Parkinson's disease–associated mutant VPS35 causes mitochondrial dysfunction by recycling DLP1 complexes

Journal name:
Nature Medicine
Volume:
22,
Pages:
54–63
Year published:
DOI:
doi:10.1038/nm.3983
Received
Accepted
Published online

Abstract

Mitochondrial dysfunction represents a critical step during the pathogenesis of Parkinson's disease (PD), and increasing evidence suggests abnormal mitochondrial dynamics and quality control as important underlying mechanisms. The VPS35 gene, which encodes a key component of the membrane protein–recycling retromer complex, is the third autosomal-dominant gene associated with PD. However, how VPS35 mutations lead to neurodegeneration remains unclear. Here we demonstrate that PD-associated VPS35 mutations caused mitochondrial fragmentation and cell death in cultured neurons in vitro, in mouse substantia nigra neurons in vivo and in human fibroblasts from an individual with PD who has the VPS35D620N mutation. VPS35-induced mitochondrial deficits and neuronal dysfunction could be prevented by inhibition of mitochondrial fission. VPS35 mutants showed increased interaction with dynamin-like protein (DLP) 1, which enhanced turnover of the mitochondrial DLP1 complexes via the mitochondria-derived vesicle–dependent trafficking of the complexes to lysosomes for degradation. Notably, oxidative stress increased the VPS35-DLP1 interaction, which we also found to be increased in the brains of sporadic PD cases. These results revealed a novel cellular mechanism for the involvement of VPS35 in mitochondrial fission, dysregulation of which is probably involved in the pathogenesis of familial, and possibly sporadic, PD.

At a glance

Figures

  1. VPS35 regulates mitochondrial dynamics in vitro.
    Figure 1: VPS35 regulates mitochondrial dynamics in vitro.

    (a) Representative three-dimensional (3D) pictures (top) of mitochondria in rat cortical neurons cotransfected with either an empty-vector control plasmid (Vec) or plasmids expressing the indicated Flag-VPS35 fusions and a construct encoding mitoDsRed2 (bottom left). A segment of axon (boxed area) was enlarged (bottom right). WT, VPS35WT; D620N, VPS35D620N; R524W, VPS35R524W. Scale bars, 10 μm (microscopy images) and 5 μm (boxed area). (b,c) Quantification of mitochondrial aspect ratio (b) and percentage of neurons with fragmented mitochondria (c) in VPS35-overexpressing rat cortical neurons (vector-only control, n = 35; VPS35WT, n = 32, VPS35D620N, n = 40; VPS35R524W, n = 43). (d) The ratio of fission and fission-plus-fusion events in live neurons expressing VPS35 and mitoDsRed2 (vector-only control, n = 43; VPS35WT, n = 33, VPS35D620N, n = 37; VPS35R524W, n = 38). (e) Measurement of neuronal viability in cortical neurons expressing GFP, VPS35 and mitoDsRed2 (vector-only control, n = 51; VPS35WT, n = 46, VPS35D620N, n = 55; VPS35R524W, n = 51). (f,g) Representative confocal images (f) and quantification of mitochondrial length (g) in PD D620N fibroblasts or in NHFs without (NHF, n = 34; VPS35D620N PD, n = 44) (f, left) or with (NHF + mdivi-1, n = 27; VPS35D620N PD + mdivi-1, n = 53) (f, right) mdivi-1 treatment. In f, enlarged images of the white boxed areas are shown to the right of each image. PD, PD D620N fibroblasts. Scale bars, 50 μm and 5 μm (enlarged images). (h,i) Representative EM micrographs of mitochondria (h) and quantification of mitochondrial length (i) in PD D620N fibroblasts (h, right) and NHFs (h, left). In h, enlarged images of the boxed areas are shown to the right. A total of 149 and 151 mitochondria from five randomly selected NHFs or PD D620N fibroblasts, respectively, were analyzed. Data are means ± s.e.m. from three independent experiments. *P < 0.05; n.s., not significant; one-way analysis of variance (ANOVA) with Tukey's multiple-comparison test.

