Ion Imaging in Neuro-Muscular Organoids (iNEMO)

The neuromuscular (NM) junction (NMJ) is the key synapse between motor neurons and skeletal muscle fibers, essential for voluntary movement. Its structural and functional impairment is central to many NM disorder. Despite recent progress, important questions remain about the molecular mechanisms that govern NMJ development, maintenance, and plasticity. In particular, emerging evidence points to noncoding RNAs as important regulators of muscle and motor neuron physiology, but their contribution to NM circuits requires deeper definitions. These challenges align with recent advances in in vitro modelling, such as NM organoids (NMOs), which provide innovative platforms to study the NMJ niche within a complex, human-derived architecture. In our work, NMO manipulations are combined with cee-type-specific molecular profiling and advanced imaging to map how perturbations in these regulatory layers reshape NM connectivity. We further couple these structural and molecular readouts with functional assessment on Multi-Electrode Array platforms, which allow monitoring of network activity, synaptic transmission and coordinated muscle contraction dynamics. Through this multimodal strategy, we begin to define how noncanonical gene networks influence NMJ assembly, maturation, and synaptic efficacy. Overall, our approach offers a refined, cell-type-resolved view of NM communication and opens new avenues to interrogate the noncoding regulatory landscape underlying human NMJ physiology and its vulnerability in disease.