Project Summary Aging skeletal muscle undergoes chronic denervation, which leads to decreased muscle mass and function in older adults. The neuromuscular junction (NMJ), the key structure that connects motor neuron nerves with muscle cells, shows increased defects with aging, and is a main contributor to chronic muscle denervation with aging. Evidence from various species indicates that with aging, NMJs from type II fast-twitch skeletal muscle fibers show more structural and functional deterioration than type I slow-twitch fibers. But how this process is regulated is largely unknown. A better understanding of the mechanisms regulating skeletal muscle type II fiber-type specific NMJ deterioration with aging could be critical to identifying novel treatments. Caspases, especially caspase-3 and caspase -9, are found at NMJ in various muscle diseases with endplate myopathies, where they lead to either subsynaptic nuclear apoptosis or the NMJ terminal Schwann cells apoptosis. Caspase-3 also plays a critical role in modulating the formation of postsynaptic nicotinic acetylcholine receptor clusters during early development of skeletal muscle, adding another non-apoptotic role of caspase in synapse refinement in the skeletal muscle. However, here the role of these caspases at the NMJ in aging skeletal muscle is still unknown. Our preliminary data revealed that caspase-3 and caspase-9 activity and intensity at NMJ increase with aging, mainly in fast-twitch skeletal muscles of C57/BL6 mice. In addition, the enrichment of these active caspases at NMJ coincides with enrichment of protein kinase A (PKA) regulatory subunits RII (PKA-RII? and PKA-RII?), protein kinase C subtypes (PKC? and PKC?), and cardiac troponin T (cTnT). Findings from our lab and others show that a) cTnT may regulate PKA, PKC intensity at NMJ, and b) both PKA and PKC regulate caspase activity. Added to this evidence, our new preliminary data strongly indicate a potential role for cTnT-PKA/PKC-caspase signaling at NMJ in aging fast skeletal muscle fibers. To extend these findings, we propose the current project, led by a New Investigator with a well-established team of co- investigators. Thus, the goals of this exploratory R21 project are to: (1) Determine whether enrichment of active caspase-3 and caspase-9 at the NMJ is aging dependent and specific to fast skeletal muscle fibers; (2) Determine the effects of caspase activation and inhibition on skeletal muscle NMJ structure and function; and (3) Determine whether cTnT-PKA/PKC signaling regulates caspase activation at NMJ. Our substantive preliminary data suggest that the proposed study, if successful, could establish proof-of-principle for a novel role for cTnT-PKA/PKC-caspase signaling in regulating NMJ structure and function. These data could suggest useful targets for future prevention and therapy of age-related decline in muscle function.