A Fluorescence Resonance Energy Transfer Assay For Monitoring α- Synclein Aggregation in a Caenorhabditis Elegans Model For Parkinson's Disease.

摘要: The aggregation of α-synuclein (Syn or S) to form insoluble fibrils is important in the pathogenesis Parkinson's disease, but key risk factors remain ill-defined. We have developed Fluorescence Resonance Energy Transfer (FRET)-based assays for aggregation, using Green Fluorescent Protein variants Cerulean (C) Venus (V), fused each other (CV, VC) human synuclein (SC, SV etc). Bacterially expressed proteins were purified homogeneity, and C-terminal fusions SC largely retained their ability aggregate vitro. FRET signals from mixtures used monitor aggregation. These fusion genes linked C. elegans unc-54 myosin promoter generate integrated transgenic strains. Increased signals, indicative S observed following treatment unc-54::SC + unc-54::SV double worms with low concentrations mercury chlorpyrifos, RNAi against hsp-70 hip-1. Opposite changes Yellow (YFP) fluorescence an strain (NL5901) are likely reflect aggregates Syn protein. This could provide basis a high throughput screening assay, which be studying effects toxic chemicals environmental pollutants on such as vivo.