Studying the formation of neuromuscular junctions in vitro

The neuromuscular junction (NMJ) is a specialised structure that acts as a chemical synapse between a motor neuron terminal and muscle fiber end plate. It allows transfer of neural signals resulting in muscular contraction. Although extensively studied, the NMJ still requires investigation, as much remains unknown regarding molecular physiology of the junction in health and in disease. This is in part a result of the challenges faced studying the NMJ in animals and lack of good in vitro models. This project describes the development of a novel co-culture system enabling the potential for the development of neuromuscular junctions in vitro. Neurons derived from pluripotent stem cells and an established myoblast lineage were used to produce a robust and reproducible co-culture model whereby neurons and muscle cells developed interactions possessing key characteristics of NMJs. Physiologically relevant structures including terminal boutons and points of co-localisation were observed and subsequently characterised in the model. At these points of co-localisation, acetylcholine receptor clustering and nuclei accumulation was evident. Evidence suggests that certain fundamental aspects of NMJ formation have recapitulated in vitro. Building on these observations, preliminary evidence of muscle cell contraction was observed after pharmacological manipulation of cultures. Other developmental aspects were also apparent, including neurite competition at the myotube surface. In addition, this project investigated the role of Rho A and ROCK signaling during the differentiation of the neurons and myotubes. These molecules are involved in actin cytoskeleton dynamics, but their involvement at the NMJ is poorly understood. This project provides evidence that ROCK-inhibition enhances the growth conditions of neurons and muscle cells whereby C2C12 myotube differentiation and neurite outgrowth was significantly enhanced. Combined, these data provide the potential to increase functional NMJ synapses per unit area, which could prove invaluable in the research of NMJ formation and the evaluation of drugs acting at these synapses.