Decoding muscle-resident Schwann cell dynamics during neuromuscular junction remodeling

Understanding neuromuscular junction (NMJ) repair mechanisms is essential for addressing degenerative neuromuscular conditions. Here, we focus on the role of muscle-resident Schwann cells in NMJ reinnervation. Using an accepted model of progressive NMJ degeneration, Sod1-/- mice, we identified a clear NMJ ‘regenerative window’ that allowed us to define cellular and molecular regulators of synapse remodeling and muscle fiber reinnervation. High-resolution imaging and single-cell RNA sequencing provide a detailed analysis of Schwann cell number, morphology, and transcriptome revealing multiple subtypes, including a previously unrecognized terminal Schwann cell (tSC) population expressing a synapse promoting signature. We also discovered a novel SPP1-driven cellular interaction between myelin Schwann cells and tSCs and show that it promotes tSC proliferation and reinnervation following nerve injury in wild type mice. Our findings offer important insights into molecular regulators critical in NMJ reinnervation that are mediated through tSCs to maintain NMJ function. S100GFPtg and S100GFPtg Sod1-/- mice were bred at the University of Michigan, were fed normal chow ad libitum, and housed 12:12 hour light-dark cycle under the UofM veterinary staff supervision. All procedures were approved by the University Committee on the Use and Care of Animals at UM and were in accordance with the U.S. National Institute of Health (NIH). We used 2-month-old male mice from each genotype (n=4 per group). We simultaneously harvested and processed hind limb muscles (Tibialis Anterior and Gastrocnemius) from both groups. Using Fluorescence-Activated Cell Sorting, we separated GFP+ and GFP- single cells. The sorted cells were then prepared for library construction and underwent single-cell RNA Sequencing, all processed in the same batch for consistency.