The long range goal of this research is to understand, in molecular terms, how synapses form during embryonic life and how they are maintained in adult animals. These events can be studied with great precision at the vertebrate neuromuscular junction, a synapse whose functional, morphological and biochemical phenotype is understood better than any other chemical synapse. This proposal is focused on ARIA, a polypeptide that was purified based on its acetylcholine receptor inducing activity in embryonic skeletal myotubes. ARIA probably acts at developing and mature junctions to promote the synthesis of ACh receptors and their accumulation in the postsynaptic muscle membrane. One immediate goal of the proposal is to study the processing of ARIA: how the synthesis is regulated in motor neurons, how it is transported to the motor axon terminals and how it is displayed in the synaptic cleft. Membrane binding sites will be studied in detail to determine which type correlates best with physiological responses. Another goal is to characterize ARIA binding proteins in the muscle membrane, and other binding proteins in the extracellular matrix that may modulate its action. Another important set of experiments is designed to define ARIA's spectrum of action at various stages of endplate development. This potent differentiation factor may regulate several genes that encode ion channels in the presynaptic and postsynaptic membrane, proteins in the postsynaptic cytoplasm, and proteins that are destined for the extracellular matrix. It may, in short, be a master switch that regulates synapse specific gene expression. ARIA is synthesized as a transmembrane precursor (proARIA), and several alternatively spliced isoforms have been uncovered. A final goal of this research is, therefore, to determine exactly which isoforms are expressed in motor neurons and the precise role for each one. The planned experiments depend on recombinant DNA, biochemical, electrophysiological and morphological techniques. Identification of ARIA as one of the first trophic factors operating at the neuromuscular junction opens new areas of study regarding the two-way, nutritive relationship between motor neurons and their synaptic partners in the periphery. ARIA was purified from the brain, and it is likely that it exerts powerful trophic effects in the central nervous system as well as in the periphery. Judging from its location in cholinergic neurons throughout the nervous system and in some non-cholinergic cells, our studies at the neuromuscular junction will have immediate import for studies of normal and abnormal development in CNS.
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