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Regulation of mitochondrial permeability transition pore by PINK1.

Gautier CA, Giaime E, Caballero E, Núñez L, Song Z, Chan D, Villalobos C, Shen J - Mol Neurodegener (2012)

Bottom Line: Enzymatic activities of the electron transport system complexes are normal in PINK1-/- cells, but mitochondrial transmembrane potential is reduced.Following FCCP treatment, calcium increases in the cytosol are higher in PINK1-/- compared to wild-type cells, suggesting that intra-mitochondrial calcium concentration is higher in the absence of PINK1.Our findings show that loss of PINK1 causes selective increases in mPTP opening and mitochondrial calcium, and that the excessive mPTP opening may underlie the mitochondrial functional defects observed in PINK1-/- cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Neurologic Diseases, Department of Neurology, Brigham & Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT

Background: Loss-of-function mutations in PTEN-induced kinase 1 (PINK1) have been linked to familial Parkinson's disease, but the underlying pathogenic mechanism remains unclear. We previously reported that loss of PINK1 impairs mitochondrial respiratory activity in mouse brains.

Results: In this study, we investigate how loss of PINK1 impairs mitochondrial respiration using cultured primary fibroblasts and neurons. We found that intact mitochondria in PINK1-/- cells recapitulate the respiratory defect in isolated mitochondria from PINK1-/- mouse brains, suggesting that these PINK1-/- cells are a valid experimental system to study the underlying mechanisms. Enzymatic activities of the electron transport system complexes are normal in PINK1-/- cells, but mitochondrial transmembrane potential is reduced. Interestingly, the opening of the mitochondrial permeability transition pore (mPTP) is increased in PINK1-/- cells, and this genotypic difference between PINK1-/- and control cells is eliminated by agonists or inhibitors of the mPTP. Furthermore, inhibition of mPTP opening rescues the defects in transmembrane potential and respiration in PINK1-/- cells. Consistent with our earlier findings in mouse brains, mitochondrial morphology is similar between PINK1-/- and wild-type cells, indicating that the observed mitochondrial functional defects are not due to morphological changes. Following FCCP treatment, calcium increases in the cytosol are higher in PINK1-/- compared to wild-type cells, suggesting that intra-mitochondrial calcium concentration is higher in the absence of PINK1.

Conclusions: Our findings show that loss of PINK1 causes selective increases in mPTP opening and mitochondrial calcium, and that the excessive mPTP opening may underlie the mitochondrial functional defects observed in PINK1-/- cells.

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Normal levels of oxidative stress markers and normal production of reactive oxygen species inPINK1−/− MEFs. A. Left panel: OxyBlot analysis of mitochondrial fractions showing levels of protein carbonyls in both genotypes. Right panel: Quantification of levels of protein carbonyls in PINK1−/− and +/+ MEFs. B. Levels of lipid peroxidation measured by the TBARS assay in PINK1−/− and +/+ MEFs. C. Kinetics of H2O2 production in isolated mitochondria measured by following Amplex Red fluorescence over time. D. Kinetics of cytosolic superoxide anion O2- production measured in resuspended MEFs by following DHEt fluorescence over time. E. Representative flow cytometry dot plots showing calcein fluorescence in PINK1−/− and +/+ MEFs in the presence of Co2+ after antioxidant treatment with Tocopherol (50 μM, 4 hr) or NAC (1 mM, 2 hr). F. Quantification of calcein fluorescence in PINK1−/− and +/+ MEFs following treatment with Tocopherol (50 μM, 4 hr) or NAC (1 mM, 2 hr). All data are expressed as mean ± SEM. *p < 0.05.
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Figure 7: Normal levels of oxidative stress markers and normal production of reactive oxygen species inPINK1−/− MEFs. A. Left panel: OxyBlot analysis of mitochondrial fractions showing levels of protein carbonyls in both genotypes. Right panel: Quantification of levels of protein carbonyls in PINK1−/− and +/+ MEFs. B. Levels of lipid peroxidation measured by the TBARS assay in PINK1−/− and +/+ MEFs. C. Kinetics of H2O2 production in isolated mitochondria measured by following Amplex Red fluorescence over time. D. Kinetics of cytosolic superoxide anion O2- production measured in resuspended MEFs by following DHEt fluorescence over time. E. Representative flow cytometry dot plots showing calcein fluorescence in PINK1−/− and +/+ MEFs in the presence of Co2+ after antioxidant treatment with Tocopherol (50 μM, 4 hr) or NAC (1 mM, 2 hr). F. Quantification of calcein fluorescence in PINK1−/− and +/+ MEFs following treatment with Tocopherol (50 μM, 4 hr) or NAC (1 mM, 2 hr). All data are expressed as mean ± SEM. *p < 0.05.

