Limits...
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.

Show MeSH

Related in: MedlinePlus

Mitochondrial effects due to loss of PINK1 can be rescued by knockdown of Drp1.(A) Exposure of control shRNA or PINK1 shRNA stable cell lines to Drp1 siRNA for four days results in an approximately 70% knockdown of Drp1 (arrow) compared to a non-specific GFP siRNA. β-actin shows equal loading. Molecular weight markers are in kilodaltons. (B) Live cell images of mito-YFP transfected control shRNA (upper panels) and PINK1 shRNA (lower panel) cells exposed to a non-specific siRNA against GFP (left panels) or an siRNA against Drp1 (right panels). Scale bar is 2 µm, applies to all fluorescence micrographs. (C) FRAP curves show that under basal situations, there was a difference between the control shRNA line and the PINK1 shRNA line. These differences were partially normalized after the cells were exposed to an siRNA against Drp1. Each point is the average of >30 separate measurements and error bars indicate the SEM. (D) Mobile fraction of mito-YFP was measured in control shRNA or PINK1 shRNA cell lines either after expression of a control siRNA (against GFP) or after expression of an siRNA against Drp1. Boxplots summarize data from n = 24–30 cells and are 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 evaluate the differences between control shRNA and PINK1 shRNA after each treatment. *P<0.05; ** P<0.01. (E) Counts of mitochondrial morphology as in figure 2 were performed on n = 60 cells from duplicate experiments. Drp1 siRNA decreased the number of cells with truncated or fragmented mitochondria. Differences were analyzed by two-way ANOVA using morphology and cell line/treatment as factors, P = 0.001 overall for the different cell groups. (F) Mitochondrial length was measured from images of unfixed, mito-YFP transfected cells. Boxes indicate the upper and lower quartiles, central line indicates the median and range bars indicate the 10th to 90th percentile range. Differences between treatments were significant overall (P<0.0001 by ANOVA, n = 14–29) and Student-Newman Kuells' post-hoc test was used to evaluate the differences between control shRNA and PINK1 shRNA after each treatment. *** P<0.001.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2683574&req=5

pone-0005701-g005: Mitochondrial effects due to loss of PINK1 can be rescued by knockdown of Drp1.(A) Exposure of control shRNA or PINK1 shRNA stable cell lines to Drp1 siRNA for four days results in an approximately 70% knockdown of Drp1 (arrow) compared to a non-specific GFP siRNA. β-actin shows equal loading. Molecular weight markers are in kilodaltons. (B) Live cell images of mito-YFP transfected control shRNA (upper panels) and PINK1 shRNA (lower panel) cells exposed to a non-specific siRNA against GFP (left panels) or an siRNA against Drp1 (right panels). Scale bar is 2 µm, applies to all fluorescence micrographs. (C) FRAP curves show that under basal situations, there was a difference between the control shRNA line and the PINK1 shRNA line. These differences were partially normalized after the cells were exposed to an siRNA against Drp1. Each point is the average of >30 separate measurements and error bars indicate the SEM. (D) Mobile fraction of mito-YFP was measured in control shRNA or PINK1 shRNA cell lines either after expression of a control siRNA (against GFP) or after expression of an siRNA against Drp1. Boxplots summarize data from n = 24–30 cells and are 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 evaluate the differences between control shRNA and PINK1 shRNA after each treatment. *P<0.05; ** P<0.01. (E) Counts of mitochondrial morphology as in figure 2 were performed on n = 60 cells from duplicate experiments. Drp1 siRNA decreased the number of cells with truncated or fragmented mitochondria. Differences were analyzed by two-way ANOVA using morphology and cell line/treatment as factors, P = 0.001 overall for the different cell groups. (F) Mitochondrial length was measured from images of unfixed, mito-YFP transfected cells. Boxes indicate the upper and lower quartiles, central line indicates the median and range bars indicate the 10th to 90th percentile range. Differences between treatments were significant overall (P<0.0001 by ANOVA, n = 14–29) and Student-Newman Kuells' post-hoc test was used to evaluate the differences between control shRNA and PINK1 shRNA after each treatment. *** P<0.001.

Mentions: We asked whether loss of Dynamin related protein 1 (Drp1), a GTPase involved in various aspects of mitochondrial morphology and function, would antagonize the effects of PINK1 deficiency. Using siRNA to Drp1, we achieved knockdown to ∼30% of controls and is similar in control or PINK1 shRNA lines (Fig. 5A). This partial knockdown resulted in an elongated mitochondrial network in both cell lines (Fig. 5B), similar to previous studies in other cell types [37]–[39]. This partial knockdown prevented mitochondrial fragmentation associated with loss of PINK1 expression in the absence of other stressors. Calculating the mobile fraction of mitoYFP, the difference between control shRNA and PINK1 shRNA lines remained significant (P<0.05 by one-way ANOVA with Student-Newmann Kuell's post-hoc test) after expression of a control siRNA directed against GFP. The mobile fraction in PINK1 shRNA cells was significantly different after Drp1 RNAi compared to GFP RNAi (P<0.01; Fig. 5D). A similar rescue of the mobile fraction deficit was seen in cell lines expressing the second PINK1 shRNA sequence (data not shown). Cell counts show a decrease in the proportion of cells with fragmented or truncated mitochondria after Drp siRNA (P = 0.001 by two-way ANOVA; Fig. 5E). Finally, mitochondrial length was measured and was lower in PINK1 deficient cells compared to control (P<0.001 by one-way ANOVA) and increased after Drp siRNA (P<0.001; Fig. 5F).


