Research ArticleParkinson’s Disease

α-Synuclein binds to TOM20 and inhibits mitochondrial protein import in Parkinson’s disease

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Science Translational Medicine  08 Jun 2016:
Vol. 8, Issue 342, pp. 342ra78
DOI: 10.1126/scitranslmed.aaf3634
  • Fig. 1. Ex vivo PL between α-synuclein and TOM20 is associated with decreased mitochondrial import of the complex I subunit Ndufs3 in nigrostriatal neurons in vivo in the rotenone and α-synuclein overexpression rat models of PD.

    (A) In a vehicle-treated rat (top row), there is little α-synuclein (α-syn)–TOM20 PL signal, and there is intense punctate staining of Ndufs3 in mitochondria of nigrostriatal neurons. In contrast, in a rotenone-treated rat (bottom row), there is a strong α-synuclein–TOM20 PL signal, which is associated with loss of mitochondrial Ndufs3 staining. In the box plot, mean nigrostriatal cellular PL fluorescence values for individual animals (vehicle- or rotenone-treated) are indicated by black circles. In each animal, PL signal was measured in 35 to 50 nigrostriatal neurons per hemisphere. P < 0.005 by two-tailed unpaired t test with Welch’s correction. TH, tyrosine hydroxylase. (B) In a rat that received a unilateral injection of AAV-shSNCA, the rotenone-induced α-synuclein–TOM20 PL signal was largely prevented, and mitochondrial Ndufs3 staining was preserved. In the box plot, mean nigrostriatal cellular PL fluorescence values for each hemisphere (control or AAV-shSNCA–injected) of individual animals are indicated by black circles. For each animal, PL signal was measured in 50 to 70 nigrostriatal neurons per hemisphere. P < 0.0005 by two-tailed paired t test with Welch’s correction. (C) In a rat that received unilateral injection of an α-synuclein overexpression vector (AAV-hSNCA), the α-synuclein–injected hemisphere showed a strong α-synuclein–TOM20 PL signal with an associated loss of Ndufs3 staining. In the box plot, mean nigrostriatal cellular PL fluorescence values for each hemisphere (control AAV-GFP or AAV-hSNCA–injected) of individual animals are indicated by black circles. For each animal, PL signal was measured in 50 to 70 nigrostriatal neurons per hemisphere. P < 0.05 by two-tailed unpaired t test with Welch’s correction. Scale bar, 30 μm. GFP, green fluorescent protein.

  • Fig. 2. Evidence of impaired mitochondrial protein import in human dopaminergic substantia nigra neurons in PD.

    (A) In TH-positive dopamine neurons from postmortem brain tissue of PD patients, there was an intense α-synuclein–TOM20 PL signal and a marked loss of Ndufs3 immunoreactivity. In PD cases, remaining Ndufs3 staining was rather diffuse instead of punctate. (B) Quantification of the α-synuclein–TOM20 PL signal in control versus PD dopamine neurons. (C) Quantification of Ndufs3 immunoreactivity in control versus PD dopamine neurons. The Ndufs3 signal was normalized to the TH signal, which tended to minimize the apparent differences. ***P < 0.0001; *P < 0.05, two-tailed unpaired t test with Welch’s correction for unequal variances.

  • Fig. 3. Posttranslationally modified α-synuclein binds to TOM20 and inhibits mitochondrial protein import.

    (A) Mitochondrial GFP (mtGFP) import in intact wild-type (WT) and TOM20-overexpressing (OE) SH-SY5Y cells exposed to various forms of α-synuclein. In WT cells treated with oligomeric, dopamine (DA)–modified, or S129E α-synuclein, note the diffuse pattern of staining compared to vehicle. In TOM20-overexpressing cells, mtGFP maintained its mitochondrial localization despite α-synuclein treatment. (B) Quantification of mtGFP import in WT and TOM20-overexpressing cells. For each condition in each experiment, mtGFP localization was determined in 5 to 10 regions of interest in zoomed confocal images from 5 to 10 cells, and three or four independent experiments were performed. (C) Autoradiographs of in vitro import of pre-OTC (pOTC) into mitochondria isolated from rat brain (top), WT SH-SY5Y cells (middle), and TOM20-overexpressing SH-SY5Y cells (bottom) after exposure to various forms of α-synuclein (30 min at 4°C). The upper band represents 35S-labeled pre-OTC, and the lower band represents imported, cleaved (mature) OTC. Mitos, mitochondria. (D) Quantification of OTC import into rat brain mitochondria. Results were normalized to the vehicle-treated control, and trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) + oligomycin was used to collapse membrane potential and define zero import. n = 3 independent experiments. (E) Quantification of OTC import into mitochondria from WT and TOM20-overexpressing SH-SY5Y cells. n = 3 to 4 independent experiments. (F) Immunolocalization of TOM20 and Ndufs3 in WT and TOM20-overexpressing HEK293 cells exposed to various forms of α-synuclein. In WT cells exposed to oligomeric, dopamine-modified, or S129E α-synuclein, Ndufs3 localization was diffuse rather than mitochondrial (that is, Ndufs3 redistributed outside of mitochondria as defined by TOM20). This effect was prevented in TOM20-overexpressing cells. (G) Correlation (Pearson index) of the localizations of TOM20 and Ndufs3 in WT versus TOM20-overexpressing cells. TOM20 overexpression rescues the normal localization of Ndufs3. For each experimental condition, at least 100 cells were analyzed in each of three or four independent experiments. Statistical analyses were by one- or two-way ANOVA followed by pairwise testing and correction for multiple comparisons. aP < 0.0001 versus monomer; bP < 0.0001 versus WT cells; cP < 0.005 versus monomer; dP < 0.05 versus WT cells; eP < 0.002 versus WT cells. Scale bars, 5 μm.

