Trophic Factors in Muscle-Nerve Cross-Talk Signaling Augment Muscle Fiber and Motor Endplate Development

Abstract

Synaptogenesis requires complex coordination between the terminating motor neuron and the developing myofiber endplate. Cross-talk research has focused on in vivo models or singular treatments with known signaling molecules identified from these animal studies. However, in vivo models are inefficient at measuring dynamic signaling changes due to assay resolution and cost. Further, despite advances in culture methods relying on microfluidic platforms, much remains unknown about the dynamic cross-talk between these two key cell types. As such, there is an unmet investigation into simple and reproducible coculture studies. In this study, we characterize both myoblast (C2C12) and motor neuron (NSC-34) changes that occur in either a conditioned media model, a transwell coculture, and a 2D migration coculture. We successfully demonstrate repeatable changes in synaptogenesis with ~38% increase in Chrng protein levels (p < 0.05) in each model, increased myotube alignment in cocultured myoblasts measured with FFT analysis, and show motor neurons are preferentially chemo-attracted to myotubes without the use of neurite-path constraining microfluidics. Lastly, we identified a potential new signaling protein responsible for motor endplate development, apolipoprotein E (ApoE). This coculture approach reveals changes to myotube myogenesis and synaptogenesis providing a consistent platform for cross-talk and pathway analysis for future studies.