A brief overview of amyloids and Alzheimer’s disease

Abstract

Amyloid fibrils are self-assembled fibrous protein aggregates that are associated with a number of presently incurable diseases such as Alzheimer’s and Parkinson’s disease. Millions of people worldwide suffer from amyloid diseases. This review summarizes the unique cross-β structure of amyloid fibrils, morphological variations, the kinetics of amyloid fibril formation, and the cytotoxic effects of these fibrils and oligomers. Alzheimer’s disease is also explored as an example of an amyloid disease to show the various approaches to treat these amyloid diseases. Finally, this review investigates the nanotechnological and biological applications of amyloid fibrils; as well as a summary of the typical biological pathways involved in the disposal of amyloid fibrils and their precursors.

Introduction

Amyloid fibril formation is associated with number of incurable diseases.¹ The first known observation of an amyloid disease was in 1639 by Nicolaus Fontanus.² Originally, they were called “amyloids” in 1838 by Matthias Scheiden as they stain blue with an iodine stain, and thus were mistakenly believed to be related to starch.² These fibrils kept the name “amyloid” even though in 1859, they were found to be chemically different from starch.² The most well-known amyloid disease is Alzheimer’s disease and it was first described by Alois Alzheimer in 1906.³

It is estimated that Alzheimer’s disease affects over 12 million individuals worldwide and is the leading cause of dementia in people over the age of 60.⁴ Although it is possible to manage the symptoms of amyloid diseases, there are presently no cures for amyloid diseases. While there is significant research is ongoing to find therapeutics to combat amyloid diseases, it is important to first understand and appreciate the various structural and physiochemical properties of amyloid fibrils in order to design better therapeutics for treating this class of diseases.

This review aims to summarize various aspects of amyloid fibrils using Alzheimer’s disease and amyloid beta (Aβ) as a case study. The review first starts with an overview of the structure of the amyloid fibrils and the kinetics of fibril formation. This is then followed by a discussion of the various effects of amyloid monomers, oligomers, and fibrils on biological systems and the difference in cytotoxic effects of amyloid oligomers and amyloid fibrils. After that, amyloid diseases and current research into amyloid disease therapeutics will be discussed, using Alzheimer’s disease as an example. Finally, the review concludes with an examination of naturally occurring amyloid fibrils, amyloid fibril management in the body and some potential uses of amyloid fibrils.

Amyloids

Amyloid fibrils are self-assembled, fibrillar structures made from proteins that fold into an alternative, β-rich form. For many proteins the most stable conformation in physiological conditions is the native state, as seen by energy surface plots.⁵ This allows the protein to refold into its native state should it be unfolded.⁵ Proteins can take on different structural conformations when subjected to denaturing conditions, and many proteins can fold into an alternative conformation that allows for self-assembly into fibrillar structures known as amyloid fibrils.^6,7 It is believed that the ability to form amyloid fibrils is a generic property of proteins, as many unrelated proteins, such as Aβ_1–40,⁸ lysozyme,² and insulin,⁹ are known to form amyloid fibrils both in nature and in vitro when exposed to the appropriate incubation conditions.¹⁰ These fibrils can accumulate to form deposits or plaques in various organs in the body¹¹ that are related to a variety of diseases.¹ Amyloid diseases are usually associated with a single amyloid fibril forming protein or peptide such as amyloid beta (Aβ) for Alzheimer’s disease.¹

Structure

Many different proteins can form amyloid fibrils and there is little similarity in the amino acid sequence of the proteins that can form amyloid fibrils.¹² The similarities between amyloid fibrils of different proteins start at the secondary structural level. Typically, these amyloid fibrils have a large amount of β-sheet structure, although some amyloid fibrils have a β-sheet content as low as 35%.¹³ The true defining structural features of an amyloid fibril are in the tertiary and quaternary structural levels.

The tertiary structure of an amyloid fibril is unique, as the fibril-forming subunits or monomers have a distinct cross-β arrangement.^1,12,14–16 This similarity in structure is also the reason why amyloid fibrils of different proteins tend to have similar diameters.¹⁵ These monomers then self-assemble into a quaternary structured amyloid protein fibril.¹⁷ This structure is unique to amyloids and produces a characteristic X-ray diffraction pattern that is illustrated in Figure 1. The use of solid state NMR to determine the structure of Aβ amyloid fibrils has revealed that the Aβ monomers adopt a β-strand-turn-β-strand structure and use various side chain interactions with neighbouring Aβ monomers to form two parallel β-sheet structures with a hydrophobic core.^18,19 The cross β structure of the Aβ_1–40 amyloid fibril is therefore believed to be a double-layered structure as seen in Figure 2 ¹⁸ and is similar to structure of Aβ_1–42 amyloid fibrils.^19,20

Figure 1.

Structure of amyloid fibrils. (a) An electron micrograph of Aβ_1–40 Amyloid fibrils. Arrowheads indicate points where fibrils crossover at regular distances. (b) Representation of fibrils consisting of two, three, and four protofilaments. (c) Left-handed fibril chirality of Aβ(1–40) amyloid fibrils observed with transmission electron microscopy (TEM) after platinum side shadowing. (d) Schematic representation of the X-ray diffraction patterns of cross-β and parallel-β structures. (e) Displays an interpretation of the proto-filament structure based on X-ray diffraction, without the twist of the fibrils. In (b–e), the main fibril axis is aligned in the vertical direction. Image taken from Ref.1 (Reproduced from Ref.1, with permission from ©Springerlink).

Figure 2.

Structure of Aβ 1–40 based on solid state NMR. (a) Arrangement of parallel β-sheets in a double-layered arrangement with the characteristic cross-β arrangement of the amyloid fibril. Orange arrows represent the β-sheet structure from residues 12 to 24 and blue arrows represent the β-sheet structure from residues 30 to 40. (b) Central Aβ molecule in an energy-minimized state. Green residues are hydrophobic, magenta residues are polar, blue residues are positively charged and red residues are negatively charged. Image from Ref.18 (Reproduced from Ref.18, with permission from © PNAS).