  2. VPS35 regulates mitochondrial dynamics in vivo.
    Figure 2: VPS35 regulates mitochondrial dynamics in vivo.

    (a) Left, schematic (top) and representative image (bottom) showing mitochondria (red) in positively transfected TH+ neurons (green) after stereotactical injection of a lentivirus (that expresses VPS35 and mitoDsRed2) unilaterally into the ventral tegmental area (VTA) of a mouse. Right, representative 3D images of mitochondria (red) in a TH+ neuron in FVB mice injected with an empty-vector control (Vec) lentivirus (top) or a lentivirus that expresses VPS35WT or VPS35D620N. DAPI staining is shown in blue. AP, anteroposterior; ML, medial-lateral; DV, dorsal-ventral. Scale bars, 200 μm (left) and 5 μm (right). (b,c) Quantification of the mitochondrial aspect ratio (b) and the percentage of neurons with fragmented mitochondria (c) in TH+ and TH neurons in 2- to 3-month-old FVB mice injected with the indicated construct (TH+ neurons: vector-only, n = 20; VPS35WT, n = 23; VPS35D620N, n = 19; VPS35R524W, n = 21; TH neurons: vector-only control, n = 31; VPS35WT, n = 36; VPS35D620N, n = 35; VPS35R524W, n = 31; from three FVB mice per group). (d) Representative images of the substantia nigra in FVB mice (n = 3) injected with the indicated construct showing immunofluorescent costaining using antibodies specific for the neuronal protein NeuN (blue) and TH (green) and using mitoDsRed2. Scale bar, 400 μm. (e) Quantification of neuronal loss in TH+ and TH neurons in c. (fh) Quantification of the mitochondrial aspect ratio (f), the percentage of neurons with fragmented mitochondria (g) and neuronal loss (h) in TH+ neurons from FVB mice that were injected with lentiviruses expressing VPS35 and treated with or without (DMSO) mdivi-1 (DMSO-treated: vector-only control, n = 25; VPS35WT, n = 26; VPS35D620N, n = 27; mdivi-1–treated: vector-only control, n = 28; VPS35WT, n = 22; VPS35D620N, n = 24; from three mice per group). Throughout, n represents number of neurons per group. Data are means ± s.e.m. *P < 0.05; n.s., not significant; one-way ANOVA with Tukey's multiple-comparison test.

  3. Inhibition of mitochondrial fission alleviates VPS35-induced mitochondrial dysfunction and neuronal deficits.
    Figure 3: Inhibition of mitochondrial fission alleviates VPS35-induced mitochondrial dysfunction and neuronal deficits.

    (ad) The intracellular levels of ATP (a,c) and mitochondrial membrane potential (MMP), as determined by a tetramethylrhodamine methyl ester (TMRM) fluorescent probe, (b,d) were measured in M17 cell lines stably expressing VPS35 (a,b) and in NHFs and PD D620N fibroblasts (c,d) in the presence or absence of mdivi-1 treatment. (eg) Representative images of dendrites (e) and quantification of dendritic spines (f) and PSD95+ puncta (g) in primary rat cortical neurons (DIV14) cotransfected with mitoDsRed2 and the indicated VPS35 construct, and treated with or without mdivi-1 (vehicle-treated: vector-only control, n = 24; VPS35WT, n = 23; VPS35D620N, n = 25; VPS35R524W, n = 28; mdivi-1–treated: vector-only control, n = 22; VPS35WT, n = 27; VPS35D620N, n = 25; VPS35R524W, n = 25). Scale bar, 1 μm. (hj) Representative images of primary cortical neurons (boxed segment of axons are enlarged below) (h), and quantification of mitochondrial aspect ratio in the axon (i) and the percentage of neurons with fragmented mitochondria (j) in primary cortical neurons expressing mitoDsRed2, dominant-negative DLP1 (encoded by DLP1K38A) and the indicated VPS35 constructs (vector-only control, n = 25; VPS35WT, n = 23; VPS35D620N, n = 26; VPS35R524W, n = 28; vector + DLP1K38A, n = 30; VPS35WT + DLP1K38A, n = 21; VPS35D620N + DLP1K38A, n = 22; VPS35R524W + DLP1K38A, n = 29; vector + mdivi-1, n = 31; VPS35D620N + mdivi-1, n = 25). Throughout, n represents the number of neurons per group. Data are means ± s.e.m. from three independent experiments. *P < 0.05; n.s., not significant; one-way ANOVA with Tukey's multiple-comparison test.

  4. VPS35 promotes clearance of the mitochondrial DLP1 complex.
    Figure 4: VPS35 promotes clearance of the mitochondrial DLP1 complex.

