Several observations have implicated oxidative stress and aggregation of the presynaptic protein ␣-synuclein in the pathogenesis of Parkinson disease. ␣-Synuclein has been shown to have affinity for unsaturated fatty acids and membranes enriched in polyunsaturated fatty acids, which are especially sensitive to oxidation under conditions of oxidative stress. One of the most important products of lipid oxidation is 4-hydroxy-2-nonenal (HNE), which has been implicated in the pathogenesis of Parkinson disease. Consequently, we investigated the effects of the interaction of HNE with ␣-synuclein. Incubation of HNE with ␣-synuclein at pH 7.4 and 37°C resulted in covalent modification of the protein, with up to six HNE molecules incorporated as Michael addition products. Fourier transform infrared and CD spectra indicated that HNE modification of ␣-synuclein resulted in a major conformational change involving increased ␤-sheet. HNE modification of ␣-synuclein led to inhibition of fibrillation in an HNE concentration-dependent manner. This inhibition of fibrillation was shown to be due to the formation of soluble oligomers based on size exclusion high pressure liquid chromatography and atomic force microscope data. Small angle x-ray scattering analysis indicated that the HNE-induced oligomers were compact and tightly packed. Treatment with guanidinium chloride demonstrated that the HNE-induced oligomers were very stable with an extremely slow rate of dissociation. Addition of 5 M HNE-modified oligomers to primary mesencephalic cultures caused marked neurotoxicity because the integrity of dopaminergic and GABAergic neurons was reduced by 95 and 85%, respectively. Our observations indicate that HNE modification of ␣-synuclein prevents fibrillation but may result in toxic oligomers, which could therefore contribute to the demise of neurons subjected to oxidative damage.

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Immunoglobulin light chain deposition diseases involve various types of extracellular deposition of light chain variable domains, including amyloid fibrils and amorphous deposits. The decreased thermodynamic stability of the light chain is believed to be the major factor leading to fibrillation. However, the differences in the nature of the deposits among the light chain deposition diseases raise the question of whether the mechanisms leading to fibrillar or amorphous aggregation is different. In this study, we generated two partially folded intermediates of the light chain variable domain SMA in the presence of guanidine hydrochloride (GuHCl) and characterized their conformations. The more unfolded intermediate formed fibrils most rapidly, while the more native-like intermediate predominantly led to amorphous deposits. The results also show that the monomeric, rather than the dimeric state, was critical for fibrillation. The data also indicate that fibril elongation involves addition of a partially unfolded intermediate, rather than the native state. We postulate that a more highly unfolded intermediate is more suited to undergo the topological rearrangements necessary to form amyloid fibrils than a more structured one and that this also correlates with increased destabilization. In the case of light chain aggregation, it appears that more native-like intermediate conformations are more prone to form amorphous deposits.

Elucidating the details of the assembly of amyloid fibrils is a key step to understanding the mechanism of amyloid deposition diseases including Parkinson's disease. Although several models have been proposed, based on analyses of polypeptides and short peptides, a detailed understanding of the structure and mechanism of alpha-synuclein fibrillation remains elusive. In this study, we used trypsin and endoproteinase GluC to digest intact alpha-synuclein fibrils and to analyze the detailed morphology of the resultant fibrils/remnants. We also created three mutants of alpha-synuclein, in which the N-terminal and C-terminal regions were removed, both individually and in combination, and investigated the detailed morphology of the fibrils from these mutants. Our results indicate that the assembly of mature alpha-synuclein fibrils is hierarchical: protofilaments --> protofibrils --> mature fibrils. There is a core region of approximately 70 amino acids, from residues approximately 32 to 102, which comprises the beta-rich core of the protofilaments and fibrils. In contrast, the two terminal regions show no evidence of participating in the assembly of the protofilament core but play a key role in the interactions between the protofilaments, which is necessary for the fibril maturation.