Mentions: Because mPTP opening can be affected by elevated oxidative stress [29], we went further to examine whether there is an accumulation of oxidative species in the mitochondrial fraction of PINK1−/− and control MEFs. We measured the levels of protein carbonyls, a marker of protein oxidation. As measured by OxyBlot, the total level of carbonyls is similar between the two genotypic groups (Figure 7A). We then measured the accumulation of another common marker of oxidative stress, thiobarbituric acid reactive substances (TBARS), which reflects lipid peroxidation, and found no significant differences between the two genotypes (Figure 7B). We further evaluated the production of oxidative species. Using the Amplex Red dye fluorescence assay we evaluated the propensity of cells to generate Reactive Oxygen Species (ROS) by measuring the production of H2O2. Because H2O2 extrusion across the plasma membrane can be kinetically limiting we measured the rate of H2O2 produced by isolated mitochondria from MEFs. Mitochondria are the main source of ROS in the cells. We found that isolated mitochondria from PINK1−/− and WT cells energized with succinate (10 mM) produce H2O2 at similar rates (Figure 7C). We also monitored the production of superoxide anion O2.-. Superoxide is the primary oxidant species generated as a byproduct of mitochondrial respiration. Using the DHEt dye fluorescence assay, we found similar kinetics of O2.- generation between PINK1−/− and WT MEFs (Figure 7D). As positive controls we used MEFs derived from our DJ-1−/− mice. Using the same assay conditions, DJ-1 MEFs displayed higher rates of H2O2 and O2.- production as monitored with the Amplex Red and the DHEt assays (data not shown). Thus, loss of PINK1 does not increase the production of reactive oxygen species.


Regulation of mitochondrial permeability transition pore by PINK1.

Gautier CA, Giaime E, Caballero E, Núñez L, Song Z, Chan D, Villalobos C, Shen J - Mol Neurodegener (2012)

Normal levels of oxidative stress markers and normal production of reactive oxygen species inPINK1−/− MEFs. A. Left panel: OxyBlot analysis of mitochondrial fractions showing levels of protein carbonyls in both genotypes. Right panel: Quantification of levels of protein carbonyls in PINK1−/− and +/+ MEFs. B. Levels of lipid peroxidation measured by the TBARS assay in PINK1−/− and +/+ MEFs. C. Kinetics of H2O2 production in isolated mitochondria measured by following Amplex Red fluorescence over time. D. Kinetics of cytosolic superoxide anion O2- production measured in resuspended MEFs by following DHEt fluorescence over time. E. Representative flow cytometry dot plots showing calcein fluorescence in PINK1−/− and +/+ MEFs in the presence of Co2+ after antioxidant treatment with Tocopherol (50 μM, 4 hr) or NAC (1 mM, 2 hr). F. Quantification of calcein fluorescence in PINK1−/− and +/+ MEFs following treatment with Tocopherol (50 μM, 4 hr) or NAC (1 mM, 2 hr). All data are expressed as mean ± SEM. *p < 0.05.
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Related In: Results  -  Collection

