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

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.

Targeting α-Synuclein as a Parkinson’s Disease Therapeutic

“…These oligomers were morphologically higherorder aggregates of heterogeneous size, such as annular and amorphous protofibrillar oligomers [122], consistent with the heterogeneity described in other studies.…”

“…Most recently it was shown that specific forms of α-synuclein oligomers, including β-sheet-containing dimers [122] and HNE-modified [248] α-synuclein, seed the formation of oligomeric aggregates. For example, when injected into the hippocampi of wild-type mice, the dimer-derived oligomers, but not freshly dissolved α-synuclein or insoluble fibrils, promoted apoptosis [122].…”

“…For example, when injected into the hippocampi of wild-type mice, the dimer-derived oligomers, but not freshly dissolved α-synuclein or insoluble fibrils, promoted apoptosis [122].…”

The Neuroprotective Effect of Dimethyl Fumarate in an MPTP-Mouse Model of Parkinson's Disease: Involvement of Reactive Oxygen Species/Nuclear Factor-κB/Nuclear Transcription Factor Related to NF-E2

“…a-Synuclein dimerization can accelerate the formation of neurotoxic aggregates and amyloid fibrils, which may be crucial to PD pathology at the molecular level (56). Western blot analysis revealed a threefold increase of a-synuclein monomer, dimer, and oligomer in MPTP-injured mice ( …”

When amyloids become prions

“…[76][77][78] Nevertheless, these transmissions are far from neuronal invasion, probably due to limitations on the spread of a-synuclein aggregates. On the one hand, the fact that both mature fibers and oligomers (the most transmissible material) display high neuronal toxicity [79][80][81][82] and on the other, the added difficulty of having to be secreted to reach distant neurons, could be key factors that impede the prion capacity of a-synuclein aggregates. Very importantly at this point, it has been shown that cells normally secrete a-synuclein into their surrounding media; this opens up the possibility that oligomers of a-synuclein could be spread and act as prions.…”