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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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PINK1 deficiency and increased Drp1 expression have additive effects.(A–D) Control shRNA (A,B) or PINK1 shRNA (C,D) were transfected with YFP-Drp1 (green in upper panels) and stained with mitotracker (red in upper panels; lower panels show an enlarged portion of the mitotracker channel only). At 24 hours after transfection, PINK1 shRNA lines (D) showed more mitochondrial damage than control shRNA lines. Arrowheads show small fragments of mitochondria that are particularly seen in PINK1 deficient cells, arrows show circular fragments that are only seen in PINK1 deficient cells. Scale bar in the lower panel of (D) is 10 µm and applies to all photomicrographs. (E) Control shRNA and PINK1 shRNA cells were transfected with YFP or YFP-Drp1. Total cell lysates or mitochondrial fractions were blotted for Drp1 or Hsp60 as a loading control. Markers on the right are in kilodaltons. (F) Control shRNA (upper panels) or PINK1 shRNA cells (lower panels) were transfected with mito-YFP without (left panels) or with Drp1 (right panels) and live images taken. As in A–D, arrowheads show small fragments and arrows circular remnants of mitochondria. Scale bar in the lower right panel is 2 µm, applies to all fluorescence micrographs. (G, H) FRAP was used to assess the effect of Drp1 overexpression as in figure 3. Open symbols are untransfected cells, closed symbols were transfected with Drp1 for 24 hours. The FRAP curves over time in (G) are the average of 30 observations, representative of duplicate experiments. Error bars indicate the SEM. (H) Summary data for the mobile fractions are shown from n = 30 cells, representative of duplicate experiments. Differences between treatments were significant overall (P<0.0001 by ANOVA) and Student-Newman Kuells' post-hoc test was used to compare each line with and without Drp1 expression. *P<0.05; ** P<0.01; ***P<0.001. (I) Counts of mitochondrial morphology as in figure 2 were performed on 60 cells from duplicate experiments in control shRNA or PINK1 shRNA cell lines. 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. (J) M17 or stable PINK1 WT cells were transfected with Drp1. FRAP curves (data not shown) were generated and mobile fractions were derived. (J) Differences between treatments were significant overall (P<0.0001 by ANOVA, n = 30 cells) and Student-Newman Kuells' post-hoc test was used to compare each line with and without Drp1 expression. *P<0.05; ** P<0.01; ***P<0.001. Data is representative of duplicate independent experiments. (K) Counts of mitochondrial morphology were performed on 60 cells from duplicate experiments in control or PINK1 overexpressing cell lines. 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.
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pone-0005701-g006: PINK1 deficiency and increased Drp1 expression have additive effects.(A–D) Control shRNA (A,B) or PINK1 shRNA (C,D) were transfected with YFP-Drp1 (green in upper panels) and stained with mitotracker (red in upper panels; lower panels show an enlarged portion of the mitotracker channel only). At 24 hours after transfection, PINK1 shRNA lines (D) showed more mitochondrial damage than control shRNA lines. Arrowheads show small fragments of mitochondria that are particularly seen in PINK1 deficient cells, arrows show circular fragments that are only seen in PINK1 deficient cells. Scale bar in the lower panel of (D) is 10 µm and applies to all photomicrographs. (E) Control shRNA and PINK1 shRNA cells were transfected with YFP or YFP-Drp1. Total cell lysates or mitochondrial fractions were blotted for Drp1 or Hsp60 as a loading control. Markers on the right are in kilodaltons. (F) Control shRNA (upper panels) or PINK1 shRNA cells (lower panels) were transfected with mito-YFP without (left panels) or with Drp1 (right panels) and live images taken. As in A–D, arrowheads show small fragments and arrows circular remnants of mitochondria. Scale bar in the lower right panel is 2 µm, applies to all fluorescence micrographs. (G, H) FRAP was used to assess the effect of Drp1 overexpression as in figure 3. Open symbols are untransfected cells, closed symbols were transfected with Drp1 for 24 hours. The FRAP curves over time in (G) are the average of 30 observations, representative of duplicate experiments. Error bars indicate the SEM. (H) Summary data for the mobile fractions are shown from n = 30 cells, representative of duplicate experiments. Differences between treatments were significant overall (P<0.0001 by ANOVA) and Student-Newman Kuells' post-hoc test was used to compare each line with and without Drp1 expression. *P<0.05; ** P<0.01; ***P<0.001. (I) Counts of mitochondrial morphology as in figure 2 were performed on 60 cells from duplicate experiments in control shRNA or PINK1 shRNA cell lines. 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. (J) M17 or stable PINK1 WT cells were transfected with Drp1. FRAP curves (data not shown) were generated and mobile fractions were derived. (J) Differences between treatments were significant overall (P<0.0001 by ANOVA, n = 30 cells) and Student-Newman Kuells' post-hoc test was used to compare each line with and without Drp1 expression. *P<0.05; ** P<0.01; ***P<0.001. Data is representative of duplicate independent experiments. (K) Counts of mitochondrial morphology were performed on 60 cells from duplicate experiments in control or PINK1 overexpressing cell lines. 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.

