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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 expression alters mitochondrial function and cellular viability.(A–C) Stable overexpression of PINK1 variants. (A) Western blot of stable cell lines generated using lentiviral transduction and selected for equal expression of PINK1. A control lentivirus expressing LacZ is shown in lane 1, lanes 2–4 are cell lines expressing wild-type (WT), G309D or kinase dead (KD) PINK1. The PINK1 in these cells is V5-tagged at the C-terminus, the precursor (arrow) and mature (closed arrowhead) forms of PINK1 are visible. In the lower panel, the blot was reprobed with β-actin to show equal loading. Molecular weight markers on the right are in kilodaltons. (B) Mitochondrial membrane potential was measured using FACS in live cells using TMRM. Results are expressed as the mean number of cells with TMRM fluorescence above threshold set by depolarizing one set of cells with CCCP (100 µM, 10 minutes; see Supplementary Fig. S1). Error bars show the SEM (n = 6–7 independent experiments per line). There are no statistically significant differences between the lines (P = 0.23 by ANOVA). (C) Cell viability was measured after exposure of cells to 100 nM rotenone for 48 hours using FACS. Viable cells were defined as AnnexinV (AnnV) and propidium iodide (PI) negative, and are expressed as percentage of all sorted cells; apoptotic cells were AnnV positive/PI negative; necrotic cells were AnnV negative/PI positive; late apoptotic cells were AnnVpositive/PI positive. Each bar is the mean of 3 experiments with 10,000 cells counted in each, and error bars indicate the SEM between experiments. The improvement in cell viability was significant for cells expressing WT PINK1 only (p = 0.03). (D–F) Stable knockdown of PINK1. (D) Bars show the mean relative PINK1 expression estimated using quantitative RT-PCR, normalized to β-actin expression. Two different PINK1 shRNA sequences, A and C, decrease endogenous mRNA expression relative to a non-specific control shRNA. The differences between the cell lines were assessed using one-way ANOVA (P<0.0001 overall) and Dunnett's multiple comparison post hoc tests when compared to the control shRNA cell line; ***, P<0.0001 (n = 6 independent experiments, error bars indicate the SEM). (E) Mitochondrial membrane potential was measured and is expressed as in (B). The differences between control and PINK1 shRNA were significant by t-test (P = 0.0008, n = 5 independent experiments). (F) Cell viability after rotenone exposure, as in (C), was lower in PINK1 deficient cells compared to control shRNA. The difference in the percentage of viable cells was significant (P = 0.009 by t-test, n = 3).
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pone-0005701-g001: PINK1 expression alters mitochondrial function and cellular viability.(A–C) Stable overexpression of PINK1 variants. (A) Western blot of stable cell lines generated using lentiviral transduction and selected for equal expression of PINK1. A control lentivirus expressing LacZ is shown in lane 1, lanes 2–4 are cell lines expressing wild-type (WT), G309D or kinase dead (KD) PINK1. The PINK1 in these cells is V5-tagged at the C-terminus, the precursor (arrow) and mature (closed arrowhead) forms of PINK1 are visible. In the lower panel, the blot was reprobed with β-actin to show equal loading. Molecular weight markers on the right are in kilodaltons. (B) Mitochondrial membrane potential was measured using FACS in live cells using TMRM. Results are expressed as the mean number of cells with TMRM fluorescence above threshold set by depolarizing one set of cells with CCCP (100 µM, 10 minutes; see Supplementary Fig. S1). Error bars show the SEM (n = 6–7 independent experiments per line). There are no statistically significant differences between the lines (P = 0.23 by ANOVA). (C) Cell viability was measured after exposure of cells to 100 nM rotenone for 48 hours using FACS. Viable cells were defined as AnnexinV (AnnV) and propidium iodide (PI) negative, and are expressed as percentage of all sorted cells; apoptotic cells were AnnV positive/PI negative; necrotic cells were AnnV negative/PI positive; late apoptotic cells were AnnVpositive/PI positive. Each bar is the mean of 3 experiments with 10,000 cells counted in each, and error bars indicate the SEM between experiments. The improvement in cell viability was significant for cells expressing WT PINK1 only (p = 0.03). (D–F) Stable knockdown of PINK1. (D) Bars show the mean relative PINK1 expression estimated using quantitative RT-PCR, normalized to β-actin expression. Two different PINK1 shRNA sequences, A and C, decrease endogenous mRNA expression relative to a non-specific control shRNA. The differences between the cell lines were assessed using one-way ANOVA (P<0.0001 overall) and Dunnett's multiple comparison post hoc tests when compared to the control shRNA cell line; ***, P<0.0001 (n = 6 independent experiments, error bars indicate the SEM). (E) Mitochondrial membrane potential was measured and is expressed as in (B). The differences between control and PINK1 shRNA were significant by t-test (P = 0.0008, n = 5 independent experiments). (F) Cell viability after rotenone exposure, as in (C), was lower in PINK1 deficient cells compared to control shRNA. The difference in the percentage of viable cells was significant (P = 0.009 by t-test, n = 3).

Mentions: To address the function of PINK1 in living cells, we generated stable dopaminergic neuroblastoma lines that express wild-type PINK1, a recessive mutant, G309D and an artificial variant lacking kinase activity [29] (Fig. 1A–C). Because PINK1 mutations are recessive, we also examined cells stably transduced with either of two shRNA sequences directed against PINK1, with a scrambled shRNA used as control (Fig. 1D–F).


