Fig 1: Effect of retromer and WIPI1 on human endosomes Colocalization of WIPI1 with Vps26A. The indicated WIPI1mCherry variants and VPS26EGFP were expressed for 18 h in HK2 cells, from which endogenous WIPI1 had been deleted (WIPI1-KO). The cells were analyzed by confocal microscopy. Scale bars: 10 µm. Insets show enlargements of the outlined areas.Colocalization between WIPI1 variants and Vps26 was quantified in the cells from A using the Manders’ colocalization coefficient M2. The red line indicates the mean; n = 120 cells per condition, pooled from three independent experiments. P values were calculated by t-test (analysis performed with 99% confidence ***P < 0.0001).Expression levels of WIPI1mCherry variants. Lysates (50 µg of protein per sample) from the cells in A were analyzed by SDS–PAGE and Western blot against WIPI1 and tubulin.Depletion of VPS35. HK2 cells expressing the indicated WIPI1eGFP variants were transfected with siRNA against VPS35 (VPS35 KD) or a control siRNA pool. Lysates (50 µg per sample) from the cells were analyzed by SDS–PAGE and Western blot against Vps35 and tubulin.Blots from D were quantified on a LICOR Odyssey fluorescence imager. n = 3. Red lines indicate the means, n = 3 independent experiments, using a Welch’s t-test statistical analysis. Bars represent the mean and error bars the SD.Cells from D were analyzed by confocal microscopy 18 h after transfection. Scale bar: 10 µm.Quantification of tubule length in the cells from F. Data are means ± SD. of n = 210 tubules per condition, from three independent experiments. P values were calculated by unpaired Student’s t-test. 99% confidence: ***P < 0.0001.
Fig 2: Heterozygous (D620N) mutation of VPS35 causes abnormal morphology and dysfunction of mitochondria.A In contrast to intact morphology of mitochondria observed in neuromelanin-positive SNpc DAergic cells of age-matched WT mice, fragmented mitochondria with a reduced size were found in neuromelanin organelle-containing SNpc DAergic neurons of heterozygous VPS35D620N/+ mice aged 16 months. Arrow and star indicate mitochondria and neuromelanin organelle, respectively. Each bar represents mean ± S.E. value of 200 mitochondria. **P < 0.01 compared with WT mice. B Confocal immunofluorescence staining of Tom20 showed that mitochondria in TH-positive SNpc DAergic neurons of WT mice exhibited a long thread-like structure. In contrast, truncated and shortened mitochondria were found in TH-positive SNpc DAergic neurons of VPS35D620N/+ mice at age of 16 months. Each bar shows mean ± S.E. value of 80 neurons from four mice. *P < 0.05 compared with WT mice. C Immunoblotting assays demonstrated that protein expression of mitochondrial mitofusin 2 was downregulated in SN of VPS35D620N/+ knockin mice aged 16 months. Downregulated expression of mitofusin 2 was not found in SN of 12-month-old VPS35D620N/+ mice. Level of mitochondrial Drp1 was not altered in SN of VPS35D620N/+ mice at age of 12 or 16 months. COX IV is a protein marker of mitochondrial fraction. Each bar shows mean ± S.E. value of six mice. ***P < 0.001 compared with WT mice. D Confocal double immunofluorescence staining demonstrated that protein expression of mitofusin 2 was downregulated in TH-positive SNpc DAergic neurons of VPS35D620N/+ knockin mice aged 16 months. Each bar represents mean ± S.E. value of 80 neurons from four mice. E Mitochondrial complex I or complex IV activity was reduced in SN of 16-month-old heterozygous VPS35D620N/+ mice. Each bar represents mean ± S.E. value of six mice. F Intracellular level of ATP was decreased in SN of VPS35D620N/+ knockin mice at age of 16 months. Each bar shows mean ± S.E. value of six mice. G Compared to WT mice, reduced basal oxygen consumption rate was observed in SN of 16-month-old VPS35D620N/+ mice. Each bar represents mean ± S.E. value of three mice.
