a b s t r a c tThe conversion of soluble peptides and proteins into amyloid fibrils and/or intermediate oligomers is believed to be the central event in the pathogenesis of most human neurodegenerative diseases. Existing treatments are at best symptomatic. Accordingly, small molecule inhibitors of amyloid fibril formation and their mechanisms are of great interest. Here we report that the conformational changes undergone by a -synuclein as it assembles into amyloid fibrils can be detected by epitopespecific antibodies. We show that the conformations of polyphenol-bound a-synuclein monomers and dimers differ from those of unbound monomers and resemble amyloid fibrils. This strongly suggests that small molecule inhibitors bind and stabilize intermediates of amyloid fibril formation, consistent with the view that inhibitor-bound molecular species are on-pathway intermediates.

Fibrillization or conformational change of ␣-synuclein is central in the pathogenesis of ␣-synucleinopathies, such as Parkinson disease. We found that the A30P mutant accelerates nucleation-dependent fibrillization of wild type (WT) ␣-synuclein. Electron microscopy observation and ultracentrifugation experiments revealed that shedding of fragments occurs from A30P fibrils and that these fragments accelerate fibrillization by serving as seeds. Immunochemical analysis using epitope-specific antibodies and biochemical analyses of protease-resistant cores demonstrated that A30P fibrils have a distinct conformation. Interestingly, WT fibrils formed with A30P seeds exhibited the same character as A30P fibrils, as did A30P fibrils formed with WT seeds, indicating that the A30P mutation affects the conformation and fibrillization of both WT and A30P. These effects of A30P mutation may explain the apparent conflict between the association of A30P with Parkinson disease and the slow fibrillization of A30P itself and therefore provide new insight into the molecular mechanisms of ␣-synucleinopathies. Parkinson disease (PD)2 is the second most common neurodegenerative disorder, after Alzheimer disease. Neuropathological features of PD are selective loss of dopaminergic neurons in the substantia nigra and appearance of intracellular inclusion bodies, referred to as Lewy bodies (LBs) and Lewy neurites. Ultrastructurally, LBs are composed of a dense core of filamentous and granular material that is surrounded by radially oriented fibrils (1, 2). Biochemical and immunochemical analyses showed that hyperphosphorylated ␣-synuclein is the major component of the fibrous structures of LBs and Lewy neurites (3).Genetic analyses of ␣-synuclein gene of familial cases of PD and dementia with LBs have demonstrated that expression of abnormal ␣-synuclein or overexpression of normal ␣-synuclein is associated with these diseases; namely, three missense mutations (A53T (4), A30P (5), and E46K (6)) and multiplication (7-12) of the ␣-synuclein gene have been found to cosegregate with the onset of PD in kindreds of autosomal dominantly inherited familial PD and dementia with LBs.␣-Synuclein is a 140-amino acid protein, harboring seven imperfect tandem repeats (KTKEGV-type) in the N-terminal half, followed by a hydrophobic central region (non-A␤ component of Alzheimer disease (NAC)) and an acidic C-terminal. The tandem repeat region has been assumed to form an amphipathic ␣-helix by binding to phospholipid (13). Circular dichroism and Fourier-transform IR analysis revealed that ␣-synuclein is a natively unfolded protein with little ordered secondary structure (14). However, recent NMR analyses have revealed three intramolecular long range interactions. These interactions are between the highly hydrophobic NAC region (residues 85-95) and the C terminus (residues 110 -130), C-terminal residues 120 -130 and residues 105-115, and the region around residue 120 and the N terminus around residue 20 (15).Recombinant ␣-synuclein in vitro assembles into fi...

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