Mentions: Next, we addressed what would happen if levels of Drp1 were increased in PINK1 deficient cells. We transiently expressed YFP-Drp1 in the same cell lines and then imaged mitochondria to detect YFP, using non-transfected (YFP negative) cells in the same cultures as controls (Fig. 6A–D). In both control and PINK1 shRNA lines, YFP-Drp1 was recruited to foci on the surface of mitochondria as described previously [40]–[42]. This punctate staining pattern was detected with both N- and C- terminal YFP fusions of Drp1, but not with YFP alone (data not shown) and represent fission competent foci of active Drp1. Twenty-four hours after transfection, PINK1 deficient lines showed more disrupted mitochondrial networks compared to control lines or untransfected cells (compare Figs. 6B and 6D). The amount of YFP-Drp1 recruited into mitochondrial fractions was similar in control and PINK1 deficient lines by immunoblot (Fig. 6E). Furthermore, we also noted that increased expression of YFP-Drp1 resulted in an accumulation of both transfected and endogenous Drp1 protein in mitochondria, again presumably due to the formation of fission-competent Drp1 oligomers at the mitochondrial surface, and again this effect is similar in both control and PINK1 shRNA cell lines.


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)

PINK1 deficiency and increased Drp1 expression have additive effects.(A–D) Control shRNA (A,B) or PINK1 shRNA (C,D) were transfected with YFP-Drp1 (green in upper panels) and stained with mitotracker (red in upper panels; lower panels show an enlarged portion of the mitotracker channel only). At 24 hours after transfection, PINK1 shRNA lines (D) showed more mitochondrial damage than control shRNA lines. Arrowheads show small fragments of mitochondria that are particularly seen in PINK1 deficient cells, arrows show circular fragments that are only seen in PINK1 deficient cells. Scale bar in the lower panel of (D) is 10 µm and applies to all photomicrographs. (E) Control shRNA and PINK1 shRNA cells were transfected with YFP or YFP-Drp1. Total cell lysates or mitochondrial fractions were blotted for Drp1 or Hsp60 as a loading control. Markers on the right are in kilodaltons. (F) Control shRNA (upper panels) or PINK1 shRNA cells (lower panels) were transfected with mito-YFP without (left panels) or with Drp1 (right panels) and live images taken. As in A–D, arrowheads show small fragments and arrows circular remnants of mitochondria. Scale bar in the lower right panel is 2 µm, applies to all fluorescence micrographs. (G, H) FRAP was used to assess the effect of Drp1 overexpression as in figure 3. Open symbols are untransfected cells, closed symbols were transfected with Drp1 for 24 hours. The FRAP curves over time in (G) are the average of 30 observations, representative of duplicate experiments. Error bars indicate the SEM. (H) Summary data for the mobile fractions are shown from n = 30 cells, representative of duplicate experiments. Differences between treatments were significant overall (P<0.0001 by ANOVA) and Student-Newman Kuells' post-hoc test was used to compare each line with and without Drp1 expression. *P<0.05; ** P<0.01; ***P<0.001. (I) Counts of mitochondrial morphology as in figure 2 were performed on 60 cells from duplicate experiments in control shRNA or PINK1 shRNA cell lines. 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. (J) M17 or stable PINK1 WT cells were transfected with Drp1. FRAP curves (data not shown) were generated and mobile fractions were derived. (J) Differences between treatments were significant overall (P<0.0001 by ANOVA, n = 30 cells) and Student-Newman Kuells' post-hoc test was used to compare each line with and without Drp1 expression. *P<0.05; ** P<0.01; ***P<0.001. Data is representative of duplicate independent experiments. (K) Counts of mitochondrial morphology were performed on 60 cells from duplicate experiments in control or PINK1 overexpressing cell lines. 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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2683574&req=5