There are several different proposed models for the self-assembly amyloid fibrils from their monomers and they are the refolding model, natively disordered and gain-of-interaction models, and they are illustrated in Figures 3 and 4.²¹ The refolding model is the most well-known model, where a protein refolds from its native state to an amyloid-forming state.²¹ Natively disordered model is a model of amyloid formation where the protein is natively disordered and folds directly into an amyloid-forming state.²¹ Gain-of-interaction model is a more complicated self-assembly model where part of one amyloid protein monomer becomes a domain in another amyloid protein monomer, and this eventually forms the amyloid fibril.²¹

Figure 3.

A cartoon representation of various models of conversion of a native protein to an amyloid fibril form. (A) Three models of conversion from the native protein to an amyloid. Refolding is where a structured native protein unfolds to an intermediate before forming an amyloid fibril. Natively disordered model occurs when the native protein has little secondary structure and folds into an amyloid, and gain-of-interaction occurs where a portion of the protein allows for interactions with other protein molecules to form the amyloid fibril. Amyloid fibrils formed by the gain-of-interaction model retain a significant amount of native protein structure. (b) A ribbon diagram of an example of a left-handed β-helix of prion fibrils and (c) shows how stacked native c-terminal domains (spheres) can pack around a β-sheet fibrillar core. Image taken with permission from Ref.21, from ©Elsevier.

Figure 4.

Various gain-of-interaction models. (a) Four types of gain-of-interaction models. Direct stacking consists of just simple stacking of the subunits. The cross-β model is where a segment of protein separates from the core to form the cross-β structure of the amyloid. Three-dimensional domain swapping model is where a portion of the protein swaps with a domain in another protein molecule to stack and form a fibril. Three-dimensional domain swapping with cross-β spine is a more complicated version of the three-dimensional domain swapping where the part of the protein connecting the two protein molecules also forms the cross-β structure of the amyloid. (b) A ribbon diagram of a crystalline filament of human superoxide dismutase mutant S134N as an example of direct stacking where three dimers stack together. (c) A ribbon diagram showing the pair sheets of a peptide with a sequence of GNNQQY as a cross-β spine with side chains represented as a ball-and-stick model. (d) A ribbon diagram of the crystal structure of a domain-swapped dimer of human crystalline showing the swapped domains of two monomers. (e) A ribbon diagram of an example of a three-dimensional domain-swapped cross-β spine model of fibrillar polyglutamine mutants of Ribonuclease A. Image taken with permission from Ref.21, from © Elsevier.

A typical fibril has a core of two to six protofilaments, and one possible structure is known as the close packed arrangement, where these protofilaments wind around each other in a rope-like manner to form a super coiled rope-like structure.¹⁷ More recent work using AFM technology has shown that the protofilaments in a fibril can also arrange into a ribbon-like lateral-packing arrangement, where the individual monomers form a ribbon of up to five monomers wide.^22,23 In this mechanism, the monomers first form protofibrils that line up into a fibril, and then twist in regular intervals to form the fibrils. This has been demonstrated using β-lactoglobulin amyloid fibrils.^22,23

Generally, proteins with large numbers of aromatic side groups can form amyloid fibrils due to the π–π interactions that occur between the π-orbitals of the benzene ring structure. These π–π interactions help to bind the amyloid monomers to each other in the amyloid fibril.¹² Other hydrophobic interactions are also believed to be important for the self-assembly of the fibril^{12,18,24–27} and this can be seen in Figure 3. More recent studies of amyloid fibrils using solid state NMR shows that Aβ amyloid fibril monomers assemble in such a way that maximizes contact between the hydrophobic side chains of the monomers.¹⁸ Apart from hydrophobic interactions, salt bridges are also known to aid in the binding between monomers in the fibril structure.¹² Amyloid fibrils usually have a left-handed fibril twist due to the left-handed twist of the β sheets^1,16 as illustrated in Figure 1. The resulting amyloid fibril structure is often resistant to proteolysis^28,29 and this is believed to be due to the amyloid monomer’s protein structure.³⁰ Furthermore, the amyloidogenic monomers that self-assemble into the amyloid fibrils are more resistant to proteolysis due to their new structural conformation; for instance, soluble Aβ amyloid monomers were found to be more protease resistant than native Aβ in vitro.³⁰ Ex vivo Aβ amyloid fibrils were also found to be more protease resistant than Aβ amyloid fibrils produced in vitro,³¹ most probably due to differences in fibrillar morphologies due to a difference in the amyloid fibril’s growth environment.

Variations in morphology

While amyloid fibrils of the same protein often form rather consistent structures, the morphology and properties of these fibrils can vary with exposure to differing environmental conditions during growth. Environmental conditions such as temperature,¹⁶ salt concentration,³² and shear forces^33–35 can affect the morphology of the fibrils in various ways. Although the full extent of how different fibril structures affect cellular and biological functions is not fully understood, it is known that variations in fibril morphology with subtle changes in growth conditions can significantly change the cytotoxicity of the amyloid fibrils formed.³⁶ A good example is that Aβ’s cytotoxicity to SH-SY5Y cells changes if the fibrils were grown with or without stirring.³³ The changes in cytotoxicity are believed to be due to the different molecular structures of the fibrils formed, hence the solvent-exposed amino acid residues would be different, leading to different cytotoxicity levels.³⁶

The presence of other chemicals during the formation of the amyloid fibril can also affect the morphology of the fibrils formed. Even if the chemicals are regarded to be inert, the molecular crowding effect from these chemicals is known to affect the kinetics of amyloid fibril formation.³⁷ For instance, the rate of amyloid fibril formation is increased in the presence of 50 mg/mL dextran due to molecular crowding effects.³⁷ Saccharides such as glucose and mannose can affect the kinetics and morphology of amyloid fibrils by replacing water in hydrogen bonding sites,³⁸ and have been shown to affect the morphology of the fibrils formed. Small carbohydrates tend to cause longer fibrils to form,³⁸ while the sulfated polysaccharide chondroitin sulfate B tends to form larger smooth fibrils by acting as a template.³⁹ An in-depth understanding of how these biopolymers grow in the presence of other seemingly inert and noninteracting biomolecules is important, as the environment that amyloid fibrils evolve in vitro is very different from the crowded environment in vivo. Hence, amyloid fibrils formed in vitro may have very different properties from those formed in vivo and those formed ex vivo, especially as ex vivo fibrils also do contain structural variations.⁴⁰ It is important to take note that ex vivo obtained fibrils may have different properties to fibrils formed in vivo due to fibril fragmentation and possible changes in morphology. This is due to postmortem events and the strong mechanical forces involved in the process of recovering the amyloid fibrils from the amyloid deposits.⁴⁰

