and find ways to block the entire process, which is an important

A detailed study of molecular and cellular processes underlying the development of protein‐ opathy, search for targets for drug development and the use of the obtained data in the development of methods for early diagnosis are essential for the successful treatment and prevention of common diseases. Due to the new generation of neuroprotective drugs acting directly on the pathogenesis of the disease, all components of the cascade of pathological key protein aggregation proteinopathy can be regarded as potential targets. We believe that the combination of theoretical and experimental approaches employed throughout our research

We thank T.B. Kuvshinkina, E.I. Grigorashvili, M. Yu. Suvorina, A.V. Finkelstein, and A.K. Surin for assistance in the manuscript preparation. This study was supported by the Russian

task in the prevention and treatment of diseases associated with amyloid deposits.

is particularly useful and productive for obtaining new important results.

nucleation core, 3.46 ± 0.28 (**Figure 5**), calculated for actin [50].

**7. Concluding remarks**

15816 Exploring New Findings on Amyloidosis

experiments.

**Acknowledgements**

Science Foundation No. 14-14-00536.

Oxana V. Galzitskaya\* , Nikita V. Dovidchenko and Olga M. Selivanova

\*Address all correspondence to: ogalzit@vega.protres.ru

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, Russia

## **References**


[11] Ivanova MI, Sawaya MR, Gingery M, Attinger A, Eisenberg D. An amyloid-forming segment of beta2-microglobulin suggests a molecular model for the fibril. Proc. Natl.

[12] Tsolis AC, Papandreou NC, Iconomidou VA, Hamodrakas SJ. A consensus method for the prediction of "aggregation-prone" peptides in globular proteins. Plos One.

[13] Walsh I, Seno F, Tosatto SCE, Trovato A. PASTA 2.0: an improved server for protein

[14] Fernandez-Escamilla A-M, Rousseau F, Schymkowitz J, Serrano L. Prediction of sequence-dependent and mutational effects on the aggregation of peptides and

[15] Emily M, Talvas A, Delamarche C. MetAmyl: a METa-predictor for AMYLoid proteins.

[16] Maurer-Stroh S, Debulpaep M, Kuemmerer N, Lopez de la Paz M, Martins IC, Reumers J, et al. Exploring the sequence determinants of amyloid structure using position-

[17] Garbuzynskiy SO, Lobanov MY, Galzitskaya OV. FoldAmyloid: a method of prediction of amyloidogenic regions from protein sequence. Bioinform. Oxf. Engl. 2010;26:326–32.

[18] Ahmed AB, Znassi N, Château M-T, Kajava AV. A structure-based approach to predict predisposition to amyloidosis. Alzheimers Dement. J. Alzheimers Assoc. 2015;11:681–

[19] Dovidchenko NV, Galzitskaya OV. Computational approaches to identification of aggregation sites and the mechanism of amyloid growth. Adv. Exp. Med. Biol.

[20] Oosawa F, Asakura S, Hotta K, Imai N, Ooi T. G–F transformation of actin as a fibrous

[21] Hofrichter J, Ross PD, Eaton WA. Kinetics and mechanism of deoxyhemoglobin S gelation: a new approach to understanding sickle cell disease. Proc. Natl. Acad. Sci. U.

[22] Ferrone FA, Hofrichter J, Sunshine HR, Eaton WA. Kinetic studies on photolysisinduced gelation of sickle cell hemoglobin suggest a new mechanism. Biophys. J.

[23] Frieden C, Goddette DW. Polymerization of actin and actin-like systems: evaluation of the time course of polymerization in relation to the mechanism. Biochemistry.

[24] Goldstein RF, Stryer L. Cooperative polymerization reactions. Analytical approxima‐ tions, numerical examples, and experimental strategy. Biophys. J. 1986;50:583–99.

aggregation prediction. Nucleic Acids Res. 2014;42:W301–7.

specific scoring matrices. Nat. Methods. 2010;7:237–42.

Acad. Sci. U. S. A. 2004;101:10584–9.

proteins. Nat. Biotechnol. 2004;22:1302–6.

condensation. J. Polym. Sci. 1959;37:323–36.

Plos One. 2013;8:e79722.

90.

2015;855:213–39.

S. A. 1974;71:4864–8.

1980;32:361–80.

1983;22:5836–43.

2013;8:e54175.

16018 Exploring New Findings on Amyloidosis


[53] Brange J, Andersen L, Laursen ED, Meyn G, Rasmussen E. Toward understanding insulin fibrillation. J. Pharm. Sci. 1997;86:517–25.

[40] Lashuel HA, Hartley DM, Petre BM, Wall JS, Simon MN, Walz T, et al. Mixtures of wild-type and a pathogenic (E22G) form of Abeta40 in vitro accumulate protofibrils,

[41] Waugh DF. Regeneration of insulin from insulin fibrils by the action of alkali. J. Am.

[42] Nielsen L, Khurana R, Coats A, Frokjaer S, Brange J, Vyas S, et al. Effect of environ‐ mental factors on the kinetics of insulin fibril formation: elucidation of the molecular

[43] Nielsen L, Frokjaer S, Brange J, Uversky VN, Fink AL. Probing the mechanism of insulin

[44] Nielsen L, Frokjaer S, Carpenter JF, Brange J. Studies of the structure of insulin fibrils by Fourier transform infrared (FTIR) spectroscopy and electron microscopy. J. Pharm.

[45] Dzwolak W, Ravindra R, Lendermann J, Winter R. Aggregation of bovine insulin probed by DSC/PPC calorimetry and FTIR spectroscopy. Biochemistry. 2003;42:11347–

[46] Jansen R, Grudzielanek S, Dzwolak W, Winter R. High pressure promotes circularly

[47] Jansen R, Dzwolak W, Winter R. Amyloidogenic self-assembly of insulin aggregates probed by high resolution atomic force microscopy. Biophys. J. 2005;88:1344–53.

[48] Selivanova OM, Suvorina MY, Dovidchenko NV, Eliseeva IA, Surin AK, Finkelstein AV, et al. How to determine the size of folding nuclei of protofibrils from the concen‐ tration dependence of the rate and lag-time of aggregation. II. Experimental application for insulin and LysPro insulin: aggregation morphology, kinetics, and sizes of nuclei.

[49] Binger KJ, Pham CLL, Wilson LM, Bailey MF, Lawrence LJ, Schuck P, et al. Apolipo‐ protein C-II amyloid fibrils assemble via a reversible pathway that includes fibril

[50] Sept D, McCammon JA. Thermodynamics and kinetics of actin filament nucleation.

[51] Brange J, Dodson GG, Edwards DJ, Holden PH, Whittingham JL. A model of insulin fibrils derived from the X-ray crystal structure of a monomeric insulin (despentapep‐

[52] Pease LF, Sorci M, Guha S, Tsai D-H, Zachariah MR, Tarlov MJ, et al. Probing the nucleus model for oligomer formation during insulin amyloid fibrillogenesis. Biophys.

fibril formation with insulin mutants. Biochemistry. 2001;40:8397–409.

including amyloid pores. J. Mol. Biol. 2003;332:795–808.

mechanism. Biochemistry. 2001;40:6036–46.

shaped insulin amyloid. J. Mol. Biol. 2004;338:203–6.

breaking and rejoining. J. Mol. Biol. 2008;376:1116–29.

J. Phys. Chem. B. 2014;118:1198–206.

tide insulin). Proteins. 1997;27:507–16.

Biophys. J. 2001;81:667–74.

J. 2010;99:3979–85.

Chem. Soc. 1948;70:1850–7.

16220 Exploring New Findings on Amyloidosis

Sci. 2001;90:29–37.

55.


## **Role of Glycation in Amyloid: Effect on the Aggregation Process and Cytotoxicity**

Clara Iannuzzi, Gaetano Irace and Ivana Sirangelo

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62995

#### **Abstract**

Although the aggregation process of amyloidogenic proteins has been widely studied in vitro and many physiological factors have been identified, the molecular mecha‐ nisms underlying the formation of aggregates in vivo and under pathological conditions are still poorly understood. Post‐translational modifications are known to affect protein structure and function. Some of these modifications might affect proteins in detrimental ways and lead to their misfolding and accumulation. Reducing sugars play an important role in modifying proteins, forming advanced glycation end‐ products (AGEs) in a nonenzymatic process, called glycation. Recently, much attention has been devoted to the role played by glycation in stimulating amyloid aggregation and cellular toxicity. Proteins in amyloid deposits are often found glycated, suggest‐ ing a direct correlation between protein glycation and amyloidosis.

AGE products increase in aging and are considered a marker for several diseases such as Alzheimer disease and diabetes. In addition to directly affecting the protein structure and function, AGEs also induce cellular toxicity.

This chapter focuses on the molecular effects induced by glycation in the amyloid aggregation of several protein models. In particular, both the structural effects induced by glycation and their consequence on cellular toxicity will be extensively described.

**Keywords:** amyloid aggregation, protein glycation, AGEs, cellular toxicity

## **1. Introduction**

Reducing sugars play an important role in modifying proteins, forming advanced glycation end‐products (AGEs) in a nonenzymatic process, called glycation. This process is different

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

from glycosylation; indeed, these two post‐translational modifications affect the structure of the target protein in a different way. Glycosylation is a selective protein modification, driven by specific enzymes, that is generally associated with a gain of function (or stabilization) of the target protein. Nonenzymatic glycation is a nonselective modification, and it is generally associated with a loss of function of the target protein due to modifications of its native structure. While glycosylation is a well‐controlled cellular mechanism, nonenzymatic glycation depends on the exposure of free amino groups in the polypeptide chain, concentra‐ tion of the sugar, and oxidative conditions. Glycation is a pathological process that is highly relevant in diabetes patients, as it plays a crucial role not only in diabetic complications but also in the normal aging process. Increasing evidence suggests a link between diabetes and neurodegenerative processes such as Alzheimer and Parkinson diseases [1]. In this respect, much attention has been recently devoted to the role played by nonenzymatic glycation of proteins in stimulating amyloid aggregation and toxicity. The observation that proteins in amyloid deposits, such as *β*‐amyloid, tau, prions,transthyretin, and *β*2‐microglobulin, are often found glycated in patients suggests a direct correlation between protein glycation and amyloid formation [2–8]. This is thought to be associated with the formation of cross‐links that stabilize protein aggregates. Indeed, AGEs formation can not only interfere with the regular function‐ ing of the proteins to which they are attached but also induce the formation of covalent cross‐ links with close proteins. In addition, glycation can affect the protein degradation process [9], and, being an abnormal modification, it has been found to induce some proteins to misfold and, thus, promote protein aggregation [10–12].

Moreover, the AGE‐modified proteins are tightly involved in physiopathological cellular mechanisms. Once formed, AGEs interact with specific cellular receptors leading to the activation of different signaling pathways. The most studied AGE‐receptor, known as RAGE, is a multiligand receptor belonging to the immunoglobulin superfamily [13, 14]. The activa‐ tion of RAGE regulates key cellular processes such as inflammation, apoptosis, proliferation, autophagy, and recently it has been associated with the pathogenesis of amyloidosis [15, 16]. In neurons, glia, and endothelial cells, RAGE is also the binding site for A*β* peptide on the cell surface and mediates Alzheimer's disease pathology [15, 17, 18].

## **2. Protein glycation**

The nonenzymatic glycation of protein amino groups by reducing sugars (also called Mail‐ lard reaction) is a chemical reaction common to all cell types: glycated products slowly ac‐ cumulate in vivo, leading to several different protein dysfunctions caused by alterations of their integrity [19, 20]. The process begins with a nucleophilic addition reaction between a free amino group of a protein and a carbonyl group of a reducing sugar, forming a reversi‐ ble intermediate product (Schiff's base). Side‐chains of arginine and lysine residues and the N‐terminal amino group of proteins are the main targets of protein glycation. The process depends on several conditions, such as the concentration and reactivity of the glycation agent, the presence of catalytic factors (metals, buffer ions, and oxygen), the physiological pH, temperature, and the half‐life of each protein. All reducing sugars can participate in

glycation reactions and, among them, D‐ribose is the most active form, and its intracellular level can be quite high. D‐glucose is the less reactive form and its intracellular concentration is negligible, while dicarbonyl compounds, such as methylglyoxal and glyoxal, are far more reactive. These compounds are intermediates of glycation reaction, but can also be generat‐ ed by various oxidative processes and be formed through other metabolic pathways such as glycolysis and catabolism of threonine and ketone bodies. The levels of D‐ribose in the blood are estimated around 20 mg/L in healthy individuals, while that of D‐glucose are around 6–10 g/L. Once formed, the Schiff's base can convert into a stable ketoamine by Amadori rearrangement (**Figure 1**). This reaction is reversible, depending on the concentra‐ tion of the reactants. The late stage of the process is an irreversible cascade of reactions in‐ volving enolization, dehydration, condensation, oxidation, fragmentation, and other rearrangements, leading to the formation of AGEs [21]. Glucose, Schiff's base, and Amadori product, can also exhibit auto‐oxidation reactions that are responsible for free radicals and highly reactive carbonyl compound production. These compounds can react with other ami‐ no acid side‐chains and further contribute to post‐translational modifications. Unlike organ‐ ic syntheses, AGEs formation does not produce well‐defined products but a large number of structures. The pathways leading to several AGEs are extensively outlined in [22]. Glycation reaction produces very reactive intermediates that can promote the formation of intramolec‐ ular and intermolecular cross‐links within AGE‐modified protein monomers. However, the reaction can also evolve into AGE protein adducts unable to form covalent cross‐links (**Figure 1**).

from glycosylation; indeed, these two post‐translational modifications affect the structure of the target protein in a different way. Glycosylation is a selective protein modification, driven by specific enzymes, that is generally associated with a gain of function (or stabilization) of the target protein. Nonenzymatic glycation is a nonselective modification, and it is generally associated with a loss of function of the target protein due to modifications of its native structure. While glycosylation is a well‐controlled cellular mechanism, nonenzymatic glycation depends on the exposure of free amino groups in the polypeptide chain, concentra‐ tion of the sugar, and oxidative conditions. Glycation is a pathological process that is highly relevant in diabetes patients, as it plays a crucial role not only in diabetic complications but also in the normal aging process. Increasing evidence suggests a link between diabetes and neurodegenerative processes such as Alzheimer and Parkinson diseases [1]. In this respect, much attention has been recently devoted to the role played by nonenzymatic glycation of proteins in stimulating amyloid aggregation and toxicity. The observation that proteins in amyloid deposits, such as *β*‐amyloid, tau, prions,transthyretin, and *β*2‐microglobulin, are often found glycated in patients suggests a direct correlation between protein glycation and amyloid formation [2–8]. This is thought to be associated with the formation of cross‐links that stabilize protein aggregates. Indeed, AGEs formation can not only interfere with the regular function‐ ing of the proteins to which they are attached but also induce the formation of covalent cross‐ links with close proteins. In addition, glycation can affect the protein degradation process [9], and, being an abnormal modification, it has been found to induce some proteins to misfold

Moreover, the AGE‐modified proteins are tightly involved in physiopathological cellular mechanisms. Once formed, AGEs interact with specific cellular receptors leading to the activation of different signaling pathways. The most studied AGE‐receptor, known as RAGE, is a multiligand receptor belonging to the immunoglobulin superfamily [13, 14]. The activa‐ tion of RAGE regulates key cellular processes such as inflammation, apoptosis, proliferation, autophagy, and recently it has been associated with the pathogenesis of amyloidosis [15, 16]. In neurons, glia, and endothelial cells, RAGE is also the binding site for A*β* peptide on the cell

The nonenzymatic glycation of protein amino groups by reducing sugars (also called Mail‐ lard reaction) is a chemical reaction common to all cell types: glycated products slowly ac‐ cumulate in vivo, leading to several different protein dysfunctions caused by alterations of their integrity [19, 20]. The process begins with a nucleophilic addition reaction between a free amino group of a protein and a carbonyl group of a reducing sugar, forming a reversi‐ ble intermediate product (Schiff's base). Side‐chains of arginine and lysine residues and the N‐terminal amino group of proteins are the main targets of protein glycation. The process depends on several conditions, such as the concentration and reactivity of the glycation agent, the presence of catalytic factors (metals, buffer ions, and oxygen), the physiological pH, temperature, and the half‐life of each protein. All reducing sugars can participate in

and, thus, promote protein aggregation [10–12].

**2. Protein glycation**

166 168Exploring New Findings on Amyloidosis

surface and mediates Alzheimer's disease pathology [15, 17, 18].

**Figure 1. Simplified reaction scheme of some chemical processes involved in AGE formation.** The reaction between a free amino group of a protein and the carbonyl group of a reducing sugar leads to the production of the Schiff's base that can turn into a stable ketoamine by Amadori rearrangement. Glycation reaction can evolve to AGE derivatives, forming, that is, di‐aminoethyl bridge, or not forming, that is, carboxymethyl‐lysine, intramolecular and intermolecu‐ lar protein cross‐links. Adapted from Glomb and Monnier [23].

## **3. Differential effects of glycation on protein aggregation and cytotoxicity**

Several proteins related and not related to misfolding diseases have been so far examined to investigate the effect of glycation on their propensity to aggregate and form amyloid structure.

#### **3.1. Proteins related to misfolding diseases**

#### *3.1.1. Aβ‐peptide*

A*β*‐peptide is crucially involved in Alzheimer's disease as the main component of the amyloid plaques found in the brains of Alzheimer patients. This peptide results from the amyloid precursor protein (APP), which is cleaved by beta‐secretase and gamma‐secretase. Although carbohydrates are directly involved in the formation of neurofibrillary tangles and senile plaques in the brains of the patients with Alzheimer disease (AD) [24–26], their influence on the mechanism of A*β* peptide aggregation and induced cell toxicity is still controversial. Vitek et al. [10] reported, for the first time, that plaque fractions of AD brains contained about threefold more AGE adducts than preparations from healthy, age‐matched controls. This observation was further corroborated by immunohistochemical studies on postmortem tissues thatidentifiedAGEs as major components of amyloid plaques [27]. Studies in vitro have shown that AGE‐modified A*β* peptide‐nucleation seeds induce fast aggregation of the soluble A*β* peptide compared to nonmodified seed material [10]. Aggregation of the A*β* peptide follows a nucleation‐dependent polymerization process, consisting of two steps, an initial slow nucleus formation, followed by a rapid growth phase. The formation of a nucleus is a reversible process dependent on the concentration of the peptide monomer; thus, it is likely that the initiation of the process at a subthreshold concentration may be started by irreversible covalent cross‐linking of the glycated monomers [10]. The AGEs species that enhance nucleation were suggested to be the relatively early glycation products as evidenced by the time course over which glucose accelerated the formation of A*β* peptide aggregates. Successively, it has been reported that glycation by fructose also promotes amyloid aggregation in vitro of A*β* pep‐ tide. During the aggregation process, both nucleus formation and aggregates' growth were accelerated by AGE‐mediated cross‐linking [28, 29]. Specifically, an increased content of oligomeric forms at the expense of fibrillar species was detected when A*β* peptide was incubated in the presence of glucose or fructose [29]. These species resulted to improve cell viability probably due to the stabilization of the oligomeric forms. Indeed, the mechanism by which A*β* induces toxicity is intimately associated to the conformational state of the peptide, the fibrillar forms rather than the soluble oligomers result more toxic to cells [30]. However, increasing evidence has recently shown that soluble oligomers are also cytotoxic, both in vitro and in vivo [31, 32]. The idea that all amyloid oligomers are intrinsically toxic has recently been questioned by a number of evidence showing that, depending on the growth conditions, the same protein/peptide can generate structurally different oligomers endowed with different stability, hydrophobic exposure, compactness, and cytotoxicity [33]. A*β* oligomers display a high degree of polymorphism with different structural, biophysical, and cytotoxic proper‐ ties. In particular, A*β* oligomers have been classified as A+ and A-, with A+ more toxic than A- oligomers, possibly as a consequence of the increased exposure of hydrophobic residues that would favor their interaction with the plasma membrane [34, 35]. In this respect, glyca‐ tion could affect the structural and physicochemical features of amyloid oligomers as well as their interaction to the cell membrane and, consequentially, induce different cytotoxicities. Also, glycation by methylglyoxal promotes the formation of *β*‐sheets, oligomers, and protofi‐ brils in A*β* peptide as well as the increase in size of the oligomers, suggesting an enhance‐ ment of intermolecular and intramolecular interactions which stabilizes the aggregate species [36].

A*β* peptide has been identified as a ligand of RAGE, and the A*β*–RAGE interaction triggers the activation of different signaling pathways responsible for the neuronal cell death [37]. RAGE is overexpressed in the AD brains, and its upregulation has been shown to mediate A*β*‐ induced oxidative stress, activation of transcription factor Nf-κB, and apoptosis [38, 39]. Recently, Li et al. [40] have reported that fully glycated A*β* peptide (long incubation with methylglyoxal) exacerbates the neuronal toxicity by the upregulation of RAGE and subse‐ quent activation of death‐signaling pathways. These apparently contrasting effects of the cytotoxic properties of the glycated A*β* could be explained, assuming that glycation induces two different mechanisms in time: an immediate cytoprotective effect, likely associated with the structural properties of the oligomers, and a toxic effect at longer times associated to AGE formation.

## *3.1.2. β2‐Microglobulin*

**3. Differential effects of glycation on protein aggregation and cytotoxicity**

Several proteins related and not related to misfolding diseases have been so far examined to investigate the effect of glycation on their propensity to aggregate and form amyloid structure.

A*β*‐peptide is crucially involved in Alzheimer's disease as the main component of the amyloid plaques found in the brains of Alzheimer patients. This peptide results from the amyloid precursor protein (APP), which is cleaved by beta‐secretase and gamma‐secretase. Although carbohydrates are directly involved in the formation of neurofibrillary tangles and senile plaques in the brains of the patients with Alzheimer disease (AD) [24–26], their influence on the mechanism of A*β* peptide aggregation and induced cell toxicity is still controversial. Vitek et al. [10] reported, for the first time, that plaque fractions of AD brains contained about threefold more AGE adducts than preparations from healthy, age‐matched controls. This observation was further corroborated by immunohistochemical studies on postmortem tissues thatidentifiedAGEs as major components of amyloid plaques [27]. Studies in vitro have shown that AGE‐modified A*β* peptide‐nucleation seeds induce fast aggregation of the soluble A*β* peptide compared to nonmodified seed material [10]. Aggregation of the A*β* peptide follows a nucleation‐dependent polymerization process, consisting of two steps, an initial slow nucleus formation, followed by a rapid growth phase. The formation of a nucleus is a reversible process dependent on the concentration of the peptide monomer; thus, it is likely that the initiation of the process at a subthreshold concentration may be started by irreversible covalent cross‐linking of the glycated monomers [10]. The AGEs species that enhance nucleation were suggested to be the relatively early glycation products as evidenced by the time course over which glucose accelerated the formation of A*β* peptide aggregates. Successively, it has been reported that glycation by fructose also promotes amyloid aggregation in vitro of A*β* pep‐ tide. During the aggregation process, both nucleus formation and aggregates' growth were accelerated by AGE‐mediated cross‐linking [28, 29]. Specifically, an increased content of oligomeric forms at the expense of fibrillar species was detected when A*β* peptide was incubated in the presence of glucose or fructose [29]. These species resulted to improve cell viability probably due to the stabilization of the oligomeric forms. Indeed, the mechanism by which A*β* induces toxicity is intimately associated to the conformational state of the peptide, the fibrillar forms rather than the soluble oligomers result more toxic to cells [30]. However, increasing evidence has recently shown that soluble oligomers are also cytotoxic, both in vitro and in vivo [31, 32]. The idea that all amyloid oligomers are intrinsically toxic has recently been questioned by a number of evidence showing that, depending on the growth conditions, the same protein/peptide can generate structurally different oligomers endowed with different stability, hydrophobic exposure, compactness, and cytotoxicity [33]. A*β* oligomers display a high degree of polymorphism with different structural, biophysical, and cytotoxic proper‐ ties. In particular, A*β* oligomers have been classified as A+ and A-, with A+ more toxic than A- oligomers, possibly as a consequence of the increased exposure of hydrophobic residues

**3.1. Proteins related to misfolding diseases**

*3.1.1. Aβ‐peptide*

168 170Exploring New Findings on Amyloidosis

*β*<sup>2</sup> microglobulin (*β*2M) is a major constituent of amyloid fibrils deposited in patients with hemodialysis‐associated amyloidosis. This type of amyloidosis is a common and serious complication in patients on long‐term hemodialysis.

Glycation seems to promote amyloid aggregation in *β*2M. In particular, D‐ribose has been shown to rapidly induce human *β*2M to generate AGEs and form aggregates in a time‐ dependent manner [41]. The process takes few days in vitro and proceeds through the formation of covalent cross‐links that are likely to favor protein aggregation. Ribosylated *β*2M was highly oligomerized compared to unglycated protein, and had a granular morphology [41]. Furthermore, once ribosylated *β*2M aggregates have been formed, they are difficult to be degraded by proteases and can persist in human tissues for a long period. These oligomeric aggregates show significant cytotoxicity to human neuroblastoma and fibroblast cells. Indeed, the exposure of cells to ribosylated *β*2M aggregates resulted in a significant increase of intracellular reactive oxygen species (ROS), and thereby induced apoptosis [41]. Inhibition of fibril extension in vitro was reported for *β*2M also upon glycation with D‐glucose [42]. As glycated *β*2M is found as a major component of the amyloid deposits in hemodialysis‐ associated amyloidosis [2], these findings suggest that glycation could promote the forma‐ tion of stable *β*2‐microglobulin aggregates in vivo that contribute to the cell dysfunction and death, thus playing an important role in the pathogenesis of *β*2M‐associated diseases.

## *3.1.3. Insulin*

Insulin is a small protein hormone that is crucial for the control of glucose metabolism. Monomeric insulin undergoes amyloid aggregation in vivo, and insulin amyloid‐like fibrils are the hallmark of a clinical condition observed in insulin‐dependent diabetic patients, called insulin injection amyloidosis.

Glycation of insulin has been reported to differentially affect protein structure, stability, and aggregation, depending on the glycating agent and/or environmental conditions. This protein is intimately associated with glycemia and is vulnerable to glycation by glucose and other highly reactive carbonyls, especially in diabetic conditions [43]. Glycated insulin is unable to regulate glucose homeostasis in vivo and to stimulate glucose transport and adipose tissue lipogenesis [44].

In vitro experiments have shown that insulin can be glycated by glucose to be able to react with Lys29 in the C‐terminal region of chain B and with N‐terminus of chains A and B [45, 46]. Glucose induces the formation of glycated insulin adducts having different structural features, depending on the experimental conditions used. In particular, glycation in reducing condi‐ tions is able to induce insulin oligomerization, thus accelerating amyloid formation. On the contrary, glycation in nonreducing conditions strongly inhibit amyloid formation in a way proportional to the glycation extent [47]. Probably, underthe latter conditions, insulin adducts possess a higher internal dynamics that prevent formation of the rigid cross‐*β* core structure, thus reducing the ability to form fibrils. Human insulin can also be glycated by methylglyox‐ al able to react with a single site, that is, Arg22 of the B‐chain. This modification promotes the formation of native‐like aggregates and reduces the ability of human insulin to form fibrils by impairing the formation of the seeding nuclei. These aggregates are small, soluble, nonfibril‐ lar, and retain a native‐like structure. The lag‐phase of the nucleation‐dependent polymeriza‐ tion process increased as a function of methylglyoxal concentration [48]. Also, using ribose as the glycating agent, the insulin's native conformation is preserved and does not evolve in amyloid aggregates, because ribosylation impairs the *α*‐helix to *β*‐sheet transition, maintain‐ ing the protein in a soluble monomeric state [49]. Again, the effects may be ascribed to a higher dynamics in glycated insulin leading to impairment in the formation of the rigid cross‐*β* structure. However, ribose‐glycated insulin strongly affects the cell viability, triggering a death pathway involving the activation of apoptotic signaling, intracellular reactive oxygen species (ROS) production, and activation of the transcription factor Nf‐κB [49].

#### *3.1.4. α‐Synuclein*

*α*‐Synuclein is a natively unfolded protein which is found in the typical amyloid fibrillar form in the intraneuronal Lewy bodies (LBs) in Parkinson's disease (PD). Several factors such as metals, oxidative stress, failure of proper protein degradation, and mutations are associated to the altered protein conformation and function [50, 51]. Post‐translational modifications like phosphorylation and glycation are known to affect the *α*‐synuclein aggregation process [52]. Glycation was first reported to be present in the substantia nigra and locus coeruleus of peripheral LBs [53]. Increased accumulation of AGEs was detected in neuronal LBs and glial cells in the frontal cortex of early‐stage PD brains, suggesting a role for AGEs in the disease [54]. Moreover, AGEs and *α*‐synuclein were found similarly distributed and colocalized in early LBs in the brains of PD patients [6, 55]. Intracellular accumulation of AGEs precedes *α*‐ synuclein‐positive inclusion body formation, and extracellular AGEs accelerate the process of intracellular *α*‐synuclein‐positive inclusion body formation [56].

In vitro studies showed that AGEs promote cross‐linking of *α*‐synuclein. The protein con‐ tains 15 lysine residues making it a target for glycation at multiple sites. Glycation with both methylglyoxal and glyoxal induces oligomerization of *α*‐synuclein and inhibits the forma‐ tion of amyloid fibrils [57, 58]. Under aggregation conditions, glycated *α*‐synuclein promot‐ ed the *β*‐sheet conversion and the formation of spherical aggregates which were similar to oligomeric intermediates in their size and morphology, but no further elongation to fibrils was observed. Moreover, protein fibrillization was significantly suppressed by the seeding of modified *α*‐synuclein species [57]. However, AGEs formation did not alter the secondary structure: the glycated *α*‐synuclein showed similar random coil conformation as the native protein [57, 58]. Similar results were obtained with D‐ribose: ribosylation of *α*‐synuclein promotes the formation of molten globule‐like aggregates and not fibrils. Moreover, these aggregates induce oxidative stress in cell models and result in high cytotoxicity. Changes in secondary structure upon ribosylation were not detected in *α*‐synuclein. However, conforma‐ tional changes in the tertiary structure occurred as suggested by change in intrinsic fluores‐ cence and exposure of hydrophobic patches [59]. The glycation‐induced folding alterations might affect the aggregation kinetics of *α*‐synuclein inducing oligomerization and stabilize oligomeric aggregates.

Choi and Lim [60] reported, in a mouse model of parkinsonism, that *α*‐synuclein is modified by AGEs in vivo. The authors showed that an oligomeric form of *α*‐synuclein is linked to N*ε*‐ (carboxymethyl)lysine (CML) and N*ε*‐(carboxyethyl)lysine (CEL) suggesting that the AGEs modification is involved in the aggregation of the *α*‐synuclein in vivo.

These results indicate that glycation of *α*‐synuclein results in the formation of oligomeric or globular structures that are the more toxic aggregate forms. Thus, it is likely that, in a glycation‐ prone environment, more cytotoxic *α*‐synuclein aggregates or oligomers are formed contri‐ buting to the pathogenesis of PD.

## *3.1.5. Lysozime*

*3.1.3. Insulin*

lipogenesis [44].

*3.1.4. α‐Synuclein*

insulin injection amyloidosis.

170 172Exploring New Findings on Amyloidosis

Insulin is a small protein hormone that is crucial for the control of glucose metabolism. Monomeric insulin undergoes amyloid aggregation in vivo, and insulin amyloid‐like fibrils are the hallmark of a clinical condition observed in insulin‐dependent diabetic patients, called

Glycation of insulin has been reported to differentially affect protein structure, stability, and aggregation, depending on the glycating agent and/or environmental conditions. This protein is intimately associated with glycemia and is vulnerable to glycation by glucose and other highly reactive carbonyls, especially in diabetic conditions [43]. Glycated insulin is unable to regulate glucose homeostasis in vivo and to stimulate glucose transport and adipose tissue

In vitro experiments have shown that insulin can be glycated by glucose to be able to react with Lys29 in the C‐terminal region of chain B and with N‐terminus of chains A and B [45, 46]. Glucose induces the formation of glycated insulin adducts having different structural features, depending on the experimental conditions used. In particular, glycation in reducing condi‐ tions is able to induce insulin oligomerization, thus accelerating amyloid formation. On the contrary, glycation in nonreducing conditions strongly inhibit amyloid formation in a way proportional to the glycation extent [47]. Probably, underthe latter conditions, insulin adducts possess a higher internal dynamics that prevent formation of the rigid cross‐*β* core structure, thus reducing the ability to form fibrils. Human insulin can also be glycated by methylglyox‐ al able to react with a single site, that is, Arg22 of the B‐chain. This modification promotes the formation of native‐like aggregates and reduces the ability of human insulin to form fibrils by impairing the formation of the seeding nuclei. These aggregates are small, soluble, nonfibril‐ lar, and retain a native‐like structure. The lag‐phase of the nucleation‐dependent polymeriza‐ tion process increased as a function of methylglyoxal concentration [48]. Also, using ribose as the glycating agent, the insulin's native conformation is preserved and does not evolve in amyloid aggregates, because ribosylation impairs the *α*‐helix to *β*‐sheet transition, maintain‐ ing the protein in a soluble monomeric state [49]. Again, the effects may be ascribed to a higher dynamics in glycated insulin leading to impairment in the formation of the rigid cross‐*β* structure. However, ribose‐glycated insulin strongly affects the cell viability, triggering a death pathway involving the activation of apoptotic signaling, intracellular reactive oxygen

species (ROS) production, and activation of the transcription factor Nf‐κB [49].

*α*‐Synuclein is a natively unfolded protein which is found in the typical amyloid fibrillar form in the intraneuronal Lewy bodies (LBs) in Parkinson's disease (PD). Several factors such as metals, oxidative stress, failure of proper protein degradation, and mutations are associated to the altered protein conformation and function [50, 51]. Post‐translational modifications like phosphorylation and glycation are known to affect the *α*‐synuclein aggregation process [52]. Glycation was first reported to be present in the substantia nigra and locus coeruleus of peripheral LBs [53]. Increased accumulation of AGEs was detected in neuronal LBs and glial cells in the frontal cortex of early‐stage PD brains, suggesting a role for AGEs in the disease Hen egg white lysozyme (HEWL) has also been used to study the impact of glycation on protein structure and aggregation. HEWL is a structural homolog of human lysozyme, responsible for systemic amyloidosis disease and, for this reason, it is considered a very good model for amyloid aggregation studies. HEWL undergoes glycation in vitro, and potential glycation sites are considered to be the N‐terminal *α*‐amino group, *ε*‐amino group of lysine residues, and guanidino group of arginine residues [61]. Glycation of HEWL has been tested over a prolonged period in the presence of D‐glucose, D‐fructose, and D‐ribose [62, 63]. Among the tested sugars, D‐ribose resulted to be the most effective one, and glycation has been found to promote formation of cross‐linked oligomers but no fibrillar species in HEWL. More recently, ribosylation of HEWL in the early phase of the process has been studied by comple‐ mentary high‐resolution techniques [64]. These studies indicate that ribosylation modifies the protein surface in HEWL without altering the overall structure but affecting its hydrophobic surface. Such modifications lead to the formation of native‐like spherical oligomers, able to affect cell viability, which further evolve into insoluble native‐like protofibrils [64].

#### **3.2. Proteins not related to misfolding diseases**

## *3.2.1. Albumin*

Human serum albumin (HSA) is the most abundant protein in human plasma or serum (around 60% of total proteins). Serum albumin is known to be capable of self‐assembling in amyloidogenic aggregates under particular experimental conditions (pH, temperature, concentration, and isoelectric point) and is a widely used model for the study of amyloid aggregation. Both HSA and bovine serum albumin (BSA) have been shown to be efficiently glycated in vitro by different glycating agents. Glycation and AGE modifications of serum albumin induce structural changes that depend on the chemical reactivity of the modifying reagent and the concentration used for in vitro glycation. However, glycation has been shown to promote strong conformational changes affecting both secondary and tertiary structures. Such modifications in tertiary structure have been revealed by complementary spectroscopi‐ cal techniques: circular dichroism (CD), Fourier transform infrared (FTIR), nuclear magnetic resonance (NMR), and fluorescence spectroscopy [65–69]. In particular, the microenviron‐ ment of Trp214 seems to be strongly affected by glycation as indicated by fluorescence and NMR spectroscopy [66, 69]. Such conformational changes in the tertiary structure could be a consequence of molecular rearrangements after the formation of AGE products. Indeed, some of these AGEs forming covalent cross‐links within adjacent protein strands require conforma‐ tional changes which produce more apolar and tight molecules with respect to the native protein. Besides, the accessibility of the hydrophobic regions in the protein has been shown to increase with glycation [67, 68]. Modification at the secondary structure level can be detect‐ ed only at longer times of incubation with glycating agents. This could be due to the fact that glycation is likely to induce loss of tertiary structure before that of secondary structure, as suggested by comparing intrinsic fluorescence and far‐UV CD results in glycated albumin [65, 66, 70].

Glycation‐induced protein misfolding promotes the formation of amyloid‐like aggregates in serum albumin [12, 71, 72]. These amyloid‐like deposits appear as densely staining granules under atomic force microscopy and are able to bind the amyloid‐specific dye thioflavin T. Also, they were shown to induce high cytotoxicity that triggers cell death by activation of cellular signaling cascades. In fact, independent experiments have shown that aggregates of glycated BSA are able to induce ROS‐mediated oxidative stress and apoptosis in both neurotypic SH‐ SY5Y and MCF‐7 cells. These results indicate that glycation of serum albumin results in the formation of oligomeric or globular structures that are the more toxic aggregate forms [68, 72].

These observations could have important implications, as serum albumin, being a circulat‐ ing protein, is likely undergoing glycative alteration in the case of diabetes pathology and hyperglycemia. Forinstance, antioxidant activities of serum albumin were impaired in patients with diabetes.

## *3.2.2. Apomyoglobin*

protein surface in HEWL without altering the overall structure but affecting its hydrophobic surface. Such modifications lead to the formation of native‐like spherical oligomers, able to

Human serum albumin (HSA) is the most abundant protein in human plasma or serum (around 60% of total proteins). Serum albumin is known to be capable of self‐assembling in amyloidogenic aggregates under particular experimental conditions (pH, temperature, concentration, and isoelectric point) and is a widely used model for the study of amyloid aggregation. Both HSA and bovine serum albumin (BSA) have been shown to be efficiently glycated in vitro by different glycating agents. Glycation and AGE modifications of serum albumin induce structural changes that depend on the chemical reactivity of the modifying reagent and the concentration used for in vitro glycation. However, glycation has been shown to promote strong conformational changes affecting both secondary and tertiary structures. Such modifications in tertiary structure have been revealed by complementary spectroscopi‐ cal techniques: circular dichroism (CD), Fourier transform infrared (FTIR), nuclear magnetic resonance (NMR), and fluorescence spectroscopy [65–69]. In particular, the microenviron‐ ment of Trp214 seems to be strongly affected by glycation as indicated by fluorescence and NMR spectroscopy [66, 69]. Such conformational changes in the tertiary structure could be a consequence of molecular rearrangements after the formation of AGE products. Indeed, some of these AGEs forming covalent cross‐links within adjacent protein strands require conforma‐ tional changes which produce more apolar and tight molecules with respect to the native protein. Besides, the accessibility of the hydrophobic regions in the protein has been shown to increase with glycation [67, 68]. Modification at the secondary structure level can be detect‐ ed only at longer times of incubation with glycating agents. This could be due to the fact that glycation is likely to induce loss of tertiary structure before that of secondary structure, as suggested by comparing intrinsic fluorescence and far‐UV CD results in glycated albumin [65,

Glycation‐induced protein misfolding promotes the formation of amyloid‐like aggregates in serum albumin [12, 71, 72]. These amyloid‐like deposits appear as densely staining granules under atomic force microscopy and are able to bind the amyloid‐specific dye thioflavin T. Also, they were shown to induce high cytotoxicity that triggers cell death by activation of cellular signaling cascades. In fact, independent experiments have shown that aggregates of glycated BSA are able to induce ROS‐mediated oxidative stress and apoptosis in both neurotypic SH‐ SY5Y and MCF‐7 cells. These results indicate that glycation of serum albumin results in the formation of oligomeric or globular structures that are the more toxic aggregate forms [68, 72].

These observations could have important implications, as serum albumin, being a circulat‐ ing protein, is likely undergoing glycative alteration in the case of diabetes pathology and hyperglycemia. Forinstance, antioxidant activities of serum albumin were impaired in patients

affect cell viability, which further evolve into insoluble native‐like protofibrils [64].

**3.2. Proteins not related to misfolding diseases**

*3.2.1. Albumin*

172 174Exploring New Findings on Amyloidosis

66, 70].

with diabetes.

Apomyoglobin (i.e., heme‐free myoglobin) is a small, soluble *α*‐helical protein able to form amyloid fibrils under particular experimental conditions. In addition, an apomyoglobin mutant, that is, W7FW14F, is able to form amyloid fibrils in physiological conditions of pH and temperature, and for this reason this protein is a good model for the study of amyloido‐ sis [73–78].

Wild‐type apomyoglobin is rapidly glycated in vitro by different glycating agents, and glycation has been shown to induce strong conformational changes, affecting both secon‐ dary and tertiary structures. In particular, glycation induces partial loss of the helical con‐ tentin apomyoglobin without promoting an *α* to *β* transition, typical ofthe amyloidaggregates. Glycation induces strong modifications in the tridimensional organization, as suggested by the loss of ability of the glycated protein to bind the prosthetic group [79]. Such modifica‐ tions eventually lead to the formation of oligomeric species, stabilized by intermolecular cross‐ links, able to affect cell viability as observed for amyloid prefibrillar oligomeric aggregates, specifically, cell exposure to fully glycated wild‐type apomyoglobin‐induced ROS‐mediated apoptosis [79].

At the same time, glycation has been shown to affect the aggregation kinetics in the W7FW14F apomyoglobin mutant, able to form amyloid fibrils in physiological conditions [73–78]. In particular, glycation accelerates the formation of harmless amyloid fibrils in the apomyoglo‐ bin this amyloidogenic mutant [76]. A plausible explanation for such faster kinetics could be related to a higher tendency of the mutant to form intermolecular links upon glycation able to reduce the flexibility of aggregation‐prone regions and thus favor the subsequent step of fibril elongation.

Although apomyoglobin is not related to any amyloidogenic diseases, it represents a suita‐ ble modelfor studying the role of glycation in amyloid aggregation. Indeed, due to the different aggregation propensities of the native protein and the W7FW14F mutant in physiological conditions, this protein model allows to dissect the effect of glycation in promoting amyloid aggregation and contributes to the aggregation kinetics. The above results indicate that glycation can be considered not only a triggering factor in amyloidosis but also a player in later stages of the aggregation process.

## **4. Molecular effects of glycation on amyloid aggregation process**

The overall evidences on several model proteins indicate that AGE modifications may alter the folding state of proteins and their solubility, thereby influencing protein aggregation. The main outcome of this study is that the effect of glycation on amyloid aggregation cannot be generalized. Indeed, being a post‐translational modification, it differentially affects the aggregation process in proteins by promoting, accelerating, and/or stabilizing on‐pathway and off‐pathway species (**Figure 2**). Molecular basis of such modulation are still poorly under‐ stood. Most of the evidence indicate that glycation strongly affects the tertiary structure of the target protein promoting the formation of globular amyloid‐like deposits [62, 72]. Depend‐ ing on the protein involved, glycation induces chemical modifications ofthe positively charged side chains (mostly lysine, arginine, and N‐terminus), thus affecting the protein charge and favoring the exposure of its hydrophobic surface. This effect could trigger native‐like aggre‐ gation favoring the formation of small oligomers that, being stabilized by the AGE‐derived covalent cross‐links, do not evolve into amyloid fibrils. Recent evidence indicates that glycation promotes the formation of the amyloid oligomeric species in several model pro‐ teins (**Figure 2B**). However, glycation does not necessarily induce protein oligomerization. Due to the complexity of the glycation reaction, some AGE adducts might not evolve to the formation of protein cross‐links. In this case, glycation seems to stabilize the monomeric form thus inhibiting the amyloid aggregation process as observed for ribosylated human insulin (**Figure 2C**). Moreover, the cross‐links of AGE‐derived oligomers do not necessarily show amyloid properties (**Figure 2D**). However, AGEs‐modified proteins are always able to affect cell viability, irrespective of amyloid properties.

**Figure 2. Effects of glycation in amyloid aggregation.** Protein monomers are shown as blue dots, AGE‐derived cross‐ links are shown in gray, and AGE modification are shown in red. (A) Typical amyloid aggregation pathway. Possible effects induced by glycation: (B) promote the formation of amyloid oligomeric species stabilized by covalent cross‐ links; (C) stabilize the monomeric form thus inhibiting amyloid aggregation; (D) promote the formation of cross‐linked nonamyloidogenic oligomers.

## **5. Role of glycation in the amyloid‐induced cell toxicity**

The effect of glycation on the aggregation process has important implications in the patho‐ logical mechanisms involved in amyloid diseases. In most proteins, glycation has been shown

to stabilize the aggregates in the oligomeric forms. This modification has important patholog‐ ical implications as oligomeric species are known to be far more toxic than the fibrillar aggregates [80, 81]. The oligomeric species may interact with the cell membrane, altering its permeability and leading to cell homeostasis imbalance and neuronal cell dysfunction [82]. Recent evidence indicates that the amyloid oligomer toxicity is not strictly related to the oligomer properties, but rather a behavior that results from a complex interplay between the structural properties of both oligomers and cell membrane taken as a whole. Indeed, oligom‐ ers of comparable size but different structure and biophysical properties can display differ‐ ent toxicities, possibly as a consequence of the increased exposure of hydrophobic residues that would destabilize them and favor the interaction with the plasma membrane [83]. In this respect, glycation could affect structural and physicochemical features of amyloid oligomers as well as theirinteraction to the cell membrane and subsequently modulate and/orinduce the cell toxicity. Also, the glycated oligomeric species can induce formation of reactive oxygen species, worsening the oxidative stress in the cell and further promoting protein glycation. Moreover, protein glycation leads to the formation of AGEs which have a pathological role in several diseases [84–86]. AGE adducts may therefore activate, through interaction with RAGE receptor, inflammatory response generally associated to amyloid toxicity [13]. The AGE– RAGE binding results in the activation of NADPH‐oxidases that leads to an increased production ofROS. A key downstream target ofRAGE is the proinflammatory Nf‐κB pathway, which in turn leads to high RAGE expression, producing a feedback loop in which continu‐ ous activation of RAGE keeps the cellular inflammatory state activated [14, 87].

target protein promoting the formation of globular amyloid‐like deposits [62, 72]. Depend‐ ing on the protein involved, glycation induces chemical modifications ofthe positively charged side chains (mostly lysine, arginine, and N‐terminus), thus affecting the protein charge and favoring the exposure of its hydrophobic surface. This effect could trigger native‐like aggre‐ gation favoring the formation of small oligomers that, being stabilized by the AGE‐derived covalent cross‐links, do not evolve into amyloid fibrils. Recent evidence indicates that glycation promotes the formation of the amyloid oligomeric species in several model pro‐ teins (**Figure 2B**). However, glycation does not necessarily induce protein oligomerization. Due to the complexity of the glycation reaction, some AGE adducts might not evolve to the formation of protein cross‐links. In this case, glycation seems to stabilize the monomeric form thus inhibiting the amyloid aggregation process as observed for ribosylated human insulin (**Figure 2C**). Moreover, the cross‐links of AGE‐derived oligomers do not necessarily show amyloid properties (**Figure 2D**). However, AGEs‐modified proteins are always able to affect

**Figure 2. Effects of glycation in amyloid aggregation.** Protein monomers are shown as blue dots, AGE‐derived cross‐ links are shown in gray, and AGE modification are shown in red. (A) Typical amyloid aggregation pathway. Possible effects induced by glycation: (B) promote the formation of amyloid oligomeric species stabilized by covalent cross‐ links; (C) stabilize the monomeric form thus inhibiting amyloid aggregation; (D) promote the formation of cross‐linked

The effect of glycation on the aggregation process has important implications in the patho‐ logical mechanisms involved in amyloid diseases. In most proteins, glycation has been shown

**5. Role of glycation in the amyloid‐induced cell toxicity**

cell viability, irrespective of amyloid properties.

174 176Exploring New Findings on Amyloidosis

nonamyloidogenic oligomers.

Glycated proteins are also resistant to proteasomal degradation; once proteins become glycated attheir exposedlysine residues, clearance by the ubiquitin–proteasome system would be impaired, because ubiquitination at lysine residues, a modification that targets proteins to the proteasome for degradation, might be impeded. Thus, accumulation of proteins as aggregates or as depositions or inclusions in tissues might be favored after glycation.

Taking into account the above considerations, protein glycation can be considered a key dynamic contributor to these multifactorial diseases. In fact, it can both promote the forma‐ tion of pathological oligomeric species and directly trigger cell dysfunction, damage, and death through the AGEs formation. For these reasons, AGEs are considered key therapeutic targets in amyloidosis, and anti‐AGEs drugs are objects ofintense ongoing research. Specifically, three main strategies have been developed to counteract the AGEs' effects: (i) to prevent the formation of AGEs; (ii) to break cross‐links aftertheirformation; (iii) to prevent AGEs' negative effects.

In vitro and in vivo experiments have shown that many compounds including aminoguani‐ dine, antioxidants such as vitamin C and vitamin E, pyridoxamine, thiamine and its synthet‐ ic derivative benfotiamine, alpha‐lipoic acid, taurine, pimagedine, aspirin, carnosine, metformin, pioglitazone, and pentoxifylline are able to inhibit AGE formation. Some of these compounds have already been used in clinical practice and some others are under clinical trials. Compounds that have been shown to break existing AGE cross‐links mainly include alagebrium (and related ALT‐462, ALT‐486, and ALT‐946) and N‐phenacylthiazolium bromide. Studies with the aim to counteract the negative effects of the AGEs mainly involve

the use of natural products as polyphenols, such as resveratrol and curcumin and some flavonoids [88–90].

However, although in vitro and in vivo studies have shown the beneficial effects of various coumpounds, the potential clinical value of these interventions remains to be established. In fact, it seems that safety and/or efficacy in clinical studies with these compounds are still a concern.

## **Author details**

Clara Iannuzzi, Gaetano Irace and Ivana Sirangelo\*

\*Address all correspondence to: ivana.sirangelo@unina2.it

Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Naples, Italy

## **References**


[7] Dukic‐Stefanovic S, Schinzel R, Riederer P, Munch G. AGES in brainageing: AGE‐ inhibitors as neuroprotective and anti‐dementia drugs? Biogerontology. 2001; 2, 19–34. doi: 10.1023/A:1010052800347.

the use of natural products as polyphenols, such as resveratrol and curcumin and some

However, although in vitro and in vivo studies have shown the beneficial effects of various coumpounds, the potential clinical value of these interventions remains to be established. In fact, it seems that safety and/or efficacy in clinical studies with these compounds are still a

Department of Biochemistry, Biophysics and General Pathology, Second University of Naples,

[1] Hassan M, Sehgal SA, Rashid S. Regulatory cascade of neuronal loss and glucose

[2] Miyata T, Oda O, Inagi R, Iida Y, Araki N, Yamada N, Horiuchi S, Taniguchi N, Maeda K, Kinoshita T. Beta 2‐microglobulin modified with advanced glycation end products is a major component of hemodialysis‐associated amyloidosis. J Clin Invest. 1993; 92,

[3] Ledesma MD, Bonay P, Colaco C, Avila J. Analysis of microtubule‐associated protein tau glycation in paired helical filaments. J Biol Chem. 1994; 269(34), 21614–19.

[4] Sasaki N, Fukatsu R, Tsuzuki K, Hayashi Y, Yoshida T, Fujii N, Koike T, Wakayama I, Yanagihara R, Garruto R, Amano N, Makita Z. Advanced glycation end products in Alzheimer's disease and other neurodegenerative diseases. Am J Pathol. 1998; 153,

[5] Kikuchi S, Ogata A, Shinpo K, Moriwaka F, Fujii F, Taniguchi N, Tashiro K. Detec‐ tion of an Amadori product, 1‐hexitol‐lysine, in the anterior horn of the amyotrophic lateral sclerosis and spinobulbar muscular atrophy spinal cord: evidence for early involvement of glycation in motoneuron diseases. Acta Neuropathol. 2000; 99, 63–6.

[6] Munch G, Luth HJ, Wong A, Arendt T, Hirsch E, Ravid R, Riederer P. Crosslinking of alpha‐synuclein by advanced glycation endproducts–an early pathophysiological step

in Lewy body formation? J Chem Neuroanat. 2000; 20, 253–7.

metabolism. CNS Neurol Disord Drug Targets. 2014; 13(7), 1232–45.

flavonoids [88–90].

176 178Exploring New Findings on Amyloidosis

**Author details**

Naples, Italy

**References**

1243–52.

1149–55.

doi: 10.1007/PL00007407.

Clara Iannuzzi, Gaetano Irace and Ivana Sirangelo\*

\*Address all correspondence to: ivana.sirangelo@unina2.it

concern.


[31] Dahlgren KN, Manelli AM, Stine WB Jr, Baker LK, Krafft GA, LaDu MJ. Oligomeric and fibrillar species of amyloid‐beta peptides differentially affect neuronal viability. J Biol Chem. 2002; 277(35), 32046–53.

[19] Miyata T, van Ypersele de Strihou C, Kurokawa K, Baynes JW. Alterations in nonen‐ zymatic biochemistry in uremia: origin and significance of "carbonyl stress" in long‐ term uremic complications. Kidney Int. 1999; 55, 389–99. doi: 10.1046/j.1523‐

[20] Gul A, Rahman MA, Salim A, Simjee SU. Advanced glycation end products in senile diabetic and nondiabetic patients with cataract. J Diabetes Complications. 2009; 23, 343–

[21] Ulrich P, Cerami A. Protein glycation, diabetes, and aging. Recent Prog Horm Res. 2001;

[22] Cho SJ, Roman G, Yeboah F, Konishi Y. The road to advanced glycation end products:

[23] Glomb MA, Monnier VM. Mechanism of protein modification by glyoxal and glyco‐ laldehyde reactive intermediates ofthe Maillard reaction. J Biol Chem. 1995; 270, 10017–

[24] Münch G, Thome J, Foley P, Schinzel R, Riederer P. Advanced glycation end prod‐ ucts in ageing and Alzheimer's disease. Brain Res Brain Res Rev. 1997; 23, 134–143.

[25] Münch G, Schinzel R, Loske C, Wong A, Durany N, Li JJ, Vlassara H, Smith MA, Perry G, Riederer P. Alzheimer's disease––synergistic effects of glucose deficit, oxidative stress and advanced glycation endproducts. J Neural Transm. 1998; 105, 439–461. [26] Münch G, Deuther‐Conrad W, Gasic‐Milenkovic J. Glycoxidative stress creates a vicious cycle of neurodegeneration in Alzheimer's disease––a target for neuroprotec‐

[27] Smith MA, Taneda S, Richey PL, Miyata S, Yan SD, Stern D, Sayre LM, Monnier VM, Perry G. Advanced Maillard reaction end products are associated with Alzheimer

[28] Münch G, Mayer S, Michaelis J, Hipkiss AR, Riederer P, MüllerR, Neumann A, Schinzel R, Cunningham AM. Influence of advanced glycation end‐products and AGEinhibi‐ tors on nucleation‐dependent polymerization of β‐amyloid peptide. Biochim Biophys

[29] Fernandez‐Busquets X, Ponce J, Bravo R, Arimon M, Martianez T, Gella A, Cladera J, Durany N. Modulation of amylois β peptide 1‐42 cytotoxicity and aggregation in vitro by glucose and chondroitin sulfate. Curr Alzheimer Res. 2010; 7, 428–38. doi:

[30] Pike CJ, Burdick D, Walencewicz AJ, Glabe CG, Cotman CW. Neurodegeneration induced by beta‐amyloid peptides in vitro: the role of peptide assembly state. J

a mechanistic perspective. Curr Med Chem. 2007; 14(15), 1653–71.

tive treatment strategies? J Neural Transm Suppl. 2002; 62, 303–307.

disease pathology. Proc Natl Acad Sci USA. 1994; 91(12), 5710–4.

1755.1999.00302.x.

178 180Exploring New Findings on Amyloidosis

Acta. 1997; 1360, 17–29.

10.2174/156720510791383787.

Neurosci. 1993; 13, 1676–87.

56, 1–2.

26.

8. doi: 10.1016/j.jdiacomp.2008.04.001.


[55] Castellani RJ, Perry G, Siedlak SL, Nunomura A, Shimohama S, Zhang J, Montine T, Sayre LM, Smith MA. Hydroxynonenal adducts indicate a role for lipid peroxidation in neocortical and brainstem Lewy bodies in humans. Neurosci Lett. 2002; 319, 25e28.

[43] Brange J, Andersen L, Laursen ED, Meyn G, Rasmussen E. Toward understanding

[44] Boyd AC, Abdel‐Wahab YH, McKillop AM, McNulty H, Barnett CR, O'Harte FP, Flatt PR. Impaired ability of glycated insulin to regulate plasma glucose and stimulate glucose transport and metabolism in mouse abdominal muscle. Biochim Biophys Acta.

[45] O'Harte FPM, Højrup P, Barnett CR, Flatt PR. Identification of the site of glycation of

[46] Guedes S, Vitorino R, Domingues MR, Amado F, Domingues P. Mass spectrometry characterization of the glycation sites of bovine insulin by tandem mass spectrometry.

[47] Alavi P, Yousefi R, Amirghofran S, Karbalaei‐Heidari HR, Moosavi‐Movahedi AA. Structural analysis and aggregation propensity of reduced and nonreduced glycated insulin adducts. Appl Biochem Biotechnol. 2013; 170, 623–38. doi: 10.1016/j.ijbiomac.

