The Neuromuscular Junction (NMJ) is a specialized synapse between the motor neurons and muscle fibers. NMJ's are formed by a series of developmental changes in neurites, extracellular matrix and myotubes involving a myriad of biological events including cell proliferation and death, elaboration and retraction of processes and development of membrane specializations. Currently, the exact mechanism of synaptogenesis remains unclear; however, it is generally accepted that factors released by nerves (e.g. Agrin, ARIA, adhesion molecules) may facilitate and co-ordinate the process. Consistent with this hypothesis, it has been demonstrated that a neurally derived molecule named Acetylcholine Receptor Inducing Activity (ARIA) induces the expression of certain NMJ-specific proteins (e.g. Acetylcholine receptors, Sodium channels) in cultured myotubes. By analyzing the biochemical associations and mechanism of actions of ARIA in muscle, we hope to make progress toward our long term goal of understanding how the complex developmental changes at the NMJ are choreographed as well as provide insights into diseases affecting the neuromuscular system. More specifically, the experiments proposed here will allow us to identify molecules that are biochemically associated with ARIA and characterize the nature of their association. This analysis will entail using biochemical methods to determine the affinity and possible structural selectivity of ARIA towards candidate proteins as well as identifying novel ARIA-binding proteins present in cultured cells as well as muscle and nerve. Additionally, we will examine the mechanisms by which ARIA influences synapse formation, both at the level of gene expression and post- translational modification of preexisting proteins as well as attempt to elucidate the signaling pathways utilized by ARIA to achieve these changes. The possibility of ARIA being important in human neuromuscular diseases will be investigated by isolating and characterizing highly informative genetic markers for the human homologues of ARIA and ARIA- receptor genes and analyzing diseases considered good candidates based on patho-physiological grounds or genetic map position.
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