Kinetics and formation

Amyloid fibrils form from partially denatured proteins that were subjected to mildly denaturing conditions. These conditions are required for the protein to unfold and re-fold into an alternative form that is capable of self-assembling into an amyloid fibril.⁴¹ Natively unfolded proteins, as discussed previously, can fold directly into an amyloid fibril as illustrated in Figure 3. As the amyloid-forming version of the protein is often thermodynamically more stable than the native state, amyloid fibril formation is usually irreversible in physiological conditions.^42,43 Individual monomers of amyloid protein (aka amyloid precursors) self-assemble to form oligomers or protofibrils that elongate and intertwine to form mature amyloid fibrils.⁴⁴ These units may even induce other native proteins to turn into more precursors as seen in prion diseases.⁴⁵

During a typical amyloid fibril formation, there is a distinct lag phase followed by an elongation phase where rapid fibril growth is observed. In the elongation phase, more amyloid monomers bind to the protofibril⁴⁶ and these protofibrils eventually form a mature amyloid fibril. The actual growth of a fibril generally occurs rapidly and it is known that the rate limiting step is the formation of the partially unfolded ensembles or nucleation.⁴⁷ Amyloid fibril formation is a first-ordered process,²⁰ and a leading theory on the kinetics of amyloid fibril formation is the “one-monomer slow continuous nucleation followed by a fast autocatalytic growth” that was suggested in the Finke-Watzky mechanism,⁴⁸ which is an empirical expression. This theory has been shown to fit aggregation kinetics data of many different amyloid proteins,⁴⁸ hence showing that for most proteins, amyloid formation is limited by the rate of formation of the monomers.

Factors affecting fibril formation

There are several conditions that are known to trigger or accelerate fibril formation, and factors that cause protein denaturation often cause amyloid fibril formation. These include denaturing temperatures,^49,50 denaturing pH conditions,⁴⁹ various solvents,⁴⁹ metal ions such as Zn²⁺ and Cu²⁺,^20,51–53 and shear forces/agitation.^33,35 It is notable, that while some of these factors are not essential to forming amyloid fibrils, they do alter the kinetics of fibril formation. For example, amyloid fibrils can form in the absence of shear and metal ions.^51–53 Some factors affect the amount of conversion of protein to amyloid fibrils. For instance, increased stirring speed results in an increased conversion of protein into amyloid fibrils combined with an increased rate of fibril formation.⁵⁴ Amyloid fibrils grown in different conditions also show different levels of cytotoxicity. It has been shown that fibrils formed in shear conditions are more toxic than those formed in quiescent conditions, however, their precursors are less cytotoxic than those formed without agitation.³³ Fragmentation of amyloid fibrils, especially due to agitation and shear forces, is known to greatly increase the formation of amyloid fibrils⁵⁵ by providing more nucleation sites for the fibrils to form. Furthermore amyloid fibrils can self-assemble by different pathways in different environments, for instance low pH environments can cause protein hydrolysis that result in different amyloid fibrils.⁵⁶ This further highlights the importance of the environmental conditions on the growth and morphology of the fibrils.

Seeding of amyloid fibrils with small amyloid fragments or “seeds” is known to greatly increase the rate of fibril formation.^{10,20,57–60} Seeding bypasses the rate-limiting step of fibril formation; which is the formation of the amyloid monomers or nuclei involving protein unfolding.⁶¹ This is evident as seeded amyloid fibril formation typically lacks a lag phase,^59,61 as seen in Figure 5, as the elongation of the fibril becomes the new rate limiting step in seeded amyloid fibril growth.^57,58 There does appear to be some specificity of seeds in amyloid fibrils, as seeding is most effective when the seed amyloid sequence is close to that of the targeted amyloid forming protein.⁵⁹ For instance, turkey lysozyme amyloid seeds accelerate the fibril formation process of hen egg white lysozyme (HEWL) more effectively than human lysozyme seeds.⁵⁹ This results in a species barrier to prevent fibril formation from amyloid fragments of other species.⁵⁹

Figure 5.

An illustration of the effects of seeding on the kinetics of amyloid fibril formation. Image based on data from Ref.61.

Furthermore, amyloid fibrils tend to form in solutions where the proteins have a net repulsion to each other.⁶² If there is a net attractive force between the monomers, such as in high salt concentration solutions, disordered aggregates will form instead of amyloid fibrils⁶² and this is likely due to the rapid association of these monomers.⁶³

Apart from the factors that affect fibril formation in vitro, there are known risk factors that can affect the chance of developing an amyloid disease such as Alzheimer’s disease. This suggests that these factors affect the amount of amyloid fibrils produced, or affect the response of the body to the amyloid fibrils, and this eventually results in the amyloid diseases. These factors are generally identified through population studies. For example, it is shown that the amount of leisure-time physical activity⁶⁴ and alcohol consumption⁶⁵ can affect the risk of contracting Alzheimer’s disease. However, most of these lifestyle factors affect multiple systems in the body and it is unclear how these will eventually affect amyloid fibril formation. Mutations in other genes are also known to increase the risk of amyloid diseases, for example; the mutation of the apolipoprotein E gene on chromosome 19 is regarded to significantly increase the risk of Alzheimer’s disease.⁶⁶ Unsurprisingly, apolipoprotein E gene mutations are commonly found in patients with late-onset Alzheimer’s disease.⁶⁶