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)

Mitochondrial effects due to loss of PINK1 can be rescued by knockdown of Drp1.(A) Exposure of control shRNA or PINK1 shRNA stable cell lines to Drp1 siRNA for four days results in an approximately 70% knockdown of Drp1 (arrow) compared to a non-specific GFP siRNA. β-actin shows equal loading. Molecular weight markers are in kilodaltons. (B) Live cell images of mito-YFP transfected control shRNA (upper panels) and PINK1 shRNA (lower panel) cells exposed to a non-specific siRNA against GFP (left panels) or an siRNA against Drp1 (right panels). Scale bar is 2 µm, applies to all fluorescence micrographs. (C) FRAP curves show that under basal situations, there was a difference between the control shRNA line and the PINK1 shRNA line. These differences were partially normalized after the cells were exposed to an siRNA against Drp1. Each point is the average of >30 separate measurements and error bars indicate the SEM. (D) Mobile fraction of mito-YFP was measured in control shRNA or PINK1 shRNA cell lines either after expression of a control siRNA (against GFP) or after expression of an siRNA against Drp1. Boxplots summarize data from n = 24–30 cells and are 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 evaluate the differences between control shRNA and PINK1 shRNA after each treatment. *P<0.05; ** P<0.01. (E) Counts of mitochondrial morphology as in figure 2 were performed on n = 60 cells from duplicate experiments. Drp1 siRNA decreased the number of cells with truncated or fragmented mitochondria. Differences were analyzed by two-way ANOVA using morphology and cell line/treatment as factors, P = 0.001 overall for the different cell groups. (F) Mitochondrial length was measured from images of unfixed, mito-YFP transfected cells. Boxes indicate the upper and lower quartiles, central line indicates the median and range bars indicate the 10th to 90th percentile range. Differences between treatments were significant overall (P<0.0001 by ANOVA, n = 14–29) and Student-Newman Kuells' post-hoc test was used to evaluate the differences between control shRNA and PINK1 shRNA after each treatment. *** P<0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005701-g005: Mitochondrial effects due to loss of PINK1 can be rescued by knockdown of Drp1.(A) Exposure of control shRNA or PINK1 shRNA stable cell lines to Drp1 siRNA for four days results in an approximately 70% knockdown of Drp1 (arrow) compared to a non-specific GFP siRNA. β-actin shows equal loading. Molecular weight markers are in kilodaltons. (B) Live cell images of mito-YFP transfected control shRNA (upper panels) and PINK1 shRNA (lower panel) cells exposed to a non-specific siRNA against GFP (left panels) or an siRNA against Drp1 (right panels). Scale bar is 2 µm, applies to all fluorescence micrographs. (C) FRAP curves show that under basal situations, there was a difference between the control shRNA line and the PINK1 shRNA line. These differences were partially normalized after the cells were exposed to an siRNA against Drp1. Each point is the average of >30 separate measurements and error bars indicate the SEM. (D) Mobile fraction of mito-YFP was measured in control shRNA or PINK1 shRNA cell lines either after expression of a control siRNA (against GFP) or after expression of an siRNA against Drp1. Boxplots summarize data from n = 24–30 cells and are 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 evaluate the differences between control shRNA and PINK1 shRNA after each treatment. *P<0.05; ** P<0.01. (E) Counts of mitochondrial morphology as in figure 2 were performed on n = 60 cells from duplicate experiments. Drp1 siRNA decreased the number of cells with truncated or fragmented mitochondria. Differences were analyzed by two-way ANOVA using morphology and cell line/treatment as factors, P = 0.001 overall for the different cell groups. (F) Mitochondrial length was measured from images of unfixed, mito-YFP transfected cells. Boxes indicate the upper and lower quartiles, central line indicates the median and range bars indicate the 10th to 90th percentile range. Differences between treatments were significant overall (P<0.0001 by ANOVA, n = 14–29) and Student-Newman Kuells' post-hoc test was used to evaluate the differences between control shRNA and PINK1 shRNA after each treatment. *** P<0.001.
Mentions: We asked whether loss of Dynamin related protein 1 (Drp1), a GTPase involved in various aspects of mitochondrial morphology and function, would antagonize the effects of PINK1 deficiency. Using siRNA to Drp1, we achieved knockdown to ∼30% of controls and is similar in control or PINK1 shRNA lines (Fig. 5A). This partial knockdown resulted in an elongated mitochondrial network in both cell lines (Fig. 5B), similar to previous studies in other cell types [37]–[39]. This partial knockdown prevented mitochondrial fragmentation associated with loss of PINK1 expression in the absence of other stressors. Calculating the mobile fraction of mitoYFP, the difference between control shRNA and PINK1 shRNA lines remained significant (P<0.05 by one-way ANOVA with Student-Newmann Kuell's post-hoc test) after expression of a control siRNA directed against GFP. The mobile fraction in PINK1 shRNA cells was significantly different after Drp1 RNAi compared to GFP RNAi (P<0.01; Fig. 5D). A similar rescue of the mobile fraction deficit was seen in cell lines expressing the second PINK1 shRNA sequence (data not shown). Cell counts show a decrease in the proportion of cells with fragmented or truncated mitochondria after Drp siRNA (P = 0.001 by two-way ANOVA; Fig. 5E). Finally, mitochondrial length was measured and was lower in PINK1 deficient cells compared to control (P<0.001 by one-way ANOVA) and increased after Drp siRNA (P<0.001; Fig. 5F).

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