Figure 4

Exosome-associated αsyn oligomers are preferentially internalized and more toxic than exosome-free αsyn oligomers. (A) Exosomal fractions and exosome-free supernatants (sup) from human H4 cells transfected with S1/S2 or mock were applied to naive H4 cells and incubated for 3 days. Uptake of αsyn oligomers into recipient cells was measured by luciferase assay. n = 9, unpaired t test with Welch's correction, ***p < 0.001. (B) Naïve H4 cells treated with exosome-associated S1/S2 oligomers and exosome-free S1/S2 oligomers (sup) were assayed for luciferase activity. Luciferase signal was normalized to the amount of αsyn oligomers in the input, n = 9, unpaired t test with Welch's correction, ***p < 0.001. (C) H4 cells treated with exosome-associated αsyn fractions conferred greater toxicity on naive H4 cells than exosome-free αsyn (sup n = 5, unpaired t test with Welch's correction, ***p < 0.001 (D) Naïve H4 cells treated with exosome-associated αsyn oligomers and exosome free αsyn oligomers (sup) were assayed for toxicity. Level of toxicity was normalized to the luciferase activity in each input n = 5, Mann Whitney test, *p < 0.05. (E) Exosomal fractions derived from primary neurons infected with AAV-S1/S2 or AAV-GFP were applied to naive neurons and incubated for 3 days. Uptake of αsyn oligomers into recipient neurons was measured performing a luciferase assay on recipient cells. n = 3, one-sample t-test, *p < 0.05. (F) Toxicity assay of naïve primary neurons treated with exosome-enriched fractions and exosome-free fractions (sup) derived from either AAV-αsyn-ires-GFP or AAV-GFP infected primary neurons. n = 9, one sample t-test, ***p < 0.001.