SUMMARY Skeletal muscle is the most abundant tissue in the human body, with a wide range of functions, from locomotion to breathing, vision, blood pressure control, metabolism, and endocrine regulation. Muscle homeostasis and regeneration are largely dependent on a small population of stem cells that reside quiescent in the muscle tissue until activated by a stimulus, typically an injury. Tight regulation of the activation process is essential for tissue homeostasis: too little activation would impair the tissue’s ability to regenerate in response to damage, while too much activation could lead to tumor formation or premature depletion of the stem cell pool. Neutrophils attracted to injured muscle secrete proteolytic enzymes, and we have recently observed that one such enzyme, neutrophil elastase, appears to promote muscle stem cell activation. Moreover, we have observed that triple knockout of all three neutrophil serine proteases practically abrogates muscle regeneration in response to acute injury. The overreaching goal of this proposal is to investigate the physiological function of neutrophil serine proteases in the muscle stem cell niche and decipher the underlying molecular mechanisms. Specifically, our first aim is to define how the muscle stem cell niche as a whole (cells, matrix and soluble factors) are affected by loss of each NSP and all three combined. Our second aim is to identify the molecular mechanisms that are regulated in a cell-autonomous manner by the NSPs in the context of MuSC activation and first division. Finally, our third and last aim is to validate in vitro and in vivo the candidate biological functions discovered in aim 1 and 2. In conclusion, we expect that, in addition to answering the main question of which processes and mechanisms neutrophil serine proteases regulate during muscle regeneration, and how, the results from this work will provide a platform for future investigations into how stem cell niches are continuously remodeled by proteases to ensure prompt and efficient regeneration as well as stem cell and tissue homeostasis.

NARRATIVE The central hypothesis of this proposal is that the muscle stem cell niche is an everchanging environment in which proteases play an important role by cleaving and activating, or neutralizing, signaling molecules, their receptors, and the extracellular matrix. Our three specific aims are to define how the muscle stem cell niche is remodeled in the early hours and days after injury by the activity of neutrophil serine proteases. We will identify the cellular and molecular mechanisms regulated by neutrophil serine proteases in the context of muscle regeneration with a special focus on muscle stem cell activation, and further validate them in appropriate cell and animal model systems.