ABSTRACT
Leigh syndrome is a fatal neurometabolic disorder caused by defects in mitochondrial function. Mechanistic target of rapamycin (mTOR) inhibition with rapamycin attenuates disease progression in a mouse model of Leigh syndrome (Ndufs4 knock-out (KO) mouse); however, the mechanism of rescue is unknown. Here we identify protein kinase C (PKC) downregulation as a key event mediating the beneficial effects of rapamycin treatment of Ndufs4 KO mice. Assessing the impact of rapamycin on the brain proteome and phosphoproteome of Ndufs4 KO mice, we find that rapamycin restores mitochondrial protein levels, inhibits signalling through both mTOR complexes and reduces the abundance and activity of multiple PKC isoforms. Administration of PKC inhibitors increases survival, delays neurological deficits, prevents hair loss and decreases inflammation in Ndufs4 KO mice. Thus, PKC may be a viable therapeutic target for treating severe mitochondrial disease.
Subject(s)
Mitochondrial Diseases/drug therapy , Protein Kinase C/biosynthesis , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Sirolimus/pharmacology , Sirolimus/therapeutic use , Animals , Brain Chemistry/drug effects , Down-Regulation/drug effects , Electron Transport Complex I/biosynthesis , Electron Transport Complex I/genetics , Leigh Disease/drug therapy , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Kinase C/genetics , Proteome/drug effects , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/antagonists & inhibitorsABSTRACT
Stimulating brown adipose tissue (BAT) activity represents a promising therapy for overcoming metabolic diseases. mTORC2 is important for regulating BAT metabolism, but its downstream targets have not been fully characterized. In this study, we apply proteomics and phosphoproteomics to investigate the downstream effectors of mTORC2 in brown adipocytes. We compare wild-type controls to isogenic cells with an induced knockout of the mTORC2 subunit RICTOR (Rictor-iKO) by stimulating each with insulin for a 30-min time course. In Rictor-iKO cells, we identify decreases to the abundance of glycolytic and de novo lipogenesis enzymes, and increases to mitochondrial proteins as well as a set of proteins known to increase upon interferon stimulation. We also observe significant differences to basal phosphorylation because of chronic RICTOR loss including decreased phosphorylation of the lipid droplet protein perilipin-1 in Rictor-iKO cells, suggesting that RICTOR could be involved with regulating basal lipolysis or droplet dynamics. Finally, we observe mild dampening of acute insulin signaling response in Rictor-iKO cells, and a subset of AKT substrates exhibiting statistically significant dependence on RICTOR.