Current Pharmaceutical Design - Volume 14, Issue 30, 2008

Amyloidosis is a pathological condition where proteins with diverse chemical composition are extracellularly deposited as fibrils in the brain, heart, kidney, liver, pancreas, nerves, and other tissues or organs resulting in their serious dysfunctions. Misfolding or conformational changes of normally soluble proteins lead to pathological deposition of fibrillar proteins with a cross-β-pleated configuration. In addition to amyloidosis characterized by extracellular fibril deposition, protein-misfolding or conformational disorders include diseases characterized by intracellular deposition of fibrillar structures (inclusion bodies) such as Lewy body diseases [Parkinson's disease (PD) and dementia with Lewy bodies (DLB)] and polyglutamine diseases of the brain. Amyloidosis is classified to (1) systemic amyloidosis that involves various organs in the body, and (2) localized amyloidosis that affects a specific organ. According to the amyloidogenic protein, systemic amyloidosis is classified to several types including immunoglobulin (AL), AA, transthyretin (TTR) [familial amyloidotic polyneuropathy (FAP) and senile systemic amyloidosis (SSA)], and β2-microglobulin (β2-m) amyloidoses (dialysis-related amyloidosis). An example of localized amyloidosis is brain amyloidosis including amyloid β-protein (Aβ) [Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA)] and prion protein amyloidoses (Creutzfeldt-Jakob disease and related disorders). A pathologic process to amyloid deposition includes: (1) production of amyloid precursor proteins, (2) processing of the precursor proteins to amyloidogenic proteins (monomers), and (3) protein misfolding (conformational change) and aggregation, finally resulting in deposition of the fibrillar proteins. The protein misfolding and aggregation is an essential process, shared by different types of amyloidoses and other protein-misfolding disorders. In this process, oligomeric forms or intermediate states have been reported to be more toxic than mature fibrils in Alzheimer's disease or other protein-misfolding disorders. Previously, a disease-modifying therapy for amyloidosis had been largely limited to the suppression of production of amyloid precursor proteins; for example, familial TTR amyloidosis caused by mutations of the TTR gene has been treated by transplantation of the liver, TTR-producing organ. Recently, however, anti-amyloidogenic or anit-protein misfolding therapies that target the process of protein misfolding and aggregation have been under development, and clinical trials with these therapies have been started in some diseases. In this issue, the experts in this research field discuss molecular basis and therapeutic potentials of anti-amyloidogenic/proteinmisfolding therapies in these disorders. In the first article, Dr. Goto and his colleagues [1] address recent advances in the structural study of structure, formation, and propagation of amyloid fibrils. On the basis of various approaches including solidstate nuclear magnetic resonance (NMR), hydrogen/deuterium exchange of amide protons, and total internal reflection fluorescence microscopy, convincing models of amyloid structures, their formation, and propagation have emerged. In the second article, Dr. Sekijima and his colleagues [2] discuss the pathogenesis of TTR amyloidosis (FAP/SSA), and suggest new strategies for therapeutic interventions for replacement of liver transplantation that is currently the only effective treatment for FAP. The new strategies include stabilization of TTR tetramer (native state) by small molecule binding in order to prevent its dissociation to misfolded monomers. In the third article, Dr. Teplow and his colleagues [3] discuss therapeutic strategies against AD, the most common neurodegerative disease in the elderly. They summarize recent efforts to develop disease-modifying therapeutic agents targeting Aβ assembly, including immunotherapy, nutraceuticals, and a variety of candidate molecules, of which some have progressed to phase III clinical trials, and the others are less mature, but have therapeutic potential. In the fourth article, Dr. Ono and our group [4] focus on aggregation of α-synuclein (αS) which is a major component of Lewy bodies, neuropathological hallmarks of PD/DLB. Some compounds such as polyphenols are found with anti-fibrillogenic, antioligomeric, and fibril-destabilizing effects for αS, indicating that they could be key molecules for development of the preventives and therapeutics for PD/DLB and other αS-related disorders (α-synucleinopathies). In the fifth article, Drs. Nagai and Popiel [5] deal with the polyglutamine (polyQ) diseases, including Huntington’s disease and spinocerebellar ataxias, which are caused by an abnormal expansion of the polyQ stretch in disease-causative proteins.

