pubmed.ncbi.nlm.nih.gov

Defective mitochondrial protein import contributes to complex I-induced mitochondrial dysfunction and neurodegeneration in Parkinson's disease - PubMed

  • ️Mon Jan 01 2018

Defective mitochondrial protein import contributes to complex I-induced mitochondrial dysfunction and neurodegeneration in Parkinson's disease

Sandra Franco-Iborra et al. Cell Death Dis. 2018.

Abstract

Mitochondria are the prime energy source in most eukaryotic cells, but these highly dynamic organelles are also involved in a multitude of cellular events. Disruption of mitochondrial homeostasis and the subsequent mitochondrial dysfunction plays a key role in the pathophysiology of Parkinson's disease (PD). Therefore, maintenance of mitochondrial integrity through different surveillance mechanisms is critical for neuronal survival. Here, we have studied the mitochondrial protein import system in in vitro and in vivo models of PD. Complex I inhibition, a characteristic pathological hallmark in PD, impaired mitochondrial protein import, which was associated with a downregulation of two key components of the system: translocase of the outer membrane 20 (TOM20) and translocase of the inner membrane 23 (TIM23), both in vitro and in vivo. In vitro, those changes were associated with OXPHOS protein downregulation, accumulation of aggregated proteins inside mitochondria and downregulation of mitochondrial chaperones. Most of these pathogenic changes, including mitochondrial dysfunction and dopaminergic cell death, were abrogated by TOM20 or TIM23 overexpression, in vitro. However, in vivo, while TOM20 overexpression exacerbated neurodegeneration in both substantia nigra (SN) pars compacta (pc) and striatum, overexpression of TIM23 partially protected dopaminergic neurons in the SNpc. These results highlight mitochondrial protein import dysfunction and the distinct role of two of their components in the pathogenesis of PD and suggest the need for future studies to further characterize mitochondrial protein import deficit in the context of PD.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Mitochondrial protein import machinery deficiency in human substantia nigra in PD.

a Representative immunoblots of TIM23 and TOM20 protein levels in substantia nigra homogenates from Ctrl (n = 4–5) and PD patients (n = 9). Protein levels were normalized relative to Ponceau-S. Quantification is depicted as fold change to Ctrl (*P < 0.05 after unpaired two-sided Student’s t-test). b) Representative immunoblots of NDUFS3, COX IV and α-tubulin protein levels in substantia nigra homogenates from control (Ctrl, n = 5) and PD patients (n = 9). Protein levels were normalized relative to α-tubulin (*P < 0.05 after unpaired two-sided Student’s t-test). Error bars indicate s.e.m.

Fig. 2
Fig. 2. Complex I inhibition impairs mitochondrial protein import activity.

a–c, e BE(2)-M17 cells were treated with MPP+ (0.25, 0.5 or 1 mM as stated) or untreated (UT) for 24 h. a Representative immunoblots of TIM23, TOM20 and VDAC protein levels normalized relative to VDAC (n = 3 independent experiments). Quantification is depicted as fold change to UT condition (*P < 0.05, **P < 0.01 after one-way ANOVA test followed by Tukey’s post hoc test). b Representative images of mitoGFP-transfected BE(2)-M17 cells. Quantification is depicted as the fold change of mitoGFP intensity per cell compared with UT condition. A minimum of 35 cells were analyzed per condition (*P < 0.05 after unpaired two-sided Student’s t-test). Scale bar: 10 μm. c Representative immunoblots of GFP and β-actin protein levels. mitoGFP import was calculated as the ratio of mature (m) GFP compared to the sum of precursor (p) and mature GFP (total GFP; n = 5 independent experiments, ***P < 0.001 after unpaired two-sided Student’s t-test). d Representative immunoblots of radiolabelled OTC in isolated mitochondria UT or treated with MPP+ (1 mM) at different time points. pOTC import was calculated as the percentage of mature (m) OTC compared to the sum of precursor (p) and mature OTC (n = 3 independent experiments, ***P < 0.001 after unpaired two-sided Mann–Whitney test). e Representative images of NDUFS3 and COX IV immunofluorescence. Quantification is represented as the fold change in NDUFS3 or COX IV intensity compared with UT condition. A minimum of 35 cells were analyzed per condition (*P < 0.05, **P < 0.01 after unpaired two-sided Student’s t-test or unpaired two-sided Mann–Whitney test). Scale bar: 10 μm. Error bars indicate s.e.m.

