Synapse formation requires induction of specialized structures in both presynaptic and postsynaptic cells. The """"""""agrin hypothesis"""""""" states that postsynaptic differentiation at the neuromuscular junction is induced by agrin, a protein secreted by the nerve terminal. Here we propose to extend the agrin hypothesis by assaying in vivo the function of agrin isoforms, domains and agrin receptors during development of embryonic neuromuscular junctions in Xenopus. Overexpression of these proteins will be accomplished by injection of RNAs into early embryos. This novel system will provide new in vivo tests of the molecular mechanisms of embryonic synapse formation.
The specific aims are as follows: (1) To determine synapse-organizing activity of agrin isoforms and domains at the embryonic neuromuscular junction. Neuronal and muscle agrin isoforms and domains with different binding sites will be overexpressed to determine their ability to organize postsynaptic molecules. (2) To examine the role of two muscle cell agrin-binding molecules, MuSK (muscle-specific kinase) and alpha-dystroglycan, in postsynaptic differentiation in vivo. These proteins are putative agrin receptor and co-receptor, respectively. Overexpression of these and a dominant negative mutant of MuSK (lacking kinase domain) will be used to assess their role in agrin-induced postsynaptic differentiation. (3) To explore the role of agrin and MuSK in presynaptic nerve terminal differentiation. Overexpression should indicate if these molecules also regulate presynaptic differentiation at the embryonic synapse. Using this novel experimental approach, we can test the function of any synaptic molecule (or parts thereof) in embryonic synapse formation. This project has relevance to molecular mechanisms of synaptic plasticity in the brain and to mental retardation often associated with Duchenne muscular dystrophy. In this disease, dystrophin is affected, and alpha-dystroglycan, with which it is associated and which also binds agrin, could be defective in a synaptic role. These molecules are concentrated in some CNS neurons. They may be involved in synapse formation and remodeling in CNS and nerve-muscle synapses.
