Aggregation and neurotoxicity of recombinant α-synuclein aggregates initiated by dimerization

Background: Aggregation of the alpha-Synuclein (alpha-Syn) protein, amyloid fibril formation and progressive neurodegeneration are the neuropathological hallmarks of Parkinson's Disease (PD). However, a detailed mechanism of alpha-Syn aggregation/fibrillogenesis and the exact nature of toxic oligomeric species produced during amyloid formation process are still unknown. Results: In this study, the rates of alpha-Syn aggregation were compared for the recombinant wild-type (WT) alpha-Syn and a structurally relevant chimeric homologous protein containing an inducible Fv dimerizing domain (alpha-Syn(Fv)), capable to form dimers in the presence of a divalent ligand (AP20187). In the presence of AP20187, we report a rapid random coil into beta-sheet conformational transformation of alpha-Syn(Fv) within 24 h, whereas WT alpha-Syn showed 24 h delay to achieve beta-sheet structure after 48 h. Fluorescence ANS and ThT binding experiments demonstrate an accelerated oligomer/amyloid formation of dimerized alpha-Syn(Fv), compared to the slower oligomerization and amyloidogenesis of WT alpha-Syn or alpha-Syn(Fv) without dimerizer AP20187. Both alpha-Syn(Fv) and alpha-Syn pre-fibrillar aggregates internalized cells and induced neurotoxicity when injected into the hippocampus of wild-type mice. These recombinant toxic aggregates further converted into non-toxic amyloids which were successfully amplified by protein misfolding cyclic amplification method, providing the first evidence for the in vitro propagation of synthetic alpha-Syn aggregates. Conclusions: Together, we show that dimerization is important for alpha-Syn conformational transition and aggregation. In addition, alpha-Syn dimerization can accelerate the formation of neurotoxic aggregates and amyloid fibrils which can be amplified in vitro. A detailed characterization of the mechanism of alpha-Syn aggregation/amyloidogenesis and toxicity is crucial to comprehend Parkinson's disease pathology at the molecular level.