The long-term goal of our research is to understand molecular mechanisms underlying the formation of the neuromuscular junction, a cholinergic synapse between motoneurons and skeletal muscle fibers. This application focuses on signaling mechanisms in regulation of nicotinic acetylcholine receptor (AChR) synthesis. AChR is a pentameric ligand-gated ion channel that is concentrated at the neuromuscular junction. AChR expression is regulated both spatially and temporally during development. For example, AChR synthesis become confined at adult neuromuscular junction and when the synapse matures, the receptor subunit composition changes from gamma-subunit containing receptor to epsilon-subunit containing receptor. Neuregulin, a factor from motoneurons, increases whereas electrical activity of muscle cells inhibits AChR subunit expression. However, molecular signaling mechanisms in the regulation of AChR synthesis remain largely unknown. Our recent studies demonstrated that although neuregulin increases both epsilon- and gamma-mRNA, the signaling mechanisms involved are different: Neuregulin-mediated epsilon-mRNA increase requires de novo protein synthesis while that of gamma-subunit does not. We have also demonstrated that neuregulin-increased transcription of epsilon-subunit gene requires c-JUN and that neuregulin increases gamma-mRNA stability. Moreover, our results, both published and preliminary, suggest that two kinase signaling pathways are involved in regulating AChR synthesis: ERK activation leads to an increase in all five subunit mRNA whereas expression of gamma-mRNA appears to be inhibited by JNK. Based on these results, we hypothesize that synapse-specific synthesis and gamma-e subunit switch are mediated by integration of ERK and JNK signaling pathways.
The specific aims of the proposed research are designed to test this hypothesis. Specifically, we will 1) determine whether transcriptions of epsilon- and gamma-subunit genes are regulated by different mechanisms; 2) determine which signaling pathways regulate AChR mRNA stability; and 3) study signaling mechanisms of electrical activity to inhibit AChR expression and the cross-talk between neuregulin- and electrical activity-activated signaling pathways. The results of proposed experiments should contribute not only to our understanding of signaling mechanisms in regulation of AChR synthesis, but also to a more general understanding of synapse formation.
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