Current Medicinal Chemistry - Immunology, Endocrine & Metabolic Agents - Volume 3, Issue 4, 2003

The term amyloid was initially generally used to describe macroscopic abnormalities which became evident only at autopsy of patients succumbing to a variety of disease states. The advent of histopathological, biochemical and molecular biological techniques, however, has resulted in great advancement in our understanding of amyloid structure. Deposits of amyloid proteins are now recognised as being of diverse origin and chemical make-up, all types of which exhibit a fibrillar structure when viewed by electron microscopy. They are further characterised by their cross-β structure, which confers specific tinctorial properties (Congo red and thioflavin S binding) and resistance to proteolytic degradation. These properties are conferred upon the different amyloids by interactions in the main backbone chain of the molecules and specific sequence effects are usually not of great significance in structure determinance. In the Neurosciences, the term “amyloid” invariably leads many to believe that it is the beta-amyloid or ABeta of Alzheimer's disease which is being discussed. In fact, a crude literature trawl shows that in the 1970-80 decade only 2% of amyloid publications described Alzheimer's disease. The following decade saw this proportion increase to 17% and then to 48% in the last decade of the 20th century; since the Millenium it stands at 56%. It is perhaps fitting, therefore, that this volume of CMC-IEMA begins with the ABeta peptide. Since its biochemical elucidation in 1984, the amyloid protein of AD plaque has been termed beta-amyloid, beta- A4, amyloid-β-peptide, Aβ and ABeta. It should be noted that much of the reported in vitro work with Aβ peptide employs something of a misnomer as the peptide monomer is neither an amyloid, in the strictest sense of the word, nor does it always exhibit beta-pleated sheet structure. Poetic licence has been permitted, however, and no attempt made to standardise (or correct) the nomenclature in the present reviews. Although the biochemical identity of the Ab peptide has been known for two decades, the precise form of the peptide and the means by which it affects its purported neurodegeneration is far from clear. Thus, the volume starts with Walsh and colleagues who explore “The Many Faces of Ab”, reviewing the literature investigating the role of oligomers, protofibrils, ADDL's and fibrils in the development of AD pathology. Even without knowledge of the precise identity of the toxic form of Aβ, most Alzheimer's literature supports The Amyloid Hypothesis, originally proposed in the early 90's. Opposing this, however, we are able to consider the evidence presented by Lee and colleagues who assert that Aβ is the innocent victim of mistaken identity and that the production of the protein is actually the body's attempt to deal with stress and injury. This is a theme that I have also attempted to explore in the final review dealing with a number of amyloidoses and protein conformational disease, how protein misfolding occurs and how the body attempts to deal with what is sees essentially as a foreign body. Overwhelming evidence shows that misfolded proteins are pathological features of their respective disease states - but are they the villains or innocent by-standers? Evidence for a direct effect of aggregated peptides on cell death is presented by Tabner et al. who propose that the production of reactive oxygen species is a fundamental pathological process in many neurodegenerative disorders. Linked to this is the role that metal ions, principally copper and zinc, play in oxidative damage. Curtain and colleagues discuss metal ions, with particular attention being paid to the emerging data with the chelating agent clioquinol. Aβ deposition is also the characteristic feature of cerebrovascular amyloid angiopathy. The importance of these vascular deposits in the pathogenesis of Alzheimer's disease and dementia in general is reviewed by Kalaria et al. Aside from Aβ, this edition of CMC-IMEA deals with a detailed consideration of a number of other misfolded proteins. Rubinsztein discusses the polyQ huntingtin protein in Huntington's disease and explores how cells deal with aggregated and misfolded proteins to protect against excitotoxicity. Neurofibrillary tangles comprising aggregates of hyperphosphorylated tau are a major pathological feature of Alzheimer's and other neurodegenerative diseases such as progressive supranuclear palsy and Pick's disease. Goedert discusses the function of tau and the role of tau aggregation in these disorders. Moving away from the CNS, Brito et al. describe the folding mechanisms underlying the amyloid formation by transthyretin in peripheral diseases such as senile systemic amyloidosis. Therapeutic agents capable of preventing the aggregation or misfolding of proteins associated with the diseases discussed above have long been a scourge of Medicinal Chemists. Difficulties in determining xray structure (particularly with Aβ) and the very nature of protein-protein interactions have hindered the development of potential therapeutics. Advances are being made, however, and Gervais et al. discuss the role of proteoglycans in promoting amyloid formation and how this knowledge is leading towards the discovery of compounds capable of interfering with proteoglycan-amyloid binding. Proteins are dynamic structures that display continuous conformational change throughout their lives from initial translation to eventual degradation. Understanding these changes and the nature of protein folding mechanisms should lead to the discovery of drugs capable of protecting cells from injury. The reviews presented in this edition of CMC-IMEA hopefully provide an insight into both the progress being made but also of the difficulties that still remain.

