The long-term goal of these studies is to determine the mechanisms by which extracellular matrix (ECM) molecules influence the development of embryonic neuromuscular junctions. Knowledge of factors that influence synapse formation may aid in understanding neuromuscular disorders such as myasthenia gravis. The specific hypothesis to be tested is that molecules in the synaptic basal lamina direct the differentiation of neuromuscular junctions in the embryo. In particular, agrin, a basal lamina protein that aggregates acetylcholine receptors (AChRs) on cultured muscle cells, may cause the formation of the postsynaptic apparatus. Although agrin-like proteins are found in most basement membranes, they also present in a population of ventral spinal cored neurons. In the chicken embryo, extracts of spinal cord and brain contain biologically active agrin, while only inactive agrin-like molecules have thus far been isolated from other tissues. Thus we hypothesize that neuronal agrin is released by nerve terminals, binds to synaptic basal lamina and aggregates postsynaptic AChRs. Neuronal agrin and agrin in basement membranes may be related isoforms with different amino acid sequences, hence different biological activities.
Specific aims are: (1) To characterize the neuronal and muscle basement membrane forms of chicken agrin. The agrin-like proteins in brain, spinal cored and muscle will be characterized by immunoblotting with antibodies against individual bands of agrin-like proteins purified from the ECM and from brain, and by comparing the AChR-aggregating activity of purified proteins from these tissues. The agrin-like proteins secreted by neurons and muscle cells will be identified by immunoprecipitation. (2) To identify cDNA sequence(s) coding for agrin, and to determine the pattern of expression of agrin mRNA(s) during development. cDNA clones coding from chicken agrin-like proteins will be obtained, for sequencing and comparison with sequences of other ECM components, for studies of developmental regulation of agrin mRNA and for in situ hybridization. (3) To explore the function of agrin and other basement membrane molecules in neuromuscular junction development. Purified proteins and antibodies against the will be assessed for their ability to enhance or inhibit AChR clustering at developing neuromuscular synapses in culture. Relative contributions of nerve and muscle to agrin in synaptic basal lamina will also be investigated by selectively labeling agrin from each tissue in nerve-muscle co-cultures.
