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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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Increased opening of the mitochondrial permeability transition pore inPINK1−/− cells. A. Representative confocal microscopic images of PINK1−/− and +/+ MEFs after incubation with calcein-AM (1 μM) and Mitotracker Deep Red (150 nM) in the presence or absence of Co2+ (1 mM), which quenches calcein fluorescence (green) outside of mitochondria. Mitotracker Deep Red allows visualization of calcein fluorescence in mitochondria. The bottom right inserts are the higher power views of the boxed areas in the same panel. The calcein fluorescence in mitochondria is lower in PINK1−/− cells in the presence of Co2+. In the absence of Co2+, calcein fluorescent signals are very intense and are present in the entire cell, and there are no genotypic differences. B. The bar graph shows quantification of calcein fluorescence in PINK1−/− and +/+ cells in the presence or absence of Co2+ 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 calcein signals in PINK1−/− and +/+ MEFs following incubation with calcein-AM (1 μM) in the presence or absence of Co2+ (1 mM). D. The bar graph of calcein signals measured by FACS analysis shows reduced calcein signals in PINK1−/− MEFs in the presence of Co2+. The number shown in the panel indicates the number of independent experiments performed. E. The bar graph of calcein fluorescent signals in PINK1−/− and +/+ neurons shows reduced calcein signals in PINK1−/− 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.
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Figure 3: Increased opening of the mitochondrial permeability transition pore inPINK1−/− cells. A. Representative confocal microscopic images of PINK1−/− and +/+ MEFs after incubation with calcein-AM (1 μM) and Mitotracker Deep Red (150 nM) in the presence or absence of Co2+ (1 mM), which quenches calcein fluorescence (green) outside of mitochondria. Mitotracker Deep Red allows visualization of calcein fluorescence in mitochondria. The bottom right inserts are the higher power views of the boxed areas in the same panel. The calcein fluorescence in mitochondria is lower in PINK1−/− cells in the presence of Co2+. In the absence of Co2+, calcein fluorescent signals are very intense and are present in the entire cell, and there are no genotypic differences. B. The bar graph shows quantification of calcein fluorescence in PINK1−/− and +/+ cells in the presence or absence of Co2+ 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 calcein signals in PINK1−/− and +/+ MEFs following incubation with calcein-AM (1 μM) in the presence or absence of Co2+ (1 mM). D. The bar graph of calcein signals measured by FACS analysis shows reduced calcein signals in PINK1−/− MEFs in the presence of Co2+. The number shown in the panel indicates the number of independent experiments performed. E. The bar graph of calcein fluorescent signals in PINK1−/− and +/+ neurons shows reduced calcein signals in PINK1−/− 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.

Mentions: Under basal conditions, calcein fluorescence measured by both microscopic and flow cytometric analyses is lower in PINK1−/− MEFs, suggesting increases in mPTP opening (Figure 3A-D). We also measured calcein fluorescence in the absence of cobalt. As expected, calcein fluorescence is much higher in the absence of Co2+ and is similar between PINK1−/− and control cells, indicating similar calcein loading (Figure 3A-D). We extended the analysis to PINK1−/− neurons to confirm if mPTP opening is also increased. Because flow cytometric analysis requires re-suspension of cultured cells and thus would damage mature neurons, we used only microscopic analysis [33]. We found marked reduction of calcein fluorescence in PINK1−/− cortical neurons, further confirming increases in mPTP opening in the absence of PINK1 (Figure 3E).


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)

Increased opening of the mitochondrial permeability transition pore inPINK1−/− cells. A. Representative confocal microscopic images of PINK1−/− and +/+ MEFs after incubation with calcein-AM (1 μM) and Mitotracker Deep Red (150 nM) in the presence or absence of Co2+ (1 mM), which quenches calcein fluorescence (green) outside of mitochondria. Mitotracker Deep Red allows visualization of calcein fluorescence in mitochondria. The bottom right inserts are the higher power views of the boxed areas in the same panel. The calcein fluorescence in mitochondria is lower in PINK1−/− cells in the presence of Co2+. In the absence of Co2+, calcein fluorescent signals are very intense and are present in the entire cell, and there are no genotypic differences. B. The bar graph shows quantification of calcein fluorescence in PINK1−/− and +/+ cells in the presence or absence of Co2+ 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 calcein signals in PINK1−/− and +/+ MEFs following incubation with calcein-AM (1 μM) in the presence or absence of Co2+ (1 mM). D. The bar graph of calcein signals measured by FACS analysis shows reduced calcein signals in PINK1−/− MEFs in the presence of Co2+. The number shown in the panel indicates the number of independent experiments performed. E. The bar graph of calcein fluorescent signals in PINK1−/− and +/+ neurons shows reduced calcein signals in PINK1−/− 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.
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Show All Figures
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Figure 3: Increased opening of the mitochondrial permeability transition pore inPINK1−/− cells. A. Representative confocal microscopic images of PINK1−/− and +/+ MEFs after incubation with calcein-AM (1 μM) and Mitotracker Deep Red (150 nM) in the presence or absence of Co2+ (1 mM), which quenches calcein fluorescence (green) outside of mitochondria. Mitotracker Deep Red allows visualization of calcein fluorescence in mitochondria. The bottom right inserts are the higher power views of the boxed areas in the same panel. The calcein fluorescence in mitochondria is lower in PINK1−/− cells in the presence of Co2+. In the absence of Co2+, calcein fluorescent signals are very intense and are present in the entire cell, and there are no genotypic differences. B. The bar graph shows quantification of calcein fluorescence in PINK1−/− and +/+ cells in the presence or absence of Co2+ 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 calcein signals in PINK1−/− and +/+ MEFs following incubation with calcein-AM (1 μM) in the presence or absence of Co2+ (1 mM). D. The bar graph of calcein signals measured by FACS analysis shows reduced calcein signals in PINK1−/− MEFs in the presence of Co2+. The number shown in the panel indicates the number of independent experiments performed. E. The bar graph of calcein fluorescent signals in PINK1−/− and +/+ neurons shows reduced calcein signals in PINK1−/− 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.
Mentions: Under basal conditions, calcein fluorescence measured by both microscopic and flow cytometric analyses is lower in PINK1−/− MEFs, suggesting increases in mPTP opening (Figure 3A-D). We also measured calcein fluorescence in the absence of cobalt. As expected, calcein fluorescence is much higher in the absence of Co2+ and is similar between PINK1−/− and control cells, indicating similar calcein loading (Figure 3A-D). We extended the analysis to PINK1−/− neurons to confirm if mPTP opening is also increased. Because flow cytometric analysis requires re-suspension of cultured cells and thus would damage mature neurons, we used only microscopic analysis [33]. We found marked reduction of calcein fluorescence in PINK1−/− cortical neurons, further confirming increases in mPTP opening in the absence of PINK1 (Figure 3E).

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