Fig 1: 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 (VPS35FLOX/WT/αSyn) versus their A53T-α-Syn littermates (VPS35WT/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 VPS35WT/WT/αSynA53T 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 VPS35WT/WT/αSynA53T or VPS3FLOX/WT/αSynA53T 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 VPS35WT/WT/αSynA53T (n = 27) and VPS35FLOX/WT/αSynA53T (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)
Fig 2: Knockdown of Retromer Assembly Subunit VPS35 by shRNA Increases Aβ and pTAU/tTAU Ratio in hiPSC-Derived Neurons(A and B) Aβ peptides (A, Aβ1-40; B, Aβ1-42) measured from the cell culture media of hiPSC-derived neurons are increased when VPS35 levels are reduced by VPS35 shRNA (two shRNA, VPS35-c and VPS35-d) compared with a scrambled shRNA (SCR shRNA).(C) pTAU levels increase in hiPSC-derived neurons when VPS35 levels are reduced by VPS35 shRNA (two shRNA, c and d) compared with scrambled shRNA (SCR shRNA).(D) VPS35 shRNA-c reduces VPS35 protein by 50%.(E) VPS 35 shRNA-c reduces VPS35 mRNA by 50%. n = 4 independent experiments per treatment/condition.(A–C) For each comparison, a one-way ANOVA with a Tukey multiple comparisons posttest was performed. (D and E) For each comparison, a two-tailed t test was performed. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p<0.001. Error bars represent SD. See also Figure S4.
Fig 3: Axonal damage in D620N VPS35 KI mice. Representative photomicrographs of striatum and substantia nigra from 13-mo-old VPS35D620N/WT, VPS35D620N/D620N, and control VPS35WT/WT mice indicating (A) APP immunohistochemistry revealing APP-positive spheroids (black arrowheads), a marker of axonal damage. (Scale bars: 100 μm.) Insets show high-power images of the boxed regions. (Scale bars: 50 μm.) (B) Gallyas silver staining revealing degenerating neuritic processes (red arrows). (Scale bar: 50 μm.) (C) Bar graph indicating quantitation of silver-positive black neurites in the striatum. Data are expressed as the percent of total pixels per image (mean ± SEM, n = 5 or 6 animals per genotype). ***P < 0.001 or ****P < 0.0001 by one-way ANOVA with Bonferroni’s post hoc test compared with WT/WT mice.
Fig 4: hVPS35 antibody screening by Western Blot.Lysates of HAP1 (WT and VPS35 KO) were prepared and 20 μg of protein were processed for Western Blot with the indicated hVPS35 antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the polyacrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. An exception was given for antibody 81453**, which was titrated to 1/500, as the signal was too weak when following the supplier’s recommendations. Antibody dilution used: GTX635821** at 1/1000, GTX108058 at 1/1000, GTX116260 at 1/1000, A9278** at 1/1000, MA5-34647** at 1/1000, PA5-21898 at 1/1000, PA5-30654 at 1/1000, ab157220** at 1/1000, ab57632* at 1/270, ab118838 at 1/900, 81453** at 1/500, 10236-1-AP at 1/500, NBP2-75710** at 1/1000. Predicted band size: 91 kDa. *=monoclonal antibody, **=recombinant antibody.
Fig 5: VPS35 D620N affects trafficking and localization of ATG9A.(a) HeLa cells were immunostained for endogenous ATG9A and VPS35 and subjected to confocal microscopy. Magnified areas are shown on the right of the pictures. (b) HeLa cells were transfected with ATG9A-GFP for 24 h, and subsequently fixed, immunostained for endogenous WASH1 and subjected to confocal microscopy. (c) HeLa cells were transfected with ATG9A-GFP as in b, but immunostained instead for endogenous FAM21. (d) HeLa cells stably expressing GFP-VPS35 WT and D620N were depleted of endogenous VPS35 using 40 nM of siRNA, and subsequently immunostained for TGN46 and endogenous ATG9A and subjected to confocal microscopy. (e) Colocalization between TGN and ATG9A is expressed in terms of the Pearson’s Coefficient. n=25 cells (WT) and 33 cells (D620N). Error bars represent s.e.m. and **P=0.01 by 2-tailed Student’s t-test. Scale bars in (a–d), 10 μm.
Supplier Page from Abcam for Anti-VPS35 antibody [2D3]