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Mitochondrial alterations in PINK1 deficient cells are influenced by calcineurin-dependent dephosphorylation of dynamin-related protein 1.

Sandebring A, Thomas KJ, Beilina A, van der Brug M, Cleland MM, Ahmad R, Miller DW, Zambrano I, Cowburn RF, Behbahani H, Cedazo-Mínguez A, Cookson MR - PLoS ONE (2009)

Bottom Line: As in previous studies, PINK1 deficient cells have lower mitochondrial membrane potential and are more sensitive to the toxic effects of mitochondrial complex I inhibitors.Accordingly, the calcineurin inhibitor FK506 blocks both Drp1 dephosphorylation and loss of mitochondrial integrity in PINK1 deficient cells but does not fully rescue mitochondrial membrane potential.We propose that alterations in mitochondrial connectivity in this system are secondary to functional effects on mitochondrial membrane potential.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neurogenetics, National Institute on Aging, Bethesda, Maryland, United States of America.

ABSTRACT
PTEN-induced novel kinase 1 (PINK1) mutations are associated with autosomal recessive parkinsonism. Previous studies have shown that PINK1 influences both mitochondrial function and morphology although it is not clearly established which of these are primary events and which are secondary. Here, we describe a novel mechanism linking mitochondrial dysfunction and alterations in mitochondrial morphology related to PINK1. Cell lines were generated by stably transducing human dopaminergic M17 cells with lentiviral constructs that increased or knocked down PINK1. As in previous studies, PINK1 deficient cells have lower mitochondrial membrane potential and are more sensitive to the toxic effects of mitochondrial complex I inhibitors. We also show that wild-type PINK1, but not recessive mutant or kinase dead versions, protects against rotenone-induced mitochondrial fragmentation whereas PINK1 deficient cells show lower mitochondrial connectivity. Expression of dynamin-related protein 1 (Drp1) exaggerates PINK1 deficiency phenotypes and Drp1 RNAi rescues them. We also show that Drp1 is dephosphorylated in PINK1 deficient cells due to activation of the calcium-dependent phosphatase calcineurin. Accordingly, the calcineurin inhibitor FK506 blocks both Drp1 dephosphorylation and loss of mitochondrial integrity in PINK1 deficient cells but does not fully rescue mitochondrial membrane potential. We propose that alterations in mitochondrial connectivity in this system are secondary to functional effects on mitochondrial membrane potential.