Fig 3: Synaptic VPS35 is functionally knocked down by three independent shRNA. (a) Representative confocal microscopy images of cortical neurons infected with control shRNA and the three shRNAs against VPS35. Left, mCherry signal reporting the expression of lentivirus containing the shRNAs coding sequences. Right, neurons immunolabeled for VPS35. Scale bar = 50 µm. (b) Representative western blot showing the knock down of VPS35 by three independent shRNAs. Original uncropped blots are shown in Figure S1. (c). Quantification of VPS35 intensity in immunostainings (n = 25 ± 1 fields of view, N = 2 animals) (d) Quantification of VPS35 levels normalized to total protein levels (assessed by TCE staining) in western blot. Values are presented as a ratio compared to the control condition. (N = 5 ± 1 blots/animals). (e) Representative confocal microscopy images of hippocampal neurons expressing either control shRNA or one of the three shRNAs against VPS35. Left, mCherry signal which reports expression of lentivirus containing the shRNAs. Right, surface immunolabelling of GluA1. Scale Bar = 5 µm. (f) Quantitative analyses of GluA1 staining intensity (n = 35 ± 1 fields of view, N = 3 animals). Detailed information (average, SEM, n and statistics) is shown in Supplementary Table S1.
Fig 4: VPS35 overexpression reduces Aß1-40 and phosphorylated tau in trisomic neurons. (A) Representative immunoblots of VPS35, VPS26, and VPS29 in euploid, trisomic, and trisomic neurons overexpressing VPS35 (trisomic + AAV-VPS35) at 25 days post-differentiation. (B) Densitometry analysis of immunoblots in panel A (n = 5, in duplicate for VPS35; n = 4, in duplicate for VPS26 and VPS29). (C) Relative expression of VPS35, VPS26, and VPS29 message in euploid, trisomic, and trisomic neurons overexpressing VPS35 (trisomic + AAV-VPS35) at 25 days post-differentiation (n = 3, in duplicate). (D) Levels of Aß1-40 detected in the culture media from euploid, trisomic, and trisomic neurons overexpressing VPS35 (trisomic + AAV-VPS35) (n = 4). (E) Representative immunoblots of HT7, AT270, and PHF1, in euploid, trisomic, and trisomic neurons overexpressing VPS35 (trisomic + AAV-VPS35). (F) Densitometry analysis of immunoblots shown in panel E (n = 3, in duplicate). Comparisons between groups were using a main-effects model two-way ANOVA. Values represent mean ± standard error of the mean, #P < .10, *P < .05, **P < .01, ***P < .001, and ****P < .0001
Fig 5: Retromer cargo recognition core proteins are decreased in trisomic neurons by 25 days post-differentiation. (A) Representative immunoblots of VPS35, VPS26, VPS29, and APP in euploid and trisomic neurons at 5 days post-differentiation. (B) Densitometry analysis of immunoblots shown in panel A (n = 3, in duplicate). (C) Representative immunoblots of VPS35, VPS26, VPS29, and APP in euploid and trisomic neurons at 15 days post-differentiation. (D) Densitometry analysis of immunoblots shown in panel C (n = 4, in duplicate). (E) Representative immunoblots of VPS35, VPS26, VPS29, and APP in euploid and trisomic neurons at 25 days post-differentiation. (F) Densitometry analysis of immunoblots shown in panel E (n = 3, in duplicate for VPS35, VPS26, and VPS29; n = 4, in duplicate for APP). (G) Relative expression of VPS35, VPS26, VPS29, and APP message in euploid and trisomic neurons at 25 days post-differentiation (n = 3, in duplicate). Comparisons between groups were made using the Student t-test. Values represent mean ± standard error of the mean, #P < .10, *P < .05, **P < .01, ***P < .001, and ****P < .0001
Supplier Page from Abcam for Anti-VPS35 antibody