Amyloid fibrils have been a critical subject in recent studies of proteins since they are associated with the pathology of more than 20 serious human diseases. Moreover, a variety of proteins and peptides not related to diseases are able to form amyloid fibrils or amyloid-like structures, implying that amyloid formation is a generic property of polypeptides. Although understanding the structure and formation of amyloid fibrils is crucial, due to the extremely high molecular weight and insolubility of amyloid fibrils, most of the conventional techniques available for soluble proteins are not directly applicable to these fibrils. However, structural studies using solid-state NMR have shown that the basic motif of amyloid fibrils is a β-strand-loop-β-strand conformation often in a parallel β-sheet assembly. From the hydrogen/ deuterium exchange of amide protons, amyloid fibrils have been shown to be stabilized by an extensive network of hydrogen bonds substantiating β-sheets. Our approach using total internal reflection fluorescence microscopy combined with thioflavin T, an amyloid-specific fluorescence dye, enabled monitoring fibril growth in real-time at single fibril level. On the basis of these various approaches, increasingly convincing models of amyloid structures, their formation and propagation are emerging.

Transthyretin (TTR) is a homotetrameric serum and cerebrospinal fluid protein that transports both thyroxine (T4) and the retinol-retinol binding protein complex (holoRBP). Rate-limiting tetramer dissociation and rapid monomer misfolding and misassembly of variant TTR results in familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), or familial central nervous system amyloidosis. Analogous misfolding of wild-type TTR results in senile systemic amyloidosis (SSA) characterized by sporadic amyloidosis in elderly populations. With the availability of genetic and immunohistochemical diagnostic tests, patients with TTR amyloidosis have been found in many nations worldwide. Recent studies indicate that TTR amyloidosis is not a rare endemic disease as previously thought. The only effective treatment for the familial TTR amyloidoses is liver transplantation; however, this strategy has a number of limitations, including a shortage of donors, a requirement for surgery for both the recipient and living donor, and the high cost. Furthermore, a large number of patients are not good transplant candidates. Recent studies focused on the TTR gene and protein have provided insight into the pathogenesis of TTR amyloidosis and suggested new strategies for therapeutic intervention. TTR tetramer (native state) kinetic stabilization by small molecule binding, immune therapy, and gene therapy with small interfering RNAs, antisense oligonucleotides, and single-stranded oligonucleotides are promising strategies based on our understanding of the pathogenesis of TTR amyloidosis. Among these, native state kinetic stabilization by diflunisal and Fx-1006A, a novel therapeutic strategy against protein misfolding diseases, are currently in Phase II/III clinical trials.

Alzheimer's disease (AD), the most common neurodegenerative disorder in the aged, is characterized by the cerebral deposition of fibrils formed by the amyloid β-protein (Aβ), a 40-42 amino acid peptide. The folding of Aβ into neurotoxic oligomeric, protofibrillar, and fibrillar assemblies is hypothesized to be the key pathologic event in AD. Aβ is formed through cleavage of the Aβ precursor protein by two endoproteinases, β-secretase and γ-secretase, that cleave the Aβ N-terminus and C-terminus, respectively. These facts support the relevance of therapeutic strategies targeting Aβ production, assembly, clearance, and neurotoxicity. Currently, no disease-modifying therapeutic agents are available for AD patients. Instead, existing therapeutics provide only modest symptomatic benefits for a limited time. We summarize here recent efforts to produce therapeutic drugs targeting Aβ assembly. A number of approaches are being used in these efforts, including immunological, nutraceutical, and more classical medicinal chemical (peptidic inhibitors, carbohydratecontaining compounds, polyamines, “drug-like” compounds, chaperones, metal chelators, and osmolytes), and many of these have progressed to phase III clinical trails. We also discuss briefly a number of less mature, but intriguing, strategies that have therapeutic potential. Although initial trials of some disease-modifying agents have failed, we argue that substantial cause for optimism exists.

