The long range objective of our research is to describe, in molecular terms, crucial events that underlie the formation and maintenance of synapses. This proposal is devoted to characterization of the structure, function, and biological significance of a protein that stimulates acetylcholine receptor (AChR) synthesis in embryonic muscle cell. The protein, named ARIA for its acetylcholine receptor inducing activity, has been embryonic chick brain, and a cDNA (designated 65-21) that probably encodes ARIA has been isolated from an embryonic chick brain cDNA library. The 65-21 cDNA predicts a protein of 267 amino acids that is homologous to the mammalian prion protein.
The first aim i s to prove that ARIA is, in fact, the protein encoded by the 65-21 cDNA. Attempts will be made to separate ARIA from the 65-21 encoded protein (recognized with an anti- fusion protein antiserum) by chromatography, electrophoresis and selective immunoprecipitation. Attempts will also be made to express ARIA by transfecting neuronal cell lines and postmitotic neurons with plasmids containing the 65-21 coding region. The role of ARIA at developing chick neuromuscular junctions will be tested in vitro and in vivo with antibodies that block the effect of ARIA on cultured myotubes and with 65-21 anti- sense constructs. Quantitative fluorescence microscopy will be used to measure the induction of AChR clusters in control and antibody blocked preparations. The spectrum of ARIA's action at the neuromuscular junction will be explored in regard to other (non-AChR) protein known to be concentrated at mature motor endplates in the postsynaptic membrane, the synaptic cleft and the postsynaptic cytoplasm. These experiments involve patch clamp microelectrode recordings of whole cell currents and individual ion channels as well as fluorescence video microscopy. The mechanism of ARIA's action will be studied by characterizing 125I ARIA binding sites and measuring cytoplasmic Ca++, a likely intracellular messenger. Results obtained at chick nerve - muscle synapses will be extended to mammalian junctions with particular attention paid to the appearance of adult - type AChRs (60 pS, 1 msec channels) and the corresponding regulation of mRNA that encodes the epsilon subunit of the AChR. Experiments are also planned to define the distribution of ARIA within the brain by immunohistochemistry, and to determine the effect of ARIA on the differentiation and survival of dissociated neurons maintained in culture. Neuronal, nicotinic AChRs will be the first assayed. Studies of trophic factors in the developing nervous system have import for mechanisms that maintain synapses throughout adult life. The likely homology to the prion protein is significant in this regard. An altered form of this protein has been implicated in the pathogenesis of a variety of neurodegenerative disorders of humans as well as animals.
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