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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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Dephosphorylation and increased GTPase activity of Drp1 in PINK1 deficient cells.(A) Lysates from control shRNA lines or either of two PINK1 shRNA lines were separated by phospho-enrichment compared to total lysates and blotted for the proteins indicated on the left of the blots. Drp1 was in both phospho-enriched and total fractions, but less phospho-Drp1 was seen in the PINK1 deficient cells. We used phospho-Drp1 to confirm the phosphopurification was efficient and DJ-1 served as a negative control. (B) Quantification of Drp1 as a ratio of the phospho-enriched and total fractions shows that there was an ∼30% decrease in phospho-Drp1 (n = 3) that is consistent between both shRNA sequences (*, P<0.05 by ANOVA). (C) Recruitment of endogenous Drp1 to mitochondrial fractions (mito) is similar in control and PINK1 shRNA cell lines (total: whole cell lysates, cyt: cytosolic fractions). Data are representative of duplicate experiments and molecular weight markers on the right of the blots are in kilodaltons. (D) Oligomerization status of Drp1 is not affected by PINK1 shRNA compared to control lines. Lysates were crosslinked with BMH or not treated to show equivalent loading of Drp1. Arrowhead shows Drp1 oligomers, arrow shows monomeric Drp1. Molecular weight markers are in kilodaltons. (E) GTPase activity of Drp1 immunopurified from control or PINK1 shRNA cell lines was followed over time and expressed as percentage of GTP converted to GDP. The difference between the lines was significant (P<0.01 by two-way ANOVA). Each point is the average of 3 replicates and is representative of two independent experiments.
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pone-0005701-g007: Dephosphorylation and increased GTPase activity of Drp1 in PINK1 deficient cells.(A) Lysates from control shRNA lines or either of two PINK1 shRNA lines were separated by phospho-enrichment compared to total lysates and blotted for the proteins indicated on the left of the blots. Drp1 was in both phospho-enriched and total fractions, but less phospho-Drp1 was seen in the PINK1 deficient cells. We used phospho-Drp1 to confirm the phosphopurification was efficient and DJ-1 served as a negative control. (B) Quantification of Drp1 as a ratio of the phospho-enriched and total fractions shows that there was an ∼30% decrease in phospho-Drp1 (n = 3) that is consistent between both shRNA sequences (*, P<0.05 by ANOVA). (C) Recruitment of endogenous Drp1 to mitochondrial fractions (mito) is similar in control and PINK1 shRNA cell lines (total: whole cell lysates, cyt: cytosolic fractions). Data are representative of duplicate experiments and molecular weight markers on the right of the blots are in kilodaltons. (D) Oligomerization status of Drp1 is not affected by PINK1 shRNA compared to control lines. Lysates were crosslinked with BMH or not treated to show equivalent loading of Drp1. Arrowhead shows Drp1 oligomers, arrow shows monomeric Drp1. Molecular weight markers are in kilodaltons. (E) GTPase activity of Drp1 immunopurified from control or PINK1 shRNA cell lines was followed over time and expressed as percentage of GTP converted to GDP. The difference between the lines was significant (P<0.01 by two-way ANOVA). Each point is the average of 3 replicates and is representative of two independent experiments.

Mentions: Next, we wanted to know if Drp1 activity was modified in PINK1 deficient cells. As Drp1 activity can be affected by serine phosphorylation [40]–[43], we examined the phosphorylation status of Drp1 in PINK1 deficient cells by enriching for phosphoproteins and blotting for endogenous Drp1. We confirmed the phospho-enrichment by blotting for p-S637 Drp1 and used DJ-1, which is not basally phosphorylated [44], as a negative control (Fig. 7A). Drp1 showed a loss of phosphorylation in the absence of PINK1 (Fig. 7A). Quantification across multiple experiments showed an ∼30% loss of endogenous Drp1 phosphorylation in PINK1 deficient cells (Fig. 7B). Phosphorylated amounts of OPA1, Mfn1 and Mfn2 and HtrA2 were also examined, but no differences between cell lines were found (Supplementary Fig. S5). Subcellular fractionation of control versus PINK1 shRNA cells were analyzed by western blotting and show mitochondrial recruitment of endogenous Drp1 is not influenced by the loss of PINK1 (Fig. 7C). BMH-crosslinking experiments did not show any alterations in Drp1 oligomerization in PINK1 deficient cells (Fig. 7D). Using in vitro GTPase assays, we found increased GTPase activity of Drp1 in cells expressing PINK1 shRNA (Fig. 7E; P<0.01 for cell lines), which may be related to the decreased phosphorylation seen in the same 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)

