During the project, we established an integrated experimental and technological framework to investigate cellular interactions in the neuromuscular system across multiple spatial scales. A central achievement was the implementation of single-cell RNA sequencing using the iCell8 platform, enabling the combined analysis of transcriptomic profiles and morphological features of individual cells. Using this approach, we generated comprehensive datasets from peripheral nerves along the proximo-distal axis and identified previously unrecognized heterogeneity among myelinating Schwann cells. Distinct Schwann cell populations were found to exhibit specific molecular programs associated with axonal caliber and functional specialization, providing a mechanistic explanation for the selective vulnerability of axons in neuropathies. To complement these findings, we established spatial transcriptomics and advanced imaging approaches, including tissue clearing and volume electron microscopy, allowing the investigation of cellular interactions within intact tissue architecture. These analyses revealed pronounced spatial organization of neuropathic pathology, including region-specific inflammatory responses with increased immune cell infiltration in proximal nerve segments. In addition, we identified transcriptional and cellular alterations in the spinal cord, including activation and spatial reorganization of microglia around motoneurons, indicating a remote central nervous system response to peripheral nerve dysfunction. To dissect causal mechanisms, we developed novel mouse models enabling spatially restricted induction of glial dysfunction, including conditional overexpression of the neuropathy-associated gene Pmp22 in defined nerve compartments. These models allow direct investigation of how local perturbations propagate along the neuromuscular axis. In parallel, we established focal demyelination paradigms to study local and distant effects of myelin damage. Building on complementary work in the central nervous system, we demonstrated that persistent myelin ensheathment by dysfunctional glial cells can compromise axonal integrity, highlighting the importance of glial support functions for neuronal survival (Schäffner et al., Nature Neuroscience, 2023). Together, these findings establish a multi-scale framework linking glial heterogeneity, spatial organization of pathology, and axonal degeneration in neuromuscular disease.