Effects of Amyloids

Effects of amyloids on cells

There is a large body of evidence that shows that the fibrils or the intermediates of fibril formation can adversely affect cells, however, there is a lack of consensus in the literature as to their precise effect on cells.¹⁴ Some researchers argue that the fibril precursors are cytotoxic to cells^14,15 and cause the damage that later manifests as the amyloid disease, while the amyloid fibrils themselves are relatively inert.^7,14,67 In these works, the soluble oligomeric fibril intermediates or precursors are presented as the primary toxic species responsible for the cytotoxic effects observed.⁶⁸ Amyloid oligomers are known to disrupt various cellular processes, such as allowing the passage of Ca²⁺ ions through cell membranes.⁶⁸

Another school of thought is that the amyloid fibrils themselves can physically disrupt cells and tissues by interacting with cellular membranes through their exposed hydrophobic surfaces¹¹ or have cytotoxic properties as well.¹⁴ Cell damage by the amyloid fibrils is achieved by the mechanical disruption of the cell membranes,⁶⁹ or by other means such as interacting with various cellular receptors.¹⁴ It is also believed that if the fibrils get large enough, they can disrupt the cell plasma membranes.⁷⁰ The lack of consensus on the cytotoxicity of the amyloid fibrils may be due to the polymorphism which exists in amyloid fibrils that results in differing cytotoxic pathways and cytotoxicity levels.¹⁴ This is further complicated as changes in the incubation conditions of the amyloid forming protein, such as shearing, can change the cytotoxicity of both oligomers and fibrils.³³ Amyloid fibrils and their intermediates have generic cytotoxicity pathways, as well as more specific cytotoxic effects from interactions with cellular receptors by amyloid precursors or fibrils of specific proteins.

The most cytotoxic species from amyloid fibril formation pathway is generally believed to be the soluble oligomeric state,²⁰ as the oligomers have been shown to be more cytotoxic than the mature fibrils for many proteins.⁶⁸ These soluble species can permeate cell membranes and cause an influx of calcium ions into the cytosol from the surroundings and the endoplasmic reticulum of the cell, this eventually results in apoptosis.^20,68 This effect is believed to be a generic, nonspecific mechanism for the cytotoxicity for all oligomeric amyloids. Other effects that these oligomers cause include binding of oligomers to the membrane resulting in generalized membrane thinning, a carpeting effect, and detergent effects where the amphipathic amyloid oligomers interact with the phospholipid bilayer membranes.^69,71 The various mechanisms of cytotoxicity from the amyloid oligomers are illustrated in Figure 6. These cytotoxic effects cause disruption to the cell’s phospholipid membranes and eventually lead to leakage of ions across the membrane.⁶⁹ As amyloid forming proteins, such as Aβ in the body, are usually found at concentrations significantly lower than that required to form fibrils in vitro, it is believed that binding of the soluble protein to certain parts of the cellular membranes helps to induce and accelerate fibril formation in vivo.⁷¹ Hence, the presence of amyloid fibrils in vivo proves that the amyloid oligomers or monomers do interact with cells in vivo.

Figure 6.

The various toxic effects of amyloid oligomers on cell plasma membranes. Effects illustrated are nonspecific interactions which are the carpeting effect, pore formation and detergent effects with cellular phospholipid bilayers that result in leakage across the membranes. Image taken from Ref.69, from ©Wiley, reproduced with permission.

Amyloid fibrils themselves have also been found to induce cell death either via apoptosis or necrosis.¹⁴ However, it is difficult to distinguish between the two forms of cell death due to the presence of both apoptotic and necrotic markers in the affected cells.¹⁴ A good example of the different effects the fibrils and oligomers have on cells, is that HEWL amyloids in fibril and oligomeric forms have been found to induce cellular death via different pathways in vitro.¹⁴ For cytotoxicity studies, cell plasma membrane damage can be tracked by the release of the enzyme lactate dehydrogenase from cells into the supernatant.¹⁴ The activation of the enzyme caspase is used as a marker to show that an apoptotic cell death pathway had occurred.¹⁴ The HEWL oligomers were found to increase caspase activity, which suggests an apoptotic pathway. This later resulted in the release of lactate dehydrogenase and eventually caused an apoptotic like cell death.¹⁴ In contrast, HEWL amyloid fibrils were found to cause mitochondrial failure and trigger the release of lactate dehydrogenase in a more nonspecific necrotic pathway.¹⁴ However, some caspase activation was also detected for fibril-induced cell death in later stages of incubation, which suggests activation of some of the apoptotic pathway components.¹⁴ The cytotoxicity of the amyloid fibrils appears to be morphologically dependent and species dependent, and this is further complicated by the heterogeneous morphology of amyloid fibrils formed. For instance, equine lysozyme protofilaments were found to be nontoxic to cells.¹⁴

There also exist more specific interactions of amyloid fibrils, whereby amyloid fibrils of a particular protein cause various effects to a target cell by interacting with specific cellular receptors. Examples are of Aβ interacting with microglial receptors to cause inflammation⁷² and transthyretin amyloid fibrils interacting with RAGE (receptor for advanced glycation end products) receptors to cause oxidative stress, inflammation and various cytotoxic effects.^14,73,74 Aβ itself is also believed to cause lipid peroxidation as well as the production of various reactive oxygen species.⁷⁴ These different mechanisms highlight different means in which amyloid fibrils and their intermediates can cause cellular damage that ultimately manifests as an amyloid disease. They also serve to illustrate the different effects that amyloid fibrils from different proteins can have on cells, thus providing an insight into why specific amyloid proteins are associated with a particular amyloid disease, such as Aβ for Alzheimer’s disease.¹

Effects on tissues and organs

Amyloidosis is defined as the deposition of amyloids in one or more organs of the body. Cellular death due to amyloid fibril deposits and their cytotoxic precursors can damage tissues and ultimately result in organ failure. Sometimes organ failure occurs before very large amyloid fibril deposits are observed in the affected organs,⁷⁵ suggesting that damage to the organs was done by the oligomers or protofilaments leading to the amyloids fibrils. Thus, it is important to differentiate amyloidosis from amyloid diseases that are due to the toxic effects of the various amyloid species.

