Neurotrophic factors support the survival, growth, and differentiation of both central and peripheral neurons. The great specificity and fidelity of the effects of neurotrophic factors are due in part to their activation of high affinity cell surface receptors. The neurotrophins, the first identified family of neurotrophic factors exemplified by the prototypical member nerve growth factor (NGF), function via activation of their receptor tyrosine kinases (RTKs), the Trks. A second family of trophic factors, the glial cell-line derived neurotrophic factor (GDNF) family ligands (GFLs), function via activation of their RTK, Ret. We have recently identified an inter-RTK signaling mechanism by which activation of the NGF receptor, TrkA, leads both in vitro and in vivo to the maturation-dependent activation Ret in the absence of GFLs. NGF-mediated Ret activation augments the trophic status of mature, but not immature, sympathetic neurons. In order to identify the mechanism by which NGF promotes Ret activation, biochemical experiments designed to systematically test the most likely hypotheses are proposed, making use of mature sympathetic neurons maintained in vitro. The identification of the mechanism by which NGF promotes Ret activation is critically important for the formation of a long-term project to reveal which receptors participate in inter-RTK signaling, and what developmental functions this process has. In order to identify both the GFL-dependent and GFL-independent (i.e. NGF-dependent) Ret functions in vivo transgenic animals will be produced that are deficient in either all Ret functions, or in only GFL-dependent Ret functions. A detailed examination of the central and peripheral nervous systems of these animals will reveal, for the first time, the postnatal functions of Ret, given the perinatal lethality of Ret deficient animals that has not allowed examination of postnatal development. As the principle investigator I will learn a new repertoire of important experimental skills, such as the production and analysis of transgenic animals at the anatomic, physiologic, and behavioral levels, as well as the production of lentiviral vectors for the expression of foreign proteins in primary neurons. The results from these proposed experiments, as well as the techniques I will learn, will provide a solid basis for me to pursue my career objective to establish an independent research program as the principle investigator of an academic laboratory.