  • Fig. 4. PL of posttranslationally modified α-synuclein and TOM20 and Ndufs3 localization in HEK293 cells.

    (A) In untransfected cells, there was PL between TOM20 and oligomeric, dopamine-modified, and S129E α-synuclein, but not monomeric or nitrated species. This was associated with a cytosolic redistribution of Ndufs3. Fibrillar α-synuclein did not interact with TOM20 (fig. S5). When cells were transfected with an MTS expression vector before treatment with α-synuclein, the TOM20–α-synuclein interaction was blocked, indicating that the α-synuclein binding site overlaps with the MTS binding site on TOM20. MTS transfection also preserved the punctate (mitochondrial) distribution of Ndufs3. (B) When cells were transfected with the MTS expression vector 24 hours after α-synuclein treatment, the TOM20–α-synuclein interaction was reversed. Bar graphs show quantification of the α-synuclein–TOM20 PL signal in mock-transfected (black bars) and MTS-overexpressing cells (white bars). At least 100 cells were analyzed for each condition in every independent experiment (n = 3). aP < 0.0001 versus vehicle; bP < 0.0001 versus mock-transfected, two-way ANOVA. Scale bar, 5 μm.

  • Fig. 5. Binding curves of TOM20 C-terminal cytosolic domain to various forms of α-synuclein.

    (A) There was saturable binding of oligomeric, dopamine-modified, and S129E α-synuclein, but not the monomeric or nitrated species. n = 3. CTD, C-terminal cytosolic domain. (B) The COX8 MTS peptide inhibited binding of oligomeric α-synuclein to TOM20. When binding was performed in the presence of excess MTS (250 μM), specific binding was markedly reduced or abolished; nonlinear curve fitting yielded an affinity of >7 × 1014 μM when the MTS was present. Similar results were obtained with dopamine-modified and S129E α-synuclein. The overall effect of the MTS was significant (P < 0.02) by two-way ANOVA. n = 3.

  • Fig. 6. α-Synuclein interaction with TOM20 prevents the normal interaction between TOM20 and TOM22 in HEK293 cells.

    (A) Under basal conditions (vehicle), PL detects an interaction between TOM20 and TOM22. This was blocked by oligomeric, dopamine-modified, and S129E α-synuclein, but not monomeric or nitrated species. Loss of the TOM20-TOM22 PL signal was associated with relocalization of Ndufs3 to the cytosol. In cells overexpressing a naked MTS (COX8 presequence), the TOM20-TOM22 PL signal was maintained even after treatment with oligomeric, dopamine-modified, and S129E α-synuclein. (B) When cells were treated with α-synuclein and then transfected with the MTS 24 hours later, the TOM20-TOM22 PL signal was restored. Graphs show quantification of TOM20-TOM22 signal in mock-transfected (black bars) and MTS-overexpressing cells (white bars). aP < 0.0001 versus vehicle; bP < 0.0001 versus mock-transfected, two-way ANOVA. At least 100 cells were analyzed per condition in each independent experiment. n = 3. Scale bar, 5 μm.

  • Fig. 7. Downstream effects of α-synuclein on mitochondria.

    (A) In WT SH-SY5Y cells, a 24-hour exposure to oligomeric or dopamine-modified α-synuclein reduced basal and FCCP-stimulated mitochondrial respiration. Monomeric α-synuclein was without effect. *P < 0.01 versus vehicle, one-way ANOVA; n = 3. OCR, oxygen consumption rate. (B) In SH-SY5Y cells overexpressing TOM20, the deleterious effects of oligomeric and dopamine-modified α-synuclein were prevented. n = 3. (C) In WT HEK293 cells, a 24-hour exposure to oligomeric, dopamine-modified, or S129E α-synuclein induced oxidation of protein thiols, whereas exposure to monomeric or nitrated α-synuclein did not. (D and E) In HEK293 cells overexpressing TOM20, α-synuclein did not induce oxidative stress. At least 100 cells were quantified per condition in each experiment. aP < 0.001 versus vehicle; bP < 0.001 versus mock-transfected cells, two-way ANOVA; n = 3. (F) TMRM fluorescence in SH-SY5Y cells (as an index of ΔΨm) was reduced by oligomeric but not monomeric α-synuclein. (G and H) In SH-SY5Y cells overexpressing TOM20, oligomeric α-synuclein did not significantly affect ΔΨm. Thirty to 50 cells were analyzed for each treatment in each of three independent experiments. aP < 0.001 versus vehicle; bP < 0.001 versus WT cells, two-way ANOVA. Scale bars, 10 μm. Oligo, oligomycin; Rot, rotenone.