Amyloid fibril deposits are also believed to disrupt organ function by physical disruption of the tissue architecture.⁷⁵ This effect is more pronounced in organs such as the heart, where very large amyloid fibril deposits can cause significant changes to the gross structural properties of the heart, leading cardiac dysfunction.⁷⁶ In particular, the inelasticity due to amyloid fibril infiltration of cardiac muscle restricts ventricular filling, and this eventually results in heart failure.¹¹ Additionally, the infiltration of tissues with amyloid fibrils can cause necrosis, as observed in the cardiac muscle in cardiac amyloidosis sufferers, and this can lead to impaired organ function and eventually death.⁷⁷

Amyloid diseases

There are large numbers of diseases that are associated with amyloid fibril deposition, and some of the more well-known diseases are shown in Figure 7. There are differing schools of thought as to how the fibrils affect the organs to cause the diseases, as there is a poor correlation between disease symptom severity (e.g. extent of dementia from Alzheimer’s disease) and the amount of amyloid plaque found in the individual’s organs (e.g. Aβ in the brain for Alzheimer’s disease).⁶⁸ In the case of Alzheimer’s disease, it was found that the soluble amyloid protein (Aβ) concentration correlated better with the symptom severity than the number of amyloid plaques.²⁰ Despite this, it is known that familial systemic amyloidosis tend to result from mutations that destabilize the folded structures of the globular proteins associated with the disease.¹⁵ This provides evidence that the amyloid species are responsible for the manifestation of the diseases.

Figure 7.

A List of some amyloid diseases and their precursor polypeptides. Data taken from Ref.1, from ©Springerlink, reproduced with permission.

Diseases associated with amyloid fibrils are broadly classified into degenerative diseases (such as Alzheimer’s disease) and prion diseases (such as bovine spongiform encephalitis or BSE). Prions are an unusual subset of amyloids that are known to induce amyloid fibril formation in vivo in other systems.^78,79 Organ failure is a common form of death from amyloid-related diseases, for instance, a third of patients with systemic amyloidosis die from a cardiac related death from congestive heart failure due to extensive amyloid fibril deposits.⁷⁷

Treatment for amyloid diseases is difficult and is presently limited to management of the symptoms of the diseases, especially for hereditary systemic amyloidosis.¹¹ Though some forms of amyloidosis can be treated through organ transplantation and chemotherapy, large amyloid fibril masses can only be removed surgically.¹¹

Alzheimer’s disease

Alzheimer’s disease one of the most well-known of all amyloid diseases and is presently the fourth most common cause of death in the developed world.⁸⁰ Due to the aging of worldwide population, Alzheimer’s disease presents a growing social and economic burden to society.⁸¹ The causes and pathology of the disease itself are still not entirely understood. Electron microscopy of the plaques from Alzheimer’s disease sufferers in 1963 showed the presence of amyloid fibrils in the brains of patients with Alzheimer’s disease,⁸² and this highlighted the link between the amyloid fibrils and the disease. There are two forms of Alzheimer’s disease: an early onset version caused by an autosomal dominant mutation, and a sporadic form of the disease.¹⁵ The disease is often studied using special transgenic mice that serve as a model for Alzheimer’s disease.⁸³

Symptoms and diagnosis

The best known symptoms of Alzheimer’s disease are memory disorders and impairment of cognitive domains. These symptoms interfere with the affected individual’s daily life.⁸⁴ The diagnosis of Alzheimer’s disease was formerly based on the previously mentioned symptoms.⁸⁴

Due to the existence of many different types of dementia associated with aging, it is difficult to accurately distinguish Alzheimer’s disease from another form of dementia. More recently, other techniques such as magnetic resonance imaging (MRI) and positron emission tomography (PET) have been used as improved diagnostic methods for Alzheimer’s disease. People with Alzheimer’s disease tend to have medial temporal lobe atrophy of the brain, as well as hypometabolism and hypoperfusion in the temporoparietal areas of the brain, and these symptoms can be detected by MRI and PET.⁸⁴ Another diagnostic method presently under investigation is biomarker sampling of the cerebrospinal fluid;²⁰ as increased levels of tau protein, increased levels of phosphorylated tau protein and a decrease in Aβ_1–42 levels in the cerebrospinal fluid are indicative of Alzheimer’s disease.⁸⁴ Despite all these advances, the only definitive diagnostic method for Alzheimer’s disease is still via a brain biopsy.⁸⁴

Established treatment methods

Currently, Alzheimer’s disease is usually managed using acetylcholinesterase inhibitors^81,85,86 or NMDA (N-methyl-d-aspartate) receptor antagonists.^20,81 Present treatment methods focus on replacing the neurotransmitters lost during the onset of Alzheimer’s disease in the brain to help alleviate the symptoms of the disease.⁸⁵

Aβ

Before going into the current research in Alzheimer’s disease therapeutics, it is important to briefly introduce Aβ, as many of the currently researched treatment strategies revolve around controlling Aβ production or Aβ amyloid fibril formation. Aβ is a small protein found in mammals that is expressed from a gene found on chromosome 21 in humans⁶⁶ and is well known to form fibrils under physiological conditions.³⁴ It is believed that the cytotoxic intermediates of Aβ fibril formation result in neuronal disruption, which in turn results in the symptoms seen in Alzheimer’s disease.⁸⁷ The neurotoxic effects of Aβ include inhibition of long-term potentiation, loss of glutamate receptors and disruption of calcium ion uptake by α7 nicotinic acetylcholine receptors.²⁰

In Alzheimer’s disease, Aβ amyloid plaques first appear in the basal part of the isocortex and spread to the rest of the isocortex as the disease progresses.⁸⁸ In late stage Alzheimer’s disease, these amyloid plaques can be found thought the brain, with the isocortex having the highest amyloid plaque density.⁸⁸

Due to its relationship with Alzheimer’s disease, Aβ is a very popular model protein to study amyloid fibril formation and inhibition in vitro.^8,89–94 The native conformation of Aβ has little secondary structure, and hence Aβ is known as an natively unfolded protein.⁹⁵ Aβ forms amyloid fibrils by folding from the native random-coil rich state to a α-helical rich intermediate, and finally to a β-sheet rich amyloid monomer that self-assembles into the fibrils.⁹⁶

