Structural remodeling during amyloidogenesis of physiological Nα-acetylated α-synuclein.

The misfolding and aggregation of the presynaptic protein α-synuclein (AS) into amyloid fibrils is pathognomonic of Parkinson's disease, though the mechanism by which this structural conversion occurs is largely unknown. Soluble oligomeric species that accumulate as intermediates in the process of fibril formation are thought to be highly cytotoxic. Recent studies indicate that oligomer-to-fibril AS transition plays a key role in cell toxicity and progression of neurodegeneration. We previously demonstrated that a subgroup of oligomeric AS species are ordered assemblies possessing a well-defined pattern of intermolecular contacts which are arranged into a distinctive antiparallel ß-sheet structure, as opposed to the parallel fibrillar fold. Recently, it was demonstrated that the physiological form of AS is N-terminally acetylated (Ac-AS). Here, we first showed that well-characterized conformational ensembles of Ac-AS, namely monomers, oligomers and fibrils, recapitulate many biophysical features of the nonacetylated protein, such as hydrodynamic, tinctorial, structural and membrane-leakage properties. Then, we relied on ATR-FTIR spectroscopy to explore the structural reorganization during Ac-AS fibrillogenesis. We found that antiparallel ß-sheet transient intermediates are built-up at early stages of aggregation, which then evolve to parallel ß-sheet fibrils through helix-rich/disordered species. The results are discussed in terms of regions of the protein that might participate in this structural rearrangement. Our work provides new insights into the complex conformational reorganization occurring during Ac-AS amyloid formation.

Two distinct ß-sheet structures in Italian-mutant amyloid-beta fibrils: a potential link to different clinical phenotypes.

Most Alzheimer's disease (AD) cases are late-onset and characterized by the aggregation and deposition of the amyloid-beta (Aß) peptide in extracellular plaques in the brain. However, a few rare and hereditary Aß mutations, such as the Italian Glu22-to-Lys (E22K) mutation, guarantee the development of early-onset familial AD. This type of AD is associated with a younger age at disease onset, increased ß-amyloid accumulation, and Aß deposition in cerebral blood vessel walls, giving rise to cerebral amyloid angiopathy (CAA). It remains largely unknown how the Italian mutation results in the clinical phenotype that is characteristic of CAA. We therefore investigated how this single point mutation may affect the aggregation of Aß1-42 in vitro and structurally characterized the resulting fibrils using a biophysical approach. This paper reports that wild-type and Italian-mutant Aß both form fibrils characterized by the cross-ß architecture, but with distinct ß-sheet organizations, resulting in differences in thioflavin T fluorescence and solvent accessibility. E22K Aß1-42 oligomers and fibrils both display an antiparallel ß-sheet structure, in comparison with the parallel ß-sheet structure of wild-type fibrils, characteristic of most amyloid fibrils described in the literature. Moreover, we demonstrate structural plasticity for Italian-mutant Aß fibrils in a pH-dependent manner, in terms of their underlying ß-sheet arrangement. These findings are of interest in the ongoing debate that (1) antiparallel ß-sheet structure might represent a signature for toxicity, which could explain the higher toxicity reported for the Italian mutant, and that (2) fibril polymorphism might underlie differences in disease pathology and clinical manifestation.

Amyloid fibrils are the molecular trigger of inflammation in Parkinson's disease.

Parkinson's disease (PD) is an age-related movement disorder characterized by a progressive degeneration of dopaminergic neurons in the midbrain. Although the presence of amyloid deposits of α-synuclein (α-syn) is the main pathological feature, PD brains also present a severe permanent inflammation, which largely contributes to neuropathology. Although α-syn has recently been implicated in this process, the molecular mechanisms underlying neuroinflammation remain unknown. In the present study, we investigated the ability of different α-syn aggregates to trigger inflammatory responses. We showed that α-syn induced inflammation through activation of Toll-like receptor 2 (TLR2) and the nucleotide oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome only when folded as amyloid fibrils. Oligomeric species, thought to be the primary species responsible for the disease, were surprisingly unable to trigger the same cascades. As neuroinflammation is a key player in PD pathology, these results put fibrils back to the fore and rekindles discussions about the primary toxic species contributing to the disease. Our data also suggest that the inflammatory properties of α-syn fibrils are linked to their intrinsic structure, most probably to their cross-ß structure. Since fibrils of other amyloids induce similar immunological responses, we propose that the canonical fibril-specific cross-ß structure represents a new generic motif recognized by the innate immune system.

ATR-FTIR: a "rejuvenated" tool to investigate amyloid proteins.

Amyloid refers to insoluble protein aggregates that are responsible for amyloid diseases but are also implicated in important physiological functions (functional amyloids). The widespread presence of protein aggregates but also, in most of the cases, their deleterious effects explain worldwide efforts made to understand their formation, structure and biological functions. We emphasized the role of FTIR and especially ATR-FTIR techniques in amyloid protein and/or peptide studies. The multiple advantages provided by ATR-FTIR allow an almost continuous structural view of protein/peptide conversion during the aggregation process. Moreover, it is now well-established that infrared can differentiate oligomers from fibrils simply on their spectral features. ATR-FTIR is certainly the fastest and easiest method to obtain this information. ATR-FTIR occupies a key position in the analysis and comprehension of the complex aggregation mechanism(s) at the oligomer and/or fibril level. These mechanism(s) seem to present strong similarities between different amyloid proteins and might therefore be extremely important to understand for both disease-associated and functional amyloid proteins. This article is part of a Special Issue entitled: FTIR in membrane proteins and peptide studies.

