Fig. 1

Characterization of α-syn aggregation in neurons isolated from M83 mice. A Recombinant human α-syn PFFs were added to DIV7 M83 neurons at 1.000,0.500, 0.250 and 0.125 ug/ml concentrations along with 1.000 ug/ml of α-syn monomer and PBS control. Cells were fixed at 7-, 14- and 21-days post-treatment. Small puncta of pS129-α-syn inclusions (green) were initially detected in neurites 7 days after α-syn PFF treatment. At 14 and 21 days of PFF treatment, pS129-α-syn aggregates become more elongated and some were also found in cell soma shown by arrows. B-D Quantification using high-content image analysis showed significantly higher total pS129-α-syn aggregate formation at 7 (B), 14 (C), and 21days (D) of PFF treatment in a dose-dependent manner and no aggregation was observed with PBS or α-syn monomer treatment. (N=6 replicates). Data are mean±SD ****p<0.0001 (Ordinary one-way ANOVA). E Immunofluorescence quantification suggested that the number of aggregates in the cell soma were higher at 14 and 21 days after PFF treatment compared to 7 days. F-G pS129-α-syn aggregates were positive for autophagy markers p62 and ubiquitin (orange). Neurons were stained with MAP2 (purple) and nucleus with Hoechst (blue). Scale bars, 50 μm