pone-0005701-g006: PINK1 deficiency and increased Drp1 expression have additive effects.(A–D) Control shRNA (A,B) or PINK1 shRNA (C,D) were transfected with YFP-Drp1 (green in upper panels) and stained with mitotracker (red in upper panels; lower panels show an enlarged portion of the mitotracker channel only). At 24 hours after transfection, PINK1 shRNA lines (D) showed more mitochondrial damage than control shRNA lines. Arrowheads show small fragments of mitochondria that are particularly seen in PINK1 deficient cells, arrows show circular fragments that are only seen in PINK1 deficient cells. Scale bar in the lower panel of (D) is 10 µm and applies to all photomicrographs. (E) Control shRNA and PINK1 shRNA cells were transfected with YFP or YFP-Drp1. Total cell lysates or mitochondrial fractions were blotted for Drp1 or Hsp60 as a loading control. Markers on the right are in kilodaltons. (F) Control shRNA (upper panels) or PINK1 shRNA cells (lower panels) were transfected with mito-YFP without (left panels) or with Drp1 (right panels) and live images taken. As in A–D, arrowheads show small fragments and arrows circular remnants of mitochondria. Scale bar in the lower right panel is 2 µm, applies to all fluorescence micrographs. (G, H) FRAP was used to assess the effect of Drp1 overexpression as in figure 3. Open symbols are untransfected cells, closed symbols were transfected with Drp1 for 24 hours. The FRAP curves over time in (G) are the average of 30 observations, representative of duplicate experiments. Error bars indicate the SEM. (H) Summary data for the mobile fractions are shown from n = 30 cells, representative of duplicate experiments. Differences between treatments were significant overall (P<0.0001 by ANOVA) and Student-Newman Kuells' post-hoc test was used to compare each line with and without Drp1 expression. *P<0.05; ** P<0.01; ***P<0.001. (I) Counts of mitochondrial morphology as in figure 2 were performed on 60 cells from duplicate experiments in control shRNA or PINK1 shRNA cell lines. 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. (J) M17 or stable PINK1 WT cells were transfected with Drp1. FRAP curves (data not shown) were generated and mobile fractions were derived. (J) Differences between treatments were significant overall (P<0.0001 by ANOVA, n = 30 cells) and Student-Newman Kuells' post-hoc test was used to compare each line with and without Drp1 expression. *P<0.05; ** P<0.01; ***P<0.001. Data is representative of duplicate independent experiments. (K) Counts of mitochondrial morphology were performed on 60 cells from duplicate experiments in control or PINK1 overexpressing cell lines. 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.
Mentions: Next, we addressed what would happen if levels of Drp1 were increased in PINK1 deficient cells. We transiently expressed YFP-Drp1 in the same cell lines and then imaged mitochondria to detect YFP, using non-transfected (YFP negative) cells in the same cultures as controls (Fig. 6A–D). In both control and PINK1 shRNA lines, YFP-Drp1 was recruited to foci on the surface of mitochondria as described previously [40]–[42]. This punctate staining pattern was detected with both N- and C- terminal YFP fusions of Drp1, but not with YFP alone (data not shown) and represent fission competent foci of active Drp1. Twenty-four hours after transfection, PINK1 deficient lines showed more disrupted mitochondrial networks compared to control lines or untransfected cells (compare Figs. 6B and 6D). The amount of YFP-Drp1 recruited into mitochondrial fractions was similar in control and PINK1 deficient lines by immunoblot (Fig. 6E). Furthermore, we also noted that increased expression of YFP-Drp1 resulted in an accumulation of both transfected and endogenous Drp1 protein in mitochondria, again presumably due to the formation of fission-competent Drp1 oligomers at the mitochondrial surface, and again this effect is similar in both control and PINK1 shRNA cell lines.

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