Figure 2

aSyn fibrils seed endogenous aSyn to different extent in primary neuronal cultures from different regions. Primary neuronal cultures from different brain regions of WT or SNCA−/− embryos were exposed to 100 nM aSyn Fibrils or to vehicle at DIV7 and observed at DIV21. (a) Fixed cells were stained for MAP2 (grey), pSyn (green) and Hoechst (blue) and imaged by epifluorescence microscopy. Scale bar represents 100 µm. (b) pSyn area/MAP2 area at fixed thresholds. 5 to 42 replicates from 2 to 11 individual experiments. Two-way ANOVA was performed on experiments means, followed by Tukey’s multiple comparisons test. Adjusted p values are shown. (c) Quantification of the percentage of neurons with somatic pSyn. 10 to 20 replicates from 3 to 5 individual experiments. Means +/− CI95 are shown. Brown-Forsythe ANOVA tests and Welch’s ANOVA test were performed, followed by Dunnett’s T3 multiple comparisons test. Adjusted p values are shown. (d) Primary neuronal cultures were exposed to 100 nM aSyn Fibrils or to vehicle at DIV7, lysed at DIV14 and the amount of pSyn and TUJ1 quantified by Western blotting. The loading control gel was not loaded in the same order as the gel for pSyn staining in order to reveal high molecular weight pSyn species. Thus, the gel containing loading controls had to be cropped and rearranged. Full length gel is available in Supp. Fig. S9.