Project Summary Inhibition of the mechanistic Target of Rapamycin (mTOR) improves prognosis in a mouse model of Leigh Syndrome (LS) and in a small cohort of Mitochondrial Encephalomyopathy with Lactic Acidosis and Stroke (MELAS) patients, two neurological mitochondrial disorders. However, its specific mechanism of action remains to be determined. We find that the mTOR inhibitor rapamycin reverses metabolic alterations in LS mice lacking the Complex I subunit Ndufs4 (Ndufs4-/-): more specifically, rapamycin reduces the accumulation of glycolytic intermediates and increases the abundance of fatty acids in both brain and liver of Ndufs4-/- mice. Loss of the mitochondrial sirtuin Sirt3 abrogates lifespan extension and the delay of neurological phenotypes in Ndufs4-/- mice treated with rapamycin. In Aim 1 of this proposal, we explore the dependency of mTOR inhibition on Sirt3 to improve disease phenotypes in these animals. Specifically, we hypothesize that expression of Sirt3 in the liver is required to promote the metabolic shift to fatty acid oxidation described above. Treatment with the Nucleoside Analog Reverse Transcriptase Inhibitor (NRTI) Adefovir Dipivoxil (ADV) delays symptoms of disease and improves survival in Ndufs4-/- mice. ADV increases the expression of C/EBP-β, a transcription factor that increases hepatic fatty acid oxidation in response to calorie restriction and mTOR inhibition. In Aim 2 we explore the hypothesis that C/EBP-β guides the metabolic shift induced by rapamycin in Ndufs4-/- mice. With a combination of genetic and pharmacological approaches, both aims will determine the exact nature of the metabolic alterations induced by loss of Complex I, the mechanisms by which this loss of metabolic homeostasis is rescued, and its importance in the etiology and progression of neurological mitochondrial disease. This project will determine the metabolic nature of these disorders and lay the foundation for novel treatments based on fine tuning of nutrient metabolism in patients affected by Complex I dysfunction.