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Regulation of mitochondrial permeability transition pore by PINK1.

Gautier CA, Giaime E, Caballero E, NΓΊΓ±ez 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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Reduced mitochondrial transmembrane potential inPINK1βˆ’/βˆ’ cells. A. Representative confocal microscopic images of PINK1βˆ’/βˆ’ and +/+ MEFs after staining with TMRM (50 nM, red) and Mitotracker Green (200 nM) in the presence or absence of oligomycin (Olig, 1 μM) or FCCP (10 μM). The intensity of TMRM reflects the level of mitochondrial transmembrane potential, whereas the intensity of Mitotracker Green is not affected by transmembrane potential. The bottom right inserts are the higher power views of the boxed areas in the same panel. The TMRM signal is reduced in PINK1βˆ’/βˆ’ cells, whereas the TMRM signal is increased and decreased similarly in both PINK1βˆ’/βˆ’ and +/+ cells following oligomycin and FCCP treatment, respectively. B. The bar graph shows quantification of TMRM signals in PINK1βˆ’/βˆ’ and +/+ MEFs in the presence or absence of oligomycin and FCCP using confocal images. The number shown in the panel indicates the number of cells used in the study. C. Representative flow cytometry dot plots show the intensity of TMRM signals in PINK1βˆ’/βˆ’ and +/+ MEFs following incubation with TMRM (50 nM) in the presence or absence of oligomycin (1 μM) or FCCP (10 μM). D. The bar graph shows quantification of TMRM signals measured by FACS analysis in PINK1βˆ’/βˆ’ and +/+ MEFs. The number shown in the panel indicates the number of independent experiments performed. E. The bar graph of TMRM fluorescent signals in PINK1βˆ’/βˆ’ and +/+ neurons shows reduced TMRM signals in PINK1βˆ’/βˆ’ neurons. The numbers shown indicate the number of neurons used (left) and the number of independent experiments performed (right) in the study. All data are expressed as mean ± SEM. Scale bar: 10 μm. * p < 0.05, ** p < 0.01, *** p < 0.001.
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Figure 2: Reduced mitochondrial transmembrane potential inPINK1βˆ’/βˆ’ cells. A. Representative confocal microscopic images of PINK1βˆ’/βˆ’ and +/+ MEFs after staining with TMRM (50 nM, red) and Mitotracker Green (200 nM) in the presence or absence of oligomycin (Olig, 1 μM) or FCCP (10 μM). The intensity of TMRM reflects the level of mitochondrial transmembrane potential, whereas the intensity of Mitotracker Green is not affected by transmembrane potential. The bottom right inserts are the higher power views of the boxed areas in the same panel. The TMRM signal is reduced in PINK1βˆ’/βˆ’ cells, whereas the TMRM signal is increased and decreased similarly in both PINK1βˆ’/βˆ’ and +/+ cells following oligomycin and FCCP treatment, respectively. B. The bar graph shows quantification of TMRM signals in PINK1βˆ’/βˆ’ and +/+ MEFs in the presence or absence of oligomycin and FCCP using confocal images. The number shown in the panel indicates the number of cells used in the study. C. Representative flow cytometry dot plots show the intensity of TMRM signals in PINK1βˆ’/βˆ’ and +/+ MEFs following incubation with TMRM (50 nM) in the presence or absence of oligomycin (1 μM) or FCCP (10 μM). D. The bar graph shows quantification of TMRM signals measured by FACS analysis in PINK1βˆ’/βˆ’ and +/+ MEFs. The number shown in the panel indicates the number of independent experiments performed. E. The bar graph of TMRM fluorescent signals in PINK1βˆ’/βˆ’ and +/+ neurons shows reduced TMRM signals in PINK1βˆ’/βˆ’ neurons. The numbers shown indicate the number of neurons used (left) and the number of independent experiments performed (right) in the study. All data are expressed as mean ± SEM. Scale bar: 10 μm. * p < 0.05, ** p < 0.01, *** p < 0.001.

Mentions: In the absence of enzymatic defects of the ETS complexes, we turned our attention towards mitochondrial transmembrane potential (ΔΨm), the electrochemical force that modulates the kinetics of proton reentry to the matrix through ATP-synthase. Using microscopic and flow cytometric analyses, we measured the transmembrane potential of MEFs stained with TMRM (50 nM). TMRM is a cationic fluorescent dye that accumulates inside the mitochondrial matrix according to the membrane potential. Interestingly, TMRM fluorescence signal is reduced in both experiments in PINK1βˆ’/βˆ’ MEFs (Figure 2A-D). To ensure that dye is equally loaded and that the TMRM signal is not auto-quenched we compared TMRM fluorescence in PINK1βˆ’/βˆ’ and control MEFs following oligomycin and FCCP treatment. Oligomycin, an inhibitor of ATP synthase, induces hyperpolarization of mitochondria and increases of TMRM fluorescence, whereas FCCP dissipates transmembrane potential. No differences in TMRM fluorescence between the two genotypes were found following either of these treatments.


