Fig 1: Molecular and phenotypic analyses of VPS35 cKO mice crossed with Synapsin-1-Cre mice. (A) Schematic indicating the targeted VPS35 cKO allele before or after Cre-mediated recombination (with synapsin-1-Cre mice) to remove exon 6 selectively in neurons. The location of PCR primers used for detecting genomic recombination in C is shown. (B) Fluorescent images indicating GFP (green) in sagittal sections of brain (upper) and coronal sections of the spinal cord (lower) induced by Cre-mediated recombination in mT/mG reporter mice crossed with Syn1-Cre mice at P10. (C) PCR analysis of genomic DNA derived from brain or liver of VPS35 cKO mice (floxed, fl) crossed with Syn1-Cre at P10 using primers flanking exon 6 (For3/Rev3) shown in A. (D) Representative images of VPS35 mRNA (red) detected by fluorescence in situ hybridization (RNAscope) co-localized with NeuN immunofluorescence (green) in the hippocampus and hindbrain (upper panels) or the cervical, thoracic and lumbar spinal cord (lower panels) of VPS35fl/fl/Cre mice at P10 compared to Cre-negative mice. (E) Confocal microscopic images of VPS35 mRNA (red) detected by RNAscope co-localized with NeuN (green) and ChAT (purple) immunofluorescence indicating VPS35 mRNA expression in ventral horn motor neurons of the lumbar spinal cord in P10 mice. Quantitation of VPS35 mRNA fluorescence intensity in NeuN-positive neurons (n = 113 cells, from three Cre-negative mice; n = 125 cells, from three VPS35fl/fl/Cre mice) and ChAT-positive neurons (n = 52 cells, from three Cre-negative mice; n = 58 cells, from three VPS35fl/fl/Cre mice) localized within the dorsal and ventral horns of the lumbar spinal cord is shown (right). Bars represent the mean ± SEM. ****P < 0.0001 by unpaired, two-tailed Student’s t-test. (F) Levels of VPS35, VPS26, VPS29 and actin proteins in soluble spinal cord extracts of VPS35 cKO mice at P12–P14 by western blot analysis. Graphs indicate densitometric quantification of VPS35, VPS26 or VPS29 levels normalized to actin from VPS35fl/fl/Cre mice (n = 3 mice) compared to Cre-negative control mice (VPS35fl/fl or VPS35fl/wt; n = 3 mice). Bars represent the mean ± SEM. **P < 0.01 by unpaired, two-tailed Student’s t-test. (G) Representative images of mouse littermates at P1 (upper) or P14 (lower) with their indicated genotypes. VPS35fl/fl/Cre mice are indicated by red arrows. (H) Graphs indicating body weight (grams, upper) and body length (cm, lower) of mice at P1, P5, P10, P12 or P15 for each genotype. Line bars represent mean ± SEM (n = 8–18 mice/genotype for P1, n = 11–15 mice for P5, n = 8–14 mice for P10). Note, n = 2–5 mice/genotype for P12 or P15 due to limited surviving animals (with error bars present for n ≥ 2). Data points are shown for individual mice as indicated by black or white shapes. White triangles indicate VPS35fl/fl/Cre mice. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with Bonferroni’s post hoc test, compared to VPS35fl/wt mice (black circles) or as indicated.
Fig 2: Normal retromer assembly in brain but reduced VPS35 and WASH complex in dopaminergic neurons of D620N VPS35 KI mice. (A) Hemibrain extracts from D620N VPS35 KI and WT mice at 3 mo (n = 2 mice per genotype) were subjected to IP with anti-VPS35 antibody (or control anti-V5 IgG). Western blot analysis of IP and input fractions reveal equivalent interactions of endogenous WT and D620N VPS35 with retromer subunit VPS26, whereas WASH complex subunits (FAM21 and WASH1) are not detected. (B) LC-MS/MS analysis of anti-VPS35 IP (or anti-V5 IP) fractions from A reveal equivalent interactions of WT and D620N VPS35 with retromer subunits VPS26A, VPS26B, and VPS29 in mouse brain. WASH complex subunits are not detected. MS/MS spectral counts for each protein are shown. (C) HEK-293T cells expressing V5-tagged human VPS35 (WT or D620N) were subjected to IP with anti-V5 antibody, and IP and input fractions were probed for VPS26, FAM21, and WASH1. WT and D620N VPS35 interact equivalently with endogenous VPS26, whereas D620N VPS35 displays an impaired interaction with endogenous FAM21 and WASH1. (D) Immunofluorescent confocal colocalization analysis reveals markedly reduced levels of VPS35 and WASH1 selectively in nigral dopaminergic neurons (TH-positive) of 3-mo-old D620N/WT and D620N/D620N KI mice. (Scale bars: 15 μm.) (E) Bar graphs indicating CTCF values (mean ± SEM, n = 4 animals per group) of VPS35 or WASH1 fluorescence intensity in TH-positive dopaminergic neurons. ***P < 0.001 by one-way ANOVA with Bonferroni’s post hoc test compared with WT/WT mice.