    (a,b) Representative western blot (left) and quantification (right) of DLP1 in mitochondrial fractions from M17 stable cell lines expressing the indicated VPS35 constructs (a) or RNAi constructs (b). ATP5a, RAB5 and tubulin were used as mitochondrial, endosomal and cytosolic markers, respectively. NcRNAi, negative-control RNAi. (c) Representative confocal images (left) of DLP1 (green) and mitochondria (red) demonstrating mitochondrial DLP1 puncta (right image is the enlarged image of the boxed area in the left image) and histograms (right) of the size distribution of mitochondrial DLP1 puncta in M17 cells expressing mitoDsRed2 and the indicated VPS35 or RNAi constructs, and immunostained for DLP1 (n = 35 for each cell line). Scale bars, 10 μm and 1 μm (enlarged images). (d) Quantification of the number of mitochondrial DLP1 puncta in the indicated M17 stable cell lines that were transfected with mitoDsRed2, after immunostaining for DLP1 (n = 35 for each cell line). (e) Quantification of the lifetime of mitochondrial GFP–DLP1 puncta in live M17 cells expressing the indicated constructs, GFP–DLP1 and mitoDsRed2 (n = 25 for each cell line). (f) Representative western blot (top) and quantification (bottom) of DLP1 in ultracentrifugation precipitates (270K pellets) from the mitochondrial fractions of the indicated M17 stable cell lines, after treatment with DTME. (g) Representative western blot and quantification of DLP1 (denoted below the blot) in the 270K pellets from mitochondrial fractions of PD D620N fibroblasts or NHFs, after treatment with DTME. (h,i) Representative western blot of DLP1 (left) and quantification (right) of oligomeric and monomeric DLP1 in the mitochondrial fraction of VPS35-expressing M17 cells (h) or M17 cells in which VPS35 was knocked down (i), after treatment with DSS. NC, negative control; KD, VPS35 knockdown. Throughout, n represents number of neurons per group. Data are means ± s.e.m. from three independent experiments from three stable M17 clonal lines. *P < 0.05; n.s., not significant; one-way ANOVA with Tukey's multiple-comparison test.

  5. The VPS35-DLP1 interaction is key to the turnover of mitochondrial DLP1 complexes.
    Figure 5: The VPS35-DLP1 interaction is key to the turnover of mitochondrial DLP1 complexes.

    (a) Representative 3D images (n = 3) showing the colocalization of endogenous VPS35 and DLP1 on mitochondria in M17 cells transfected with mitoDsRed2 and immunostained with anti-DLP1 and anti-VPS35. Boxed area in the left image was enlarged and viewed at different angles. Scale bars, 10 μm (left image) and 2 μm (enlarged images). (b) Yeast two-hybrid analysis of VPS35-DLP1 interaction. All positively cotransformed cells grew on SD plates lacking leucine and tryptophan (left). After being streaked onto high-stringency plates (SD plates lacking leucine, tryptophan, histidine and adenine), only clones cotransformed with pGBKT7-DLP1 and pGADT7-DLP1 (positive control) or pGBKT7-VPS35 and pGADT7-DLP1 could grow (right) (n = 3). pGBKT7 and pGADT7 are the empty vectors. (c,d) Representative western blot analyses (n = 3) for the presence of DLP1 and VPS35 in VPS35 immunoprecipitates (c) or DLP1 immunoprecipitates (d) from normal M17 cells. HC, heavy chain; IgG, isotype control antibody. (e) Representative western blot analysis (n = 3) for the presence of endogenous DLP1 and VPS35 in mitochondrial fractions from M17 cells. COX II and NDUFB8 (NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 8) were used as mitochondrial markers; tubulin and RAB5 were used as cytosolic and endosomal markers, respectively. (f) Representative western blot (left; n = 3) and quantification (right) of DLP1 in Flag immunoprecipitates of the highly purified mitochondrial fraction prepared from M17 cells expressing Flag-tagged VPS35 variants. (g) Representative western blot (n = 3) of VPS35 and DLP1 in DLP1 immunoprecipitates from PD D620N fibroblasts and NHFs. (h) Representative western blot analysis (left) and quantification (right) of VPS35 in DLP1 immunoprecipitates from homogenates of the midbrains of individuals with sporadic PD and age-matched controls (n = 5 per group). (i) Representative western blot analysis (left) and quantification (right; n = 3) of DLP1 in Flag immunoprecipitate from M17 cells expressing the indicated Flag-tagged VPS35 mutants. D620A, VPS35D620A; D620E; VPS35D620E; D620I, VPS35D620I; D620R; VPS35D620R; D620Y; VPS35D620Y. (j) Representative western blot analysis (left) and quantification (right; n = 3) of DLP1 in the 270K pellets from mitochondrial fractions of M17 cell lines knocked down for endogenous VPS35 and expressing the indicated Flag-tagged VPS35 mutants. Throughout, data are means ± s.e.m. from three independent experiments. *P < 0.05, as compared with KD + Vector; #P < 0.05 as compared with KD + WT; one-way ANOVA with Tukey's multiple-comparison test (except for h, which used Student's t-test).