Although converging lines of evidence suggest a central role for amyloid ß-protein (Aß) in the etiology of Alzheimer's disease (AD), the active form(s) of Aβ that leads to altered neuronal function and death have not been definitively identified. Indeed, until recently, efforts at understanding Aβ aggregation and toxicity focused primarily on fibrillar forms of Aβ akin to those detected in senile plaques. But plaque density and severity of dementia correlate weakly and recent studies report more robust correlations between the levels of soluble Aβ and the extent of synaptic loss and severity of cognitive impairment. Moreover, the identification of pre-fibrillar intermediates in vitro and the detection of SDS-stable oligomers of Aβ in human brain and CSF and in the medium of cells expressing human Aβ demonstrate the existence of soluble, non-fibrillar Aβ assemblies. Because great effort is currently being expended on the development of anti-amyloid therapeutics, it is crucial that remaining concerns about a causative role for Aß in AD be rigorously addressed. Here we review evidence that Aβ toxicity is likely to be mediated by multiple different Aβ assembly forms and discuss possible therapeutic strategies designed to remove or prevent the formation of soluble toxic assemblies.

Protein aggregation and misfolding are two of the pathological hallmarks that are common to many neurodegenerative diseases including Alzheimer disease, Parkinson disease and Huntington disease. While it has generally been assumed that protein aggregation is responsible for neurodegeneration in these disorders, we suspect that protein aggregation, rather than being a major killer of neurons, is, in fact, an attempt to protect neurons from stressful, disease-causing conditions. In this review, we weigh the evidence of whether amyloids, aggregates and neuronal inclusions are good or bad news for neurons.

The deposition of abnormal protein fibrils is a prominent pathological feature of many different 'protein conformational' diseases, including some important neurodegenerative diseases. Some of the fibril-forming proteins or peptides associated with these diseases have been shown to be toxic to cells in culture. A clear understanding of the molecular mechanisms responsible for this toxicity should shed light on the probable link between protein deposition and cell loss in these diseases. In the case of the β-amyloid (Aβ) peptide, which accumulates in the brain in Alzheimer's disease, there is good evidence that the toxic mechanism involves the production of reactive oxygen species (ROS). By means of an electron spin resonance (ESR) spin-trapping method, we have shown that solutions of Aβ liberate hydroxyl radicals when incubated in vitro, upon the addition of small amounts of Fe(II). We have also obtained similar results with α-synuclein, which accumulates in Lewy bodies in Parkinson's disease, and with the PrP (106-126) toxic fragment of the prion protein. It is becoming clear that some transition metal ions, especially Fe(III) and Cu(II), can bind to these aggregating peptides, and that some of them can reduce the oxidation state of Fe(III) and / or Cu(II). The data suggest that hydrogen peroxide accumulates during incubation of these various proteins and peptides, and is subsequently converted to hydroxyl radicals in the presence of redox-active transition metal ions. Consequently, a fundamental molecular mechanism underlying the pathogenesis of cell death in several different neurodegenerative diseases could be the direct production of ROS during formation of the abnormal protein aggregates.