[48] Oliveira LM, Lages A, Gomes RA, Neves H, Família C, Coelho AV, Quintas A. Insulin glycation by methylglyoxal results in native‐like aggregation and inhibition of fibril

[49] Iannuzzi C, Borriello M, Carafa V, Altucci L, Vitiello M, Balestrieri ML, Ricci G, Irace G, Sirangelo I. D‐ribose‐glycation of insulin prevents amyloid aggregation and produces cytotoxic adducts. Biochim Biophys Acta. 2016; 1862(1), 93–104. doi: 10.1016/

[50] Hashimoto M, Hsu LJ, XiaY, Takeda A, Sisk A, Sundsmo M, Masliah E. Oxidative stress induces amyloid‐like aggregate formation of NACP/α‐synuclein in vitro. Neuro

[51] Paik SR, Shin HJ, Lee JH. Metal catalyzed oxidation of α‐synuclein in the presence of copper (II) and hydrogen peroxide. Arch Biochem Biophys. 2000; 378, 269–77.

[52] Guerrero E, Vasudevaraju P, Hegde ML, Britton GB, Rao KS. Recent advances in α‐ synuclein functions, advanced glycation, and toxicity: implications for Parkinson's

[53] Castellani R, Smith MA, Richey PL, Perry G. Glycoxidation and oxidative stress in Parkinson disease and diffuse Lewy body disease. Brain Res. 1996; 737: 195–200. [54] Dalfo E, Portero‐Otin M, Ayala V, Martinez A, Pamplona R, Ferrer I. Evidence of oxidative stress in the neocortex in incidental Lewy body disease. J Neuropathol Exp

formation. BMC Biochem. 2011; 12, 41. doi: 10.1186/1471‐2091‐12‐41.

J Am Soc Mass Spectrom. 2009; 20, 1319–26. doi: 10.1016/j.jasms.2009.03.004.

insulin fibrillation. J Pharm Sci. 1997; 86, 517–25. doi: 10.1021/js960297s.

2000; 1523(1), 128–34.

180 182Exploring New Findings on Amyloidosis

2012.05.021.

j.bbadis.2015.10.021.

Report. 1999; 10, 717–21.

Neurol. 2005; 64: 816–30.

disease. Mol Neurobiol. 2013: 47(2), 525–36.

human insulin. Peptides. 1996; 17, 1323–30.


[78] Iannuzzi C, Irace G, Sirangelo I. Differential effects of glycation on protein aggrega‐ tion and amyloid formation. Front Mol Biosci. 2014; 1, 9. doi: 10.3389/fmolb.2014.00009.

[67] Rondeau P, Navarra G, Cacciabaudo F, Leone M, Bourdon E, Militello V. Thermal aggregation of glycated bovine serum albumin. Biochim Biophys Acta. 2010; 1804, 789–

[68] Khan MS, Dwivedi S, Priyadarshini M, Tabrez S, Siddiqui MA, Jagirdar H, Al‐Senaidy AM, Al‐Khedhairy AA, MusarratJ. Ribosylation of bovine serum albumin inducesROS accumulation and cell death in cancer line (MCF‐7). Eur Biophys J. 2013; 42, 811–18.

[69] Szkudlarek A, Sułkowska A, Maciążek‐Jurczyk M, Chudzik M, Równicka‐Zubik J. Effects of non‐enzymatic glycation in human serum albumin. Spectroscopic analysis. Spectrochim Acta A Mol Biomol Spectrosc. 2016; 152, 645–53. doi: 10.1016/j.saa.

[70] Vetter SW, Indurthi VS. Moderate glycation of serum albumin affects folding, stabili‐ ty, and ligand binding. Clin Chim Acta. 2011; 412, 2105–16. doi: 10.1016/j.cca.

[71] Sattarahmady N, Moosavi‐Movahedi AA, Habibi‐Rezaei M, Ahmadian S, Saboury AA, Heli H, Sheibani N. Detergency effects of nanofibrillar amyloid formation on glyca‐ tion of human serum albumin. Carbohydr Res. 2008; 343, 2229–34. doi: 10.1016/j.carres.

[72] Wei Y, Chen L, Chen J, Ge L, He R Q. Rapid glycation with D‐ribose induces globular amyloid‐like aggregations of BSA with high cytotoxicity to SH‐SY5Y cells. BMC Cell

[73] Infusini G, Iannuzzi C, Vilasi S, Maritato R, Birolo L, Pagnozzi D, Pucci P, Irace G, Sirangelo I. W‐F substitutions in apomyoglobin increase the local flexibility of the N‐ terminalregion causing amyloid aggregation: a H/D exchange study. Protein Pept Lett.

[74] Vilasi A, Vilasi S, Romano R, Acernese F, Barone F, Balestrieri ML, Maritato R, Irace G, Sirangelo I. Unraveling amyloid toxicity pathway in NIH3T3 cells by a combined proteomic and 1 H‐NMR metabonomic approach. J Cell Physiol. 2013; 228(6), 1359–67.

[75] Sirangelo I, Giovane A, Maritato R, D'Onofrio N, Iannuzzi C, Giordano A, Irace G, Balestrieri ML. Platelet‐activating factor mediates the cytotoxicity induced by W7FW14F apomyoglobin amyloid aggregates in neuroblastoma cells. J Cell Biochem.

[76] Iannuzzi C, Maritato R, Irace G, Sirangelo I. Glycation accelerates fibrillization of the amyloidogenic W7FW14F apomyoglobin. PLoS One. 2013; 8(12), e80768. doi: 10.1371/

[77] Iannuzzi C, Maritato R, Irace G, Sirangelo I. Misfolding and amyloid aggregation of apomyoglobin. Int J Mol Sci. 2013; 14(7), 14287–300. doi: 10.3390/ijms140714287.

98. doi: 10.1016/j.bbapap.2009.12.003.

Biol. 2009; 10, 10. doi: 10.1186/1471‐2121‐10‐10.

2014; 115(12), 2116–22. doi: 10.1002/jcb.24888.

doi: 10.1007/s00249‐013‐0929‐6.

2015.01.120.

182 184Exploring New Findings on Amyloidosis

2011.07.022.

2008.04.036.

2013; 20(8), 898–904.

doi: 10.1002/jcp.24294.

journal.pone.0080768.


## **Is Alzheimer's Associated Amyloid Beta an Innate Immune Protein**

Ruth Kandel, Mitchell R White, I-Ni Hsieh and Kevan L. Hartshorn

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63021

#### **Abstract**

There is now abundant evidence that chronic inflammation in the brain is central to the pathogenesis of Alzheimer's disease (AD) and that this is precipitated through accumulation of amyloid beta (Aβ) peptides. In this review, we first outline this evidence and how specific receptors on microglia and monocyte/macrophages determine whether extracellular Aβ peptides can be cleared through non‐inflammato‐ ry phagocytosis or instead result in pro‐inflammatory cytokine generation. Most efforts of treatment for AD so far have focused on reduction of Aβ levels (in particular neurotoxic oligomers of Aβ1‐42) in the brain. Recent findings suggest an alternative approach in which pro‐inflammatory responses to Aβ peptides are targeted to reduce injury. Most recently evidence has come to light that Aβ peptides resemble anti‐ microbial peptides which are part of the innate defense system against infection. Such peptides act both by directly inactivating pathogens, but also by modulating respons‐ es of innate immune cells, including phagocytes. Indeed, Aβ peptides, particularly Aβ1‐42, do inhibit a range of potential pathogens, including bacteria, fungi, and viruses. Coupling this with evidence linking chronic viral, bacteria, or fungal infection to AD suggests that production of Aβ peptides in the brain is part of an innate immune response which might normally be beneficial, but which leads to harm when it is chronic or uncontrolled. This suggests that discovery of the original possibly infectious (or other inflammatory) stimulus for Aβ production would allow early intervention to prevent development of AD.

**Keywords:** Inflammasome, TREM2, Microglia, antimicrobial peptide

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

Aβ accumulation is believed to contribute strongly to the pathogenesis of AD, although the actual physiological function and reason for accumulation of Aβ in the brain are not known. Aβ is a fragment of the larger β amyloid precursor protein (APP) which is a transmembrane protein which can be broken down by various proteases into a variety of fragments, includ‐ ing extracellular and intracellular fragments and the peptide fragments Aβ1‐42 and Aβ1‐40 which are composed partly of the extracellular and partly of the transmembrane domain of APP. Aβ1‐40 is more abundant than Aβ1‐42, but Aβ1‐42 is the more amyloidogenic and neurotoxic species [1–3]. The neurotoxicity of Aβ1‐42 has been shown to depend on the ability of this peptide to form unstable oligomers (pentamers mainly), whereas the protofibrils or fibrils formed from the peptide are less neurotoxic. Recent studies are at last starting to elucidate why accumulation of Aβ, especially the 1–42 form leads to brain injury. These studies focus on the role of Aβ as a trigger of inflammation and emphasize its interaction with glial cells in the brain. A vicious cycle appears to occur in which Aβ peptides activate glial and other phagocytic cells which in turn impairs the ability of these cells to clear Aβ peptides and plaques from the brain. The reasons for production of Aβ in the brain in the first place are less clear. Recent findings that Aβ peptides function as antibacterial and antimicrobial peptides have given rise to the hypothesis that production of Aβ peptides may have evolved as part of the innate host defense system.

**I. Evidence for a link between chronic inflammation and AD—**At the outset, it is impor‐ tant to distinguish between early onset AD and late onset AD. Both forms of AD are strong‐ ly linked to excess accumulation of Aβ in plaques in the brain; however, the causes of Aβ accumulation may differ. In the case of early onset disease, there is a link to actual mutations in the Aβ gene or in genes involved in proteolytic processing of the precursor protein to form Aβ peptides. The most commonly used mouse model, the APP‐PS1 model, of AD is based on over‐expression of Aβ peptides in the brain in a similar manner. Late onset AD appears mainly to result from impaired clearance of Aβ peptides (ratherthan increased production per se) and is linked to polymorphisms of several genes as outlined below. There is strong and growing evidence for a link between chronic inflammation and the development of both forms of AD and some other dementing illnesses. We refer the reader to several recent excellent reviews forin depth consideration this topic [4–6]. A link between inflammation and AD has long been suspected in part based on clinical findings (e.g., the apparent protective effect of long‐term non‐steroidal anti‐inflammatory use against development of sporadic AD) [7, 8]. In addition, pathological studies have shown evidence of inflammation surrounding neuritic plaques in AD. Complement factors, clusters of activated microglia and cytokines has been found in and near Aβ plaques [5, 9]. These findings of inflammation were also noted as early events in the brains of patients with AD. Expression of genes associated with inflammation in brain is increased in aging, and this effect is accentuated in patients with AD [10, 11].

**II. Microglia and monocyte/macrophages as pivotal cells in mediating clearance or inflam‐ matory responses to Aβ peptides**—Microglia are resident phagocytic cells in the brain that plays a key role in maintaining the health of neurons and responding to sterile or infectious injury. Evidence is now converging from genome‐wide association studies (GWAS), in vitro cell biologic experiments and mouse models of excess Aβ accumulation, that microglia and to an extent recruited blood monocyte macrophages, are critical in mediating either protective clearance of Aβ or damage through inflammatory activation by Aβ peptides.

**1. Introduction**

186 188Exploring New Findings on Amyloidosis

the innate host defense system.

Aβ accumulation is believed to contribute strongly to the pathogenesis of AD, although the actual physiological function and reason for accumulation of Aβ in the brain are not known. Aβ is a fragment of the larger β amyloid precursor protein (APP) which is a transmembrane protein which can be broken down by various proteases into a variety of fragments, includ‐ ing extracellular and intracellular fragments and the peptide fragments Aβ1‐42 and Aβ1‐40 which are composed partly of the extracellular and partly of the transmembrane domain of APP. Aβ1‐40 is more abundant than Aβ1‐42, but Aβ1‐42 is the more amyloidogenic and neurotoxic species [1–3]. The neurotoxicity of Aβ1‐42 has been shown to depend on the ability of this peptide to form unstable oligomers (pentamers mainly), whereas the protofibrils or fibrils formed from the peptide are less neurotoxic. Recent studies are at last starting to elucidate why accumulation of Aβ, especially the 1–42 form leads to brain injury. These studies focus on the role of Aβ as a trigger of inflammation and emphasize its interaction with glial cells in the brain. A vicious cycle appears to occur in which Aβ peptides activate glial and other phagocytic cells which in turn impairs the ability of these cells to clear Aβ peptides and plaques from the brain. The reasons for production of Aβ in the brain in the first place are less clear. Recent findings that Aβ peptides function as antibacterial and antimicrobial peptides have given rise to the hypothesis that production of Aβ peptides may have evolved as part of

**I. Evidence for a link between chronic inflammation and AD—**At the outset, it is impor‐ tant to distinguish between early onset AD and late onset AD. Both forms of AD are strong‐ ly linked to excess accumulation of Aβ in plaques in the brain; however, the causes of Aβ accumulation may differ. In the case of early onset disease, there is a link to actual mutations in the Aβ gene or in genes involved in proteolytic processing of the precursor protein to form Aβ peptides. The most commonly used mouse model, the APP‐PS1 model, of AD is based on over‐expression of Aβ peptides in the brain in a similar manner. Late onset AD appears mainly to result from impaired clearance of Aβ peptides (ratherthan increased production per se) and is linked to polymorphisms of several genes as outlined below. There is strong and growing evidence for a link between chronic inflammation and the development of both forms of AD and some other dementing illnesses. We refer the reader to several recent excellent reviews forin depth consideration this topic [4–6]. A link between inflammation and AD has long been suspected in part based on clinical findings (e.g., the apparent protective effect of long‐term non‐steroidal anti‐inflammatory use against development of sporadic AD) [7, 8]. In addition, pathological studies have shown evidence of inflammation surrounding neuritic plaques in AD. Complement factors, clusters of activated microglia and cytokines has been found in and near Aβ plaques [5, 9]. These findings of inflammation were also noted as early events in the brains of patients with AD. Expression of genes associated with inflammation in brain is

increased in aging, and this effect is accentuated in patients with AD [10, 11].

**II. Microglia and monocyte/macrophages as pivotal cells in mediating clearance or inflam‐ matory responses to Aβ peptides**—Microglia are resident phagocytic cells in the brain that plays a key role in maintaining the health of neurons and responding to sterile or infectious **A. GWAS studies**—The first gene to show strong linkage to development of AD was the ε4 variant of apolipoprotein E (APOE). Although APOE is mainly known forits ability to regulate cholesterol and lipid transport, the APOEε4 allele is linked to accumulation of Aβ in plaques in humans and mouse models [12]. In addition, there is evidence that APOEε4 may be linked to inflammatory responses in the brain through interaction with receptors on microglia [13]. For instance, crossing APOEε4 over‐expressing mice with APP/PS1 mice results in worsen‐ ing inflammatory responses to lipopolysaccharide (LPS) as compared to APP/PS1 mice over‐ expressing APOEε3 [7]. Other proteins which modulate lipid metabolism, for instance surfactant protein D, have innate immune activity as well [14]. Of interest, surfactant protein D has also been linked to dementia [15]. Several other gene variants which are primarily expressed on microglia or other myeloid cells have been linked to AD. The most prominent of these is the triggering receptor expressed on myeloid cells 2 (TREM2) [6, 16–18]. This recep‐ tor mediates phagocytic activity and cytokine responses of myeloid cells and polymorphic forms of this receptor are linked to development of late onset AD with an effect size similar to APOEε4. Similar polymorphisms of the complementreceptor CR‐1 and an additional myeloid cellreceptor, CD33, have been linked to development of AD [19, 20]. The contributions of these and other myeloid receptors to accumulation of Aβ and neuronal injury are now being elucidated through in vivo and in vitro studies.

**B. Cell biology and mouse model studies**—There is abundant evidence that accumulation of Aβ1‐42 itself activates microglia and monocyte macrophages through binding to various receptors on these cells, either directly or through binding to other proteins (e.g., comple‐ ment). A key hypothesis which brings together the various studies is that the ability of microglia to ingest Aβ1‐42, or to degrade it through proteolysis is protective, whereas production of pro‐inflammatory cytokines (e.g., TNF, IL‐1 or IL‐18) in response to Aβ1‐42 is harmful. Microglia and macrophages can have a variety of phenotypes, with two major categories being the MI and M2 phenotypes. The MI phenotype is associated with pro‐ inflammatory cytokine generation as well as production of nitric oxide or superoxide radicals. In contrast, the M2 phenotype (presumably the more beneficial phenotype in the context of Aβ accumulation) is associated with enhanced phagocytosis and reduced pro‐inflammatory signaling. Given the sensitive nature of the brains and neurons, clearance of pathogens, harmful proteins, or cellular debris ideally would proceed with minimal inflammation. The beneficial or harmful effects of various myeloid receptors have been categorized according to whether they mediate either M1 or M2 like activities [5, 21].

**Microglial scavenger receptors**—Receptors shown to promote phagocytosis and non‐ inflammatory clearance of Aβ1‐42 include the scavenger receptors SR‐A or Scara‐A [22, 23]. In contrast, CD36, which is another scavenger receptor, appears to mediate pro‐inflammatory responses to Aβ1‐42 [24, 25]. Crossing of APP/PS1 with mice‐lacking SR‐A leads to greater Aβ accumulation and worsened survival, whereas crossing with mice‐lacking CD36 causes reduced brain cytokine production and Aβ accumulation and improved survival. CD33 is another receptor that is involved in uptake of Aβ peptides by microglia. CD33 is over‐ expressed in microglia of humans with AD and the CD33 mutations that were found to be protective vs AD caused decreased expression of CD33 [19, 26]. Crossing of mice‐lacking CD33 with APP/PS1 mice leads to reduced Aβ peptide accumulation and plaque burden [19]. In vitro studies showed that CD33 actually reduces Aβ1‐42 uptake by microglial cells.

**Triggering receptor expressed on myeloid cells 2 (TREM2)**—TREM2 has a more complex role in that it can mediate either phagocytic clearance or pro‐inflammatory cytokine responses by myeloid cells [6]. Like CD33, it is highly expressed in microglia and monocytes in the brain. In particular, it is highly expressed in microglia surrounding amyloid plaques in APP/PS1 mice [16]. In one study using this mouse model, deletion of TREM2 reduced inflammatory pathology in the brain [27]. In contrast, in another study, over‐expression of TREM2 in these mice improved pathology [28]. The GWAS studies suggest that polymorphisms associated with decreased TREM2 production increase risk of development of AD or other neuro‐ degenerative diseases [16]. Based on this, it has been postulated that loss of TREM2 impairs non‐inflammatory phagocytic clearance of Aβ peptides or of damaged neurons. TREM2 is well described as a phagocytic receptor for bacteria. Further studies will be needed to understand the complex role of TREM2 in AD. Of interest, two recent studies provide potential links between the contributions of APOEε4 and TREM2 to inflammation in AD. APOE was shown to bind to wild‐type (but not mutant) TREM2 [29] and the APOEε4 variant has distinctive effects (as compared to other APOE subtypes) in modulating microglial prostaglandin production and TREM2 expression [30].

**Complement and complement receptors**—The complement receptor CR‐1 also appears to have a complex role in AD [20]. CR‐1 is the receptor for complement factors C3b and C4b. Aβ can activate the complement cascade and bind to C3b. This in turn leads to binding of Aβ peptides to CR‐1. CR‐1 is expressed on myeloid cells and erythrocytes. In the case of erythro‐ cytes, it serves to mediate clearance of complement bound proteins or organisms from the circulation. There is some evidence that this may promote clearance of Aβ outside the brain [31]. CR‐1 also mediates phagocytosis of complement bound proteins and pathogens by phagocytes. Aβ can activate the complement system via the alternative pathway. This could conceivably lead to increased inflammation in the brain. However, it has been found that C3 deficiency or inhibition of complement worsens Aβ accumulation and neurodegeneration in mice [32]. These finding suggest that the complement activation may overall be beneficial vs AD. A possible explanation for the role of CR‐1 is that the polymorphism most tightly linked to increased risk for AD (CR1‐S) has an increased C3b/C4b binding site and that CR‐1 actually acts to inhibit further activation of the complement cascade after binding to C3b/C4b [20].

**Toll like receptors**—Toll‐like receptors (TLRs) and the associated adaptor protein also have been found to mediate phagocyte activation by Aβ [24, 33–37]. Once again the exact role of TLRs in AD pathology is unclear. A simplistic hypothesis would assume that TLR activation should worsen AD pathology; however, there is also evidence that the reverse may be true [33, 38]. Of interest, IL‐10 which is predominantly an anti‐inflammatory cytokine has been found to have adverse effects in AD mouse models [39].

**Inflammasomes**—Anotherimportant line of evidence relates to the role of nucleotide‐binding domain leucine‐rich repeat containing 3 (NLRP3) inflammasomes in AD [4, 40, 41]. Inflam‐ masomes are multi‐molecular complexes in phagocytic cells which mediate production of the pro‐inflammatory cytokines IL‐1 and IL‐18 through the action of caspase 1 and induction of an inflammatory form of cell death called pyroptosis [42, 43]. Inflammasomes are involved in phagocyte mediated host defense against various pathogens. In the case of bacteria, the inflammasomes are activated by pathogen‐associated molecular patterns (or PAMPs), like LPS. Increasingly, however, inflammasomes have been implicated in various inflammatory states triggered by self molecules termed damage‐associated molecular patterns or DAMPs. NLRP3 inflammasomes activation has been linked to inflammatory bowel diseases, celiac disease, gout, multiple sclerosis, and type II diabetes mellitus. It appears that NLRP3 inflam‐ masomes also mediate chronic inflammatory responses to Aβ peptides. NLRP3 inflamma‐ somes are one subtype among a variety of inflammasomes. **Figure 1** illustrates the potential mechanism of assembly and activation of NLRP3 inflammasomes by Aβ peptides. Aβ peptides appearto act as a DAMP. As noted, two signals are required for activation, both of which could be triggered by Aβ peptides. The NLRP3 inflammasome complex consists of oligomeric assemblies of the NLRP3 protein, the apoptosis‐associated speck‐like protein containing a caspase activation and recruitment domain (ASC) protein and caspase 1. Crossing of mice‐ lacking caspase 1 or NLRP3 with APP/PS1 mice results in increased clearance of Aβ pepti‐ des and plaques, reduced neurodegeneration and a skewing of microglia to the M2 phenotype. Pro‐inflammatory signaling in response to Aβ peptides mediated through inflammasomes appears to lead to a vicious cycle in which microglia acquire and M1 phenotype and cause increasing injury and decreased Aβ clearance [21].

reduced brain cytokine production and Aβ accumulation and improved survival. CD33 is another receptor that is involved in uptake of Aβ peptides by microglia. CD33 is over‐ expressed in microglia of humans with AD and the CD33 mutations that were found to be protective vs AD caused decreased expression of CD33 [19, 26]. Crossing of mice‐lacking CD33 with APP/PS1 mice leads to reduced Aβ peptide accumulation and plaque burden [19]. In vitro

**Triggering receptor expressed on myeloid cells 2 (TREM2)**—TREM2 has a more complex role in that it can mediate either phagocytic clearance or pro‐inflammatory cytokine responses by myeloid cells [6]. Like CD33, it is highly expressed in microglia and monocytes in the brain. In particular, it is highly expressed in microglia surrounding amyloid plaques in APP/PS1 mice [16]. In one study using this mouse model, deletion of TREM2 reduced inflammatory pathology in the brain [27]. In contrast, in another study, over‐expression of TREM2 in these mice improved pathology [28]. The GWAS studies suggest that polymorphisms associated with decreased TREM2 production increase risk of development of AD or other neuro‐ degenerative diseases [16]. Based on this, it has been postulated that loss of TREM2 impairs non‐inflammatory phagocytic clearance of Aβ peptides or of damaged neurons. TREM2 is well described as a phagocytic receptor for bacteria. Further studies will be needed to understand the complex role of TREM2 in AD. Of interest, two recent studies provide potential links between the contributions of APOEε4 and TREM2 to inflammation in AD. APOE was shown to bind to wild‐type (but not mutant) TREM2 [29] and the APOEε4 variant has distinctive effects (as compared to other APOE subtypes) in modulating microglial prostaglandin

**Complement and complement receptors**—The complement receptor CR‐1 also appears to have a complex role in AD [20]. CR‐1 is the receptor for complement factors C3b and C4b. Aβ can activate the complement cascade and bind to C3b. This in turn leads to binding of Aβ peptides to CR‐1. CR‐1 is expressed on myeloid cells and erythrocytes. In the case of erythro‐ cytes, it serves to mediate clearance of complement bound proteins or organisms from the circulation. There is some evidence that this may promote clearance of Aβ outside the brain [31]. CR‐1 also mediates phagocytosis of complement bound proteins and pathogens by phagocytes. Aβ can activate the complement system via the alternative pathway. This could conceivably lead to increased inflammation in the brain. However, it has been found that C3 deficiency or inhibition of complement worsens Aβ accumulation and neurodegeneration in mice [32]. These finding suggest that the complement activation may overall be beneficial vs AD. A possible explanation for the role of CR‐1 is that the polymorphism most tightly linked to increased risk for AD (CR1‐S) has an increased C3b/C4b binding site and that CR‐1 actually acts to inhibit further activation of the complement cascade after binding to C3b/C4b [20].

**Toll like receptors**—Toll‐like receptors (TLRs) and the associated adaptor protein also have been found to mediate phagocyte activation by Aβ [24, 33–37]. Once again the exact role of TLRs in AD pathology is unclear. A simplistic hypothesis would assume that TLR activation should worsen AD pathology; however, there is also evidence that the reverse may be true [33, 38]. Of interest, IL‐10 which is predominantly an anti‐inflammatory cytokine has been found

studies showed that CD33 actually reduces Aβ1‐42 uptake by microglial cells.

production and TREM2 expression [30].

188 190Exploring New Findings on Amyloidosis

to have adverse effects in AD mouse models [39].

**Chemokines**—Chemokines and their receptors also appear to modulate Aβ‐related patholo‐ gy. The chemokine CXCL10 is expressed at high levels in AD brain. Deletion or inhibition of the receptor for this and other CXCL chemokines (CXCR3) increased microglial uptake of Aβ in vitro and in vivo [44]. Deletion of CXCR1 had a similar effect [45]. These findings imply that these chemokines worsen the inflammatory effects which lead to increased injury or im‐ paired clearance of Aβ. An active area of investigation as well is the role of recruited mono‐ cyte macrophages to AD pathology or Aβ clearance. Deletion of the monocyte receptor CCR2 in mice reduces monocyte recruitment and increases amyloid pathology in APP/PS1 mice [46], indicating that these cells play a role in clearance. Similarly CCR5 deletion increased Aβ deposition and neurological loss [47]. Other cytokines, like IL‐12, are linked to exacerbation of AD as is activation of oxidant production by microglia [48–50].

**C. Epidemiological studies**—Life‐style or other factors which are known to increase AD incidence also may mediate their effects through chronic inflammation. Examples include obesity, lack of exercise, peri‐odontitis, or diabetes [5]. Overall these studies lend strong supportto the hypothesis thatthe innate immune system, and specifically, resident orrecruited phagocytic cells play a pivotalrole in determining the balance between protective Aβ clearance or damaging Aβ peptide‐induced inflammation.

**Figure 1.** Microglial NLRP3 inflammasome activation by Aβ oligomers—the NLRP3 inflammasome can be activated by various PAMPs or DAMPS. Aβ oligomers can be considered as a DAMP which leads to NLRP3 activation in micro‐ glia. To induce NLRP3 activation, there needs to be at least two signals. The first signal causes increased production of the NLRP3 protein, and the second signal induces assembly of the multimolecular inflammasome complex. Other pro‐ teins involved in this complex include ASC and caspase 1. Activation of caspase 1 results in cleavage of pro‐IL‐1β and pro‐IL‐18 to form active IL‐1β and IL‐18. An additional effect of inflammasome activation is induction of a form of cell death caused pyroptosis. The result is a significant induction of local inflammation but also impairment of microglial phagocytosis, reducing further clearance of Aβ. The exact triggers of signal 1 and 2 are have not been fully elucidated. Since Aβ can trigger TLR activation, it is possible that this provides signal 1. In the case of some bacteria, rupture of phagosomes appears to provide signal 2, possibly by release of cathepsin B. We speculate that this could be involved in NLRP3 inflammasome activation by Aβ. Other possible mediators of the second signal include reactive oxygen spe‐ cies release or activation of plasma membrane ion channels.

**III. What is the physiological stimulus for Aβ production?—**Thus far most of the studies we, we have discussed consider the downstream effects of Aβ accumulation on inflammation or cellular dysfunction. For early onset AD, and for the APP/PS1 mice, increased Aβ accumula‐ tion is the result of alterations of the Aβ protein itself or of the enzymes involved in cleavage of the precursor protein. It is unclear, however, what triggers Aβ production under normal circumstances or in late onset AD. Attempts to directly reduce Aβ levels through the use of antibodies against Aβ peptides have not been highly successful, although it is possible that use in earlier stages of the disease (prior to major cognitive impairment) may be beneficial. There is also hope that intervention to reduce inflammatory response to Aβ may be benefi‐ cial, although here again it may be necessary to act early in the course of disease since the inflammatory phenotype seems to precede clinical AD.

If it was possible to determine the initial causes for Aβ accumulation in the brain, this might provide another approach to early intervention. One hypothesis is that infection initiates or sustains the process of Aβ accumulation. Excess accumulation of Aβ has been linked to Human Immunodeficiency Virus‐related dementia [51, 52], and the virus can cause Aβ accumula‐ tion in vitro as well [53, 54]. Similarly Herpes Simplex Virus (HSV) induced encephalitis, and HSV infection in vitro is associated with Aβ accumulation [55–59], again implying that viruses may be a stimulus of Aβ production orimpaired clearance. These findings suggest that viruses that infect the brain could be triggers for accumulation of Aβ, perhaps as part of an aberrant or sustained innate immune response. Antibodies to Cytomegalovirus, Epstein Barr Virus, or Human Herpes Virus 6 (HHV6) have also been associated with AD [60, 61] in some studies. In contrast, another study showed no link between AD and antibodies to HHV6 [62]. A variety of studies have also linked bacterial infection, including with chlamydia to development of AD [63, 64]. Of great interest, recent studies found fungal forms and sequences in brains of AD patients but not in controls [65–67]. Of course of a causal connection between these infec‐ tions and AD is far from proven.

**IV. Aβ peptides as antimicrobial agents**—An alternative hypothesis to explain Aβ peptide and ultimately plaque production is that it is part of a host defense response to infectious or traumatic injury. Aβ peptides resemble some anti‐microbial peptides or AMPs in their structure [68, 69]. Aβ peptides are similar to the porcine AMP, protegrin, in ability to form channels in membrane structures which is believed to be one of the anti‐bacterial and anti‐ fungal mechanisms of AMPs. Recently, Soscia et al. demonstrated antibacterial and antifun‐ gal activity for Aβ peptides [70]. In addition, this study showed that Aβ isolated from the brain of AD patients had antimicrobial activity and that incubation of these brain‐derived samples with antibodies to Aβ ablated the antimicrobial activity. More recently, we demonstrated that Aβ peptides also have antiviral activity using influenza A virus as a model [71]. In our study and that of Soscia et al., Aβ1‐42 was found to have greater antimicrobial or antiviral activity than Aβ1‐40. We demonstrated that Aβ1‐42, but not Aβ1‐40, caused viral aggregation which appears to contribute to its antiviral effects. This implies a possible connection between the ability of Aβ1‐42 to assemble into oligomers and its antiviral activity, since this peptide has a greater propensity to form oligomers and fibrils than Aβ1‐40.

**Figure 1.** Microglial NLRP3 inflammasome activation by Aβ oligomers—the NLRP3 inflammasome can be activated by various PAMPs or DAMPS. Aβ oligomers can be considered as a DAMP which leads to NLRP3 activation in micro‐ glia. To induce NLRP3 activation, there needs to be at least two signals. The first signal causes increased production of the NLRP3 protein, and the second signal induces assembly of the multimolecular inflammasome complex. Other pro‐ teins involved in this complex include ASC and caspase 1. Activation of caspase 1 results in cleavage of pro‐IL‐1β and pro‐IL‐18 to form active IL‐1β and IL‐18. An additional effect of inflammasome activation is induction of a form of cell death caused pyroptosis. The result is a significant induction of local inflammation but also impairment of microglial phagocytosis, reducing further clearance of Aβ. The exact triggers of signal 1 and 2 are have not been fully elucidated. Since Aβ can trigger TLR activation, it is possible that this provides signal 1. In the case of some bacteria, rupture of phagosomes appears to provide signal 2, possibly by release of cathepsin B. We speculate that this could be involved in NLRP3 inflammasome activation by Aβ. Other possible mediators of the second signal include reactive oxygen spe‐

**III. What is the physiological stimulus for Aβ production?—**Thus far most of the studies we, we have discussed consider the downstream effects of Aβ accumulation on inflammation or cellular dysfunction. For early onset AD, and for the APP/PS1 mice, increased Aβ accumula‐ tion is the result of alterations of the Aβ protein itself or of the enzymes involved in cleavage of the precursor protein. It is unclear, however, what triggers Aβ production under normal circumstances or in late onset AD. Attempts to directly reduce Aβ levels through the use of antibodies against Aβ peptides have not been highly successful, although it is possible that use in earlier stages of the disease (prior to major cognitive impairment) may be beneficial. There is also hope that intervention to reduce inflammatory response to Aβ may be benefi‐ cial, although here again it may be necessary to act early in the course of disease since the

cies release or activation of plasma membrane ion channels.

190 192Exploring New Findings on Amyloidosis

inflammatory phenotype seems to precede clinical AD.

The finding that Aβ peptides, especially, Aβ1‐42 act like other cationic antimicrobial pepti‐ des may also explain its ability to activate phagocytic cells. AMPs have direct antimicrobial and antiviral activities but they also triggerrecruitment and activation of immune cells [34, 36, 72]. We also recently showed that Aβ1‐42 modulates responses of neutrophils and mono‐ cytes to the influenza virus [71]. Aβ1‐42 increased neutrophil uptake of influenza A virus and potentiated neutrophil respiratory burst and neutrophil extracellular trap (NET) formation in response to the virus. Aβ1‐42 also reduced inflammatory cytokine production triggered by influenza virus in monocytes. The opsonizing activity of Aβ1‐42 was again notreplicated with Aβ1‐40. More recently, we found that Aβ peptides can increase neutrophil uptake of bacte‐ ria as well (unpublished data). Overall, these studies lend support to the hypothesis that Aβ peptides serve a host defense role and that chronic infectious or inflammatory stimuli may result in an aberrant prolongation of what normally would be a helpful response.

## **2. Conclusions**

There is now abundant evidence from a variety of sources that AD is characterized by a chronic inflammatory response in the brain. The key elements in this process include the ability of Aβ peptides, especially Aβ1‐42, to directly activate phagocytic cells, most notably microglia and, to a lesser extent, monocyte/macrophages. **Figure 2** summarizes the microglial receptors, cytokines, and signaling mechanisms known to be linked to responses to Aβ peptides. These phagocytic cells are atthe cross‐roads ofinnate immune responses in the brain, and they appear to play a pivotal role in determining whether the response to Aβ peptide accumulation is non‐ inflammatory phagocytosis or pro‐inflammatory cytokine production. One conclusion from these studies is that inhibition of pro‐inflammatory responses early in the evolution of Aβ related pathology could be protective. For example, inhibition of inflammasome activation has been proposed as an approach to treatment. One dilemma is that there is not a simple correlation between processes normally thought of as pro‐inflammatory and reduction of neuronal injury in AD models. As prime examples, activation of the complement system or of toll‐like receptor pathways appears to be protective in some studies. In addition, the role of TREM2, while clearly important, is not as simply as initially expected. The recent findings that Aβ peptides (especially Aβ1‐42) function like other AMPs suggest that Aβ peptides may play a beneficial physiological role in vivo and may actually be part of an innate immune re‐ sponse to infection. If this is so then discovery of underlying infectious triggers of AD might provide a different modality of treatment.

**Figure 2.** Microglial receptors and signaling pathways that are involved in response to Aβ—receptors, signaling path‐ ways or extracellular cytokines shown to promote neuronal injury are shown in red, whereas those shown in green are protective vs neuronal injury or progression of AD like pathology. Receptors shown in as a mixture of green and red have been found to have both beneficial or adverse effects in various studies.

## **Author details**

**2. Conclusions**

192 194Exploring New Findings on Amyloidosis

provide a different modality of treatment.

There is now abundant evidence from a variety of sources that AD is characterized by a chronic inflammatory response in the brain. The key elements in this process include the ability of Aβ peptides, especially Aβ1‐42, to directly activate phagocytic cells, most notably microglia and, to a lesser extent, monocyte/macrophages. **Figure 2** summarizes the microglial receptors, cytokines, and signaling mechanisms known to be linked to responses to Aβ peptides. These phagocytic cells are atthe cross‐roads ofinnate immune responses in the brain, and they appear to play a pivotal role in determining whether the response to Aβ peptide accumulation is non‐ inflammatory phagocytosis or pro‐inflammatory cytokine production. One conclusion from these studies is that inhibition of pro‐inflammatory responses early in the evolution of Aβ related pathology could be protective. For example, inhibition of inflammasome activation has been proposed as an approach to treatment. One dilemma is that there is not a simple correlation between processes normally thought of as pro‐inflammatory and reduction of neuronal injury in AD models. As prime examples, activation of the complement system or of toll‐like receptor pathways appears to be protective in some studies. In addition, the role of TREM2, while clearly important, is not as simply as initially expected. The recent findings that Aβ peptides (especially Aβ1‐42) function like other AMPs suggest that Aβ peptides may play a beneficial physiological role in vivo and may actually be part of an innate immune re‐ sponse to infection. If this is so then discovery of underlying infectious triggers of AD might

**Figure 2.** Microglial receptors and signaling pathways that are involved in response to Aβ—receptors, signaling path‐ ways or extracellular cytokines shown to promote neuronal injury are shown in red, whereas those shown in green are protective vs neuronal injury or progression of AD like pathology. Receptors shown in as a mixture of green and red

have been found to have both beneficial or adverse effects in various studies.

Ruth Kandel1 , Mitchell R White2 , I-Ni Hsieh2 and Kevan L. Hartshorn2\*

\*Address all correspondence to: Khartsho@bu.edu

1 Hebrew Senior Life, Harvard Medical School, Boston, Massachusetts, USA

2 Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA

## **References**


receptor 1 increasing C3b/C4b binding sites. Mol Psychiatry. 2012;17(2):223–33. doi: 10.1038/mp.2011.24. PubMed PMID: 21403675; PubMed Central PMCID: PMC3265835.

[21] Heneka MT, Golenbock DT, Latz E. Innate immunity in Alzheimer's disease. Nat Immunol. 2015;16(3):229–36. doi:10.1038/ni.3102. PubMed PMID: 25689443.

[9] Rogers J, Cooper NR, Webster S, Schultz J, McGeer PL, Styren SD, et al. Complement activation by beta‐amyloid in Alzheimer disease. Proc Natl Acad Sci USA. 1992;89(21):

[10] Barrientos RM, Kitt MM, Watkins LR, Maier SF. Neuroinflammation in the normal aging hippocampus. Neuroscience. 2015;309:84–99. doi:10.1016/j.neuroscience. 2015.03.007. PubMed PMID: 25772789; PubMed Central PMCID: PMC4567963.

[11] Jiang T, Yu JT, Zhu XC, Tan MS, Gu LZ, Zhang YD, et al. Triggering receptor ex‐ pressed on myeloid cells 2 knockdown exacerbates aging‐related neuroinflammation and cognitive deficiency in senescence‐accelerated mouse prone 8 mice. Neurobiol Aging. 2014;35(6):1243–51. doi:10.1016/j.neurobiolaging.2013.11.026. PubMed PMID:

[12] Liu CC, Kanekiyo T, Xu H, Bu G. Apolipoprotein E and Alzheimer disease: risk, mechanisms and therapy. Nat Rev Neurol. 2013;9(2):106–18. doi:10.1038/nrneurol.

[13] Holtzman DM, Herz J, Bu G. Apolipoprotein E and apolipoprotein E receptors: normal biology and roles in Alzheimer disease. Cold Spring Harbor Perspect Med. 2012;2(3):a006312. doi:10.1101/cshperspect.a006312. PubMed PMID: 22393530;

[14] Hartshorn KL. Role of surfactant protein A and D (SP‐A and SP‐D) in human antivi‐ ral host defense. Front Biosci (Schol Ed). 2010;2:527–46. PubMed PMID: 20036966. [15] Nybo M, Andersen K, Sorensen GL, Lolk A, Kragh‐Sorensen P, Holmskov U. Serum surfactant protein D is correlated to development of dementia and augmented

[16] Neumann H, Daly MJ. Variant TREM2 as risk factor for Alzheimer's disease. N Engl J Med. 2013;368(2):182–4. doi:10.1056/NEJMe1213157. PubMed PMID: 23151315.

[17] Jonsson T, Stefansson H, Steinberg S, Jonsdottir I, Jonsson PV, Snaedal J, et al. Variant of TREM2 associated with the risk of Alzheimer's disease. N Engl J Med. 2013;368(2): 107–16. doi:10.1056/NEJMoa1211103. PubMed PMID: 23150908; PubMed Central

[18] Guerreiro R, Wojtas A, Bras J, Carrasquillo M, Rogaeva E, Majounie E, et al. TREM2 variants in Alzheimer's disease. N Engl J Med. 2013;368(2):117–27. doi:10.1056/ NEJMoa1211851. PubMed PMID: 23150934; PubMed Central PMCID: PMC3631573.

[19] Griciuc A, Serrano‐Pozo A, Parrado AR, Lesinski AN, Asselin CN, Mullin K, et al. Alzheimer's disease risk gene CD33 inhibits microglial uptake of amyloid beta. Neuron. 2013;78(4):631–43. doi:10.1016/j.neuron.2013.04.014. PubMed PMID:

[20] Brouwers N, Van Cauwenberghe C, Engelborghs S, Lambert JC, Bettens K, Le Bastard N, et al. Alzheimer risk associated with a copy number variation in the complement

23623698; PubMed Central PMCID: PMC3706457.

mortality. Clin Immunol. 2007;123(3):333–7. PubMed PMID: 17449329.

2012.263. PubMed PMID: 23296339; PubMed Central PMCID: PMC3726719.

10016–20. PubMed PMID: 1438191; PubMed Central PMCID: PMC50268.

24368090.

194 196Exploring New Findings on Amyloidosis

PubMed Central PMCID: PMC3282491.

PMCID: PMC3677583.


[40] Sheedy FJ, Grebe A, Rayner KJ, Kalantari P, Ramkhelawon B, Carpenter SB, et al. CD36 coordinates NLRP3 inflammasome activation by facilitating intracellular nucleation of soluble ligands into particulate ligands in sterile inflammation. Nat Immunol. 2013;14(8):812–20. doi:10.1038/ni.2639. PubMed PMID: 23812099; PubMed Central PMCID: PMC3720827.

[30] Li X, Montine KS, Keene CD, Montine TJ. Different mechanisms of apolipoprotein E isoform‐dependent modulation of prostaglandin E2 production and triggering receptor expressed on myeloid cells 2 (TREM2) expression after innate immune activation of microglia. FASEB J. 2015;29(5):1754–62. doi:10.1096/fj.14‐262683. PubMed

[31] Rogers J, Li R, Mastroeni D, Grover A, Leonard B, Ahern G, et al. Peripheral clear‐ ance of amyloid beta peptide by complement C3‐dependent adherence to erythro‐ cytes. Neurobiol Aging. 2006;27(12):1733–9. doi:10.1016/j.neurobiolaging.2005.09.043.

[32] Wyss‐Coray T, Yan F, Lin AH, Lambris JD, Alexander JJ, Quigg RJ, et al. Prominent neurodegeneration and increased plaque formation in complement‐inhibited Alzheimer's mice. Proc Natl Acad Sci USA. 2002;99(16):10837–42. doi:10.1073/pnas.

[33] Michaud JP, Richard KL, Rivest S. MyD88‐adaptor protein acts as a preventive mechanism for memory deficits in a mouse model of Alzheimer's disease. Mol Neurodegener. 2011;6(1):5. doi:10.1186/1750‐1326‐6‐5. PubMed PMID: 21235801;

[34] Jana M, Palencia CA, Pahan K. Fibrillar amyloid‐beta peptides activate microglia via TLR2: implications for Alzheimer's disease. J Immunol. 2008;181(10):7254–62. PubMed

[35] Tang SC, Lathia JD, Selvaraj PK, Jo DG, Mughal MR, Cheng A, et al. Toll‐like recep‐ tor‐4 mediates neuronal apoptosis induced by amyloid beta‐peptide and the mem‐ brane lipid peroxidation product 4‐hydroxynonenal. Exp Neurol. 2008;213(1):114–21.

[36] Richard KL, Filali M, Prefontaine P, Rivest S. Toll‐like receptor 2 acts as a natural innate immune receptorto clear amyloid beta 1–42 and delay the cognitive decline in a mouse model of Alzheimer's disease. J Neurosci. 2008;28(22):5784–93. PubMed PMID:

[37] Chen K, Iribarren P, Hu J, Chen J, Gong W, Cho EH, et al. Activation of Toll‐like receptor 2 on microglia promotes cell uptake of Alzheimer disease‐associated amy‐

[38] Michaud JP, Halle M, Lampron A, Theriault P, Prefontaine P, Filali M, et al. Toll‐like receptor 4 stimulation with the detoxified ligand monophosphoryl lipid A improves Alzheimer's disease‐related pathology. Proc Natl Acad Sci USA. 2013;110(5):1941–6. doi:10.1073/pnas.1215165110. PubMed PMID: 23322736; PubMed Central PMCID:

[39] Michaud JP, Rivest S. Anti–inflammatory signaling in microglia exacerbates Alzheim‐ er's disease‐related pathology. Neuron. 2015;85(3):450–2. doi:10.1016/j.neuron.

loid beta peptide. J Biol Chem. 2006;281(6):3651–9. PubMed PMID: 16339765.

162350199. PubMed PMID: 12119423; PubMed Central PMCID: PMC125059.

PMID: 25593125; PubMed Central PMCID: PMC4415020.

PubMed PMID: 16290270.

196 198Exploring New Findings on Amyloidosis

PMID: 18981147.

18509040.

PMC3562771.

2015.01.021. PubMed PMID: 25654250.

PubMed PMID: 18586243.

PubMed Central PMCID: PMC3030527.


protein. J Neurosci. 2007;27(20):5394–404. doi:10.1523/JNEUROSCI.5047‐06.2007. PubMed PMID: 17507561.


[61] Carbone I, Lazzarotto T, Ianni M, Porcellini E, Forti P, Masliah E, et al. Herpes virus in Alzheimer'sdisease: relation to progression ofthedisease. NeurobiolAging. 2013;35(1): 122–9. PubMed PMID: 23916950.

protein. J Neurosci. 2007;27(20):5394–404. doi:10.1523/JNEUROSCI.5047‐06.2007.

[51] Zhang J, Liu J, KatafiaszB, Fox H, Xiong H. HIV‐1 gp120‐inducedaxonal injurydetected by accumulation of beta‐amyloid precursor protein in adult rat corpus callosum. J

[52] Andras IE, Eum SY, Toborek M. Lipid rafts and functional caveolae regulate HIV‐ induced amyloid beta accumulation in brain endothelial cells. Biochem Biophys Res

[53] Lan X, Kiyota T, Hanamsagar R, Huang Y, Andrews S, Peng H, et al. The effect of HIV protease inhibitors on amyloid‐beta peptide degradation and synthesis in human cells and Alzheimer's disease animal model. J Neuroimmune Pharmacol. 2012;7(2):412–23. doi:10.1007/s11481‐011‐9304‐5. PubMed PMID: 21826404; PubMed Central PMCID:

[54] Lan X, Xu J, Kiyota T, Peng H, Zheng JC, Ikezu T. HIV‐1 reduces Abeta‐degrading enzymatic activities in primary human mononuclear phagocytes. J Immunol. 186(12):

[55] Wozniak MA, Itzhaki RF, Shipley SJ, Dobson CB. Herpes simplex virus infection causes cellular beta‐amyloid accumulation and secretase upregulation. Neurosci Lett.

[56] Wozniak MA, Frost AL, Preston CM, Itzhaki RF. Antivirals reduce the formation of key Alzheimer's disease molecules in cell cultures acutely infected with herpes simplex

[57] De Chiara G, Marcocci ME, Civitelli L, Argnani R, Piacentini R, Ripoli C, et al. APP processing induced by herpes simplex virus type 1 (HSV‐1) yields several APP fragments in human and rat neuronal cells. PLoS One. 2010;5(11):e13989. PubMed

[58] Lukiw WJ, Cui JG, Yuan LY, Bhattacharjee PS, Corkern M, Clement C, et al. Acyclo‐ vir or Abeta42 peptides attenuate HSV‐1‐induced miRNA‐146a levels in human primary brain cells. Neuroreport. 2010;21(14):922–7. PubMed PMID: 20683212. [59] Piacentini R, Civitelli L, Ripoli C, Marcocci ME, De Chiara G, Garaci E, et al. HSV‐1 promotes Ca2+ ‐mediated APP phosphorylation and Abeta accumulation in rat cortical

neurons. Neurobiol Aging. 2010;32(12):2323 e13–26. PubMed PMID: 20674092. [60] Lurain NS, Hanson BA, Martinson J, Leurgans SE, Landay AL, Bennett DA, et al. Virological and immunological characteristics of human cytomegalovirus infection associated with Alzheimer disease. J Infect Dis. 2013;208(4):564–72. PubMed PMID:

virus type 1. PLoS One. 2011;6(10):e25152. PubMed PMID: 22003387.

Neuroimmune Pharmacol. 2011;6(4):650–7. PubMed PMID: 21286834.

Commun. 2012;421(2):177–83. PubMed PMID: 22490665.

PubMed PMID: 17507561.

198 200Exploring New Findings on Amyloidosis

PMC3223330.

PMID: 21085580.

23661800.

6925–32. PubMed PMID: 21551363.

2007;429(2–3):95–100. PubMed PMID: 17980964.


## **Amyloidogenesis and Responses to Stress**

Magda de Eguileor, Rossana Girardello, Annalisa Grimaldi, Laura Pulze and Gianluca Tettamanti

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64479

#### **Abstract**

Amyloidogenesis is a primitive, physiological response that seems to be an ancient process widely distributed in different cell types of evolutionary distant organisms. The amyloid fibril synthesis is part of a more general inflammatory response to stressful conditions all entailing overproduction of reactive oxygen species (ROS). Interesting‐ ly, this event has been integrated into additional physiological functions: (i) the formation of a scaffold promoting the activation and packaging of melanin; (ii) the formation of a scaffold to compartmentalize hormones in the cytoplasm; (iii) the ability to reversibly link different types of molecules to drive close to the nonself; (iv) the construction of a framework to close body lesions. Amyloid fibril formation is a cellular response harmonically integrated with the stress response but for a deregulation in assembling/dismantling, dangerous depots, as in a lot of pathologies, can occur.

**Keywords:** amyloidogenesis, ROS, melanin, invertebrates, vertebrates

## **1. Introduction**

Bacteria, fungi, yeasts, unicellular algae, plants, invertebrates, and vertebrates can synthe‐ size functional amyloids and physiological amyloidogenesis is a key process in response to stress. Even though the aggregation of proteins into amyloid fibrils is associated with human neurodegenerative diseases [1], including Alzheimer's, Parkinson's, and prion disease, it has been evidenced that amyloid is also a fundamental nonpathological protein folding process

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

that spans from bacteria to humans [2–8]. There are many examples of functional and physiological amyloidogenesis: the bacterial biofilm, the insect silk to form the cocoon, the spider silk for constructing their webs, producing egg sacs, and wrapping in their prey, etc., the scaffold to package melanin in human melanocytes when activated by UV, etc.

The various proteins forming amyloids do not necessary display the same primary structure and/or the same biochemical function and/or similarities in biophysical properties: irrespective of the nature of the polypeptide, the proteins, under defined conditions, share the ability to change folding and adopt cross-β sheet conformation with high resistance to enzymatic cleavage, and denaturant. Amyloids can be easily recognized due to their ability to bind to specific dyes such as Thioflavin T/S and Congo Red (CR) and due to their ultrastructural features.

Once the aggregates, in form of amyloid fibrillar material, are formed and tailor-made for the intra- or extracellular space, they can contribute in numerous ways to cellular/tissue/organism activities.

In functional amyloidogenesis, the synthesis of fibrils, occurring in a controlled folding pathway, is balanced by mechanisms involved in their clearance through enzymatic cleavage. When there is a slow down or a total block in this complex balance between assembling and dismantling, a spatial or temporal accumulation of fibrillar material, responsible of deleteri‐ ous/pathological state, occurs.

The exposure to any kind of chemical or physical nonlethal stress is responsible of an excessive production of cytoplasmic reactive oxygen species (ROS) that, if overpowering the endoge‐ nous antioxidant mechanisms, leads to a progressive cellular damage that, in turn, impairs the survival of the cell. In other words, transient or persistent increase of ROS, as a harmful bioproduct deriving from regular cellular metabolism, is coped in all cellular types with the same sequence of events. Constant basal ROS concentration is strictly maintained with the activation of enzymatic and nonenzymatic defences such as production of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), methionine reductase, ascorbic acid (vitamin C), glutathione (GSH), and vitamin A. Uncontrolled, imbalanced production of ROS, and an inefficient free radical scavenging systems may result in inflammation, hypersensitivity, and in the pathogenesis of numerous human diseases such as neurodegenerative diseases, atherosclerosis, allergy, and cancer [9]. In healthy cells, ROS are produced in a small amount in mitochondria but the dysfunction of these organelles, resulting in increased ROS produc‐ tion, can be caused by rough endoplasmic reticulum (RER) stress. In stressed cells, RER cisternae release Ca2+ that in turn is taken up by mitochondria where determine the opening of membrane pores with the consequent enhancement of ROS production and release in cytoplasm. Resulted vicious cycle leads to impaired cellular homeostasis up to cellular death [10].

ROS overexpression, responsible of a change in cytoplasmic pH, can be considered the earliest event and an absolute prerequisite to induce proteins to adopt a β-sheet conformation, thus the amyloid fibril production is strictly linked to this event [2, 4, 5, 11].

## **1.1. Types of cells and subcellular districts involved in amyloid fibril synthesis**

that spans from bacteria to humans [2–8]. There are many examples of functional and physiological amyloidogenesis: the bacterial biofilm, the insect silk to form the cocoon, the spider silk for constructing their webs, producing egg sacs, and wrapping in their prey, etc.,

The various proteins forming amyloids do not necessary display the same primary structure and/or the same biochemical function and/or similarities in biophysical properties: irrespective of the nature of the polypeptide, the proteins, under defined conditions, share the ability to change folding and adopt cross-β sheet conformation with high resistance to enzymatic cleavage, and denaturant. Amyloids can be easily recognized due to their ability to bind to specific dyes such as Thioflavin T/S and Congo Red (CR) and due to their ultrastructural

Once the aggregates, in form of amyloid fibrillar material, are formed and tailor-made for the intra- or extracellular space, they can contribute in numerous ways to cellular/tissue/organism

In functional amyloidogenesis, the synthesis of fibrils, occurring in a controlled folding pathway, is balanced by mechanisms involved in their clearance through enzymatic cleavage. When there is a slow down or a total block in this complex balance between assembling and dismantling, a spatial or temporal accumulation of fibrillar material, responsible of deleteri‐

The exposure to any kind of chemical or physical nonlethal stress is responsible of an excessive production of cytoplasmic reactive oxygen species (ROS) that, if overpowering the endoge‐ nous antioxidant mechanisms, leads to a progressive cellular damage that, in turn, impairs the survival of the cell. In other words, transient or persistent increase of ROS, as a harmful bioproduct deriving from regular cellular metabolism, is coped in all cellular types with the same sequence of events. Constant basal ROS concentration is strictly maintained with the activation of enzymatic and nonenzymatic defences such as production of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), methionine reductase, ascorbic acid (vitamin C), glutathione (GSH), and vitamin A. Uncontrolled, imbalanced production of ROS, and an inefficient free radical scavenging systems may result in inflammation, hypersensitivity, and in the pathogenesis of numerous human diseases such as neurodegenerative diseases, atherosclerosis, allergy, and cancer [9]. In healthy cells, ROS are produced in a small amount in mitochondria but the dysfunction of these organelles, resulting in increased ROS produc‐ tion, can be caused by rough endoplasmic reticulum (RER) stress. In stressed cells, RER cisternae release Ca2+ that in turn is taken up by mitochondria where determine the opening of membrane pores with the consequent enhancement of ROS production and release in cytoplasm. Resulted vicious cycle leads to impaired cellular homeostasis up to cellular death

ROS overexpression, responsible of a change in cytoplasmic pH, can be considered the earliest event and an absolute prerequisite to induce proteins to adopt a β-sheet conformation, thus

the amyloid fibril production is strictly linked to this event [2, 4, 5, 11].

the scaffold to package melanin in human melanocytes when activated by UV, etc.

features.

activities.

[10].

ous/pathological state, occurs.

2022 Exploring New Findings on Amyloidosis

Even though the formation of amyloid or amyloid-like structure under physiological condition is a generic property of polypeptides across all kingdoms of life and can be considered as a response to different types of stimulations, it is of paramount importance to have an overview about which kinds of organisms, which types of cells, and for which purpose amyloid fibrils are synthesized.

