Developing Novel FRET Based Biosensors that Monitor α-Synuclein Assembly for Use in High Throughput Screening

Abstract

Aggregation of endogenous α-synuclein is the hallmark of Parkinson's disease pathology. Under normal conditions, α-synuclein plays an important role in neurological function. Under pathologic conditions, misfolded α-synuclein forms cytotoxic oligomers and fibrils. There has been significant effort to identify small molecule inhibitors of α-synuclein fibrillization. However, recent evidence suggests the kinetically unstable oligomeric species, and not fibrils, are the source of α-synuclein cytotoxicity. We interrogated the self-association of the oligomeric species under aggregation prone conditions using FRET. To achieve this, we developed a set of cell-free FRET based biosensors that monitor oligomer-oligomer interactions and fibril formation through fluorescence lifetime. Using fluorescence lifetime provides a 30-fold increase in sensitivity over more traditional FRET measurements. We have achieved an optimal FRET of 14.99% for oligomeric interactions and a seeded induced change in FRET of 15.34%. We are currently optimizing these biosensors for scale up to facilitate high throughput screening. Hits will be validated for FRET dose response and biophysical changes in aggregation kinetics via thioflavin T assays and AFM characterization.