Schwann Cell Derived Signals Enhance AChR Clustering in C2C12 Myotubes

Abstract

The neuromuscular junction (NMJ) is a tripartite structure made up of the pre‐synaptic motor neuron terminal, the post‐synaptic muscle end plate, and the perisynaptic Schwann cells (SC). Degeneration of the NMJ is strongly associated with age‐associated muscle mass and strength declines as well as other neurodegenerative diseases; however, the cellular interactions and signals that initiate the degeneration are poorly understood. In particular, understanding of the role of perisynaptic SCs in NMJ maintenance is scant, whereas the signaling between motor neurons and muscle fibers has been extensively studied. For example, agrin is a motor neuron derived secreted factor that is the primary signal for the induction of acetylcholine receptor (AChR) clustering in myofibers, and treatment of myotubes in culture with agrin has been shown to similarly promote AChR clustering in the absence of neurons. The presence of SCs at motor nerve terminals implicates signaling from these cells in both NMJ formation and maintenance. Thus, the goal was to determine if SC derived signals impact AChR clustering. We hypothesized that SC derived factors would potentiate agrin‐induced aneural AChR clustering in C2C12 myotubes. Differentiated C2C12 myotubes were treated with agrin for either 10 minutes or 4 hours to minimally or maximally induce AChR clustering, respectively. Additional myotubes were treated with agrin for 10 minutes followed by treatment with conditioned media (CM) derived from SW10 Schwann cell line cultures for the remaining 4 hours. Finally, additional myotube cultures were treated for 4 hours with CM alone or normal complete media as controls. The number of clusters and mean cluster area were increased in myotube cultures treated with the combination of a 10‐minute agrin pulse followed by and CM from SW10 cells compared to cultures treated for either 10 minutes or 4 hours with agrin. Neither cluster number nor area were elevated compared with controls in cultures treated with CM alone. These data suggest that SC‐derived signals can potentiate agrin‐induced post‐synaptic AChR clustering in vitro. We are currently investigating the critical SC‐derived signals that potentiate AChR clustering to determine factors that may provide therapeutic benefit in neurodegenerative phenotypes including aging.