Dephosphorylation and increased GTPase activity of Drp1 in PINK1 deficient cells.(A) Lysates from control shRNA lines or either of two PINK1 shRNA lines were separated by phospho-enrichment compared to total lysates and blotted for the proteins indicated on the left of the blots. Drp1 was in both phospho-enriched and total fractions, but less phospho-Drp1 was seen in the PINK1 deficient cells. We used phospho-Drp1 to confirm the phosphopurification was efficient and DJ-1 served as a negative control. (B) Quantification of Drp1 as a ratio of the phospho-enriched and total fractions shows that there was an ∼30% decrease in phospho-Drp1 (n = 3) that is consistent between both shRNA sequences (*, P<0.05 by ANOVA). (C) Recruitment of endogenous Drp1 to mitochondrial fractions (mito) is similar in control and PINK1 shRNA cell lines (total: whole cell lysates, cyt: cytosolic fractions). Data are representative of duplicate experiments and molecular weight markers on the right of the blots are in kilodaltons. (D) Oligomerization status of Drp1 is not affected by PINK1 shRNA compared to control lines. Lysates were crosslinked with BMH or not treated to show equivalent loading of Drp1. Arrowhead shows Drp1 oligomers, arrow shows monomeric Drp1. Molecular weight markers are in kilodaltons. (E) GTPase activity of Drp1 immunopurified from control or PINK1 shRNA cell lines was followed over time and expressed as percentage of GTP converted to GDP. The difference between the lines was significant (P<0.01 by two-way ANOVA). Each point is the average of 3 replicates and is representative of two independent experiments.
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Related In: Results  -  Collection

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pone-0005701-g007: Dephosphorylation and increased GTPase activity of Drp1 in PINK1 deficient cells.(A) Lysates from control shRNA lines or either of two PINK1 shRNA lines were separated by phospho-enrichment compared to total lysates and blotted for the proteins indicated on the left of the blots. Drp1 was in both phospho-enriched and total fractions, but less phospho-Drp1 was seen in the PINK1 deficient cells. We used phospho-Drp1 to confirm the phosphopurification was efficient and DJ-1 served as a negative control. (B) Quantification of Drp1 as a ratio of the phospho-enriched and total fractions shows that there was an ∼30% decrease in phospho-Drp1 (n = 3) that is consistent between both shRNA sequences (*, P<0.05 by ANOVA). (C) Recruitment of endogenous Drp1 to mitochondrial fractions (mito) is similar in control and PINK1 shRNA cell lines (total: whole cell lysates, cyt: cytosolic fractions). Data are representative of duplicate experiments and molecular weight markers on the right of the blots are in kilodaltons. (D) Oligomerization status of Drp1 is not affected by PINK1 shRNA compared to control lines. Lysates were crosslinked with BMH or not treated to show equivalent loading of Drp1. Arrowhead shows Drp1 oligomers, arrow shows monomeric Drp1. Molecular weight markers are in kilodaltons. (E) GTPase activity of Drp1 immunopurified from control or PINK1 shRNA cell lines was followed over time and expressed as percentage of GTP converted to GDP. The difference between the lines was significant (P<0.01 by two-way ANOVA). Each point is the average of 3 replicates and is representative of two independent experiments.
Mentions: Next, we wanted to know if Drp1 activity was modified in PINK1 deficient cells. As Drp1 activity can be affected by serine phosphorylation [40]–[43], we examined the phosphorylation status of Drp1 in PINK1 deficient cells by enriching for phosphoproteins and blotting for endogenous Drp1. We confirmed the phospho-enrichment by blotting for p-S637 Drp1 and used DJ-1, which is not basally phosphorylated [44], as a negative control (Fig. 7A). Drp1 showed a loss of phosphorylation in the absence of PINK1 (Fig. 7A). Quantification across multiple experiments showed an ∼30% loss of endogenous Drp1 phosphorylation in PINK1 deficient cells (Fig. 7B). Phosphorylated amounts of OPA1, Mfn1 and Mfn2 and HtrA2 were also examined, but no differences between cell lines were found (Supplementary Fig. S5). Subcellular fractionation of control versus PINK1 shRNA cells were analyzed by western blotting and show mitochondrial recruitment of endogenous Drp1 is not influenced by the loss of PINK1 (Fig. 7C). BMH-crosslinking experiments did not show any alterations in Drp1 oligomerization in PINK1 deficient cells (Fig. 7D). Using in vitro GTPase assays, we found increased GTPase activity of Drp1 in cells expressing PINK1 shRNA (Fig. 7E; P<0.01 for cell lines), which may be related to the decreased phosphorylation seen in the same 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