The molecular mechanisms by which synapses form during development and are modified in the adult are largely unknown. Many higher brain functions such as learning and memory are thought to involve changes at the synapse. Therefore understanding the mechanisms by which cell-cell interactions at the synapse influence synaptic protein expression will provide insight into synaptogenesis and synapse modulation. The long term goal of this research proposal is to characterize these mechanisms. The neuromuscular junction provides an ideal system for studying these processes since it is homogeous, accessible to experimental manipulation and is the best studied synapse in the nervous system. The nicotinic acetylcholine receptor (nAChR) mediates communication across this synapse and serves as an excellent. marker for studying how pre- and post-synaptic cell interactions regulate synaptic protein expression. During development of the neuromuscular junction the level, distribution and properties of this receptor change. Many of these changes are a result of muscle innervation influencing the pattern of nAChR gene expression. This proposal addresses the molecular mechanisms by which muscle innervation suppresses expression of extrajunctional nAChRs and induces expression of nAChRs at the endplate. Gene transfer experiments win be used to identify cis-acting elements of the nAChR genes mediating electrical regulation and endplate-specific expression. These regulatory cis-acting sequences will be further characterized by mutagenesis and DNA-protein interaction studies. The trans-acting proteins that bind these DNA sequences will be purified by ligand-affinity chromatography or expression library screening. Second messenger systems mediating the effects of muscle activity and innervation on nAChR gene expression will be characterized by perturbing these systems using pharmacological agents and introducing genes into muscle cells that encode protein kinase enzymes or their peptide inhibitors.