  6. The VPS35-containing retromer mediates mitochondrial DLP1 complex degradation through an MDV-to-lysosome pathway.
    Figure 6: The VPS35-containing retromer mediates mitochondrial DLP1 complex degradation through an MDV-to-lysosome pathway.

    (a) Representative western blot analysis (n = 3) for the presence of DLP1 in in vitro–reconstituted MDVs. TOM20 and COX IV were used as positive and negative controls for MDVs, respectively. (b,c) Representative western blot analysis (n = 3) (b) and quantification (c) of DLP1 in the in vitro–reconstituted MDVs in the presence of cytosolic fractions from the indicated M17 cells. (d) Representative electron micrographs (n = 7) of purified mitochondria highlighting double-membraned (left) and single-membraned (right) MDVs still attached to the mitochondrion. Scale bars, 100 nm. (e) Representative immuno-EM micrographs (n = 5) showing the specific labeling of DLP1-immunoreactive gold particles associated with MDVs. Scale bars, 100 nm. (f) Representative images (n = 3) showing the colocalization of DLP1 puncta and MDVs (TOM20+mitoDsRed2) (arrows) in M17 cells. Scale bars, 5 μm and 1 μm (enlarged images). (g,h) Representative confocal images (g) and quantification (h) of the colocalization of DLP1 puncta and MDVs (as highlighted in circles) in the indicated M17 stable cell lines. (vector-only control, n = 31; VPS35WT, n = 35; VPS35D620N, n = 37; negative control (nc) RNAi, n = 28; VPS35 RNAi, n = 31). Scale bars, 1 μm. (i) Representative 3D-reconstructed images (n = 3) of an M17 cell (left) showing the colocalization of DLP1 puncta and MDVs in the lysosome (left, boxed area). Enlarged views of the boxed area are shown to the right. Arrows point to MDVs-associated DLP1 puncta in a lysosome. Scale bars, 1 μm (left) and 1 μm (enlarged images). (j) Quantification of the percentage of MDVs associated with DLP1 puncta within lysosomes with or without bafilomycin A1 treatment (DMSO-treated: vector-only control, n = 33; VPS35WT, n = 28; VPS35D620N, n = 35; ncRNAi, n = 37; VPS35 RNAi, n = 31; bafilomycin A1–treated: vector-only control, n = 31; VPS35WT, n = 35; VPS35D620N, n = 36; ncRNAi, n = 27; VPS35 RNAi, n = 31). Data are means ± s.e.m. from three independent experiments. *P < 0.05; one-way ANOVA with Tukey's multiple-comparison test.

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Author information

  1. These authors contributed equally to this work.

    • Wenzhang Wang &
    • Xinglong Wang

Affiliations

  1. Department of Pathology, Case Western Reserve University, Cleveland, Ohio, USA.

    • Wenzhang Wang,
    • Xinglong Wang &
    • Xiongwei Zhu
  2. Electron Microscopy Core Facility, Case Western Reserve University, Cleveland, Ohio, USA.

    • Hisashi Fujioka
  3. Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, USA.

    • Charles Hoppel
  4. Center for Mitochondrial Diseases, Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.

    • Charles Hoppel
  5. Institute of Clinical Neurosciences, Southmead Hospital, University of Bristol, Bristol, UK.

    • Alan L Whone
  6. Trinity College Institute for Neuroscience, Trinity College Dublin, Dublin, Ireland.

    • Maeve A Caldwell
  7. The Henry Wellcome Integrated Signaling Laboratories, School of Biochemistry, University of Bristol, Bristol, UK.

    • Peter J Cullen
  8. Department of Neurology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

    • Jun Liu

Contributions

X.Z. and X.W. conceived and directed the project, interpreted the results and wrote the manuscript. W.W. and X.W. designed and carried out experiments, analyzed results and generated figures. H.F. helped with electron microscopy (EM) and the immuno-EM study; C.H. helped with bioenergetics measurements; A.L.W., M.A.C. and P.J.C. contributed fibroblasts from the individual with PD who has the VPS35D620N mutation and provided feedback on the manuscript; and J.L. contributed to the conception of the project, design of the experiments and the interpretation of results, and provided feedback on the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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