Investigating Mechanisms of Mitochondrial Regulation in Muscle Stem Cells and Neuromuscular Regeneration in Aging

Sarcopenia, the age-associated atrophy of muscle tissue, affects a significant proportion of elderly patients, leading to significant loss of muscle mass, strength, and motor function. In the interest of promoting healthier aging, it is necessary to research the mechanisms that fail in aged muscle and uncover therapeutic strategies to restore function. One hallmark of muscle aging is the decline of muscle stem cells (MuSCs), the resident adult stem cell population that is responsible for regenerating muscle fibers upon injury. In age, MuSCs experience defects in their ability to maintain quiescence, a dormant state that preserves the stem cell reservoir for regenerative needs across lifespan. The dysregulation of MuSC quiescence leads to the eventual exhaustion of the stem cell pool and a decrease in its regenerative capacity. The neuromuscular junction (NMJ) is the connection point between a muscle fiber and its respective motor neuron that enables muscle contraction. In age, NMJs become denervated due to mounting defects in pre-synaptic motor neurons and post-synaptic myonuclei (PSMs), leading to a decline in motor units and force generation. The age-related decline of both MuSCs and NMJs is associated with defective mitochondrial function and oxidative stress, however there is still much to uncover about the mechanisms that drive these defects. The primary objective of this dissertation is to elucidate novel pathways and genes through which mitochondrial function contributes to the disruption of MuSC quiescence and maintenance of the NMJ by PSMs. The first aim identifies the attenuation of Vitamin A metabolism in age as a potential driver of mitochondrial dysfunction(definition) and loss of quiescence in MuSCs. In this study, we administered a Vitamin A-deficient diet to young mice to recapitulate an aged-like phenotype in young MuSCs, which hampered mitochondrial metabolism and induced oxidative stress, leading to premature activation in freshly isolated MuSCs. Next, we set out to overcome the effects of reduced Vitamin A metabolism in aged MuSCs by stimulating the downstream receptors of Vitamin A’s active metabolite, retinoic acid. In doing so, we were able to augment mitochondrial respiration in myoblasts, restrain aged MuSCs from activation, and reduce mitochondrial reactive oxygen species. Stimulating this pathway in vivo similarly enhance quiescence regulation, as well as fatty acid oxidation and NAD metabolism. Next, we investigated the ability of STRA6, a molecule in the retinoic acid signaling pathway, as a potential modulator of mitochondrial function and oxidative stress. Knocking down gene expression of Stra6 in C2C12 myoblasts prompted a depolarization of the mitochondrial membrane potential, stark increases in oxidative stress, defects in mitochondrial respiration, and a metabolic switch to glycolysis. In the second aim, we focus on the role of PSMs and their mitochondria in the regulation of NMJ stability following denervation injury in age. Using single-nucleus RNA sequencing (snRNA-seq) we identified age-associated defects in mitochondrial function in PSMs that hindered the denervation response. We then identified two downregulated genes, Chchd10 and Chchd2, and performed in vivo CRISPR-Cas9 knockdown in myonuclei to probe their role in muscle denervation. We found that knocking these genes down significantly hindered mitochondrial function and altered NMJ morphology in muscle before injury, leading to decreases in muscle mass. These findings fill existing knowledge gaps regarding the regulation of mitochondrial function in aged muscle and may help inform new rejuvenation strategies that aim to restore MuSC function and improve NMJ integrity.