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Calcineurin-mediated dephosphorylation of Drp1 contributes to mitochondrial phenotypes related to loss of PINK1.(A) Calcineurin enzyme activity was measured in postnuclear supernatants of extracts from control and PINK1 shRNA lines. Recombinant calcineurin (CaN) was used as a positive control. Activity is expressed as the release of Pi from a calcineurin substrate peptide, corrected for activity seen in the presence of EDTA to chelate calcium (***, P<0.001.) (B) Cell extracts from PINK1 shRNA lines, treated either with DMSO as a vehicle or 1 µM FK506 for 1 h and enrichment of Drp1 in the phosphorylated fraction was measured as in figure 6B. Quantification (n = 6) confirmed a lower relative amount of phospho-Drp1 in PINK1 shRNA cells compared to controls and a significant increase comparing PINK1 shRNA cells treated with DMSO to those treated with FK506 (*, P<0.05 by ANOVA). All other comparisons were not significant (P>0.05). (C) Mitochondrial morphology in control shRNA and PINK1 shRNA cells was assessed by mito-YFP expression. Cells were treated with vehicle only or 1 µM FK506 for 1 h or 3 h. Increasing length of FK506 treatment improves mitochondrial connectedness, especially in the PINK1 shRNA line. Scale bar is 2 µm and applies to all panels. (D) Mobile fraction values were estimated from FRAP curves either without treatment or after 1 h or 3 h treatment with 1 µM FK506. Each box is the average of 60 measurements from cells across duplicate experiments. *, P<0.05; **, P<0.01, ***, P<0.001 by one-way ANOVA with Student-Newman Kuells' post-hoc test. For clarity, non-significant differences (P>0.05) are not indicated. (E) Counts of mitochondrial morphology as in figure 2 were performed on n = 60 cells from duplicate experiments in control shRNA or PINK1 shRNA cell lines after treatment with FK506 for 1 or 3 hours. Differences were analyzed by two-way ANOVA using morphology and cell line/treatment as factors and P values for cell groups are given above each graph. (F) Mitochondrial membrane potential was estimated in PINK1 knockdown cells using TMRM staining and FACS analysis with and without 1 or 3 h treatment with 1 µM FK506. There is a significant (*, P<0.05; ***, P<0.001, n = 6 independent experiments) difference between the lines under basal conditions or after 1 h treatment by one-way ANOVA with Student-Newman Kuells' post-hoc test, although this is not significant (ns) at 3 h.
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pone-0005701-g008: Calcineurin-mediated dephosphorylation of Drp1 contributes to mitochondrial phenotypes related to loss of PINK1.(A) Calcineurin enzyme activity was measured in postnuclear supernatants of extracts from control and PINK1 shRNA lines. Recombinant calcineurin (CaN) was used as a positive control. Activity is expressed as the release of Pi from a calcineurin substrate peptide, corrected for activity seen in the presence of EDTA to chelate calcium (***, P<0.001.) (B) Cell extracts from PINK1 shRNA lines, treated either with DMSO as a vehicle or 1 µM FK506 for 1 h and enrichment of Drp1 in the phosphorylated fraction was measured as in figure 6B. Quantification (n = 6) confirmed a lower relative amount of phospho-Drp1 in PINK1 shRNA cells compared to controls and a significant increase comparing PINK1 shRNA cells treated with DMSO to those treated with FK506 (*, P<0.05 by ANOVA). All other comparisons were not significant (P>0.05). (C) Mitochondrial morphology in control shRNA and PINK1 shRNA cells was assessed by mito-YFP expression. Cells were treated with vehicle only or 1 µM FK506 for 1 h or 3 h. Increasing length of FK506 treatment improves mitochondrial connectedness, especially in the PINK1 shRNA line. Scale bar is 2 µm and applies to all panels. (D) Mobile fraction values were estimated from FRAP curves either without treatment or after 1 h or 3 h treatment with 1 µM FK506. Each box is the average of 60 measurements from cells across duplicate experiments. *, P<0.05; **, P<0.01, ***, P<0.001 by one-way ANOVA with Student-Newman Kuells' post-hoc test. For clarity, non-significant differences (P>0.05) are not indicated. (E) Counts of mitochondrial morphology as in figure 2 were performed on n = 60 cells from duplicate experiments in control shRNA or PINK1 shRNA cell lines after treatment with FK506 for 1 or 3 hours. Differences were analyzed by two-way ANOVA using morphology and cell line/treatment as factors and P values for cell groups are given above each graph. (F) Mitochondrial membrane potential was estimated in PINK1 knockdown cells using TMRM staining and FACS analysis with and without 1 or 3 h treatment with 1 µM FK506. There is a significant (*, P<0.05; ***, P<0.001, n = 6 independent experiments) difference between the lines under basal conditions or after 1 h treatment by one-way ANOVA with Student-Newman Kuells' post-hoc test, although this is not significant (ns) at 3 h.

Mentions: Calcineurin (CaN) increases Drp1 activity via its phosphatase activity [41]. Neither calcineurin catalytic α subunit, regulatory calcineurin-β subunit or calmodulin protein levels were altered between PINK1 deficient cells and controls (data not shown). However, cellular CaN activity was significantly increased in PINK1 deficient cells (Fig. 8A; P<0.001 by ANOVA, n = 5). To address whether CaN activity influences Drp1, we treated cells with 1 µM of the CaN inhibitor FK506 for 1 hour and separated phosphorylated and total proteins and immunoblotted for Drp1 as in figure 7B. Quantification of multiple experiments confirmed the decreased ratio of phosphorylated to total Drp1 in PINK1 deficient cells compared to control cells and showed that this measure of Drp1 phosphorylation was increased to a level similar to controls after FK506 treatment (P<0.05 by ANOVA, n = 6; Fig. 8B).


