Project Summary Altered mitochondrial function, a hallmark of aging, contributes to age-related diseases, in part, due to energetic failure. Maintenance of mitochondrial energetics requires proper fission and fusion that are in turn regulated by proteins at endoplasmic reticulum-mitochondria contacts (a.k.a. mitochondria-associated membranes or MAMs). Fasting stimulates mitochondrial fatty acid utilization, and it is thought that the benefits of fasting are due to adaptive changes in mitochondrial dynamics and respiration. How fasting impacts mitochondrial dynamics and MAMs in intact mammalian systems, and whether these fundamental mechanisms are altered with age and dietary stress remain unknown. The mechanisms by which upstream signaling cascades regulate mitochondrial dynamics is becoming an area of great interest, with recent work showing AMPK as a regulator of fission. Our new data show that the typically nutrient-responsive mTORC2 is paradoxically reactivated by fasting, and that mTORC2 reactivation supports mitochondrial fission. Indeed, livers silenced for mTORC2 regulator Rictor show impaired fission and dampened respiration; and mTORC2 activity is suppressed with age. These data raise a key point: since clinically available mTOR inhibitors (e.g., rapamycin) block both mTORC1 and mTORC2, we argue that benefits of long-term mTOR inhibition with rapamycin are counterbalanced by mitochondrial dysfunction due to concomitant mTORC2 inhibition. This forms that basis to explore if blocking mTORC1—in conjunction with maintaining mTORC2 activity—will lead to healthspan and lifespan extension. Phosphorylation is a well-established signal in regulation of mitochondrial dynamics. Our phosphoproteomics data suggest that paradoxical reactivation of mTORC2 kinase during fasting stimulates mitochondrial fission by phosphorylating novel protein targets at MAMs, including NDRG1, N-Myc downstream regulated 1. Interestingly, the NDRG1S336A phosphorylation-deficient mutant recapitulates the effects of loss of mTORC2 on mitochondrial fission/respiration, while phosphomimetic NDRG1S336D stimulates mitochondrial fission. Intriguingly NDRG1, but not the NDRG1S336A phosphorylation-deficient mutant, interacts with Cdc42, a cytokinetic protein with the intrinsic GTP hydrolysis activity required for mitochondrial fission. Based on our preliminary data, we propose that activation of the mTORC2-phosphorylated NDRG1Ser336-Cdc42 axis at MAMs regulates mitochondrial fission during fasting, and that dysregulation of the mTORC2-NDRG1Ser336-Cdc42 axis leads to age-related loss of mitochondrial fission and respiration. Three aims are proposed that will characterize (Aim 1) mitochondrial dynamics and MAMs in aged and stressed livers; (Aim 2) determine if and how dysregulation of the mTORC2- NDRG1Ser336-Cdc42 axis causes age-related loss of mitochondrial fission; and (Aim 3) explore if synergistic use of rapamycin and co-expression of phosphomimetic NDRG1S336D will lead to gre...