Bacteria are able to produce and utilize amyloid material in adhesion to solid surfaces as a starting point to form the biofilm that is, for the most part of microorganisms, the guarantee to live in a consortium spatially and metabolically organized [12]. Amyloid proteins, contri‐ buting with exopolysaccharides to form the biofilm, provide a key component of this protective scaffold [13, 14].

**Figure 1. Unstimulated and stimulated H. virescens circulating cells**. Optical and ultrastructural images (A–H). Scan‐ ning electron micrographs (SEMs) (A, B): the surface of several granulocytes shows bowl-shaped depression with exo‐ cytosed fibrillar material (white arrowheads). Transmission electron micrographs (TEMs) (C, E, H): the fibrillar material (white arrowhead) is spatially organized close to an electron dense core (E) in dilated RER cisternae surround‐ ing the central nucleus (C). The Thioflavin positivity (green/yellow fluorescence), localized within RER cisternae (D, arrowhead), identify the amyloid fibrils. Melanin is ultrastructurally identified by using specific technique (the period‐ ic acid-silver methenamine staining (PASM). Pigment deposition is localized on exocytosed amyloid fibrils (H, arrow‐ head). ROS generation is detected by monitoring the increase in fluorescence of the oxidized dye H2DCFDA (F, G). The increase in ROS level is evident comparing control granulocytes (F) with those from activated hemocytes (G). Immuno‐ cytochemical characterizations for ACTH (I, J), α-MSH (K, L), and IL-18 (M, N) show higher positivity in activated granulocytes (J, L, N).

Moreover, many bacterial species, under stress condition, are able to induce not only the biofilm production but also melanin synthesis [15].

As in bacteria, many protozoa are able to secrete huge amounts of melanin and generally, the production of the pigment is strictly linked to the presence of an amyloid scaffold.

In 2006, Fowler et al. [4] demonstrated that, in mammals, nonpathogenic amyloids are involved in melanin synthesis in melanocytes where the glycoprotein Pmel17 polymerizes into amyloid fibrils forming a framework on which pigment assembled.

Our data (based on light microscopy (LM), transmission and scanning ultrastructural analysis paralleled by immunocytochemical ones) (see Section 3) about activated hemocytes in the lepidopteran-moth *Heliothis virescens* [7, 16], underline the relationship between melanin synthesis and the production of amyloid fibrils to template the pigment. Moreover, we have described and characterized the morpho-functional events sustaining the amyloid fibril assembly in relation to melanin production, in immune cells under stress condition, not only in insects such as *H. virescens* and *Spodoptera elicoverpa*, but also in different invertebrates (protostomes and deuterostomes) such as *Hirudo medicinalis* (Annelid), *Helix pomatia* (Mollusc), *Ciona intestinalis*, and *Botrillus schlosseri* (Ascidian). In all these animals, melanin synthesis/ amyloid fibril assemblage always starts with ROS overproduction that, in turn, causes a cytoplasmic pH variation [8]. Amyloidogenesis takes place in RER where the large amount of fibrils, spatially organized, dilate them (**Figure 1**, panels **A** and **B**). RER is specialized in folding and maturation of a considerable number of secreted proteins and it is highly sensitive to external (chemical or physical) and internal stimuli (such as change in the redox state) [10].

This amyloid production due to RER deregulation/accumulation can be morphologically evidenced by Thioflavine S staining and by ultrastructural analysis. During this amyloid/ pigment productive phase, a cross-talk between immune and endocrine systems, able to mutually influencing, occurs. These intercommunications are mediated by neuromodulators with the activation of stress-sensoring circuits to produce and release molecules such as adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormone (α-MSH), and neutral endopeptidase (NEP) [7, 8, 16] (**Figure 1**, panels **I**–**N**). These molecules have a direct role in immune defences. ACTH is widely expressed in various tissues (as well demonstrated in immunocytochemical localizations) and it is synthesized not only by cells of nervous system but also by nonneural cells [17–19]. ACTH expression is induced in stress conditions (such as ROS increase, lipopolysaccharide (LPS) administration, presence of bacteria and proinflam‐ matory cytokine administration) to regulate the immune cellular functions influencing proliferation, migration, production of immune mediators, and trafficking of immunocytes.

The temporal extend of ACTH action is restrained by the production of a neutral endopepti‐ dase (NEP) that is concomitantly synthesized. This protease inactivates ACTH and the released cleaved product, α-MSH, is able to inhibit chemotaxis, production of proinflammatory cytokines (nuclear factor kappa-light-chain-enhancer of activated B cells) NFkB stimulation, as well as to stimulate melanin synthesis.

The pigment is accumulated in specific organelles where an amyloid scaffold sustains its packaging. Moreover, NEP can be also involved in the degradation of amyloid fibrils [20, 21].

Furthermore, the activation of amyloidogenesis is accompanied by the overexpression of a furin-like proprotein convertase, a specific enzyme that is activated in melanosomal biogenesis to liberate a fibrillogenic fragment [22] and by the overexpression of proinflammatory cytokines such as interleukin-18 (IL-18) [4, 8, 23, 24]. In particular, this is an important molecule that cooperates both in the activation of innate immune system and in amyloidogenic processes in Alzheimer disease (AD) [23, 24].

Moreover, many bacterial species, under stress condition, are able to induce not only the

As in bacteria, many protozoa are able to secrete huge amounts of melanin and generally, the

In 2006, Fowler et al. [4] demonstrated that, in mammals, nonpathogenic amyloids are involved in melanin synthesis in melanocytes where the glycoprotein Pmel17 polymerizes into amyloid

Our data (based on light microscopy (LM), transmission and scanning ultrastructural analysis paralleled by immunocytochemical ones) (see Section 3) about activated hemocytes in the lepidopteran-moth *Heliothis virescens* [7, 16], underline the relationship between melanin synthesis and the production of amyloid fibrils to template the pigment. Moreover, we have described and characterized the morpho-functional events sustaining the amyloid fibril assembly in relation to melanin production, in immune cells under stress condition, not only in insects such as *H. virescens* and *Spodoptera elicoverpa*, but also in different invertebrates (protostomes and deuterostomes) such as *Hirudo medicinalis* (Annelid), *Helix pomatia* (Mollusc), *Ciona intestinalis*, and *Botrillus schlosseri* (Ascidian). In all these animals, melanin synthesis/ amyloid fibril assemblage always starts with ROS overproduction that, in turn, causes a cytoplasmic pH variation [8]. Amyloidogenesis takes place in RER where the large amount of fibrils, spatially organized, dilate them (**Figure 1**, panels **A** and **B**). RER is specialized in folding and maturation of a considerable number of secreted proteins and it is highly sensitive to external (chemical or physical) and internal stimuli (such as change in the redox state) [10]. This amyloid production due to RER deregulation/accumulation can be morphologically evidenced by Thioflavine S staining and by ultrastructural analysis. During this amyloid/ pigment productive phase, a cross-talk between immune and endocrine systems, able to mutually influencing, occurs. These intercommunications are mediated by neuromodulators with the activation of stress-sensoring circuits to produce and release molecules such as adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormone (α-MSH), and neutral endopeptidase (NEP) [7, 8, 16] (**Figure 1**, panels **I**–**N**). These molecules have a direct role in immune defences. ACTH is widely expressed in various tissues (as well demonstrated in immunocytochemical localizations) and it is synthesized not only by cells of nervous system but also by nonneural cells [17–19]. ACTH expression is induced in stress conditions (such as ROS increase, lipopolysaccharide (LPS) administration, presence of bacteria and proinflam‐ matory cytokine administration) to regulate the immune cellular functions influencing proliferation, migration, production of immune mediators, and trafficking of immunocytes. The temporal extend of ACTH action is restrained by the production of a neutral endopepti‐ dase (NEP) that is concomitantly synthesized. This protease inactivates ACTH and the released cleaved product, α-MSH, is able to inhibit chemotaxis, production of proinflammatory cytokines (nuclear factor kappa-light-chain-enhancer of activated B cells) NFkB stimulation,

The pigment is accumulated in specific organelles where an amyloid scaffold sustains its packaging. Moreover, NEP can be also involved in the degradation of amyloid fibrils [20, 21].

production of the pigment is strictly linked to the presence of an amyloid scaffold.

biofilm production but also melanin synthesis [15].

2044 Exploring New Findings on Amyloidosis

as well as to stimulate melanin synthesis.

fibrils forming a framework on which pigment assembled.

It is important to emphasize that in immune cells from phylogenetically distant metazoans (viz. molluscs, annelids, insects, ascidians, and vertebrates) [8, 16], the amyloid scaffold is produced to package melanin and to convey the pigment in close contact to the invader with a twofold advantage: to isolate the nonself from host tissues and to focus the toxic bio-product [7, 8, 16], thus minimizing the diffusion of highly reactive, toxic melanin precursors. This association may occur in intra- or extracell compartments. In several invertebrates, stimulated granulocytes (granular circulating cells) produce an amyloid lattice into the RER, that is exocytosed in the body cavity where it works as a template for melanin resulting from humoral prophenoloxidase (proPO) system reactions [25, 26]. proPOs synthesized primarily by hemocytes are present after their lysis in the hemolymph. The proPO activation cascade leads to the formation of quinones that undergo additional reactions leading to synthesis of melanin. This pigment is deposited on the parasite surface, on nodules made by hemocytes, and in wound sites.

In other invertebrates and in vertebrates, melanin and amyloid fibril synthesis are not disjoined and the molecules are produced and assembled in the same cell (melanocytes) [4]. In any case, amyloid fibrils, synthesized by circulating immunocytes (we use this generic term to identify the cells with the same morpho-functional characteristic that, in invertebrates, have been called, phagocytes, amebocytes, hemocytes), are a shuttle of melanin made to trap and isolate the nonself in capsules where the killing of pathogens is facilitated.

So far, we have described the formation of amyloid fibrils as an event absolutely necessary to activate, support, and store pigments, but this is also true, always as response to stress and due to its intrinsic adhesiveness [27], for the assembly and the compartmentalization of hormones such as ACTH in the cytoplasm [28].

The activation of vertebrates neutrophils, due to the presence of bacteria or fungi, culminates with the production of the neutrophil extracellular trap (NET). NETs, as we have immunocy‐ tochemically demonstrated, are characterized by a fibrillar backbone for addressing cytotoxic proteins and DNA against the nonself, that shows amyloid birefringence upon Congo red staining, Thioflavin S positivity, and harbors a basic mammalian protein, Pmel17 [4], as validated by the colocalization of signals [29] (**Figure 2**, panels **D**–**J**). In stimulated neutrophils, there is a correlation among amyloidogenesis and ROS generation, change in cytoplasmic pH, enhanced expression of the ACTH/α-MSH loop, synthesis of specific cytokine, and also autophagy and exosome release (**Figure 2**, panel **C**). These closely related partners appear to play a key role in the management of the amyloid fibrils that constitute the scaffold of NET [29].

**Figure 2. Unstimulated and stimulated human neutrophils**. Thin and semithin sections of resting (A) and activated neutrophils (B, C). The unstimulated cells are roundish but after stimulation with LPS (B, C) they lose their globoid shape acquiring migratory phenotype (B, C). (D–F) Identification of amyloid fibrils with Thioflavin S (ThS): unstimu‐ lated neutrophils (D) in comparison with stimulated cells (E, F) from LPS administration. Within cells (E) and extracell‐ ularly (F), amyloid fibrils are localized by ThS brightly fluorescence. Nuclei and DNA material in NETs are marked in brilliant blue with DAPI staining. The blue (DAPI) and green (ThS) in NETs, released on stimulation, co-localize (white arrowhead). (G–J) Immunolocalization with a specific antibody directed against Pmel17 (a mammalian protein in‐ volved in amyloidogenesis): immunofluorescence staining show the increased Pmel17 expression that is present in the cytoplasm of stimulated neutrophils (H) and in the NETs (H, I) (arrowheads). DAPI (blue), staining for DNA, is local‐ ized in nuclei, as well as extracellularly in NETs. (J) Double labeling of neutrophils with ThS (green) and antibody against Pmel17 (red). Note the colocalization of two signals (merge in yellow). Nuclei (blue) are stained with DAPI.

Amyloid fibrils are also synthesized in inflammatory response induced by the presence of abiotic material such as nanotubes that, as waste, can be found in the air, in the soil, and into the water having a harmful effect to the health of many animals, including humans [30, 31]. Leeches, as animal model, exposed to multiwall carbon nanotubes (MWCNTs) powder dispersed in water, reflect the situation of aquatic animals subjected to uncontrolled carbon nanotube (CNT) exposition. MWCNTs, showing an average 9.5 nm external diameter and 1.5 μm mean length, are able to across the epithelial superficial barrier promoting inflammatory responses. MWCNTs in the leech body wall provoke nonspecific responses characterized by proliferation and migration of macrophages toward the stimulated area. Girardello et al. [32] show that these immune cells, recruited in the area of inflammation, produce amyloid material, as demonstrated by Thioflavin-T staining. In addition, the authors correlated this synthesis with a concomitant overexpression of IL-18, confirming the importance of this cytokine in the process. Extracellular amyloid fibrils entrap and isolate the nanotube aggregation but, forming a kind of spongy coating, can have an additional function in concentrating the cytokines produced by neighbor activated cells thus attracting and driving the macrophages engaged in immune responses [32] (**Figure 3**, panels **A**–**E**).

**Figure 2. Unstimulated and stimulated human neutrophils**. Thin and semithin sections of resting (A) and activated neutrophils (B, C). The unstimulated cells are roundish but after stimulation with LPS (B, C) they lose their globoid shape acquiring migratory phenotype (B, C). (D–F) Identification of amyloid fibrils with Thioflavin S (ThS): unstimu‐ lated neutrophils (D) in comparison with stimulated cells (E, F) from LPS administration. Within cells (E) and extracell‐ ularly (F), amyloid fibrils are localized by ThS brightly fluorescence. Nuclei and DNA material in NETs are marked in brilliant blue with DAPI staining. The blue (DAPI) and green (ThS) in NETs, released on stimulation, co-localize (white arrowhead). (G–J) Immunolocalization with a specific antibody directed against Pmel17 (a mammalian protein in‐ volved in amyloidogenesis): immunofluorescence staining show the increased Pmel17 expression that is present in the cytoplasm of stimulated neutrophils (H) and in the NETs (H, I) (arrowheads). DAPI (blue), staining for DNA, is local‐ ized in nuclei, as well as extracellularly in NETs. (J) Double labeling of neutrophils with ThS (green) and antibody against Pmel17 (red). Note the colocalization of two signals (merge in yellow). Nuclei (blue) are stained with DAPI.

Amyloid fibrils are also synthesized in inflammatory response induced by the presence of abiotic material such as nanotubes that, as waste, can be found in the air, in the soil, and into the water having a harmful effect to the health of many animals, including humans [30, 31]. Leeches, as animal model, exposed to multiwall carbon nanotubes (MWCNTs) powder dispersed in water, reflect the situation of aquatic animals subjected to uncontrolled carbon nanotube (CNT) exposition. MWCNTs, showing an average 9.5 nm external diameter and 1.5 μm mean length, are able to across the epithelial superficial barrier promoting inflammatory responses. MWCNTs in the leech body wall provoke nonspecific responses characterized by proliferation and migration of macrophages toward the stimulated area. Girardello et al. [32] show that these immune cells, recruited in the area of inflammation, produce amyloid material, as demonstrated by Thioflavin-T staining. In addition, the authors correlated this synthesis with a concomitant overexpression of IL-18, confirming the importance of this cytokine in the process. Extracellular amyloid fibrils entrap and isolate the nanotube aggregation but, forming a kind of spongy coating, can have an additional function in concentrating the cytokines produced by neighbor activated cells thus attracting and driving the macrophages engaged in

immune responses [32] (**Figure 3**, panels **A**–**E**).

2066 Exploring New Findings on Amyloidosis

**Figure 3. Effects of carbonanotube exposure and wound healing in leeches**. Optical (A, C, D, E) and ultrastructural (B) images. Leech body wall sectioned in wound healing area (A, C, D, E). The specific Thioflavin-T colorimetric meth‐ od evidences the presence of amyloid fibrils close to the aggregates of nanotubes (arrowheads). Ultrastructural (B) im‐ age of carbon nanotubes (CNTs) crude powder (used as control). The fibrillar material, Thioflavin-T positive, produced by migrating cells (E) contribute to form a plug (D) in the area of lesion (arrowheads).

Amyloids are also synthesized by the cells that are recruited in the lesioned body wall of diverse invertebrates to work as a "collision mat," the strategic solution patterned to close a boat leak, by forming a clot. The fibrillar material, Thioflavin-T positive, deposited in correspondence of damaged area, contributes to block the ingress of water/pathogens/external materials and prevent the loss of cells and hemolimph/blood from the body (unpublished data).

We have shown (combining light and electron microscopy, staining reactions, and immuno‐ cytochemical characterization—see paragraph about methodologies) that different types of cells characterized by different lineages (i.e., from mesoderm and ectoderm) and from diverse organisms, such as larval hemocytes, IPLB-LdFB and Drosophila Schneider's S2 cells from insects; NIH3T3 embryonic fibroblasts from mouse; human umbilical vein endothelial cells (HUVEC), and mesenchymal stem cells from human, under stress condition (for instance, LPS stimulation) are characterized by an increase of cytoplasmic ROS [33].

After stimulation, these cells are characterized by a morphological change and in the cyto‐ plasm, empty vacuoles or RER cisternae filled with material showing staining properties typical of amyloid fibrils are visible (**Figure 4**, panels **A**–**L**). Moreover, the activation of amyloidogenesis is also linked with an extensive production of ACTH and α-MSH in all cultured cell types. These data suggest that amyloidogenesis could be a common, physiological cellular response to weak ROS, starting when other antistress cellular systems failed to restore homeostasis. Then the morphological evidence and/or functional characterization of synthe‐ sized amyloid fibrils could be an early indicator of oxidative stress that may lead to a general inflammatory process.

**Figure 4. Effects of LPS stimulation on different cell types**. Optical (A–C, E–G, I–K) and ultrastructural (D, H, I) im‐ ages of S2 cells from *Drosophila melanogaster* (A–D), IPLB cells from insect (E–H) and NIH3T3 murine fibroblast (I–L). Amyloid fibril presence is detected with Thioflavin T. The yellow-green brightly fluorescence is more evident in stimu‐ lated cells (C, G, K) as compared to controls (B, F, J). Nuclei are stained with DAPI and marked in brilliant blue. TEM analysis: thin sections of LPS activated cells (D, H, L) show the presence of dilated reticulum cisternae filled with fibril‐ lar material (arrowheads).

## **2. Conclusions**

It is intriguing to underline that amyloid fibril synthesis is a cellular dynamic and versatile system evolutionary conserved. Recent evidence strengthen the idea that:


## **3. Methodologies**

We have shown (combining light and electron microscopy, staining reactions, and immuno‐ cytochemical characterization—see paragraph about methodologies) that different types of cells characterized by different lineages (i.e., from mesoderm and ectoderm) and from diverse organisms, such as larval hemocytes, IPLB-LdFB and Drosophila Schneider's S2 cells from insects; NIH3T3 embryonic fibroblasts from mouse; human umbilical vein endothelial cells (HUVEC), and mesenchymal stem cells from human, under stress condition (for instance, LPS

After stimulation, these cells are characterized by a morphological change and in the cyto‐ plasm, empty vacuoles or RER cisternae filled with material showing staining properties typical of amyloid fibrils are visible (**Figure 4**, panels **A**–**L**). Moreover, the activation of amyloidogenesis is also linked with an extensive production of ACTH and α-MSH in all cultured cell types. These data suggest that amyloidogenesis could be a common, physiological cellular response to weak ROS, starting when other antistress cellular systems failed to restore homeostasis. Then the morphological evidence and/or functional characterization of synthe‐ sized amyloid fibrils could be an early indicator of oxidative stress that may lead to a general

**Figure 4. Effects of LPS stimulation on different cell types**. Optical (A–C, E–G, I–K) and ultrastructural (D, H, I) im‐ ages of S2 cells from *Drosophila melanogaster* (A–D), IPLB cells from insect (E–H) and NIH3T3 murine fibroblast (I–L). Amyloid fibril presence is detected with Thioflavin T. The yellow-green brightly fluorescence is more evident in stimu‐ lated cells (C, G, K) as compared to controls (B, F, J). Nuclei are stained with DAPI and marked in brilliant blue. TEM analysis: thin sections of LPS activated cells (D, H, L) show the presence of dilated reticulum cisternae filled with fibril‐

It is intriguing to underline that amyloid fibril synthesis is a cellular dynamic and versatile

system evolutionary conserved. Recent evidence strengthen the idea that:

stimulation) are characterized by an increase of cytoplasmic ROS [33].

inflammatory process.

2088 Exploring New Findings on Amyloidosis

lar material (arrowheads).

**2. Conclusions**

## **3.1. Light microscopy (LM) and transmission electron microscopy (TEM)**

LM and TEM analysis give important information about morphology of cell and tissues.

Collected samples were fixed in 4% glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.2) and washed in 0.1 M Na-cacodylate buffer at pH 7.2. The initial fixation is followed by a postfixation in 1% osmic acid in cacodylate buffer (pH 7.2) for 20 min. After standard dehydration in an ethanol series, samples were embedded in an Epon-Araldite 812 mixture and sectioned with a Reichert Ultracut S ultratome (Leica, Nussloch, Germany). Semithin sections were stained by conventional methods utilizing histological dyes such as crystal violet and basic fuchsine and then observed with a light microscope (Eclipse Nikon, Amsterdam, Netherlands). Thin sections were stained by uranyl acetate and lead citrate and observed with a Jeol 1010 electron microscope (Jeol, Tokyo, Japan).

#### **3.2. Intracellular reactive oxygen species evaluation**

Oxidative stress can induce proteins to adopt an insoluble β-pleated sheet conformation, and according to numerous authors, oxidative damage appears to be the earliest events preceding amyloid fibril formation. Thus, it is important to evaluate the overproduction of ROS in relation to LPS activation responsible for amyloid fibril production. ROS production and its derivation was validated by use of 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, Molecular Probes, Eugene, OR, USA), a fluorigenic probe commonly used to detect the overall degree of intracellular level of ROS. H2DCFDA is a nonfluorescent compound that readily crosses cell membranes. It is hydrolyzed to 2′,7′-dichlorofluorescein (DCF) within cells and becomes fluorescent when it is oxidized by ROS. Oxidation can be detected by monitoring the increase in fluorescence. Fluorescence was determined by excitation at 488 nm and emission at wavelength of 525 nm; fluorescent images visualized on a fluorescence Eclipse Nikon micro‐ scope, were acquired with a DS-5 M-L1 Nikon digital camera system.

## **3.3. Amyloid fibril characterization**

Amyloid structures were identified by staining cells/tissues with Thioflavin S and visualizing the amyloid-specific green/yellow fluorescence with an Eclipse Nikon microscope. Images were acquired with a DS-5 M-L1 Nikon digital camera system. Amyloid fibrils were also characterized with Congo red staining, according to published methods and observed under cross-polarized light with an Axioskop 2 microscope (Carl Zeiss, Jena, Germany), equipped with a MC 80 DX camera (Carl Zeiss).

Amyloid fibrils (see the paragraph below) were also localized immunocytochemically using an antibody directed against Pmel17, protein that has amyloid characteristics and contributes to form fibrillar structures in mammals.

## **3.4. Immunocytochemistry for ACTH, α-MSH, NEP, interleukin 18, and Pmel17 localization**

The presence of amyloid fibrils was confirmed by using the primary antibody antihuman Pmel17 (H-300) polyclonal antibody (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The presence of ACTH and its cleavage product (due to degrading enzyme NEP), α-MSH, and of the proinflammatory interleukin 18 (IL-18) were assessed by using the following primary antibodies: antihuman ACTH polyclonal antibody (1:100 dilution, Abcam, Cambridge, UK); antihuman α-MSH polyclonal antibody (1:100 dilution, Abcam); antihuman IL-18 polyclonal antibody (1:100 dilution, Abnova, Taipei City, Taiwan); antihuman CD10/ CALLA (NEP) monoclonal antibody (1:100 dilution, GeneTex, Hsinchu City, Taiwan). Incubations with suitable secondary antibodies conjugated with Cyanine5 (Cy5; 1:50 dilution, Abcam) were performed for 1 h in a dark humid chamber at room temperature. Nuclei were stained with 4′,6′-diamino-2-phenylindole (DAPI, Sigma-Aldrich). The PBS buffer used for washing steps and antibodies dilutions contained 2% bovine serum albumin (BSA, Sigma-Aldrich) and 0.1% Tween20.

In colocalization experiments, the cells were first stained with Thioflavin S (as described above) and then incubated with anti-Pmel17 antibody (as described above).

In control samples, primary antibodies were omitted, and samples were treated with BSA/ Tween20-containing PBS. Coverslips were mounted in citifluor (citifluor Ltd., London, UK). Slides were observed under an Eclipse Nikon microscope.

## **Acknowledgements**

R.G. is participants in the Biotechnology, Biosciences, and Surgical Technologies Doctoral programs of the University of Insubria. This work was supported by grant from FOCOVA (Fondazione Comunitaria del Varesotto, 2015).

## **Author details**

in fluorescence. Fluorescence was determined by excitation at 488 nm and emission at wavelength of 525 nm; fluorescent images visualized on a fluorescence Eclipse Nikon micro‐

Amyloid structures were identified by staining cells/tissues with Thioflavin S and visualizing the amyloid-specific green/yellow fluorescence with an Eclipse Nikon microscope. Images were acquired with a DS-5 M-L1 Nikon digital camera system. Amyloid fibrils were also characterized with Congo red staining, according to published methods and observed under cross-polarized light with an Axioskop 2 microscope (Carl Zeiss, Jena, Germany), equipped

Amyloid fibrils (see the paragraph below) were also localized immunocytochemically using an antibody directed against Pmel17, protein that has amyloid characteristics and contributes

**3.4. Immunocytochemistry for ACTH, α-MSH, NEP, interleukin 18, and Pmel17 localization** The presence of amyloid fibrils was confirmed by using the primary antibody antihuman Pmel17 (H-300) polyclonal antibody (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The presence of ACTH and its cleavage product (due to degrading enzyme NEP), α-MSH, and of the proinflammatory interleukin 18 (IL-18) were assessed by using the following primary antibodies: antihuman ACTH polyclonal antibody (1:100 dilution, Abcam, Cambridge, UK); antihuman α-MSH polyclonal antibody (1:100 dilution, Abcam); antihuman IL-18 polyclonal antibody (1:100 dilution, Abnova, Taipei City, Taiwan); antihuman CD10/ CALLA (NEP) monoclonal antibody (1:100 dilution, GeneTex, Hsinchu City, Taiwan). Incubations with suitable secondary antibodies conjugated with Cyanine5 (Cy5; 1:50 dilution, Abcam) were performed for 1 h in a dark humid chamber at room temperature. Nuclei were stained with 4′,6′-diamino-2-phenylindole (DAPI, Sigma-Aldrich). The PBS buffer used for washing steps and antibodies dilutions contained 2% bovine serum albumin (BSA, Sigma-

In colocalization experiments, the cells were first stained with Thioflavin S (as described above)

In control samples, primary antibodies were omitted, and samples were treated with BSA/ Tween20-containing PBS. Coverslips were mounted in citifluor (citifluor Ltd., London, UK).

R.G. is participants in the Biotechnology, Biosciences, and Surgical Technologies Doctoral programs of the University of Insubria. This work was supported by grant from FOCOVA

and then incubated with anti-Pmel17 antibody (as described above).

Slides were observed under an Eclipse Nikon microscope.

(Fondazione Comunitaria del Varesotto, 2015).

scope, were acquired with a DS-5 M-L1 Nikon digital camera system.

**3.3. Amyloid fibril characterization**

21010 Exploring New Findings on Amyloidosis

with a MC 80 DX camera (Carl Zeiss).

to form fibrillar structures in mammals.

Aldrich) and 0.1% Tween20.

**Acknowledgements**

Magda de Eguileor\* , Rossana Girardello, Annalisa Grimaldi, Laura Pulze and Gianluca Tettamanti

\*Address all correspondence to: magda.deeguileor@uninsubria.it

Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy

## **References**


[12] Nikolaev IuA and Plakunov VK. Biofilm—"City of microbes" or an analogue of

[13] Hufnagel DA, Tϋkel C, Chapman MR. Disease to dirt: the biology of microbial amy‐

[14] Romero D, Sanabria-Valentin E, Vlamakis H, Kolter R. Biofilm inhibitors that target

[15] Plonka PM, Grabacka M. Melanin synthesis in microorganisms—biotechnological and

[16] Grimaldi A, Tettamanti G, Congiu T, Girardello R, Malagoli D, Falabella P, et al. The main actors involved in parasitization of *Heliotis virescens* larva. Cell Tissue Res. 350:

[17] Slominski A, Zbytek B, Szczesniewski A, Semak I, Kaminski J, Sweatman T, et al. CRH stimulation of corticosteroids production in melanocytes is mediated by ACTH. Am.

[18] Ottaviani E, Malagoli D, Franceschi C. Common evolutionary origin of the immune and neuroendocrine systems: from morphological and functional evidence to in silico

[19] Caruso C, Carniglia L, Durand D, Scimonelli TN, Lasaga M. Melanocortins: Antiinflammatory and neuroprotective peptides. In: Martins LM et al., eds. Neurodegen‐

[20] Marr RA, Rockenstein E, Mukherjee A, Kindy MS, Hersh LB, Gage FH, et al. Neprilysin gene transfer reduces human amyloid pathology in transgenic mice. J. Neurosci. 23:

[21] Turner AJ, Nalivaeva NN New insights into the roles of metalloproteinases in neuro‐ degeneration and neuroprotection. Int. Rev. Neurobiol. 82: 113–135, 2007.

[22] Berson JF, Theos AC, Harper DC, Tenza D, Raposo G, Marks MS. Proprotein convertase cleavage liberates a fibrillogenic fragment of a resident glycoprotein to initiate mela‐

[23] Bossù P, Ciaramella A, Salani F, Vanni D, Palladino I, Caltagirone C, et al. Interleukin-18 from neuroinflammation to Alzheimer's disease. Curr. Pharm. Des. 16: 4213–4224, 2010.

[24] Alboni S, Montanari C, Benatti C, Blom JM, Simone ML, Brunello N, et al. Constitutive and LPS-regulated expression of interleukin-18 receptor beta variants in the mouse

[25] Duval C, Regnier M, Schmidt R. Distinct melanogenic response of human melanocytes in mono-culture, in co-culture with keratinocytes and in reconstructed epidermis, to

multicellular organisms?. Mikrobiologiia. 76: 149–163, 2007.

loids. PLoS Pathog. 9: e1003740, 2013.

491–502, 2012.

21212 Exploring New Findings on Amyloidosis

1992–1996, 2003.

amyloid proteins. Chem. Biol. 20: 102–110, 2013.

medical aspects. Acta Biochim. Pol. 53: 429–443, 2006.

J. Physiol. Endocrinol. Metab. 288: E701–E706, 2005.

approaches. Trends Immunol. 28: 497–502, 2007.

nosome biogenesis. J. Cell Biol. 161: 521–533, 2003.

brain. Brain Behav. Immun. 25: 483–493, 2011.

UV exposure. Pigment Cell Res. 14: 348–355, 2001.

eration, InTech, pp. 93–121, 2012.


## **Developments in the Treatment of Amyloid A Amyloidosis Secondary to Rheumatoid Arthritis**

Tadashi Nakamura

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63132

#### **Abstract**

Amyloidosis refers to a heterogeneous group of diseases in which a soluble precursor, misfolded protein, and subsequently aggregates into highly structured protein fibrils with a cross-β-pleated structure. Of these diseases, amyloid A (AA) amyloidosis is a complication of long-standing inflammatory diseases such as rheumatoid arthritis (RA). Treatment of this amyloidosis with RA aims to stop serum AA protein production. Immunosuppressants have reportedly been useful for both RA inflammation and AA amyloidosis. Also, biologics are effective for these specific pathological processes by targeting key players in each inflammation. In addition to the above-mentioned medications, agents both inhibiting AA fibrillogenesis and destabilizing AA fibrils have recently been employed. Phagocytes play importantroles in the regression of AA fibrils. Renal involvement is the most common complication in AA amyloidosis. Peritoneal dialysis, hemodialysis, and even renal transplantation are available for patients with end-stage renal disease and AA amyloidosis. This chapter thus discusses current developments in the treatment of AA amyloidosis secondary to RA.

**Keywords:** AA amyloidosis, rheumatoid arthritis, *SAA1.3* allele, biologics, fibrinolysis

## **1. Introduction**

Amyloidosis is a rare disorder in which extracellular amyloid fibrils are deposited in vari‐ ous tissues. Those fibrils derive from the misfolding of precursor proteins, the result being multiple organs dysfunction. Systemic amyloidosis is thus characterized by failure of all sorts

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of organs and the presence of amyloid precursor protein in the serum. Reactive amyloid A (AA) amyloidosis is one of the most severe complications of a number of chronic disorders, especially rheumatoid arthritis (RA); most patients with this amyloidosis have an underly‐ ing rheumatic disease. AA amyloidosis, an extra-articular complication of RA, is a serious disorder, possibly life-threatening, that is caused by deposition in multiple organs of AA amyloid fibrils which originate from the circulatory acute-phase reactant, serum amyloid A protein (SAA) [1–4].

Both treatment and understanding of the roles of cytokines in RA have resulted in consider‐ able progress. Remarkable advances, which not only provide insight into the pathophysiolo‐ gy of the disease but also aid discovery of new therapies to fight the deadly disease, have recently been made [5, 6]. For example, the introduction of biological therapies targeting specific inflammatory mediators revolutionized RA treatment. Focusing on essential compo‐ nents of the immune system allows effective suppression of the pathological inflammatory cascade that produces RA symptoms and the resulting joint destruction [7–10]. Several new biologics may permit AA amyloidosis secondary to RA to become a treatable, even manage‐ able, disease. Furthermore, that the allele *SAA1.3* is not only a univariate predictor of surviv‐ al but also a risk factor for the association of AA amyloidosis with RA in Japanese patients is very interesting [11].

Patients with RA, who have a less-than-optimal response to or cannot tolerate conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) [12], such as methotrexate (MTX) [13, 14], are often prescribed biological DMARDs (bDMARDs). Treatment of AA amyloidosis caused by RA seeks to stop SAA production [15]. This approach to AA amyloi‐ dosis treatment is the most common and best-studied therapy; it interferes with synthesis of the precursor protein, with the goal of preventing continued amyloid fibril formation. Similarly, cytotoxic immunosuppressants, such as chlorambucil and cyclophosphamide (CYC), have reportedly been useful for both RA inflammation and AA amyloidosis [16–20].

An alternative approach to therapy involves developing drugs to inhibit amyloid fibrillogen‐ esis. One technique targets AA amyloid deposits directly, by destabilizing AA amyloid fibrils so that they cannot maintain their structural configuration. Treatment with the drug (*R*)-1-[6- [(*R*)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC), a novel hexanoyl bis(D-proline), effectively removes the serum amyloid P component (SAP) from plasma but leaves some SAP in amyloid deposits that can then be specific targets of therapeutic IgG anti-SAP antibodies [21]. Also, eprodisate, which binds to glycosaminogly‐ can-binding sites on AA amyloid fibrils and in theory would destabilize them in tissues, may thereby cause regression of AA amyloidosis [22]. Although the mechanisms by which amyloid deposits are cleared are not well known, they supposedly involve breakdown of amyloid fibrils and associated molecules by macrophages and/or parenchymal cells [23, 24].

This chapter aims to review the advances in the treatment of AA amyloidosis secondary to RA and to describe the latest therapeutic developments based on our reports and literature reviews.

## **2.** *SAA1.3* **genotype and SAA concentration as predictive and prognostic factors of AA amyloidosis outcome**

of organs and the presence of amyloid precursor protein in the serum. Reactive amyloid A (AA) amyloidosis is one of the most severe complications of a number of chronic disorders, especially rheumatoid arthritis (RA); most patients with this amyloidosis have an underly‐ ing rheumatic disease. AA amyloidosis, an extra-articular complication of RA, is a serious disorder, possibly life-threatening, that is caused by deposition in multiple organs of AA amyloid fibrils which originate from the circulatory acute-phase reactant, serum amyloid A

Both treatment and understanding of the roles of cytokines in RA have resulted in consider‐ able progress. Remarkable advances, which not only provide insight into the pathophysiolo‐ gy of the disease but also aid discovery of new therapies to fight the deadly disease, have recently been made [5, 6]. For example, the introduction of biological therapies targeting specific inflammatory mediators revolutionized RA treatment. Focusing on essential compo‐ nents of the immune system allows effective suppression of the pathological inflammatory cascade that produces RA symptoms and the resulting joint destruction [7–10]. Several new biologics may permit AA amyloidosis secondary to RA to become a treatable, even manage‐ able, disease. Furthermore, that the allele *SAA1.3* is not only a univariate predictor of surviv‐ al but also a risk factor for the association of AA amyloidosis with RA in Japanese patients is

Patients with RA, who have a less-than-optimal response to or cannot tolerate conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) [12], such as methotrexate (MTX) [13, 14], are often prescribed biological DMARDs (bDMARDs). Treatment of AA amyloidosis caused by RA seeks to stop SAA production [15]. This approach to AA amyloi‐ dosis treatment is the most common and best-studied therapy; it interferes with synthesis of the precursor protein, with the goal of preventing continued amyloid fibril formation. Similarly, cytotoxic immunosuppressants, such as chlorambucil and cyclophosphamide (CYC), have reportedly been useful for both RA inflammation and AA amyloidosis [16–20].

An alternative approach to therapy involves developing drugs to inhibit amyloid fibrillogen‐ esis. One technique targets AA amyloid deposits directly, by destabilizing AA amyloid fibrils so that they cannot maintain their structural configuration. Treatment with the drug (*R*)-1-[6- [(*R*)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC), a novel hexanoyl bis(D-proline), effectively removes the serum amyloid P component (SAP) from plasma but leaves some SAP in amyloid deposits that can then be specific targets of therapeutic IgG anti-SAP antibodies [21]. Also, eprodisate, which binds to glycosaminogly‐ can-binding sites on AA amyloid fibrils and in theory would destabilize them in tissues, may thereby cause regression of AA amyloidosis [22]. Although the mechanisms by which amyloid deposits are cleared are not well known, they supposedly involve breakdown of amyloid fibrils

This chapter aims to review the advances in the treatment of AA amyloidosis secondary to RA and to describe the latest therapeutic developments based on our reports and literature

and associated molecules by macrophages and/or parenchymal cells [23, 24].

protein (SAA) [1–4].

216 218Exploring New Findings on Amyloidosis

very interesting [11].

reviews.

The frequency of *SAA1* gene polymorphism and that of *SAA1* alleles differ among races and regions worldwide. Three main *SAA1* alleles—*SAA1.1, SAA1.3*, and *SAA1.5*—are defined by two single-nucleotide polymorphisms (SNPs) in exon 3, which result in two amino acid differences at positions 52 and 57, respectively, [25]. In Japanese people, the three alleles occur approximately at the same frequency. The association between AA amyloidosis and the *SAA1* genotype was first observed in Japanese patients with RA, in whom *SAA1.3* allele homo‐ zygosity proved to be a risk factor [26]. The *SAA1.3/1.3* genotype in Japanese patients with RA was related to a shorter latency before the onset of AA amyloidosis and more severe AA amyloidosis-related symptoms [27, 28]. In addition, it was a univariate predictor of survival (**Figure 1**). Thus, the *SAA1.3* allele was a risk factor for AA amyloidosis, was associated with clinical severity of the disease, and poor prognosis [11]. In Caucasians, AA amyloidosis was often found in *SAA1.1* homozygous individuals, and the *SAA1.1* allele was believed to be a risk factor for AA amyloidosis [29]. As for SNPs of the *SAA1* gene promoter region, –13T was found to be a high-risk factor for AA amyloidosis in Japanese patients with RA, and –13T/T and –13T/C were more closely correlated with AA amyloidosis compared with –13C/C [30]. *SAA1* gene polymorphism affects both SAA transcriptional activity in hepatocytes and blood SAA levels, so differences in enzymatic SAA1 proteolysis have demonstrated a close associa‐ tion between *SAA1* gene polymorphism and the onset of AA amyloidosis [31]. However, the mechanism by which *SAA1* gene polymorphism is associated with of AA amyloidosis onset and the reason for ethnic differences in disease-susceptible SNPs are still unknown.

**Figure 1.** Kaplan-Meier survival curves for RA patients with and without *SAA1.3/1.3*. Statistical analysis of a large number of RA patients with AA amyloidosis who carried the *SAA1.3* allele revealed that the risk for association with AA amyloidosis was about eight times higher for *SAA1.3* homozygotes than for the control group and that homozy‐ gotes could develop AA amyloidosis very early after RA onset. Quoted from Nakamura et al. Rheumatology (Oxford) 2006; 45: 43–49.

**Figure 2.** Kaplan-Meier survival curves for patients with systemic AA amyloidosis stratified according to whether the SAA concentration was above or below 10 μg/ml. During follow-up, the proportion of patients with AA amyloidosis who remained alive at 10 years was 90% in the group with median SAA values below 10 μg/ml and 40% in the group with SAA values above that value. Quoted and modified from Gillmore et al. Lancet 2011; 358: 24–29.

The current primary objective of therapy for all forms of amyloidosis was the reduction of the precursor protein supply [32]. In AA amyloidosis, long-term suppression of SAA levels is critical for patient and disease outcomes and for AA amyloidosis in patients with RA. The degree to which SAA concentration increased during follow-up was a strong predictor of outcome [33]. Sustained complete suppression of RA disease activity with the normalization of SAA levels should be the treatment aims in patients with AA amyloidosis, and monitor‐ ing the SAA levels is a vital part of patient management (**Figure 2**) [34].

**Figure 3.** Biological versatility of SAA. SAA has important roles in high-grade and low-grade inflammation. Similar to cytokines, it utilizes autocrine, endocrine, and paracrine mechanisms. SAA, as a precursor protein of AA amyloidosis, induces this amyloidosis. Using different modes of action, SAA also affects metabolic syndrome. These humoral and cellular inflammatory events interact, with SAA as an essential factor. RAGE: receptor for advanced glycation end products; FPRL1: formyl peptide receptor-like 1; TLR2, 4: toll-like receptors 2 and 4; CLA-1: CD36, and LIMPII analo‐ gous-1, a human ortholog of the scavenger receptor class B type I (SR-BI); AGEs: advanced glycation end products. Quoted from Nakamura T. Clin Exp Rheumatol 2011; 29: 850–857.

SAA plays important roles in both high-grade and low-grade inflammation (**Figure 3**) [35]. Similar to cytokines, SAA operates by means of autocrine, endocrine, and paracrine mecha‐ nisms. SAA is a precursor protein of AA amyloid fibrils, and it induces AA amyloidosis. SAA also functions in metabolic syndrome by means of different modes of action. As a critical factor in the inflammatory interactions, SAA acts mutually among humoral and cellular events within inflammation.

## **3. Treatment with conventional synthetic DMARDs for AA amyloidosis secondary to RA**

Treatment of patients with RA has focused on using immunosuppressants as conventional synthetic DMARDs (csDMARDs). Although case reports and studies of small series of patients showed that immunosuppressants can reverse nephrotic syndrome [36] and can even lead to complete resolution of proteinuria [37, 38], as **Table 1** shows, management of AA amyloido‐ sis has focused on the RA process causing the inflammation as the underlying disease. We cannot, therefore, determine a clear difference in the effectiveness of therapies for RA and AA amyloidosis.

**Figure 2.** Kaplan-Meier survival curves for patients with systemic AA amyloidosis stratified according to whether the SAA concentration was above or below 10 μg/ml. During follow-up, the proportion of patients with AA amyloidosis who remained alive at 10 years was 90% in the group with median SAA values below 10 μg/ml and 40% in the group

The current primary objective of therapy for all forms of amyloidosis was the reduction of the precursor protein supply [32]. In AA amyloidosis, long-term suppression of SAA levels is critical for patient and disease outcomes and for AA amyloidosis in patients with RA. The degree to which SAA concentration increased during follow-up was a strong predictor of outcome [33]. Sustained complete suppression of RA disease activity with the normalization of SAA levels should be the treatment aims in patients with AA amyloidosis, and monitor‐

**Figure 3.** Biological versatility of SAA. SAA has important roles in high-grade and low-grade inflammation. Similar to cytokines, it utilizes autocrine, endocrine, and paracrine mechanisms. SAA, as a precursor protein of AA amyloidosis, induces this amyloidosis. Using different modes of action, SAA also affects metabolic syndrome. These humoral and cellular inflammatory events interact, with SAA as an essential factor. RAGE: receptor for advanced glycation end products; FPRL1: formyl peptide receptor-like 1; TLR2, 4: toll-like receptors 2 and 4; CLA-1: CD36, and LIMPII analo‐ gous-1, a human ortholog of the scavenger receptor class B type I (SR-BI); AGEs: advanced glycation end products.

with SAA values above that value. Quoted and modified from Gillmore et al. Lancet 2011; 358: 24–29.

218 220Exploring New Findings on Amyloidosis

ing the SAA levels is a vital part of patient management (**Figure 2**) [34].

Quoted from Nakamura T. Clin Exp Rheumatol 2011; 29: 850–857.


AA: amyloid A; RA: rheumatoid arthritis; JIA: juvenile idiopathic arthritis; CYC: cyclophosphamide.

**Table 1.** Reported cases in the treatment for AA amyloidosis secondary to RA/JIA with immunosuppressants.

The efficacy of corticosteroid treatment with regard to AA amyloidosis secondary to RA is still controversial [39]. Corticosteroids can reduce the magnitude of acute phase reactions includ‐ ing synthesis of C-reactive protein (CRP) and SAA. In cultures of human hepatocyte, cortico‐ steroids stimulated SAA production but not CRP production. Although corticosteroids reduced SAA and CRP levels in longitudinal studies of patients with RA, the effect was somewhat greaterfor CRP than for SAA [40]. Growing evidence suggests that SAA is sensitive to change, achieves much higher levels than CRP, declines rapidly, and may therefore accurately reflect disease activity. The advantages of SAA as a biomarker of disease activity include the rapid production and exceptionally wide dynamic range of the inflammatory response. During acute inflammation, serum SAA levels may rise up to 1000-fold and the biologic half-life of SAA levels are significantly shorter than that of CRP [41]. Monitoring SAA instead of CRP levels would thus be advisable, especially if corticosteroids are used. Treating patients with AA amyloidosis secondary to RA using cytotoxic drugs either alone or togeth‐ er with prednisolone, which is a synthetic glucocorticoid and a cortisol derivative, seems reasonable [42]. Because the effect of immunosuppressants may require weeks or months to be obvious, giving steroids in addition to immunosuppressants is recommended to ensure an immediate reduction in the acute phase response and, in particular, SAA synthesis [43].

**Figure 4.** Differences between CYC and methotrexate (MTX) treatments in RA patients with AA amyloidosis. The de‐ ducted value (in the figure) was determined by subtracting the starting CRP and serum creatinine values from the corresponding ending values in each treatment. Quoted from Nakamura et al. Rheumatology (Oxford) 2006; 45: 43–49.

Although no evidence is available that csDMARDs have a particular effect on amyloidogen‐ esis and AA amyloidosis in RA, reports provided encouraging description of the beneficial results of alkylating agents in clinical trials in RA patients with AA amyloidosis [44–46]. Using immunosuppressive agents may improve prognosis, and CYC has proved to be superior compared with methotrexate (MTX) for the treatment of RA patients with AA amyloidosis (**Figure 4**) [11]. Because between before and during CYC treatment, the values of Lansbury index, which implies a statistical approach to indices of disease activity, have lowered more; CYC may be more effective mainly in patients with *SAA1.3* homozygosity than in patients with *SAA1.3* heterozygosity, which suggests that *SAA1.3* homozygosity is a CYC treatmentsusceptible factor (**Figure 5**) [20].

The efficacy of corticosteroid treatment with regard to AA amyloidosis secondary to RA is still controversial [39]. Corticosteroids can reduce the magnitude of acute phase reactions includ‐ ing synthesis of C-reactive protein (CRP) and SAA. In cultures of human hepatocyte, cortico‐ steroids stimulated SAA production but not CRP production. Although corticosteroids reduced SAA and CRP levels in longitudinal studies of patients with RA, the effect was somewhat greaterfor CRP than for SAA [40]. Growing evidence suggests that SAA is sensitive to change, achieves much higher levels than CRP, declines rapidly, and may therefore accurately reflect disease activity. The advantages of SAA as a biomarker of disease activity include the rapid production and exceptionally wide dynamic range of the inflammatory response. During acute inflammation, serum SAA levels may rise up to 1000-fold and the biologic half-life of SAA levels are significantly shorter than that of CRP [41]. Monitoring SAA instead of CRP levels would thus be advisable, especially if corticosteroids are used. Treating patients with AA amyloidosis secondary to RA using cytotoxic drugs either alone or togeth‐ er with prednisolone, which is a synthetic glucocorticoid and a cortisol derivative, seems reasonable [42]. Because the effect of immunosuppressants may require weeks or months to be obvious, giving steroids in addition to immunosuppressants is recommended to ensure an immediate reduction in the acute phase response and, in particular, SAA synthesis [43].

220 222Exploring New Findings on Amyloidosis

**Figure 4.** Differences between CYC and methotrexate (MTX) treatments in RA patients with AA amyloidosis. The de‐ ducted value (in the figure) was determined by subtracting the starting CRP and serum creatinine values from the corresponding ending values in each treatment. Quoted from Nakamura et al. Rheumatology (Oxford) 2006; 45: 43–49.

Although no evidence is available that csDMARDs have a particular effect on amyloidogen‐ esis and AA amyloidosis in RA, reports provided encouraging description of the beneficial results of alkylating agents in clinical trials in RA patients with AA amyloidosis [44–46]. Using immunosuppressive agents may improve prognosis, and CYC has proved to be superior compared with methotrexate (MTX) for the treatment of RA patients with AA amyloidosis (**Figure 4**) [11]. Because between before and during CYC treatment, the values of Lansbury index, which implies a statistical approach to indices of disease activity, have lowered more; CYC may be more effective mainly in patients with *SAA1.3* homozygosity than in patients

**Figure 5.** Changes in Lansbury index after CYC or prednisolone treatment in RA patients with AA amyloidosis with *SAA1.3* heterozygosity or homozygosity. In *SAA1.3* homozygous patients, a statistically significant difference occurred between before and during, whereas *SAA1.3* heterozygous patients showed no such significant difference. Quoted from Nakamura et al. Clin Rheumatol 2003; 22: 371–375.

During signal transduction, interleukin-6 (IL-6) binds to the membrane-bound IL-6 receptor gp80 [47], and after which the IL-6-gp80 dimer interacts with gp130. Formation of gp130 containing complexes activates Janus kinases, which stimulates signal transducers and activators of transcription (STATs) [48–50]. Some evidence suggests that STAT3 is the critical transcription factor that is responsible for IL-6 activation of SAA gene transcription [51]. The function of Janus kinase inhibition in the IL-6-signaling pathway will thus be one target of RA treatment. Suppressing IL-6-mediated pro-inflammatory signaling pathways using Janus kinase inhibitors may be a novel anti-inflammatory therapeutic strategy for RA and AA amyloidosis.

Another agent, tacrolimus, may inhibit T-cell function in the pathogenesis of AA amyloido‐ sis. In experimental murine models of AA amyloidosis, blocking the function of T lympho‐ cytes with the calcineurin inhibitor tacrolimus showed that it inhibited deposition of AA amyloid fibril in a dose-dependent manner. Also, the location of CD4+ T lymphocytes in the spleen was identical to that of AA amyloid fibril deposits, which suggests that T lympho‐ cytes have a role in the pathogenesis of AA amyloidosis [52].

## **4. Treatment with biological DMARDs for AA amyloidosis secondary to RA**

Tight control of RA during treatment is important for obtaining clinical remission or for low disease activity [53]. This control is achieved via periodic assessments of RA disease activity and aggressive investigation of additional more effective treatments [54]. Biological DMARDs (bDMARDs) therapy is expected to be effective against systemic inflammation and local inflammation such as those occurring in RA.


DMARDs: disease-modifying antirheumatic drugs; AA: amyloid A; RA: rheumatoid arthritis; TNFα: tumor necrosis factor α; IFX: infliximab; ETN: etanercept; ADA: adalimumab; IL-6: interleukin6; TCZ: tocilizumab; ABT: abatacept; CD: cluster of differentiation; RTX: rituximab.

**Table 2.** Biological DMARDs for patients with AA amyloidosis secondary to RA.

Etanercept (ETN) and infliximab (IFX), both tumor necrosis factor α (TNFα) antagonists, can lower SAA levels in RA patients with AA amyloidosis [55, 56]. This effect ameliorates both RA

inflammation and AA amyloidosis, reduces the number of swollen and tender joints, lowers or normalizes proteinuria, and improves renal function [57, 58]. Although a small number of patients with AA amyloidosis secondary to RA received ETN, this drug had benefits for both RA inflammation and AA amyloidosis, even in *SAA1.3/1.3* allele-carrying RA patients (**Table 2**) [59–62]. Such benefits were determined by evaluating the surrogate markers disease activity score 28-erythrocyte sedimentation rate, CRP, SAA, and proteinuria (**Table 3**). Also, patients with mild RA disease and renal dysfunction demonstrated significantly improved serum creatinine levels. This result suggests that an earlier intervention with bDMARDs produces a better outcome for RA patients with AA amyloidosis (**Table 4**) [63, 64].


ETN: etanercept; AA: amyloid A; RA: rheumatoid arthritis; DAS: disease activity score; ESR: erythrocyte sedimentation rate; CRP: C-reactive protein; SAA: serum amyloid A protein.

\*Serum levels.

and aggressive investigation of additional more effective treatments [54]. Biological DMARDs (bDMARDs) therapy is expected to be effective against systemic inflammation and

> IFX Arthritis Rheum 2002; 46: 2571 ETN/IFX Arthritis Rheum 2003; 48: 2019 ETN/IFX Rheumatology (Oxford) 2003; 42:1425

ETN Intern Med J 2004; 34: 570

ETN/IFX Am J Med 2005; 118: 552

ETN/IFX Rheumatology (Oxford) 2004; 43: 669

ETN Clin Exp Rheumatol 2007; 25: 518 IFX Rheumatol Int 2008; 28: 1155 ETN/IFX J Rheumatol 2009; 36: 2409 ETN Clin Rheumatol 2010; 29: 1395 ETN Rev Bras Reumatol 2010; 50: 205 ETN Rheumatol Int 2011; 31: 247 ETN/ADA Joint Bone Spine 2013; 80: 223

TCZ Arthritis Rheum 2006; 54: 2997 TCZ Clin Rheumatol 2009; 28: 1113 TCZ Clin Rheumatol 2010; 29: 1195 TCZ Mod Rheumatol 2014; 24: 405

TCZ Clin Exp Rheumatol 2015; 33 (Suppl. 94): S46

ABT Clin Exp Rheumatol 2014; 32: 501

RTX Joint Bone Spine 2011; 78: 98

DMARDs: disease-modifying antirheumatic drugs; AA: amyloid A; RA: rheumatoid arthritis; TNFα: tumor necrosis factor α; IFX: infliximab; ETN: etanercept; ADA: adalimumab; IL-6: interleukin6; TCZ: tocilizumab; ABT: abatacept;

Etanercept (ETN) and infliximab (IFX), both tumor necrosis factor α (TNFα) antagonists, can lower SAA levels in RA patients with AA amyloidosis [55, 56]. This effect ameliorates both RA

TCZ Amyloid 2015; 22:84

local inflammation such as those occurring in RA.

TNFα antagonist

222 224Exploring New Findings on Amyloidosis

IL-6 receptor antagonist

Anti-CD20 antibody

Selective costimulation modulator of T-cell function

CD: cluster of differentiation; RTX: rituximab.

**Table 2.** Biological DMARDs for patients with AA amyloidosis secondary to RA.

**Type of agent Biologic References**

ETN was significantly effective for RA patients with AA amyloidosis. DAS28-ESR and CRP indicated RA inflammation, and SAA and proteinuria-indicated AA amyloidosis. Quoted and modified from Nakamura et al. Clin Rheumatol 2010; 29: 1395–1401.

**Table 3.** Effect of ETN on AA amyloidosis secondary to RA.


ETN: etanercept; AA: amyloid A; RA: rheumatoid arthritis.

Although a statistical significance of serum levels of creatinine was not observed as shown in **Table 3**, when cutting levels of serum creatinine by 2.0 mg/dl, the group with values lower than 2.0 mg/dl revealed to be significant. Regarding with renal dysfunction because of AA amyloidosis secondary to RA, the earlier the intervention, the better the outcome was suggested. Quoted and modified from Nakamura et al. Clin Rheumatol 2010; 29: 1395–1401. a P = 0.021, b not significant.

**Table 4.** Effect of ETN on renal dysfunction in AA amyloidosis secondary to RA.

Tocilizumab, an IL-6 receptor antagonist, also produced excellent SAA suppression and may show promise as a therapeutic agent for AA amyloidosis [65–67]. Circulating SAA levels usually indicate changes in CRP, and levels of these acute phase reactants usually increase at the same time, but certain differences can occur. SAA and CRP seem to be somewhat affect‐ ed by different cytokines [68, 69]. As mentioned earlier, therapy in which IL-6 is blocked, rather than therapy utilizing TNF-α blockade, should influence multiple signal transduction systems and may normalize SAA levels in RA patients (**Figure 6**) [70].

**Figure 6.** Changes in serum values of SAA between the first and last observations for each biologic. SAA values showed more significant suppression in the tocilizumab group than in the TNF inhibitor group (P = 0.0194, Wilcoxon rank sum test). Quoted and modified from Okuda et al. Mod Rheumatol 2014; 24: 137–143.


