Figure 4. Inhibitors do not aggregate by themselves and do not seed α-syn aggregation in cells.

(A) Inhibitors, S61, S62, S37 and S71 incubated alone (at 500 µM concentration) did not show any increase in ThT fluorescence over a time period of 2 days whereas α-syn showed a time dependent increase in fluorescence indicative of fibril formation. Each curve represents average of 3 data sets and error bars represent standard deviation. (B) Electron micrographs of ThT assay samples at the end of 2 days of incubation. Sparse, small amorphous aggregates are observed for any of the inhibitors, whereas abundant fibrils are observed for α-syn. Scale 200 nm. (C) Fluorescence micrographs of WT α-syn expressing HEK293 cells transfected with α-syn/inhibitor incubated under shaking conditions for 2 days. Cells were imaged after 1 day of transfection. Inhibitor transfected cells did not have any puncta, whereas α-syn transfected cells had abundant puncta (shown by white arrows). Scale 100 um. (D) Quantification of puncta formed in different conditions. In agreement with the visual findings, the quantification of puncta shows that inhibitor/buffer transfected cells did not have puncta whereas α-syn fibrils cause abundant puncta formation.

Figure 4—figure supplement 1. Scrambling the inhibitor sequence abolishes its inhibitory effect on α-syn aggregation and seeding.

We created a scrambled peptide (SP) by scrambling the binding motif sequence of S61, keeping its cell penetration tag sequence intact. To test the sequence specificity of the inhibitory activity of our designed inhibitors we tested the efficacy of our SP in inhibiting α-syn aggregation and cell seeding. (A) In vitro aggregation monitored by the increase in fluorescence intensity of ThT showed rapid fibril growth in α-syn incubated alone as well as in presence of SP (α-syn:SP molar ratio, 1:2). However, α-syn incubated in presence of our inhibitor, S61 (α-syn:S61 molar ratio, 1:2) had about 10-fold less ThT fluorescence. Electron micrographs of ThT assay samples at the end of 2 days of incubation show abundant fibrils were observed for α-syn incubated alone and in presence of SP, whereas, sparse, thick, bundled fibrils are observed in α-syn incubated in presence of S61. Scale bars in electron micrographs are 200 nm. (B) WT α-syn expressing HEK293 cells were transfected with α-syn incubated alone or in presence of either SP or S61. Bar graph showing the average number of puncta formed in cells per well measured using imaging cytometer. Transfection of α-syn+SP does not cause a reduction in puncta whereas the inhibitor S61 causes significant in puncta per well. (C) Representative fluorescence micrographs of cells transfected with α-syn, α-syn+S61, α-syn+SP and buffer. Cells were imaged after 1 day of transfection. α-syn and α-syn+SP transfected cells had abundant puncta (shown by white arrows), whereas α-syn+S61 transfected cells had significantly lower amounts of puncta. This suggests that scrambling the binding motif sequence results in loss of inhibitory effect of our designed peptides. Scale 100 µm.