Aβ is derived from the membrane-bound amyloid precursor protein (APP).^20,97 There are two possible cleavage sites for APP in a human cell; one site is acted on by α-secretase that produces the peptides αAPPs and C83. The other site is cleaved by β-secretase which produces βAPPs and C99. Following the initial cleavage by α or β-secretase, a large multiprotein complex known as γ-secretase acts on both possible APPs to produce peptide P3 for αAPP and Aβ1–40 or Aβ1–42 for βAPPs.^66,97 In Alzheimer’s disease sufferers, the rate of production of Aβ is unchanged while the clearance rate of Aβ is reduced.²⁰

The normal functionality of Aβ is not fully understood to date. Aβ is known to have roles in kinase activation, protection from metal oxidation damage, cholesterol transport and ion channels.⁹⁸ APP is a highly conserved protein that is expressed in almost all mammalian tissues, where the highest levels appear in the brain and kidney.⁶⁶ Similar proteins are found in Drosophila and Ceanorhabditis elegans,⁶⁶ hence it is believed that this protein plays a crucial role in the development of organisms. Some in vitro studies show that the various products of the cleavage of APP help mediate cell adhesion⁸⁰ and growth of neurons.⁶⁶ It also is known that knockouts of the γ-secretase enzyme results in a lethal phenotype as γ-secretase is known to have some relationship with the notch receptors.^4,97

Some evidence that Aβ plays a significant part in the loss of mental function observed in Alzheimer’s disease can be inferred from studying traumatic brain injury. Aβ plaques are often observed forming in humans as well as animal models after brain injury, and the plaques formed appear diffused and similar to those of early Alzheimer’s disease.⁹⁹ Mice with a β-amyloid converting enzyme impairment were shown to have reduced Aβ plaques and subsequently, significantly better behavioral outcomes after injury,⁹⁹ hence suggesting that the Aβ itself does cause impaired brain function in some way.

Tau Protein

Another protein closely linked to Aβ is the tau protein, a protein that is involved in the stabilization of microtubules.¹⁰⁰ In Alzheimer’s disease, hyperphosphorylation of tau protein occurs which causes the tau proteins to fold from an unfolded monomer to a more structured form that is capable of self-assembly into neurofibrillary tangles (NFTs).¹⁰¹ These tangles occur largely in the stoma of neurons in a few types of neurons, such as the pyramidal cells.¹⁰² In the brain, NFTs tend to be concentrated in the transentorhinal and entorhinal layer, but can be found thought the brain in late stage Alzheimer’s disease.⁸⁸ The deletion of the N and C terminus of the tau protein is known to accelerate aggregation of the protein.¹⁰¹ These tau protein aggregates are cytotoxic, especially when formed from the truncated tau proteins, and the eventual neuronal death is believed to lead to Alzheimer’s disease.¹⁰¹ As the density of these tau incursions corresponds well with the severity of cognitive decline in Alzheimer’s disease,¹⁰¹ these NFTs are believed to play a critical role in the progression of the disease. The production of the NFTs appears to be intrinsic and unaffected by the presence of Aβ amyloid deposits,¹⁰² although it is believed that Aβ has some synergistic effects with tau protein that enhances neurodegeneration.²⁰

Current Research into Amyloid Disease Therapeutics

There are many different approaches to developing a cure or therapeutic for amyloid-based diseases. To illustrate the various approaches we shall use the treatment of Alzheimer’s disease as a case study, as it is the most well-known of the amyloid diseases.

Inhibitors of Amyloid Fibril Formation

There have been many attempts at developing an inhibitor to stop amyloid fibril formation. Chelators of metal ions, particularly copper and iron ions, have been investigated as a possible treatment as copper and iron ions play an important role in Aβ fibril formation.^{20,89,103,104} Another approach was the use of antibodies raised against the amyloid fibrils such as antibodies that bind to Aβ.^81,90,104 This approach was used by the company, Elan, in 1996 and reached a stage 2 clinical trial.¹⁰⁵ However, the trial was halted due to 6% of the patients developing meningoencephalitis.^20,106 Other methods include using synthetic peptides that bind to fibrils to inhibit fibril formation^8,27 and using small ligands to stabilize Aβ in an α-helical conformation to prevent amyloid fibril formation.^20,94

As the self-assembly of amyloid fibril relies on the interactions between the exposed hydrophobic surfaces of the amyloid fibril precursors,¹² surfactants can be used to bind to these surfaces and inhibit amyloid fibril formation. Amphiphilic surfactants have been used to inhibit model amyloid fibril formation.⁹² Some examples of amphiphilic inhibitors for Aβ are the surfactants 1,2-dihexanoyl-sn-glycero-3-phosphocholine (di-C6-PC),1,2-diheptanoyl-sn-glycero-3-phosphocholine (di-C7-PC)⁹² and hexadecyl-N-methylpiperidinium (HMP) bromide.⁹¹ Inhibitors that target residues involved in aggregation, such as lysine, have also been shown to prevent amyloid formation¹⁰⁷ by binding to parts of the Aβ that are used for fibril assembly, hence preventing the self-assembly of the fibril.

Other approaches to treating Alzheimer’s disease

Other than targeting amyloid plaque formation itself, other possible approaches to combating amyloid diseases are also being investigated, such as modifying the production of the amyloidogenic protein and targeting Serum Amyloid Protein (SAP).⁸⁵ Another approach would be to decrease the production of Aβ. Hence, drugs that inhibit β-secretase or γ-secretase, or increase the activity of α-secretase would reduce the amount of Aβ and this could possibly halt the progression of Alzheimer’s disease.^{4,81,104,108,109}

β-secretase and γ-secretase inhibitors are a very popular choice for an Alzheimer’s disease therapeutic and several of these inhibitors are being investigated, with some reaching various stages of clinical trials.^4,81,93,109 γ-secretase is a difficult protein to target for a therapeutic against Alzheimer’s disease, due to the involvement of γ-secretase in many other signaling pathways in the cell, such as its role in notch receptor release.^97,104 Inhibiting the activity of γ-secretase may result in side effects such as hematological and gastrointestinal toxicity, skin reactions, and even changes to hair colour.⁸¹ Second-generation γ-secretase inhibitors that do not affect notch signaling are currently being investigated, and some of these inhibitors show promising results as a therapeutics.⁸¹

