In vitro modeling of p-α-syn and p-tau copathology recapitulates in vivo data. (A–I) IF double labeling shows p-α-syn pathology (red) and increased p-tau pathology (green) in neurons transduced with AD-tau–enriched extracts and α-syn mpffs combined (C, H, and I) compared with α-syn mpffs (A and G) or AD-tau–enriched extracts (B) alone. Scale bar, 50 µm. P-tau pathology is seen in both α-syn mpff–transduced neurons (G) and after combined treatment with α-syn mpffs/AD-tau–enriched extracts (H and I); in combined treatment, p-tau pathology can be seen in cells where p-α-syn pathology is present (H) or absent (I). (G–I) Arrows point to colocalized α-syn and tau pathology in primary neurons while arrowheads point to the presence of tau pathology in the absence of α-syn pathology. Quantification of cell body tau (D), insoluble neuritic tau (E), and p-α-syn (F) pathology seen in A–C. One-way ANOVA followed by Tukey’s post-hoc analysis was used to analyze the data (three repeats). (J) Immunoelectron microscopy shows tau (arrows) and α-syn (arrow heads) in proximity. Scale bar, 100 nm. (K and L) IF for insoluble tau (green) of WT mouse neurons treated with human AD-tau–enriched extracts and either sham shRNA (K) or α-syn shRNA (L). (M) Quantification of p-tau pathology showing a significant reduction in insoluble tau pathology in α-syn shRNA-treated neurons. A two-tailed t test was performed to calculate the difference between groups; *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Data are presented as mean ± SEM (n = 6–7 replicates). All experimental data were verified in at least two independent experiments.