Mitochondrially localized PKA reverses mitochondrial pathology and dysfunction in a cellular model of Parkinson's disease

Abstract

Mutations in PTEN-induced kinase 1 (PINK1) are associated with a familial syndrome related to Parkinson’s disease (PD). We previously reported that stable neuroblastoma SH-SY5Y cell lines with reduced expression of endogenous PINK1 exhibit mitochondrial fragmentation, increased mitochondria-derived superoxide, induction of compensatory macroautophagy/mitophagy and a low level of ongoing cell death. Here, we investigated the ability of protein kinase A (PKA) to confer protection in this model, focusing on its subcellular targeting. Either: 1) treatment with pharmacological PKA activators; 2) transient expression of a constitutively active form of mitochondria-targeted PKA; or 3) transient expression of wild-type AKAP1, a scaffold that targets endogenous PKA to mitochondria, reversed each of the phenotypes attributed to loss of PINK1 in SH-SY5Y cells, and rescued parameters of mitochondrial respiratory dysfunction. Mitochondrial and lysosomal changes in primary cortical neurons derived from PINK1 knockout mice or subjected to PINK1 RNAi were also reversed by activation of PKA. PKA phosphorylates the rat dynamin-related protein 1 isoform 1 (Drp1) at serine 656 (homologous to human serine 637), inhibiting its pro-fission function. Mimicking phosphorylation of Drp1 recapitulated many of the protective effects of AKAP1/PKA. These data indicate that redirecting endogenous PKA to mitochondria can compensate for deficiencies in PINK1 function, highlighting the importance of compartmentalized signaling networks in mitochondrial quality control.

Mitochondrial O-GlcNAc Transferase (mOGT) Regulates Mitochondrial Structure, Function, and Survival in HeLa Cells

“…We analyzed mitochondrial morphology and mitochondrial content in cells transfected with the validated mOGT siRNAs 1 and 2 ( Fig. 1) by using well characterized parameters of mitochondrial morphology (28,29) as described under "Experimental Procedures." In control cells transfected with NT siRNA, mitochondria predominantly showed a tubular and elongated morphology, some of which colocalized with OGT ( Fig.…”

“…Because siRNA-mediated reduction of endogenous mOGT levels remodels mitochondria and increases mitochondrial respiration per mitochondrion (Figs. 2 and 3), we investigated the effects of reducing endogenous levels of mOGT on the mitochondrial transmembrane potential, which is an index of mitochondrial health (28,35). The transmembrane potential was 2J).…”

“…These macros allow for semi-automated image analysis of individual mitochondrial particles and mitochondrial content (28,54). To analyze the effects of reducing endogenous mOGT on cell number, the number of DAPI-stained nuclei per epifluorescence field in NT siRNA-or mOGT siRNA-transfected HeLa cells was manually counted by an observer who was blind to the transfection status of the cells, and the data were expressed as number of DAPI-stained cells/epifluorescence field.…”

“…Cells seeded in either high glucose (25 mM) or galactose (10 mM) were supplemented with 2 mM glutamine and 1 mM sodium pyruvate and grown at 37°C and 5% CO 2 for 24 h before the assay. OCRs in transfected HeLa cells were analyzed at baseline and following the sequential addition of 1 M oligomycin, 300 nM FCCP, and 1 M rotenone/antimycin A to determine the ATP-linked OCRs, maximal OCRs, and mitochondrially derived OCRs, respectively, as published previously (28). Spare respiratory capacity was calculated as described previously (56,57).…”

“…Mitochondrial Enrichment and Western Blotting-Mitochondria enriched fractions derived from HeLa cells transfected with NT, mOGT1, mOGT2, or ncOGT siRNAs were isolated using a mitochondrial isolation kit (Sigma) per the manufacturer's recommendation and as previously published (28,31). The purity of the mitochondrial and cytosolic fractions was assessed by Western blotting.…”