Other viable therapeutic strategies to combating Alzheimer’s disease that are currently being investigated include the clearance of Aβ deposits, prevention of mitochondrial dysfunction, targeting of tau proteins, as well as neurotrophins that prevent neuronal cell death.⁸¹ The use of antioxidants for neuroprotective effects is a viable strategy to combat the oxidative stresses associated with Aβ amyloid formation.²⁰ As Aβ amyloid formation causes a variety of toxic effects as previously discussed, combating these toxic effects would also be a viable treatment strategy for Alzheimer’s disease as well. A final option to treating Alzheimer’s disease is targeting known risk factors that are known to increase the chance of contracting the disease such as cholesterol levels.¹⁰⁴ One advantage of this method is that cholesterol lowering statin drugs are presently widely prescribed, well tolerated, and that lowering cholesterol levels have some generic health benefits beyond prevention of amyloid diseases.¹⁰⁴ However, it is still not known how cholesterol affects the production of Aβ or the progression of Alzheimer’s disease.¹⁰⁴

Use of natural products to treat amyloid diseases

There are several natural products that are capable of inhibiting amyloid fibril formation. For example, a natural product of soy beans, Genistein, is capable of inhibiting amyloid fibril formation of transthyretin.¹¹⁰ Therefore, increasing the oral uptake of such products can potentially reduce the effect of amyloid fibril formation in people.¹¹⁰ A large number of other natural products are also being investigated as leads in developing drugs to treat Alzheimer’s disease.¹¹¹ The effects of these natural products vary greatly and target different aspects of Alzheimer’s disease by acting as acetylcholinesterase inhibitors, secretase inhibitors, amyloid fibril inhibitors, amyloid fibril removers, neuroregenerators or antioxidants.¹¹¹ Examples of these are the alkaloid acetylcholinesterase inhibitor physostigmine (eserine) from Physostigma venenosum, Terpenoid inhibitors of beta-secretase 1 derived from the edible herb Aralia cordata, and the Aβ amyloid inhibitor curcumin from the turmeric plant.¹¹¹

Other impacts of amyloids

Apart from amyloid diseases, amyloid fibril formation also causes problems in industries that work with proteins, such as the insulin manufacturing industry^112,113 and the dairy industry.¹¹⁴ Proteins that usually do not form amyloid fibrils under physiological conditions can form amyloid fibrils during processing, as the high temperatures and shear forces involved in processing these proteins tend to cause the formation of amyloid fibrils. For instance, the purification of insulin often involves steps with very low pH conditions, and this tends to promote amyloid fibril formation.¹¹³ This is undesirable as the presence of amyloid fibrils will reduce the quality of product, cause a wastage of protein as well as cause fouling in various parts of the processing machines.¹¹⁴ Amyloid fibril formation also limits the shelf life of proteins such as insulin.¹¹³

Uses of amyloids

Like many other naturally occurring protein structures, amyloid fibrils are used in nature and they can be referred to as “Functional Amyloids.” There are also some more recent studies that investigate the use of amyloid fibrils for other purposes.

Natural occurring functional amyloids

Amyloid fibrils do occur naturally in many biological systems, performing various different roles in the organisms using them. In mammals, one example is the proteins of the interior lens fiber cells of the eye.^7,115 It is believed that amyloid-like structures of the crystallin proteins of the lens are responsible for the lens picking up amyloid-specific dyes such as Congo red and Thioflavin T, as well as showing other amyloid traits in Raman and infrared spectroscopy.¹¹⁵ It is believed that the stability and protease resistance of the β-sheet rich amyloid fibrils plays an important role in these function of these proteins.¹¹⁶ Another notable use of amyloid fibrils in humans is the production of the pigment melanin. This pigment is produced in specialized lysosomes that contain an amyloid fibril that acts to sequester toxic intermediates involved in the production of melanin, hence acting as a template for the formation of melanin.¹¹⁶

It has been also suggested that amyloid fibrils may be deposited as a means to seal off damaged capillaries and arterioles. Aβ deposits have been observed forming in the brains of people who survived severe head trauma,⁷ and it is believed that these deposits play some role in vascular integrity.¹¹⁷ Aβ production is known to be increased in cells under oxidative stress due to the up-regulation of the production of the Aβ from the APP, even though the production of APP remains constant.⁸⁷ Damage to the nerve axons can also cause an accumulation of APP,⁹⁹ which can also result in increased Aβ production. It is also notable that people who suffer from strokes have a slightly increased chance of getting Alzheimer’s disease.¹¹⁸ Similarly, Aβ plaques have been observed in the autopsy of 30% of patients from all age groups after death by a traumatic brain injury.⁹⁹ It is likely that the increase in amyloid fibril deposition may have occurred in an attempt to contain the blood loss from damaged blood vessels from a stroke or similar injury to the brain.¹¹⁷ Additionally, factor XII of the clotting cascade, as well as plasmin is known to be activated by amyloids,¹¹⁶ providing further evidence in the use of amyloids to control blood loss.

Other than mammalian systems, there are many other organisms that employ functional amyloid-like systems including insects, fungi and bacteria.⁷ In bacteria, a well-known extracellular amyloid fibril is the curli, and these proteins are involved in surface adhesion and colony formation.¹¹⁶ Various yeast prions are known to exist naturally and are believed to play a part in causing phenotypic variations without causing changes in genotypes. This allows the yeast to change its phenotype and survive when changes in environmental conditions occur, yet allowing it to rapidly switch back to its original phenotype when the original environmental conditions return.¹¹⁹ Even fungi hydrophobins that are involved in production of fungi fruiting bodies, spidroins from spider silk and proteins in insect eggshells are known to be a type of amyloid.¹¹⁶ These proteins most likely evolved to use high yield strength and protease-resistant nature of the amyloid fibril itself.¹¹⁶