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Figure 7: Normal levels of oxidative stress markers and normal production of reactive oxygen species inPINK1−/− MEFs. A. Left panel: OxyBlot analysis of mitochondrial fractions showing levels of protein carbonyls in both genotypes. Right panel: Quantification of levels of protein carbonyls in PINK1−/− and +/+ MEFs. B. Levels of lipid peroxidation measured by the TBARS assay in PINK1−/− and +/+ MEFs. C. Kinetics of H2O2 production in isolated mitochondria measured by following Amplex Red fluorescence over time. D. Kinetics of cytosolic superoxide anion O2- production measured in resuspended MEFs by following DHEt fluorescence over time. E. Representative flow cytometry dot plots showing calcein fluorescence in PINK1−/− and +/+ MEFs in the presence of Co2+ after antioxidant treatment with Tocopherol (50 μM, 4 hr) or NAC (1 mM, 2 hr). F. Quantification of calcein fluorescence in PINK1−/− and +/+ MEFs following treatment with Tocopherol (50 μM, 4 hr) or NAC (1 mM, 2 hr). All data are expressed as mean ± SEM. *p < 0.05.
Mentions: Because mPTP opening can be affected by elevated oxidative stress [29], we went further to examine whether there is an accumulation of oxidative species in the mitochondrial fraction of PINK1−/− and control MEFs. We measured the levels of protein carbonyls, a marker of protein oxidation. As measured by OxyBlot, the total level of carbonyls is similar between the two genotypic groups (Figure 7A). We then measured the accumulation of another common marker of oxidative stress, thiobarbituric acid reactive substances (TBARS), which reflects lipid peroxidation, and found no significant differences between the two genotypes (Figure 7B). We further evaluated the production of oxidative species. Using the Amplex Red dye fluorescence assay we evaluated the propensity of cells to generate Reactive Oxygen Species (ROS) by measuring the production of H2O2. Because H2O2 extrusion across the plasma membrane can be kinetically limiting we measured the rate of H2O2 produced by isolated mitochondria from MEFs. Mitochondria are the main source of ROS in the cells. We found that isolated mitochondria from PINK1−/− and WT cells energized with succinate (10 mM) produce H2O2 at similar rates (Figure 7C). We also monitored the production of superoxide anion O2.-. Superoxide is the primary oxidant species generated as a byproduct of mitochondrial respiration. Using the DHEt dye fluorescence assay, we found similar kinetics of O2.- generation between PINK1−/− and WT MEFs (Figure 7D). As positive controls we used MEFs derived from our DJ-1−/− mice. Using the same assay conditions, DJ-1 MEFs displayed higher rates of H2O2 and O2.- production as monitored with the Amplex Red and the DHEt assays (data not shown). Thus, loss of PINK1 does not increase the production of reactive oxygen species.

Bottom Line: Enzymatic activities of the electron transport system complexes are normal in PINK1-/- cells, but mitochondrial transmembrane potential is reduced.Following FCCP treatment, calcium increases in the cytosol are higher in PINK1-/- compared to wild-type cells, suggesting that intra-mitochondrial calcium concentration is higher in the absence of PINK1.Our findings show that loss of PINK1 causes selective increases in mPTP opening and mitochondrial calcium, and that the excessive mPTP opening may underlie the mitochondrial functional defects observed in PINK1-/- cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Neurologic Diseases, Department of Neurology, Brigham & Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT

Background: Loss-of-function mutations in PTEN-induced kinase 1 (PINK1) have been linked to familial Parkinson's disease, but the underlying pathogenic mechanism remains unclear. We previously reported that loss of PINK1 impairs mitochondrial respiratory activity in mouse brains.

Results: In this study, we investigate how loss of PINK1 impairs mitochondrial respiration using cultured primary fibroblasts and neurons. We found that intact mitochondria in PINK1-/- cells recapitulate the respiratory defect in isolated mitochondria from PINK1-/- mouse brains, suggesting that these PINK1-/- cells are a valid experimental system to study the underlying mechanisms. Enzymatic activities of the electron transport system complexes are normal in PINK1-/- cells, but mitochondrial transmembrane potential is reduced. Interestingly, the opening of the mitochondrial permeability transition pore (mPTP) is increased in PINK1-/- cells, and this genotypic difference between PINK1-/- and control cells is eliminated by agonists or inhibitors of the mPTP. Furthermore, inhibition of mPTP opening rescues the defects in transmembrane potential and respiration in PINK1-/- cells. Consistent with our earlier findings in mouse brains, mitochondrial morphology is similar between PINK1-/- and wild-type cells, indicating that the observed mitochondrial functional defects are not due to morphological changes. Following FCCP treatment, calcium increases in the cytosol are higher in PINK1-/- compared to wild-type cells, suggesting that intra-mitochondrial calcium concentration is higher in the absence of PINK1.

Conclusions: Our findings show that loss of PINK1 causes selective increases in mPTP opening and mitochondrial calcium, and that the excessive mPTP opening may underlie the mitochondrial functional defects observed in PINK1-/- cells.

Show MeSH
Related in: MedlinePlus