Age-associated dysregulation of post-synaptic mitochondria perturbs re-innervation kinetics

Age-associated degeneration of neuromuscular junctions (NMJs) contributes to sarcopenia and motor decline, yet the mechanisms linking mitochondrial dysfunction to impaired NMJ regeneration in aging remain poorly defined. Here, we demonstrate that post-synaptic mitochondria are significantly diminished in both quantity and bioenergetic function in aged skeletal muscle, correlating with increased denervation and delayed reinnervation following nerve injury. Single-nucleus RNA sequencing of myofibers before and after sciatic nerve crush revealed that sub-synaptic myonuclei in aged muscle exhibit attenuated induction of mitochondrial gene programs, including oxidative phosphorylation, biogenesis, and import. To test whether these deficits are causal, we developed a muscle-specific CRISPR genome editing approach targeting CHCHD2 and CHCHD10—two nuclear-encoded mitochondrial proteins that localize to the intermembrane space and interact with the mitochondrial contact site and cristae organizing system (MICOS). Knockout of CHCHD2/CHCHD10 in young muscle recapitulated aged phenotypes, including mitochondrial disorganization, reduced ATP production, NMJ fragmentation, and delayed reinnervation. Transcriptional profiling of NMJ nuclei from knockout muscles revealed impaired pseudotime progression, failure to activate mitochondrial remodeling programs, and elevated stress signatures. These findings establish a critical role for sub-synaptic mitochondrial integrity in sustaining NMJ stability and regenerative capacity and identify CHCH domain-containing proteins as key regulators of post-synaptic mitochondrial function during aging and injury. Overall design: Tibialis anterior muscles from young (2–5 months) and aged (23–29 months) mice were collected at 0, 7, and 14 days after sciatic nerve crush injury. Samples include uninjured and post-injury timepoints. Experimental groups consist of age-matched controls and injured muscles, with additional groups receiving intramuscular injections of AAV targeting CHCHD2/CHCHD10 or saline (PBS). All conditions include biological replicates. Young and aged timepoints include 2–3 biological replicates per group, and AAV vs. PBS conditions include 3–4 replicates per timepoint. Both multiplexed and non-multiplexed libraries were generated across batches.