Stem cells are responsible for homeostasis and repair of many tissues in the body, and stem cell exhaustion is one of the hallmarks of aging. In recent years, work from our group and others has drawn attention to the mechanisms by which the resilience of muscle stem cells (MuSCs) declines with age at the population level and at the single cell level. As one example, we have shown that a signaling pathway involving Notch activation and increased p53 activity prevents MuSCs from undergoing a form of cell death, mitotic catastrophe, as they activated out of quiescence and enter the cell cycle. This Notch/p53 axis declines with age and leads to an increased propensity of aged MuSCs to undergo mitotic catastrophe, leading to a decline in MuSCs over time. Furthermore, in preliminary studies, we have found that quiescent MuSCs exhibit evidence of replicative stress and that an ATR response to that stress prevents cell cycle entry and preserves the MuSC population. We have also found that dietary interventions, in particular fasting and a ketogenic diet, enhance MuSC resilience, perhaps mediated by HDAC activity and p53 acetylation. Together, these observations highlight robust processes to maintain MuSC resilience and prevent stem cell depletion, processes that go awry during the aging process. The primary goals of this proposal are to explore these processes in more detail, to identify the molecular mediators of each, to use unbiased screens to identify as yet unknown mediators, and to pursue rejuvenating interventions that restore resiliency to aged MuSCs. To address these issues, this proposal is divided into three Specific Aims. Aim 1: To examine changes of the Notch/p53 axis as a cause of the age-related reduction of MuSC resilience. We will use novel genetic models to modulate Notch signaling in MuSCs and test for resilience signatures of cells protected against mitotic catastrophe. We will also assess resilient cells for evidence of mediators downstream of p53 using single cell RNA-seq. Aim 2: To examine replicative stress and the ATR response in young and old MuSCs. We will examine a potential downstream mediator of ATR, CDK12, identified in a phosphoproteomic screen, in preserving resilience of the population. We will also test whether this replicate stress response pathway changes with age and protect MuSCs from undergoing mitotic catastrophe when they activate out of quiescence. Aim 3: To elucidate the mechanisms by which ketosis promotes MuSC resilience. We will test for enhancement of resilience using three different ketosis-inducing interventions, and we will test for mechanisms of action based on the well- documented role of the major circulating ketone body, beta-hydroxybutyrate (βHB), as an inhibitor of histone deacetylases (HDACs). We will also test whether ketosis enhances MuSC resilience, at least in part, by promoting p53 activity and preventing mitotic catastrophe. Together, these studies will advance our understanding of the mechanisms of stem cell resiliency and how to enhance the resilience of aged stem cells to promote tissue homeostasis and repair across the lifespan.
