α-Synuclein-Confocal Nanoscanning (ASYN-CONA), a Bead-Based Assay for Detecting Early-Stage α-Synuclein Aggregation

alpha-Synuclein fibrils are considered a hallmark of Parkinson's disease and other synucleinopathies. However, small oligomers that formed during the early stages of alpha-synuclein aggregation are thought to be the main toxic species causing disease. The formation of alpha-synuclein oligomers has proven difficult to follow, because of the heterogeneity and transient nature of the species formed. Here, a novel bead-based aggregation assay for monitoring the earliest stages of alpha-synuclein oligomerization, alpha-Synuclein-Confocal Nanoscanning (ASYN-CONA), is presented. The alpha-synuclein A91C single cysteine mutant is modified with a trifunctional chemical tag, which allows simultaneous fluorescent labeling with a green dye (tetramethylrhodamine, TMR) and attachment to microbeads. Beads with bound TMR-labeled alpha-synuclein are then incubated with a red dye (Cy5)-labeled variant of alpha-synuclein A91C, and EtOH (20%) to induce aggregation. Aggregation is detected by confocal scanning imaging, below the equatorial plane of the beads, which is known as the CONA technique. On-bead TMR-labeled alpha-synuclein and aggregated Cy5-labeled alpha-synuclein from the solution are quantitatively monitored in parallel by detection of fluorescent halos or rings. alpha-Synuclein on-bead oligomerization results in a linear increase of red bead ring fluorescence intensity over a period of 5 h. Total internal reflection fluorescence microscopy was performed on oligomers cleaved from the beads, and it revealed that (i) oligomers are sufficiently stable in solution to investigate their composition, consisting of 6 +/- 1 monomer units, and (ii) oligomers containing a mean of 15 monomers bind Thioflavin-T. Various known inhibitors of alpha-synuclein aggregation were used to validate the ASYN-CONA assay for drug screening. Baicalein, curcumin, and rifampicin showed concentration-dependent inhibition of the alpha-synuclein aggregation and the IC50 (the concentration of the compound at which the maxiumum intensity was reduced by one-half) were calculated.