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