Fig. 3

Accumulation of pSer129-αSyn and APP in the substantia nigra of WT mice induced by D620N VPS35 expression. A) Immunohistochemical staining of substantia nigra from WT and SNCA KO mice at 12 weeks after intranigral delivery of AAV2/6 vector expressing human D620N VPS35 (versus MCS) reveals the specific accumulation of pSer129-αSyn and APP in WT but not KO mice. Levels/distribution of total αSyn are normal in WT mice but absent from KO mice. Higher power images of the injected nigra (boxed) are shown. B) Western blot analysis of injected ventral midbrain and striatal extracts (1% Triton-X100 or 2% SDS fractions) from WT or SNCA KO mice at 12 weeks following the intranigral delivery of AAV2/6 vectors (MCS, WT VPS35 or D620N VPS35). Blots were probed with antibodies to V5 (VPS35), pS129-αSyn, total αSyn and APP, with β-tubulin as a loading control. C-D) Densitometric analysis of human VPS35 (V5), total αSyn, pS129-αSyn and APP levels in ventral midbrain and striatal extracts of WT or KO mice normalized to β-tubulin levels. pS129-αSyn levels were normalized to total αSyn. Bars represent mean ± SEM (n = 3–4 animals/group). *P < 0.05 or ***P < 0.001 by one-way ANOVA with Dunnett’s post-hoc analysis, as indicated. n.s., non-significant