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

Mol Neurodegener. 2013 Jan 22;8:5. doi: 10.1186/1750-1326-8-5.

Abstract

Background: Aggregation 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.

Results: In 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.

Conclusions: Together, 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Hippocampus / pathology*
  • In Situ Nick-End Labeling
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Parkinson Disease / pathology
  • Protein Multimerization*
  • Protein Structure, Secondary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / toxicity
  • alpha-Synuclein / chemistry*
  • alpha-Synuclein / toxicity*

Substances

  • Recombinant Proteins
  • alpha-Synuclein