Fig. 3
Fig. 3. Complex I inhibition leads to a mitochondrial dysfunction and cell death.

a–f BE(2)-M17 cells were treated with MPP+ (1 mM, 24 h) or untreated (UT). a Representative immunoblots of ATP5A (complex V), UQCRC2 (complex III), SDHB (complex II), COX II (complex IV), NDUFB8 (complex I) and VDAC protein levels normalized relative to VDAC (n = 3 independent experiments). Quantification is depicted as fold change to UT condition (*P < 0.05, **P < 0.01, ***P < 0.001 compared with UT condition after unpaired two-sided Student’s t-test). b Representative immunoblots of SDHB and ATP5A protein levels in soluble and insoluble mitochondria-isolated fractions (n = 3 independent experiments). Quantification is depicted as fold change to UT condition (*P < 0.05, **P < 0.01 after unpaired two-sided Student’s t-test). c CLPX and HSP9 gene expression levels normalized relative to RPLP0 (n = 3 independent experiments). Quantification is depicted as fold change to UT condition (**P < 0.01, ***P < 0.001 after unpaired two-sided Student’s t-test). d Representative immunoblots of CLPX, GRP75 and VDAC protein levels normalized relative to VDAC (n = 4 independent experiments). Quantification is depicted as fold change to UT condition (*P < 0.05 after unpaired two-sided Student’s t-test). e Mitochondrial membrane potential measured as TMRM fluorescence intensity. Quantification is depicted as the fold change in fluorescence intensity compared with UT condition (n = 3 independent experiments, ***P < 0.001 after unpaired two-sided Student’s t-test). f ROS production measured as CM-H2DCFDA fluorescence intensity. Quantification is depicted as the fold change in fluorescence intensity compared with UT condition (n = 3 independent experiments, **P < 0.01 after unpaired two-sided Student’s t-test). g In vitro sensitivity of BE(2)-M17 cells to increasing MPP+ concentrations for 24 h (n = 4 independent experiments). Cell survival was determined by MTT assay. Quantification is depicted as the % of cell survival relative to UT condition (*P < 0.05 after one-way ANOVA test followed by Tukey’s post hoc test). Error bars indicate s.e.m.

Fig. 4
Fig. 4. TOM20 overexpression restores mitochondrial dysfunction and prevents cell death in vitro.

Vehicle- or pCMV6-XL5-TOM20-transfected (TOM20) BE(2)-M17 cells were untreated (UT) or treated with a–c 1 mM MPP+ for 24 h or d 5 mM for 24 h. a Representative images of COX IV and TOM20 immunostained cells. Quantification is depicted as the fold change in COX IV intensity compared with vehicle-transfected UT condition. Dashed line shows TOM20-overexpressing cells. A minimum of 15 cells were analyzed per condition (***P < 0.001 after two-way ANOVA followed by Tukey’s post hoc test). Scale bar: 10 μm. b ROS production measured as CM-H2DCFDA fluorescence intensity. Quantification is depicted as the fold change in fluorescence intensity compared with vehicle-transfected UT condition (n = 3 independent experiments, *P < 0.05, **P < 0.01 after one-way ANOVA followed by Tukey’s post hoc test). c Mitochondrial membrane potential measured as TMRM fluorescence intensity. Quantification is depicted as the fold change in fluorescence intensity compared with vehicle-transfected UT condition (n = 5 independent experiments, *P < 0.05, **P < 0.01 after one-way ANOVA followed by Tukey’s post hoc test). d Cell death measured as propidium iodide (PI)-positive cells (n = 5 independent experiments). Quantification is depicted as the fold change in the percentage of PI-positive cells compared with each control condition (**P < 0.01 after unpaired two-sided Student’s t-test). Error bars indicate s.e.m.