**Figure 7.** Clinical course of two RA patients with AA amyloidosis who received abatacept treatment. Clinical parame‐ ters were related to RA inflammation and AA amyloidosis after treatment. DAS28-CRP: disease activity score in 28 joints based on the CRP level; HAQ: Health Assessment Questionnaire; eGFR: estimated glomerular filtration rate; Uprotein: qualitative protein analysis of spot urine. Quoted and modified from Nakamura et al. Clin Exp Rheumatol 2014; 32: 501–508.

Abatacept (ABT) is a soluble fusion protein consisting of the extracellular domain of recombi‐ nant human cytotoxic T lymphocyte-associated antigen 4 plus a fragment of the Fc domain human immunoglobulin IgG1 (CTLA-4Ig) [71]. CTLA-4Ig may reduce T lymphocyte respons‐ es by competing with CD80/CD86 to access CD28 and thus limit the CD28 signaling that T lymphocyte activation requires [72]. ABT may also affect more than just T lymphocytes [73]. Whether intracellular signaling or other CTLA-4Ig-mediated effects contribute to a favorable

outcome or a poor outcome, especially during the treatment of RA patients, is not entirely clear, and the exact role of CTLA-4Ig in biological systems, including patients with AA amyloidosis secondary to RA, is also unresolved. Although accumulating clinical data on ABT treatment suggest an advantage for ABT in RA management [74, 75], the safety and efficacy of ABT in patients with AA amyloidosis secondary to RA have not yet been studied. **Figure 7** illustrates that in two patients, who had more than 20 years of RA history and who carried the *SAA1.3* allele, which is a risk factor for AA amyloidosis in Japanese RA patients, ABT gradually improved RA disease activities, proteinuria, and various gastrointestinal symp‐ toms and was clinically effective to some degree in one case and completely in the other for both RA inflammation and AA amyloidosis. Study results also suggest that ABT targeting of costimulatory molecules may be useful for treating patients with AA amyloidosis secondary to RA and that ABT may be an alternative to anti-cytokine therapies for AA amyloidosis complicating RA [76].

than therapy utilizing TNF-α blockade, should influence multiple signal transduction systems

**Figure 6.** Changes in serum values of SAA between the first and last observations for each biologic. SAA values showed more significant suppression in the tocilizumab group than in the TNF inhibitor group (P = 0.0194, Wilcoxon

**Figure 7.** Clinical course of two RA patients with AA amyloidosis who received abatacept treatment. Clinical parame‐ ters were related to RA inflammation and AA amyloidosis after treatment. DAS28-CRP: disease activity score in 28 joints based on the CRP level; HAQ: Health Assessment Questionnaire; eGFR: estimated glomerular filtration rate; Uprotein: qualitative protein analysis of spot urine. Quoted and modified from Nakamura et al. Clin Exp Rheumatol

Abatacept (ABT) is a soluble fusion protein consisting of the extracellular domain of recombi‐ nant human cytotoxic T lymphocyte-associated antigen 4 plus a fragment of the Fc domain human immunoglobulin IgG1 (CTLA-4Ig) [71]. CTLA-4Ig may reduce T lymphocyte respons‐ es by competing with CD80/CD86 to access CD28 and thus limit the CD28 signaling that T lymphocyte activation requires [72]. ABT may also affect more than just T lymphocytes [73]. Whether intracellular signaling or other CTLA-4Ig-mediated effects contribute to a favorable

rank sum test). Quoted and modified from Okuda et al. Mod Rheumatol 2014; 24: 137–143.

2014; 32: 501–508.

and may normalize SAA levels in RA patients (**Figure 6**) [70].

224 226Exploring New Findings on Amyloidosis

Rituximab, an anti-CD20 monoclonal antibody, was effective for treating patients with severe active RA who had an inadequate response to TNFα inhibitor (or inhibitors) [77]. The efficacy and safety of rituximab for patients with AA amyloidosis secondary to RA together with substantial clinical improvement in articular symptoms, marked reduction in acute phase reactants, and stabilization of renal function and proteinuria were demonstrated [78].

## **5. Inhibiting AA amyloid fibrillogenesis in AA amyloidosis secondary to RA**

Highly sulfated glycosaminoglycans, especially heparan sulfate and dermatan sulfate proteoglycans, are universal constituents of amyloid deposits and promote fibril assembly and help maintain conformational changes related to amyloidogenesis [79, 80]. Eprodisate is a negatively charged, sulfonated low molecular weight molecule that has a structure which is similar to that of heparan sulfate [81]. It binds to the SAA binding site to prevent interaction of SAA with glycosaminoglycans and thereby inhibits a conformational change required to cause SAA to become amyloidogenic. In in vivo studies with murine models, eprodisate inhibited the development of amyloid deposits [82, 83].

Eprodisate is still the only drug that was tested in a phase II/III multicenter, placebo-control‐ led, double-blinded study of amyloidosis [84]. Patients were stratified according to the presence of nephrotic syndrome and the treatment center, after of which they were random‐ ized to receive eprodisate or placebo twice daily for up to 2 years. Outcome measures were a composite endpoint of serum creatinine, creatinine clearance, and progression to end-stage renal disease (ESRD) or death. Secondary outcome measures included the creatinine clear‐ ance slope, change in proteinuria, improvement in diarrhea, and alteration in amyloid content of abdominal fat. The study demonstrated that eprodisate may have contributed to the failure to achieve the study's primary endpoints, although the eprodisate-treated group did show renal benefits (**Figure 8**). The study, however, did not demonstrate a significant benefit from active therapy on progression to ESRD or risk of death, although a trend to benefit was seen. A phase III clinical study is currently under way in Japan.

**Figure 8.** Kaplan-Meier survival curves for patients with AA amyloidosis who were given eprodisate or placebo. In terms of event-free survival, eprodisate demonstrated superior effectiveness compared with placebo, but this effect was not statistically significant. An event was defined as any component of the composite endpoint of worsened dis‐ ease. Quoted and modified from Dember et al. N Engl J Med 2007; 356: 2349–2360.

## **6. Immunotherapy for AA amyloidosis secondary to RA**

An alternative therapeutic approach was to target other components of amyloid deposits to destabilize amyloid fibrils. SAP is a normal plasma component and is a universal constitu‐ ent of amyloid deposits. Its presence may therefore mask the presence of amyloid deposits and inhibit effective clearance of amyloid [85]. In fact, SAP knockout mice showed inhibited amyloid formation [86]. SAP was identified as a therapeutic target, which then led to devel‐ opment of CPHPC, adrug thatinhibits the binding of SAP to amyloiddeposits [87]. The activity of this agent relates to its ability to cross-link SAP molecule pairs face to face, which results in rapid hepatic clearance and completely blocks the binding face of the SAP molecule [88]. A preliminary study demonstrated that regular administration resulted in sustained and profound SAP depletion. Several patients received the drug for many years with no obvious adverse effects, although the degree of potential clinical benefit was not great enough to be determined in this open, non-controlled study (**Table 5**) [89].

Antibodies recognizing a common fibril epitope were raised and given to mice [90] with systemic AA amyloidosis, which resulted in reduced amyloid levels. CPHPC effectively removed SAP from the blood, but only very slowly from amyloid deposits, which allowed the development of an antibody directed at SAP. CPHPC was used to remove the SAP from plasma followed by use of an anti-SAP antibody, which led to rapid clearance by macrophages of experimentally induced amyloid deposits; this method begin developed for use in patients [91].


AA: amyloid A; SAP: serum amyloid P component; CPHPC: (*R*)-1-[6-[(*R*)-2-carboxy-pyrrolidin-1-yl]-6-oxohexanoyl]pyrrolidine-2-carboxylic acid; AApoA1: apolipoprotein A1.

active therapy on progression to ESRD or risk of death, although a trend to benefit was seen.

**Figure 8.** Kaplan-Meier survival curves for patients with AA amyloidosis who were given eprodisate or placebo. In terms of event-free survival, eprodisate demonstrated superior effectiveness compared with placebo, but this effect was not statistically significant. An event was defined as any component of the composite endpoint of worsened dis‐

An alternative therapeutic approach was to target other components of amyloid deposits to destabilize amyloid fibrils. SAP is a normal plasma component and is a universal constitu‐ ent of amyloid deposits. Its presence may therefore mask the presence of amyloid deposits and inhibit effective clearance of amyloid [85]. In fact, SAP knockout mice showed inhibited amyloid formation [86]. SAP was identified as a therapeutic target, which then led to devel‐ opment of CPHPC, adrug thatinhibits the binding of SAPto amyloiddeposits [87]. The activity of this agent relates to its ability to cross-link SAP molecule pairs face to face, which results in rapid hepatic clearance and completely blocks the binding face of the SAP molecule [88]. A preliminary study demonstrated that regular administration resulted in sustained and profound SAP depletion. Several patients received the drug for many years with no obvious adverse effects, although the degree of potential clinical benefit was not great enough to be

Antibodies recognizing a common fibril epitope were raised and given to mice [90] with systemic AA amyloidosis, which resulted in reduced amyloid levels. CPHPC effectively removed SAP from the blood, but only very slowly from amyloid deposits, which allowed the development of an antibody directed at SAP. CPHPC was used to remove the SAP from plasma followed by use of an anti-SAP antibody, which led to rapid clearance by macrophages of experimentally induced amyloid deposits; this method begin developed for use in patients

A phase III clinical study is currently under way in Japan.

226 228Exploring New Findings on Amyloidosis

ease. Quoted and modified from Dember et al. N Engl J Med 2007; 356: 2349–2360.

determined in this open, non-controlled study (**Table 5**) [89].

[91].

**6. Immunotherapy for AA amyloidosis secondary to RA**

Improved SAP scan: an improved scan was defined as a 123I-SAP scintigram revealing a reduction in organ amyloid load at day 42 after SAP-antibody administration.

Combination treatment with CPHPC and anti-SAP antibody was effective to some extent for AA amyloidosis. Quoted and modified from Richards et al. N Engl J Med 2015; 373: 1106–1114.

**Table 5.** Response of patients with AA amyloidosis to anti-SAP antibody after depletion of plasma SAP with CPHPC.

## **7. Biomarkers predicting effectiveness of treatment for AA amyloidosis secondary to RA**

Because renal dysfunction is the most common symptom in AA amyloidosis secondary to RA, surrogate markers representing the effectiveness of each treatment were investigated, with a focus on kidney function, in RA patients with AA amyloidosis who carried the *SAA 1.3* allele and who were treated with CYC or ETN focusing [92]. Identifying patients with a poor prognosis when it may be possible to modify the disease process and in whom any therapy may be justified is important. The presence of *SAA1.3* allele in Japanese RA patients may be a critical indicator for maintaining tight control of RA inflammation via vigorous treatment during the early phase of the RA disease course [11]. The rationale for using biologics in AA amyloidosis relates to their ability to lowerlevels of serum pro-inflammatory cytokines, which regulate SAA synthesis [93, 94]. A retrospective study reported the efficacy and safety of ETN for patients with AA amyloidosis secondary to RA who carried *SAA1.3* allele [64]. Using ETN for RA patients with AA amyloidosis may be possible, even for those undergoing dialysis [95, 96]. The efficacy of ETN was compared with that of CYC for treating AA amyloidosis secondary to RA as related to the *SAA1.3* allele, which was not a factor affecting therapeutic susceptibility (**Figure 9**). Demonstrable endpoints included recovery of serum albumin biosynthesis, improvement in the acute phase response, and amelioration of estimated glomerular filtration rate (eGFR). *SAA1.3* allele polymorphism was not affected on these parameters (**Table 6**). Albumin in factreflects the severity of AA amyloidosis [97]. The changes in CRP and albumin were influenced by the difference between therapies rather than *SAA1.3* allele polymorphism (**Figure 10**). In contrast, the eGFRin patients with end-stage renal damage may reflect diminished urinary flow and may indicate improvement in renal function. Only ETN aided the amelioration of the eGFR, which indicated the greater efficacy of ETN com‐ pared with CYC for treating AA amyloidosis secondary to RA.

**Figure 9.** Kaplan-Meier survival curves after the treatment with ETN or CYC. ETN clearly demonstrated more effec‐ tiveness for RA patients with AA amyloidosis than did CYC. Quoted and modified from Nakamura et al. Rheumatolo‐ gy (Oxford) 2012; 51: 2064–2069.


ETN: etanercept; CYC: cyclophosphamide; CRP: C-reactive protein; Alb: albumin; eGFR: estimated glomerular filtration rate; Crea: creatinine.

Except creatinine, recovery of serum albumin biosynthesis, improvement in the acute phase response, and amelioration of estimated glomerular filtration rate (eGFR) were valuable parameters to show effectiveness in the treatment with etanercept. *SAA1.3* allele polymorphism was not affected on these parameters. Quoted from Nakamura et al. Rheumatology (Oxford) 2012; 51: 2064–2069.

**Table 6.** Parameters showing effectiveness of treatment with ETN or CYC.

**Figure 10.** Changes in the eGFR between initial and last visits according to the effectiveness of treatment with ETN or CYC (A) and as a function of the *SAA1.3* allele homozygosity or other polymorphisms (B). ETN increased the eGFR, which improved the reduced renal function caused by AA amyloidosis, more than did CYC, and *SAA1.3* did not affect treatment in both groups of patients. Quoted from Nakamura et al. Rheumatology (Oxford) 2012; 51: 2064–2069.

## **8. End-stage renal disease treatment in AA amyloidosis secondary to RA**

Kidneys that have extensive AA amyloid deposits are extremely susceptible to intercurrent insults such as hypoperfusion, hypertension, nephrotoxic drug effects, and surgical injuries, which should all be avoided in AA amyloidosis [98, 99]. Renal involvement is the most common problem in AA amyloidosis, and patients with AA amyloidosis frequently prog‐ ress to end-stage renal disease (ESRD) and poor prognosis (**Figure 11**). When evaluating therapies for renal dysfunction related to AA amyloidosis, renal transplantation has been thought to be a suitable method [100]. However, existing data on patient survival and graft prognosis do not provide conclusive results about whether renal plantation is suitable for patients with AA amyloidosis [101]. An alternative to transplantation is chronic dialysis, but experience with dialysis in patients with AA amyloidosis has not yet as been encouraging [102, 103]. Peritoneal dialysis increases the susceptibility to infection and protein loss [104]. Hypoalbuminemia is well known to predict overall mortality in hemodialysis patients [105]. An elevated CRP value is associated with an increased risk of death in dialysis patients [106], and CRP has been found to be an independent predictor of major adverse cardiac events [107]. Because patients with AA amyloidosis who have progressed to ESRD have already been exposed to significant levels of inflammation, which may be ongoing, they may have an additional cardiovascular risk [108]. Patients with marked proteinuria have an increased risk of thrombosis, and the decision to use anticoagulants must be made on an individual basis [109]. General management principles apply to patients with AA amyloidosis secondary to RA for lowering high lipid levels and modifying diet as other causes of renal dysfunction.

**Figure 9.** Kaplan-Meier survival curves after the treatment with ETN or CYC. ETN clearly demonstrated more effec‐ tiveness for RA patients with AA amyloidosis than did CYC. Quoted and modified from Nakamura et al. Rheumatolo‐

**Category CRP Alb eGFR Crea** Within-subject <0.01 <0.01 0.035 0.085

*SAA1.3* allele polymorphism 0.777 0.715 0.465 0.228 Treatment (ETN/CYC) <0.01 <0.01 0.032 0.148 ETN: etanercept; CYC: cyclophosphamide; CRP: C-reactive protein; Alb: albumin; eGFR: estimated glomerular

Except creatinine, recovery of serum albumin biosynthesis, improvement in the acute phase response, and amelioration of estimated glomerular filtration rate (eGFR) were valuable parameters to show effectiveness in the treatment with etanercept. *SAA1.3* allele polymorphism was not affected on these parameters. Quoted from Nakamura

**Figure 10.** Changes in the eGFR between initial and last visits according to the effectiveness of treatment with ETN or CYC (A) and as a function of the *SAA1.3* allele homozygosity or other polymorphisms (B). ETN increased the eGFR, which improved the reduced renal function caused by AA amyloidosis, more than did CYC, and *SAA1.3* did not affect treatment in both groups of patients. Quoted from Nakamura et al. Rheumatology (Oxford) 2012; 51: 2064–2069.

gy (Oxford) 2012; 51: 2064–2069.

228 230Exploring New Findings on Amyloidosis

filtration rate; Crea: creatinine.

et al. Rheumatology (Oxford) 2012; 51: 2064–2069.

**Table 6.** Parameters showing effectiveness of treatment with ETN or CYC.

Interaction with

**Figure 11.** Kaplan-Meier survival curves after diagnosis of AA amyloidosis for patients with serum creatinine values of >2.5 or ≤2.5 mg/dl. Renal dysfunction, the most common symptom in AA amyloidosis secondary to RA, is a factor indicating a poor prognosis for survival. Quoted from Nakamura et al. Rheumatology (Oxford) 2006; 45: 43–49.

## **9. Possible involvement of phagocytes in degeneration of AA amyloid fibrils**

Macrophages participate in SAA processing and deposition [110, 111], and cell surfaceexpressed heparan sulfate proteoglycans have an essential function in amyloidogenesis through the binding of high-density lipoprotein-associated SAA [112]. In addition, Fcreceptor-positive macrophages have been implicated in the reduction of the amyloid load after inflammation has resolved [113]. Phagocytes such as neutrophils and macrophages will serve an important function for reducing AA amyloid deposits during ABT treatment (**Figure 12**).

**Figure 12.** Immunohistochemical analysis of a biopsy specimen from a patient with RA and AA amyloidosis who re‐ ceived abatacept (ABT). (A) Stained with the antibody anti-formyl peptide receptor-like 1 (fPRL-1, the receptor in‐ volved in production of reactive oxygen species, degradation, and phagocyte migration) and (B) anti-human CD68 antibody staining. Phagocytes, polymorphonuclear leukocytes, and macrophages stained positively in the upper gas‐ trointestinal mucosa specimen. Quoted and modified from Nakamura et al. Clin Exp Rheumatol 2014; 32: 501–508.

**Figure 13.** Relation between macrophages and AA amyloid deposits. AA amyloid deposits showed positive Congo-red staining (A), and anti-CD68 antibody-positive macrophages (arrows) surrounded AA amyloid deposits (B). These find‐ ings suggest an interaction between macrophages and amyloid deposits for the reduction in AA amyloid deposits. Quoted and modified from Nakamura et al. Clin Exp Rheumatol 2014; 32: 501–508.

T lymphocytes may influence the formation or metabolism of these amyloid fibrils. These cells colocalized within AA amyloid deposits, which indicates that phagocytes may participate in the metabolism or turnover of these amyloid deposits (**Figure 13**). Involvement of macro‐ phages in AA amyloid reduction was proposed [114], and this hypothesis was supported by observations that macrophage-derived proteases completely degraded AA amyloid [115]. Resolution of AA amyloid deposits appears to start when inflammation subsides and SAA levels normalize. Additional data on the natural clearance of AA amyloid are vital [116], for both a better understanding of the dynamics of amyloidogenesis and the development of effective treatments for patients with AA amyloidosis secondary to RA.

## **10. Conclusion**

**9. Possible involvement of phagocytes in degeneration of AA amyloid**

Macrophages participate in SAA processing and deposition [110, 111], and cell surfaceexpressed heparan sulfate proteoglycans have an essential function in amyloidogenesis through the binding of high-density lipoprotein-associated SAA [112]. In addition, Fcreceptor-positive macrophages have been implicated in the reduction of the amyloid load after inflammation has resolved [113]. Phagocytes such as neutrophils and macrophages will serve an important function for reducing AA amyloid deposits during ABT treatment (**Figure 12**).

**Figure 12.** Immunohistochemical analysis of a biopsy specimen from a patient with RA and AA amyloidosis who re‐ ceived abatacept (ABT). (A) Stained with the antibody anti-formyl peptide receptor-like 1 (fPRL-1, the receptor in‐ volved in production of reactive oxygen species, degradation, and phagocyte migration) and (B) anti-human CD68 antibody staining. Phagocytes, polymorphonuclear leukocytes, and macrophages stained positively in the upper gas‐ trointestinal mucosa specimen. Quoted and modified from Nakamura et al. Clin Exp Rheumatol 2014; 32: 501–508.

**Figure 13.** Relation between macrophages and AA amyloid deposits. AA amyloid deposits showed positive Congo-red staining (A), and anti-CD68 antibody-positive macrophages (arrows) surrounded AA amyloid deposits (B). These find‐ ings suggest an interaction between macrophages and amyloid deposits for the reduction in AA amyloid deposits.

Quoted and modified from Nakamura et al. Clin Exp Rheumatol 2014; 32: 501–508.

**fibrils**

230 232Exploring New Findings on Amyloidosis

AA amyloidosis is an uncommon yet important complication of chronic inflammatory conditions. Significant progress has been made in understanding of pathology, pathogenesis, and clinical treatment of AA amyloidosis secondary to RA, but AA amyloidosis is still a serious problem that deserves continued investigation. The *SAA1.3* allele is both a risk factor for AA amyloidosis and a factorrelated to poor prognosis and shortened survival in Japanese patients with RA. The incidence of AA amyloidosis secondary to RA will likely decrease because of remarkable advances in RA treatments such as bDMARDs and intracellular signal transduc‐ tion inhibitors. However, when rheumatologists first meet patients with AA amyloidosis secondary to RA, the patients may be facing a serious, life-threatening disorder such as ESRD and/or cardiac complications, even given the present medical milieu in developed countries. The pathological process in RA patients with AA amyloidosis is likely to be more complicat‐ ed and subtle than previously realized. Clarification of the formation and degeneration of AA amyloid fibrils induced by not only drugs but also cellular mechanisms and elucidation of the biological significance of SAA in health and disease is indispensable prerequisites to the management of AA amyloidosis secondary to RA. Novel therapies that target AA fibril formation and immunotherapy are currently under investigation and will lead to improved prognosis in the near future.

## **Acknowledgements**

The author would like to thank his colleagues Hirokazu Takaoka, MD, and Tetsuhiro Maesaki, MD, at Kumamoto Shinto General Hospital; Masahiro Shono, MD, Ph.D., at Kumamoto Yuge Hospital; Michishi Tsukano, MD, Ph.D., Kunihiro Tomoda, MD, Ph.D., at Kumamoto Orthopaedic Hospital; Takeshi Kambara, MD, Ph.D., Tetsuro Yamamoto, MD, Ph.D., Yukio Ando, MD, Ph.D., and Mitsuharu Ueda, MD, Ph.D., at Kumamoto University; Takami Matsuyama, MD, Ph.D., at Kagoshima University, in Japan; and Laurence A. Boxer, MD, Ph.D., at University of Michigan Medical Center, Ann Arbor, MI, USA. This work was supported in part by a Grant-in-Aid for scientific research from the Japanese Ministry of Health, Labor, and Welfare and the AmyloidosisResearch Committee forIntractable Diseases, Epochal Diagnosis, and Treatment in Japan.

#### **Conflict of interest**

The author has declared no conflicts of interest.

## **Author details**

Tadashi Nakamura

Address all correspondence to: nakamura@k-shinto.or.jp

Director, Section of Clinical Rheumatology, Kumamoto Shinto General Hospital, and Clinical Professor, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan

## **References**


[10] Saleem B, Keen H, Goeb V, Parmar R, Nizam S, Hensor EMA, Churchman SM, Quinn M, Wakefield R, Conaghan PG, Ponchel F, Emery P. Patients with RA in remission on TNF blockers: when and in whom can TNF blocker therapy be stopped? Ann Rheum Dis 2010; 69: 1636–1642.

Welfare and the AmyloidosisResearch Committee forIntractable Diseases, Epochal Diagnosis,

Director, Section of Clinical Rheumatology, Kumamoto Shinto General Hospital, and Clinical Professor, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan

[1] Pinney JH, Hawkins PN. Amyloidosis. Ann Clin Biochem 2012; 49: 229–241.

targeted therapy. Annu Rev Pathol 2015; 10: 321–344.

to prevention. Nat Rev Rheumatol 2014; 10: 521–530.

nisms and solutions. Curr Opin Rheumatol 2005; 17: 286–292.

Rheumatol 1998; 25: 417–426.

[2] Hazenberg BPC. Amyloidosis: a clinical overview. Rheum Dis Clin N Am 2013; 39: 323–

[3] de Asua DR, Costa R, Galvan JM, Filigheddu MT, Trujillo D, Cadinanos J. Systemic AA amyloidosis: epidemiology, diagnosis, and management. Clin Epidemiol 2014; 6: 369–

[4] Westermark GT, Fändrich M, Westermark P. AA amyloidosis: pathogenesis and

[5] Mijinheer G, Prakken BJ, van Wijk F. The effect of autoimmune arthritis treatment strategies on regulatory T-cell dynamics. Curr Opin Rheumatol 2013; 25: 260–267.

[6] Hunt L, Emery P. Defining populations at risk of rheumatoid arthritis: the first steps

[7] Plant MJ, Jones PW, Saklavala J, Ollier WE, Dawes PT. Patterns of radiographic progression in early rheumatoid arthritis: results of an 8 year prospective study. J

[8] Chiche L, Jorde-Chiche N, Pascual V, Chaussabel D. Current perspectives on system‐ ic immunology approaches to rheumatic diseases. ArthritisRheum 2013; 65: 1407–1417.

[9] Sattar N, McInnes IB. Vascular comorbidity in rheumatoid arthritis: potential mecha‐

and Treatment in Japan.

232 234Exploring New Findings on Amyloidosis

The author has declared no conflicts of interest.

Address all correspondence to: nakamura@k-shinto.or.jp

**Conflict of interest**

**Author details**

Tadashi Nakamura

**References**

345.

377.


[36] Pamuk ON, Donmez S, Pamuk GE, Puyan FO, Keystone EC. Turkish experience in rheumatoid arthritis patients with clinical apparent amyloid deposition. Amyloid 2013; 20: 245–250.

[22] Rumjon A, Coats T, Javaid MM. Review of eprodisate forthe treatment ofrenal disease

[23] Nystrom SN, Westermark GT. AA-amyloid is cleared by endogenous immunological

[24] Wynn TA, Chawla A, Pollard JW. Macrophage biology in development, homeostasis

[25] Nakamura T. Amyloid A amyloidosis secondary to rheumatoid arthritis: an uncom‐

[26] Baba S, Masago SA, Takahashi T, Kasama T, Sugimura H, Tsugane S, Tsutsui Y, Shirasawa H. A novel allelic variant of serum amyloid A, SAA1γ: genomic evidence, evolution, frequency, and implication as a risk factor for reactive systemic AA-

[27] Nakamura T, Yamamura Y, Tomoda K, Tsukano M, Baba S. Massive hematuria due to bladder amyloidosis in patients with rheumatoid arthritis: three case reports. Clin Exp

[28] Nakamura T, Tomoda K, Tsukano M, Yamamura Y, Baba S. Gustatory sweating due to autonomic neuropathy in a patient with amyloidosis secondary to rheumatoid

[29] Utku U, Dilek M, Akpolat I, Bedir A, Akpolat T. *SAA1* α/α alleles in Behçet's disease

[30] Moriguchi M, Kaneko H, Terai C, KosekiY, Kajiyama H, Inada S, KitamuraY, Kamatani N. Relative transcriptional activities of *SAA1* promoters polymorphic at position -13(T/ C): potential association between increased transcription and amyloidosis. Amyloid

[31] Turesson C, Weyand CM, Matteson EL. Genetics of rheumatoid arthritis: is there a pattern predicting extraarticular manifestations? Arthritis Rheum 2004; 51: 853–863.

[32] Ebert EC, Nagar MN. Gastrointestinal manifestations of amyloidosis. Am J Gastroen‐

[33] Herrera GA, Teng J, Turbat-Herrera EA. Renal amyloidosis: current views on patho‐ genesis and impact on diagnosis. In: Herrera GA, editor. Experimental Models for Renal Diseases: Pathogenesis and Diagnosis. Contrib Nephrol. Basel: Karger; 2011,

[34] Gillmore JD, Lovat LB, Persey MR, Pepys MB, Hawkins PN. Amyloid load and clinical outcome in AA amyloidosis in relation to circulating concentration of serum amyloid

[35] Nakamura T. Amyloid A amyloidosis secondary to rheumatoid arthritis: pathophysi‐

ology and treatment. Clin Exp Rheumatol 2011; 29: 850–857.

mon yet important complication. Curr Rheumatol Rev 2007; 3: 231–241.

in AA amyloidosis. Int J Nephrol Renovasc Dis 2012; 5: 37–43.

mechanisms. Amyloid 2012; 19: 138–145.

234 236Exploring New Findings on Amyloidosis

and disease. Nature 2013; 496: 445–455.

Rheumatol 2003; 21: 673–674.

2005; 12: 26–32.

terol 2008; 103: 776–787.

vol. 169, pp 232–246.

A protein. Lancet 2001; 358: 24–29.

amyloidosis. Hum Mol Genet 1995; 4: 1083–1087.

arthritis. Mod Rheumatol 2004; 14: 498–501.

related amyloidosis. Clin Rheumatol 2007; 26: 927–929.


interleukin-6 receptor antibody: a multicenter, double-blind, placebo-controlled trial. Arthritis Rheum 2004; 50: 1761–1769.


[57] Roque R, Ramiro S, Corderio A, Goncalves P, da Canas S, Santos MJ. Development of amyloidosis in patients with rheumatoid arthritis under TNF-blocking agents. Clin Rheumatol 2011; 30: 869–870.

interleukin-6 receptor antibody: a multicenter, double-blind, placebo-controlled trial.

[48] Hagiwara K, Nishikawa T, Isobe T, Song J, SugamataY, Yoshizaki K. IL-6 plays a critical role in the synergistic induction of human serum amyloid A (SAA) gene when stimulated with proinflammatory cytokines as analyzed with an SAA isoform real-time quantitative RT-PCR assay system. Biochem Biophys Res Commun 2004; 314: 363–369.

[49] Kishimoto T, Akira S, Taga T. IL-6 receptor and mechanism of signal transduction. Int

[50] Hirano T, Ishihara K, Hibi M. Roles of STAT3 in mediating the cell growth, differen‐ tiation and survival signals relayed through the IL-6 family of cytokine receptors.

[51] Cutolo M, Soldona S, Contini P, Sulli A, Seriolo B, Montagna P, Brizzolara R. Intracel‐ lular NF-κB-decrease and IκBα increase in human macrophages following CTLA4-Ig

[52] Ueda M, Ando Y, Nakamura M, Yamashita T, Himeno S, Kim J, Sun X, Saito S, Tateishi T, Bergström J, Uchino M. FK506 inhibits murine AA amyloidosis: possible involve‐

[53] Aletaha D, Neogi T, Silman AJ, Funovits J, Felson DT, Bingham CO, III, Birnbaum NS, Burmester GR, Bykerk VP, Cohen MD, Combe B, Costenbader KH, Dougados M, Emery P, Ferraccioli G, Hazes JM, Hobbs K, Huizinga TW, Kavanaugh A, Kay J, Kvien TK, Laing T, Mease P, Ménard HA, Moreland LW, Naden RL, Pincus T, Smolen JS, Stanislawska-Biernat E, Symmons D, Tak PP, Upchurch KS, Vencovský J, Wolfe F, Hawker G. 2010 Rheumatoid arthritis classification criteria. An American College of Rheumatology/European League Against Rheumatism Collaborative Initiative.

[54] Smolen JS, Landewé R, Breedveld FC, Buch M, Bumester G, Dougados M, Emery P, Gaujoux-Viala C, Gossec L, Nam J, Ramiro S, Winthrop K, de Wit M, Aletaha D, Betteridge N, Bijlsma JW, Boers M, Buttgereit F, Combe B, Cutolo M, Damjanov N, Hazes JM, Kouloumas M, Kvien TK, Mariette X, Pavelka K, van Riel PL, Rubbert-Roth A, Scholte-Voshaar M, Scott DL, Sokka-Isler T, Wong JB, van der Heijde D. EULAR recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2013 update. Ann Rheum Dis 2014;

[55] Pettersson T, Konttinen YT, Maury CPJ. Treatment strategies for amyloid A amyloi‐

[56] Dember LM. Modern treatment of amyloidosis: unsolved questions. J Am Soc

dosis. Expert Opin Pharmacother 2008; 9: 2117–2128.

ment of T cells in amyloidogenesis. J Rheumatol 2006; 33: 2260–2270.

Arthritis Rheum 2004; 50: 1761–1769.

236 238Exploring New Findings on Amyloidosis

J Immunopharmacol 1992; 14: 431–438.

Arthritis Rheum 2010; 62: 2569–2581.

73: 492–509.

Nephrol 2009; 20: 469–472.

treatment. Clin Exp Rheumatol 2013; 31: 943–946.

Oncogene 2000; 19: 2548–2556.


[80] Snow AD, Kisilevsky R, Sobh MA, Mohamed NA, Sheashaa HA, Ghoneim MA. Longterm outcome of live donor kidney transplantation for renal amyloidosis. Am J Kidney Dis 2003; 42: 370–375.

[68] Fernández-Nebro A, Olivé A, Castro MC, Varela AH, Riera E, Irigoyen MV, García de Yébenes MJ, García-Vicuña R. Long-term TNF-α blockade in patients with amyloid A

[69] Miyagawa I, Nakayamada S, Saito K, Hanami K, Nawata M, Sawamukai N, Nakano K, Yamaoka K, Tanaka Y. Study on the safety and efficacy of tocilizumab, an anti-IL-6 receptor antibody, in patients with rheumatoid arthritis complicated with AA amyloi‐

[70] Okuda Y, Ohnishi M, Matoba K, Jouyama K, Yamada A, Sawada N, Mokuda S, Murata Y, Takasugi K. Comparison of the clinical utility of tocilizumab and anti-TNF thera‐ py in AA amyloidosis complicating rheumatic diseases. Mod Rheumatol 2014; 24: 137–

[71] Haggerty HG, AbbottMA, Reilly TP, DeVona DA, Gleason CR, Tay L, DodgeR, Aranda R. Evaluation of immunogenicity of T cell costimulation modulator abatacept in

[72] Fiocco U, Sfriso P, Oliviero F, Pagnin E, Scaglioni E, Campana C, Dainese S, Cozzi L, Punzi L. Co-stimulatory modulation in rheumatoid arthritis: the role of (CTLA4-Ig)

[73] Cope AP, Schulze-Koops H, Aringger M. The central role of T cells in rheumatoid

[74] BenucciM, StamWB, Gilloteau I, Sennfält K, LeclercA, MaetzelA, Lucioni C. Abatacept or infliximab for patients with rheumatoid arthritis and inadequate response to methotrexate: an Italian trial-based and real-life cost-consequence analysis. Clin Exp

[75] Innone F, Lapadula G. The inhibition of costimulation of T cells: abatacept. J Rheuma‐

[76] Nakamura T, Kumon Y, Hirata S, Takaoka H. Abatacept may be effective and safe in patients with amyloid A amyloidosis secondary to rheumatoid arthritis. Clin Exp

[77] Rubber-Roth A, Finckh A. Treatment options in patients with rheumatoid arthri‐ tis failing TNF inhibitor therapy: a critical review. Arthritis Res Ther 2009; 11 (Suppl.

[78] Narváez J, Hernández MV, Ruiz JM, Vaquero CG, Juanola X, Nollaa JM. Rituximab therapy for AA-amyloidosis secondary to rheumatoid arthritis. Joint Bone Spine 2011;

[79] Kisilevsky R, Lemieux LJ, Fraser PE, Kong X, Hultin PG, Szarek WA. Arresting amyloidosis in vivo using small molecule anionic sulphonates or sulphate: implica‐

tions for Alzheimer's disease. Nat Med 1995; 1: 143–148.

patients treated for rheumatoid arthritis. J Rheumatol 2007; 34: 2365–2373.

amyloidosis complicating rheumatic diseases. Am J Med 2010; 123: 454–461.

dosis. Mod Rheumatol 2014; 24: 405–409.

abatacept. Autoimmun Rev 2008; 8: 76–82.

Rheumatol 2013; 31: 575–583.

Rheumatol 2014; 32: 501–508.

tol 2012; 89: 100–102.

1): S1.

48: 101–103.

arthritis. Clin Exp Rheumatol 2007; 25 (Suppl. 46): S4–S11.

143.

238 240Exploring New Findings on Amyloidosis


Antibodies to human serum amyloid P component eliminate visceral amyloid deposits. Nature 2010; 468: 93–97.


Antibodies to human serum amyloid P component eliminate visceral amyloid deposits.

[92] Nakamura T, Higashi S, Tomoda K, Tsukano M, Shono M. Effectiveness of etaner‐ cept *vs* cyclophosphamide as treatment of patients with amyloid A amyloidosis secondary to rheumatoid arthritis. Rheumatology (Oxford) 2012; 51: 2064–2069.

[93] Chiche L, Jorde-Chiche N, Pascual V, Chaussabel D. Current perspectives on systems immunology approaches to rheumatic diseases. Arthritis Rheum 2013; 65: 1407–1417.

[94] Obici L, Raimondi S, Lavetelli F, Bellotti V, Merlini G. Susceptibility to AA amyloido‐ sis in rheumatic diseases: a critical overview. Arthritis Care Res 2009; 61: 1435–1440.

[95] Don BR, Spin G, Nestorov I, Hutmacher M, Rose A, Kaysen GA. The pharmacokinet‐ ics of etanercept in patients with end-stage renal disease on haemodialysis. J Pharm

[96] Nakamura T, Higashi S, Tomda K, Tsukano M, Arizono K, Nakamura T. Etanercept treatment in patients with rheumatoid arthritis on dialysis. Rheumatol Int 2010; 30:

[97] Maury CPJ, Teppo A-M. Mechanism of reduced amyloid-A-degrading activity in

[98] Kuroda K, Tanabe N, Sato H, Ajiro J, Wada Y, Murakami S, Hasegawa H, Sakatsume M, Nakano M, Gejyo F. Outcome of patients with reactive amyloidosis associated with

[99] Sahin S, Sahin GM, Ergin H, Kantarci G. The effect of dialytic modalities on clinical outcomes in ESRD patients with familial Mediterranean fever. Ren Fail 2007; 29: 315–

[100] Heering P, Hetzel R, Grabensee B, Opelz G. Renal transplantation in secondary

[101] Kofman T, Grimbert P, Canouï-Poitrine F, Zuber J, Garrigue V, Mousson C, Frimat L, Kamar N, Couvrat G, Bouvier N, Albano L, Le Thuaut A, Pillebout E, Choukroun G, Couzi L, PeltierJ, Mariat C, Delahousse M, Buchler M, Le Pogamp P, Bridoux F, Pouteil-Noble C, Lang P, Audard V. Renal transplantation in patients with AA amyloidosis nephropathy: results from a French multicenter study. Am J Transpl 2011; 11: 2423–

[102] Iwamoto M, Honma S, Asano Y, Minota S. Effective and safe administration of tocilizumab to a patient with rheumatoid arthritis on haemodialysis. Rheumatol Int

[103] Kuroda T, Tanabe N, Kobayashi D, Sato H, Wada Y, Murakami S, Sakatsume M, Nakano M, Narita I. Programmed initiation of hemodialysis for systemic amyloidosis patients associated with rheumatoid arthritis. Rheumatol Int 2011; 31: 1177–1182.

systemic amyloidosis. Clin Transpl 1998; 12: 159–164.

rheumatoid arthritis in dialysis treatment. Rheumatol Int 2006; 26: 1147–1153.

serum of patients with secondary amyloidosis. Lancet 1982; 2: 234–237.

Nature 2010; 468: 93–97.

240 242Exploring New Findings on Amyloidosis

Pharmacol 2005; 57: 1407–1413.

1527–1528.

319.

2431.

2011; 31: 559–560.


## **The Role of Human IAPP in Stress and Inflammatory Processes in Type 2 Diabetes**

Joel Montane and Anna Novials

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/102425

#### **Abstract**

Understanding the mechanisms regulating whole-body glucose homeostasis is important in order to understand what happens in a disease such as type 2 diabetes (T2D). Insulin resistance, inflammation, dysfunction of islet β-cells, and the presence of amyloid deposits in the pancreas are some of the major causes involved in the process of β-cell deterioration. The unique peptide constituent of amyloid deposits, human islet amyloid polypeptide (hIAPP), is capable of inducing endoplasmic reticulum (ER) stress and the resulting unfolded-protein response activation. Additionally, hIAPP has been shown to induce interleukin-1β expression, the main cytokine involved in inflamma‐ tion and T2D causing inflammation and eventually, inducing apoptosis. Nevertheless, the mechanisms behind the process of hIAPP aggregation and amyloid formation are still unknown. In this chapter, we describe the different mechanisms by which hIAPP induces ER stress and inflammation. This should open the door for designing therapeutic interventions aimed at modulating the immune system and the ER stress response.

**Keywords:** Diabetes, pancreatic amyloid, inflammation, ER stress, IAPP, chaperones, diabetes

## **1. Introduction**

Type 2 diabetes (T2D) is a chronic metabolic disease characterized by hyperglycemia result‐ ing from defects in insulin secretion, insulin action, or both. The chronic hyperglycemia is associated with long-term damage, dysfunction and failure of different organs, especially the eyes, kidneys, nerves, heart and blood vessels. The genetic and molecular basis of the patho‐ genesis of T2D is not completely elucidated; however, a growing body of evidence has reported

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

thattheprogressionfromnormalto impairedbloodglucose levelregulationinindividualswith T2D is mostly influenced by insulin resistance and β-cell dysfunction. During the last decade, the reduction in β-cell function has been attributed mostly to a decrease in β-cell mass; howev‐ er, its precise role for the etiology of T2D remains controversial due to the lack of longitudinal studies.Inspite ofthis, several studies inpatients withT2Dhave showna significantβ-cellmass reduction ranging from 20 to 65% [1, 2]. Although, the reasons underlying this deficit are not clearly understood, some factors responsible for this decline have been suggested, for exam‐ ple, metabolic abnormalities (gluco- and lipotoxicity), hormonal changes (inadequate incretin secretion and action), aging, genetic abnormalities, etc. [3]. Additionally, a growing body of evidence has demonstrated the contribution of the hypersecretion of islet amyloid polypep‐ tide (IAPP), together with amyloid deposition for the establishment of T2D.

The process of amyloid deposition is a remarkable physiopathological finding in individuals with T2D (**Figure 1**). The term amyloid emerged from the Latin word *amylum*, which means starch. For a long time, it was thought that these deposits were starch-like, but later it was discovered that they were actually a mass of proteins with a particular β-sheet structure. From that, pathologies with conformational changes in normally soluble proteins or peptides that result in the formation of intermolecular hydrogen bonds, β-sheet conformation, and fibril formation are namely conformational diseases [4]. Besides T2D, these conditions have been also implicated in different human disorders, including Alzheimer's disease (AD), Parkinson's disease, and Huntington's disease.

**Figure 1.** Amyloid deposits from cadaveric human pancreata. Amyloid deposits are observed in human pancreas by (A) hematoxylin/eosin staining, (B) Congo Red staining, or (C) electron microscopy, kindly provided by Anne Clark (unpublished data).

The contribution of IAPP in T2D is still controversial; several studies question whether amyloid deposition is a cause or a consequence of islet decline and whether it occurs intra- or extracellularly [5, 6]. However, numerous evidences correlate the role of IAPP with the severity of the disease. The facts indicate that amyloid deposits are seen in 90% of patients with T2D at autopsy and can possibly correspond to stages of the pathology [7, 8].

While IAPP and amyloid formation seem to have a substantial relation in T2D, it is unknown whether aggregation of IAPP plays any role in the development of type 1 diabetes (T1D). One can assume that during β-cell destruction, as in T2D, cells are exposed to high levels of IAPP. Indeed, children with new onset of T1D presented with high levels of IAPP [9]. Nevertheless, further studies need to be done in order to determine the importance of IAPP in the develop‐ ment of T1D.

## **2. IAPP and T2D**

thattheprogressionfromnormalto impairedbloodglucose levelregulationinindividualswith T2D is mostly influenced by insulin resistance and β-cell dysfunction. During the last decade, the reduction in β-cell function has been attributed mostly to a decrease in β-cell mass; howev‐ er, its precise role for the etiology of T2D remains controversial due to the lack of longitudinal studies.Inspite ofthis, several studies inpatients withT2Dhave showna significantβ-cellmass reduction ranging from 20 to 65% [1, 2]. Although, the reasons underlying this deficit are not clearly understood, some factors responsible for this decline have been suggested, for exam‐ ple, metabolic abnormalities (gluco- and lipotoxicity), hormonal changes (inadequate incretin secretion and action), aging, genetic abnormalities, etc. [3]. Additionally, a growing body of evidence has demonstrated the contribution of the hypersecretion of islet amyloid polypep‐

The process of amyloid deposition is a remarkable physiopathological finding in individuals with T2D (**Figure 1**). The term amyloid emerged from the Latin word *amylum*, which means starch. For a long time, it was thought that these deposits were starch-like, but later it was discovered that they were actually a mass of proteins with a particular β-sheet structure. From that, pathologies with conformational changes in normally soluble proteins or peptides that result in the formation of intermolecular hydrogen bonds, β-sheet conformation, and fibril formation are namely conformational diseases [4]. Besides T2D, these conditions have been also implicated in different human disorders, including Alzheimer's disease (AD), Parkinson's

**Figure 1.** Amyloid deposits from cadaveric human pancreata. Amyloid deposits are observed in human pancreas by (A) hematoxylin/eosin staining, (B) Congo Red staining, or (C) electron microscopy, kindly provided by Anne Clark

The contribution of IAPP in T2D is still controversial; several studies question whether amyloid deposition is a cause or a consequence of islet decline and whether it occurs intra- or extracellularly [5, 6]. However, numerous evidences correlate the role of IAPP with the severity of the disease. The facts indicate that amyloid deposits are seen in 90% of patients with T2D

at autopsy and can possibly correspond to stages of the pathology [7, 8].

tide (IAPP), together with amyloid deposition for the establishment of T2D.

disease, and Huntington's disease.

2442 Exploring New Findings on Amyloidosis

(unpublished data).

#### **2.1. Islet amyloid polypeptide**

Islet amyloid polypeptide or IAPP, also known as amylin, is a normal product of pancreatic β-cells. It is stored along with insulin in secretory granules and co-secreted in response to nutrient stimuli. This 37-amino acid peptide was identified in 1987, although the gene was isolated and characterized in 1989 [7]. Nishi et al. located it in chromosome 12, containing three exons and two introns that codified for an 89-amino acid precursor termed preproIAPP with an amino-terminal signal sequence. This signal peptide is then cleaved from the precursor to generate a 67-amino acid propeptide termed proIAPP. This peptide undergoes further translational modifications by the prohormone convertases, which include the formation of disulfide bridges between cysteine residues and an amidation of a C-terminal tyrosine. These prohormone convertases are as well responsible for proteolytic conversion of proinsulin to insulin, supporting the idea that the processing of proIAPP might also be impaired in T2D [10].

The function of IAPP has been suggested to be involved with glucose homeostasis. In general, this hormone inhibits gastric emptying and is important in controlling and delaying the rate of meal-derived glucose. It has also been shown to inhibit secretion of other pancreatic hormones, such as glucagon and somatostatin. Indeed, physiological concentrations of IAPP are responsible for the regulation of food intake and body weight. Several other effects have been described, including the regulation of renal filtration [11], calcium homeostasis [12], and vasodilatation [13]. Nevertheless, a critical role positions IAPP as the main responsible for the pathogenesis of T2D through formation of amyloid deposits and destruction of pancreatic βcells.

Non-toxic bioactive variants of IAPP have been shown to be clinically important for the treatment of T1D, T2D, and obesity. For example, co-administration of modified non-toxic variants of IAPP and insulin helped normalization of oscillating glucose levels to a greater extent than insulin alone [14, 15]. Furthermore, combinations of IAPP and leptin have also been used for the treatment of obesity [16]. Nevertheless, aggregation, engineering, and solubility problems at physiological pHs have been affecting the different approaches.

#### **2.2. Molecular mechanisms of IAPP aggregation and amyloid formation**

Although there has been considerable progress, the exact mechanism of abnormal aggregation of IAPP is still largely unknown; however, several studies pointed the overproduction or mutations in IAPP as the main causes of amyloid formation. The role of overproduction of IAPP is associated with the increased secretory demand for insulin due to insulin resistance and increasing hyperglycemia [17]. Because IAPP and insulin are co-secreted in β-secretory granules, this increased production and secretion could result in accumulation and aggrega‐ tion of IAPP. Many studies have reported that transgenic mice overexpressing hIAPP develop islet amyloid deposits [18, 19]. However, other studies contradicted this hypothesis, claiming that IAPP levels are several times higher than normally. To confirm these findings, Kahn et al. demonstrated that non-diabetic obese and/or insulin-resistant individuals with elevated IAPP levels do not develop amyloid deposits per se. However, in both cases, the presence of some factors, such as genetic predisposition, high fat diet, and obesity, might be critical for the development of extensive islet amyloid [18, 19].

Another possible mechanism for amyloid formation concerns the mutations in the IAPP gene or promoter regions, by producing more fibrillogenic forms [20]. For example, based on several studies, the S20G mutation in the IAPP gene appears to be associated with an early onset and more severe form of T2D [21].

In addition, the existence of hydrophobic amino acids in the mid-portion of IAPP could also be responsible for its propensity to aggregate into β-pleated sheets. Several algorithms, such as discrete molecular dynamics simulations [22] or specifically designed software such as TANGO [23–25], have been developed to identify the amyloidogenic regions of hIAPP. In this line, the residues at positions 20–29 of the polypeptide chain have been determined to be the amyloidogenic region of the peptide. Accordingly, the proline substitutions in the 24–29 regions of rodent IAPP are thought to prevent amyloid fibril formation completely [26]. Proline is known to be a β-sheet breaker, and a total inhibition of amyloid formation was seen when substitutions in the 20–29 wild-type area were performed [27]. Proteoglycans, the major components of the extracellular matrix, have been implicated with several pathologies, including AD and T2D, and have been associated with amyloid deposits in the human body [28]. In particular, together with IAPP, heparan sulfate proteoglycan perlecan is the main component of amyloid deposits in pancreatic islets. Several hypotheses indicate that the impaired processing of proIAPP may result in an elevated secretion of the IAPP precursor with a strong affinity for heparan sulfate proteoglycans, which could eventually result in a gener‐ ation of a nucleus from amyloid formation [17, 18].

## **2.3. IAPP toxicity**

Aggregated hIAPP has cytotoxic properties and is believed to be of critical importance for the progression in patients with T2D. Early studies have shown that the formation of islet amyloid is strongly associated with reduction of insulin secretion and with loss of approximately 50% of the β-cell mass [29, 30]. Human IAPP aggregation has been suggested to occur in a stepwise manner, with soluble monomeric hIAPP forming oligomeric structures, protofibrils, and eventually amyloid fibrils (**Figure 2**).

mutations in IAPP as the main causes of amyloid formation. The role of overproduction of IAPP is associated with the increased secretory demand for insulin due to insulin resistance and increasing hyperglycemia [17]. Because IAPP and insulin are co-secreted in β-secretory granules, this increased production and secretion could result in accumulation and aggrega‐ tion of IAPP. Many studies have reported that transgenic mice overexpressing hIAPP develop islet amyloid deposits [18, 19]. However, other studies contradicted this hypothesis, claiming that IAPP levels are several times higher than normally. To confirm these findings, Kahn et al. demonstrated that non-diabetic obese and/or insulin-resistant individuals with elevated IAPP levels do not develop amyloid deposits per se. However, in both cases, the presence of some factors, such as genetic predisposition, high fat diet, and obesity, might be critical for the

Another possible mechanism for amyloid formation concerns the mutations in the IAPP gene or promoter regions, by producing more fibrillogenic forms [20]. For example, based on several studies, the S20G mutation in the IAPP gene appears to be associated with an early onset and

In addition, the existence of hydrophobic amino acids in the mid-portion of IAPP could also be responsible for its propensity to aggregate into β-pleated sheets. Several algorithms, such as discrete molecular dynamics simulations [22] or specifically designed software such as TANGO [23–25], have been developed to identify the amyloidogenic regions of hIAPP. In this line, the residues at positions 20–29 of the polypeptide chain have been determined to be the amyloidogenic region of the peptide. Accordingly, the proline substitutions in the 24–29 regions of rodent IAPP are thought to prevent amyloid fibril formation completely [26]. Proline is known to be a β-sheet breaker, and a total inhibition of amyloid formation was seen when substitutions in the 20–29 wild-type area were performed [27]. Proteoglycans, the major components of the extracellular matrix, have been implicated with several pathologies, including AD and T2D, and have been associated with amyloid deposits in the human body [28]. In particular, together with IAPP, heparan sulfate proteoglycan perlecan is the main component of amyloid deposits in pancreatic islets. Several hypotheses indicate that the impaired processing of proIAPP may result in an elevated secretion of the IAPP precursor with a strong affinity for heparan sulfate proteoglycans, which could eventually result in a gener‐

Aggregated hIAPP has cytotoxic properties and is believed to be of critical importance for the progression in patients with T2D. Early studies have shown that the formation of islet amyloid is strongly associated with reduction of insulin secretion and with loss of approximately 50% of the β-cell mass [29, 30]. Human IAPP aggregation has been suggested to occur in a stepwise manner, with soluble monomeric hIAPP forming oligomeric structures, protofibrils, and

development of extensive islet amyloid [18, 19].

ation of a nucleus from amyloid formation [17, 18].

eventually amyloid fibrils (**Figure 2**).

**2.3. IAPP toxicity**

more severe form of T2D [21].

2464 Exploring New Findings on Amyloidosis

**Figure 2.** Proposed model of amyloid formation. (A) Primary sequences of human and murine islet amyloid polypep‐ tide. Amyloidogenic region predicted by intrinsic propensities for aggregation is indicated in gray. Note the presence of proline residues in rodent IAPP sequence. (B) Folding or trafficking alterations can induce hIAPP misfolding lead‐ ing to aggregation. Misfolded monomers aggregate to oligomers that eventually form the characteristic amyloid pla‐ ques from T2D.

Initially, there was general acceptance about the concept that the fibrillar forms of hIAPP are the toxic species [31]. Moreover, amyloid fibrils are less toxic than small oligomers formed by aggregates of IAPP. Studies have shown a strong correlation between islet amyloidosis and hIAPP cytotoxicity and eventually β-cell death. Yanker et al. have demonstrated that toxicity is mediated by IAPP fibrils by direct contact of fibrils with the cell surface causing DNA fragmentation, chromatin condensation, and protuberances in the plasma membrane leading to islet cell apoptosis [32]. The common feature for hIAPP fibrils lies on the classical cross βstructure, polymorphic, and typically unbranched [33]. Additionally, in vitro studies have provided evidences that synthetic hIAPP readily forms fibrils and amyloid deposits, which allowed studying the overall morphology and formation process [33, 34].

According to the structural information available for hIAPP fibrils and oligomers, it is clear that hIAPP as an amyloid protein has shown to be toxic through similar mechanisms as other amyloid proteins. One of the most widely accepted mechanisms refers to membrane interac‐ tion which leads to cell membrane permeabilization or disruptions [35]. Concerning hIAPP oligomers, the membrane leakage occurs via direct interaction and/or formation of ionic pores and depends on the lipid composition, peptide ratio, pH, and ionic strength. In the case of fibril formation, the damage in the membrane may happen through interaction of fibrils with specific channels located on the cell surface, such as potassium channels [36]. Moreover, oligomers of hIAPP have been shown to increase inflammation in β-cells via the inflamma‐ some, a large group of intracellular multiprotein complexes that induce inflammation and play a central role in immunity [37].

Comparably, hIAPP can form oligomers and fibrils that contribute to islet inflammation. In this line, hIAPP oligomers and fibrils (but not rodent IAPP) have been shown to induce synthesis of interleukins and other inflammatory mediators by pancreatic islets that recruit and activate macrophages in vivo [38]. Further, endoplasmic reticulum (ER) stress has been proposed to be an important contributor to hIAPP-induced-β-cell death since exogenously added hIAPP has been confirmed to induce ER stress. Some reports showed that ER stressmediated apoptosis is exacerbated in rodent β-cells expressing amyloidogenic isoforms of hIAPP, leading to a reduction of β-cell mass in hIAPP transgenic mice and rats [6, 39]. In addition, Casas et al. [40] demonstrated that extracellular hIAPP aggregation is associated with ER stress responses in mouse β-cells. Although the mechanism responsible for β-cell cytotox‐ icity during the process of hIAPP aggregation is still not well defined, a growing body of evidence firmly indicates that IAPP fibrils or oligomers have a crucial role in the progressive β-cell dysfunction in T2D.

Prevention of IAPP amyloid formation may represent a potential treatment for T2D. Thus, the use of several small-molecule inhibitors is being exploited. Several small inhibitory peptides [26, 41], natural polyphenols (such as resveratrol and epigallocatechin gallate; EGCG) [42, 43] or specific antibodies [44, 45] have been successfully used to validate the application of antiamyloid compounds. For example, the peptide D-ANFLVH inhibited the formation of islet amyloid deposits and contributed to the preservation of β-cell area and improved glucose tolerance in mice [46]. These results validate the application of anti-amyloid compounds as therapeutic strategies to maintain β-cell function in patients with T2D.