Mitochondrial alterations in PINK1 deficient cells are influenced by calcineurin-dependent dephosphorylation of dynamin-related protein 1.

Sandebring A, Thomas KJ, Beilina A, van der Brug M, Cleland MM, Ahmad R, Miller DW, Zambrano I, Cowburn RF, Behbahani H, Cedazo-Mínguez A, Cookson MR - PLoS ONE (2009)

Calcineurin-mediated dephosphorylation of Drp1 contributes to mitochondrial phenotypes related to loss of PINK1.(A) Calcineurin enzyme activity was measured in postnuclear supernatants of extracts from control and PINK1 shRNA lines. Recombinant calcineurin (CaN) was used as a positive control. Activity is expressed as the release of Pi from a calcineurin substrate peptide, corrected for activity seen in the presence of EDTA to chelate calcium (***, P<0.001.) (B) Cell extracts from PINK1 shRNA lines, treated either with DMSO as a vehicle or 1 µM FK506 for 1 h and enrichment of Drp1 in the phosphorylated fraction was measured as in figure 6B. Quantification (n = 6) confirmed a lower relative amount of phospho-Drp1 in PINK1 shRNA cells compared to controls and a significant increase comparing PINK1 shRNA cells treated with DMSO to those treated with FK506 (*, P<0.05 by ANOVA). All other comparisons were not significant (P>0.05). (C) Mitochondrial morphology in control shRNA and PINK1 shRNA cells was assessed by mito-YFP expression. Cells were treated with vehicle only or 1 µM FK506 for 1 h or 3 h. Increasing length of FK506 treatment improves mitochondrial connectedness, especially in the PINK1 shRNA line. Scale bar is 2 µm and applies to all panels. (D) Mobile fraction values were estimated from FRAP curves either without treatment or after 1 h or 3 h treatment with 1 µM FK506. Each box is the average of 60 measurements from cells across duplicate experiments. *, P<0.05; **, P<0.01, ***, P<0.001 by one-way ANOVA with Student-Newman Kuells' post-hoc test. For clarity, non-significant differences (P>0.05) are not indicated. (E) Counts of mitochondrial morphology as in figure 2 were performed on n = 60 cells from duplicate experiments in control shRNA or PINK1 shRNA cell lines after treatment with FK506 for 1 or 3 hours. Differences were analyzed by two-way ANOVA using morphology and cell line/treatment as factors and P values for cell groups are given above each graph. (F) Mitochondrial membrane potential was estimated in PINK1 knockdown cells using TMRM staining and FACS analysis with and without 1 or 3 h treatment with 1 µM FK506. There is a significant (*, P<0.05; ***, P<0.001, n = 6 independent experiments) difference between the lines under basal conditions or after 1 h treatment by one-way ANOVA with Student-Newman Kuells' post-hoc test, although this is not significant (ns) at 3 h.
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getmorefigures.php?uid=PMC2683574&req=5