Over a decade of studies have pointed to metal mediated neural oxidative damage as an attractive target for the treatment of Alzheimer's disease. Because of the nature of the blood brain barrier, systemic depletion of the metals, copper, zinc and possibly iron, is not a viable approach. However preliminary studies with CQ, a blood brain barrier penetrating chelating agent, are showing promise. CQ probably works by combining with the metal centres, primarily copper and zinc complexes of Aβ, in the neuropil. This review discusses some of the background that resulted in CQ becoming a lead compound and how we might advance our understanding of its action

Cerebrovascular amyloidosis occurs increasingly in older age. The amyloid β (Aβ) protein type of cerebral amyloid angiopathy (CAA) is the most common form of this microangiopathy, evident in virtually all cases of Alzheimer's disease (AD). CAA may range from focal deposits to widespread infiltration of amyloid in walls of perforating and meningeal arteries, capillaries and diffuse perivascular plaques. Prior to their degeneration vascular smooth muscle cells may be sensitised and stimulated by the aggregated amyloid peptide itself and cytokines. Two patterns of CAA namely arteriolar and capillary types have recently been recognized. CAA also occurs in other dementing conditions including Down's syndrome and dementia with Lewy bodies. It is the principal feature of the hereditary amyloid angiopathies such as hereditary cerberal haemorrhage with amyloidosis of the Dutch type and familial British dementia. Varying degrees of CAA have been recorded in early onset familial AD. Mutations in the amyloid precursor protein (APP) gene that lie in codons within the Aβ domain may result in a phenotype characterised by severe CAA, cerebral infarction and white matter disease. The apolipoprotein E ε4 allele is a strong factor in the development of Aβ CAA, which may progress to lobar or intracerebral hemorrhages. At least two different transgenic mice models over-expressing human APP implicate neuronal origin of the Aβ within vascular deposits. CAA may largely develop due to lack of clearance by reduced proteolytic degradation and progressive blockage of the interstitial drainage pathways via the brain vascular routes superimposed by age-related arteriosclerotic changes. Current observations from both sporadic and familial cases suggest CAA to be an independent factor for cognitive impairment and dementia.

Huntington's disease (HD) is one of nine known neurodegenerative conditions caused by CAG trinucleotide repeat expansions that are translated into abnormally long polyglutamine tracts in the mutant protein. This review describes the clinical and pathological features of HD and considers the genetic and transgenic / knockout mouse data supporting gain-of-function vs. loss-of-function mechanisms whereby the mutation may cause disease. Intraneuronal aggregates (also known as inclusions) are one of the pathological hallmarks of all of the polyglutamine expansion diseases. A major focus of the review is a detailed consideration of the debate as to whether aggregates / aggregation are pathogenic, deleterious or epiphenomena, drawing on data from cell-based and animal models of different polyglutamine diseases and also from the polyalanine codon reiteration disease, oculopharyngeal muscular dystrophy, which manifests intramuscular nuclear inclusions. I will describe how cells deal with aggregateprone and misfolded proteins using chaperones and various degradation pathways. Using data from animal and cell-based models, the review considers some of the different but non-mutually exclusive mechanisms whereby the HD mutation may cause disease, including early changes in gene transcription, production of reactive oxygen species, aberrant proteinprotein interactions and abnormal cellular susceptibility to glutamate. Understanding possible pathogenic mechanisms for HD has provided a rational basis for intervention strategies, ranging from antibodies / peptides that prevent mutant protein aggregation to drugs that enhance mitochondrial function and protect against excitotoxicity.

Tau protein is the major component of the intracellular filamentous deposits that define a number of neurodegenerative diseases. They include the largely sporadic Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration, Pick's disease and argyrophilic grain disease, as well as the inherited frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Until recently, it was unclear whether the dysfunction of tau protein follows disease or whether disease follows the dysfunction of tau protein. The identification of mutations in Tau as the cause of FTDP-17 has resolved this issue by showing that the dysfunction of tau protein is sufficient to cause neurodegeneration and dementia. About half of the known mutations have their primary effect at the protein level. They reduce the ability of tau protein to interact with microtubules and increase its propensity to assemble into abnormal filaments. Surprisingly, the other mutations have their primary effect at the RNA level, thus perturbing the normal ratio of three-repeat to four-repeat tau isoforms. Where studied, this resulted in the relative overproduction of tau protein with four microtubule-binding repeats in brain. Several Tau mutations give rise to diseases that resemble progressive supranuclear palsy, corticobasal degeneration or Pick's disease. Moreover, the H1 haplotype of Tau has been shown to be a significant risk factor for progressive supranuclear palsy and corticobasal degeneration. At an experimental level, the work on FTDP-17 is rapidly leading to the development of good transgenic mouse models for the human tauopathies.