Fig. 5
Fig. 5. TIM23 overexpression restores mitochondrial dysfunction and prevents cell death in vitro.

Vehicle- or pCMV6-XL5-TIM23-transfected (TIM23) BE(2)-M17 cells were untreated (UT) or treated with a–c 1 mM MPP+ for 24 h or d 5 mM for 24 h. a Representative images of COX IV and TIM23 immunofluorescence. Quantification is depicted as the fold change in COX IV intensity compared with vehicle-transfected UT condition. Dashed line shows TIM23-overexpressing cells. In all, 15 to 60 cells were analyzed per condition (*P < 0.05, ***P < 0.001 after two-way ANOVA followed by Tukey’s post hoc test). Scale bar: 10 μm. b ROS production measured as CM-H2DCFDA fluorescence intensity. Quantification is depicted as the fold change in fluorescence intensity compared with vehicle-transfected UT condition (n = 3 independent experiments, **P < 0.01 after one-way ANOVA followed by Tukey’s post hoc test). c Mitochondrial membrane potential measured as TMRM fluorescence intensity. Quantification is depicted as the fold change in fluorescence intensity compared with vehicle-transfected UT condition (n = 5 independent experiments, **P < 0.01 after one-way ANOVA followed by Tukey’s post hoc test). d Cell death measured as propidium iodide (PI)-positive cells (n = 5 independent experiments). Quantification is depicted as the fold change in the percentage of PI-positive cells compared with each control condition (**P < 0.01 after unpaired two-sided Student’s t-test). Error bars indicate s.e.m.

Fig. 6
Fig. 6. Mitochondrial translocases loss precedes dopaminergic neuron degeneration upon MPTP intoxication.

a Representative immunoblots of TIM23, TOM20 and VDAC protein levels in ventral midbrain of vehicle- (Veh, n = 5) and MPTP-treated (n = 6–7) mice euthanized at different time points. Protein levels were normalized relative to VDAC and quantification is depicted as fold change to vehicle condition (*P < 0.05, ***P < 0.001 after one-way ANOVA followed by Tukey’s post hoc test). b Representative images of tyrosine hydroxylase (TH) and COX IV immunostained in ventral midbrain sections from vehicle- (n = 5) or MPTP-treated (n = 5) mice euthanized at day 2 after the last injection. Quantification is depicted as the fold change of COX IV intensity in TH-positive neurons compared to vehicle condition. White arrows indicate representative neurons. A minimum of 36 neurons were analyzed per animal (*P < 0.05 after unpaired two-sided Student’s t-test). Error bars indicate s.e.m.

Fig. 7
Fig. 7. TOM20 overexpression cannot attenuate MPTP-induced dopaminergic neuron injury in vivo.