Toxic prefibrillar α-synuclein amyloid oligomers adopt a distinctive antiparallel ß-sheet structure.

Parkinson's disease is an age-related movement disorder characterized by the presence in the mid-brain of amyloid deposits of the 140-amino-acid protein AS (α-synuclein). AS fibrillation follows a nucleation polymerization pathway involving diverse transient prefibrillar species varying in size and morphology. Similar to other neurodegenerative diseases, cytotoxicity is currently attributed to these prefibrillar species rather than to the insoluble aggregates. Nevertheless, the underlying molecular mechanisms responsible for cytotoxicity remain elusive and structural studies may contribute to the understanding of both the amyloid aggregation mechanism and oligomer-induced toxicity. It is already recognized that soluble oligomeric AS species adopt ß-sheet structures that differ from those characterizing the fibrillar structure. In the present study we used ATR (attenuated total reflection)-FTIR (Fourier-transform infrared) spectroscopy, a technique especially sensitive to ß-sheet structure, to get a deeper insight into the ß-sheet organization within oligomers and fibrils. Careful spectral analysis revealed that AS oligomers adopt an antiparallel ß-sheet structure, whereas fibrils adopt a parallel arrangement. The results are discussed in terms of regions of the protein involved in the early ß-sheet interactions and the implications of such conformational arrangement for the pathogenicity associated with AS oligomers.

Transformation of amyloid ß(1-40) oligomers into fibrils is characterized by a major change in secondary structure.

Alzheimer's disease (AD) is a neurodegenerative disorder occurring in the elderly. It is widely accepted that the amyloid beta peptide (Aß) aggregation and especially the oligomeric states rather than fibrils are involved in AD onset. We used infrared spectroscopy to provide structural information on the entire aggregation pathway of Aß(1-40), starting from monomeric Aß to the end of the process, fibrils. Our structural study suggests that conversion of oligomers into fibrils results from a transition from antiparallel to parallel ß-sheet. These structural changes are described in terms of H-bonding rupture/formation, ß-strands reorientation and ß-sheet elongation. As antiparallel ß-sheet structure is also observed for other amyloidogenic proteins forming oligomers, reorganization of the ß-sheet implicating a reorientation of ß-strands could be a generic mechanism determining the kinetics of protein misfolding. Elucidation of the process driving aggregation, including structural transitions, could be essential in a search for therapies inhibiting aggregation or disrupting aggregates.

Antiparallel beta-sheet: a signature structure of the oligomeric amyloid beta-peptide.

AD (Alzheimer's disease) is linked to Abeta (amyloid beta-peptide) misfolding. Studies demonstrate that the level of soluble Abeta oligomeric forms correlates better with the progression of the disease than the level of fibrillar forms. Conformation-dependent antibodies have been developed to detect either Abeta oligomers or fibrils, suggesting that structural differences between these forms of Abeta exist. Using conditions which yield well-defined Abeta-(1-42) oligomers or fibrils, we studied the secondary structure of these species by ATR (attenuated total reflection)-FTIR (Fourier-transform infrared) spectroscopy. Whereas fibrillar Abeta was organized in a parallel beta-sheet conformation, oligomeric Abeta displayed distinct spectral features, which were attributed to an antiparallel beta-sheet structure. We also noted striking similarities between Abeta oligomers spectra and those of bacterial outer membrane porins. We discuss our results in terms of a possible organization of the antiparallel beta-sheets in Abeta oligomers, which may be related to reported effects of these highly toxic species in the amyloid pathogenesis associated with AD.

Modulation of amyloid-ß 1-42 structure and toxicity by proline-rich whey peptides.

A proline-rich peptide product prepared from bovine whey protein that was enriched in several hydrophobic amino acids including proline (whey proline-rich peptide, wPRP) was shown to modulate the folding pathway of human amyloid beta peptide 1-42 (Aß42) into oligomers. Concentration-dependent changes in ThT-binding to Ab42 by wPRP indicated suppression of oligomerisation, that was supported by Transmission Electron Microscopy. Suppression of ß-sheet and specifically, anti-parallel ß-sheet structures by wPRP was demonstrated by ATR-FTIR spectroscopy, where evidence for capacity of wPRP to dissociate pre-existing ß-sheet structures in Aß42 was also apparent. Suppression of anti-parallel ß-sheets of oligomeric Aß42 was associated with rescue of yeast and SH-SY5Y neuronal cells providing important evidence for the association between anti-parallel ß-sheet structure and oligomer toxicity. It was proposed that the interaction of wPRP with Aß42 interfered with the anti-parallel folding pathway of oligomeric Aß42 and ultimately produced 'off-pathway' structures of lowered total ß-sheet content, with attenuated cellular toxicity.

A comparative analysis of the aggregation behavior of amyloid-ß peptide variants.

Aggregated forms of the amyloid-ß peptide are hypothesized to act as the prime toxic agents in Alzheimer disease (AD). The in vivo amyloid-ß peptide pool consists of both C- and N-terminally truncated or mutated peptides, and the composition thereof significantly determines AD risk. Other variations, such as biotinylation, are introduced as molecular tools to aid the understanding of disease mechanisms. Since these modifications have the potential to alter key aggregation properties of the amyloid-ß peptide, we present a comparative study of the aggregation of a substantial set of the most common in vivo identified and in vitro produced amyloid-ß peptides.