## **3. The endoplasmic reticulum**

## **3.1. Physiological role**

Protein folding begins as the nascent polypeptide chain is co-translationally translocated through the ER membrane into the ER lumen. The unique environment of the ER lumen allows for both oxidative protein folding and post-translational modification such as glycosylation and disulfide bond formation, and accounts for approximately one-third of all proteins in eukaryotic cells. Since ER is mainly associated with protein synthesis, if the protein is not properly folded/maturated, it will remain in the ER and will eventually be degraded without reaching its normal cellular site of action [47]. This sophisticated supervision carried by the ER is regulated by sensitive quality control systems that can discriminate between the proper folded proteins from the misfolded ones. For example, folding chaperones consist of a considerable number of proteins that have the capacity to recognize properties common to non-native proteins, such as exposed hydrophobic areas and in most cases through the expenditure of ATP [48]. Thus, chaperones have the role of correctly folding and assembling secreted proteins, aiding oligomerization, and performing post-translational modifications [49]. The other quality control system is the ubiquitin-proteasome system (UPS) in which irreparably damaged proteins are identified and sorted for degradation. This system is responsible for the clearance of intracellular misfolded and aggregated proteins [50]. Many stimuli can disrupt this process, and its failure can give rise to the malfunctioning of living systems leading to the development of an increasing number of disorders, including Parkin‐ son, AD, Huntington, and T2D [51].

#### **3.2. ER stress and the unfolded protein response**

and activate macrophages in vivo [38]. Further, endoplasmic reticulum (ER) stress has been proposed to be an important contributor to hIAPP-induced-β-cell death since exogenously added hIAPP has been confirmed to induce ER stress. Some reports showed that ER stressmediated apoptosis is exacerbated in rodent β-cells expressing amyloidogenic isoforms of hIAPP, leading to a reduction of β-cell mass in hIAPP transgenic mice and rats [6, 39]. In addition, Casas et al. [40] demonstrated that extracellular hIAPP aggregation is associated with ER stress responses in mouse β-cells. Although the mechanism responsible for β-cell cytotox‐ icity during the process of hIAPP aggregation is still not well defined, a growing body of evidence firmly indicates that IAPP fibrils or oligomers have a crucial role in the progressive

Prevention of IAPP amyloid formation may represent a potential treatment for T2D. Thus, the use of several small-molecule inhibitors is being exploited. Several small inhibitory peptides [26, 41], natural polyphenols (such as resveratrol and epigallocatechin gallate; EGCG) [42, 43] or specific antibodies [44, 45] have been successfully used to validate the application of antiamyloid compounds. For example, the peptide D-ANFLVH inhibited the formation of islet amyloid deposits and contributed to the preservation of β-cell area and improved glucose tolerance in mice [46]. These results validate the application of anti-amyloid compounds as

Protein folding begins as the nascent polypeptide chain is co-translationally translocated through the ER membrane into the ER lumen. The unique environment of the ER lumen allows for both oxidative protein folding and post-translational modification such as glycosylation and disulfide bond formation, and accounts for approximately one-third of all proteins in eukaryotic cells. Since ER is mainly associated with protein synthesis, if the protein is not properly folded/maturated, it will remain in the ER and will eventually be degraded without reaching its normal cellular site of action [47]. This sophisticated supervision carried by the ER is regulated by sensitive quality control systems that can discriminate between the proper folded proteins from the misfolded ones. For example, folding chaperones consist of a considerable number of proteins that have the capacity to recognize properties common to non-native proteins, such as exposed hydrophobic areas and in most cases through the expenditure of ATP [48]. Thus, chaperones have the role of correctly folding and assembling secreted proteins, aiding oligomerization, and performing post-translational modifications [49]. The other quality control system is the ubiquitin-proteasome system (UPS) in which irreparably damaged proteins are identified and sorted for degradation. This system is responsible for the clearance of intracellular misfolded and aggregated proteins [50]. Many stimuli can disrupt this process, and its failure can give rise to the malfunctioning of living systems leading to the development of an increasing number of disorders, including Parkin‐

therapeutic strategies to maintain β-cell function in patients with T2D.

β-cell dysfunction in T2D.

2486 Exploring New Findings on Amyloidosis

**3. The endoplasmic reticulum**

son, AD, Huntington, and T2D [51].

**3.1. Physiological role**

Some situations (oxidative stress, energy deprivation, metabolic challenge, or inflammatory stimuli) can represent a major problem for the cell due to a probable production of unfolded or misfolded proteins. These physiological and pathological conditions may interfere with protein maturation and trafficking processes, leading to the accumulation of unfolded and/or misfolded proteins.

A complex homeostatic mechanism known as the unfolded protein response (UPR) has evolved linking the load of newly synthesized proteins with the capacity of the ER to mature them. The UPR works as a multifaceted strategy to protect the integrity of the ER and the associated functionality of the secretory pathway. In mammalians, the first response consists in attenuating the translation of most peptides, followed by an induction of ER chaperone translation that promotes the correct folding [52] and finally, the activation of the ER-associated degradation (ERAD), in which misfolded proteins are retrotranslocated from the ER lumen to the cytosol and degraded by the ubiquitin-proteasome [52, 53]. Conversely, if this mechanism of adaptation and survival fails to relieve ER stress, a continued accumulation of misfolded proteins takes place within the ER, and consequently UPR will generate pro-apoptotic signals to eliminate the diseased cell [52].

In mammals, the UPR consists of three main classes of proteins that act as sensors of ER stress: inositol-requiring enzyme 1 (IRE1), double-stranded RNA-activated 9 protein kinase RNA (PKR)-like ER kinase (PERK), and activating transcription factor 6 (ATF6) (**Figure 3**). Despite the difference, each of these proteins activates different signaling pathways and activates transcription factors that mediate the induction of several ER stress genes. IRE1 is considered a central regulator of the ER stress signaling and plays an important role in protein biosyn‐ thesis. Under non-stressed conditions, IRE1 remains in an inactive monomeric form. Upon accumulation of unfolded proteins, IRE1 is activated and released by the binding immuno‐ globulin protein or BiP, an endogenous chaperone located in the lumen of the ER that binds newly synthesized proteins and helps them through the process of folding and maturation. Subsequently, IRE1 activates a transcription factor named X-box-binding protein (XPB1), which once translocated into the nucleus initiates several transcriptional programs that upregulate UPR-associated genes. In a similar way, once chaperone BiP releases from its interaction with PERK, PERK is able to dimerize, promoting autophosphorylation and activation [54]. Once activated, PERK phosphorylates eukaryotic initiation factor 2a (elF2α), its only recognized target. Once activated, elF2α leads to a rapid reduction in the number of proteins entering the already overwhelmed ER [55]. However, in some circumstances, other transcription factors such as activating transcription factor 4 (ATF4) are translated and modulate the expression of C/EBP homologous protein (CHOP). CHOP acts by inducing apoptosis [56]. A major mediator of transcriptional induction by ER stress is the basic leucine zipper domain transcription factor ATF6. This protein is also regulated not only by BiP but also by intra- and inter-molecular disulfide bridges which are thought to keep ATF6 inactive [57, 58]. In response to ER stress, BiP is released from ATF6 and disulfide bonds are reduced, which eventually target the expression of transcription chaperones, upregulation of XBP1, and transcription of elements of the ERAD (**Figure 3**).

**Figure 3.** Branches of the UPR and inflammation signaling pathways. The three sensor-transducers of the UPR are ino‐ sitol-requiring protein-1 (IRE-1), PERK, and ATF6. These three sensor-transducers determine the state of unfolded pro‐ teins in the ER lumen. If persistent, NF-κβ and JNK become activated, leading to inflammation and apoptosis.

## **3.3. Link between ER stress and apoptosis**

A growing number of studies implicate ER stress with β-cell death during the evolution of T2D [59, 60]. Several physiological, environmental, and genetic factors can provoke alterations in ER homeostasis leading to a state of stress. Evidences suggest that a continuous increase in insulin biosynthesis might overwhelm the folding capacity of the ER, leading to a chronic state. As a consequence, the UPR signaling pathways are triggered with the objective of maintaining β-cell function and promoting β-cell survival. In general, cells have the capacity to adapt to substantial ER stress, but if the ER stress is too severe and long-standing, the UPR-mediated efforts ultimately fail and the apoptotic pathway is activated in order to protect the organism by eliminating damaged cells (**Figure 3**). At least, three parallel pathways are involved in the stress-mediated apoptosis: activation of CHOP (recognized as a key mediator of apoptosis in ER stress), activation of IRE1–JNK pathway, and activation of caspase 12 [61, 62]. β-Cell apoptosis is also observed in human pancreatic sections and post-mortem islet grafts in correlation with amyloid deposition levels [6, 63, 64].

As previously discussed, ER stress-mediated apoptosis is exacerbated in rodent β-cells expressing hIAPP in β-cells, leading to a reduction of β-cell mass [39]. In addition, extracellular hIAPP aggregation is associated with ER stress, contributing to β-cell apoptosis [40, 65]. Nevertheless, in a rat pancreatic β-cell line overexpressing hIAPP, the detection of toxic intracellular oligomers, which lead to defective insulin and IAPP secretion levels in response to glucose, did not change the expression of genes involved in ER stress and apoptosis was not induced [36]. These results agree with other findings with hIAPP transgenic mice, in which the authors demonstrated that amyloid formation was not associated with significant increases in the expression of ER stress markers [66]. As discussed elsewhere, the discrepancy in these results may be explained by differences in the ratio of IAPP and insulin produced by the different models used in vitro and in vivo, ranging from low to significantly high levels of hIAPP [67].

## **4. ER stress and inflammation**

**Figure 3.** Branches of the UPR and inflammation signaling pathways. The three sensor-transducers of the UPR are ino‐ sitol-requiring protein-1 (IRE-1), PERK, and ATF6. These three sensor-transducers determine the state of unfolded pro‐ teins in the ER lumen. If persistent, NF-κβ and JNK become activated, leading to inflammation and apoptosis.

A growing number of studies implicate ER stress with β-cell death during the evolution of T2D [59, 60]. Several physiological, environmental, and genetic factors can provoke alterations in ER homeostasis leading to a state of stress. Evidences suggest that a continuous increase in insulin biosynthesis might overwhelm the folding capacity of the ER, leading to a chronic state. As a consequence, the UPR signaling pathways are triggered with the objective of maintaining β-cell function and promoting β-cell survival. In general, cells have the capacity to adapt to substantial ER stress, but if the ER stress is too severe and long-standing, the UPR-mediated efforts ultimately fail and the apoptotic pathway is activated in order to protect the organism by eliminating damaged cells (**Figure 3**). At least, three parallel pathways are involved in the stress-mediated apoptosis: activation of CHOP (recognized as a key mediator of apoptosis in ER stress), activation of IRE1–JNK pathway, and activation of caspase 12 [61, 62]. β-Cell apoptosis is also observed in human pancreatic sections and post-mortem islet grafts in

As previously discussed, ER stress-mediated apoptosis is exacerbated in rodent β-cells expressing hIAPP in β-cells, leading to a reduction of β-cell mass [39]. In addition, extracellular hIAPP aggregation is associated with ER stress, contributing to β-cell apoptosis [40, 65]. Nevertheless, in a rat pancreatic β-cell line overexpressing hIAPP, the detection of toxic intracellular oligomers, which lead to defective insulin and IAPP secretion levels in response to glucose, did not change the expression of genes involved in ER stress and apoptosis was not induced [36]. These results agree with other findings with hIAPP transgenic mice, in which

**3.3. Link between ER stress and apoptosis**

2508 Exploring New Findings on Amyloidosis

correlation with amyloid deposition levels [6, 63, 64].

The three branches of UPR response can trigger inflammatory signals through different branches that converge in signaling pathways involving c-Jun N-terminal kinases (JNKs) and the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κβ). JNK is considered to play an important role in ER stress in mouse models of diabetes. For instance, an increase in JNK activity promotes insulin resistance in peripheral tissues and in pancreatic β-cells without affecting cell viability [68]. The importance of the JNK pathway in stress has also been observed in knock-out (KO) mice where suppression of the JNK pathway protects β-cells against oxidative stress induction [69].

The pathway of the UPR involving IRE1 can, by different mechanisms, trigger an inflammatory signaling pathway through the activation of JNK (**Figure 3**) [70]. In addition, through multiple mechanisms, both the IRE1 and PERK pathways can also lead to the activation of the NF-κβ pathway, which also plays a critical role in the induction of multiple inflammatory mediators and has been implicated in insulin resistance [60, 71]. ATF6 has also been linked to inflamma‐ tory signaling. Genetic and pharmacological inhibition of ATF6 significantly suppresses NFκβ activation, which can transcriptionally regulate many other inflammatory genes [72]. Activation of either JNK or NF-κB pathways in pancreatic β-cells has been reported to cause increased expression of proinflammatory molecules that can act as mediators, such as inflam‐ matory interleukins 8 (IL8) and 6 (IL6), monocyte chemotactic protein-1 (MCP-1 or CCL2), and the cytotoxin tumor necrosis factor-α (TNF-α), which may have a detrimental effect on cell survival and function [73, 74]. Local chemokine and cytokine release can also contribute to the inflammatory milieu, attracting host macrophages to the pancreatic β-cells, which further propagate local inflammation [38, 75]. In addition, the NF-κβ pathway has been shown to activate the NLRP3 inflammasome, a multiprotein, cytosolic molecular platform that controls the activation of caspase 1, and the secretion of other proinflammatory cytokines such as interleukin-1β (IL-1β) and interleukin 18 (IL-18) in metabolic stress [76]. Inflammation induced by inflammasome-dependent proinflammatory cytokines may produce insulin resistance or cause the death of pancreatic β-cells, leading to the development of diabetes [37, 77].

NLRP3 inflammasome formation is reported to be induced by a variety of compounds, including hIAPP [37]. Oligomeric hIAPP has been shown to induce inflammasome activation and subsequent production of IL-1β. This is supported by observations with a transplantation model, in which hIAPP-expressing islets were transplanted into immunodeficient NOD-SCID mice treated with and without the IL-1β receptor agonist (IL1Ra) [38]. In this study, IL-1Ra was able to protect transplanted hIAPP-expressing islets from impaired glucose tolerance. Islet grafts expressing hIAPP contained amyloid deposits in close association with macrophages. Moreover, early aggregates of hIAPP induced production of inflammatory cytokines and chemokines, such as CCL2, TNF-α, IL-1α, IL-1β, chemokine (C–C motif) ligand 3 (CCL3), and the chemokine (C–X–C motif) ligand 1, 2, and 10 (CXCL1, CXCL2, and CXCL10, respectively) [38].

## **5. The role of hIAPP in ER stress, inflammation, and apoptosis**

Multiple physiological and pathological conditions, including the accumulation of misfolded proteins, such as insulin or hIAPP, are responsible for the loss of ER homeostasis in β-cells [6, 60].

## **5.1. Exogenous hIAPP induces ER stress and apoptosis in vitro**

Several approaches have been applied to study amyloid toxicity in vitro; synthetic peptides, corresponding to either fragments or the whole protein, have been useful attempt in defining the amyloidogenic pathology. Several studies have reported that amyloid peptide is proficient to induce cytotoxic cell death by external addition of synthetic hIAPP [32, 40, 78–80]. Although the precise mechanism by which IAPP aggregates lead to β-cell death is still unknown, it has been recognized that this aggregation is a concentration dependent on synthetic hIAPP in vitro. Bailey et al. suggested a progressive increase in cell toxicity according to the initial peptide concentration, as well as the time exposed for the process of IAPP fibrillation [34]. In addition, Casas et al. demonstrated that extracellular hIAPP aggregation is associated with ER stress responses in mouse β-cells, by an intracellular signaling that involves downstream inhibition of the ubiquitin-proteasome pathway, contributing to β-cell apoptosis [40]. In line with these studies, evidences have demonstrated that in aqueous solution, synthetic hIAPP spontane‐ ously forms β-sheets and aggregates, whereas synthetic rat IAPP does not [81, 82], and the aggregation process seems to be extremely sensitive to amyloid concentrations [83].

## **5.2. Intracellular hIAPP**

The mechanism by which amyloid oligomers and/or fibrils are formed within the β-cell is not completely understood. For that reason, many attempts to express this protein in various vectors and hosts have been designed. O'Brien et al. [84] were able to transfect fibroblast-like cell line (COS-1) cells with vectors expressing amyloidogenic IAPP; however, those cells containing amyloid fibrils were degenerated or dead when compared to rat IAPP overexpres‐ sion. Years later, the same group, with the effort to understand the mechanism by which intracellular hIAPP causes cell death, demonstrated that in transfected COS-1 cells, the accumulation of hIAPP initiates a cascade of intracellular signaling events that trigger the apoptotic pathway [85]. However, this cell line was not expected to prevent such event due to the lack of the cellular machinery needed for the processing and trafficking of immature IAPP, such as secretory granules or the presence of prohormone convertases.

Recent studies have also reported successful cloning and expression of recombinant hIAPP in cultured mammalian cells. Several in vitro approaches allowed the successful expression, purification, and characterization of the amyloidogenicity and cytotoxicity of the human mature IAPP in sufficient amounts using, for example, the Lacl-T7 RNA polymerase-based heterologous expression system for *Escherichia coli*. This *E. coli* expression system has been shown to remove potential toxic proteins and, at the same time, generate high levels of recombinant proteins [86]. Likewise, other studies were capable to clone the hIAPP full-length peptide into not only COS-1 cells but also in rat insulinoma (RIN) and Chinese hamster ovary (CHO) cells [87]. Nevertheless, when studies were performed in INS1E cells, a β-cell line with all the equipment for the processing and regulation of IAPP, the expression of hIAPP by adenovirus has not resulted in cell death unless hIAPP was high enough to cause impaired proIAPP processing [88].

Furthermore, Soty et al. established an in vitro model in which INS1E cells were stably transfected with hIAPP cDNA under the cytomegalovirus promoter (CMV). Under hIAPP overexpression, these cells showed intracellular oligomers and a strong alteration of glucosestimulated insulin and IAPP secretion. Moreover, inhibition of insulin and secretion of IAPP affected the activity of KATP channels, leading to an increased mitochondrial metabolism in order to counteract the secretory defects of the β-cells [36]. Nevertheless, hIAPP-expressing INS1E cells were able to completely restore insulin secretion and prevent ER stress upon treatment with molecular (BiP and protein disulfide isomerase; PDI) and chemical (taurour‐ sodeoxycholic acid; TUDCA and 4-phenylbutyrate; PBA) chaperones [89]. Amelioration of insulin secretion upon high glucose stimulation and prevention of β-cell death was further confirmed by the same group using hIAPP transgenic mouse islets and molecular chaperone PDI [90].

#### **5.3. In vivo models of hIAPP overexpression**

grafts expressing hIAPP contained amyloid deposits in close association with macrophages. Moreover, early aggregates of hIAPP induced production of inflammatory cytokines and chemokines, such as CCL2, TNF-α, IL-1α, IL-1β, chemokine (C–C motif) ligand 3 (CCL3), and the chemokine (C–X–C motif) ligand 1, 2, and 10 (CXCL1, CXCL2, and CXCL10, respectively)

Multiple physiological and pathological conditions, including the accumulation of misfolded proteins, such as insulin or hIAPP, are responsible for the loss of ER homeostasis in β-cells [6,

Several approaches have been applied to study amyloid toxicity in vitro; synthetic peptides, corresponding to either fragments or the whole protein, have been useful attempt in defining the amyloidogenic pathology. Several studies have reported that amyloid peptide is proficient to induce cytotoxic cell death by external addition of synthetic hIAPP [32, 40, 78–80]. Although the precise mechanism by which IAPP aggregates lead to β-cell death is still unknown, it has been recognized that this aggregation is a concentration dependent on synthetic hIAPP in vitro. Bailey et al. suggested a progressive increase in cell toxicity according to the initial peptide concentration, as well as the time exposed for the process of IAPP fibrillation [34]. In addition, Casas et al. demonstrated that extracellular hIAPP aggregation is associated with ER stress responses in mouse β-cells, by an intracellular signaling that involves downstream inhibition of the ubiquitin-proteasome pathway, contributing to β-cell apoptosis [40]. In line with these studies, evidences have demonstrated that in aqueous solution, synthetic hIAPP spontane‐ ously forms β-sheets and aggregates, whereas synthetic rat IAPP does not [81, 82], and the

aggregation process seems to be extremely sensitive to amyloid concentrations [83].

such as secretory granules or the presence of prohormone convertases.

The mechanism by which amyloid oligomers and/or fibrils are formed within the β-cell is not completely understood. For that reason, many attempts to express this protein in various vectors and hosts have been designed. O'Brien et al. [84] were able to transfect fibroblast-like cell line (COS-1) cells with vectors expressing amyloidogenic IAPP; however, those cells containing amyloid fibrils were degenerated or dead when compared to rat IAPP overexpres‐ sion. Years later, the same group, with the effort to understand the mechanism by which intracellular hIAPP causes cell death, demonstrated that in transfected COS-1 cells, the accumulation of hIAPP initiates a cascade of intracellular signaling events that trigger the apoptotic pathway [85]. However, this cell line was not expected to prevent such event due to the lack of the cellular machinery needed for the processing and trafficking of immature IAPP,

**5. The role of hIAPP in ER stress, inflammation, and apoptosis**

**5.1. Exogenous hIAPP induces ER stress and apoptosis in vitro**

[38].

25210 Exploring New Findings on Amyloidosis

60].

**5.2. Intracellular hIAPP**

One of the most active research areas that have contributed substantially to our current understanding of the molecular basis in a multifactorial disease such as T2D is the creation and development of diverse animal models. Nowadays, in vivo studies of human pancreas morphology are not possible by obvious ethical considerations, and the collective human material comes from either autopsy or surgical resection from pancreatic cancer. It is interest‐ ing that apart from humans, the only species capable of spontaneously developing T2D are non-human primates and cats; nevertheless, besides the cost of working with such big species, these models not always progress toward T2D, making the use of these models not optimal for research [91]. Studies performed with rodent models of diabetes are then greatly useful and advantageous, especially regarding islet amyloidosis studies. As previously mentioned, unlike the human IAPP, the rodent IAPP is not amyloidogenic due to the proline substitutions in the 20–29 amino acid region. This lack of amyloid development in these models makes impossible to assess the role of IAPP aggregation in islet physiology. Since only a limited number of species spontaneously form islet amyloid, several groups have developed trans‐ genic mice strains choosing hIAPP as a model for islet amyloidogenesis [91].

Nonetheless, some reports showed that the mere hIAPP overproduction did not lead to amyloid formation or deposition despite elevated plasma concentration of hIAPP. Thus, other factors beyond overexpression had to be involved in the mechanism of islet amyloid formation since these mice were normoglycemic and normoinsulinemic (94–96). Islet amyloid was reported in transgenic mice fed a diet high in fat. Verchere et al. [18] have shown that approx‐ imately 80% of male transgenic mice (<13 months old) presented amyloid deposits and were always associated with severe hyperglycemia. In the case of hemizygous transgenic mice for hIAPP, the treatment with growth hormone or dexamethasone induced small intra- and extracellular amyloid deposits [92].

Another strategy used to overexpress hIAPP was to cross-bread hIAPP mice onto a mouse with obese background (ob/ob) [19] or obese Agouti viable yellow (Avy/Agouti) [93]. These mice developed amyloid formation and loss of β-cells, which was associated with progression of diabetes (**Figure 4**).

**Figure 4.** Amyloid formation in hIAPP transgenic mouse islets in obese Agouti yellow mice. Amyloid staining of hI‐ APP Tg mice in (A) FVB background, Avy/Agouti background at (B) 16 weeks of age and (C) 22 weeks of age. Note the presence of amyloid deposits in Avy/Agouti background as shown by Thioflavin S staining (unpublished data).

Moreover, it was found that female transgenic mice do not increase the occurrence of amyloid when oophorectomized, suggesting a protective role of ovarian hormones in islet amyloidosis. In recent years, Butler et al. showed that transgenic β-cell expression of human proIAPP in rats (HIP rats) that are homozygous for hIAPP develop diabetes within 5-10 months, together with the presence of extracellular amyloid, decreased β-cell mass, and increased β-cell apoptosis [94]. In addition, the loss of approximately 60% of β-cell mass at diabetes onset is comparable to the loss observed in humans with T2D [1].

In conclusion, with a variety of transgenic hIAPP models, it has been possible to clearly highlight that the process of islet amyloid formation is a complex event associated with a great number of factors considered important in the pathogenesis of T2D.

## **Acknowledgements**

This work was supported by grants PI11/00679 and PI14/00447, integrated in the Plan Estatal I+D+I 2013-2016, and co-financed by the ISCIII-Subdirección General de Evaluación y Fomento de la investigación el Fondo Europeo de Desarrollo Regional (FEDER), by Centro de Investi‐ gación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM) and with the support of project number 2014\_SGR\_520 of the Department of Universities, Research and Information Society of the Government of Catalonia.

## **Author details**

Nonetheless, some reports showed that the mere hIAPP overproduction did not lead to amyloid formation or deposition despite elevated plasma concentration of hIAPP. Thus, other factors beyond overexpression had to be involved in the mechanism of islet amyloid formation since these mice were normoglycemic and normoinsulinemic (94–96). Islet amyloid was reported in transgenic mice fed a diet high in fat. Verchere et al. [18] have shown that approx‐ imately 80% of male transgenic mice (<13 months old) presented amyloid deposits and were always associated with severe hyperglycemia. In the case of hemizygous transgenic mice for hIAPP, the treatment with growth hormone or dexamethasone induced small intra- and

Another strategy used to overexpress hIAPP was to cross-bread hIAPP mice onto a mouse with obese background (ob/ob) [19] or obese Agouti viable yellow (Avy/Agouti) [93]. These mice developed amyloid formation and loss of β-cells, which was associated with progression

**Figure 4.** Amyloid formation in hIAPP transgenic mouse islets in obese Agouti yellow mice. Amyloid staining of hI‐ APP Tg mice in (A) FVB background, Avy/Agouti background at (B) 16 weeks of age and (C) 22 weeks of age. Note the presence of amyloid deposits in Avy/Agouti background as shown by Thioflavin S staining (unpublished data).

Moreover, it was found that female transgenic mice do not increase the occurrence of amyloid when oophorectomized, suggesting a protective role of ovarian hormones in islet amyloidosis. In recent years, Butler et al. showed that transgenic β-cell expression of human proIAPP in rats (HIP rats) that are homozygous for hIAPP develop diabetes within 5-10 months, together with the presence of extracellular amyloid, decreased β-cell mass, and increased β-cell apoptosis [94]. In addition, the loss of approximately 60% of β-cell mass at diabetes onset is comparable

In conclusion, with a variety of transgenic hIAPP models, it has been possible to clearly highlight that the process of islet amyloid formation is a complex event associated with a great

This work was supported by grants PI11/00679 and PI14/00447, integrated in the Plan Estatal I+D+I 2013-2016, and co-financed by the ISCIII-Subdirección General de Evaluación y Fomento de la investigación el Fondo Europeo de Desarrollo Regional (FEDER), by Centro de Investi‐

number of factors considered important in the pathogenesis of T2D.

extracellular amyloid deposits [92].

to the loss observed in humans with T2D [1].

**Acknowledgements**

of diabetes (**Figure 4**).

25412 Exploring New Findings on Amyloidosis

Joel Montane1,2 and Anna Novials1,2\*

\*Address all correspondence to: anovials@clinic.ub.es

1 The August Pi i Sunyer Biomedical Research Institute (IDIBAPS), Barcelona, Spain

2 Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBER‐ DEM), Barcelona, Spain

## **References**


hIAPP in type 2 diabetes mellitus. Exp Diabetes Res. 2008;2008: 697035. doi: 10.1155/2008/697035

[20] Novials a, Mato E, Lucas M, Franco C, Rivas M, Santisteban P, et al. Mutation at position-132 in the islet amyloid polypeptide (IAPP) gene promoter enhances basal transcriptional activity through a new CRE-like binding site. Diabetologia. 2004;47: 1167–1174. doi:10.1007/s00125-004-1439-y

[8] Marzban L, Trigo-Gonzalez G, Verchere CB. Processing of pro-islet amyloid polypep‐ tide in the constitutive and regulated secretory pathways of beta cells. Mol Endocrinol.

[9] Paulsson JF, Ludvigsson J, Carlsson A, Casas R, Forsander G, Ivarsson SA, et al. High plasma levels of islet amyloid polypeptide in young with new-onset of type 1 diabetes mellitus. PLoS One. 2014 Mar 26;9(3):e93053. doi:10.1371/journal.pone.0093053

[10] Higham CE, Hull RL, Lawrie L, Shennan KIJ, Morris JF, Birch NP, et al. Processing of synthetic pro-islet amyloid polypeptide (proIAPP) "amylin" by recombinant prohor‐ mone convertase enzymes, PC2 and PC3, in vitro. Eur J Biochem. 2000;267: 4998–5004.

[11] Harris PJ, Cooper ME, Hiranyachattada S, Berka JL, Kelly DJ, Nobes M, et al. Amylin stimulates proximal tubular sodium transport and cell proliferation in the rat kidney.

[12] Dacquin R, Davey RA, Laplace C, Levasseur R, Morris HA, Goldring SR, et al. Amylin inhibits bone resorption while the calcitonin receptor controls bone formation in vivo.

[13] Chin SY, Hall JM, Brain SD, Morton IK. Vasodilator responses to calcitonin gene-related peptide (CGRP) and amylin in the rat isolated perfused kidney are mediated via CGRP1

[14] Ratner RE, Dickey R, Fineman M, Maggs DG, Shen L, Strobel S a, et al. Amylin replacement with pramlintide as an adjunct to insulin therapy improves long-term glycaemic and weight control in type 1 diabetes mellitus: a 1-year, randomized controlled trial. Diabet Med. 2004;21: 1204–1212. doi:10.1111/j.1464-5491.2004.01319.x

[15] Lebovitz HE. Adjunct therapy for type 1 diabetes mellitus. Nat Rev Endocrinol. 2010;6:

[16] Roth JD, Roland BL, Cole RL, Trevaskis JL, Weyer C, Koda JE, et al. Leptin responsive‐ ness restored by amylin agonism in diet-induced obesity: evidence from nonclinical and clinical studies. Proc Natl Acad Sci USA. 2008;105: 7257–7262. doi:10.1073/pnas.

[17] Marzban L, Park K, Verchere CB. Islet amyloid polypeptide and type 2 diabetes. Exp

[18] Verchere CB, D'Alessio D a, Palmiter RD, Weir GC, Bonner-Weir S, Baskin DG, et al. Islet amyloid formation associated with hyperglycemia in transgenic mice with pancreatic beta cell expression of human islet amyloid polypeptide. Proc Natl Acad Sci

[19] Höppener JWM, Jacobs HM, Wierup N, Sotthewes G, Sprong M, de Vos P, et al. Human islet amyloid polypeptide transgenic mice: in vivo and ex vivo models for the role of

Gerontol. 2003;Apr;38(4):347–351. doi:10.1016/S0531-5565(03)00004-4

USA. 1996;93: 3492–3496. doi:10.1073/pnas.93.8.3492

2005;19: 2154–2163. doi:10.1210/me.2004-0407

25614 Exploring New Findings on Amyloidosis

doi:10.1046/j.1432-1327.2000.01548.x

326–34. doi:10.1038/nrendo.2010.49

0706473105

Am J Physiol Ren Physiol. 1997;272: F13–F21.

J Cell Biol. 2004;164: 509–514. doi:10.1083/jcb.200312135

receptors. J Pharmacol Exp Ther. 1994;269: 989–992.


[42] Feng Y, Wang X ping, Yang S gao, Wang Y jiong, Zhang X, Du X ting, et al. Resveratrol inhibits beta-amyloid oligomeric cytotoxicity but does not prevent oligomer formation. Neurotoxicology. 2009;30: 986–995. doi:10.1016/j.neuro.2009.08.013

[31] Green JD, Goldsbury C, Kistler J, Cooper GJS, Aebi U. Human amylin oligomer growth and fibril elongation define two distinct phases in amyloid formation. J Biol Chem.

[32] Lorenzo a, Razzaboni B, Weir GC, Yankner B a. Pancreatic islet cell toxicity of amylin associated with type-2 diabetes mellitus. Nature. 1994;368: 756–760. doi:

[33] Makin OS, Serpell LC. Structural characterisation of islet amyloid polypeptide fibrils.

[34] Bailey J, Potter KJ, Verchere CB, Edelstein-Keshet L, Coombs D. Reverse engineering an amyloid aggregation pathway with dimensional analysis and scaling. Phys Biol.

[35] Abedini A, Schmidt AM. Mechanisms of islet amyloidosis toxicity in type 2 diabetes.

[36] Soty M, Visa M, Soriano S, Carmona MDC, Nadal Á, Novials A. Involvement of ATPsensitive potassium (K(ATP)) channels in the loss of beta-cell function induced by human islet amyloid polypeptide. J Biol Chem. 2011;286: 40857–40866. doi:10.1074/

[37] Masters SL, Dunne A, Subramanian SL, Hull RL, Tannahill GM, Sharp FA, et al. Activation of the NLRP3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced IL-1β in type 2 diabetes. Nat Immunol. 2010;11: 897–904. doi:

[38] Westwell-Roper C, Dai DL, Soukhatcheva G, Potter KJ, van Rooijen N, Ehses JA, et al. IL-1 Blockade attenuates islet amyloid polypeptide-induced proinflammatory cytokine release and pancreaticislet graft dysfunction. J Immunol. 2011;187: 2755–2765. doi:

[39] Huang CJ, Lin CY, Haataja L, Gurlo T, Butler AE, Rizza RA, et al. High expression rates of human islet amyloid polypeptide induce endoplasmic reticulum stress mediated beta-cell apoptosis, a characteristic of humans with type 2 but not type 1 diabetes.

[40] Casas S, Gomis R, Gribble FM, Altirriba J, Knuutila S, Novials A. Impairment of the ubiquitin-proteasome pathway is a downstream endoplasmic reticulum stress re‐ sponse induced by extracellular human islet amyloid polypeptide and contributes to pancreatic beta-cell apoptosis. Diabetes. 2007;56: 2284–2294. doi:10.2337/db07-0178

[41] Potter KJ, Scrocchi LA, Warnock GL, Ao Z, Younker MA, Rosenberg L, et al. Amyloid inhibitors enhance survival of cultured human islets. Biochim Biophys Acta Gen Subj.

FEBS Lett. 2013;Apr 17;587(8):1119–1127. doi:10.1016/j.febslet.2013.01.017

2004;279: 12206–12212. doi:10.1074/jbc.M312452200

J Mol Biol. 2004;335: 1279–1288. doi:10.1016/j.jmb.2003.11.048

2011;Dec;8(6) 066009. doi:10.1088/1478-3975/8/6/066009

Diabetes. 2007;56: 2016–2027. doi:10.2337/db07-0197

2009;1790: 566–574. doi:10.1016/j.bbagen.2009.02.013

10.1038/368756a0

25816 Exploring New Findings on Amyloidosis

jbc.M111.232801

10.1038/ni.1935

10.4049/jimmunol.1002854


[67] Montane J, Cadavez L, Novials A. Stress and the inflammatory process: a major cause of pancreatic cell death in type 2 diabetes. Diabetes Metab Syndr Obes. 2014;Feb 3;7:25– 34. doi:10.2147/DMSO.S37649

[55] Hetz C, Chevet E, Harding HP. Targeting the unfolded protein response in disease. Nat

[56] Eizirik DL, Cardozo AK, Cnop M. The role for endoplasmic reticulum stress in diabetes

[57] Nadanaka S, Okada T, Yoshida H, Mori K. Role of disulfide bridges formed in the luminal domain of ATF6 in sensing endoplasmic reticulum stress. Mol Cell Biol.

[58] Shen J, Chen X, Hendershot L, Prywes R. ER stress regulation of ATF6 localization by dissociation of BiP/GRP78 binding and unmasking of Golgi localization signals. Dev

[59] Oslowski CM, Hara T, O'Sullivan-Murphy B, Kanekura K, Lu S, Hara M, et al. Thio‐ redoxin-interacting protein mediates ER stress-induced β cell death through initiation of the inflammasome. Cell Metab. 2012;16: 265–273. doi:10.1016/j.cmet.2012.07.005 [60] Hotamisligil GS. Endoplasmic reticulum stress and the inflammatory basis of metabolic

[61] Oyadomari S, Mori M. Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell

[62] Morishima N, Nakanishi K, Takenouchi H, Shibata T, Yasuhiko Y. An endoplasmic reticulum stress-specific caspase cascade in apoptosis. Cytochrome c-independent activation of caspase-9 by caspase-12. J Biol Chem. 2002;277: 34287–34294. doi:10.1074/

[63] Potter KJ, Abedini A, Marek P, Klimek AM, Butterworth S, Driscoll M, et al. Islet amyloid deposition limits the viability of human islet grafts but not porcine islet grafts.

[64] Andersson A, Bohman S, Borg LA, Paulsson JF, Schultz SW, Westermark GT, et al. Amyloid deposition in transplanted human pancreatic islets: a conceivable cause of their long-term failure. Exp Diabetes Res. 2008;2008: 562985. doi:10.1155/2008/562985

[65] Casas S, Novials A, Reimann F, Gomis R, Gribble FM. Calcium elevation in mouse pancreatic beta cells evoked by extracellular human islet amyloid polypeptide involves activation of the mechanosensitive ion channel TRPV4. Diabetologia. 2008;51: 2252–

[66] Hull RL, Zraika S, Udayasankar J, Aston-Mourney K, Subramanian SL, Kahn SE. Amyloid formation in human IAPP transgenic mouse islets and pancreas, and human pancreas, is not associated with endoplasmic reticulum stress. Diabetologia. 2009;52:

Proc Natl Acad Sci USA. 2010;107: 4305–4310. doi:10.1073/pnas.0909024107

disease. Cell. 2010;Mar 19;140(6):900–917. doi:10.1016/j.cell.2010.02.034

mellitus. Endocr Rev. 2008;Feb;29(1):42–61. doi:10.1210/er.2007-0015

Rev Drug Discov. 2013;12: 703–19. doi:10.1038/nrd3976

Cell. 2002;3: 99–111. doi:10.1016/S1534-5807(02)00203-4

Death Differ. 2004;11: 381–389. doi:10.1038/sj.cdd.4401373

jbc.M204973200

26018 Exploring New Findings on Amyloidosis

2262. doi:10.1007/s00125-008-1111-z

1102–1111. doi:10.1007/s00125-009-1329-4

2007;27: 1027–1043. doi:10.1128/MCB.00408-06


human islet amyloid polypeptide. Mol Cell Endocrinol. 2016;420: 57–65. doi:10.1016/ j.mce.2015.11.018

[91] Matveyenko A V, Butler PC. Islet amyloid polypeptide (IAPP) transgenic rodents as models for type 2 diabetes. ILAR J. 2006;47: 225–233.

[79] Saafi EL, Konarkowska B, Zhang S, Kistler J, Cooper GJ. Ultrastructural evidence that apoptosis is the mechanism by which human amylin evokes death in RINm5F pancre‐

[80] Macgibbon GA, Cooper GJS, Dragunow M. Acute application of human amylin, unlike beta-amyloid peptides, kills undifferentiated pc12 cells by apoptosis. Neuroreport.

[81] Konarkowska B, Aitken JF, Kistler J, Zhang S, Cooper GJS. The aggregation potential of human amylin determines its cytotoxicity towards islet beta-cells. FEBS J. 2006;273:

[82] Goldsbury C, Goldie K, Pellaud J, Seelig J, Frey P, Müller SA, et al. Amyloid fibril formation from full-length and fragments of amylin. J Struct Biol. 2000;130: 352–362.

[83] Aitken JF, Loomes KM, Scott DW, Reddy S, Phillips ARJ, Prijic G, et al. Tetracycline treatment retards the onset and slows the progression of diabetes in human amylin/ islet amyloid polypeptide transgenic mice. Diabetes. 2010;59: 161–171. doi:10.2337/

[84] O'Brien TD, Butler PC, Kreutter DK, Kane LA, Eberhardt NL. Human islet amyloid polypeptide expression in COS-1 cells. A model of intracellular amyloidogenesis. Am

[85] Hiddinga HJ, Eberhardt NL. Intracellular amyloidogenesis by human islet amyloid polypeptide induces apoptosis in COS-1 cells. Am J Pathol. 1999;154: 1077–1088. doi:

[86] Lopest DHJ, Colin C, Degaki TL, De Sousa AC V, Vieira MNN, Sebollela A, et al. Amyloidogenicity and cytotoxicity of recombinant mature human islet amyloid polypeptide (rhIAPP). J Biol Chem. 2004;279: 42803–42810. doi:10.1074/jbc.M406108200

[87] Jyoti S, Satendra S, Sushma S, Anjana T, Shashi S. Antistressor activity of *Ocimum sanctum* (Tulsi) against experimentally induced oxidative stress in rabbits. Methods Find Exp Clin Pharmacol. 2007;29: 411–416. doi:1118135 [pii]\r10.1358/mf.

[88] Marzban L, Rhodes CJ, Steiner DF, Haataja L, Halban PA, Verchere CB. Impaired NH2 terminal processing of human proislet amyloid polypeptide by the prohormone convertase PC2 leads to amyloid formation and cell death. Diabetes. 2006;55: 2192–2201.

[89] Cadavez L, Montane J, Alcarraz-Vizán G, Visa M, Vidal-Fàbrega L, Servitja JM, et al. Chaperones ameliorate beta cell dysfunction associated with human islet amyloid polypeptide overexpression. PLoS One. 2014;9: 1–11. doi:10.1371/journal.pone.0101797

[90] Montane J, de Pablo S, Obach M, Cadavez L, Castaño C, Alcarraz-Vizán G, et al. Protein disulfide isomerase ameliorates β-cell dysfunction in pancreatic islets overexpressing

atic islet beta-cells. Cell Biol Int. 2001;25: 339–50. doi:10.1006/cbir.2000.0643

1997;8: 3945–3949.

26220 Exploring New Findings on Amyloidosis

doi:10.1006/jsbi.2000.4268

J Pathol. 1995;147: 609–616.

10.1016/S0002-9440(10)65360-6

2007.29.6.1118135

doi:10.2337/db05-1566

db09-0548

3614–3624. doi:10.1111/j.1742-4658.2006.05367.x


## **Proteasome Inhibitors to Treat AL Amyloidosis**

James J. Driscoll and Saulius Girnius

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63467

#### **Abstract**

Amyloidoses represent a highly heterogeneous group of diseases characterized by the abnormal production and accumulation of abnormal, insoluble amyloid proteins in various tissues leading to organ dysfunction. Light‐chain (AL) amyloidosis is the most common form of systemic amyloidosis and is characterized by extracellular deposi‐ tion of pathologic insoluble fibrillar proteins in organs and tissues. Primary systemic AL amyloidosis (AL) arises from the production of abnormal immunoglobulins (Igs) by clonal plasma cells, such as those associated with the plasma cell dyscrasia multiple myeloma (MM). AL amyloidosis can affect a wide range of organs, most commonly the kidneys, and consequently presents with a range of symptoms. Currently, the most effective treatment is autologous bone marrow transplants with stem cell rescue, but many patients are too weak to tolerate this approach and are ineligible. Novel therapeutic strategies recently used include forms of chemotherapy and targeted therapy similar to those used to treat MM. As a single agent, the proteasome inhibitor bortezomib has notable activity in selected populations of patients with relapsed AL. Here, we discuss recent advances using proteasome inhibitors to improve the outcome of AL amyloidosis patients.

**Keywords:** Amyloid, light chain, plasma cell, proteasome inhibitor, bortezomib

## **1. Introduction**

Light‐chain amyloidosis (AL) is a dyscrasia of clonal origin that results in amyloid fibril deposition within vital organs leading to their progressive dysfunction and ultimately death. The precise molecular events that lead to AL amyloidosis are poorly understood and treat‐ ment options based upon the biology of disease that improve patient survival are limited. AL amyloidosis is frequently a challenge to diagnose because of its broad spectrum of symptoms. Clinical manifestations include nephrotic‐range proteinuria, hepatomegaly, congestive heart

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

failure (CHF), and autonomic and sensory neuropathy. Diagnostic advances include develop‐ mentofaserum‐freelight‐chainassay,cardiacmagneticresonanceimaging(MRI),andserologic cardiac biomarkers. Treatment advances include the inclusion of the proteasome inhibitor bortezomib. Here, we discuss current and emerging treatment strategies, many focused on proteasomeinhibitors,thathaveevolvedorareevolvingtoprolongsurvivalandpreserveorgan function in patients with this disease. Finally, we discuss emerging strategies designed to eradicate the clonal cell of origin that may provide further clinical benefit for AL amyloidosis patients.

## **2. Targeting the ubiquitin + proteasome system in AL amyloidosis**

Protein degradation is a highly complex, temporally controlled, highly regulated process that maintains proteostasis in eukaryotic cells [1–5]. In normal and transformed cells, protein degradation pathways fulfill an essential role to maintain many critical pro‐survival path‐ ways [1]. Studies by Schoenheimer [1] in the 1930s described the dynamic turnover of individual cellular proteins. Subsequently, it was shown that protein half‐lives required energy, in the form of ATP, and that the half‐lives of individual proteins varied widely within mammalian cells2 . Previously, the lysosome had been considered the central site of intracel‐ lular proteolysis [3]. Discovery of the small polypeptide protein ubiquitin (Ub) followed as well as experiments demonstrating that Ub is covalently conjugated to target proteins to direct their proteasomal degradation then greatly advanced understanding protein degradation (**Figure 1**) [4, 5]. Ub is covalently linked to protein targets in three sequential steps to target proteins to direct their rapid, ATP‐dependent proteasomal degradation. In the first step, Ub is activated by an enzyme referred to as E1. Ub is then transferred from E1 to an E2 Ub‐ conjugating enzyme, and an isopeptide bond is formed between a lysine residue on the substrate target and the carboxy‐terminus glycine of the Ub moiety. E3 Ub protein ligases then recognize Ub‐conjugated target proteins [4, 5].

The vast majority of intracellular proteins are degraded by proteasomes in eukaryotic cells. The 26S proteasome is high‐molecular‐weight, ATP‐dependent structure that consists of a 20S catalytic core particle (CP) capped at the ends by 19S regulatory particles (RP) (**Figure 2**) [6–10]. The proteasome serves as the catalytic core of the Ub‐proteasome system (UPS) to degrade short‐lived and denatured proteins and was the first component of the Ub‐protea‐ some pathway to be targeted therapeutically. Bortezomib is a selective, boron‐containing reversible inhibitor of the proteasome that induces apoptosis in a number of different cancer cells. Bortezomib is a potent small molecule that binds reversibly to the proteasome β‐5 subunit to inhibit the chymotryptic‐like (Ct‐L) activity (**Figure 3**). The anti‐tumor effect of bortezo‐ mib was evident in a multitude of cell lines and xenograft models from different cancer types [11–15], including malignant plasma cells. Bortezomib has demonstrated substantial benefit in monotherapy or in combinations that induce chemo‐ or radiosensitization [11]. Federal Drug Administration (FDA)‐approval of bortezomib (Velcade, Millennium‐Takeda Oncology Co., Cambridge, MA) represented a major advance in the treatment of MM [11, 12]. Bortezomib is the first proteasome inhibitor to change the natural history of a hematologic malignancy. However, clinical efficacy in the treatment of solid tumors has not been achieved.

failure (CHF), and autonomic and sensory neuropathy. Diagnostic advances include develop‐ mentofaserum‐freelight‐chainassay,cardiacmagneticresonanceimaging(MRI),andserologic cardiac biomarkers. Treatment advances include the inclusion of the proteasome inhibitor bortezomib. Here, we discuss current and emerging treatment strategies, many focused on proteasomeinhibitors,thathaveevolvedorareevolvingtoprolongsurvivalandpreserveorgan function in patients with this disease. Finally, we discuss emerging strategies designed to eradicate the clonal cell of origin that may provide further clinical benefit for AL amyloidosis

**2. Targeting the ubiquitin + proteasome system in AL amyloidosis**

Protein degradation is a highly complex, temporally controlled, highly regulated process that maintains proteostasis in eukaryotic cells [1–5]. In normal and transformed cells, protein degradation pathways fulfill an essential role to maintain many critical pro‐survival path‐ ways [1]. Studies by Schoenheimer [1] in the 1930s described the dynamic turnover of individual cellular proteins. Subsequently, it was shown that protein half‐lives required energy, in the form of ATP, and that the half‐lives of individual proteins varied widely within

lular proteolysis [3]. Discovery of the small polypeptide protein ubiquitin (Ub) followed as well as experiments demonstrating that Ub is covalently conjugated to target proteins to direct their proteasomal degradation then greatly advanced understanding protein degradation (**Figure 1**) [4, 5]. Ub is covalently linked to protein targets in three sequential steps to target proteins to direct their rapid, ATP‐dependent proteasomal degradation. In the first step, Ub is activated by an enzyme referred to as E1. Ub is then transferred from E1 to an E2 Ub‐ conjugating enzyme, and an isopeptide bond is formed between a lysine residue on the substrate target and the carboxy‐terminus glycine of the Ub moiety. E3 Ub protein ligases then

The vast majority of intracellular proteins are degraded by proteasomes in eukaryotic cells. The 26S proteasome is high‐molecular‐weight, ATP‐dependent structure that consists of a 20S catalytic core particle (CP) capped at the ends by 19S regulatory particles (RP) (**Figure 2**) [6–10]. The proteasome serves as the catalytic core of the Ub‐proteasome system (UPS) to degrade short‐lived and denatured proteins and was the first component of the Ub‐protea‐ some pathway to be targeted therapeutically. Bortezomib is a selective, boron‐containing reversible inhibitor of the proteasome that induces apoptosis in a number of different cancer cells. Bortezomib is a potent small molecule that binds reversibly to the proteasome β‐5 subunit to inhibit the chymotryptic‐like (Ct‐L) activity (**Figure 3**). The anti‐tumor effect of bortezo‐ mib was evident in a multitude of cell lines and xenograft models from different cancer types [11–15], including malignant plasma cells. Bortezomib has demonstrated substantial benefit in monotherapy or in combinations that induce chemo‐ or radiosensitization [11]. Federal Drug Administration (FDA)‐approval of bortezomib (Velcade, Millennium‐Takeda Oncology Co., Cambridge, MA) represented a major advance in the treatment of MM [11, 12].

. Previously, the lysosome had been considered the central site of intracel‐

patients.

266 268Exploring New Findings on Amyloidosis

mammalian cells2

recognize Ub‐conjugated target proteins [4, 5].

**Figure 1.** Schematic representation of the Ub + proteasome system. The UPS pathway for protein degradation in eu‐ karyotic cells comprises: (1) a series of enzymes [E1, activation; E2, conjugation; E3, ligation] that covalently modify proteins with a polyubiquitin tag for recognition and targeted degradation and (2) the 26S proteasome, a 2 MDa multi‐ catalytic enzyme complex that hydrolyzes the polyubiquitin‐tagged proteins into short polypeptides, typically seven to nine amino acids in length. The proteasome degrades unwanted proteins by recognizing specific polyubiquitin tags covalently attached to these proteins. Inhibition of the proteasome catalytic core by bortezomib leads to the unwanted accumulation of ubiquitinated proteins and culminates in apoptosis.

**Figure 2.** Subunit organization of the 26S proteasome. The 26S proteasome is a eukaryotic ATP‐dependent, dumbbell‐ shaped protease complex with a molecular mass of approximately 2000 kDa. It consists of a central 20S proteasome, functioning as a catalytic machine, and two large V‐shaped terminal modules, having possible regulatory roles, com‐ posed of multiple subunits of 25–110 kDa attached to the central portion in opposite orientations. Shown are the 20S proteasome catalytic particles (CP) and the 19S regulatory particles (RP). The 19S regulator is bound to either one or both ends of the 20S proteasome and stimulates hydrolytic activity of the 20S proteasome. The 19S RP enables ATP‐ dependent degradation of ubiquitinated proteins and supports elevated peptidase activity but not ubiquitin‐conjugate degradation.

**Figure 3.** The 20S proteasome CP houses three pairs of catalytically active subunits, β1, β2, and β5, that exhibit protein substrate cleavage preferences referred to as caspase‐like (C‐L), trypsin‐like (T‐L), and chymotrypsin‐like (CT‐L), re‐ spectively, and which work in concert to degrade protein substrates. Substrate hydrolysis by the 20S CP commences with recognition of amino acid side chains by sequential binding pockets proximal to the proteolytic active site. Borte‐ zomib forms a reversible, non‐covalent adduct at the active site of the β‐5 subunit. Carfilzomib has irreversible binding properties for the same active site of the β‐5 subunit. Marizomib is a β‐lactone‐γ‐lactam that binds slowly with sus‐ tained inhibition of the proteasome β‐1, β‐2, and β‐5 active sites.

The aim of current treatment strategies for AL is to inhibit production of insoluble amyloido‐ genic immunoglobulin light‐chain fragments and ultimately restore organ function [16–18]. AL and MM are both clonal plasma cell dyscrasias and, therefore, AL treatment is typically based on therapies that have shown efficacy in MM [19, 20]. The depth of hematologic response and complete response (CR) has been linked with improved organ function in AL patients and improved overall survival (OS) in MM [21–23]. Intensive therapy with high‐dose melphalan and stem cell transplantation has been shown to be highly effective in AL, but a risk‐adapt‐ ed strategy may be needed to reduce mortality and toxicity [24, 25]. In addition, similarto MM, side effects with treatment are evident and drug resistance emerges even in patients that do respond initially.

Second‐generation proteasome inhibitors offer a number of potential advantages over bortezomib. The newer proteasome inhibitors may provide improved target specificity, safety, tolerability, and the capacity to overcome bortezomib resistance (**Table 1**). Second‐genera‐ tion proteasome inhibitors include the recently FDA‐approved epoxyketone carfilzomib [26], and agents in clinical development that include ONX‐0912 [27], Ixazomib (Ninlaro) [28] (Millennium‐Takeda), Marizomib [29] (NPI‐0052), and CEP‐18870 [30] (Cephalon). In contrast to bortezomib, carfilzomib is an irreversible proteasome inhibitor. Ixazomib is an orally bioavailable reversible proteasome inhibitorthat is immediately hydrolyzed to its active form, MLN2238 after conversion in aqueous solutions. Ixazomib binds preferentially to the protea‐ some β‐5 active site to inhibit the chymotrypsin‐like activity. Ixazomib and CEP‐18770 are reversible inhibitors of the proteasome Ct‐L activity that exhibit inhibitory activity compara‐ ble to bortezomib. The differences improved anti‐tumor activity.


**Table 1.** Agents to target the proteolytic activities within the proteasome complex.

**Figure 3.** The 20S proteasome CP houses three pairs of catalytically active subunits, β1, β2, and β5, that exhibit protein substrate cleavage preferences referred to as caspase‐like (C‐L), trypsin‐like (T‐L), and chymotrypsin‐like (CT‐L), re‐ spectively, and which work in concert to degrade protein substrates. Substrate hydrolysis by the 20S CP commences with recognition of amino acid side chains by sequential binding pockets proximal to the proteolytic active site. Borte‐ zomib forms a reversible, non‐covalent adduct at the active site of the β‐5 subunit. Carfilzomib has irreversible binding properties for the same active site of the β‐5 subunit. Marizomib is a β‐lactone‐γ‐lactam that binds slowly with sus‐

The aim of current treatment strategies for AL is to inhibit production of insoluble amyloido‐ genic immunoglobulin light‐chain fragments and ultimately restore organ function [16–18]. AL and MM are both clonal plasma cell dyscrasias and, therefore, AL treatment is typically based on therapies that have shown efficacy in MM [19, 20]. The depth of hematologic response and complete response (CR) has been linked with improved organ function in AL patients and improved overall survival (OS) in MM [21–23]. Intensive therapy with high‐dose melphalan and stem cell transplantation has been shown to be highly effective in AL, but a risk‐adapt‐ ed strategy may be needed to reduce mortality and toxicity [24, 25]. In addition, similarto MM, side effects with treatment are evident and drug resistance emerges even in patients that do

Second‐generation proteasome inhibitors offer a number of potential advantages over bortezomib. The newer proteasome inhibitors may provide improved target specificity, safety, tolerability, and the capacity to overcome bortezomib resistance (**Table 1**). Second‐genera‐ tion proteasome inhibitors include the recently FDA‐approved epoxyketone carfilzomib [26], and agents in clinical development that include ONX‐0912 [27], Ixazomib (Ninlaro) [28] (Millennium‐Takeda), Marizomib [29] (NPI‐0052), and CEP‐18870 [30] (Cephalon). In contrast to bortezomib, carfilzomib is an irreversible proteasome inhibitor. Ixazomib is an orally bioavailable reversible proteasome inhibitorthat is immediately hydrolyzed to its active form, MLN2238 after conversion in aqueous solutions. Ixazomib binds preferentially to the protea‐ some β‐5 active site to inhibit the chymotrypsin‐like activity. Ixazomib and CEP‐18770 are reversible inhibitors of the proteasome Ct‐L activity that exhibit inhibitory activity compara‐

ble to bortezomib. The differences improved anti‐tumor activity.

tained inhibition of the proteasome β‐1, β‐2, and β‐5 active sites.

respond initially.

268 270Exploring New Findings on Amyloidosis

Carfilzomib is a tetrapeptide epoxyketone that also irreversibly inhibits the Ct‐L activity of proteasomes [31–36]. Carfilzomib yields a more sustained inhibition compared to bortezo‐ mib and has been shown to promote cell death in bortezomib‐resistant cells. ONX‐0912 is an orally bioavailable analog of carfilzomib that has been investigated in early phase trials to study the effect on solid tumors. ONX‐0912 irreversibly inhibits β‐5 activity and in xenograft models was shown to reduce tumor growth and prolong survival. ONX‐0914 is an immuno‐ proteasome‐specific inhibitor with potential to treat both cancers and autoimmune diseases. Marizomib (NPI‐0052) irreversibly inhibits the β‐5 subunit and is a natural β‐lactone derived from *Salinospora tropica.* Marizomib inhibits the major catalytic activities (Ct‐L, tryptic‐like, and caspase‐like) of proteasomes which may yield a long‐term benefit to preclude resistance (**Figure 3**). Thus, the benefit of second‐generation proteasome inhibitors may be due to their ability to act as irreversible inhibitors and to inhibit multiple active sites within the proteasome.