a Representative images of TOM20 and TH immunofluorescence in AAV-hTOM20 mice. Scale bar: 150 μm. b–d Sham- and AAV-hTOM20-injected mice were treated with vehicle or MPTP. b Representative images of TH and COX IV immunofluorescence in ventral midbrain sections (Sham-Vehicle n = 5; Sham-MPTP n = 6; AAV-hTOM20-Vehicle n = 4; AAV-hTOM20-MPTP n = 4). Quantification is depicted as the fold change in COX IV intensity compared with sham-injected vehicle-treated group (**P < 0.01, ***P < 0.001 after two-way ANOVA followed by Tukey’s post hoc test). Scale bar: 50 μm. c Representative photomicrographs of TH immunohistochemistry in striatum (Sham-Vehicle n = 7; Sham-MPTP n = 5; AAV-hTOM20-Vehicle n = 8; AAV-hTOM20-MPTP n = 7). Quantification is depicted as the optical densitometry of striatal TH immunoreactivity in the different experimental groups at day 21 post MPTP (*P < 0.05, **P < 0.01 after two-way ANOVA followed by Tukey’s post hoc test). Scale bar: 500 μm. d Representative photomicrographs of TH immunohistochemistry in SNpc (Sham-Vehicle n = 14; Sham-MPTP n = 10; AAV-hTOM20-Vehicle n = 7; AAV-hTOM20-MPTP n = 7). Quantification is depicted as the stereological cell counts of SNpc TH-immunoreactive neurons in the different experimental groups at day 21 post MPTP (*P < 0.05, ***P < 0.001 after two-way ANOVA followed by Tukey’s post hoc test). Scale bar: 500 μm. Error bars indicate s.e.m.

Fig. 8
Fig. 8. TIM23 overexpression partialy attenuates MPTP-induced dopaminergic neuron injury in vivo.

a Representative images of TIM23 and TH immunofluorescence in ventral midbrain sections from AAV-hTIM23 mice (n = 3). Scale bar: 150 μm. b–d Sham- and AAV-hTIM23-injected mice were treated with vehicle or MPTP. b Representative images of TH and COX IV immunofluorescence in ventral midbrain sections (Sham-Vehicle n = 5; Sham-MPTP n = 6; AAV-hTIM23-Vehicle n = 5; AAV-hTIM23-MPTP n = 6). Quantification is depicted as the fold change in COX IV intensity compared with sham-injected vehicle-treated group (*P < 0.05, ***P < 0.001 after two-way ANOVA followed by Tukey’s post hoc test). Scale bar: 50 μm. c Representative photomicrographs of TH immunohistochemistry in striatum (Sham-Vehicle n = 7; Sham-MPTP n = 5; AAV-hTIM23-Vehicle n = 8; AAV-hTIM23-MPTP n = 8). Quantification is depicted as the optical densitometry of striatal TH immunoreactivity in the different experimental groups at day 21 post MPTP (**P < 0.01 after two-way ANOVA followed by Tukey’s post hoc test). Scale bar: 500 μm. d Representative photomicrographs of TH immunohistochemistry SNpc (Sham-Vehicle n = 14; Sham-MPTP n = 10; AAV-hTIM23-Vehicle n = 7; AAV-hTIM23-MPTP n = 8). Quantification is depicted as the stereological cell counts of SNpc TH-immunoreactive neurons in the different experimental groups at day 21 post MPTP (*P < 0.05, ***P < 0.001 after two-way ANOVA followed by Tukey’s post hoc test). Scale bar: 500 μm. Error bars indicate s.e.m.

Similar articles

Cited by

References

    1. Franco-Iborra S, Vila M, Perier C. The Parkinson disease mitochondrial hypothesis. Neurosci. 2016;22:266–277. - PubMed
    1. Schapira AH, et al. Mitochondrial complex I deficiency in Parkinson’s disease. J. Neurochem. 1990;54:823–827. doi: 10.1111/j.1471-4159.1990.tb02325.x. - DOI - PubMed
    1. Anderson S, et al. Sequence and organization of the human mitochondrial genome. Nature. 1981;290:457–465. doi: 10.1038/290457a0. - DOI - PubMed
    1. Wiedemann N, Pfanner N. Mitochondrial machineries for protein import and assembly. Annu. Rev. Biochem. 2017;86:685–714. doi: 10.1146/annurev-biochem-060815-014352. - DOI - PubMed
    1. Kiebler M, et al. Identification of a mitochondrial receptor complex required for recognition and membrane insertion of precursor proteins. Nature. 1990;348:610–616. doi: 10.1038/348610a0. - DOI - PubMed

Publication types

MeSH terms

Substances