pone-0005701-g008: Calcineurin-mediated dephosphorylation of Drp1 contributes to mitochondrial phenotypes related to loss of PINK1.(A) Calcineurin enzyme activity was measured in postnuclear supernatants of extracts from control and PINK1 shRNA lines. Recombinant calcineurin (CaN) was used as a positive control. Activity is expressed as the release of Pi from a calcineurin substrate peptide, corrected for activity seen in the presence of EDTA to chelate calcium (***, P<0.001.) (B) Cell extracts from PINK1 shRNA lines, treated either with DMSO as a vehicle or 1 µM FK506 for 1 h and enrichment of Drp1 in the phosphorylated fraction was measured as in figure 6B. Quantification (n = 6) confirmed a lower relative amount of phospho-Drp1 in PINK1 shRNA cells compared to controls and a significant increase comparing PINK1 shRNA cells treated with DMSO to those treated with FK506 (*, P<0.05 by ANOVA). All other comparisons were not significant (P>0.05). (C) Mitochondrial morphology in control shRNA and PINK1 shRNA cells was assessed by mito-YFP expression. Cells were treated with vehicle only or 1 µM FK506 for 1 h or 3 h. Increasing length of FK506 treatment improves mitochondrial connectedness, especially in the PINK1 shRNA line. Scale bar is 2 µm and applies to all panels. (D) Mobile fraction values were estimated from FRAP curves either without treatment or after 1 h or 3 h treatment with 1 µM FK506. Each box is the average of 60 measurements from cells across duplicate experiments. *, P<0.05; **, P<0.01, ***, P<0.001 by one-way ANOVA with Student-Newman Kuells' post-hoc test. For clarity, non-significant differences (P>0.05) are not indicated. (E) Counts of mitochondrial morphology as in figure 2 were performed on n = 60 cells from duplicate experiments in control shRNA or PINK1 shRNA cell lines after treatment with FK506 for 1 or 3 hours. Differences were analyzed by two-way ANOVA using morphology and cell line/treatment as factors and P values for cell groups are given above each graph. (F) Mitochondrial membrane potential was estimated in PINK1 knockdown cells using TMRM staining and FACS analysis with and without 1 or 3 h treatment with 1 µM FK506. There is a significant (*, P<0.05; ***, P<0.001, n = 6 independent experiments) difference between the lines under basal conditions or after 1 h treatment by one-way ANOVA with Student-Newman Kuells' post-hoc test, although this is not significant (ns) at 3 h.
Mentions: Calcineurin (CaN) increases Drp1 activity via its phosphatase activity [41]. Neither calcineurin catalytic α subunit, regulatory calcineurin-β subunit or calmodulin protein levels were altered between PINK1 deficient cells and controls (data not shown). However, cellular CaN activity was significantly increased in PINK1 deficient cells (Fig. 8A; P<0.001 by ANOVA, n = 5). To address whether CaN activity influences Drp1, we treated cells with 1 µM of the CaN inhibitor FK506 for 1 hour and separated phosphorylated and total proteins and immunoblotted for Drp1 as in figure 7B. Quantification of multiple experiments confirmed the decreased ratio of phosphorylated to total Drp1 in PINK1 deficient cells compared to control cells and showed that this measure of Drp1 phosphorylation was increased to a level similar to controls after FK506 treatment (P<0.05 by ANOVA, n = 6; Fig. 8B).

Bottom Line: As in previous studies, PINK1 deficient cells have lower mitochondrial membrane potential and are more sensitive to the toxic effects of mitochondrial complex I inhibitors.Accordingly, the calcineurin inhibitor FK506 blocks both Drp1 dephosphorylation and loss of mitochondrial integrity in PINK1 deficient cells but does not fully rescue mitochondrial membrane potential.We propose that alterations in mitochondrial connectivity in this system are secondary to functional effects on mitochondrial membrane potential.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neurogenetics, National Institute on Aging, Bethesda, Maryland, United States of America.

ABSTRACT
PTEN-induced novel kinase 1 (PINK1) mutations are associated with autosomal recessive parkinsonism. Previous studies have shown that PINK1 influences both mitochondrial function and morphology although it is not clearly established which of these are primary events and which are secondary. Here, we describe a novel mechanism linking mitochondrial dysfunction and alterations in mitochondrial morphology related to PINK1. Cell lines were generated by stably transducing human dopaminergic M17 cells with lentiviral constructs that increased or knocked down PINK1. As in previous studies, PINK1 deficient cells have lower mitochondrial membrane potential and are more sensitive to the toxic effects of mitochondrial complex I inhibitors. We also show that wild-type PINK1, but not recessive mutant or kinase dead versions, protects against rotenone-induced mitochondrial fragmentation whereas PINK1 deficient cells show lower mitochondrial connectivity. Expression of dynamin-related protein 1 (Drp1) exaggerates PINK1 deficiency phenotypes and Drp1 RNAi rescues them. We also show that Drp1 is dephosphorylated in PINK1 deficient cells due to activation of the calcium-dependent phosphatase calcineurin. Accordingly, the calcineurin inhibitor FK506 blocks both Drp1 dephosphorylation and loss of mitochondrial integrity in PINK1 deficient cells but does not fully rescue mitochondrial membrane potential. We propose that alterations in mitochondrial connectivity in this system are secondary to functional effects on mitochondrial membrane potential.

Show MeSH
Related in: MedlinePlus