## **3. Clinical evaluation of proteasome inhibitors for AL amyloidosis treatment**

Intensive therapy with high‐dose melphalan (a chemotherapy drug belonging to the class of nitrogen mustard alkylating agents) and stem cell transplantation (SCT) have been com‐ bined as a regimen (MEL‐SCT) and used as an effective therapy in AL amyloidosis. MEL‐SCT is used as a risk‐adapted approach that is necessary to minimize treatment‐related mortality (TRM). Therefore, up to 82% of persons with AL amyloidosis may be ineligible for MEL‐SCT based upon age, performance status, and severity of cardiac involvement. Wechalekar et al. [37] reported preliminary observations with bortezomib and demonstrated its effectiveness in a heavily pre‐treated population, with an 80% hematologic overall response (OR) rate, including 15% hematologic CR rates. A subsequent phase I/II study of single agent bortezo‐ mib in relapsed/refractory aL amyloidosis showed excellent tolerance to both once‐ and twice‐ weekly [38]. Importantly, the median time to response in the twice‐weekly arm was only 0.7 months. One‐year hematologic response rates were 72.2 and 74.6%, one‐year response rates were 93.8 and 84.0%, and one‐third of patients exhibited a CR. The median OS was 61.1 months [39]. Rates of neuropathy were as high as 35% in the twice‐weekly, with 9% rate of discontinuation and 6% rate of dose reductions.

Deregulating the ubiquitin‐proteasome pathway may affect the heart, causing plaque instability, altered intracellular signal transduction resulting in decreased myocardial cytoprotection, desensitization of adrenergic receptors, and accumulation of unfolded proteins impairing cardiac function [40]. Although cardiac toxicity was described in small series, case reports, and clinical trials, subsequent meta‐analysis did not support an increase in cardio‐ toxicity in multiple myeloma (MM) [41]. Early sudden death has been described in amyloid cardiomyopathy in persons treated with bortezomib. The Greek and UK groups demonstrat‐ ed increased rates of early cardiac death in those treated with bortezomib, when compared to lenalidomide [42, 43]. However, sudden death is not uncommon in advanced amyloid cardiomyopathy. Since bortezomib typically has a shorter time to hematologic response, it is used more frequently than lenalidomide in advanced cardiac amyloidosis. Therefore, increased rates of sudden death may be related to patient selection, rather than a class effect from proteasome inhibitors.

Since bortezomib demonstrated such great promise, several studies explored the clinical efficacy in triplets, including cyclophosphamide, bortezomib, and dexamethasone (CyBorD). These retrospective studies reported on 17 and 43 patients, with hematologic response rates of 81–94% and hematologic CR rates of 39.5–71.5% [44, 45]. At least half of the patients had symptomatic cardiac involvement and many had a cardiac‐specific response. However, a retrospective study from the UK demonstrated poorer outcomes than the two aforemen‐ tioned studies [46]. Of 230 patients, a hematologic response rate was 60%, which decreased to 42% in those with advanced cardiac stage III patients. Cardiac response was achieved in only 17% of patients. Unfortunately, CyBorD is associated with grade 3 and grade 4 toxici‐ ties in 50% of patients.

Bortezomib has been combined with melphalan and dexamethasone, possibly with more promising outcomes than CyBorD (**Table 2**). A prospective, multi‐center phase II trial of bortezomib, melphalan, and dexamethasone (VMD) in newly diagnosed or relapsed AL amyloidosis showed hematologic response rates of 94%, with CR of 38%. However, this was a small trial and 52% of patients required dose reduction despite an excellent performance status, questioning whether such an impressive response can be replicated off trial [47]. A subsequent phase I trial demonstrated improved safety and greater tolerability [48]. Using prospectively collected data, the Pavia group demonstrated higher response rates with VMD,

when compared to CyBorD (p=0.033) or melphalan/dexamethasone alone (*p* = 0.010) [49]. While the exposure to upfront bortezomib is associated with longer OS, a difference be‐ tween CyBorD and VMD could not be detected. A bortezomib/dexamethasone backbone is necessary in persons ineligible for melphalan‐stem cell transplantation (MEL‐SCT), and it is not clear whether melphalan or cyclophosphamide should be added. The addition of melpha‐ lan is supported with more prospective data, but higher cumulative doses are associated with leukemia, and melphalan should be avoided in persons who are borderline candidates for autologous stem cell transplantation.

(TRM). Therefore, up to 82% of persons with AL amyloidosis may be ineligible for MEL‐SCT based upon age, performance status, and severity of cardiac involvement. Wechalekar et al. [37] reported preliminary observations with bortezomib and demonstrated its effectiveness in a heavily pre‐treated population, with an 80% hematologic overall response (OR) rate, including 15% hematologic CR rates. A subsequent phase I/II study of single agent bortezo‐ mib in relapsed/refractory aL amyloidosis showed excellent tolerance to both once‐ and twice‐ weekly [38]. Importantly, the median time to response in the twice‐weekly arm was only 0.7 months. One‐year hematologic response rates were 72.2 and 74.6%, one‐year response rates were 93.8 and 84.0%, and one‐third of patients exhibited a CR. The median OS was 61.1 months [39]. Rates of neuropathy were as high as 35% in the twice‐weekly, with 9% rate of

Deregulating the ubiquitin‐proteasome pathway may affect the heart, causing plaque instability, altered intracellular signal transduction resulting in decreased myocardial cytoprotection, desensitization of adrenergic receptors, and accumulation of unfolded proteins impairing cardiac function [40]. Although cardiac toxicity was described in small series, case reports, and clinical trials, subsequent meta‐analysis did not support an increase in cardio‐ toxicity in multiple myeloma (MM) [41]. Early sudden death has been described in amyloid cardiomyopathy in persons treated with bortezomib. The Greek and UK groups demonstrat‐ ed increased rates of early cardiac death in those treated with bortezomib, when compared to lenalidomide [42, 43]. However, sudden death is not uncommon in advanced amyloid cardiomyopathy. Since bortezomib typically has a shorter time to hematologic response, it is used more frequently than lenalidomide in advanced cardiac amyloidosis. Therefore, increased rates of sudden death may be related to patient selection, rather than a class effect

Since bortezomib demonstrated such great promise, several studies explored the clinical efficacy in triplets, including cyclophosphamide, bortezomib, and dexamethasone (CyBorD). These retrospective studies reported on 17 and 43 patients, with hematologic response rates of 81–94% and hematologic CR rates of 39.5–71.5% [44, 45]. At least half of the patients had symptomatic cardiac involvement and many had a cardiac‐specific response. However, a retrospective study from the UK demonstrated poorer outcomes than the two aforemen‐ tioned studies [46]. Of 230 patients, a hematologic response rate was 60%, which decreased to 42% in those with advanced cardiac stage III patients. Cardiac response was achieved in only 17% of patients. Unfortunately, CyBorD is associated with grade 3 and grade 4 toxici‐

Bortezomib has been combined with melphalan and dexamethasone, possibly with more promising outcomes than CyBorD (**Table 2**). A prospective, multi‐center phase II trial of bortezomib, melphalan, and dexamethasone (VMD) in newly diagnosed or relapsed AL amyloidosis showed hematologic response rates of 94%, with CR of 38%. However, this was a small trial and 52% of patients required dose reduction despite an excellent performance status, questioning whether such an impressive response can be replicated off trial [47]. A subsequent phase I trial demonstrated improved safety and greater tolerability [48]. Using prospectively collected data, the Pavia group demonstrated higher response rates with VMD,

discontinuation and 6% rate of dose reductions.

from proteasome inhibitors.

270 272Exploring New Findings on Amyloidosis

ties in 50% of patients.


AL = light chain; ORR = overall response rate; CR = hematologic complete response; BD = bortezomib/dexamethasone; ND = newly diagnosed; CyBorD = cyclophosphamide/bortezomib/dexamethasone; VMD= bortezomib/melphalan/ dexamethasone; ID= ixazomib/dexamethasone; KD= carfilzomib/dexamethasone

**Table 2.** Proteasome inhibitors used in treatment regimens for AL amyloidosis.

The use of ixazomib in a phase I study in relapsed/refractory AL amyloidosis was reported only in abstract form [50]. Of the 22 patients enrolled, 11 patients were previously treated with bortezomib and 15 with cardiac involvement. Seventy‐seven percent were able to reach maximum‐tolerated dose. A hematologic response was obtained in 53%, including 11% CR. An organ‐specific cardiac response was seen in three of nine patients. The 2‐year OS was 57%, up to 85% in proteasome inhibitor naïve patients. Tourmaline‐AL1, a phase III trial for ixazomib/dexamethasone vs. physician's choice is currently ongoing and accruing patients.

Carfilzomib is another second‐generation proteasome inhibitor that is being investigated as a potential treatment in AL amyloidosis. A phase I dose escalation study of carfilzomib in AL

amyloidosis in a bortezomib‐exposed population enrolled 12 patients [51]. A hematologic OR rate of 77% was reported and no dose‐related toxicity was noted in carfilzomib doses as high as 36 mg/m<sup>2</sup> . However, three significant cardiac events were noted, including ventricular tachycardia, grade 4 restrictive cardiomyopathy, and a grade 3 drop in the left ventricular ejection fraction. Although promising, second‐generation proteasome inhibitors may not provide significant long‐term benefit and overcome therapeutic resistance in refractory patients. Numerous mechanisms of resistance have been proposed, for example, mutations of PSMB5, upregulation of other proteasome subunits, alterations of gene and protein expres‐ sion in stress response pathways, induction of autophagy, and an increase in anti‐apoptotic pathways as well as multidrug resistance pathways. Hence, a number of novel therapeutic strategies for AL amyloidosis are under development [52, 53]. Currently, treatment choices remain highly individualized and are dependent on a careful assessment of performance status and organ function.

## **4. Targeting the clonal cell of origin as a treatment strategy for AL amyloidosis**

The incidence of AL amyloidosis is similar to that of Hodgkin's lymphoma and chronic myelogenous leukemia (CML). Approximately 5–12 individuals/million/year are affected, although autopsy studies suggest a higherincidence. Amyloidosis is a monoclonal plasma cell disorder in which the secreted monoclonal Ig protein forms insoluble fibrillar deposits in one or more organs. In nearly all cases, the deposits contain Ig light (L) chains or L‐chain frag‐ ments. AL is related to both MM and monoclonal gammopathy of undetermined signifi‐ cance (MGUS), a pre‐malignant condition that nearly uniformly precedes MM. These monoclonal plasma cell disorders are categorized according to the total body burden of monoclonal plasma cells. When this burden is large, the diagnostic criteria for MM are fulfilled; when this burden is lower, MGUS is diagnosed. The plasma cell burden is typically low at 5– 10%, and in ∼10–15% of patients, AL amyloidosis occurs in association with MM.

## **5. Concluding remarks**

While targeting proteostasis is a highly effective strategy to treat plasma cell dyscrasias such as AL, more effective agents are needed to improve organ dysfunction and advance patients to SCT. Moreover, similar to MM, high‐risk forms of disease exist which do not respond to bortezomib and for those that do respond drug resistance eventually emerges. While the explosion of novel agents with activity in MM holds promise for the care of patients with AL amyloidosis, a commitment specifically to the clinical investigation of AL amyloidosis is needed to improve patient outcomes. Therefore, there is an urgent and unmet need for more effective therapeutic agents based upon the biology of the disease that increase patient survival.

## **Author details**

amyloidosis in a bortezomib‐exposed population enrolled 12 patients [51]. A hematologic OR rate of 77% was reported and no dose‐related toxicity was noted in carfilzomib doses as high

tachycardia, grade 4 restrictive cardiomyopathy, and a grade 3 drop in the left ventricular ejection fraction. Although promising, second‐generation proteasome inhibitors may not provide significant long‐term benefit and overcome therapeutic resistance in refractory patients. Numerous mechanisms of resistance have been proposed, for example, mutations of PSMB5, upregulation of other proteasome subunits, alterations of gene and protein expres‐ sion in stress response pathways, induction of autophagy, and an increase in anti‐apoptotic pathways as well as multidrug resistance pathways. Hence, a number of novel therapeutic strategies for AL amyloidosis are under development [52, 53]. Currently, treatment choices remain highly individualized and are dependent on a careful assessment of performance status

**4. Targeting the clonal cell of origin as a treatment strategy for AL**

10%, and in ∼10–15% of patients, AL amyloidosis occurs in association with MM.

While targeting proteostasis is a highly effective strategy to treat plasma cell dyscrasias such as AL, more effective agents are needed to improve organ dysfunction and advance patients to SCT. Moreover, similar to MM, high‐risk forms of disease exist which do not respond to bortezomib and for those that do respond drug resistance eventually emerges. While the explosion of novel agents with activity in MM holds promise for the care of patients with AL amyloidosis, a commitment specifically to the clinical investigation of AL amyloidosis is needed to improve patient outcomes. Therefore, there is an urgent and unmet need for more effective therapeutic agents based upon the biology of the disease that increase patient

The incidence of AL amyloidosis is similar to that of Hodgkin's lymphoma and chronic myelogenous leukemia (CML). Approximately 5–12 individuals/million/year are affected, although autopsy studies suggest a higherincidence. Amyloidosis is a monoclonal plasma cell disorder in which the secreted monoclonal Ig protein forms insoluble fibrillar deposits in one or more organs. In nearly all cases, the deposits contain Ig light (L) chains or L‐chain frag‐ ments. AL is related to both MM and monoclonal gammopathy of undetermined signifi‐ cance (MGUS), a pre‐malignant condition that nearly uniformly precedes MM. These monoclonal plasma cell disorders are categorized according to the total body burden of monoclonal plasma cells. When this burden is large, the diagnostic criteria for MM are fulfilled; when this burden is lower, MGUS is diagnosed. The plasma cell burden is typically low at 5–

. However, three significant cardiac events were noted, including ventricular

as 36 mg/m<sup>2</sup>

272 274Exploring New Findings on Amyloidosis

and organ function.

**amyloidosis**

**5. Concluding remarks**

survival.

James J. Driscoll1,2\* and Saulius Girnius1,2

\*Address all correspondence to: driscojs@uc.edu

1 The Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, Cincinnati, OH, USA

2 Division of Hematology and Oncology, University of Cincinnati College of Medicine, Cin‐ cinnati, OH, USA

## **References**


[25] Gertz MA, Lacy MQ, Dispenzieri A. Myeloablative chemotherapy with stem cellrescue for the treatment of primary systemic amyloidosis: a status report. Bone Marrow Transpl. 2000;25(5):465–70.

[11] Chen D, Dou QP. The ubiquitin‐proteasome system as a prospective molecular target for cancer treatment and prevention. Curr. Protein Pept. Sci. 2010;11(6):459–70. [12] Orlowski RZ, Kuhn DJ. Proteasome inhibitors in cancer therapy: lessons from the first

[13] Nawrocki ST, Bruns CJ, Harbison MT, et al. Effects of the proteasome inhibitor PS‐341 on apoptosis and angiogenesis in orthotopic human pancreatic tumor xenografts. Mol.

[14] Williams S, Pettaway C, Song R. Differential effects of the proteasome inhibitor bortezomib on apoptosis and angiogenesis in human prostate tumor xenografts. Mol.

[15] Hideshima T, Richardson P, Chauhan D, et al. The proteasome inhibitor PS‐341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple

[16] Reece DE, Hegenbart U, Sanchorawala V, Merlini G, Palladini G, Blade J et al. Efficacy and safety of once‐weekly and twice‐weekly bortezomib in patients with relapsed systemic AL amyloidosis: results of a phase 1/2 study. Blood. 2011;118(4):865–73. [17] Comenzo RL. Managing systemic light‐chain amyloidosis. J. Natl. Compr. Cancer

[18] Guidelines Working Group of UK MM Forum, British Committee for Standards in Hematology of the British Society for Haematology. Guidelines on the diagnosis and

[19] Falk RH, Comenzo RL, Skinner M. The systemic amyloidoses. N. Engl. J. Med.

[20] Rosen PJ, Gordon M, Lee PN, Sausville E, Papadopoulos KP, Wong AF, J Clin Oncol

[21] Merlini G, Palladini G. Amyloidosis: is a cure possible? Ann. Oncol. 2008;19(suppl

[22] Gertz MA, Lacy MQ, Dispenzieri A, et al. Effect of hematologic response on outcome of patients undergoing transplantation for primary amyloidosis: importance of

[23] Jaccard A, Moreau P, Leblond V, et al. High‐dose melphalan versus melphalan plus dexamethasone for AL amyloidosis. N. Engl. J. Med. 2007; 357(11):1083–93.

[24] Skinner M, Sanchorawala V, Seldin DC, et al. High‐dose melphalan and autologous stem‐cell transplantation in patients with AL amyloidosis: an 8‐year study. Ann. Intern.

achieving a complete response. Haematologica. 2007;92(10):1415–18.

management of AL amyloidosis. Br. J. Haematol. 2004;125(6):681–700.

decade. Clin. Cancer Res. 2008;15;14(6):1649-57.

myeloma cells. Cancer Res. 2006;61:3071–76.

Cancer Ther. 2002;1:1243–53.

274 276Exploring New Findings on Amyloidosis

Cancer Ther. 2003;2:835–43.

Netw. 2007;5(2):179–87.

1997;337(13):898–909.

Med. 2004; 140(2):85–93.

4):iv63–66.

27:15s, 2009 (suppl; abstr 3515).


with relapsed or refractory systemic light‐chain (AL) amyloidosis (RRAL). American Society of Hematology. San Francisco, CA; 2014.

[51] AD C. A phase I dose‐escalation study of Carfilzomib in patients with previously‐ treated systemic light‐chain (AL) amyloidosis. American Society of Hematology. San Francisco, CA; 2014.

[38] Reece DE, Hegenbart U, Sanchorawala V, et al. Efficacy and safety of once‐weekly and twice‐weekly bortezomib in patients with relapsed systemic AL amyloidosis: results

[39] Reece DE, Hegenbart U, Sanchorawala V, et al. Long‐term follow‐up from a phase 1/2 study of single‐agent bortezomib in relapsed systemic AL amyloidosis. Blood.

[40] Orciuolo E, Buda G, Cecconi N, et al. Unexpected cardiotoxicity in haematological

[41] Xiao Y, Yin J, Wei J, Shang Z. Incidence and risk of cardiotoxicity associated with bortezomib in the treatment of cancer: a systematic review and meta‐analysis. PLoS

[42] Kastritis E. Outcomes of primary systemic light chain (AL) amyloidosis in patients treated upfront with bortezomib of lenalidomide and the importance of risk adapted strategies. XIVth International Symposium on Amyloidosis. Indianapolis, IN; 2014.

[43] Wechalekar AD. Characterisitcs and outcomes of 714 patients with systemic AL amyloidosis – analysis of prospective study (ALChemy Study). XIVth International

[44] Mikhael JR, Schuster SR, Jimenez‐Zepeda VH, et al. Cyclophosphamide‐bortezomib‐ dexamethasone (CyBorD) produces rapid and complete hematologic response in

[45] Venner CP, Lane T, Foard D, et al. Cyclophosphamide, bortezomib, and dexametha‐ sone therapy in AL amyloidosis is associated with high clonal response rates and

[46] Palladini G, Sachchithanantham S, Milani P, et al. A European collaborative study of cyclophosphamide, bortezomib, and dexamethasone in upfront treatment of system‐

[47] Zonder J. Melphalan and dexamethasone plus bortezomib induces hematologic and organ responses in AL‐amyloidosis with tolerable neurotoxicity. American Society of

[48] Shimazaki C, Fuchida S, Suzuki K, et al. Phase 1 study of bortezomib in combination with melphalan and dexamethasone in Japanese patients with relapsed AL amyloido‐

[49] Palladini G, Milani P, Riva E, et al: Accurate risk stratification identifies patients with AL amyloidosis benefiting most from upfront bortezomib combinations: A study of treatment outcomes in 984 patients. 57th American Society of Hematology Annual

[50] Merlini G. Long‐term outcome of a Phase 1 study of the investigational oral protea‐ some inhibitor (PI) ixazomib at the recommended phase 3 dose (RP3D) in patients (Pts)

Meeting and Exposition. Orlando, FL, December 5-8, 2015 (abstr 190).

bortezomib treated patients. Br. J. Haematol. 2007;138(3):396–97.

of a phase 1/2 study. Blood. 2011;118(4):865–73.

Symposium of Amyloidosis. Indianapolis, IN; 2014.

ic AL amyloidosis. Blood. 2015;126(5):612–15.

Hematology. New Orleans, LA; 2009.

sis. Int. J. Hematol. 2016;103(1):79–85.

patients with AL amyloidosis. Blood. 2012;119(19):4391–94.

prolonged progression‐free survival. Blood. 2012;119(19):4387–90.

2014;124(16):2498–506.

276 278Exploring New Findings on Amyloidosis

One. 2014;9(1):e87671.


## **Amyloid Nephropathy: A Practical Diagnostic Approach and Review on Pathogenesis**

Paisit Paueksakon

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64083

#### **Abstract**

Amyloidosis comprises a group of protein-folding disorders in which extracellular deposits share unique Congo red staining properties and fibrillary ultrastructural appearance. These fibrillary deposits ultimately cause tissue destruction and progres‐ sive disease. Amyloidosis can be either systemic affecting multiple organs or local‐ ized. Renal involvement by amyloidosis (amyloid nephropathy) is a frequent manifestation of systemic amyloidosis. Immunofluorescence, immunohistochemistry (IHC), and more recently laser microdissection and mass spectrometry (LMD/MS) are important techniques in typing of amyloid nephropathy. This in-depth review discusses practical diagnostic approach and pathogenesis of amyloid nephropathy and includes discussion of treatment and prognosis.

**Keywords:** amyloid nephropathy, light microscopy, immunofluorescence, immuno‐ histochemistry, electron microscopy, proteomics

## **1. Introduction**

Amyloidosis comprises a group of protein-folding disorders in which extracellular deposits share unique Congo red staining properties and fibrillary ultrastructural appearance [1]. These fibrillary deposits ultimately cause tissue destruction and progressive disease. Amyloid consists of randomly arranged, nonbranching fibrils that measure 8–12 nm in diameter [1]. Amyloidosis is a rare group of diseases, with an estimated prevalence of 1: 60,000 [2]. In the USA and Europe, amyloidosis derived from immunoglobulin (Ig) light chain (AL) secon‐ dary to plasma cell dyscrasia is the most prevalent form, followed by reactive AA amyloido‐

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

sis (AA) derived from serum amyloid A (SAA), which is typically associated with chronic inflammatory conditions. This is in contrast to developing countries in Africa and Middle East of Asia, where AA is much more common than AL [3–5]. Leukocyte chemotactic factor 2 amyloidosis (ALECT2), derived from leukocyte chemotactic factor 2 protein (LECT2), is newly recognized as a common type of amyloid, with unknown etiology [6]. Familial amyloidosis is diagnosed with increasing frequency with the use of new immunohistochemical studies and mass spectrometry [7]. Amyloidosis can be either systemic affecting multiple organs or localized. Renal disease is a frequent manifestation of systemic amyloidosis with AL, previously called primary amyloidosis, the most common type involving the kidney.

## **2. Amyloid nephropathy**

The types of amyloid are categorized by the chemical composition of the proteins. There are more than 25 precursor proteins identified as major proteins in amyloid deposition so far. A large study from the Mayo clinic [8] showed that among 474 patients with amyloid diag‐ nosed by renal biopsies between 2007 and 2011, Ig-related amyloidosis (AIg) was the most common form involving the kidney (85.9%), followed by AA (7.0%), ALECT2 (2.7%), fibrino‐ gen A α chain amyloidosis (AFib) (1.3%), apolipoprotein AI amyloidosis (AApo AI), apolipo‐ protein AII amyloidosis (AApo AII), or apolipoprotein AIV amyloidosis (AApo AIV) (0.6%), AA/light and heavy chain amyloidosis (AHL) (0.2%), and unclassified amyloidosis (2.3%). AIg in most cases is derived from fragments of monoclonal light-chain protein (AL). However, in rare cases, AIg is derived from truncated Ig heavy chain and light chain (AHL) or isolated truncated Ig heavy chain [8, 9]. AIg is associated with B-cell lymphoproliferative disorders including multiple myeloma and plasma cell dyscrasia, B-cell lymphoma, and Walden‐ ström's macroglobulinemia [8]. In contrast, AA is characterized by tissue deposition of serum amyloid A protein, which is derived from an acute-phase reactant protein synthesized by the liver [10]. AA is seen in conditions associated with chronic inflammation including rheuma‐ toid arthritis, ankylosing spondylitis, chronic draining infections (i.e., osteomyelitis, chronic skin infections including decubitus ulcers, bronchiectasis, and chronic sinusitis), Crohn disease, tuberculosis, and familial Mediterranean fever (FMF) [10]. Familial amyloidosis comprises another group of amyloid thatis now being diagnosed more frequently and includes amyloid derived from transthyretins (TTR) [11], fibrinogen [12], lysozyme (Lys) [13], gelso‐ lin (Gel) [14], Apo AI [15], Apo AII [16], and Apo AIV [17]. ALECT2 nephropathy was first described in 2008 [6, 18]. LECT2 is a chemotactic factor for neutrophils and has other physio‐ logic functions, including cell proliferation, immunomodulation, repair after injury, tumor suppression, and glucose metabolism [6, 18]. ALECT2 is now the third most common type of amyloid nephropathy in the USA and accounts for 2.5–2.7% of amyloid nephropathy cases [6, 18]. ALECT2 involves mainly in the kidney and liver but rarely involve other organs such as spleen, adrenal, and myocardium [6, 18]. Type and precursor protein of amyloid nephrop‐ athy are summarized in **Table 1**.


**Table 1.** Type and precursor protein of amyloid nephropathy.

## **3. Clinical features**

sis (AA) derived from serum amyloid A (SAA), which is typically associated with chronic inflammatory conditions. This is in contrast to developing countries in Africa and Middle East of Asia, where AA is much more common than AL [3–5]. Leukocyte chemotactic factor 2 amyloidosis (ALECT2), derived from leukocyte chemotactic factor 2 protein (LECT2), is newly recognized as a common type of amyloid, with unknown etiology [6]. Familial amyloidosis is diagnosed with increasing frequency with the use of new immunohistochemical studies and mass spectrometry [7]. Amyloidosis can be either systemic affecting multiple organs or localized. Renal disease is a frequent manifestation of systemic amyloidosis with AL,

previously called primary amyloidosis, the most common type involving the kidney.

The types of amyloid are categorized by the chemical composition of the proteins. There are more than 25 precursor proteins identified as major proteins in amyloid deposition so far. A large study from the Mayo clinic [8] showed that among 474 patients with amyloid diag‐ nosed by renal biopsies between 2007 and 2011, Ig-related amyloidosis (AIg) was the most common form involving the kidney (85.9%), followed by AA (7.0%), ALECT2 (2.7%), fibrino‐ gen A α chain amyloidosis (AFib) (1.3%), apolipoprotein AI amyloidosis (AApo AI), apolipo‐ protein AII amyloidosis (AApo AII), or apolipoprotein AIV amyloidosis (AApo AIV) (0.6%), AA/light and heavy chain amyloidosis (AHL) (0.2%), and unclassified amyloidosis (2.3%). AIg in most cases is derived from fragments of monoclonal light-chain protein (AL). However, in rare cases, AIg is derived from truncated Ig heavy chain and light chain (AHL) or isolated truncated Ig heavy chain [8, 9]. AIg is associated with B-cell lymphoproliferative disorders including multiple myeloma and plasma cell dyscrasia, B-cell lymphoma, and Walden‐ ström's macroglobulinemia [8]. In contrast, AA is characterized by tissue deposition of serum amyloid A protein, which is derived from an acute-phase reactant protein synthesized by the liver [10]. AA is seen in conditions associated with chronic inflammation including rheuma‐ toid arthritis, ankylosing spondylitis, chronic draining infections (i.e., osteomyelitis, chronic skin infections including decubitus ulcers, bronchiectasis, and chronic sinusitis), Crohn disease, tuberculosis, and familial Mediterranean fever (FMF) [10]. Familial amyloidosis comprises another group of amyloid thatis now being diagnosed more frequently and includes amyloid derived from transthyretins (TTR) [11], fibrinogen [12], lysozyme (Lys) [13], gelso‐ lin (Gel) [14], Apo AI [15], Apo AII [16], and Apo AIV [17]. ALECT2 nephropathy was first described in 2008 [6, 18]. LECT2 is a chemotactic factor for neutrophils and has other physio‐ logic functions, including cell proliferation, immunomodulation, repair after injury, tumor suppression, and glucose metabolism [6, 18]. ALECT2 is now the third most common type of amyloid nephropathy in the USA and accounts for 2.5–2.7% of amyloid nephropathy cases [6, 18]. ALECT2 involves mainly in the kidney and liver but rarely involve other organs such as spleen, adrenal, and myocardium [6, 18]. Type and precursor protein of amyloid nephrop‐

**2. Amyloid nephropathy**

280 282Exploring New Findings on Amyloidosis

athy are summarized in **Table 1**.

The clinical features of patients with amyloidosis vary, depending on the varying predilec‐ tion of different types of amyloid for specific organ involvement. As described above, renal involvement is a frequent manifestation of amyloidosis, more often in AIg nephropathy, AA nephropathy, and some forms of familial amyloidosis [10, 12]. Amyloid nephropathy of any type is more common in men than in women [8]. A large retrospective review of patients with amyloidosis evaluated from 1981 to 1992 at the Mayo Clinic in Rochester, Minnesota, re‐ vealed 1315 patients, including 918 patients withALamyloidosis (69.8%). Ofthese, 474 patients had new-onset AL amyloidosis, including 69% men and 31% women with median age of 64 years [19]. At the time of diagnosis, about 17% of AL patients had either multiple myelo‐ ma or smoldering myeloma and about 16% had history of monoclonal gammopathy of undetermined significance (MGUS). On presentation, about 51% of men and 47% of women had renal failure, and about 73% of patients had proteinuria. The median 24-h urine protein was 1.2 g/d and in the patients with full nephrotic syndrome, about a quarter of patients, the mean 24-h urine protein excretion was 7.0 g/d. Serum protein electrophoresis (SPEP) re‐ vealed a monoclonal protein in about 48% of patients and when serum immunoelectrophore‐ sis was tested the percentage increased to about 72%. A monoclonal light chain was detected in about 73% of the patients by urine immunoelectrophoresis or immunofixation, including about 68% with lambda and about 32% with kappa. Overall, about 89% of the patients with AL amyloidosis showed a serum or urine monoclonal spike (M spike).

Patients with AA nephropathy usually present at younger age than AL patients, whereas ALECT2 is more common in older patients. ALECT2 nephropathy mainly affects patients of Hispanic or Mexican origin and rarely affects Caucasian patients, whereas AL and AA do not show evident ethnic differences [6, 18]. As in AL amyloid, proteinuria is also the most common clinical presentation in AA, ALECT2, and familial amyloid nephropathy [8]. Extrarenal manifestations of amyloidosis include congestive heart failure caused by myocardial infiltra‐ tion by amyloid, orthostatic hypotension, bladder dysfunction, and dysesthesias caused by amyloid infiltration into autonomic and peripheral nerves, lymphadenopathy, hepatomega‐ ly, splenomegaly, and macroglossia [18, 20]. The treatment and prognosis of amyloid nephr‐ opathy will be discussed below.

## **4. Molecular mechanisms and pathogenesis of amyloidosis**

Misfolding of extracellular proteins has a prominent role in the molecular mechanisms and pathogenesis of amyloidosis [21, 22]. The misfolded proteins are highly prone to self-aggre‐ gation. There are different mechanisms of formation of pathologic misfolded proteins [22]. The first is based on intrinsic propensity of the protein to assume pathologic conformation, which becomes more evident with aging. Examples of these mechanisms are evident when molecu‐ larly normal transthyretin misfolds in patients with senile systemic amyloidosis [22]. The second way is due to alteration of the normal protein sequence, as seen with the replace‐ ment of a single amino acid in protein, e.g., in AL and familial amyloidosis [22]. Only a small portion of Ig light chains is amyloidogenic; thus, AL nephropathy occurs only in about 12–15% ofthe patients with multiple myeloma [23]. The amyloid fibrils in amyloid nephropathy consist of either intact light chains containing the variable and constant domains or solely the variable domains, which contains the N-terminus portion of the light-chain molecule. The VλVI subgroup of the light chain preferentially leads to amyloid formation in patients with AL nephropathy [23, 24]. Therefore, it appears that germ line sequences, which enhanced near the N-terminus of the lambda light chain and in the VλVI of the light chain may be prone to mutations that generate amyloidogenic light chains. These altered proteins lead to destabili‐ zation of light chain and increase the likelihood of fibril formation [25]. In familial amyloido‐ sis, such as Transthyretin-related hereditary amyloidosis (ATTR) and Lysozyme-related amyloidosis (ALys), the substitution of a single amino acid transforms a normal protein into an amyloidogenic protein [26, 27]. The unstable protein produced by amino acid substitu‐ tion may allow the protein to precipitate when stimulated by the local physicochemical milieu, such as local surface pH, electric field, and hydration forces on the cellular surfaces [22]. A third mechanism contributing to amyloid formation is proteolytic remodeling of the protein precursor, as seen in Alzheimer's disease, in which the fibrils are composed of proteolytic fragments of 39–43 residues derived from 753-residue β-amyloid precursor protein (APP) [28]. This type of amyloid does not affect the kidney. In addition, excessive concentration of amyloidogenic protein due to unregulated production of high local concentration can promote amyloidosis [29]. When the protein precursor reaches a critical point of local concentration, it triggers fibril formation. This mechanism is enhanced further by environmental physicochem‐ ical factors and interaction with extracellular matrix. This mechanism may occur in AA amyloidosis associated with chronic inflammatory conditions or familial Mediterranean fever [30]. The excessive concentration of amyloidogenic protein may also be caused by decrease in clearance from the body; e.g., Beta-2 microglobulin associated amyloidosis (Aβ2M) develops in end-stage renal disease patients who received long-term dialysis because of ineffective clearance of β2M from circulation during dialysis or due to inability of the body to destroy the accumulation by natural mechanism [31]. Regardless of mechanism of misfold‐ ing, the misfolded proteins are prone to self-aggregate, generating protofilaments that interact to form fibrils [22].

Hispanic or Mexican origin and rarely affects Caucasian patients, whereas AL and AA do not show evident ethnic differences [6, 18]. As in AL amyloid, proteinuria is also the most common clinical presentation in AA, ALECT2, and familial amyloid nephropathy [8]. Extrarenal manifestations of amyloidosis include congestive heart failure caused by myocardial infiltra‐ tion by amyloid, orthostatic hypotension, bladder dysfunction, and dysesthesias caused by amyloid infiltration into autonomic and peripheral nerves, lymphadenopathy, hepatomega‐ ly, splenomegaly, and macroglossia [18, 20]. The treatment and prognosis of amyloid nephr‐

Misfolding of extracellular proteins has a prominent role in the molecular mechanisms and pathogenesis of amyloidosis [21, 22]. The misfolded proteins are highly prone to self-aggre‐ gation. There are different mechanisms of formation of pathologic misfolded proteins [22]. The first is based on intrinsic propensity of the protein to assume pathologic conformation, which becomes more evident with aging. Examples of these mechanisms are evident when molecu‐ larly normal transthyretin misfolds in patients with senile systemic amyloidosis [22]. The second way is due to alteration of the normal protein sequence, as seen with the replace‐ ment of a single amino acid in protein, e.g., in AL and familial amyloidosis [22]. Only a small portion of Ig light chains is amyloidogenic; thus, AL nephropathy occurs only in about 12–15% ofthe patients with multiple myeloma [23]. The amyloid fibrils in amyloid nephropathy consist of either intact light chains containing the variable and constant domains or solely the variable domains, which contains the N-terminus portion of the light-chain molecule. The VλVI subgroup of the light chain preferentially leads to amyloid formation in patients with AL nephropathy [23, 24]. Therefore, it appears that germ line sequences, which enhanced near the N-terminus of the lambda light chain and in the VλVI of the light chain may be prone to mutations that generate amyloidogenic light chains. These altered proteins lead to destabili‐ zation of light chain and increase the likelihood of fibril formation [25]. In familial amyloido‐ sis, such as Transthyretin-related hereditary amyloidosis (ATTR) and Lysozyme-related amyloidosis (ALys), the substitution of a single amino acid transforms a normal protein into an amyloidogenic protein [26, 27]. The unstable protein produced by amino acid substitu‐ tion may allow the protein to precipitate when stimulated by the local physicochemical milieu, such as local surface pH, electric field, and hydration forces on the cellular surfaces [22]. A third mechanism contributing to amyloid formation is proteolytic remodeling of the protein precursor, as seen in Alzheimer's disease, in which the fibrils are composed of proteolytic fragments of 39–43 residues derived from 753-residue β-amyloid precursor protein (APP) [28]. This type of amyloid does not affect the kidney. In addition, excessive concentration of amyloidogenic protein due to unregulated production of high local concentration can promote amyloidosis [29]. When the protein precursor reaches a critical point of local concentration, it triggers fibril formation. This mechanism is enhanced further by environmental physicochem‐ ical factors and interaction with extracellular matrix. This mechanism may occur in AA amyloidosis associated with chronic inflammatory conditions or familial Mediterranean

**4. Molecular mechanisms and pathogenesis of amyloidosis**

opathy will be discussed below.

282 284Exploring New Findings on Amyloidosis

In AL nephropathy, amyloid formation in the kidney first begins in the mesangium. The specific uptake of light-chain protein by mesangial cells underlies the predominant kidney tropism and is an important step in amyloid fibril formation. There are several factors that might promote or decelerate amyloid deposition in glomeruli, including the negative ion charge and the high concentration of glycosaminoglycan of the glomerular basement mem‐ branes with the presence of enzyme proteases that could render protein amyloidogenic [32]. Mesangial cells are modified smooth muscle cells, which have phagocytic activity [33]. Upon interaction with light-chain proteins, the light-chain proteins are avidly internalized and delivered to the primary lysosomes where the amyloid fibrils are primarily formed [34]. During this process, the mesangial cells transform into a macrophage phenotype with prominent primary lysosomes, making them more capable of processing of internalized amyloidogenic light-chain proteins and fibril formation [34]. The amyloid deposition within the mesangium further stimulates metalloproteinase enzymes, causing mesangial matrix degradation, inhibiting transforming growth factor β (TGF-β) and thus impairing mesangial matrix repair [33, 35] and increasing apoptosis, finally leading to significant mesangiolysis and replacement of the mesangial area by amyloid deposits [36]. These changes result in the absence of mesangial argyrophilia andsignificant mesangial celldeletion in the advancedstage of glomerular amyloidosis [34].

LECT2 has multiple functions including as a cytokine involved in chemotaxis of neutrophils and a growth factor involved in cell proliferation and regulation of repair after injury, immunomodulation, tumor suppression, and glucose metabolism [37]. The pathogenesis of ALECT2 is not well understood, but may be due to increased synthesis of LECT2 by hepato‐ cytes or secondary to hepatocellular damage or involve interference, possible due to genetic defect, in the LECT2 catabolism or LECT2 transportation, which may give rise to local LECT2 tissue concentration and finally lead to amyloid fibril formation [6, 18, 37].

The normal plasma protein serum amyloid P component (SAP), a glycoprotein that belongs to the pentraxin family, independently binds to fibrils in all types of amyloid and contrib‐ utes to pathogenesis of amyloidosis [38]. SAP is highly protected against proteolysis, and a binding of SAP to amyloid fibrils in vitro protect them from proteolytic enzymes and degradation by phagocytic cells. Moreover, SAP is a stubborn glycoprotein that can persist within human amyloid deposits for prolonged periods and is completely unmodified with respect to circulating SAP [38]. It is suggested that SAP may contribute to the failure to clear amyloid deposition in vivo, leading to tissue architecture destruction and organ dysfunction.

Furthermore, amyloid can be deposited in multiple organs, and this tendency might depend on several physicochemical factors, such as high concentration of local protein, low pH, presence of proteolytic processing, and seeding of protofilaments [22]. The amyloid deposits cause tissue architecture destruction, presumed to cause organ dysfunction [22]. Further‐ more, the amyloidogenic precursor proteins, folding intermediates, and protofilaments have toxicities independent of the amyloid deposits and these toxicities can also contribute to disease manifestations [39].

## **5. Gross findings**

Postmortem examination of patients with amyloidosis generally shows enlarged kidneys, unlike other causes of chronic kidney disease (CKD) where kidneys are small and shrunken. In addition, cut surfaces of kidney in patients with amyloid nephropathy are pale, firm, waxy, and flat [38] (**Figure 1a**), which are different from the normal kidney or the kidney in the patients with CKD (**Figure 1b**), which bulges slightly causing in slight curvature of the cut surfaces.

**Figure 1.** At autopsy of a patient with amyloid nephropathy, the kidney is enlarged and pale with waxy and flat cut surface (a, arrow), which is different from the kidney in a patient with chronic kidney disease with a simple cyst, which bulges slightly (arrow) resulting in slight curvature of the cut surfaces (b).

## **6. Histologic diagnosis and identification of amyloid**

#### **6.1. Light microscopy**

presence of proteolytic processing, and seeding of protofilaments [22]. The amyloid deposits cause tissue architecture destruction, presumed to cause organ dysfunction [22]. Further‐ more, the amyloidogenic precursor proteins, folding intermediates, and protofilaments have toxicities independent of the amyloid deposits and these toxicities can also contribute to

Postmortem examination of patients with amyloidosis generally shows enlarged kidneys, unlike other causes of chronic kidney disease (CKD) where kidneys are small and shrunken. In addition, cut surfaces of kidney in patients with amyloid nephropathy are pale, firm, waxy, and flat [38] (**Figure 1a**), which are different from the normal kidney or the kidney in the patients with CKD (**Figure 1b**), which bulges slightly causing in slight curvature of the cut

**Figure 1.** At autopsy of a patient with amyloid nephropathy, the kidney is enlarged and pale with waxy and flat cut surface (a, arrow), which is different from the kidney in a patient with chronic kidney disease with a simple cyst,

which bulges slightly (arrow) resulting in slight curvature of the cut surfaces (b).

disease manifestations [39].

284 286Exploring New Findings on Amyloidosis

**5. Gross findings**

surfaces.

The light microscopic (LM) findings of amyloidosis are identical regardless of the type of amyloid. Glomerular amyloid deposition begins with progressive accumulation of acellular amorphous eosinophilic material (cotton candy appearance) on hematoxylin and eosin (H&E) stained sections (**Figure 2**) that appear pale with the periodic acid Schiff (PAS) stain (**Figure 3a** and **b**). In the advanced stage, the mesangial matrix is extensively replaced by acellular amorphous material exhibiting uniform expansion with compression of the capilla‐ ry spaces and occasional nodular formation, resembling diabetic nephropathy or monoclo‐ nal immunoglobulin deposition disease. The nodular pattern is seen more frequently in AA nephropathy. The amyloid deposits are paler on PAS stain than the Kimmelstiel-Wilson nodules in diabetic nephropathy, or deposits in light-chain deposition disease or fibrillary glomerulonephritis. Further, fibrillary glomerulonephritis typically is proliferative, with increased cellularity seen by light microscopy. The amyloid deposits stain weakly or are negative with Jones methenamine silver stain (JMS) and appear as negative defects in the expanded mesangium and thick glomerular basement membranes (**Figure 4a** and **b**). Occasionally, there is a focal parallel alignment of amyloid fibrils in the subepithelial zone that results in focal capillary wall spikes, which are best visualized by JMS (**Figure 5**). These spikes are focal, closely clustered, and longer than the typical spikes of membranous nephropathy. The focal clusters of spikes may resemble feathers, so-called feathery spikes. Amyloid feathery spikes are more common in AL than AA or familial amyloidosis [7]. Rarely crescent forma‐ tion can be seen in both AA and AL [7], indicating glomerular basement membrane breaks, the injury that triggers crescent formation, can occur. Vascular involvement by amyloidosis is common, frequently involving arteriolar walls, followed by arteries, peritubular capillaries, and veins. In rare cases, particularly in AL or in ATTR nephropathy, the vessels are the only part in the kidney where amyloid is demonstrated. Interstitial amyloid deposition is seen in about 50% of cases but is present in most cases of ALECT2 nephropathy (**Figure 6**). Glomer‐ ular involvement is not extensive in ALECT2 and apolipoprotein AI (AApo AI/), AII (AApoAII), and AIV (AApoAIV) nephropathy in comparison with other types of amyloid. In addition, the histology of AFib nephropathy is very characteristic, showing glomerular enlargement with near-totalreplacement ofthe glomeruli by amyloid, with little or no vascular or interstitial involvement [10]. Special IF and immunohistochemistry (IHC) together with mass spectrometry analyses are typically used to identify the type of amyloid (see below). However, potassium permanganate treatment prior to Congo red staining has been used to distinguish between AA and AL nephropathy: after this treatment, birefringent Congo red staining is lost by AA but retained by AL. This technique is not commonly used and has been replaced by more specific SAA immunohistochemistry (IHC).

Amyloid is defined by its tinctorial characteristics, which include Congo red positivity (orange, salmon pink, or pale rose staining, **Figure 7**). The Congo red stain gives apple-green or traffic light green birefringence under polarized light (**Figure 8**). The mesangial expan‐ sion can be focal and may be segmental, global, or even nodular. However, the degree of

proteinuria does not correlate with the amount of amyloid deposition; as a result, cases with early amyloidosis with scant and segmental amyloid deposition can be mistakenly diag‐ nosed as minimal change disease if no Congo red stain, immunofluorescence (IF) and electron microscopy (EM) is performed. To demonstrate small amounts of amyloid deposits in tissue sections, it is recommended that the sections be cut thicker than normal (6 μm, instead of customary 2 μm) [40]. Proper detection of apple green birefringence depends on the intensi‐ ty of the transmitted light and, thus, a strong light source is strongly suggested to maximize results. In early amyloidosis with small amounts of amyloid, it might be difficult to demon‐ strate Congo red positivity. Therefore, an alternative way of identifying a small amount of amyloid deposits by Congo red stain is to place the stained section under fluorescent light; amyloid then is bright red [41] (**Figure 9**). Of note, false positivity may result from overstain‐ ing, and this technique is not specific for amyloid. Thus, amyloid must be confirmed by apple green polarization, immunofluorescence, IHC, and/or electron microscopy. An alternative option to demonstrate amyloid deposits in cases with no paraffin embedded tissue available is to perform Congo red stain on frozen tissue (**Figure 10**). This technique is more sensitive than a Congo red stain performed on paraffin embedded tissue from our experience. Of note, positive controls should then, of course, also be performed on frozen tissues.

**Figure 2.** In amyloid nephropathy, there is extensive mesangial expansion by amorphous eosinophilic acellular materi‐ al ("cotton candy" appearance), which segmentally extends to the glomerular capillary loops (H&E stain, original mag‐ nification ×400).

Amyloid Nephropathy: A Practical Diagnostic Approach and Review on Pathogenesis http://dx.doi.org/10.5772/64083 287 289

proteinuria does not correlate with the amount of amyloid deposition; as a result, cases with early amyloidosis with scant and segmental amyloid deposition can be mistakenly diag‐ nosed as minimal change disease if no Congo red stain, immunofluorescence (IF) and electron microscopy (EM) is performed. To demonstrate small amounts of amyloid deposits in tissue sections, it is recommended that the sections be cut thicker than normal (6 μm, instead of customary 2 μm) [40]. Proper detection of apple green birefringence depends on the intensi‐ ty of the transmitted light and, thus, a strong light source is strongly suggested to maximize results. In early amyloidosis with small amounts of amyloid, it might be difficult to demon‐ strate Congo red positivity. Therefore, an alternative way of identifying a small amount of amyloid deposits by Congo red stain is to place the stained section under fluorescent light; amyloid then is bright red [41] (**Figure 9**). Of note, false positivity may result from overstain‐ ing, and this technique is not specific for amyloid. Thus, amyloid must be confirmed by apple green polarization, immunofluorescence, IHC, and/or electron microscopy. An alternative option to demonstrate amyloid deposits in cases with no paraffin embedded tissue available is to perform Congo red stain on frozen tissue (**Figure 10**). This technique is more sensitive than a Congo red stain performed on paraffin embedded tissue from our experience. Of note,

positive controls should then, of course, also be performed on frozen tissues.

**Figure 2.** In amyloid nephropathy, there is extensive mesangial expansion by amorphous eosinophilic acellular materi‐ al ("cotton candy" appearance), which segmentally extends to the glomerular capillary loops (H&E stain, original mag‐

nification ×400).

286 288Exploring New Findings on Amyloidosis

**Figure 3.** In amyloid nephropathy (a), mesangial areas are globally expanded by weak PAS positive acellular deposits (arrow) in comparison to a glomerulus (b) in normal kidney biopsy (PAS stain, original magnification ×400).

**Figure 4.** In amyloid nephropathy (a), mesangial amyloid deposits are nonargyrophilic (silver negative, arrow) in com‐ parison with a glomerulus in the normal kidney biopsy (silver positive, b) (JMS stain, original magnification ×400).

**Figure 5.** Feathery spikes (arrow) are a characteristic features of amyloid nephropathy seen on JMS, and are seen more frequently in AIg than other forms of amyloid nephropathy (JMS stain, original magnification ×400).

(b)

288 290Exploring New Findings on Amyloidosis

**Figure 4.** In amyloid nephropathy (a), mesangial amyloid deposits are nonargyrophilic (silver negative, arrow) in com‐ parison with a glomerulus in the normal kidney biopsy (silver positive, b) (JMS stain, original magnification ×400).

**Figure 6.** ALECT2 amyloid nephropathy with intense interstitial amyloid (arrow) seen by immunohistochemistry staining (anti-LECT2 IHC, original magnification ×400).

**Figure 7.** Congo red positive mesangial deposits of amyloid with orange or salmon pink staining (Congo red stain, original magnification ×400).

**Figure 8.** Amyloid deposits with characteristic apple green birefringence when viewed by polarized light (Congo red stain, original magnification ×400).

**Figure 9.** A small amount of amyloid deposits can be detected by placing the Congo red stained section under fluores‐ cent light (Congo red stain, original magnification ×400).

**Figure 7.** Congo red positive mesangial deposits of amyloid with orange or salmon pink staining (Congo red stain,

**Figure 8.** Amyloid deposits with characteristic apple green birefringence when viewed by polarized light (Congo red

original magnification ×400).

290 292Exploring New Findings on Amyloidosis

stain, original magnification ×400).

**Figure 10.** Congo red stain performed on frozen tissue with green birefringence (arrow) when viewed by polarized light (Congo red stain, original magnification ×400).

Thioflavin T is an additional sensitive but nonspecific fluorescent stain for amyloid, which is activated by blue light to emit yellow green fluorescence staining upon binding to amyloid [42]. This testis more sensitive than Congo red in detecting a small amount of amyloid deposits. However, due to lack of specificity, it must be confirmed by Congo red apple green birefrin‐ gence, IF, IHC, and/or EM.

AL amyloid nephropathy may coexist with other manifestations of the monoclonal protein, such as light-chain cast nephropathy, light-chain proximal tubulopathy, and rarely monoclo‐ nal immunoglobulin deposition disease. Any amyloid type may have coexisting other conditions in the kidney biopsy, such as acute tubular necrosis, diabetic nephropathy, thin basement membrane lesion, or arterionephrosclerosis.

## **6.2. Immunofluorescence microscopy**

Immunofluorescence (IF) microscopy or immunohistochemistry is crucial for confirming the presence and for typing of amyloid nephropathy. IF is useful for detection of amyloid derived from Ig light and heavy chains. Amyloid deposits have a distinctive smudgy appearance of staining (**Figure 11a**). Restriction for light chain, either kappa or more commonly lambda, or for heavy-chain determents (γ or μ) must be demonstrated, in association with Congo red positivity, for a diagnosis of AIg amyloidosis. Therefore, IF staining for kappa and lambda should be routinely performed on all native renal biopsies. The findings of strong staining for a monoclonal Ig light chain with negativity for Ig heavy chains is diagnostic of AL nephrop‐ athy (**Figure 11a** and **b**). About 75% of AL nephropathy cases are due to monoclonal lambda deposits. The diagnosis of AH nephropathy is based on the presence of strong staining for a single Ig heavy chain with negativity for kappa and lambda light chains (**Figure 12**). AHL nephropathy shows intense staining for Ig heavy chain and a single Ig light chain (**Figure 13a** and **b**).

**Figure 11.** AL nephropathy with smudgy mesangial, vascular, and interstitial staining for lambda (a) and negative kappa (b) (IF, original magnification ×400).

In addition to specific staining for proteins that comprise the amyloid, amyloid deposits often have low levels of nonspecific trapping with several reagent antibodies, including Ig and complements (**Figure 14a** and **b**). This varies, with different reagent antibodies and different specimens, and is more common with AA nephropathy than AL nephropathy. The basis for this low level of staining is not well understood, but may result from charge interaction between the amyloid and the reagent antibodies, contamination with serum proteins, and/or humoral reaction directed against amyloid fibrils. These low levels of nonspecific trapping sometime render the distinction between AA nephropathy and AL/AH/AHL nephropathy difficult. About 21% of AA nephropathy cases from a study by Nasr et al. [8] could not be diagnosed with certainty by IF and needed confirmation of amyloid type by mass spectrom‐ etry.

Thioflavin T is an additional sensitive but nonspecific fluorescent stain for amyloid, which is activated by blue light to emit yellow green fluorescence staining upon binding to amyloid [42]. This testis more sensitive than Congo red in detecting a small amount of amyloid deposits. However, due to lack of specificity, it must be confirmed by Congo red apple green birefrin‐

AL amyloid nephropathy may coexist with other manifestations of the monoclonal protein, such as light-chain cast nephropathy, light-chain proximal tubulopathy, and rarely monoclo‐ nal immunoglobulin deposition disease. Any amyloid type may have coexisting other conditions in the kidney biopsy, such as acute tubular necrosis, diabetic nephropathy, thin

Immunofluorescence (IF) microscopy or immunohistochemistry is crucial for confirming the presence and for typing of amyloid nephropathy. IF is useful for detection of amyloid derived from Ig light and heavy chains. Amyloid deposits have a distinctive smudgy appearance of staining (**Figure 11a**). Restriction for light chain, either kappa or more commonly lambda, or for heavy-chain determents (γ or μ) must be demonstrated, in association with Congo red positivity, for a diagnosis of AIg amyloidosis. Therefore, IF staining for kappa and lambda should be routinely performed on all native renal biopsies. The findings of strong staining for a monoclonal Ig light chain with negativity for Ig heavy chains is diagnostic of AL nephrop‐ athy (**Figure 11a** and **b**). About 75% of AL nephropathy cases are due to monoclonal lambda deposits. The diagnosis of AH nephropathy is based on the presence of strong staining for a single Ig heavy chain with negativity for kappa and lambda light chains (**Figure 12**). AHL nephropathy shows intense staining for Ig heavy chain and a single Ig light chain (**Figure 13a**

**Figure 11.** AL nephropathy with smudgy mesangial, vascular, and interstitial staining for lambda (a) and negative

In addition to specific staining for proteins that comprise the amyloid, amyloid deposits often have low levels of nonspecific trapping with several reagent antibodies, including Ig and complements (**Figure 14a** and **b**). This varies, with different reagent antibodies and different

gence, IF, IHC, and/or EM.

292 294Exploring New Findings on Amyloidosis

and **b**).

basement membrane lesion, or arterionephrosclerosis.

**6.2. Immunofluorescence microscopy**

kappa (b) (IF, original magnification ×400).

**Figure 12.** AH nephropathy. Intense glomerular staining for IgG heavy chain. Kappa and lambda are negative (IF, original magnification ×400).

**Figure 13.** AHL nephropathy. Intense glomerular, vascular and tubular basement membrane staining for IgG heavy chain (a) and lambda light chain (b). Kappa is negative (IF, original magnification ×400).

**Figure 14.** Low level of non-specific staining for Immunoglobulin M (IgM) (a, arrow) and complement 3 (C3) (b, ar‐ row) in AA nephropathy (IF, original magnification ×400).

#### **6.3. Electron microscopy**

Transmission electron microscopy (TEM) is helpful in confirming the diagnosis of amyloido‐ sis, but it is not helpful in determining the type of amyloid. At low magnification, amyloid deposits have a characteristic cottony appearance (**Figure 15**) that should alert the patholo‐ gist to investigate at higher magnification the possibility of amyloidosis. At high magnifica‐ tion, amyloid is composed of randomly arranged, elongated fibrils that measure 8–12 nm in diameter (**Figure 16**). Amyloid fibrils are associated with smaller, round structures, namely, amyloid p component. The fibrils are extracellular and located near mesangial cells in glomeruli and myocytes in the media of vessel walls in amyloid nephropathy. The amyloid

**Figure 15.** Extensive expansion of the mesangium by amyloid, which shows lumpy cottony appearance (arrow) on low power electron microscopy (transmission electron microscopy (TEM), original magnification, ×5600).

**Figure 16.** AL nephropathy. Amyloid fibrils replacing normal mesangium. Typical amyloid fibrils are randomly ar‐ ranged, nonbranching and measure 8–12 nm in diameter (TEM, original magnification, ×14,000).

fibrils may extend into the capillary walls and thus, foot process effacement is commonly seen in the segments of glomerular basement membranes overlying amyloid deposits. The podocyte cytoplasm adjacent to the amyloid deposits often shows condensation of actin cytoskeleton. When amyloid deposits extend into the subepithelial zone, the fibrils usually show parallel alignment (**Figure 17**). This is the basis for the JMS-positive feathery spikes that may be seen by light microscopy (LM).

