The perijunctional zone is a molecularly distinct muscle subdomain altered in Duchenne muscular dystrophy

The neuromuscular junction (NMJ) is a well-established model for synapse development, structure, and function. Surrounding the NMJ is a narrow perijunctional zone (PJZ), enriched in muscle-specific voltage-gated sodium channels that prevent synaptic fatigue. Despite this role, the PJZ remains poorly characterized. To determine its molecular composition, we engineered mice to express the biotin ligase TurboID fused to the cell adhesion molecule neurofascin (Nfasc), and that localizes to the PJZ through ankyrin scaffolding proteins. Using proximity proteomics, we identify numerous PJZ-associated proteins, including Perilipin 4 (Plin4), that are highly enriched and clustered at the PJZ. We also perform proximity proteomics on the PJZ of mdx mice, a model of Duchenne muscular dystrophy. We find broad changes in PJZ composition, including significantly reduced PJZ Plin4. Although Plin4 is linked to lipid droplet storage and autosomal dominant myopathy, Plin4 knockout mice exhibit no obvious neuromuscular phenotype or changes in lipid droplet distribution, suggesting a gain-of-function disease mechanism. These findings establish the PJZ as a molecularly distinct subdomain of skeletal muscle and provide insight into its potential roles in neuromuscular function and disease. Proximity proteomics of the neuromuscular junction perijunctional zone (PJZ) reveals a distinct protein network enriched in Plin4 and altered in dystrophic muscle, identifying the PJZ as a specialized subdomain with potential roles in neuromuscular function and disease.