Our long-term goal is to understand the molecular basis of synapse formation in vertebrates. Currently, no target-derived signals have been firmly established as regulators of nerve terminal formation in vertebrates. This application focuses on a muscle-derived cue that controls nerve terminal formation at the neuromuscular junction, the most-studied synapse. Previous studies identified an activity in the basal lamina lining the synaptic cleft that induces presynaptic differentiation in reinnervating axons. The best candidate for mediating that activity is laminin-11, a muscle-derived component of the synaptic basal lamina. Laminin-11 is a heterotrimer, composed of three homologous laminin """"""""chains"""""""": a5, b2, and g1. Mice lacking laminin-11 through targeted deletion of b2 (s-laminin) die of neuromuscular failure at one month of age. Motor nerve terminals in the b2-knock-out are poorly differentiated, and cut axons fail to reinnervate b2-deficient synaptic sites properly. Purified laminin-11 (but not other laminins) acts as a """"""""stop"""""""" signal to cultured motor neurites, suggesting laminin-11 directly regulates axons in vivo. Previously, laminin-b2 was thought to contain the active sites in laminin-11. However, this simple hypothesis is now insufficient, because another b2-containing laminin (laminin-3, a2b2g1) was found to promote rather than stop neurite outgrowth. We now hypothesize that laminin-a5 bears much of the inhibitory activity of laminin-11, and that presynaptic defects in b2-knock-out mice are largely due to the accompanying loss of laminin-a5 at b2-deficient synapses. We propose to test this hypothesis in three ways. We will determine if laminin-a5 is required in vivo for synapse formation by characterizing laminin a5-knock-out mice. We will determine if laminin-a5 directly regulates axons, using in vitro assays of neurite outgrowth on substrates containing purified laminin trimers that contain a5 (but not b2). Finally, because Schwann cells that surround nerve terminals are also regulated by laminin-11, we will determine whether laminin-a5 directly regulates nerve terminal formation by assaying synaptogenesis in nerve-muscle co-cultures, where Schwann cells can be eliminated. These studies will provide insight into the mechanisms by which neuronal targets foster and control the pattern of innervation they receive, and these insights will advance methods of treating neuromuscular disorders and injuries.
