04ODenervated human muscle fibers promote reinnervation via neurotrophic factor release in SMA and ALS

Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS) are neuromuscular disorders characterized by the progressive degeneration of motor neurons, leading to skeletal muscle denervation, weakness, and atrophy. However, the molecular pathways underlying the muscle response to denervation in human tissue remain poorly defined. We performed single-nucleus RNA sequencing (snRNA-seq) and ATAC-seq on muscle biopsies from patients with SMA type 3 (n = 7), ALS (n = 4), and age- and sex-matched healthy controls (n = 5). Data were analyzed using a custom bioinformatics pipeline based on Seurat and Harmony. Key findings were validated by immunofluorescence (IF) on additional muscle samples. To assess functional implications, rat spinal cord neurons were cultured in vitro with factors identified from muscle transcriptomic analyses. Fibro-adipogenic progenitor (FAP) cells were isolated from patient and control samples and subjected to single-cell RNA sequencing to identify transcriptionally distinct subpopulations. snRNA-seq revealed a significant increase in satellite and inflammatory cell populations, along with a reduction in myofiber and smooth muscle cell numbers in SMA and ALS muscle. Pseudobulk analysis showed that genes upregulated in SMA and ALS were enriched for pathways related to vascular development, cell projection organization, cell adhesion, synaptic remodelling, and neuronal development. A subpopulation of muscle fibers was identified in both SMA and ALS, characterized by elevated expression of genes involved in synaptic maintenance and axonal guidance, including MUSK, LRP4, COL19A1, EFNA5, and NTN1. ATAC-seq confirmed accessible chromatin regions at loci related to neuromuscular junction maintenance and neuronal growth, suggesting transcriptional readiness for protein expression. Immunofluorescence confirmed increased expression of these proteins in patient biopsies. Furthermore, rat spinal cord neurons cultured with muscle-derived neurotrophic factors such as EFNA, NTN1, and BDNF exhibited enhanced dendrite outgrowth. FAPs from patient muscle displayed upregulation of extracellular matrix components and neurotrophic factors implicated in neuronal support and synaptic repair. Our study reveals that denervated muscle fibers actively engage in a compensatory, pro-regenerative response by releasing neurotrophic factors aimed at restoring neuromuscular connectivity. These factors may serve as valuable biomarkers for disease progression and represent promising therapeutic targets for enhancing reinnervation in SMA and ALS. Future work should explore their potential in combination therapies aimed at modulating the muscle microenvironment to support motor neuron recovery.