Abstract
BackgroundAggregation of the α-Synuclein (α-Syn) protein, amyloid fibril formation and progressive neurodegeneration are the neuropathological hallmarks of Parkinson's Disease (PD). However, a detailed mechanism of α-Syn aggregation/fibrillogenesis and the exact nature of toxic oligomeric species produced during amyloid formation process are still unknown.ResultsIn this study, the rates of α-Syn aggregation were compared for the recombinant wild-type (WT) α-Syn and a structurally relevant chimeric homologous protein containing an inducible Fv dimerizing domain (α-SynFv), capable to form dimers in the presence of a divalent ligand (AP20187). In the presence of AP20187, we report a rapid random coil into β-sheet conformational transformation of α-SynFv within 24 h, whereas WT α-Syn showed 24 h delay to achieve β-sheet structure after 48 h. Fluorescence ANS and ThT binding experiments demonstrate an accelerated oligomer/amyloid formation of dimerized α-SynFv, compared to the slower oligomerization and amyloidogenesis of WT α-Syn or α-SynFv without dimerizer AP20187. Both α-SynFv and α-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 α-Syn aggregates.ConclusionsTogether, we show that dimerization is important for α-Syn conformational transition and aggregation. In addition, α-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 α-Syn aggregation/amyloidogenesis and toxicity is crucial to comprehend Parkinson's disease pathology at the molecular level.
Highlights
Aggregation of the α-Synuclein (α-Syn) protein, amyloid fibril formation and progressive neurodegeneration are the neuropathological hallmarks of Parkinson's Disease (PD)
The diagnostic feature common to protein misfolding disorders involves the deposition of insoluble protein aggregates and amyloid fibrils caused by accumulation of homologous proteins which are abnormally folded into β-sheet structures [10,11]
An FK506 binding domain (Fv) dimerizing domain was fused to the C-terminal of α-Syn, thereby creating α-SynFv with the capacity to homodimerize in the presence of AP20187
Summary
Aggregation of the α-Synuclein (α-Syn) protein, amyloid fibril formation and progressive neurodegeneration are the neuropathological hallmarks of Parkinson's Disease (PD). Expression of α-Syn soluble oligomers with reduced fibrillization propensity induced neurotoxicity in worms, flies and mammalian neurons [16] These pre-fibrillar aggregate species are readily formed in vitro from α-Syn mutants such as A30P and A53T, which are associated with neurotoxicity and early-onset PD [17,18]. Interaction of α-Syn fibrils either with recombinant or cell-expressed αSyn converts soluble homologous proteins into a misfolded state through a nucleation-dependent polymerization process [22,23] These results suggest that the mechanism underlying propagation of α-Syn fibrils is reminiscent of those by which tau aggregates and prions spread and transmit the pathology through the brain tissue in Alzheimer’s and prion diseases, respectively [24]
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