Figure 6

αSyn oligomer secretion is modulated by autophagic activity. (A) Human H4 cells were transfected with αsyn complementation pair S1/S2 and treated with DMSO, rapamycin or bafilomycin A1. After 48 h cells CM was assayed for luciferase activity and a ratio of luciferase activity in media compared to cells was expressed. n = 4, One way ANOVA, Bonferroni’s Multiple Comparison Test, **p < 0.001. (B) Immunoblot levels of autophagosome marker LC3.II and macrophagy substrate p62 in H4 cells treated with autophagy inhibitor Bafilomycin A1, autophagy inducer rapamycin or DMSO. (C) Densiometric analysis of immunoblots probed with LC3.II normalized to ß-actin[n = 3 (Baf A1) p < 0.0001, t-test; n = 3 (rapa), p = 0.0021, t-test ] and p62 normalized α-tubulin [n = 3 (Baf A1) p = 0.022, t-test; n = 3 (rapa), p = 0.0022, t-test ](D) Exosomal fractions from H4 cells transfected with S1/S2 and treated with DMSO, rapamycin or bafilomycin A1 were assayed for luciferase activity. n = 4, One way ANOVA, Bonferroni’s Multiple Comparison Test, ***p < 0.001 (E) Exosomal fractions from primary neurons infected with AAV-S1/S2 and treated with DMSO, rapamycin or bafilomycin A1 were assayed for luciferase activity. n = 4, One way ANOVA, Bonferroni’s Multiple Comparison Test, p = 0.17, ns = non-significant.