**Figure 17.** Alignment of amyloid fibrils (arrow) in the subepithelial zone of a glomerular basement membrane. This is the basis for the JMS-positive feathery spikes that may be seen by LM (TEM, original magnification, ×5600).

#### **6.4. Differential diagnosis**

**6.3. Electron microscopy**

294 296Exploring New Findings on Amyloidosis

Transmission electron microscopy (TEM) is helpful in confirming the diagnosis of amyloido‐ sis, but it is not helpful in determining the type of amyloid. At low magnification, amyloid deposits have a characteristic cottony appearance (**Figure 15**) that should alert the patholo‐ gist to investigate at higher magnification the possibility of amyloidosis. At high magnifica‐ tion, amyloid is composed of randomly arranged, elongated fibrils that measure 8–12 nm in diameter (**Figure 16**). Amyloid fibrils are associated with smaller, round structures, namely, amyloid p component. The fibrils are extracellular and located near mesangial cells in glomeruli and myocytes in the media of vessel walls in amyloid nephropathy. The amyloid

**Figure 15.** Extensive expansion of the mesangium by amyloid, which shows lumpy cottony appearance (arrow) on low

**Figure 16.** AL nephropathy. Amyloid fibrils replacing normal mesangium. Typical amyloid fibrils are randomly ar‐

ranged, nonbranching and measure 8–12 nm in diameter (TEM, original magnification, ×14,000).

power electron microscopy (transmission electron microscopy (TEM), original magnification, ×5600).

The differential diagnosis and distinction from other glomerular diseases are summarized in **Table 2**. By LM, the differential diagnosis includes any disease process that induces accumu‐ lation of acellular eosinophilic material in glomeruli, blood vessels, and interstitium. Accumulations of collagenous matrix, as in Kimmelstiel-Wilson nodules in diabetic nephrop‐ athy or sclerosis secondary to chronic immune complex glomerulonephritis or hypertensive nephrosclerosis, usually can be readily distinguished from amyloid by routine staining. As mentioned earlier, amyloid stains weakly with PAS and very little or negative with JMS, whereas sclerotic matrix is strongly PAS and JMS positive. Extensive glomerular deposits present in immune complex glomerulonephritis, fibrillary glomerulonephritis, immunotac‐ toid glomerulopathy, and collagenofibrotic glomerulopathy may mimic amyloidosis on H&E stained sections; however, special stains, IHC, and EM demonstrate the distinctive features of each of these diseases. The Congo red stain is then necessary to confirm the diagnosis of amyloid nephropathy.

Light-chain deposition disease (LCDD) shares monoclonal immunoglobulin light-chain staining with AL nephropathy, but the pattern of staining is much different. LCDD typically has a more diffuse and regular distribution of staining that appears linear or ribbon-like along glomerular and tubular basement membranes. The expanded mesangium also shows globular staining in LCDD, in contrast to AL nephropathy, which typically shows a smudgy pattern of lambda or kappa light-chain staining with fuzzy border in the mesangium or along glomeru‐ lar basement membranes, but no ribbon-like tubular basement membrane deposits.



**Table 2.** Histologic differential diagnosis of amyloid nephropathy.

staining in LCDD, in contrast to AL nephropathy, which typically shows a smudgy pattern of lambda or kappa light-chain staining with fuzzy border in the mesangium or along glomeru‐

**LM IF EM**

Diffuse intense staining of single light chain (more often kappa) along

Diffuse intense staining of single Ig heavy chain (more often IgG) and a single light chain along tubular and/or glomerular basement membranes (ribbon-like), involving mesangium (globular), and blood

Diffuse intense staining of single Ig heavy chain (more IgG3) along tubular and/or glomerular basement membranes (ribbon-like), involving mesangium (globular), and blood vessels, no kappa or lambda staining

Smudgy staining for IgG (more often IgG4), C3, kappa, and lambda in mesangium and along capillary loops, about 10% show restriction of light-chain staining

Smudgy staining for IgG (more often IgG1), C3, with light chain

Finely granular (punctate) or amorphous electron dense deposits along the inner aspect of glomerular basement membranes and outer aspect of tubular basement membranes

Finely granular (punctate) or amorphous electron dense deposits along the inner aspect of glomerular basement membranes and outer aspect of tubular basement membranes, occasional immune complextype deposits, or rarely no electron deposits detected

Finely granular (punctate) or amorphous electron dense deposits along the inner aspect of glomerular basement membranes and outer aspect of tubular basement membranes, occasional immune complextype deposits, or rarely no electron deposits detected

Randomly arranged nonbranching fibrils size of 12-27 nm in diameter within mesangium, along outer aspect of glomerular basement membranes, and rarely along tubular basement membranes

Microtubular or cylindrical structure with hollow cores with a size from 20 to 90 nm in

tubular and/or glomerular basement membranes (ribbon-like), involving mesangium (globular), and blood

vessels

vessels

lar basement membranes, but no ribbon-like tubular basement membrane deposits.

**Differential diagnosis**

Light-chain deposition disease

Light- and heavychain deposition disease

Heavy-chain deposition disease

Immunotactoid glomerulopathy **Congo red stain**

296 298Exploring New Findings on Amyloidosis

Negative Deposition of PAS positive

Negative Deposition of PAS positive and argyrophilic material in mesangium with occasional nodular

> formation, more mesangial hypercellularity

Negative Deposition of PAS positive and argyrophilic material in mesangium with occasional nodular formation, more mesangial hypercellularity

> positive and nonargyrophilic in mesangium and occasionally along glomerular basement membranes

Negative Deposition of PAS positive and nonargyrophilic in

Fibrillary GN Negative Deposition of PAS

and argyrophilic material in mesangium with occasional nodular formation

> By EM, the differential diagnosis includes other renal diseases that have deposits containing organized fibrillary or microtubular substructures, including fibrillary glomerulonephritis, immunotactoid glomerulopathy, and collagenofibrotic glomerulopathy. Fibrillary glomeru‐ lonephritis is characterized by glomerular deposits composed of randomly arranged, non‐ branching fibrils that usually are 12–27 nm in diameter (**Figure 18**). The diagnosis of amyloidosis is unlikely if fibrils are larger than 20 nm. Immunotactoid glomerulopathy has organized microtubular substructures with hollow cores, measuring 20–90 nm (**Figure 19**). Collagenofibrotic glomerulopathy shows collagen fibrils deposited predominantly in the mesangium but sometimes can extend to subendothelial zone. The collagen fibrils appear curved and often frayed with periodicity of 43–65 nm. In diabetic fibrillosis, the fibrils are 10– 25 nm, negative for Congo red and Thioflavin T. These fibrils tend to be shorter and more often aligned in parallel, slightly curved, and sometimes are oriented at right angles to mesangial cell surfaces, and may touch plasma membranes (**Figure 20**). Of note, the presence of diabet‐ ic fibrillosis in diabetic nephropathy does not appear to have any specific clinical connota‐ tions because a patient with this disorder behaves similarly clinically to those without fibrils [36]. For each of the above conditions with substructure of material by EM, IF and light microscopic appearances, and negative Congo red stain, allow distinction from amyloidosis.

**Figure 18.** Fibrillary glomerulonephritis with randomly arranged nonbranching fibrils that measure 12–27 nm in diam‐ eter (TEM, original magnification, ×14,000).

**Figure 19.** Immunotactoid glomerulopathy. Replacement of mesangial matrix by microtubular substructures (arrow) with hollow cores that measure 20–90 nm in diameter (TEM, original magnification, ×14, 000).

**Figure 20.** Diabetic fibrillosis. Replacement of mesangial matrix by short nonbranching fibrils that measure 10–25 nm in diameter. These fibrils tend to be more often aligned in parallel, slightly curved, and sometimes are oriented at right angles to mesangial cell surfaces (TEM, original magnification, ×14,000).

## **6.5. Typing of amyloid does matter**

**Figure 18.** Fibrillary glomerulonephritis with randomly arranged nonbranching fibrils that measure 12–27 nm in diam‐

**Figure 19.** Immunotactoid glomerulopathy. Replacement of mesangial matrix by microtubular substructures (arrow)

with hollow cores that measure 20–90 nm in diameter (TEM, original magnification, ×14, 000).

eter (TEM, original magnification, ×14,000).

298 300Exploring New Findings on Amyloidosis

Amyloid typing is absolutely crucial for clinical management in order to avoid misdiagnosis and inappropriate, potentially harmful treatment, to assess prognosis and to offer genetic counseling if relevant. The classification is based on the nature of amyloid precursor plasma proteins. The initial step is IF, which is an important technique for AL typing. In AL nephr‐ opathy, there is restriction for either kappa or lambda light chain. In AH nephropathy, the deposits stain only a single subclass of Ig heavy chain, whereas a single heavy-chain and a single light-chain stain positive in AHL nephropathy. In cases where no frozen tissue is available, IF can be performed on paraffin embedded tissue by a pronase-digested technique. The paraffin-embedded tissue pronase-digested technique has less sensitivity than frozen tissue IF. Thus, negative IF staining for Ig light chains or heavy chain does not completely rule out AL nephropathy, AH nephropathy, or AHL nephropathy, as some monoclonal proteins may be mutated and not recognized by commercial antibodies. However, a negative IF study does typically suggest another type of amyloid nephropathy and requires further explora‐ tion by other modalities to diagnose the type of amyloid.

Current diagnostic methods, such as IHC technique using antibody-based amyloid typing, have limited ability to detect the full spectrum of amyloid forming proteins. Although IHC can be performed using commercially available antibodies against amyloid of the classes AL, AH, AA, AFib, ATTR, AApo AI, and Aβ2M [10], of these antibodies, serum amyloid A (SAA) IHC on paraffin-embedded tissue is the most extensively used and is considered the gold

standard technique for diagnosing AA (**Figure 21**). AA IHC reveals a pattern of distribution matching that seen by LM. From our study, IHC on paraffin-embedded tissue for LECT2 is sensitive; however, it is not entirely specific for ALECT2 [41]. It is recommended that the diagnosis of ALECT2 nephropathy should be confirmed by mass spectrometry analysis of renal tissues when the staining pattern shows equivocal Intensity [41].

**Figure 21.** AA nephropathy with intense glomerular and vascular staining for SAA IHC (anti-SAA IHC, original mag‐ nification ×200).

In spite of the availability of IF and IHC, there are multiple drawbacks of these methods, such as sensitivity and specificity of commercial antibodies that are designed against only nonmu‐ tated proteins. Mutant proteins and proteins with conformational changes might be less reactive to commercial antibodies [39]. For example, in 7–35% of the AL nephropathy cases, the amyloid deposits were negative for both kappa and lambda IF staining on frozen tissue [7]. Therefore, be aware that a negative staining for light- and/or heavy-chain IF does not ex‐ clude AIg nephropathy. Of note, rare cases of amyloid nephropathy show more than one type of amyloid protein, leading to difficulty in typing. The drawbacks of these techniques may be solved by mass spectrometry, which is discussed below.

#### **6.6. Proteomics**

The direct technique of amyloid demonstration is based on proteomic analysis using laser microdissection/mass spectrometry (LMD/MS), which has the capability for identification of the many different proteins [43]. This technique has a high sensitivity and specificity and solves many of the problems presented by other techniques [44].

LMD/MS has proved to be useful to determine the nature and type of the amyloid precursor protein in cases that could not be typed by routine IHC panels [45, 46]. The major advantage of LMD/MS over conventional IHC techniques in typing of amyloid is that LMD/MS is a single test that can identify and type the amyloid protein, in contrast to IHC that may require several antibodies staining multiple sections [47]. Furthermore, LMD/MS is performed from paraf‐ fin-embedded tissue and requires no special treatment. The common indications for LMD/MS in amyloid nephropathy include amyloid type confirmation, insufficient tissue sample for IF or IHC studies but with remaining tissue in paraffin block, difficult cases on routine renal biopsy studies such as heavy-chain amyloidosis and familial and hereditary forms of amyloi‐ dosis [17, 48, 49]. The diagnosis of amyloidosis at the proteomic level using LMD/MS is purely based on the presence of large spectra of proteins, which have amyloidogenic properties, in addition to apolipoprotein E (Apo E) and SAP that are commonly present in all types of amyloid [40]. Therefore, in AA nephropathy, large spectra numbers of serum amyloidassociated protein along with Apo E and SAP are present. In contrast, in ALECT2 and AGel nephropathy, large spectra numbers of LECT2 and gelsolin are present along with Apo E and SAP.

LMD/MS has been very helpful in achieving amyloid typing from formalin-fixed paraffinembedded tissue. LMD/MS has also been critical for the typing of cases with limited anti‐ body reactivity and in the discovery of new protein types of amyloid. From a study by Said et al., the type of amyloid in 16% of amyloid nephropathy cases could not have been typed without LMD/MS [8]. In spite of these major advantages, LMD/MS also has a few draw‐ backs. As mentioned above, identification of amyloid proteins is purely based on the pres‐ ence of large spectra for proteins, which have amyloidogenic properties within the analyzed sample. In a case of focal amyloidosis, a small amount of amyloid deposit may be obscured by Apo E, SAP, and various serum proteins. Furthermore, the observed peptide fragmentation data must be matched to known protein sequences that are available in the public data base, and thus, unknown protein sequences due to certain germ line polymorphisms or somatic mutations, may not be identified. Despite these drawbacks, the number of cases of amyloid with undetermined type has markedly decreased with use of this technique to 7%, with 3% of the sample insufficient for analysis [7, 8, 50]

## **6.7. Treatment and prognosis**

standard technique for diagnosing AA (**Figure 21**). AA IHC reveals a pattern of distribution matching that seen by LM. From our study, IHC on paraffin-embedded tissue for LECT2 is sensitive; however, it is not entirely specific for ALECT2 [41]. It is recommended that the diagnosis of ALECT2 nephropathy should be confirmed by mass spectrometry analysis of

**Figure 21.** AA nephropathy with intense glomerular and vascular staining for SAA IHC (anti-SAA IHC, original mag‐

In spite of the availability of IF and IHC, there are multiple drawbacks of these methods, such as sensitivity and specificity of commercial antibodies that are designed against only nonmu‐ tated proteins. Mutant proteins and proteins with conformational changes might be less reactive to commercial antibodies [39]. For example, in 7–35% of the AL nephropathy cases, the amyloid deposits were negative for both kappa and lambda IF staining on frozen tissue [7]. Therefore, be aware that a negative staining for light- and/or heavy-chain IF does not ex‐ clude AIg nephropathy. Of note, rare cases of amyloid nephropathy show more than one type of amyloid protein, leading to difficulty in typing. The drawbacks of these techniques may be

The direct technique of amyloid demonstration is based on proteomic analysis using laser microdissection/mass spectrometry (LMD/MS), which has the capability for identification of the many different proteins [43]. This technique has a high sensitivity and specificity and solves

solved by mass spectrometry, which is discussed below.

many of the problems presented by other techniques [44].

nification ×200).

300 302Exploring New Findings on Amyloidosis

**6.6. Proteomics**

renal tissues when the staining pattern shows equivocal Intensity [41].

The goal of current treatment approaches for AIg nephropathy is to eradicate the clone of plasma cells that produce amyloidogenic monoclonal light chains, Ig heavy chain, and Ig heavy and light chain. Patients with AIg nephropathy are usually treated with chemotherapeutic regimens appropriate for B-cell or plasma cell neoplasm. Melphalan-dexamethasone-borte‐ zomib is commonly used as frontline regimen for patients with Stages I and II cardiac involvement [51]. Patients with Stage III cardiac involvement or advanced chronic kidney disease (CKD) patients are generally treated with cyclophosphamide-bortezomib-dexametha‐ sone [51, 52], which showed overall 81.4% hematologic response [53]. High-dose intrave‐ nous melphalan followed by autologous stem cell transplantation (HDM/ASCT) to support bone marrow recovery has emerged as the most likely to eradicate the clonal plasma cells [39].

Experiences from several treatment centers has suggested that 25–50% of patients who undergo such treatment have complete hematologic response, indicating that there is no evidence of ongoing production of monoclonal light chains, Ig heavy chain, or Ig heavy chain and light chain [52–54]. HDM/ASCT is generally offered to patients with Stages I or II cardiac involvement patients who have glomerular filtration rate (GFR) ≥ 30 mL/min in the absence of advanced other organ involvement [54–56].

Treatment of the underlying chronic inflammatory diseases, such as immunosuppressive agents for rheumatoid arthritis and inflammatory bowel diseases is beneficial in patients with AA nephropathy. The basis of modern treatment in AA nephropathy is still reduction of SAA production. Additionally, steroids and cytotoxic drugs, treatment with monoclonal antibod‐ ies against cytokines, particularly tumor necrotic factors (TNF) and interleukin-6 (IL-6), are effective in many cases [57]. This approach of treatment is targeted not only at amyloid fibrils themselves but also at messengers in the acute-phase response. Other therapies, such as antisense oligonucleotides, have been suggested to specifically lower the expression of SAA in the liver [57]. AA nephropathy caused by familial Mediterranean fever responds to colchicine [58]. In familial amyloidosis, including AFib, ATTR, and AApo AI, in which the liver is the source of precursor protein, patients are treated with liver transplantation [57]. A novel drug, R-1-[6-[R-2carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid is a competitive inhibitor of SAP binding to amyloid fibrils [59, 60]. This palindromic compound also cross-links and dimerizes SAP molecules, leading to their very rapid clear‐ ance by the liver, and hence produces a marked reduction in human SAP in the circulation [61]. This mechanism of drug action potently removes SAP from the deposits of amyloid in human tissue and may provide a new therapeutic approach to AA amyloidosis and diseases associ‐ ated with local amyloid deposition, including Alzheimer's disease [61].

The prognosis for patients with AL nephropathy is generally poor and depends on systemic organ involvement, but survival can be prolonged with treatment [62, 63]. Patients with AL nephropathy have a median overall survival of 1–2 years [62, 63]. Patients with AA nephrop‐ athy have a better prognosis than those with AL nephropathy with the median survival varies between 2 and 10 years [13, 64]. In AL nephropathy and AA nephropathy, cardiac involve‐ ment is an independent negative predictive factor of patient survival [13]. Patients with AA, AH, AHL, AFib, and ALECT2 nephropathy have longer overall survival compared with AL nephropathy, largely because of a lower rate of cardiac involvement [13, 65, 66]. The mean survival for AFib is 15 years [10], and the median survival for ALECT nephropathy is 62 months [66].

In AL nephropathy, kidney transplantation is ideally performed after production of amyloi‐ dogenic light-chain proteins has been eradicated. Kidney transplantation for systemic AL patients has a relative good outcome with 1- and 5-year patient survival of 75% and 67% and median graft survival of 5.8 years [67–69]. However, amyloidosis may recur in kidney transplants, depending on the amyloid type and progression of the underlying disease. In a large series from the UK, among 246 patients with AL nephropathy with renal failure, only 10% received kidney transplants [69], and of these, amyloidosis recurred in 28%; however, no graft was lost due to recurrent AL amyloidosis [67]. Patient survival was best among pa‐

tients who had achieved partialremission before kidney transplantation (8.9 years). In a French series of 59 recipients with AA amyloidosis, recurrence was documented in 14% at a median of 10 years posttransplant and was often associated with nephrotic range proteinuria [70]. Among 10 recipients with hereditary AFib amyloidosis who received kidney transplantation only, recurrent AFib amyloidosis was detected in 70% and median graft survival was 7.3 years [69]. In contrast, no recurrence was detected in the nine patients who received combined kidney and liver transplantation, but the mean renal graft survival was 6.4 years [62]. Hereditary AApo I amyloidosis has recurred in 30% who received kidney transplanta‐ tion only, and of these, one graft was lost to recurrent AApo I amyloidosis [68]. There is no effective therapy for ALECT2 nephropathy [37]. Because the precursor protein in ALECT2 is nonmutant, liver transplantation is likely ineffective [26]. Kidney transplantation has been suggested as a treatment option for ALECT2 nephropathy patients with advanced renal failure [35]. Although the disease may recur in about 20% of allografts, no graft was lost to recurrent ALECT2 nephropathy [69]. Possible future therapies for ALECT2 nephropathy include reducing the supply of LECT2 (e.g., inhibiting the Wnt/β-catenin signal pathway), inhibiting amyloid fibril formation by blocking the binding of glycoaminoglycans to amy‐ loid fibrils, promoting clearance of amyloid by immunotherapy, and promoting amyloid regression by SAP-targeted therapy.

## **Acknowledgements**

Experiences from several treatment centers has suggested that 25–50% of patients who undergo such treatment have complete hematologic response, indicating that there is no evidence of ongoing production of monoclonal light chains, Ig heavy chain, or Ig heavy chain and light chain [52–54]. HDM/ASCT is generally offered to patients with Stages I or II cardiac involvement patients who have glomerular filtration rate (GFR) ≥ 30 mL/min in the absence

Treatment of the underlying chronic inflammatory diseases, such as immunosuppressive agents for rheumatoid arthritis and inflammatory bowel diseases is beneficial in patients with AA nephropathy. The basis of modern treatment in AA nephropathy is still reduction of SAA production. Additionally, steroids and cytotoxic drugs, treatment with monoclonal antibod‐ ies against cytokines, particularly tumor necrotic factors (TNF) and interleukin-6 (IL-6), are effective in many cases [57]. This approach of treatment is targeted not only at amyloid fibrils themselves but also at messengers in the acute-phase response. Other therapies, such as antisense oligonucleotides, have been suggested to specifically lower the expression of SAA in the liver [57]. AA nephropathy caused by familial Mediterranean fever responds to colchicine [58]. In familial amyloidosis, including AFib, ATTR, and AApo AI, in which the liver is the source of precursor protein, patients are treated with liver transplantation [57]. A novel drug, R-1-[6-[R-2carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid is a competitive inhibitor of SAP binding to amyloid fibrils [59, 60]. This palindromic compound also cross-links and dimerizes SAP molecules, leading to their very rapid clear‐ ance by the liver, and hence produces a marked reduction in human SAP in the circulation [61]. This mechanism of drug action potently removes SAP from the deposits of amyloid in human tissue and may provide a new therapeutic approach to AA amyloidosis and diseases associ‐

The prognosis for patients with AL nephropathy is generally poor and depends on systemic organ involvement, but survival can be prolonged with treatment [62, 63]. Patients with AL nephropathy have a median overall survival of 1–2 years [62, 63]. Patients with AA nephrop‐ athy have a better prognosis than those with AL nephropathy with the median survival varies between 2 and 10 years [13, 64]. In AL nephropathy and AA nephropathy, cardiac involve‐ ment is an independent negative predictive factor of patient survival [13]. Patients with AA, AH, AHL, AFib, and ALECT2 nephropathy have longer overall survival compared with AL nephropathy, largely because of a lower rate of cardiac involvement [13, 65, 66]. The mean survival for AFib is 15 years [10], and the median survival for ALECT nephropathy is

In AL nephropathy, kidney transplantation is ideally performed after production of amyloi‐ dogenic light-chain proteins has been eradicated. Kidney transplantation for systemic AL patients has a relative good outcome with 1- and 5-year patient survival of 75% and 67% and median graft survival of 5.8 years [67–69]. However, amyloidosis may recur in kidney transplants, depending on the amyloid type and progression of the underlying disease. In a large series from the UK, among 246 patients with AL nephropathy with renal failure, only 10% received kidney transplants [69], and of these, amyloidosis recurred in 28%; however, no graft was lost due to recurrent AL amyloidosis [67]. Patient survival was best among pa‐

ated with local amyloid deposition, including Alzheimer's disease [61].

of advanced other organ involvement [54–56].

302 304Exploring New Findings on Amyloidosis

62 months [66].

The author thanks Dr. Agnes Fogo and Dr. Mark Lusco for their helpful advice.

## **Author details**

Paisit Paueksakon

Address all correspondence to: paisit.paueksakon@vanderbilt.edu

Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA

## **References**


[15] Soutar AK, Hawkins PN, Vigushin DM, Tennent GA, Booth SE, Hutton T, Nguyen O, Totty NF, Feest TG, Hsuan JJ, et al. Apolipoprotein AI mutation Arg-60 causes autosomal dominant amyloidosis. Proc Natl Acad Sci U S A. 1992; 89(16): 7389–93.

[3] Tuglular S, Yalcinkaya F, Paydas S, Oner A, Utas C, Bozfakioglu S, Ataman R, Akpolat T, Ok E, Sen S, Düsünsel R, Evrenkaya R, Akoglu E. A retrospective analysis for aetiology and clinical findings of 287 secondary amyloidosis cases in Turkey. Nephrol

[4] Ensari C, Ensari A, Tümer N, Ertug E. Clinicopathological and epidemiological analysis of amyloidosis in Turkish patients. Nephrol Dial Transplant. 2005; 20(8): 1721–5. [5] Lekpa FK, Ndongo S, Pouye A, Tiendrebeogo JW, Ndao AC, Ka MM, Diop TM.

[6] Larsen CP, Walker PD, Weiss DT, Solomon A. Prevalence and morphology of leuko‐ cyte chemotactic factor 2-associated amyloid in renal biopsies. Kidney Int. 2010 77: 816–

[7] Sethi S, Vrana JA, Theis JD, Dogan A. Mass spectrometry based proteomics in the diagnosis of kidney disease. Curr Opin Nephrol Hypertens. 2013; 22(3):273–80.

[8] Said SM, Sethi S, Valeri AM, Leung N, Cornell LD, Fidler ME, Herrera Hernandez L, Vrana JA, Theis JD, Quint PS, Dogan A, Nasr SH. Renal amyloidosis: origin and clinicopathologic correlations of 474 recent cases. Clin J Am Soc Nephrol. 2013; 8(9):

[9] Manabe S, Hatano M, Yazaki M, Nitta K, Nagata M. Renal AH amyloidosis associat‐ ed with a truncated immunoglobulin heavy chain undetectable by immunostaining.

[10] Markowitz GS. Dysproteinemia and the kidney. Adv Anat Pathol. 2004; 11(1): 49–63. [11] von Hutten H, Mihatsch M, Lobeck H, Rudolph B, Eriksson M, Röcken C. Prevalence and origin of amyloid in kidney biopsies. Am J Surg Pathol. 2009; 33: 1198–1205. [12] Gillmore JD, Lachmann HJ, Rowczenio D, Gilbertson JA, Zeng CH, Liu ZH, Li LS, Wechalekar A, Hawkins PN. Diagnosis, pathogenesis, treatment, and prognosis of hereditary fibrinogen A alpha-chain amyloidosis. J Am Soc Nephrol. 2009; 20(2): 444–

[13] Bergesio F, Ciciani AM, Manganaro M, Palladini G, Santostefano M, Brugnano R, Di Palma AM, Gallo M, Rosati A, Tosi PL, Salvadori M.; Immunopathology Group of the Italian Society of Nephrology. Renal involvement in systemic amyloidosis: an Italian collaborative study on survival and renal outcome. Nephrol Dial Transplant. 2008;

[14] Sethi S, Thesis JD, Quint P, Maierhofer W, Kurtin PJ, Dogan A, Highsmith EW Jr. Renal amyloidosis associated with a novel sequence variant of gelsolin. Am J Kidney Dis.

Amyloidosis in sub-Saharan Africa. Med Sante Trop. 2012; 22(3): 275–8.

Dial Transplant. 2002; 17(11): 2003–5.

Am J Kidney Dis. 2015; 66(6): 1095–100.

819.

304 306Exploring New Findings on Amyloidosis

1515–23.

51.

23(3): 941–51.

2013; 1: 161–166.


[42] Westermark GT, Johnson KH, Westermark P. Staining methods for identification of amyloid in tissue. Methods Enzymol. 1999; 309: 3–25.

[28] Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer's disease: progress and

[29] McLaurin J, Yang D, Yip CM, Fraser PE. Review: modulating factors in amyloid-beta

[30] Kisilevsky R, Manley PN. Acute-phase serum amyloid A: perspectives on its physio‐

[31] Verdone G, Corazza A, Viglino P, Pettirossi F, Giorgetti S, Mangione P, Andreola A, Stoppini M, Bellotti V, Esposito G. The solution structure of human beta2-microglobu‐ lin reveals the prodromes of its amyloid transition. Protein Sci. 2002; 11(3): 487–99.

[32] Scholefield Z, Yates EA, Wayne G, Amour A, McDowell W, Turnbull JE. Heparan sulfate regulates amyloid precursor protein processing by BACE1, the Alzheimer's

[33] Ding Y, Kim JK, Kim SI, Na HJ, Jun SY, Lee SJ, Choi ME. TGF-{beta}1 protects against mesangial cell apoptosis via induction of autophagy. J Biol Chem. 2010; 285(48): 37909–

[34] Teng J, Russell WJ, Gu X, Cardelli J, Jones ML, Herrera GA. Different types of glomer‐ ulopathic light chains interact with mesangial cells using a common receptor but exhibit

[35] Teng J, Turbat-Herrera EA, Herrera GA. Extrusion of amyloid fibrils to the extracellu‐ lar space in experimental mesangial AL-amyloidosis: transmission and scanning electron microscopy studies and correlation with renal biopsy observations. Ultras‐

[36] Herrera GA, Turbat-Herrera EA. Renal diseases with organized deposits: an algorith‐ mic approach to classification and clinicopathologic diagnosis. Arch Pathol Lab Med.

[37] Nasr SH, Dogan A, Larsen CP. Leukocyte cell-derived chemotaxin 2-associated amyloidosis: a recently recognized disease with distinct clinicopathologic characteris‐

[38] Richards DB, Cookson LM, Berges AC, Barton SV, Lane T, Ritter JM, Fontana M, Moon JC, Pinzani M, Gillmore JD, Hawkins PN, Pepys MB. Therapeutic clearance of amyloid by antibodies to serum amyloid P component. N Engl J Med. 2015; 373(12): 1106–14.

[39] Dember LM. Amyloidosis-associated kidney disease. J Am Soc Nephrol. 2006; 17(12):

[40] Dikman SH, Churg J, Kahn T. Morphologic and clinical correlates in renal amyloido‐

[41] Elghetany MT, Saleem A, Barr K. The Congo red stain revisited. Ann Clin Lab Sci. 1989;

different intracellular trafficking patterns. Lab Invest. 2004; 84(4): 440–51.

problems on the road to therapeutics. Science. 2002; 297(5580): 353–6.

fibril formation. Struct Biol. 2000; 130(2–3): 259–70.

beta-secretase. J Cell Biol. 2003; 163(1): 97–107.

tics. Clin J Am Soc Nephrol. 2015; 10(11): 2084–93.

truct Pathol. 2014;38(2):104–15.

sis. Hum Pathol. 1981; 12(2): 160–9.

2010; 134(4): 512–31.

3458–71.

19(3) :190–5.

19.

306 308Exploring New Findings on Amyloidosis

logical and pathological roles. Amyloid. 2012;19 (1): 5–14.


melphalan and autologous stem-cell transplantation in patients with AL amyloidosis: an 8-year study. Ann Intern Med. 2004; 140(2): 85–93.


[66] Said SM, Sethi S, Valeri AM, Chang A, Nast CC, Krahl L,Molloy P, Barry M, Fidler ME, Cornell LD, Leung N, Vrana JA, Theis JD, Dogan A, Nasr SH. Characterization and outcomes of renal leukocyte chemotactic factor 2-associated amyloidosis. Kidney Int. 2014; 86(2): 370–7.

melphalan and autologous stem-cell transplantation in patients with AL amyloidosis:

[55] Gertz MA, Lacy MQ, Dispenzieri A, Gastineau DA, Chen MG, Ansell SM, Inwards DJ, Micallef IN, Tefferi A, Litzow MR. Stem cell transplantation for the management of

[56] Moreau P, Leblond V, Bourquelot P, Facon T, Huynh A, Caillot D, Hermine O, Attal M, Hamidou M, Nedellec G, Ferrant A, Audhuy B, Bataille R, Milpied N, Harous‐ seau JL. Prognostic factors for survival and response after high-dose therapy and autologous stem cell transplantation in systemic AL amyloidosis: a report on 21

[57] Westermark GT, Fändrich M, Westermark P. AA amyloidosis: pathogenesis and

[58] Meneses CF, Egües CA, Uriarte M, Belzunegui J, Rezola M. Colchicine use in isolated

[59] Sahota T, Berges A, Barton S, Cookson L, Zamuner S, Richards D. Target mediated drug disposition model of CPHPC in patients with systemic amyloidosis. CPT Pharmacom

[60] Kolstoe SE, Mangione PP, Bellotti V, Taylor GW, Tennent GA, Deroo S, Morrison AJ, Cobb AJ, Coyne A, McCammon MG, Warner TD, Mitchell J, Gill R, Smith MD, Ley SV, Robinson CV, Wood SP, Pepys MB. Trapping of palindromic ligands within native transthyretin prevents amyloid formation. Proc Natl Acad Sci U S A. 2010; 107(47):

[61] Pepys MB, Herbert J, Hutchinson WL, Tennent GA, Lachmann HJ, Gallimore JR, Lovat LB, Bartfai T, Alanine A, Hertel C, Hoffmann T, Jakob-Roetne R, Norcross RD, Kemp JA, Yamamura K, Suzuki M, Taylor GW, Murray S, Thompson D, Purvis A, Kolstoe S, Wood SP, Hawkins PN. Targeted pharmacological depletion of serum amyloid P component for treatment of human amyloidosis. Nature. 2002; 417(6886): 254–9. [62] Kyle RA, Linos A, Beard CM, Linke RP, Gertz MA, O'Fallon WM, Kurland LT. Incidence and natural history of primary systemic amyloidosis in Olmsted County,

[63] Falk RH, Comenzo RL, Skinner M. The systemic amyloidoses. N Engl J Med. 1997;

[64] Gillmore JD, Hawkins PN, Pepys MB. Amyloidosis: a review of recent diagnostic and

[65] Nasr SH, Said SM, Valeri AM, Sethi S, Fidler ME, Cornell LD, Gertz MA, Dispenzieri A, Buadi FK, Vrana JA, Theis JD, Dogan A, Leung N. The diagnosis and characteris‐ tics ofrenal heavy-chain and heavy/light-chain amyloidosis and their comparison with

an 8-year study. Ann Intern Med. 2004; 140(2): 85–93.

patients. Br J Haematol. 1998; 101(4):766–9.

Syst Pharmacol. 2015; 4(2): e15.

20483–8.

308 310Exploring New Findings on Amyloidosis

337(13): 898–909.

targeted therapy. Annu Rev Pathol. 2015; 10: 321–44.

renal AA amyloidosis. Reumatol Clin. 2015; 11(4): 242–3.

Minnesota, 1950 through 1989. Blood. 1992; 79(7): 1817–22.

therapeutic developments. Br J Haematol. 1997; 99(2): 245–56.

renal light-chain amyloidosis. Kidney Int. 2013; 83(3): 463–70.

primary systemic amyloidosis. Am J Med. 2002; 113(7): 549–55.


## **A Nanobody‐Based Approach to Amyloid Diseases, the Gelsolin Case Study**

Adriaan Verhelle and Jan Gettemans

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63981

#### **Abstract**

Gelsolin amyloidosis (AGel) is an autosomal‐dominant inherited disease caused by point mutations in the gelsolin gene. At the protein level, these mutations result in the loss of a Ca2+‐binding site, crucial for the correct folding and function. In the trans‐Golgi network, this mutant plasma gelsolin is cleaved by furin, giving rise to a 68 kDa Cterminal fragment. When secreted in the extracellular matrix, this fragment under‐ goes proteolysis by MT1‐MMP–like proteases, resulting in the production of 8 and 5  kDa amyloidogenic peptides. Nanobodies, the variable part of the heavy chain of heavy‐ chain antibodies, have been used as molecular chaperones for mutant plasma gelsolin and the 68 kDa C‐terminal fragment in an attempt to inhibit their pathogenic proteoly‐ sis. Furthermore, these nanobodies have also been tested and applied as a 99mTc‐based imaging agent in the gelsolin amyloidosis mouse model.

**Keywords:** AGel, Gelsolin, Nanobody, SPECT/CT, VHH, FAF

## **1. Introduction**

Gelsolin amyloidosis is an autosomal‐dominant inherited disease caused by a point mutation inthegelsolingene;G640AandG640Taremost common;G580AandC633Aweremore recently discovered [1–3]. At the protein level, these mutations result in the amino acid substitutions D187N, D187Y, G167R and N184K in gelsolin domain 2. Consequently, a Ca2+‐binding site, crucial for the correct folding and function, is lost. As a result, mutant plasma gelsolin (PG\*) adopts an intermediate state between active and inactive, thereby negatively influencing the overall structural stabilityoftheproteinandexposingacryptic,otherwiseburied,furincleavage

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

site [4]. In the trans‐Golgi network, this intermediate form of mutant plasma gelsolin is susceptible to furin cleavage, giving rise to a 68 kDa C‐terminal fragment (C68) [5]. In its turn, C68 is cleaved by MT1‐MMP–like proteases during secretion into the extracellular matrix. This MMP activity results in the formation of 8 and 5 kDa amyloidogenic peptides, which polymer‐ ize into mature amyloid fibrils [6]. Patients are generally heterozygous for the mutations and experience a triad of neurological, ophthalmological and dermatological symptoms starting from their thirties [7]. Given the importance of furin and MT1‐MMP–like proteases in cell homeostasis, a classical small compound therapy targeting these proteases is unlikely to be successful. Moreover, the gain‐of‐toxicity nature of this disease rules out replacement gene therapy. In gelsolin amyloidosis, the mutant gelsolin gene still undergoes normal expression. Curing gelsolin amyloidosis through gene therapy would therefore require the deletion of the mutant gene from every single cell. This is (at the moment) not possible with the available gene therapy technology. Shielding the mutant gelsolin from furin and MT1‐MMP–like proteases, however,provedtobeaworthwhileapproachtotacklethisdisease.Oneofthatmaybeapplicable to other similar amyloid diseases.

Using nanobodies, the variable part of the heavy chain of heavy‐chain antibodies, two routes to address AGel have been explored. In a first approach, nanobodies, which partly protect C68 against MT1‐MMP, were intraperitoneally injected in AGel mice [8]. In a second approach, a mouse model expressing Nb11 was developed [9]. Nb11 binds to mutant plasma gelsolin (PG\*) and shields it from furin degradation. The mouse model secretes this nanobody in its bloodstream. These mice were crossed with AGel mice. During the secretion pathway, Nb11 encounters PG\* in the trans-Golgi network. Both techniques resulted in a reduced deposi‐ tion of amyloidogenic gelsolin.

The nanobodies binding to C68 are also capable of recognizing the 8 kDa amyloidogenic fragment. This characteristic was further explored to develop a 99mTc‐based imaging agent.

## **2. Gelsolin amyloidosis**

## **2.1. Discovery**

In 1969, the Finnish ophthalmologist Jouko Meretoja [10] first described a new familial amyloidosis syndrome characterized by corneal dystrophy, cranial neuropathies and skin affliction. Over the years, this syndrome came to be known under many different names: Meretoja's disease, amyloid polyneuropathy type IV, gelsolin‐related amyloidosis, familial amyloidosis of the Finnish type (FAF), corneal lattice dystrophy type II and gelsolin amyloi‐ dosis (AGel).

It was not until 1990 that the role of gelsolin in AGel was demonstrated. Two independent research groups had isolated and sequenced the amyloid fibrils from patient tissues [11, 12]. The fragments appeared to be an internal part of a gelsolin variant having an aspartate to asparagine mutation at position 187 ofthe primary structure of gelsolin. AGeltherefore seemed to arise from an abnormal internal degradation from this gelsolin variant.

In the following years, the role of gelsolin, the genetic cause and pathogenic pathway were further elaborated. At the moment, there are four known mutations, which cause gelsolin‐ related amyloidosis: D187N, D187Y, G167R and the recently discovered N184K [1–3].

## **2.2. Biochemistry and genetics of gelsolin**

site [4]. In the trans‐Golgi network, this intermediate form of mutant plasma gelsolin is susceptible to furin cleavage, giving rise to a 68 kDa C‐terminal fragment (C68) [5]. In its turn, C68 is cleaved by MT1‐MMP–like proteases during secretion into the extracellular matrix. This MMP activity results in the formation of 8 and 5 kDa amyloidogenic peptides, which polymer‐ ize into mature amyloid fibrils [6]. Patients are generally heterozygous for the mutations and experience a triad of neurological, ophthalmological and dermatological symptoms starting from their thirties [7]. Given the importance of furin and MT1‐MMP–like proteases in cell homeostasis, a classical small compound therapy targeting these proteases is unlikely to be successful. Moreover, the gain‐of‐toxicity nature of this disease rules out replacement gene therapy. In gelsolin amyloidosis, the mutant gelsolin gene still undergoes normal expression. Curing gelsolin amyloidosis through gene therapy would therefore require the deletion of the mutant gene from every single cell. This is (at the moment) not possible with the available gene therapy technology. Shielding the mutant gelsolin from furin and MT1‐MMP–like proteases, however,provedtobeaworthwhileapproachtotacklethisdisease.Oneofthatmaybeapplicable

Using nanobodies, the variable part of the heavy chain of heavy‐chain antibodies, two routes to address AGel have been explored. In a first approach, nanobodies, which partly protect C68 against MT1‐MMP, were intraperitoneally injected in AGel mice [8]. In a second approach, a mouse model expressing Nb11 was developed [9]. Nb11 binds to mutant plasma gelsolin (PG\*) and shields it from furin degradation. The mouse model secretes this nanobody in its bloodstream. These mice were crossed with AGel mice. During the secretion pathway, Nb11 encounters PG\* in the trans-Golgi network. Both techniques resulted in a reduced deposi‐

The nanobodies binding to C68 are also capable of recognizing the 8 kDa amyloidogenic fragment. This characteristic was further explored to develop a 99mTc‐based imaging agent.

In 1969, the Finnish ophthalmologist Jouko Meretoja [10] first described a new familial amyloidosis syndrome characterized by corneal dystrophy, cranial neuropathies and skin affliction. Over the years, this syndrome came to be known under many different names: Meretoja's disease, amyloid polyneuropathy type IV, gelsolin‐related amyloidosis, familial amyloidosis of the Finnish type (FAF), corneal lattice dystrophy type II and gelsolin amyloi‐

It was not until 1990 that the role of gelsolin in AGel was demonstrated. Two independent research groups had isolated and sequenced the amyloid fibrils from patient tissues [11, 12]. The fragments appeared to be an internal part of a gelsolin variant having an aspartate to asparagine mutation at position 187 ofthe primary structure of gelsolin. AGeltherefore seemed

to arise from an abnormal internal degradation from this gelsolin variant.

to other similar amyloid diseases.

312 314Exploring New Findings on Amyloidosis

tion of amyloidogenic gelsolin.

**2. Gelsolin amyloidosis**

**2.1. Discovery**

dosis (AGel).

The gelsolin superfamily of actin‐binding proteins comprises gelsolin, villin, supervillin, advillin, villin‐like protein, severin, fragmin CapG (gCap39) and flightless I [13–16]. These proteins are highly conserved in the animal kingdom, and all contain three or more prototyp‐ ical 'gelsolin‐like' domains. A 'gelsolin‐like' domain consists of five or six beta‐sheets packed between a long roughly parallel and a short roughly perpendicular alpha helix [17, 18].

Gelsolin was described in 1979 as a protein able to transform solid actin gels into a soluble solution: the gel‐sol reaction, hence the name. Present in almost every human tissue, gelsolin is capable of binding, severing and capping actin filaments and plays a pivotal role in cytoskeletal homeostasis [19]. It comes in three isoforms, all derived from the same gelsolin gene located at chromosome 9q34. Alternative splicing gives rise to cytoplasmic gelsolin (CG, 82 kDa), plasma gelsolin (PG, 84 kDa) and gelsolin‐3 (82 kDa). The first two are the main isoforms, with PG differing from CG by a 24 amino acid, N‐terminal extension, which remains present after cleavage of the signal sequence, and the presence of a disulfide bond. The third variant has only been detected in brain oligodendrocytes, lungs and testis tissue and is characterized by 11 additional residues at the N‐terminus [20].

**Figure 1.** Schematic representation of gelsolin. Black boxes represent the six gelsolin domains. Calcium‐binding sites (grey and black dots), actin‐binding sites (red), PIP2‐-binding sites (yellow) and caspase‐3–sensitive linker (blue arrow) are indicated.

Gelsolin is composed of six domains (G1–6) of 120–130 amino acids which, in a calcium‐free environment, are closely packed together. This inactive state is controlled by three latches: the tail latch, formed by the C‐terminus and G2 domain, the G1–G3 latch and the G4–G6 latch [21].

Upon binding of calcium, the three latches open up and expose the actin‐binding surfaces of domains 2, 1 and 4, respectively. Following this activation, gelsolin will bind and subsequent‐ ly sever F‐actin [4]. The sequence homology between G1–3 and G4–6 suggests that the protein arose from a gene triplication followed by a duplication [22]. The two homologous halves are connected through a caspase‐3–sensitive linker (**Figure 1**). Overall, stability and function of gelsolin are regulated through Ca2+ and phosphatidylinositol 4,5‐biphosphate (PIP2). Each domain contains a Ca2+‐binding site, and G1 and G2 also have PIP2‐binding capacity (**Figure 1**) [14].

The G2 domain has two major foci of stability. First, there is the Ca2+‐binding pocket (Kd 650  nM) formed by residues D187, E209 and D259 [23]. Second, L166, Q164, N184 and D187 form cooperative hydrogen bonds with one another [18]. Mutation of any of these residues leads to a destabilization of G2 and consequently of gelsolin as a whole. This is actually what hap‐ pens in AGel. Three out of four of the known mutations (D187N, D187Y and N184K) can be directly traced back to one of these two foci of stability. The fourth mutation, G167R, possi‐ bly has a destabilizing effect on hydrogen bonding as it is close to Q164. It should be noted that both the N184K and G167R mutations have only recently been discovered in two isolated patients and their kindred [2, 3]. The overall majority of patients carry the D187N/Y muta‐ tion, but it is nevertheless noteworthy to observe that these pathological mutations are all in G2 of gelsolin, pointing to this region as a gelsolin stability sensor.

Although the causative mutations of AGel are present in all three gelsolin isoforms, the plasma variant is the sole source of gelsolin amyloidogenic peptides [24]. As mentioned earlier, the overall stability of gelsolin is highly regulated by Ca2+ binding. The AGel mutant plasma gelsolin form can no longer properly bind Ca2+ through its G2 domain. Consequently, this aberrant variant gives rise to a structurally intermediate state between active and inactive. This exposes an otherwise internal inaccessible furin cleavage site (R‐X‐X‐R) at the surface of gelsolin.

Furin is a membrane‐bound member of the proprotein convertase family, active in the endosomal and lysosomal pathways. It shuttles between the trans‐Golgi network and the cell surface while activating a wide range of serum proteins, hormones and receptors [25–27]. During secretion, PG naturally encounters furin without any consequence since wild‐type gelsolin is already correctly folded due to high calcium levels in the ER/Golgi compartments. PG\* on the other hand is susceptible to pathological furin processing due to its induced structural intermediate state. The scissile bond between R172 and A173 is cleaved, thereby releasing a 68 kDa C‐terminal fragment [28]. During secretion, this aberrant C68 constitutes a substrate for MT1‐MMP–like proteases present in the extracellular matrix. MT1‐MMP–like proteases are members of the matrix metalloprotease family [29]. They play a pivotal role in protein degradation processes during embryonic development and tissue remodelling. Here, however, 8 kDa (AA 173–243) and 5 kDa (AA 173–225) gelsolin amyloidogenic peptides are generated (**Figure 2**). Over time, the 8 and 5 kDa fragments start to aggregate in a cross‐beta sheet configuration, a hallmark of amyloid fibrils with the former being the major compo‐ nent of gelsolin amyloid in patients.

A Nanobody‐Based Approach to Amyloid Diseases, the Gelsolin Case Study http://dx.doi.org/10.5772/63981 315 317

**Figure 2.** Molecular characteristics of AGel: non‐pathological versus pathological plasma gelsolin processing. The left‐ hand side depicts the non-pathological synthesis and secretion of wild‐type plasma gelsolin through the ER and Golgi‐ network. On the right hand, the mechanism for mutant plasma gelsolin is depicted. Furin cleaves PG\* in the trans‐ Golgi network, thereby releasing a C68 fragment. During secretion, C68 is cleaved by MT1‐MMP–like proteases, forming 8 and 5 kDa amyloidogenic fragments in the extracellular matrix.

#### **2.3. Diagnosis and treatment**

Upon binding of calcium, the three latches open up and expose the actin‐binding surfaces of domains 2, 1 and 4, respectively. Following this activation, gelsolin will bind and subsequent‐ ly sever F‐actin [4]. The sequence homology between G1–3 and G4–6 suggests that the protein arose from a gene triplication followed by a duplication [22]. The two homologous halves are connected through a caspase‐3–sensitive linker (**Figure 1**). Overall, stability and function of gelsolin are regulated through Ca2+ and phosphatidylinositol 4,5‐biphosphate (PIP2). Each domain contains a Ca2+‐binding site, and G1 and G2 also have PIP2‐binding capacity (**Figure 1**)

The G2 domain has two major foci of stability. First, there is the Ca2+‐binding pocket (Kd 650  nM) formed by residues D187, E209 and D259 [23]. Second, L166, Q164, N184 and D187 form cooperative hydrogen bonds with one another [18]. Mutation of any of these residues leads to a destabilization of G2 and consequently of gelsolin as a whole. This is actually what hap‐ pens in AGel. Three out of four of the known mutations (D187N, D187Y and N184K) can be directly traced back to one of these two foci of stability. The fourth mutation, G167R, possi‐ bly has a destabilizing effect on hydrogen bonding as it is close to Q164. It should be noted that both the N184K and G167R mutations have only recently been discovered in two isolated patients and their kindred [2, 3]. The overall majority of patients carry the D187N/Y muta‐ tion, but it is nevertheless noteworthy to observe that these pathological mutations are all in

Although the causative mutations of AGel are present in all three gelsolin isoforms, the plasma variant is the sole source of gelsolin amyloidogenic peptides [24]. As mentioned earlier, the overall stability of gelsolin is highly regulated by Ca2+ binding. The AGel mutant plasma gelsolin form can no longer properly bind Ca2+ through its G2 domain. Consequently, this aberrant variant gives rise to a structurally intermediate state between active and inactive. This exposes an otherwise internal inaccessible furin cleavage site (R‐X‐X‐R) at the surface of

Furin is a membrane‐bound member of the proprotein convertase family, active in the endosomal and lysosomal pathways. It shuttles between the trans‐Golgi network and the cell surface while activating a wide range of serum proteins, hormones and receptors [25–27]. During secretion, PG naturally encounters furin without any consequence since wild‐type gelsolin is already correctly folded due to high calcium levels in the ER/Golgi compartments. PG\* on the other hand is susceptible to pathological furin processing due to its induced structural intermediate state. The scissile bond between R172 and A173 is cleaved, thereby releasing a 68 kDa C‐terminal fragment [28]. During secretion, this aberrant C68 constitutes a substrate for MT1‐MMP–like proteases present in the extracellular matrix. MT1‐MMP–like proteases are members of the matrix metalloprotease family [29]. They play a pivotal role in protein degradation processes during embryonic development and tissue remodelling. Here, however, 8 kDa (AA 173–243) and 5 kDa (AA 173–225) gelsolin amyloidogenic peptides are generated (**Figure 2**). Over time, the 8 and 5 kDa fragments start to aggregate in a cross‐beta sheet configuration, a hallmark of amyloid fibrils with the former being the major compo‐

G2 of gelsolin, pointing to this region as a gelsolin stability sensor.

[14].

314 316Exploring New Findings on Amyloidosis

gelsolin.

nent of gelsolin amyloid in patients.

The clinical diagnosis of AGel most commonly starts with the detection of corneal lattice dystrophy [30]. Together with cutis laxis and bilateral facial pareses, it forms a triad of consistent features differentiating AGel from other amyloid disorders. The diagnosis can be easily confirmed using molecular genetics. In older or homozygous patients, renal amyloido‐ sis, resulting in proteinuria, can also be a first clue [7].

No specific treatment is currently available. Only symptomatic treatments are being offered to improve the overall quality of life. Most important is good ophthalmological care ranging from eye drops to corneal transplantation. Next to that, aesthetic surgery is often needed to ameliorate the patients' overall confidence as the facial pareses and cutis laxis burden the patients with a constant droopy facial expression [31].

Although therapeutic strategies involving the inhibition of protease activity have been suggested, unwanted side effects are to be expected given the major physiologicalroles offurin and MT1‐MMP [28, 29]. Tackling the problem the other way round, directly shielding PG\* from degradation, may prove to be a more valuable route, as will be discussed later in this chapter.

## **2.4. A gelsolin amyloidosis mouse model**

To further study the molecular aspects of the disease and to test the different therapeutic avenues, two animal models were set up. In a first attempt, the gene coding for the 8 kDa amyloidogenic fragment was successfully transferred into the genome of *Drosophila mela‐ nogaster*. Unfortunately, AGel amyloid deposition could not be detected [7].

As of 2009, a mouse model is available that faithfully recapitulates the entire proteolytic cascade of human PG\* [32]. Following the electroporation of the human plasma GSN cDNA, carrying the D187N mutation, into mouse ES cells, a transgenic animal was obtained where gelsolin is expressed under control of a muscle creatine kinase (MCK) promoter. PG\*, C68 and the 8 and 5 kDa amyloidogenic fragments were detected in the heart, skeletal muscle, diaphragm and skin. Plasma samples contain both full length PG\* and C68. With age, increasing amounts of gelsolin‐positive amyloid depositions can be seen in the endomysium. Eventually, the phenotype of old homozygous D187N mice starts to resemble sporadic inclusion body myositis (sIBM) with accompanied muscle weakness.

## **3. Nanobodies**

## **3.1. Research and clinic applications of conventional antibodies**

Since the introduction of hybridoma technology in 1975, monoclonal antibodies (mAb) have become an indispensable tool in fundamentalresearch and all sorts of clinical applications [33]. The ability to mass produce antibodies against a plethora of proteins, carbohydrates, nucleic acids and haptens gave rise to molecular diagnostic tests, such as ELISA, Western blotting, immunohistochemistry (IHC) and immunofluorescence. Antibodies in the clinic are used in diagnostic applications by linkage to radioactive or fluorescent moieties. Furthermore, they are also being applied therapeutically, be it directly or as a route for drug delivery. As the applications became more and more sophisticated and diversified, the potential pitfalls of this technology also surfaced. First, the production of vast amounts of mAbs is very expensive. Second, the mAb format is hard to manipulate to obtain site‐specific linkage to other mole‐ cules such as fluorophores and radioactive nuclides. Last but not least, mAb hold the poten‐ tial of evoking an immune response rendering the therapy ineffective. This last hurdle has been addressed with the production of so‐called humanized antibodies. These second‐ generation antibodies contain a humanized antigen‐binding fragment (Fab). Third‐genera‐ tion antibodies go one step further and also include an engineered Fc domain to improve the therapeutic activity in patients, particularly in subpopulations expressing low affinity variants of the Fc receptor [34].

Conventional antibodies are comprised of 2 heavy chains and 2 light chains. This multichain nature is one of the major hurdles when trying to manipulate mAb. Attempts have been made to bypass this by producing mAb fragments such as antigen‐binding fragments and single‐ chain variable fragments (scFv). The first, Fab, is composedofthe variable andconstantdomain of a heavy chain, combined with the variable and constant domain of a light chain. The second, scFv, only contains the variable parts of the heavy and light chain, but these are interconnect‐ ed through a linker. Although these smaller formats ameliorated some of the problems posed by classical mAbs, their production still proved to be troublesome and the functionality of the original mAb was (partly) lost in some cases.

## **3.2. Discovery of nanobodies**

**2.4. A gelsolin amyloidosis mouse model**

316 318Exploring New Findings on Amyloidosis

**3. Nanobodies**

of the Fc receptor [34].

To further study the molecular aspects of the disease and to test the different therapeutic avenues, two animal models were set up. In a first attempt, the gene coding for the 8 kDa amyloidogenic fragment was successfully transferred into the genome of *Drosophila mela‐*

As of 2009, a mouse model is available that faithfully recapitulates the entire proteolytic cascade of human PG\* [32]. Following the electroporation of the human plasma GSN cDNA, carrying the D187N mutation, into mouse ES cells, a transgenic animal was obtained where gelsolin is expressed under control of a muscle creatine kinase (MCK) promoter. PG\*, C68 and the 8 and 5 kDa amyloidogenic fragments were detected in the heart, skeletal muscle, diaphragm and skin. Plasma samples contain both full length PG\* and C68. With age, increasing amounts of gelsolin‐positive amyloid depositions can be seen in the endomysium. Eventually, the phenotype of old homozygous D187N mice starts to resemble sporadic

Since the introduction of hybridoma technology in 1975, monoclonal antibodies (mAb) have become an indispensable tool in fundamentalresearch and all sorts of clinical applications [33]. The ability to mass produce antibodies against a plethora of proteins, carbohydrates, nucleic acids and haptens gave rise to molecular diagnostic tests, such as ELISA, Western blotting, immunohistochemistry (IHC) and immunofluorescence. Antibodies in the clinic are used in diagnostic applications by linkage to radioactive or fluorescent moieties. Furthermore, they are also being applied therapeutically, be it directly or as a route for drug delivery. As the applications became more and more sophisticated and diversified, the potential pitfalls of this technology also surfaced. First, the production of vast amounts of mAbs is very expensive. Second, the mAb format is hard to manipulate to obtain site‐specific linkage to other mole‐ cules such as fluorophores and radioactive nuclides. Last but not least, mAb hold the poten‐ tial of evoking an immune response rendering the therapy ineffective. This last hurdle has been addressed with the production of so‐called humanized antibodies. These second‐ generation antibodies contain a humanized antigen‐binding fragment (Fab). Third‐genera‐ tion antibodies go one step further and also include an engineered Fc domain to improve the therapeutic activity in patients, particularly in subpopulations expressing low affinity variants

Conventional antibodies are comprised of 2 heavy chains and 2 light chains. This multichain nature is one of the major hurdles when trying to manipulate mAb. Attempts have been made to bypass this by producing mAb fragments such as antigen‐binding fragments and single‐ chain variable fragments (scFv). The first, Fab, is composedofthe variable andconstantdomain of a heavy chain, combined with the variable and constant domain of a light chain. The second,

*nogaster*. Unfortunately, AGel amyloid deposition could not be detected [7].

inclusion body myositis (sIBM) with accompanied muscle weakness.

