Figure 5. Inhibitors reduce the seeding ability of α-syn fibrils in cells.
(A) Experimental design of cell culture seeding assay. Pre-formed α-syn fibrils (green) were incubated with various amounts of inhibitors (red) for 3–4 hr to allow binding. The mixture was transfected in HEK293 cells expressing YFP-labelled WT/A53T α-syn. After 1 day of transfection, cells were imaged using fluorescence microscopy and the bright fluorescent puncta were counted using imaging cytometer (B and D) Representative fluorescence micrographs of HEK293 cells expressing YFP labeled WT (B) and A53T (D) α-syn transfected with seeds and seeds pre-incubated with inhibitors. Transfection of α-syn seeds induced endogenous α-syn to form large aggregates seen as bright puncta (white arrows). Transfection of α-syn seeds pre-incubated with inhibitors S62 and S61 induced fewer puncta (white arrows) in both WT and A53T α-syn expressing HEK293 cells but S37 and S71 do not have a significant effect on puncta formation. Scale 100 µm. (C and E) Quantification of puncta formed in different conditions. In agreement to the visual findings the quantification of puncta shows that S62 and S61 significantly reduce the number of puncta in both cell lines. This means that pre-incubation of α-syn fibrils with inhibitors (S62 and S61) that have higher binding affinity for α-syn fibrils reduces cell seeding ability. In contrast, the inhibitors S71 and S37, which have lower affinity for α-syn fibrils do not have a significant effect on cell seeding ability of α-syn fibrils. Results shown as Mean + SD (n = 3) of technical replicates. Statistical significance was analyzed by two-way ANOVA. (**p<0.01,***p<0.001,****p<0.0001).
Figure 5—figure supplement 1. Incubation with inhibitors causes morphological changes in α-syn fibrils.
