The aim of this project is to investigate the mechanisms controlling formation of the neuromuscular junction in the in vitro innervated human muscle. This experimental model system permits the experimental manipulation of the neuromuscular junction and is virtually the only model for the investigation of synaptogenesis in humans. The roles of recently discovered muscle specific kinase, MuSK, and its activator, nerve-derived factor agrin, reported to control many aspects of synaptogenesis, will be investigated.
The specific aims of this proposal are to investigate the role of agrin and the muscle specific kinase, MuSK, in the accumulation of acetylcholinesterase (AChE), myonuclei, and mitochondria at the neuromuscular junction. Furthermore, the contribution of the nerve-derived AChE to the synaptic accumulation of AChE will also be examined. The numbers and distribution of nuclei and mitochondria will be examined at various stages of myo- and synaptogenesis. The localization of these organelles with respect to the neuromuscular junction will be determined. Furthermore, the localization of AChE will be examined during the process of synaptogenesis by histochemical and immunohistochemical staining, and the localization of the AChE mRNA by in situ hybridization. The role of agrin and MuSK in this process will be determined in the experimental model system. The activity of the agrin-MuSK signaling pathway will be blocked to examine if these molecules are involved in the relocalization of cellular organelles or AChE. Agrin will be added to aneural myotubes to investigate if it can fully simulate the effects of the nerve in the formation of the neuromuscular junction. If successful, these experiments will help define the role of agrin and MUSK in the organization of the neuromuscular apparatus and will contribute to our understanding of synaptogenesis and synaptic plasticity.
