Neurons and their targets exchange information of many sorts as synapses are formed, maintained and modified. We are using the skeletal neuromuscular junction, the best-studied of all synapses, to identify some of the signals and signal transduction mechanisms that regulate synaptic differentiation. Previous studies revealed that some of the cues elaborated by motoneurons and myotubes are stably associated with the basal lamina (BL) that occupies the synaptic cleft between these two cells. Subsequently, we and others identified molecules that were concentrated in synaptic portions of the BL and that were capable of influencing pre- or postsynaptic differentiation in vitro. During the past few years, we have used gene targeting in mice to ask whether any of these molecules play critical roles in vivo. This genetic analysis revealed that agrin is a critical nerve-derived organizer of postsynaptic differentiation and that synaptic (beta2) laminins are muscle-derived organizers of presynaptic differentiation. Based on these results, we now propose to learn more about how agrin and synaptic laminins function. First we will use mutant and transgenic mice to assess individual roles of several beta2 laminins (called laminins -4, -9, and -11) that occupy the synaptic cleft. Second, we will use a panel of new motoneuron-derived cell lines to seek neuronal receptors responsible for the presynaptic effects of the beta2 laminins. Third, we will elucidate the mechanisms by which agrin activates its receptor, the muscle specific kinase (MuSK), and organizes postsynaptic differentiation. Fourth, building on the observation that some synaptogenesis does occur in the absence of agrin and laminins, we will identify additional factors that cooperate with these critical signals to organize pre- and postsynaptic differentiation. Finally, we will ask whether agrin or laminin-like proteins play roles in synaptogenesis in the brain. Through this work, we hope to gain insight into molecules and mechanisms necessary for construction of synapses, which are the fundamental information-processing units of the nervous system.
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