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 expression alters mitochondrial function and cellular viability.(A–C) Stable overexpression of PINK1 variants. (A) Western blot of stable cell lines generated using lentiviral transduction and selected for equal expression of PINK1. A control lentivirus expressing LacZ is shown in lane 1, lanes 2–4 are cell lines expressing wild-type (WT), G309D or kinase dead (KD) PINK1. The PINK1 in these cells is V5-tagged at the C-terminus, the precursor (arrow) and mature (closed arrowhead) forms of PINK1 are visible. In the lower panel, the blot was reprobed with β-actin to show equal loading. Molecular weight markers on the right are in kilodaltons. (B) Mitochondrial membrane potential was measured using FACS in live cells using TMRM. Results are expressed as the mean number of cells with TMRM fluorescence above threshold set by depolarizing one set of cells with CCCP (100 µM, 10 minutes; see Supplementary Fig. S1). Error bars show the SEM (n = 6–7 independent experiments per line). There are no statistically significant differences between the lines (P = 0.23 by ANOVA). (C) Cell viability was measured after exposure of cells to 100 nM rotenone for 48 hours using FACS. Viable cells were defined as AnnexinV (AnnV) and propidium iodide (PI) negative, and are expressed as percentage of all sorted cells; apoptotic cells were AnnV positive/PI negative; necrotic cells were AnnV negative/PI positive; late apoptotic cells were AnnVpositive/PI positive. Each bar is the mean of 3 experiments with 10,000 cells counted in each, and error bars indicate the SEM between experiments. The improvement in cell viability was significant for cells expressing WT PINK1 only (p = 0.03). (D–F) Stable knockdown of PINK1. (D) Bars show the mean relative PINK1 expression estimated using quantitative RT-PCR, normalized to β-actin expression. Two different PINK1 shRNA sequences, A and C, decrease endogenous mRNA expression relative to a non-specific control shRNA. The differences between the cell lines were assessed using one-way ANOVA (P<0.0001 overall) and Dunnett's multiple comparison post hoc tests when compared to the control shRNA cell line; ***, P<0.0001 (n = 6 independent experiments, error bars indicate the SEM). (E) Mitochondrial membrane potential was measured and is expressed as in (B). The differences between control and PINK1 shRNA were significant by t-test (P = 0.0008, n = 5 independent experiments). (F) Cell viability after rotenone exposure, as in (C), was lower in PINK1 deficient cells compared to control shRNA. The difference in the percentage of viable cells was significant (P = 0.009 by t-test, n = 3).
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pone-0005701-g001: PINK1 expression alters mitochondrial function and cellular viability.(A–C) Stable overexpression of PINK1 variants. (A) Western blot of stable cell lines generated using lentiviral transduction and selected for equal expression of PINK1. A control lentivirus expressing LacZ is shown in lane 1, lanes 2–4 are cell lines expressing wild-type (WT), G309D or kinase dead (KD) PINK1. The PINK1 in these cells is V5-tagged at the C-terminus, the precursor (arrow) and mature (closed arrowhead) forms of PINK1 are visible. In the lower panel, the blot was reprobed with β-actin to show equal loading. Molecular weight markers on the right are in kilodaltons. (B) Mitochondrial membrane potential was measured using FACS in live cells using TMRM. Results are expressed as the mean number of cells with TMRM fluorescence above threshold set by depolarizing one set of cells with CCCP (100 µM, 10 minutes; see Supplementary Fig. S1). Error bars show the SEM (n = 6–7 independent experiments per line). There are no statistically significant differences between the lines (P = 0.23 by ANOVA). (C) Cell viability was measured after exposure of cells to 100 nM rotenone for 48 hours using FACS. Viable cells were defined as AnnexinV (AnnV) and propidium iodide (PI) negative, and are expressed as percentage of all sorted cells; apoptotic cells were AnnV positive/PI negative; necrotic cells were AnnV negative/PI positive; late apoptotic cells were AnnVpositive/PI positive. Each bar is the mean of 3 experiments with 10,000 cells counted in each, and error bars indicate the SEM between experiments. The improvement in cell viability was significant for cells expressing WT PINK1 only (p = 0.03). (D–F) Stable knockdown of PINK1. (D) Bars show the mean relative PINK1 expression estimated using quantitative RT-PCR, normalized to β-actin expression. Two different PINK1 shRNA sequences, A and C, decrease endogenous mRNA expression relative to a non-specific control shRNA. The differences between the cell lines were assessed using one-way ANOVA (P<0.0001 overall) and Dunnett's multiple comparison post hoc tests when compared to the control shRNA cell line; ***, P<0.0001 (n = 6 independent experiments, error bars indicate the SEM). (E) Mitochondrial membrane potential was measured and is expressed as in (B). The differences between control and PINK1 shRNA were significant by t-test (P = 0.0008, n = 5 independent experiments). (F) Cell viability after rotenone exposure, as in (C), was lower in PINK1 deficient cells compared to control shRNA. The difference in the percentage of viable cells was significant (P = 0.009 by t-test, n = 3).
Mentions: To address the function of PINK1 in living cells, we generated stable dopaminergic neuroblastoma lines that express wild-type PINK1, a recessive mutant, G309D and an artificial variant lacking kinase activity [29] (Fig. 1A–C). Because PINK1 mutations are recessive, we also examined cells stably transduced with either of two shRNA sequences directed against PINK1, with a scrambled shRNA used as control (Fig. 1D–F).

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