Fig 3: Pharmacological chaperone affects retromer complex levels in 3xTg mice. a Representative western blot analysis of VPS35, VPS26, VPS29, Cl-MPR and CTSD proteins in brain cortex homogenates from wild type (WT) and 3xTg mice treated with TPT or control (WT, 3xTg). b Densitometry of the immunoreactivities shown in the previous panel. Values represent mean ± standard error of the mean (*p < 0.05, WT Control vs WT/TPT, n = 3; #p < 0.05, WT Control vs 3xTg Control, n = 3; ^p < 0.05, 3xTg Control vs 3xTg/TPT, n = 3). c. Representative images of brain cortex sections of 3xTg receiving vehicle (3xTg) or TPT (3xTg/TPT) immunostained for VPS35 (scale bar 10 μm). d Quantification of the immune-fluorescent signal for VPS35 as observed in the previous panel. Values represent mean ± standard error of the mean (*p < 0.05 n = 3 per group)
Fig 4: Generation and molecular analyses of D620N VPS35 KI mice. (A) Floxed VPS35 mice were developed by introducing a loxP-flanked WT “minigene” (containing a splice acceptor, WT VPS35 exons 15–17, and a polyA signal) downstream of intron 14 and replacing endogenous exon 15 (with a D620N mutant version) by homologous recombination in ES cells. WT VPS35 is expected to be expressed (via splicing to the minigene) but after Cre-mediated recombination D620N VPS35 is expressed from the targeted allele. (B) Germline D620N KI mice were developed by crossing floxed VPS35 mice with CMV-Cre mice to remove the floxed WT minigene. KI mice contain a single loxP site and exon 15 harboring the D620N mutation. (C) PCR genotyping of tail genomic DNA for floxed VPS35 mice (Left) or germline VPS35 KI mice (Right) using PCR primers (arrows) as indicated in A or B. (D) Genomic PCR from liver DNA of germline D620N/WT and WT/WT mice using primers flanking exon 15. PCR products were cloned and sequenced to confirm the presence of the D620N mutation (GAT → AAT) in D620N/WT mice (Lower) compared with WT/WT mice (Upper). (E) Genotype frequencies from crosses of heterozygous (VPS35D620N/WT) mice. Percentages are from 605 F1 progeny from 70 litters. (F) Kaplan–Meier survival curves of germline VPS35 KI mice were generated by monitoring cohorts of all genotypes (VPS35WT/WT, n = 139; VPS35D620N/WT, n = 302; VPS35D620N/D620N, n = 125). (G) Western blot analysis of ventral midbrain extracts derived from 3-mo-old D620N VPS35 KI mice using antibodies to endogenous VPS35 (Ct, residues 697–797), VPS26, VPS29, or β-tubulin. (H) Western blot analysis of hemibrains from floxed VPS35 mice using antibodies to VPS35 (Nt, residues 1–311 or Ct), VPS26, VPS29, or β-tubulin. (I) Northern blot analysis of total brain RNA using a VPS35-specific DNA probe (nucleotides 163–984) confirms normal VPS35 mRNA expression in the D620N/D620N KI mice but reduced VPS35 mRNA in FLOX/WT mice relative to their WT/WT littermates.
Fig 5: VPS35 D620N impairs autophagy, while VPS35 knockdown has only modest effects.(a) HeLa cells stably expressing GFP-VPS35 WT and D620N were treated with bafilomycin A1 or DMSO vehicle control. Endogenous LC3-II and tubulin levels were examined by western blot. A representative experiment of six experiments is shown. (b) Quantification of the representative experiment in triplicate shown in a, in which endogenous LC3-II levels are normalized to tubulin and expressed as a ratio of levels in WT. ***P=7.86 × 10−6 (DMSO) and 3.81 × 10−5 (Baf) by 2-tailed Student’s t-test. (c) HeLa cells stably expressing GFP-VPS35 WT and D620N were transfected with mRFP-LC3. The number of LC3 vesicles was quantified by Cellomics automated fluorescence microscopy. A representative experiment of three independent experiments is shown, with 344 (WT) and 401 (D620N) cells analysed. ***P<0.0001 by 2-tailed Student’s t-test. (d) GFP-VPS35 WT and D620N-expressing cells were transfected with HA-Q74 and immunostained for HA. The percentage of transfected cells with aggregates was counted by a blinded experimenter. The quantification shows the mean of three experiments in triplicate with minimum 200 cells per replicate. ***P=0.00086 by 1-tailed Student’s t-test. (e) Confocal images representative of the experiment described in d. Scale bar, 20 μm. (f) Cells stably expressing WT and D620N GFP-VPS35 were transfected with GFP-α-synuclein A53T and GFP for 48 h and analyzed by western blotting. (g) Quantification of the representative experiment in triplicate in f, in which the level of α-synuclein was expressed as a ratio to GFP. A representative experiment of two independent experiments is shown. **P=0.0047 by 2-tailed Student’s t-test. (h) VPS35 was knocked down with two individual siRNA nucleotides in HeLa cells, and cells were treated with bafilomycin A1 and lysed as in a. A representative experiment is shown in triplicate. (i) Quantification of three independent experiments in triplicate. *P=0.026; other results non-significant by 2-tailed Student’s t-test. (j) Protein levels of CSC, including VPS35, VPS26 and VPS29), were assessed upon VPS35 knockdown, confirming previous results that knockdown of one component destabilizes the CSC. All error bars indicate s.e.m.
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