Using amyloid fibrils in normal cellular function is risky, due to the toxic oligomeric intermediates involved in forming the fibrils, as well as cytotoxicity of the fibrils themselves.¹¹⁶ It is for that reason that naturally occurring, functional amyloid fibrils form much faster than amyloid fibrils that are associated with diseases (such as Aβ), as it is believed that the rapid amyloid fibril formation prevents the build-up of toxic oligomeric species involved in amyloid fibril formation.¹¹⁶ Furthermore, cells have developed a variety of other safeguards, such as use of chaperones and compartmentalization, to prevent toxic effects from these functional amyloid fibrils and their oligomers.¹¹⁶

Potential applications of amyloids

Amyloid fibrils are a very attractive option as a template for various nano-structures¹²⁰ and have been put into many applications such as improving the efficiency of solar cells.¹²¹ Protein amyloid fibrils can form under relatively mild conditions, hence making them an attractive choice for bio-templating applications. Furthermore, amyloid fibrils have a very high yield strength that is comparable to that of steel,¹¹⁶ and this has potential applications in the fields of nanotechnology and material science. The ability for amyloid fibrils to form into different morphologies can also open up many other possible applications in nanotechnology.

Amyloid fibril removal in the body

The body’s main defenses against misfolded proteins are chaperones and the ubiquitination-proteasome pathway. These processes are part of proteostasis, i.e. the control of folding and abundance of proteins in the body. However, as a person ages, these processes become less efficient and become overwhelmed by the quantity of misfolded proteins present, thus allowing the amyloid fibrils to accumulate.^7,15 This is also the reason why the majority of these amyloid diseases are associated with the elderly.

Chaperones are proteins that aid protein folding and encourage proteins to fold into a particular conformation,²⁸ and that conformation is usually the protein’s native structure. There are many chaperones known in the body, many of them are heat-shock proteins or HSPs¹⁷ and they are each associated with a small set of proteins. Chaperones are largely grouped into hsp60 and hsp70 for eukaryotes²⁸ based on their structure and function. Chaperones have an affinity for exposed hydrophobic patches on proteins and often hydrolyse ATP to help fold the target protein to the desired conformation.²⁸ Some chaperones, such as Clustrin, are ATP independent and act as stabilizers to stressed proteins for refolding by other proteins.¹²²

While most chaperones are intracellular, there are a few extracellular chaperones identified to date. Examples of extracellular chaperones are: clustrin, haptoglobin, α2-macroglobulin, and serum amyloid P component (SAP).¹⁷ These extracellular chaperones are unusual in that they are often found associated with the amyloid plaques.¹⁷ Apart from refolding denatured proteins of typical intracellular chaperons, extracellular chaperones also perform a variety of other functions as well.¹⁷ Some extracellular chaperones mediate the destruction of amyloidogenic particles. Clustrin is known to remove extracellular cell debris and bind to Aβ to facilitate Aβ uptake and degradation by cells using megalin.¹⁷ α2-macroglobulin is both a protease inhibitor and a chaperone that binds to various hydrophobic molecules including amyloidogenic proteins such as Aβ, prion protein and β_s macroglobulin.¹⁷ Some of these chaperones are also known to inhibit the amyloid fibril formation of many different proteins in vitro. For instance, clustrin is found to bind to Aβ oligomers and inhibit fibril formation while leaving native Aβ and mature Aβ fibrils alone.¹⁷

The second process to regulate misfolded proteins in proteostasis is ubiquitination and it involves a small 76 residue protein known as ubiquitin.²⁸ When chaperones fail to correct misfolding in a protein, or when the protein ages, a series of enzymes attach ubiquitin molecules to the protein. These ubiquitin molecules then signal the proteasome; a large protein complex made of multiple different proteins, to degrade the targeted protein.^7,28 However, with age, many of these defenses get overwhelmed and proteasomes are often found buried in amyloid fibril aggregates as they are unable to degrade all of the amyloid protein.⁷ Furthermore, proteins can undergo oxidation with age as a consequence of the production of reactive oxygen species due to aerobic respiration.¹²³ It is also found that oxidized proteins are more resistant to ubiquitination and proteasome degradation and the build-up of these proteins is believed to contribute to amyloid diseases.¹²³

Once amyloid plaques are formed, they are sometimes removed by phagocytosis. For instance in the brain, Aβ fibrils can be removed via phagocytosis by the microglia cells and subsequently degraded by cellular enzymes.^124,125 However, the build-up of oligomeric Aβ is known to inhibit this process, resulting in further build-up of Aβ when the person ages.¹²⁵ Hence, although there are systems in the body that prevent the formation and build-up of amyloid fibrils, they can be overwhelmed with age and result in the build-up of amyloid fibrils in the body.

Conclusions

Amyloid fibrils are fibrillar protein aggregates that are self-assembled from β-sheet rich, misfolded proteins that are associated with several incurable diseases. Many unrelated proteins can form amyloid fibrils, and it is believed that amyloid fibril formation is a universal property of proteins. The self-assembly of these fibrils typically follows three phases: a lag-phase, an elongation phase, and a plateau phase. Many factors, such as stirring speed, metal ions, temperature and pH, can affect the kinetics of formation and the morphology of these fibrils. Functional amyloid fibrils occur naturally in various bodily systems, such as melanin production. The regulation of amyloid fibril production is achieved using chaperones and ubiquitination. Mature fibrils are difficult to remove but they can be removed by phagocytosis. Amyloid fibrils can affect cells and organs in many different detrimental ways, and this is believed to eventually result in various amyloid diseases. The most well-known amyloid disease is Alzheimer’s disease, though the pathology of Alzheimer’s disease and its relation to the Aβ amyloid fibrils is still not fully understood. There is no cure for Alzheimer’s disease at present and treatment of Alzheimer’s disease is largely limited to symptom management. Several different approaches to treating amyloid diseases are being investigated such as amyloid fibril inhibitors and regulating the production of Aβ. Although many of these drugs are in clinical trials, to date there has yet been a satisfactory therapeutic developed to combat these amyloid diseases. As more resources are spent on the development of therapeutics to treat amyloid diseases, an understanding of the various structural, kinetic, biological and cytotoxic aspects of the amyloid fibrils and their precursors is essential to design the next generation of therapeutic compounds to treat these presently incurable diseases.