Regulation of mitochondrial permeability transition pore by PINK1.

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

Reduced mitochondrial transmembrane potential inPINK1βˆ’/βˆ’ cells. A. Representative confocal microscopic images of PINK1βˆ’/βˆ’ and +/+ MEFs after staining with TMRM (50 nM, red) and Mitotracker Green (200 nM) in the presence or absence of oligomycin (Olig, 1 μM) or FCCP (10 μM). The intensity of TMRM reflects the level of mitochondrial transmembrane potential, whereas the intensity of Mitotracker Green is not affected by transmembrane potential. The bottom right inserts are the higher power views of the boxed areas in the same panel. The TMRM signal is reduced in PINK1βˆ’/βˆ’ cells, whereas the TMRM signal is increased and decreased similarly in both PINK1βˆ’/βˆ’ and +/+ cells following oligomycin and FCCP treatment, respectively. B. The bar graph shows quantification of TMRM signals in PINK1βˆ’/βˆ’ and +/+ MEFs in the presence or absence of oligomycin and FCCP using confocal images. The number shown in the panel indicates the number of cells used in the study. C. Representative flow cytometry dot plots show the intensity of TMRM signals in PINK1βˆ’/βˆ’ and +/+ MEFs following incubation with TMRM (50 nM) in the presence or absence of oligomycin (1 μM) or FCCP (10 μM). D. The bar graph shows quantification of TMRM signals measured by FACS analysis in PINK1βˆ’/βˆ’ and +/+ MEFs. The number shown in the panel indicates the number of independent experiments performed. E. The bar graph of TMRM fluorescent signals in PINK1βˆ’/βˆ’ and +/+ neurons shows reduced TMRM signals in PINK1βˆ’/βˆ’ neurons. The numbers shown indicate the number of neurons used (left) and the number of independent experiments performed (right) in the study. All data are expressed as mean ± SEM. Scale bar: 10 μm. * p < 0.05, ** p < 0.01, *** p < 0.001.
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Figure 2: Reduced mitochondrial transmembrane potential inPINK1βˆ’/βˆ’ cells. A. Representative confocal microscopic images of PINK1βˆ’/βˆ’ and +/+ MEFs after staining with TMRM (50 nM, red) and Mitotracker Green (200 nM) in the presence or absence of oligomycin (Olig, 1 μM) or FCCP (10 μM). The intensity of TMRM reflects the level of mitochondrial transmembrane potential, whereas the intensity of Mitotracker Green is not affected by transmembrane potential. The bottom right inserts are the higher power views of the boxed areas in the same panel. The TMRM signal is reduced in PINK1βˆ’/βˆ’ cells, whereas the TMRM signal is increased and decreased similarly in both PINK1βˆ’/βˆ’ and +/+ cells following oligomycin and FCCP treatment, respectively. B. The bar graph shows quantification of TMRM signals in PINK1βˆ’/βˆ’ and +/+ MEFs in the presence or absence of oligomycin and FCCP using confocal images. The number shown in the panel indicates the number of cells used in the study. C. Representative flow cytometry dot plots show the intensity of TMRM signals in PINK1βˆ’/βˆ’ and +/+ MEFs following incubation with TMRM (50 nM) in the presence or absence of oligomycin (1 μM) or FCCP (10 μM). D. The bar graph shows quantification of TMRM signals measured by FACS analysis in PINK1βˆ’/βˆ’ and +/+ MEFs. The number shown in the panel indicates the number of independent experiments performed. E. The bar graph of TMRM fluorescent signals in PINK1βˆ’/βˆ’ and +/+ neurons shows reduced TMRM signals in PINK1βˆ’/βˆ’ neurons. The numbers shown indicate the number of neurons used (left) and the number of independent experiments performed (right) in the study. All data are expressed as mean ± SEM. Scale bar: 10 μm. * p < 0.05, ** p < 0.01, *** p < 0.001.
Mentions: In the absence of enzymatic defects of the ETS complexes, we turned our attention towards mitochondrial transmembrane potential (ΔΨm), the electrochemical force that modulates the kinetics of proton reentry to the matrix through ATP-synthase. Using microscopic and flow cytometric analyses, we measured the transmembrane potential of MEFs stained with TMRM (50 nM). TMRM is a cationic fluorescent dye that accumulates inside the mitochondrial matrix according to the membrane potential. Interestingly, TMRM fluorescence signal is reduced in both experiments in PINK1βˆ’/βˆ’ MEFs (Figure 2A-D). To ensure that dye is equally loaded and that the TMRM signal is not auto-quenched we compared TMRM fluorescence in PINK1βˆ’/βˆ’ and control MEFs following oligomycin and FCCP treatment. Oligomycin, an inhibitor of ATP synthase, induces hyperpolarization of mitochondria and increases of TMRM fluorescence, whereas FCCP dissipates transmembrane potential. No differences in TMRM fluorescence between the two genotypes were found following either of these treatments.

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