In recent years the issues of protein stability, folding and aggregation have become central in several pathological conditions and in particular in amyloid diseases. Here, we review the recent developments on the molecular mechanisms of amyloid formation by transthyretin (TTR), in particular, in what concerns to protein conformational stability, protein folding and aggregation. Transthyretin has been implicated in pathologies such as senile systemic amyloidosis (SSA), familial amyloid polyneuropathy (FAP) and familial amyloid cardiomyopathy (FAC) which are characterized by extracellular deposition of insoluble amyloid fibrils. SSA is generally a mild disorder and affects predominantly individuals over 80 years of age. In contrast, FAP is an autossomal dominant lethal disease, characterized by peripheral neuropathy, which may affect individuals from their twenties. While in SSA WT-TTR and its fragments are the major constituents of the amyloid fibrils, in FAP and FAC the amyloid fibrils are mostly constituted by variants of TTR. Today, more than 80 TTR mutations throughout the TTR sequence are known. Transthyretin is a homotetrameric protein found in the plasma and in the cerebral-spinal fluid, it is synthesized in the liver and in the choroid plexus of the brain, it has a total molecular mass of 55kDa and a high percentage of β-sheet. Current views on amyloid fibril formation by TTR state that, depending on the protein variant or solution conditions, the native tetrameric protein might dissociate to non-native or partially unfolded monomeric (or even dimeric) species with a high tendency for ordered aggregation into soluble oligomers which grow into insoluble oligomers and eventually mature amyloid fibrils. Thus, issues such as dissociation thermodynamics and dissociation kinetics of the native tetrameric TTR and thermodynamic stability and conformational fluctuations of the non-native TTR molecular species are essential in determining the amyloidogenic potential of different TTR variants. In addition, several other cellular and tissue factors must be involved in modulating the penetrance and age of onset of amyloid pathologies by TTR.

Amyloidogenic proteins have the characteristic of adopting a β-sheet conformation and assembling into fibrils. Although similar in fibrillar appearance, each type of peripheral amyloid deposits differs in the nature of the amyloidogenic protein forming fibrils. Other elements, known as the common structural elements of the amyloid deposits, also contribute to amyloidogenic process in vivo. Among these elements, heparan sulfate proteoglycans (HSPGs) have been shown to bind to different types of amyloidogenic proteins and to promote the formation of β-sheet secondary structure. Once fibrils are formed, HSPGs protect the fibrils from proteolytic degradation, which lead to the accumulation of the deposits in the targeted organs. Understanding the regulation of protein folding by proteoglycans can lead to the development of low molecular weight compounds, which bind to the amyloidogenic proteins prior to their organization as fibrils. Such binding would interfere with the natural association of amyloidogenic protein with HSPGs and maintain the amyloid protein in a non-fibrillar structure (either random coil or a mix of α-helix and β-sheet structure). It would also favor their clearance, and thereby inhibit or completely block the formation of amyloid deposits. Since HSPGs interact with several types of amyloidogenic proteins, such an approach may be beneficial for the treatment of systemic and localized types of amyloidosis.

Misfolding of newly formed proteins not only results in a loss of physiological function of the protein but also may lead to the intra- or extra- cellular accumulation of that protein. A number of diseases have been shown to be characterised by the accumulation of misfolded proteins, notable examples being Alzheimer's disease and the tauopathies. The obvious inference is that these proteinaceous deposits are pathogenic features of the disease. However, systems such as the unfolded protein response and ubiquitin-proteasome complex are in place in the cell to target misfolded proteins for degradation and clearance. Evidence suggests that in disease states, these protein-handling systems may be overwhelmed and the misfolded proteins accumulate as either extracellular deposits (eg. senile plaques in Alzheimer's disease) or intracellular inclusions (as in Lewy bodies in Parkinson's disease). These accumulations may be the direct cause of the particular pathology associated with the diseases or they may be inert “packages” designed to protect the cell from toxic insult.