**3.1. Research and clinic applications of conventional antibodies**

By a stroke of luck in 1993, a new type of antibody was discovered in the blood of Camelus dromedarius [35]. These antibodies consist solely oftwo heavy chains; hence, they were named heavy‐chain antibodies (HCAbs). Their paratope is no longer formed by a combination of a heavy and light chain variable fragment but consists exclusively of the variable fragment of a heavy chain. The single‐domain nature of this paratope offered the natural solution for which researchers had been looking for. It has since been named variable domain of the heavy chain of heavy‐chain antibodies, VHH in short or Nanobody® (www.ablynx.com) (**Figure 3**). Since their discovery, these HCAbs have been detected in the blood of all Camelidae family members, nurse sharks, wobbegongs and ratfish.

**Figure 3.** Schematic representation of a conventional antibody, heavy‐chain antibody and a nanobody. A conventional antibody (left) consists of two heavy chains (CH3, CH2, CH1 and VH) and two light chains (CL and V<sup>l</sup> ). Heavy‐chain antibodies (middle) only consist of two heavy chains (CH3, CH2 and VHH). A nanobody (right) corresponds with the variable part of the heavy chain of heavy‐chain antibodies (VHH).

#### **3.3. Properties and production of nanobodies**

The most important novelty about nanobodies was, and still is, the fact that they are the smallest, single‐domain, natural, antigen‐binding fragment available. Their complementary determining regions 3 (CDR3) sequences are on average longer (16–18 AA) compared those of regular human antibodies (12 AA). Not only does this compensate for the absence of a light chain, but it also allows the CDR3 to form protruding loops that are able to bind cryptic epitopes. The fact that they are encoded by a single short gene fragments allows easy and straightforward protein engineering, thereby overcoming one of the mayor hurdles of the multichain nature of conventional Abs. By the same token, it allows relatively easy produc‐ tion in bacteria and yeast cells. With yields of up to 40–70 mg/l, nanobodies can be produced at a fraction of the cost of mAbs. When produced, their high resistance towards chaotropic reagents or prolonged high temperatures and stability at high concentrations allows easy storage without any functionality loss.

## **3.4. Nanobody applications**

Nanobodies, being small and stable, are ideal candidates for various research purposes, diagnostic or clinical applications. Equipped with a radionuclide, they can be applied as imaging tracers in a single‐photon emission computed tomography (SPECT) or positron emission tomography (PET)‐based set‐up allowing in vivo visualization of tumours, arthero‐ sclerotic plaques and rheumatoid arthritis‐associated inflammation sites [36–38]. Nanobod‐ ies are also capable of stabilizing proteins in a particular conformation and of minimizing structural flexibility. This has been nicely demonstrated in the case of human lysozyme amyloidosis, a hereditary systemic disease associated with at least seven amyloidogenic lysozyme variants [39, 40]. Aggregation of these lysozyme variants leads to the accumula‐ tion of amyloid in the extracellular matrix of several tissues and organs. Two nanobodies have been identified who, upon binding mutant amyloidogenic lysozyme (D67H and I56T), restored the global cooperativity characteristics of the wild‐type protein through direct contact and long‐range conformational effects. What's more, this stabilizing capacity can also be condu‐ cive to crystallizing proteins. β2‐microglobulin–specific nanobodies have been shown to serve as efficient crystallization chaperones fortransientintermediate species during fibrillation [41]. This type of antibody chaperone co‐crystallization was also used to unravel the structure of the G protein‐coupled receptor β‐2 adrenergic receptor (β2AR), research which received the 2012 Nobel Prize in Chemistry [42].

Another major field where the potential use of nanobodies is currently being explored is amyloidosis. In Alzheimer's research, nanobodies have been found which inhibit beta‐ secretase 1 (BACE1) activity, the first step in amyloid beta formation, both in vitro and in vivo [43]. Alternatively, nanobodies could also be raised against amyloid precursor protein (APP) to protect it from BACE1 degradation, as direct inhibition of the enzyme could evoke unwanted side effects. We present here the case of gelsolin amyloidosis where this approach has proven successful both in vitro and in vivo. The remainder of this chapter will focus on the obtained results, current status and future prospects regarding this AGel research arena.

## **4. Extracellular targeting of the AGel pathological pathway**

Consecutive aberrant cleavage of PG\* by furin and MT1‐MMP–like proteases leads to the production of amyloidogenic fragments which eventually polymerize into fibrils, causing gelsolin amyloidosis pathology. But these two proteases are active in different compart‐ ments. Indeed, whereas furin is active in the (intracellular) ER‐Golgi compartments, MT1‐ MMP is inserted in the plasma membrane and oriented to the extracellular environment. This has far‐reaching consequences in terms of molecular therapeutic avenues that should be followed when gelsolin has to be protected from cleavage by either of these proteases. As the second cleavage step occurs in the extracellular matrix, we reasoned that it might be possi‐ ble to block this process by intraperitoneal administration of C68 chaperone nanobodies [8].

multichain nature of conventional Abs. By the same token, it allows relatively easy produc‐ tion in bacteria and yeast cells. With yields of up to 40–70 mg/l, nanobodies can be produced at a fraction of the cost of mAbs. When produced, their high resistance towards chaotropic reagents or prolonged high temperatures and stability at high concentrations allows easy

Nanobodies, being small and stable, are ideal candidates for various research purposes, diagnostic or clinical applications. Equipped with a radionuclide, they can be applied as imaging tracers in a single‐photon emission computed tomography (SPECT) or positron emission tomography (PET)‐based set‐up allowing in vivo visualization of tumours, arthero‐ sclerotic plaques and rheumatoid arthritis‐associated inflammation sites [36–38]. Nanobod‐ ies are also capable of stabilizing proteins in a particular conformation and of minimizing structural flexibility. This has been nicely demonstrated in the case of human lysozyme amyloidosis, a hereditary systemic disease associated with at least seven amyloidogenic lysozyme variants [39, 40]. Aggregation of these lysozyme variants leads to the accumula‐ tion of amyloid in the extracellular matrix of several tissues and organs. Two nanobodies have been identified who, upon binding mutant amyloidogenic lysozyme (D67H and I56T), restored the global cooperativity characteristics of the wild‐type protein through direct contact and long‐range conformational effects. What's more, this stabilizing capacity can also be condu‐ cive to crystallizing proteins. β2‐microglobulin–specific nanobodies have been shown to serve as efficient crystallization chaperones fortransientintermediate species during fibrillation [41]. This type of antibody chaperone co‐crystallization was also used to unravel the structure of the G protein‐coupled receptor β‐2 adrenergic receptor (β2AR), research which received the

Another major field where the potential use of nanobodies is currently being explored is amyloidosis. In Alzheimer's research, nanobodies have been found which inhibit beta‐ secretase 1 (BACE1) activity, the first step in amyloid beta formation, both in vitro and in vivo [43]. Alternatively, nanobodies could also be raised against amyloid precursor protein (APP) to protect it from BACE1 degradation, as direct inhibition of the enzyme could evoke unwanted side effects. We present here the case of gelsolin amyloidosis where this approach has proven successful both in vitro and in vivo. The remainder of this chapter will focus on the obtained results, current status and future prospects regarding this AGel research arena.

Consecutive aberrant cleavage of PG\* by furin and MT1‐MMP–like proteases leads to the production of amyloidogenic fragments which eventually polymerize into fibrils, causing gelsolin amyloidosis pathology. But these two proteases are active in different compart‐ ments. Indeed, whereas furin is active in the (intracellular) ER‐Golgi compartments, MT1‐ MMP is inserted in the plasma membrane and oriented to the extracellular environment. This

**4. Extracellular targeting of the AGel pathological pathway**

storage without any functionality loss.

2012 Nobel Prize in Chemistry [42].

**3.4. Nanobody applications**

318 320Exploring New Findings on Amyloidosis

A dromedary was simultaneously immunized with the G2 domain and the 8 kDa fragment of PG\*. Phage panning was also performed with both gelsolin fragments. This yielded three different nanobodies, termed FAF Nb1‐3. All three bind to PG, PG\*, C68 and the 8 kDa peptide

**Figure 4.** Overview of different nanobody‐based strategies to diagnose and to counter amyloidogenesis in transgenic mice. A, Schematic representation of the steps leading from mutant plasma gelsolin to the 8 and 5 kDa amyloidogenic fragments. B (upper panel), Gelsolin Nb11 reduces C68 formation in vitro—lane 1: PG incubated with furin, lane 2: PG\* incubated with furin and lane 3: PG\* incubated with furin and Nb11. B (second panel), Nb11 co‐localizes with mutant gelsolin in the secretory pathway of HEK293T cells. B (third panel), **Nb 11** decreases C68 formation in HEK293T cells: lane 1: medium from cells transfected with PG, lane 2: medium from cells transfected with PG\* and lane 3: medium from cells transfected with PG\* and Nb11. This led to designing the Nb11‐expressing mouse model. C, Representative images obtained with 99mTc‐labeled FAF Nb1 in 9‐month‐old AGel mice. The heart and front and hind leg muscles show a clear signal (blue). Kidneys and bladder signal represents unbound nanobody which is cleared through the urinary system (green and red). FAF Nb1 can also be used as a primary antibody to stain amyloidogenic gelsolin build‐up in AGel mice tissue (lower panel). D (upper panel), FAF Nb1‐3 partly inhibits C68 proteolysis in vi‐ tro. For each nanobody, from left till right, after incubation with MT1‐MMP: PG, PG\*, PG\* + 0.5, 1.0 and 2.0 X FAF Nb. Numbers indicate the Nb/PG\* molar ratios. D (middle panel), Linkage with the albumin‐binding MSA21 nanobody increases serum half‐life from 4 h to more than 1 week (middle panel). D (lower panel), In vivo analysis was performed through intraperitoneal injection in AGel mice.

when tested through immunoprecipitation on crude bacterial lysates, but they did not cross‐ react with CapG, a protein that is structurally related to gelsolin. Unlike gelsolin, CapG is only capable of capping actin filaments but does not sever. It is the member of the gelsolin superfamily most closely resembling gelsolin. It possesses 49% identity with the N‐terminal half of gelsolin.

The hypothesis was that if one of these FAF nanobodies bound to C68 in the vicinity of the MT1‐MMP cleavage site or an important docking area, it would be able to (at least partly) inhibit the production of the 8 kDa fragments. This was indeed confirmed by in vitro experi‐ ments. C68 was preincubated with one of the three FAF Nbs after which MT1‐MMP was added to the mixture. Formation of the 8 kDa fragment could be followed by Western blotting, and this showed a 70–80% reduction in the presence of a FAF nanobody, attesting to their ability to indirectly prevent MT1‐MMP activity on gelsolin (**Figure 4A** and **D**). Nb13, a nanobody that binds to the linker between gelsolin domains 4 and 5, did not show the same effect. Further‐ more, the FAF Nbs had no effect on the proteolysis of collagen, another substrate of MT1‐MMP. Therefore, it was concluded that a nanobody can be used as a chaperone to prevent degrada‐ tion of a structural protein by an enzyme without directly inhibiting the activity of the enzyme. In doing so, potential side effects may be prevented. Whether the observed effects are due to a direct inhibition of MT1‐MMP docking on C68 or shielding the cleavage site or rather due to an indirect structural affect caused by the binding of the Nb with C68 still remains to be clarified. A crystal structure of MT1‐MMP bound to C68 would bring clarity in this matter.

While these results were promising at first, a new caveat presented itself when considering that nanobodies, because of their small size, are characterized by rapid blood clearance. This could impede their use as a therapeutic in vivo. Indeed, although their small size is mostly advantageous, when used as a therapeutic, it is rather a burden. Thus, it seemed necessary to increase their half‐life in the circulation as this will improve their probability of binding C68 gelsolin. To achieve this goal, FAF Nb1‐3 were coupled to the albumin‐binding nanobody MSA21 through a short Gly‐Ser linker. This linkage did not greatly affect the binding affini‐ ty between the Nb and C68 (FAF2 = Kd: 8.4 × 10‐7 ± 4.7 × 10‐8 M, FAF2‐MSA21 = Kd: 5.4 × 10‐7 ±  0.6 × 10‐7 M, determined by isothermal titration calorimetry). The bispecific FAF2‐MSA21 Nb binds albumin in the bloodstream, and the formation of this larger complex should extend the half‐life, resulting in a higher efficiency of the FAF Nb1‐3 therapeutic intervention. Serum analysis of injected mice confirmed this assumption. While the monovalent FAF Nb1‐3 were no longer detectable after 4 h, the bispecific format could still be clearly visualized up until one week post‐injection (**Figure 4D**). Additionally, the linkage between FAFNb1‐3 and MSA21 did not offset the MT1‐MMP–inhibiting potential in vitro. Hence, all requirements were fulfilled to test their effect in vivo.

The in vitro results were validated using the available AGel mouse model. Over a period of 12 weeks, mice were weekly injected with 100 μg of either FAF Nb1, FAF Nb2‐MSA21 or phosphate‐buffered saline (PBS). The trial started at the age of 4 weeks, and the nanobodies used were the ones which performed best during the in vitro set‐up. End‐stage analysis consisted of immunohistochemistry (IHC) and ex vivo muscle contractility measurements. IHC revealed a decrease in amyloidogenic gelsolin staining for FAF Nb1 of 15% and FAF Nb2‐

MSA21 of 30%. Both results were statistically significant. The difference between both nanobody formats shows that a longer half‐life is definitely desirable in a therapeutic approach such as this. The reduction seen with IHC in the FAF Nb2‐MSA21–treated mice translated into improved muscle contractility and relaxation speed function in the extensor digitorum longus (EDL). Therefore, these nanobodies are endowed with a therapeutic quality. It should be noted that the FAF Nbs display an intermediate affinity fortheirtarget. Although clear data in support of this contention are wanting, it seems likely that stronger binders might even have stronger effects in vivo. Further exploration of the FAF Nb2‐MSA21 potential in human gelsolin amyloidosis patients requires two important modification to be made. Firstly, the MSA21 nanobody would have to be replaced by a human serum albumin binder. The longer serum half‐life of human versus mouse albumin (19 days versus 35 h) may further lower the administration frequency. Secondly, although nanobodies are known for their low immuno‐ genicity, humanizing their framework regions can further decrease the risk of an undesira‐ ble immune response during therapeutic intervention. More specifically, a universal humanized nanobody scaffold has already been proposed onto which all the CDR antigen‐ binding loops could be grafted [44].

## **5. Intracellular targeting of the AGel pathological pathway**

when tested through immunoprecipitation on crude bacterial lysates, but they did not cross‐ react with CapG, a protein that is structurally related to gelsolin. Unlike gelsolin, CapG is only capable of capping actin filaments but does not sever. It is the member of the gelsolin superfamily most closely resembling gelsolin. It possesses 49% identity with the N‐terminal

The hypothesis was that if one of these FAF nanobodies bound to C68 in the vicinity of the MT1‐MMP cleavage site or an important docking area, it would be able to (at least partly) inhibit the production of the 8 kDa fragments. This was indeed confirmed by in vitro experi‐ ments. C68 was preincubated with one of the three FAF Nbs after which MT1‐MMP was added to the mixture. Formation of the 8 kDa fragment could be followed by Western blotting, and this showed a 70–80% reduction in the presence of a FAF nanobody, attesting to their ability to indirectly prevent MT1‐MMP activity on gelsolin (**Figure 4A** and **D**). Nb13, a nanobody that binds to the linker between gelsolin domains 4 and 5, did not show the same effect. Further‐ more, the FAF Nbs had no effect on the proteolysis of collagen, another substrate of MT1‐MMP. Therefore, it was concluded that a nanobody can be used as a chaperone to prevent degrada‐ tion of a structural protein by an enzyme without directly inhibiting the activity of the enzyme. In doing so, potential side effects may be prevented. Whether the observed effects are due to a direct inhibition of MT1‐MMP docking on C68 or shielding the cleavage site or rather due to an indirect structural affect caused by the binding of the Nb with C68 still remains to be clarified. A crystal structure of MT1‐MMP bound to C68 would bring clarity in this matter.

While these results were promising at first, a new caveat presented itself when considering that nanobodies, because of their small size, are characterized by rapid blood clearance. This could impede their use as a therapeutic in vivo. Indeed, although their small size is mostly advantageous, when used as a therapeutic, it is rather a burden. Thus, it seemed necessary to increase their half‐life in the circulation as this will improve their probability of binding C68 gelsolin. To achieve this goal, FAF Nb1‐3 were coupled to the albumin‐binding nanobody MSA21 through a short Gly‐Ser linker. This linkage did not greatly affect the binding affini‐ ty between the Nb and C68 (FAF2 = Kd: 8.4 × 10‐7 ± 4.7 × 10‐8 M, FAF2‐MSA21 = Kd: 5.4 × 10‐7 ±  0.6 × 10‐7 M, determined by isothermal titration calorimetry). The bispecific FAF2‐MSA21 Nb binds albumin in the bloodstream, and the formation of this larger complex should extend the half‐life, resulting in a higher efficiency of the FAF Nb1‐3 therapeutic intervention. Serum analysis of injected mice confirmed this assumption. While the monovalent FAF Nb1‐3 were no longer detectable after 4 h, the bispecific format could still be clearly visualized up until one week post‐injection (**Figure 4D**). Additionally, the linkage between FAFNb1‐3 and MSA21 did not offset the MT1‐MMP–inhibiting potential in vitro. Hence, all requirements were fulfilled

The in vitro results were validated using the available AGel mouse model. Over a period of 12 weeks, mice were weekly injected with 100 μg of either FAF Nb1, FAF Nb2‐MSA21 or phosphate‐buffered saline (PBS). The trial started at the age of 4 weeks, and the nanobodies used were the ones which performed best during the in vitro set‐up. End‐stage analysis consisted of immunohistochemistry (IHC) and ex vivo muscle contractility measurements. IHC revealed a decrease in amyloidogenic gelsolin staining for FAF Nb1 of 15% and FAF Nb2‐

half of gelsolin.

320 322Exploring New Findings on Amyloidosis

to test their effect in vivo.

Thanks to their excellent stability, nanobodies are also valuable compounds to be used as intrabodies. The reducing environment of the cytoplasm seems to have little effect on their functionality [45, 46]. As mentioned above, the FAF Nb approach significantly reduced AGel amyloid build‐up in vivo, but it did not completely halt the 8 kDa peptide production. AGel pathogenesis is a two step process; a furin cleavage of mutant plasma gelsolin produces C68, which is then on its turn cleaved by MT1‐MMP, thereby releasing 8 kDa amyloidogenic peptides. This opens the opportunity for a double‐hit approach.

For that reason, we tested whether a different set of gelsolin nanobodies could be applied as a chaperone for gelsolin to divert furin activity in the same manner as we had implemented the FAF Nb1‐3 towards C68 [9].

The nanobodies used in this study were not specifically designed for this purpose. Instead, they had been raised against wild‐type gelsolin and characterized some time ago. Through in vitro epitope mapping experiments, Nb11 was shown to interact with the G2–G3 domains (N‐ terminal half), whereas Nb13 interacts with G4–G5 (C‐terminal half). Furthermore, both nanobodies interact with different populations of gelsolin in cells. Indeed, Nb11 binds human gelsolin with high affinity (Kd = 3.65 × 10‐9 ± 0.54 × 10-9 M, determined by isothermal titration calorimetry), irrespective of whether calcium is present or not, whereas Nb13 strongly interacts with gelsolin (Kd = 9.26 × 10-9 ± 1.61 × 10-9 M, determined by isothermal titration calorimetry), only when the latter is activated by calcium. Both nanobodies were further shown to act as reliable tracers of gelsolin in MCF‐7 cells when expressed as intrabodies. Both gelsolin and nanobody are abundantly present in the cytoplasm of unstimulated MCF‐7 cells, preventing straightforward assessment of their co‐localization. However, when the cells were stimulat‐ ed with epidermal growth factor (EGF), both endogenous gelsolin and the nanobodies extensively decorated membrane ruffles [47]. In view of the binding region of Nb11 in gelsolin, it was surmised that it could interfere with furin‐mediated degradation. In analogy with the MT1‐MMP study, these gelsolin nanobodies were incubated with PG\*, and Western blotting showed that only Nb11 reduced furin cleavage of PG\* by 34% when added in concentrations equimolar to PG\* (**Figure 4A** and **B**).

This finding, however, does not guarantee that the same result will be obtained in mammali‐ an cells. Plasma gelsolin will naturally travel through the secretory pathway, but simple expression of the nanobody will result in its cytoplasmic and nuclear localization in cells. Hence, the nanobody will not be able to protect gelsolin. For this reason, the nanobody was equipped with an ER signal peptide to ensure its secretion through the Golgi apparatus. Furin naturally resides in the Golgi apparatus, and the first step in the AGel pathology takes place in this compartment. By quantifying the amount of C68 that is secreted in the extracellular environment, it could be confirmed that Nb11 exerts a protective effect. The cell media from HEK cells transfected with PG\* and Nb11 contained 80% less C68 compared to HEK cells solely transfected with PG\* (**Figure 4B**).

Although nanobodies can, and are, currently being applied in research involving intracellu‐ lar perturbation of protein‐protein interactions, at the moment there is no efficient, fail proof method to introduce them into cells in a recombinant format. Therefore, to test whether the observed effect of Nb11 in vitro could be reproduced in vivo, a Nb11‐expressing mouse model had to be created. To our knowledge, this is the first transgenic mammal that contains a therapeutic nanobody in its genome. The ER‐directed Nb11 cDNA was cloned in the pROSA‐ DV2 vector, which targets the ROSA26 locus. This locus was identified in 1991 using gene‐trap mutagenesis screening on embryonic stem cells. Thanks to its ubiquitous expression in both embryonic and adult tissues, over 130 knock‐in mouse lines have been created using this cloning site. G4 ES cells were electroporated, and positive colonies were selected through Southern blotting. Next, cells were aggregated with Swiss inner cell mass cells. These blastocysts were transferred into pseudopregnant mice uteri, and the resulting chimeric offspring was backcrossed with wild‐type C57BL/6 to check for germline transmission. Finally, these mice could then be crossed with a non‐tissue specific Cre/lox‐deleter mouse strain. This resulted in CAGG promoter‐driven Nb11 expression, resulting in the first Nb11‐expressing mice. Nb11 could be visualized and quantified in the serum through co‐immunoprecipita‐ tion and Western blotting.

The newly developed Nb11 mouse model was crossed with AGel mice. Double positive offspring was evaluated at three distinct time points: 3, 6 and 9 months. Gastrocnemius muscle tissue was stained for AGel build‐up. A costaining for laminin was used as an internal control and to discern any potential artefacts. The AGel staining was homogenous in every age group, allowing quantification. Compared to AGel mice or littermates not expressing Nb11, a reduction in AGel staining of 27 and 28%, respectively, could be detected at 3 and 9 months of age. For the group of 6 months, no significant reduction could be found. The reason for this is unclear. The group of 9‐month‐old mice was also subjected to a muscle performance evalua‐ tion. The extensor digitorum longus (EDL) muscle showed a strong attenuation of the typical

decrease in contraction speed during the fatiguing protocol. This therapeutic effect was not mimicked by Nb13 that binds to another region in gelsolin (G4–G5 linker) and that had no effect on gelsolin degradation by furin. Neither Nb11 nor Nb13 showed any cross‐reactivity with endogenous mouse gelsolin. Thus, Nb11 attenuates amyloid build‐up by (partly) protecting gelsolin against degradation by furin. One might wonder why the reduction in AGel staining was not as high as expected, given that Nb11 is a strong binder (Kd ∼ 5 nM) where calcium moreover has no effect on binding. One possibility involves the relative expression levels of gelsolin and Nb11. Western blotting with internal gelsolin and nanobody standards indicated that both are present at roughly equimolar levels. In the AGel mouse model, however, gelsolin is secreted from muscle, whereas nanobody expression is thought to be secreted from multiple organs, tissues and cells, because no tissue‐specific promoter was used to drive expression of the nanobody. Therefore, the nanobody that was detected in the serum of these animals has multiple origins suggesting that its secretion from muscle is significant‐ ly lower as compared to gelsolin secretion. This contention however needs to be examined experimentally.

## **6. In vivo visualization of AGel build‐up**

ed with epidermal growth factor (EGF), both endogenous gelsolin and the nanobodies extensively decorated membrane ruffles [47]. In view of the binding region of Nb11 in gelsolin, it was surmised that it could interfere with furin‐mediated degradation. In analogy with the MT1‐MMP study, these gelsolin nanobodies were incubated with PG\*, and Western blotting showed that only Nb11 reduced furin cleavage of PG\* by 34% when added in concentrations

This finding, however, does not guarantee that the same result will be obtained in mammali‐ an cells. Plasma gelsolin will naturally travel through the secretory pathway, but simple expression of the nanobody will result in its cytoplasmic and nuclear localization in cells. Hence, the nanobody will not be able to protect gelsolin. For this reason, the nanobody was equipped with an ER signal peptide to ensure its secretion through the Golgi apparatus. Furin naturally resides in the Golgi apparatus, and the first step in the AGel pathology takes place in this compartment. By quantifying the amount of C68 that is secreted in the extracellular environment, it could be confirmed that Nb11 exerts a protective effect. The cell media from HEK cells transfected with PG\* and Nb11 contained 80% less C68 compared to HEK cells solely

Although nanobodies can, and are, currently being applied in research involving intracellu‐ lar perturbation of protein‐protein interactions, at the moment there is no efficient, fail proof method to introduce them into cells in a recombinant format. Therefore, to test whether the observed effect of Nb11 in vitro could be reproduced in vivo, a Nb11‐expressing mouse model had to be created. To our knowledge, this is the first transgenic mammal that contains a therapeutic nanobody in its genome. The ER‐directed Nb11 cDNA was cloned in the pROSA‐ DV2 vector, which targets the ROSA26 locus. This locus was identified in 1991 using gene‐trap mutagenesis screening on embryonic stem cells. Thanks to its ubiquitous expression in both embryonic and adult tissues, over 130 knock‐in mouse lines have been created using this cloning site. G4 ES cells were electroporated, and positive colonies were selected through Southern blotting. Next, cells were aggregated with Swiss inner cell mass cells. These blastocysts were transferred into pseudopregnant mice uteri, and the resulting chimeric offspring was backcrossed with wild‐type C57BL/6 to check for germline transmission. Finally, these mice could then be crossed with a non‐tissue specific Cre/lox‐deleter mouse strain. This resulted in CAGG promoter‐driven Nb11 expression, resulting in the first Nb11‐expressing mice. Nb11 could be visualized and quantified in the serum through co‐immunoprecipita‐

The newly developed Nb11 mouse model was crossed with AGel mice. Double positive offspring was evaluated at three distinct time points: 3, 6 and 9 months. Gastrocnemius muscle tissue was stained for AGel build‐up. A costaining for laminin was used as an internal control and to discern any potential artefacts. The AGel staining was homogenous in every age group, allowing quantification. Compared to AGel mice or littermates not expressing Nb11, a reduction in AGel staining of 27 and 28%, respectively, could be detected at 3 and 9 months of age. For the group of 6 months, no significant reduction could be found. The reason for this is unclear. The group of 9‐month‐old mice was also subjected to a muscle performance evalua‐ tion. The extensor digitorum longus (EDL) muscle showed a strong attenuation of the typical

equimolar to PG\* (**Figure 4A** and **B**).

322 324Exploring New Findings on Amyloidosis

transfected with PG\* (**Figure 4B**).

tion and Western blotting.

AGel is a chronic, gain of toxicity, affliction. Accordingly, any therapy will have to be administered throughout the entire lifetime of the patients. It is therefore important, during therapy development, to test promising compounds over a long period of time and assess the effects at different time points during the course of the trial. During the Nb11 experiments, this had to be done by using different groups for every time point since the current methods of AGel analysis are end‐stage [8, 9]. Not only does this greatly augment the amount of mice needed, it also makes it impossible to evaluate the drug effect and amyloid build‐up in a continuous manner. In the hope of resolving this drawback, we set out to develop a nano‐ body‐based imaging agent. The ultimate goal was to be able to visualize the AGel amyloid build‐up in vivo in a non‐invasive manner. This would facilitate evaluation of drug respons‐ es at different time points over the entire course of treatment in the same animal.

## **6.1. Labelling of nanobody through 99mTc linkage to His6 tag**

Thanks to their excellent stability, small size and single‐chain nature, nanobodies represent an ideal antibody derived format for transformation into a radionuclide‐based amyloid imag‐ ing agent. 99mTc was the radionuclide of choice being readily available and displaying a half‐ life long enough to allow good quality imaging, but short enough to minimize the overall radiation burden during the trial. Linking it to a nanobody is also a straightforward process. Simply equipping the nanobody with a N‐ or C‐terminal His6 tag allows chelation with 99mTc via the tricarbonyl method [48]. Furthermore, 99mTc is compatible with SPECT, the imaging modality most ideal for visualization in small laboratory animals used during research trials such as this.

FAF Nb1‐3 were the prime candidates to be explored for their ability to serve as an imaging tool. When administered as in vivo imaging agents, FAF Nb1‐3 are required to remain stable for some time in the complex environment of the blood and extracellular matrix. In a prelimi‐ nary test phase, FAF Nb1‐3 were therefore incubated in C57BL/6 mouse serum at 37°C and found to remain stable for up to 24 h without deterioration.

As a last test before the first in vivo trials, a test labelling was performed. Using instant thin‐ layer chromatography (ITLC) and reverse‐phase high‐performance liquid chromatography (RP‐HPLC), the radiochemical purities were determined to be 99.7 ± 1.3%, 99.7 ± 0.9% and 98.3 ± 1.0% for FAF Nb1‐3, respectively.

## **6.2. Specificity and background signal determination**

One of the key aspects of a good imaging agent is the capability of specifically generating a high signal wherever its target is present without provoking a significant background signal. As discussed earlier, FAF Nb1‐3 are capable of binding their target with great specificity in vitro. To test whether this remained true in vivo, a group of 9‐month‐old WT C57BL/6 mice were injected with 99mTc‐FAF Nb1‐3. Nanobody BcII10 against β‐lactamase of Bacillus cereus was used as a negative control [49]. SPECT/CT images and dissection analysis revealed the classic high kidney and bladder uptake of renal‐filtered small hydrophilic proteins of which nanobodies are a textbook example. All the other organs showed no significant high signal when comparing the FAF Nb1‐3 with the BcII10 control. From these data, it can be conclud‐ ed that the FAF Nb1‐3 do not show any nonspecific binding when administered to mice (and they do not cross‐react with mouse gelsolin either).

The experiment was repeated in 9‐month‐old AGel mice (**Figure 4C**). At this age, the ani‐ mals show considerable gelsolin amyloid build‐up in the muscle tissue. Both FAF Nb1 and 2 showed significant uptake in skeletal muscle, heart and diaphragm. In the AGel mouse model, these represent three out of the four tissues in which gelsolin amyloid builds up over time. The fourth, the skin, could not be identified with the FAF Nbs, although it is possible that this organ only becomes affected later in life. Overall, this experiment showed that FAF Nb1‐2 are capable of specifically recognizing gelsolin amyloid build‐up in the AGel model. FAF Nb3 on the contrary did not seem able to provoke a significant signal in the heart nor diaphragm.

The two experiments described above were used to calculate both a signal‐to‐noise and a signal‐specificity score. The first is defined as the signals gained with FAF Nb1‐3 in AGel mice muscle over the signal obtained in AGel mice liver. The latter is defined as the signal gained with FAF Nb1‐3 in AGel mice over the signal gained with Nb BcII10 in AGel mice. On both characteristics, FAF Nb1 scored the best. Combined with the fact that it also has a slightly better affinity for the 8 kDa peptide (FAF Nb1: 3.8 × 10‐7 ± 1.2 × 10‐8 M, FAF Nb2: Kd: 8.4 × 10‐7 ± 4.7 ×  10‐8 M and FAF Nb3: Kd: 6.6 × 10‐7 ± 1.7 × 10‐7 M, determined by isothermal titration calorime‐ try), FAF Nb1 was appointed as the best candidate for further development as a AGel 99mTc‐ based imaging agent.

## **6.3. Qualitative and quantitative characteristics**

FAF Nb1‐3 were the prime candidates to be explored for their ability to serve as an imaging tool. When administered as in vivo imaging agents, FAF Nb1‐3 are required to remain stable for some time in the complex environment of the blood and extracellular matrix. In a prelimi‐ nary test phase, FAF Nb1‐3 were therefore incubated in C57BL/6 mouse serum at 37°C and

As a last test before the first in vivo trials, a test labelling was performed. Using instant thin‐ layer chromatography (ITLC) and reverse‐phase high‐performance liquid chromatography (RP‐HPLC), the radiochemical purities were determined to be 99.7 ± 1.3%, 99.7 ± 0.9% and

One of the key aspects of a good imaging agent is the capability of specifically generating a high signal wherever its target is present without provoking a significant background signal. As discussed earlier, FAF Nb1‐3 are capable of binding their target with great specificity in vitro. To test whether this remained true in vivo, a group of 9‐month‐old WT C57BL/6 mice were injected with 99mTc‐FAF Nb1‐3. Nanobody BcII10 against β‐lactamase of Bacillus cereus was used as a negative control [49]. SPECT/CT images and dissection analysis revealed the classic high kidney and bladder uptake of renal‐filtered small hydrophilic proteins of which nanobodies are a textbook example. All the other organs showed no significant high signal when comparing the FAF Nb1‐3 with the BcII10 control. From these data, it can be conclud‐ ed that the FAF Nb1‐3 do not show any nonspecific binding when administered to mice

The experiment was repeated in 9‐month‐old AGel mice (**Figure 4C**). At this age, the ani‐ mals show considerable gelsolin amyloid build‐up in the muscle tissue. Both FAF Nb1 and 2 showed significant uptake in skeletal muscle, heart and diaphragm. In the AGel mouse model, these represent three out of the four tissues in which gelsolin amyloid builds up over time. The fourth, the skin, could not be identified with the FAF Nbs, although it is possible that this organ only becomes affected later in life. Overall, this experiment showed that FAF Nb1‐2 are capable of specifically recognizing gelsolin amyloid build‐up in the AGel model. FAF Nb3 on the contrary did not seem able to provoke a significant signal in the heart nor diaphragm.

The two experiments described above were used to calculate both a signal‐to‐noise and a signal‐specificity score. The first is defined as the signals gained with FAF Nb1‐3 in AGel mice muscle over the signal obtained in AGel mice liver. The latter is defined as the signal gained with FAF Nb1‐3 in AGel mice over the signal gained with Nb BcII10 in AGel mice. On both characteristics, FAF Nb1 scored the best. Combined with the fact that it also has a slightly better affinity for the 8 kDa peptide (FAF Nb1: 3.8 × 10‐7 ± 1.2 × 10‐8 M, FAF Nb2: Kd: 8.4 × 10‐7 ± 4.7 ×  10‐8 M and FAF Nb3: Kd: 6.6 × 10‐7 ± 1.7 × 10‐7 M, determined by isothermal titration calorime‐ try), FAF Nb1 was appointed as the best candidate for further development as a AGel 99mTc‐

found to remain stable for up to 24 h without deterioration.

**6.2. Specificity and background signal determination**

(and they do not cross‐react with mouse gelsolin either).

based imaging agent.

98.3 ± 1.0% for FAF Nb1‐3, respectively.

324 326Exploring New Findings on Amyloidosis

The ultimate goal was to see whether the FAF Nbs could be implemented as imaging tools during the screening of potential AGel therapeutics. This of course means that not only do we want to qualitatively image the amyloid build‐up, but we also want the rendered signal to hold quantitative information. Only then would it be possible to track the therapeutic effect in a longitudinal follow‐up study.

The intervention study with the gelsolin Nb11‐expressing mouse model presented an ideal opportunity to test 99mTc‐FAF Nb1 for any quantitative properties. As stated above, by crossing the Nb11 expressing mice with the AGel mice, we were able to reduce AGel amyloid burden as detected by IHC. The experiment was repeated, but now, starting at the age of 3 months, the mice underwent a SPECT/CT scan with 99mTc‐FAF Nb1 every two months. Two groups were analyzed with IHC: one at 9 months of age and another at 11 months.

In the SPECT/CT images, the signal coming from the heart and hind leg muscle was quanti‐ fied using AMIDE software. The same size region of interest was used for every animal at every time point. No significant differences could be discerned in the heart tissue. This is not so surprising since IHC had revealed earlier that the heart only seems to be homogeneously affected from the age of 7–9 months onwards. Therefore, a good quantification is not possi‐ ble before that time. In the muscle tissue, however, the SPECT/CT analysis revealed a significant difference at the age of 7 and 9 months. Why the same was not true at 11 months, further research will have to reveal.

Atthe end stages of 9 and 11 months, mice were dissected right after SPECT/CTimaging. Signal strengths were determined for hind leg muscle and the heart. The same pattern as with SPECT/ CT analysis could be found, a difference at 9 months but not at 11 months. The same tissue also underwent IHC, which still is the golden standard when it comes to amyloid detection and quantification. The IHC analysis confirmed that gelsolin staining in the heart tissue is not yet homogenous so these samples were not quantified. The muscle on the other hand showed good overall staining. Hence, IHC confirmed the result obtained with SPECT/CT imaging and dissection analysis, a significant difference at the age of 9 months but no longer at 11 months.

## **7. Future prospects**

## **7.1. Combined therapy**

Gelsolin amyloidosis is caused by a point substitution in the GSN gene which has a destabi‐ lizing effect on the second gelsolin domain. As a result a cryptic, otherwise buried, furin cleavage site is exposed at the surface. The plasma isoform encounters furin along the secretory pathway in the trans‐Golgi network, at which point it is cleaved, thereby releasing a 68 kDa C-terminal fragment. In its turn, this C68 fragment is processes by MT1‐MMP–like proteases leading to the production of 8 and 5 kDa amyloidogenic fragments.

This two‐step pathological process allows intervention at two very distinct levels. The furin section takes place intracellularly, inside the trans-Golgi network, a site which is still quite troublesome to target with non‐small‐compound therapeutics. The MT1‐MMP section takes place in the extracellular matrix, a region easily accessible through injections.

Both routes were explored, and the results are described above. Although both approaches proved to hold a therapeutic potential, none of them completely solved the AGel pathogene‐ sis. Therefore, the next logical step is to combine them in a single therapeutic format. Once again the single‐domain nature of nanobodies came in handy. Using straightforward clon‐ ing techniques, FAF Nb1 was linked to the C terminus of Nb11 which contains a secretion signal. The linker between both nanobodies contains a MT1‐MMP–sensitive linker. The idea behind the construct is that when expressed the bispecific nanobody will perform its PG\* chaperone function in the trans‐Golgi network. During secretion, the MT1‐MMP–sensitive linker will lure MT1‐MMP away from the already lower amount of C68. This process will also release FAF Nb1 from Nb11 in situ, making it available to chaperone C68 and further diminish the overall production of the 8 kDa amyloidogenic pathway (**Figure 5**).

**Figure 5.** Envisioned molecular mechanism of the therapeutic bispecific Nb11‐FAF1. The left‐hand side depicts the pathological PG\* processing with the formation of C68 in the trans‐Golgi network and the 8 and 5 kDa amyloidogenic fragments in the extracellular matrix. On the right‐hand side, the intervention with bispecific Nb11‐FAF1 is shown. In the trans‐Golgi network, the nanobody uses its Nb11 moiety to bind PG\* and (partly) protect it from furin degradation. In the extracellular matrix, the MT1‐MMP–sensitive linker between Nb11 and FAF1 serves as a decoy. At the same time, FAF Nb1 is released and is able to perform its C68 chaperoning effect, thereby further decreasing the overall formation of 8 and 5 kDa amyloidogenic fragments.

This hypothesis has already been tested in vitro. A combined Furin/MT1‐MMP degradation assay was set up. Quantification of the amount of 8 kDa peptide showed Nb11‐FAF1 outper‐

formed both Nb11 and the individual FAF Nb1‐3. Meaning that, in vitro, the double‐hit approach shows a synergistic effect compared to the individual therapies.

For in vivo testing, a gene therapy approach could be chosen. Adeno‐associated viruses (AAVs) are naturally occurring, non‐pathogenic, replication‐deficient viruses. They are capable of infecting non‐dividing cells and reside in the nucleus under the form of episomal concatemers. This makes them ideal candidates to be used as gene therapy vectors. Their most important downside is the limited packaging capacity of about 5 kb [50]. Given its small size, this should not pose a problem for our Nb11‐FAF1 application. Since AGel expression in the transgenic model is driven from muscle, a serotype with a muscle tropism would be prefer‐ ential.

## **7.2. Beyond AGel**

troublesome to target with non‐small‐compound therapeutics. The MT1‐MMP section takes

Both routes were explored, and the results are described above. Although both approaches proved to hold a therapeutic potential, none of them completely solved the AGel pathogene‐ sis. Therefore, the next logical step is to combine them in a single therapeutic format. Once again the single‐domain nature of nanobodies came in handy. Using straightforward clon‐ ing techniques, FAF Nb1 was linked to the C terminus of Nb11 which contains a secretion signal. The linker between both nanobodies contains a MT1‐MMP–sensitive linker. The idea behind the construct is that when expressed the bispecific nanobody will perform its PG\* chaperone function in the trans‐Golgi network. During secretion, the MT1‐MMP–sensitive linker will lure MT1‐MMP away from the already lower amount of C68. This process will also release FAF Nb1 from Nb11 in situ, making it available to chaperone C68 and further diminish

**Figure 5.** Envisioned molecular mechanism of the therapeutic bispecific Nb11‐FAF1. The left‐hand side depicts the pathological PG\* processing with the formation of C68 in the trans‐Golgi network and the 8 and 5 kDa amyloidogenic fragments in the extracellular matrix. On the right‐hand side, the intervention with bispecific Nb11‐FAF1 is shown. In the trans‐Golgi network, the nanobody uses its Nb11 moiety to bind PG\* and (partly) protect it from furin degradation. In the extracellular matrix, the MT1‐MMP–sensitive linker between Nb11 and FAF1 serves as a decoy. At the same time, FAF Nb1 is released and is able to perform its C68 chaperoning effect, thereby further decreasing the overall

This hypothesis has already been tested in vitro. A combined Furin/MT1‐MMP degradation assay was set up. Quantification of the amount of 8 kDa peptide showed Nb11‐FAF1 outper‐

formation of 8 and 5 kDa amyloidogenic fragments.

place in the extracellular matrix, a region easily accessible through injections.

326 328Exploring New Findings on Amyloidosis

the overall production of the 8 kDa amyloidogenic pathway (**Figure 5**).

The studies performed with the AGel mouse model have shown that nanobodies are useful, potent therapeutics for both extra‐ and intracellular targets. They also highlighted that nanobodies can be used as an imaging tool in AGel, thereby facilitating the screening of potentially new therapeutic compounds. While AGel is an orphan disease, it should be emphasized that this strategy can be extrapolated to other amyloidogenic afflictions. The most infamous type of amyloidosis, Alzheimer's, actually has a quite similar pathogenesis; the sequential proteolysis of Alzheimer's precursor protein by β‐secretase and γ‐secretase results in the release of Aβ amyloidogenic peptides. Nanobodies against this β‐secretase have already proven to be therapeutically active in a transgenic AD mouse model [43]. As already stated earlier, we believe that in the long run, it would prove more beneficial to target the APP instead of the secretases. Inhibiting a protease will most likely provoke unwanted side effects, whereas chaperoning the target, as in the AGel research discussed in this chapter, is more unlikely to do so.

Raising nanobodies against full length APP, s‐APP β or Aβ, may result in nanobodies that bind nearthe β‐secretase cleavage or docking site and that block the formation of s‐APP β. A double‐ hit strategy as in the AGel case will most probably be more difficult to achieve. The gamma secretase is a multiunit integral membrane protein. It cleaves transmembrane passes. At the moment, there are no reports of nanobodies being able to integrate into the plasma mem‐ brane and remain functional. In addition, gamma secretase proteolysis also takes place in the non‐pathogenic processing of APP. As the transmembrane part of C‐terminal fragment‐alpha and C‐terminal fragment‐beta is identical, even a chaperone‐based intervention may pro‐ voke unwanted side effects.

Up until now, we have only discussed preventing the formation of amyloidogenic peptides. But directly targeting the amyloidogenic peptides, after their formation, also holds great therapeutic potential. The deposition of amyloid fibrils is the primary pathological feature of this group of afflictions. In recent years, however, scientific research towards the initial processes leads to fibrillation, implicated that the initial oligomeric aggregates may be the most toxic species in amyloid pathogenesis and mature fibrils may be far less pathogenic than previously thought [51, 52]. These new results hint to new therapeutic strategies. First, one could try to stimulate fibrillation, thus attempting to lower the half‐life of small oligomeric

aggregates. Secondly, one could try to remove the amyloidogenic peptides altogether as soon as they are formed. Most likely, the latter will prove to be the most rewarding in the long run. Because although evidence points at intermediate oligomeric species as highly toxic, the accumulation of amyloidogenic plaques would still impede normal homeostasis ofthe affected tissue from a certain point in time onwards.

FAF Nb1‐3 are currently being tested for their ability to prevent 8 kDa AGel fibrillation. With transmission electron microscopy (TEM) and/or atomic‐force microscopy (AFM), the differ‐ ences in fibrillation could then be visualized in order to link reduced toxicity to a specific shift in amyloid oligomerization or fibril species.

Once more Alzheimer's researchers are also exploring this route. Studying the cytotoxic response of SH‐SY5Y cells through LDL release, they found that oligomeric Aβ provoked a higher cytotoxic reaction compared to monomeric or fibrillar Aβ [53]. Next, the cells were incubated with oligomeric Aβ formed in the presence of anti‐Aβ nanobodies. So far, two distinct nanobodies have been discovered, each binding a specific oligomeric species. Both are able to lower the intrinsic toxicity in SH‐SY5Y cells. The first, Nb A4, does so by inhibiting further aggregation of its target. The second, Nb E1, acts in a different manner by binding to smaller Aβ species than Nb A4, thereby interfering earlier in the fibrillation process. Binding its target stabilizes the formation of small non‐toxic low‐n Aβ species.

It remains to be demonstrated if these approaches and beneficial effects can be translated in model organisms or even in patients, the current findings and results obtained with nano‐ bodies in several amyloid diseases indicate that they could represent an instrument of choice in the diagnosis and/or treatment of these debilitating disorders.

## **Acknowledgements**

This work was supported by the 'Stichting Alzheimer Onderzoek' (SAO‐FRA Belgium), the Queen Elisabeth Medical Foundation (GSKE, Belgium) and the Amyloidosis Foundation (USA). A. Verhelle is supported by the Agency for Innovation by Science and Technology in Flanders.

## **Author details**

Adriaan Verhelle and Jan Gettemans\*

\*Address all correspondence to: jan.gettemans@ugent.be

Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium

## **References**

aggregates. Secondly, one could try to remove the amyloidogenic peptides altogether as soon as they are formed. Most likely, the latter will prove to be the most rewarding in the long run. Because although evidence points at intermediate oligomeric species as highly toxic, the accumulation of amyloidogenic plaques would still impede normal homeostasis ofthe affected

FAF Nb1‐3 are currently being tested for their ability to prevent 8 kDa AGel fibrillation. With transmission electron microscopy (TEM) and/or atomic‐force microscopy (AFM), the differ‐ ences in fibrillation could then be visualized in order to link reduced toxicity to a specific shift

Once more Alzheimer's researchers are also exploring this route. Studying the cytotoxic response of SH‐SY5Y cells through LDL release, they found that oligomeric Aβ provoked a higher cytotoxic reaction compared to monomeric or fibrillar Aβ [53]. Next, the cells were incubated with oligomeric Aβ formed in the presence of anti‐Aβ nanobodies. So far, two distinct nanobodies have been discovered, each binding a specific oligomeric species. Both are able to lower the intrinsic toxicity in SH‐SY5Y cells. The first, Nb A4, does so by inhibiting further aggregation of its target. The second, Nb E1, acts in a different manner by binding to smaller Aβ species than Nb A4, thereby interfering earlier in the fibrillation process. Binding

It remains to be demonstrated if these approaches and beneficial effects can be translated in model organisms or even in patients, the current findings and results obtained with nano‐ bodies in several amyloid diseases indicate that they could represent an instrument of choice

This work was supported by the 'Stichting Alzheimer Onderzoek' (SAO‐FRA Belgium), the Queen Elisabeth Medical Foundation (GSKE, Belgium) and the Amyloidosis Foundation (USA). A. Verhelle is supported by the Agency for Innovation by Science and Technology in

Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University,

its target stabilizes the formation of small non‐toxic low‐n Aβ species.

in the diagnosis and/or treatment of these debilitating disorders.

tissue from a certain point in time onwards.

328 330Exploring New Findings on Amyloidosis

in amyloid oligomerization or fibril species.

**Acknowledgements**

Flanders.

**Author details**

Ghent, Belgium

Adriaan Verhelle and Jan Gettemans\*

\*Address all correspondence to: jan.gettemans@ugent.be


[26] Molloy SS, Bresnahan PA, Leppla SH, Klimpel KR, Thomas G. Human furin is a calcium‐dependent serine endoprotease thatrecognizes the sequence Arg‐X‐X‐Arg and efficiently cleaves anthrax toxin protective antigen. The Journal of Biological Chemistry. 1992;267(23):16396–402.

[12] Haltia M, Prelli F, Ghiso J, Kiuru S, Somer H, Palo J, et al. Amyloid protein in familial amyloidosis (Finnish type) is homologous to gelsolin, an actin‐binding protein.

[13] Kwiatkowski DJ. Functions of gelsolin: motility, signaling, apoptosis, cancer. Current

[14] Silacci P, Mazzolai L, Gauci C, Stergiopulos N, Yin HL, Hayoz D. Gelsolin superfami‐ ly proteins: key regulators of cellular functions. Cellular and Molecular Life Sciences:

[15] Khurana S, George SP. Regulation of cell structure and function by actin‐binding

[16] Dominguez R, Holmes KC. Actin structure and function. Annual Review of Biophy‐

[17] McLaughlin PJ, Gooch JT, Mannherz HG, Weeds AG. Structure of gelsolin segment 1‐ actin complex and the mechanism of filament severing. Nature. 1993;364(6439):685–92.

[18] Burtnick LD, Koepf EK, Grimes J, Jones EY, Stuart DI, McLaughlin PJ, et al. The crystal structure of plasma gelsolin: implications for actin severing, capping, and nucleation.

[19] Yin HL, Albrecht JH, Fattoum A. Identification of gelsolin, a Ca2+‐dependent regula‐ tory protein of actin gel‐sol transformation, and its intracellular distribution in a variety

[20] Vouyiouklis DA, Brophy PJ. A novel gelsolin isoform expressed by oligodendrocytes in the central nervous system. Journal of Neurochemistry. 1997;69(3):995–1005.

[21] Choe H, Burtnick LD, Mejillano M, Yin HL, Robinson RC, Choe S. The calcium activation of gelsolin: insights from the 3A structure of the G4‐G6/actin complex.

[22] Bucki R, Levental I, Kulakowska A, Janmey PA. Plasma gelsolin: function, prognostic value, and potential therapeutic use. Current Protein & Peptide Science. 2008;9(6):541–

[23] Page LJ, Huff ME, Kelly JW, Balch WE. Ca2+ binding protects against gelsolin amyloi‐ dosis. Biochemical and Biophysical Research Communications. 2004;322(4):1105–10.

[24] Kangas H, Paunio T, Kalkkinen N, Jalanko A, Peltonen L. In vitro expression analysis shows thatthe secretory form of gelsolin is the sole source of amyloid in gelsolin‐related

[25] Creemers JW, Siezen RJ, Roebroek AJ, Ayoubi TA, Huylebroeck D, Van de Ven WJ. Modulation of furin‐mediated proprotein processing activity by site‐directed muta‐

of cells and tissues. The Journal of Cell Biology. 1981;91(3 Pt 1):901–6.

Journal of Molecular Biology. 2002;324(4):691–702.

amyloidosis. Human Molecular Genetics. 1996;5(9):1237–43.

genesis. The Journal of Biological Chemistry. 1993;268(29):21826–34.

proteins: villin's perspective. FEBS Letters. 2008;582(14):2128–39.

Biochemical and Biophysical Research Communications. 1990;167(3):927–32.

Opinion in Cell Biology. 1999;11(1):103–8.

CMLS. 2004;61(19–20):2614–23.

sics. 2011;40:169–86.

330 332Exploring New Findings on Amyloidosis

Cell. 1997;90(4):661–70.

51.


[49] Conrath KE, Lauwereys M, Galleni M, Matagne A, Frere JM, Kinne J, et al. Beta‐ lactamase inhibitors derived from single‐domain antibody fragments elicited in the camelidae. Antimicrobial Agents and Chemotherapy. 2001;45(10):2807–12.

[38] Vaneycken I, Devoogdt N, Van Gassen N, Vincke C, Xavier C, Wernery U, et al. Preclinical screening of anti‐HER2 nanobodies for molecular imaging of breast cancer. FASEB Journal: Official Publication of the Federation of American Societies for

[39] Dumoulin M, Last AM, Desmyter A, Decanniere K, Canet D, Larsson G, et al. A camelid antibody fragment inhibits the formation of amyloid fibrils by human lysozyme.

[40] Pain C, Dumont J, Dumoulin M. Camelid single‐domain antibody fragments: uses and prospects to investigate protein misfolding and aggregation, and to treat diseases

[41] Domanska K, Vanderhaegen S, Srinivasan V, Pardon E, Dupeux F, Marquez JA, et al. Atomic structure of a nanobody‐trapped domain‐swapped dimer of an amyloidogen‐ ic beta2‐microglobulin variant. Proceedings of the National Academy of Sciences of the

[42] Rasmussen SG, DeVree BT, Zou Y, Kruse AC, Chung KY, Kobilka TS, et al. Crystal structure of the beta2 adrenergic receptor‐Gs protein complex. Nature. 2011;477(7366):

[43] Dorresteijn B, Rotman M, Faber D, Schravesande R, Suidgeest E, van der Weerd L, et al. Camelid heavy chain only antibody fragment domain against beta‐site of amyloid precursor protein cleaving enzyme 1 inhibits beta‐secretase activity in vitro and in vivo.

[44] Vincke C, Loris R, Saerens D, Martinez‐Rodriguez S, Muyldermans S, Conrath K. General strategy to humanize a camelid single‐domain antibody and identification of a universal humanized nanobody scaffold. The Journal of Biological Chemistry.

[45] Van Audenhove I, Debeuf N, Boucherie C, Gettemans J. Fascin actin bundling controls podosome turnover and disassembly while cortactin is involved in podosome assembly by its SH3 domain in THP‐1 macrophages and dendritic cells. Biochimica et

[46] Bethuyne J, De Gieter S, Zwaenepoel O, Garcia‐Pino A, Durinck K, Verhelle A, et al. A nanobody modulates the p53 transcriptional program without perturbing its function‐

[47] Van den Abbeele A, De Clercq S, De Ganck A, De Corte V, Van Loo B, Soror SH, et al. A llama‐derived gelsolin single‐domain antibody blocks gelsolin‐G‐actin interaction.

[48] Xavier C, Devoogdt N, Hernot S, Vaneycken I, D'Huyvetter M, De Vos J, et al. Site‐ specific labeling of his‐tagged Nanobodies with (9)(9)mTc: a practical guide. Methods

al architecture. Nucleic Acids Research. 2014;42(20):12928–38.

Cellular and Molecular Life Sciences: CMLS. 2010;67(9):1519–35.

associated with these phenomena. Biochimie. 2015;111:82–106.

Experimental Biology. 2011;25(7):2433–46.

United States of America. 2011;108(4):1314–9.

The FEBS Journal. 2015;282(18):3618–31.

Biophysica Acta. 2015;1853(5):940–52.

in Molecular Biology. 2012;911:485–90.

Nature. 2003;424(6950):783–8.

332 334Exploring New Findings on Amyloidosis

549–55.

2009;284(5):3273–84.


## *Edited by Ana-Maria Fernandez-Escamilla*

Amyloid protein aggregates are involved in ''protein-misfolding diseases'' of enormous social and economic impact, still with no effective therapies. The most prevalent amyloid pathologies are related to neurodegenerative diseases, but amyloidosis also affects other organs. The majority of the studies includes serious health connotations on amyloids. However, not all amyloid fibers play a detrimental role in host. An increasing number of studies shows an important beneficial role as ''functional amyloids''. This book opens an exciting door to provide up-to-date information about the function and the mechanisms of the amyloid formation process from the structural, biophysical, biomedical, and nanotechnological perspective, combining the new findings on toxic and functional amyloids studies using theoretical and experimental approaches to fight against amyloid-based diseases.

Exploring New Findings on Amyloidosis

Exploring New Findings on

Amyloidosis

*Edited by Ana-Maria Fernandez-Escamilla*

Photo by dkidpix / iStock