Fig. 5

Heterozygous VPS35 deletion fails to modify α-Syn levels and pathology or premature survival in human A53T-α-Syn transgenic mice. A) Western blot analyses of 1% Triton-soluble extracts from ventral midbrain, striatum, spinal cord or brain stem from ~ 13-month old heterozygous VPS35 null mice crossed with human A53T-α-Syn transgenic mice (VPS35^FLOX/WT/αSyn) versus their A53T-α-Syn littermates (VPS35^WT/WT/αSyn). Blots were probed for retromer subunits (VPS35, VPS26 and VPS29), total (Syn1), human (Syn211) and pathological (pS129-αSyn) α-synuclein or β-tubulin. B) Densitometric analysis of pS129-αSyn levels normalized to total αSyn, or total αSyn normalized to β-tubulin, expressed as a proportion of VPS35^WT/WT/αSyn^A53T mice (mean ± SEM, n = 3 mice/genotype). P > 0.05 by one-way ANOVA with Bonferroni’s post-hoc test, as indicated. n.s., non-significant. C) Densitometric analysis of VPS35, VPS29 or VPS26 levels normalized to β-tubulin levels in the ventral midbrain and striatum (mean ± SEM, n = 3 mice/genotype). *P < 0.05; n.s., non-significant by unpaired, two-tailed Student’s t-test. D) Representative images of immunohistochemical staining of pS129-αSyn (white arrowheads) in the substantia nigra (SNpc) of ~ 6-month old pre-symptomatic VPS35^WT/WT/αSyn^A53T or VPS3^FLOX/WT/αSyn^A53T mice. Non-transgenic (NTG) littermate mice do not exhibit specific α-Syn pathology, as expected. E) Digital pathology quantification of pS129 α-synuclein immunostaining in SNpc using the object colocalization module in HALO analysis software which detects pS129 α-synuclein-positive cells based on their size and shape. Regions with pS129 α-synuclein pathology are shown with or without an analysis overlay (red). F) Quantitation of total number of pS129 α-synuclein-positively stained cells per mm² of tissue area. Bars represent mean ± SEM (n = 2–5 animals/group). n.s., non-significant by one-way ANOVA with Tukey’s multiple comparison test. G) Lethal neurodegenerative phenotype of human A53T-α-Syn transgenic mice is independent of VPS35 expression. Kaplan-Meier survival curves were generated by monitoring cohorts of VPS35^WT/WT/αSyn^A53T (n = 27) and VPS35^FLOX/WT/αSyn^A53T (n = 24) mice over 18 months until animals had to be euthanized due to the onset of terminal disease. There is no significant difference in survival between the two genotypes by log-rank (Mantel-Cox) test (P = 0.2947)