Genetic approaches to neurotrophin signaling

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DESCRIPTION (provided by applicant): This proposal is aimed at determining the role of neurotrophins that signal through Trk receptors in modulating synapse structure and function in the developing and adult nervous system. Mouse neuromuscular synapses will be used as a model system. The cell types that comprise neuromuscular junctions, the perisynaptic Schwann cells, presynaptic motor neuron terminals, and postsynaptic muscle fibers, each express a complement of neurotrophins and Trks, suggesting that neurotrophin signaling at this synapse is multi-directional and involves all three cell types. Similarly, CNS neurons express several neurotrophins and Trks, but the relative roles of each signaling pathway in synaptic maturation and maintenance are unclear. Previous work and preliminary results from our lab showed that TrkB isoforms (which bind the neurotrophins BDNF and NT4/5) are expressed primarily postsynaptically, in the muscle fiber membrane in and around acetylcholine receptor (AChR) rich regions, while TrkC isoforms are localized to perisynaptic Schwann cells and TrkA is not localized to neuromuscular junctions. Down-regulation of TrkB signaling in muscle fibers, via adenovirus-mediated over-expression of a truncated, non-signaling form of TrkB (TrkB.t1), induced the dismantling of postsynaptic AChR rich regions. In contrast, preliminary results show that TrkC modulates the extension of processes by these cells that play important roles in axon sprouting and reinnervation. These observations lead to the hypothesis that exchange of ligands that signal through TrkB or TrkC receptors play functionally distinct roles in synaptic maturation and maintenance. While adenoviral methods are useful, and are the supported by another grant, to determine the role of these signaling molecules at synapses, it will be essential to selectively delete neurotrophin or Trk genes from one of the cell types at neuromuscular synapses. Because many of the relevant mutations in mice result in perinatal lethality, we will use cre-mediated recombination to delete neurotrophins or Trks from cells of interest in a spatially and temporally controlled fashion. The effect of neurotrophin (BDNF, NT3) or TrkB deletion on neuromuscular synaptic structure and function will be analyzed with in vivo imaging, immunostaining, confocal microscopy and electrophysiological characterization of synaptic strength. The results of these experiments will provide new insights into the functional role(s) of neurotrophin and Trk-mediated signaling at developing and adult synapses, and extend our understanding of the relative roles of antero- and retrograde synaptic signaling in the peripheral as well as central nervous system.