Lewy bodies (LBs) and Lewy neurites (LNs) in the brain constitute the main histopathological features of Parkinson's disease (PD) and dementia with Lewy bodies (DLB), and are comprised of amyloid-like fibrils composed of a small protein (∼14 kDa) named alpha-synuclein (αS). As the aggregation of αS in the brain has been implicated as a critical step in the development of the diseases, the current search for disease-modifying drugs is focused on modification of the process of αS deposition in the brain. In this article, the recent developments on the molecules that inhibit the formation of α-synuclein fibrils (fαS) as well as the oligomerization of αS are reviewed. Recently, various compounds such as curcumin, nicotine and wine-related polyphenols have been reported to inhibit the formation of fαS, and to destabilize preformed fαS at pH 7.5 at 37°C in vitro. Although the mechanisms by which these compounds inhibit fαS formation from fαS, and destabilize preformed fαS are still unclear, they could be key molecules for the development of preventives and therapeutics for PD and other α-synucleinopathies.

The polyglutamine (polyQ) diseases, including Huntington's disease and spinocerebellar ataxias, are classified as the protein misfolding neurodegenerative diseases like Alzheimer's and Parkinson's diseases, and they are caused by an abnormal expansion of the polyQ stretch in disease-causative proteins. Expanded polyQ stretches have been shown to undergo a conformational transition to a β-sheet-dominant structure, leading to assembly of the host proteins into insoluble β-sheet-rich amyloid fibrillar aggregates and their subsequent accumulation as inclusion bodies in affected neurons, eventually resulting in neurodegeneration. Based on cytotoxicity of the soluble β-sheet monomer of the expanded polyQ protein, we propose the “Exposed β-sheet hypothesis”, in which both the toxic β-sheet conformational transition and misassembly into amyloid fibrils of the disease-causative proteins contribute to the pathogenesis of the polyQ diseases, and possibly the other protein misfolding neurodegenerative diseases. Among the various therapeutic targets, the toxic conformational changes and aggregation of the expanded polyQ proteins are most ideal since they are the earliest events in the pathogenic cascade, and therapeutic approaches using molecular chaperones, intrabodies, peptides, and small chemical compounds have been developed to date. Furthermore, high-throughput screening approaches to identify polyQ aggregate inhibitors are in progress. We hope that protein aggregate inhibitors which are widely effective not only for the polyQ diseases, but also for many neurodegenerative diseases will be discovered in the near future.

Amyloidosis is a clinical disorder caused by deposition of proteins that abnormally self-assemble into insoluble fibrils and impair organ function. More than 20 unrelated precursor proteins lose their native structure and misfold, leading to the formation of amyloid fibrils. The latter share cross-β core structure in vivo and in vitro and gain abnormal functions. Local amyloid deposition occurs in the central nervous system in Alzheimer's disease (AD) and cerebral amyloid angiopathy. AD is the most common form of neurodegenerative disorder, with dementia in the elderly as well as dementia with Lewy bodies (DLB). Extracellular deposition of amyloid β-peptide (Aβ) has been implicated as a critical step in the pathogenesis of AD. Involvement of neuroinflammation and microglial activation has been emphasized in the AD brain. Recent epidemiological studies have shown that long-term therapeutic use of non-steroidal anti-inflammatory drugs (NSAIDs) reduces the risk of developing AD and delayed the onset of AD. We review epidemiological studies of anti-AD effects of NSAIDs, experimental studies of anti-amyloidogenic as well as anti-inflammatory effects of NSAIDs, and recent clinical trials for AD with NSAIDs. We refer to the anti-fibrillogenic and fibril-destabilizing activities of NSAIDs for other proteins that can aggregate and form amyloid-like fibrils, including α-synuclein in DLB. The anti-amyloidogenic properties of some NSAIDs provide new insights for future therapeutic and preventative opportunities for AD and other amyloidoses, and protein-misfolding disorders.