Roles Of Neuregulin1 In Neuromuscular Junction Development

Neuromuscular junction (NMJ) development is a multistep process mediated by coordinated interactions between nerve terminals, target muscles, and peri-synaptic glial cells, and thus requires reciprocal signals derived from every cell type. Neuregulin1s (NRG1s) are a family of predominantly neuronal growth and differentiation factors that are important for many aspects of nervous system development. In this thesis, both the effects of NRG1 on NMJ development and reciprocal effects of neurotrophic factors on NRG1 expression were studied as a means to define the complex regulatory communication at the NMJ. Using the chicken embryo as a model, methods were developed to study the effects of NRG1 signaling on NMJ development in ovo using sequential, quantitative measures of NMJ formation including AChR cluster size and density, pre- and post-synaptic apposition, and alignment of peri-synaptic Schwann cells. Over-expression of soluble NRG1 increased AChR cluster density, but not size, in the early developmental stage, whereas, blocking NRG1 activity with a targeted antagonist had little effect. In the middle stage, the NRG1 antagonist led to a decrease of AChR cluster size in a temporal-specific and muscle type-dependent fashion. The NRG1 antagonist also altered the distribution of synaptic vesicles, suggesting NRG1 signaling can modulate the assembly of the pre-synaptic apparatus as well. In the late stage, pre- and post-synaptic apposition, and peri-synaptic Schwann cell alignment were modestly affected by the antagonist. In motor neurons, NRG1 expression at the axonal-Schwann cell and neuromuscular junctions is regulated by synaptic activity and neurotrophic factors, however, little is known about the mechanisms that control NRG1 isoform-specific transcription. Here we show that NRG1 expression in the chick embryo increases in motor neurons that have extended their axons and that limb bud ablation before motor axon outgrowth prevents this induction, suggesting a trophic role from the developing limb. Consistently, NRG1 induction after limb bud ablation can be rescued by adding back the neurotrophic factors BDNF and GDNF. Mechanistically, BDNF induces a rapid and transient increase in types I and III NRG1 I mRNAs that peak at 4 h in rat embryonic ventral spinal cord cultures. Blocking MAPK or PI3K signaling or blocking transcription with Actinomycin D blocks BDNF induced NRG1 gene induction. BDNF had no effects on mRNA degradation, suggesting that transcriptional activation rather than message stability is important. Furthermore, BDNF activates a reporter construct that includes 700bp upstream of the type I NRG1 start site. Protein synthesis is also required for type I NRG1 mRNA transcription as cycloheximide produced a super-induction of type I, but not type III NRG1 mRNA, possibly through a mechanism involving sustained activation of MAPK and PI3K. These findings suggest that, while not critical for development, NRG1 signaling can have important roles in fine-tuning multiple stages of NMJ development and that NRG1 isoform expression can be differentially modulated by highly responsive, transient transcriptional regulatory mechanisms mediated by neurotrophic factors and axon-target interactions. Understanding these mechanisms will be important for elucidating the role of NRG1 in both development and in pathological disorders of the nervous system