The proposed research is designed to elucidate the cellular and molecular mechanisms underlying the recently identified roles of the different isoforms of NCAM in bringing about the structural and functional maturation of the neuromuscular junction required for normal motor function. It is specifically designed to define the role of the 140 and/or 120 kD isoform of NCAM in the down regulation of the immature, brefeldin A and L-type Ca+2 channel sensitive, immature vesicle cycling/release mechanism from the axon. It will also explore how the 180 kD isoform results in the appropriate localization of presynaptic molecules required for mature, effective transmission. Finally, it will elucidate the role of the highly conserved C-terminal domain of NCAM, which in concert with myosin light chain kinase and PCKe, has been shown by this work to be required to sustain effective transmission at adult synapses in response to repetitive stimulation. These goals will be achieved by electrical recordings and FM1-43 imaging of synapse formation in motoneuron myotube cultures from wild type mice or those lacking specific NCAM isoforms. Intracellular signaling and protein-protein interactions in isolated adult nerve-muscle preparations will be investigated by the introduction of specific blocking peptides into the presynaptic terminal. The formation of effective synapses is essential for normal neural function in both the peripheral and central nervous system. Mutant mice lacking NCAM, while viable and fertile, display motor defects as well as alterations in learning and memory. Similar defects are likely to occur in humans with mutations in the NCAM gene. In addition, these studies will provide insight into steps in presynaptic maturation and vesicle cycling mechanisms that are required for junctions to function at high but physiological repetition rates.
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