Fig. 7: α-Syn-induced exosomal secretion is further increased by ATG5 siRNA.

The effect of α-Syn overexpression and ATG5 siRNA on the release of α-Syn from cells was analyzed by Western blot in the conditioned medium of naïve control cells (Ctrl), cells overexpressing α-Syn, cells overexpressing the control protein GFP, cells overexpressing α-Syn treated with ATG5 siRNA, and naïve cells treated with ATG5 siRNA with or without GW4869 inhibitor. Furthermore, the effect of ATG5 siRNA combined with exosome inhibition to LDH release was analyzed. a Representative Western blot for α-Syn protein in the medium, showing the monomer at 15 kDa and an oligomer at 35 kDa. The faint band at 50 kDa is unchanged across the experimental groups and considered to be nonspecific. b Quantification of α-Syn protein (15 kDa band) from Western blots as shown in a, showing release of α-Syn from α-Syn overexpressing cells, which is further increased by additional ATG5 siRNA. c Representative Western blot for intracellular α-Syn protein. β-actin was used as loading control. d Quantification of α-Syn protein from Western blots as shown in c, showing no significant effect of naïve control cells (Ctrl), cells overexpressing α-Syn, and cells overexpressing α-Syn treated with ATG5 siRNA with GW4869 inhibitor compared to respective control cells. Intracellular levels of α-Syn significantly increased upon overexpression by comparison to naïve cells. e Representative Western blot for the exosomal marker CD81 in the vesicle enriched fraction of the medium. f Quantification of CD81 protein from Western blots as shown in c, showing an increase in exosomes in the medium by ATG5 siRNA. A further inhibition of exosome biogenesis lead to a decrease in exosomes in the medium by ATG5 siRNA. g Representative Western blot for α-Syn in the vesicle enriched fraction. h Quantification of α-Syn protein from Western blots as shown in g, showing exosomal secretion of α-Syn from α-Syn overexpressing cells, which is further increased by additional ATG5 siRNA. Furthermore, an inhibition of exosome biogenesis lead to a decrease in exosomal secretion of α-Syn in the medium by ATG5 siRNA. i Representative Western blot for the exosomal marker CD81, showing the absence of exosomes in the vesicle-free fraction of the medium. j Representative Western blot for α-Syn in the vesicle-free fraction. k Quantification of α-Syn protein from Western blots as shown in j, showing release of free α-Syn from α-Syn overexpressing cells, which is not altered by additional ATG5 siRNA. Inhibition of exosome biogenesis together with inhibition of autophagosome formation by ATG5 siRNA increased release of free α-Syn in the medium. l Quantification of lactate dehydrogenase (LDH) released into the culture medium. Data are expressed as percentage of α-Syn. Naïve control cells and α-Syn overexpressing cells treated with GW4869 did not show elevated toxicity levels compared to respective control cells. ATG5 silencing combined with GW4869 treatment significantly increased α-Syn-induced toxicity. Data in b, d, f, h, k, l are mean ± standard error from n ≥ 3 biological replicates. n.s., not significant, *p < 0.05, **p < 0.01, ***p < 0.001; one-way analysis of variance with Bonferroni’s post hoc test