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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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Blockade of mPTP opening by CsA attenuates the respiratory defect inPINK1−/− MEFs. A. Representative oxygraphs of PINK1−/− and +/+ MEFs energized with glucose (10 mM) in the presence of CsA (1 μM). The arrows indicate the time MEFs are added to the chamber. B. Oxygen consumption, which represents the endogenous respiratory activity in PINK1−/− and +/+ MEFs after treatment with CsA (1 μM). C. Representative oxygraphs of PINK1−/− and +/+ MEFs energized with 10 mM glutamate/malate (complex I substrate), 10 mM succinate (complex II substrate) or 1 mM TMPD/1 mM ascorbate (complex IV substrate) in the presence of CsA (1 μM). Arrows indicate the time of the addition of either the substrate or oligomycin (2 μM). D. Graph showing State 3 respiratory activity for complex I, complex II and complex IV in PINK1−/− and +/+ MEFs permeabilized with digitonin after treatment with CsA (1 μM). E. Representative oxygraphs of PINK1−/− and +/+ MEFs energized with 10 mM glutamate/malate (complex I substrate), 10 mM succinate (complex II substrate) or 1 mM TMPD/1 mM ascorbate (complex IV substrate) after treatment with FK-506 (5 μM). Arrows indicate the time of the addition of either the substrate or oligomycin (2 μM). F. Graph showing State 3 respiratory activity for complex I, complex II and complex IV in PINK1−/− and +/+ MEFs permeabilized with digitonin after treatment with FK-506 (5 μM). All data are expressed as mean ± SEM. * p < 0.05.
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Figure 6: Blockade of mPTP opening by CsA attenuates the respiratory defect inPINK1−/− MEFs. A. Representative oxygraphs of PINK1−/− and +/+ MEFs energized with glucose (10 mM) in the presence of CsA (1 μM). The arrows indicate the time MEFs are added to the chamber. B. Oxygen consumption, which represents the endogenous respiratory activity in PINK1−/− and +/+ MEFs after treatment with CsA (1 μM). C. Representative oxygraphs of PINK1−/− and +/+ MEFs energized with 10 mM glutamate/malate (complex I substrate), 10 mM succinate (complex II substrate) or 1 mM TMPD/1 mM ascorbate (complex IV substrate) in the presence of CsA (1 μM). Arrows indicate the time of the addition of either the substrate or oligomycin (2 μM). D. Graph showing State 3 respiratory activity for complex I, complex II and complex IV in PINK1−/− and +/+ MEFs permeabilized with digitonin after treatment with CsA (1 μM). E. Representative oxygraphs of PINK1−/− and +/+ MEFs energized with 10 mM glutamate/malate (complex I substrate), 10 mM succinate (complex II substrate) or 1 mM TMPD/1 mM ascorbate (complex IV substrate) after treatment with FK-506 (5 μM). Arrows indicate the time of the addition of either the substrate or oligomycin (2 μM). F. Graph showing State 3 respiratory activity for complex I, complex II and complex IV in PINK1−/− and +/+ MEFs permeabilized with digitonin after treatment with FK-506 (5 μM). All data are expressed as mean ± SEM. * p < 0.05.

Mentions: We further tested whether inhibition of mPTP opening reverses the mitochondrial respiration impairment in PINK1−/− cells. Treatment with CsA reduced the genotypic difference between PINK1−/− and control cells to the extent that endogenous respiratory activity is similar (Figure 6A and 6B). Moreover, CsA treatment almost fully rescued complex I driven respiration in PINK1−/− cells (Figure 6C and 6D). Treatment with FK-506 was not able to rescue the respiratory impairment in PINK1−/− cells, indicating that the effect of CsA on respiration was specific for its inhibitory effect on mPTP (Figure 6E and 6F).


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)

Blockade of mPTP opening by CsA attenuates the respiratory defect inPINK1−/− MEFs. A. Representative oxygraphs of PINK1−/− and +/+ MEFs energized with glucose (10 mM) in the presence of CsA (1 μM). The arrows indicate the time MEFs are added to the chamber. B. Oxygen consumption, which represents the endogenous respiratory activity in PINK1−/− and +/+ MEFs after treatment with CsA (1 μM). C. Representative oxygraphs of PINK1−/− and +/+ MEFs energized with 10 mM glutamate/malate (complex I substrate), 10 mM succinate (complex II substrate) or 1 mM TMPD/1 mM ascorbate (complex IV substrate) in the presence of CsA (1 μM). Arrows indicate the time of the addition of either the substrate or oligomycin (2 μM). D. Graph showing State 3 respiratory activity for complex I, complex II and complex IV in PINK1−/− and +/+ MEFs permeabilized with digitonin after treatment with CsA (1 μM). E. Representative oxygraphs of PINK1−/− and +/+ MEFs energized with 10 mM glutamate/malate (complex I substrate), 10 mM succinate (complex II substrate) or 1 mM TMPD/1 mM ascorbate (complex IV substrate) after treatment with FK-506 (5 μM). Arrows indicate the time of the addition of either the substrate or oligomycin (2 μM). F. Graph showing State 3 respiratory activity for complex I, complex II and complex IV in PINK1−/− and +/+ MEFs permeabilized with digitonin after treatment with FK-506 (5 μM). All data are expressed as mean ± SEM. * p < 0.05.
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Figure 6: Blockade of mPTP opening by CsA attenuates the respiratory defect inPINK1−/− MEFs. A. Representative oxygraphs of PINK1−/− and +/+ MEFs energized with glucose (10 mM) in the presence of CsA (1 μM). The arrows indicate the time MEFs are added to the chamber. B. Oxygen consumption, which represents the endogenous respiratory activity in PINK1−/− and +/+ MEFs after treatment with CsA (1 μM). C. Representative oxygraphs of PINK1−/− and +/+ MEFs energized with 10 mM glutamate/malate (complex I substrate), 10 mM succinate (complex II substrate) or 1 mM TMPD/1 mM ascorbate (complex IV substrate) in the presence of CsA (1 μM). Arrows indicate the time of the addition of either the substrate or oligomycin (2 μM). D. Graph showing State 3 respiratory activity for complex I, complex II and complex IV in PINK1−/− and +/+ MEFs permeabilized with digitonin after treatment with CsA (1 μM). E. Representative oxygraphs of PINK1−/− and +/+ MEFs energized with 10 mM glutamate/malate (complex I substrate), 10 mM succinate (complex II substrate) or 1 mM TMPD/1 mM ascorbate (complex IV substrate) after treatment with FK-506 (5 μM). Arrows indicate the time of the addition of either the substrate or oligomycin (2 μM). F. Graph showing State 3 respiratory activity for complex I, complex II and complex IV in PINK1−/− and +/+ MEFs permeabilized with digitonin after treatment with FK-506 (5 μM). All data are expressed as mean ± SEM. * p < 0.05.
Mentions: We further tested whether inhibition of mPTP opening reverses the mitochondrial respiration impairment in PINK1−/− cells. Treatment with CsA reduced the genotypic difference between PINK1−/− and control cells to the extent that endogenous respiratory activity is similar (Figure 6A and 6B). Moreover, CsA treatment almost fully rescued complex I driven respiration in PINK1−/− cells (Figure 6C and 6D). Treatment with FK-506 was not able to rescue the respiratory impairment in PINK1−/− cells, indicating that the effect of CsA on respiration was specific for its inhibitory effect on mPTP (Figure 6E and 6F).

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