Mitochondrial alterations in PINK1 deficient cells are influenced by calcineurin-dependent dephosphorylation of dynamin-related protein 1.
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.
Sandebring
Anna
A
Laboratory of Neurogenetics, National Institute on Aging, Bethesda, Maryland, United States of America.
Thomas
Kelly Jean
KJ
Beilina
Alexandra
A
van der Brug
Marcel
M
Cleland
Megan M
MM
Ahmad
Rili
R
Miller
David W
DW
Zambrano
Ibardo
I
Cowburn
Richard F
RF
Behbahani
Homira
H
Cedazo-Mínguez
Angel
A
Cookson
Mark R
MR
D019703:Calcineurin / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D034281:Dynamins; D004789:Enzyme Activation / Q000187:drug effects; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D024101:Mitochondrial Proteins / Q000378:metabolism*; D008954:Models, Biological; D010641:Phenotype; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000172:deficiency* / Q000378:metabolism; D012402:Rotenone / Q000494:pharmacology; D000099202:PTEN-Induced Putative Kinase
D019703:Calcineurin; D034281:Dynamins; D020558:GTP Phosphohydrolases; D008869:Microtubule-Associated Proteins; D024101:Mitochondrial Proteins; D011494:Protein Kinases; D012402:Rotenone; D000099202:PTEN-Induced Putative Kinase; C110768:DNM1L protein, human