  • Fig. 8. The normal TOM20-TOM22 PL signal seen in most neurons is absent in rat nigrostriatal dopamine neurons in vivo but is restored by knockdown of endogenous α-synuclein.

    (A) In the untreated hemisphere (top row), MAP2+ (microtubule-associated protein 2)/TH nondopaminergic neurons (arrows) showed a strong TOM20-TOM22 PL signal, which was absent in TH+ dopaminergic cells (asterisks). In the hemisphere that received AAV2-shSNCA (bottom row), there was emergence of a strong TOM20-TOM22 PL signal in the TH+ dopaminergic neurons. Scale bar, 30 μm. (B) Consistent with the in vitro data (Fig. 7, C to E), α-synuclein knockdown was associated with decreased basal protein thiol oxidation in otherwise untreated rats. Filled circles, −S-S−/−SH ratio of nigral neurons in the control hemisphere; half-filled circles, −S-S−/−SH ratio of nigral neurons in the SNCA knockdown hemisphere. Lines connect the means from each hemisphere in each animal. *P < 0.05, Wilcoxon matched-pairs signed-rank test.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/342/342ra78/DC1

    Materials and Methods

    Fig. S1. Amounts of α-synuclein and S129-phosphorylated α-synuclein in vivo.

    Fig. S2. Positive and negative PL interactions with α-synuclein in substantia nigra pars compacta.

    Fig. S3. Rotenone induces a loss of mitochondrial localization of the nuclear-encoded, imported protein Ndufs3.

    Fig. S4. All species of α-synuclein used in this study enter cells to an equivalent extent, and when added at 200 nM, they do not change intracellular concentrations of α-synuclein or its localization.

    Fig. S5. Fibrillar α-synuclein does not affect mitochondrial protein import.

    Fig. S6. Overexpression of TOM20 and TOM5.

    Fig. S7. Time course of isolated brain mitochondrial protein import in the absence or presence of monomeric and oligomeric α-synuclein.

    Fig. S8. Lack of mitochondrial depolarization by α-synuclein during import assays.

    Fig. S9. Effects of α-synuclein on import and localization of other mitochondrial proteins.

    Fig. S10. The α-synuclein–TOM20 PL signal colocalizes with mitochondria.

    Fig. S11. Downstream effects of α-synuclein on mitochondria are blocked by MTS overexpression.

    Fig. S12. The protective effects of TOM20 overexpression on mitochondrial protein import can be overcome by increased concentrations of α-synuclein.

    Fig. S13. Structural analysis of the α-synuclein species used in this study.

  • Supplementary Material for:

    α-Synuclein binds to TOM20 and inhibits mitochondrial protein import in Parkinson's disease

    Roberto Di Maio, Paul J. Barrett, Eric K. Hoffman, Caitlyn W. Barrett, Alevtina Zharikov, Anupom Borah, Xiaoping Hu, Jennifer McCoy, Charleen T. Chu, Edward A. Burton, Teresa G. Hastings, J. Timothy Greenamyre*

    *Corresponding author. Email: jgreena{at}pitt.edu

    Published 8 June 2016, Sci. Transl. Med. 8, 342ra78 (2016)
    DOI: 10.1126/scitranslmed.aaf3634

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Amounts of α-synuclein and S129-phosphorylated α-synuclein in vivo.
    • Fig. S2. Positive and negative PL interactions with α-synuclein in substantia nigra pars compacta.
    • Fig. S3. Rotenone induces a loss of mitochondrial localization of the nuclear-encoded, imported protein Ndufs3.
    • Fig. S4. All species of α-synuclein used in this study enter cells to an equivalent extent, and when added at 200 nM, they do not change intracellular concentrations of α-synuclein or its localization.
    • Fig. S5. Fibrillar α-synuclein does not affect mitochondrial protein import.
    • Fig. S6. Overexpression of TOM20 and TOM5.
    • Fig. S7. Time course of isolated brain mitochondrial protein import in the absence or presence of monomeric and oligomeric α-synuclein.
    • Fig. S8. Lack of mitochondrial depolarization by α-synuclein during import assays.
    • Fig. S9. Effects of α-synuclein on import and localization of other mitochondrial proteins.
    • Fig. S10. The α-synuclein–TOM20 PL signal colocalizes with mitochondria.
    • Fig. S11. Downstream effects of α-synuclein on mitochondria are blocked by MTS overexpression.
    • Fig. S12. The protective effects of TOM20 overexpression on mitochondrial protein import can be overcome by increased concentrations of α-synuclein.
    • Fig. S13. Structural analysis of the α-synuclein species used in this study.

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