Dissecting the role of tissue-specific lncRNAs in muscle-nerve interaction using human neuromuscular organoids

The neuromuscular junction (NMJ) is the key synapse between motor neurons and skeletal muscle fibers, essential for voluntary movement. Its structural and functional impairments are central to various neuromuscular disorders, often requiring lifelong treatments. While recent advances have improved our understanding of NMJ biology, many questions remain unresolved, particularly regarding the molecular mechanisms governing neuromuscular development and maintenance. Among these, long noncoding RNAs (lncRNAs) have emerged as crucial regulators of muscle and motor neuron physiology, although their roles in neuromuscular systems are still poorly defined. This project aims to dissect the functional relevance of lncRNAs in neuromuscular communication. By integrating CRISPR-Cas9 genome editing with induced pluripotent stem cell (iPSC) technology and molecular interactome analyses, we investigate the roles of lnc-HSCHARME—previously identified as a myogenic regulator in murine models (1,2) —and lnc-nHOTAIRM1, a motoneuron-specific lncRNA (3). Using engineered iPSC lines with a targeted knockout of these lncRNAs, we generate co-culture systems of spinal motoneurons and myotubes, as well as advanced three-dimensional neuromuscular organoids (NMOs), which recapitulate the structural and functional complexity of the neuromuscular junction (NMJ). Through this model, we explore how muscle- and neuron-derived lncRNAs influence the NMJ's formation, maturation, and functionality. Our approach enables the analysis of cell-type-specific contributions to neuromuscular connectivity, offering novel insights into the noncoding regulatory landscape of human NMJ physiology.