Brian A. Pierchala, Ph.D - US grants

DESCRIPTION (provided by applicant): The functions of GDNF family of ligands (GFLs) in postnatal development of the nervous system is largely unknown because deletion of the RTK through which GFLs function, c-Ret, causes perinatal lethality. In sympathetic neurons both in culture and in vivo the amount of Ret phosphorylation increases with postnatal age, reaching a maximum by P21. Surprisingly, this increase in Ret phosphorylation is not mediated by GFLs. Rather, nerve growth factor (NGF), a neurotrophic factor critical for sympathetic neuron development, regulates Ret phosphorylation via a novel mechanism. The goal of Aim 1 is to determine the mechanism by which NGF regulates Ret phosphorylation. The tyrosine residues in the NGF receptor TrkA, and thus the signaling pathways, responsible for NGF-dependent Ret phosphorylation will be identified by expressing TrkA point mutants in the highly transfectable Neuro 2A neuroblastoma cell line. The goal of Aim 2 is to determine the function of NGF-dependent Ret phosphorylation using two complementary approaches. First, the function of NGF-dependent Ret phosphorylation in postnatal neurons will be determined by examining cultured 21 DIV sympathetic neurons from ret-/- animals. Preliminary data suggests that Ret-1 neurons have metabolic deficiencies. Second, transgenic mice will be generated that express dominant-negative forms of Ret in the postnatal nervous system, thereby avoiding abnormalities that lead to early lethality in order to determine the function of Ret in the postnatal nervous system.

Neurotrophlc tactors support the survival, growth, and d]tterentxation of both central and peripheral neurons. The great specificity and fidelity of the effects ofneurotrophic 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 oftrophic factors, the glial cell-line derived nenrotrophic 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 transgenie 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 leam a new repertoire of important experimental skills, such as the production and analysis oftransgenic 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.

? DESCRIPTION (provided by applicant): In the developing nervous system, excess neurons are generated which are nonessential, or inappropriately connected, and are eliminated by programmed cell death. In peripheral neuronal populations, the extent of apoptosis is governed by both a limited supply of survival-promoting neurotrophic factors supplied by targets of innervation and by apoptosis-inducing competition factors secreted by neurons that successfully compete for neurotrophic factors, or winning neurons. One of the prevalent families of neurotrophic factors is the glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs), which support the survival and axon guidance of autonomic, somatosensory and spinal motor neurons. Recently we have discovered that GFL activation of their common signal transducing receptor tyrosine kinase, Ret, causes the association of Ret with p75, a member of the TNF family of death receptors, in sympathetic and sensory neurons. Importantly, we discovered that p75 was critical for GFL-mediated activation of Ret and survival of sensory neurons in vitro. Remarkably, we also found that Ret interacts with p75 upon BDNF stimulation of sympathetic neurons, which triggers the apoptotic death of these neurons. Ret deletion impaired BDNF-induced apoptosis of sympathetic neurons. Our overarching hypothesis is that the p75-Ret receptor complex is a switch regulating survival and apoptosis, depending upon which ligand promotes the assembly of this complex, and serves to integrate coincident survival and death signals. The objectives of this application are two-fold: (1) to test the hypothesis that p75 is critical for the GFL-mediated survival of nociceptive neurons; (2) to test the hypothesis that Ret is necessary for the apoptotic function of the death receptor p75 in sympathetic neurons. In order to accomplish these objectives we will use a combination of biochemical and cell biological techniques in primary neurons and transgenic mice. These experiments are critical for delineating the molecular mechanisms by which the opposing actions of neurotrophic factors and competition factors sculpt peripheral sensory and autonomic circuits. Furthermore, the determination of these receptor mechanisms that create an equilibrium between survival and death will enable a more rational approach for the development of therapeutic strategies for nervous system injuries and diseases.

Throughout the developing nervous system excess neurons are generated which are nonessential, or inappropriately connected, and are eliminated by programmed cell death (PCD). In the PNS, the extent of apoptosis is governed by both a limited supply of survival-promoting neurotrophic factors provided by targets of innervation, and by apoptosis-inducing competition factors secreted by ?winning neurons? that have successfully competed for these neurotrophic factors. The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are a family of potent growth factors that support the survival of autonomic, somatosensory and spinal motor neurons. The GFLs promote survival and growth through a common signal-transducing receptor tyrosine kinase, Ret. During this grant period we discovered that Ret interacts with p75, a member of the TNF family of death receptors, and p75 enhances GDNF-mediated Ret activation and survival. When p75 is deleted specifically in sensory neurons, approximately 20% are lost between P14 and adulthood, and these losses selectively occur in Ret+ nonpeptidergic nociceptors. These results indicate that p75 is required for the development of the nonpeptidergic nociceptor lineage by fine-tuning Ret-mediated trophic support. We also found that during PCD in sympathetic neurons of the superior cervical ganglion (SCG) Ret is restricted to a subset of degenerating neurons that rapidly undergo apoptosis. Pro-apoptotic conditions induce the association of Ret with p75, thereby enhancing the regulated intramembrane proteolysis (RIP) cleavage of p75 and activation of downstream apoptotic effectors. Deletion of p75 in Ret+ neurons, and deletion of Ret, specifically during PCD, inhibits apoptosis both in vitro and in vivo. These results indicate that Ret acts non-canonically to augment p75-mediated apoptosis. The molecular mechanisms that underlie the ability of p75 to enhance Ret signaling, and for Ret to enhance p75 mediated death, are not well understood, and are the subject of Aim 1. We also discovered recently that semaphorin 3A (Sema3A), a secreted repulsive axon guidance molecule, induces apoptosis of primary SCG neurons, and that deletion of its receptor components, Neuropilin-1 (Npn-1) and PlexinA3 (PA3), significantly reduce PCD in the SCG. Sema3A induces apoptosis via the extrinsic pathway, requiring caspase-8, as opposed to the intrinsic pathway triggered by NGF withdrawal in sympathetic neurons that requires caspase-9. The combination of apoptosis induced by neurotrophic factor deprivation and death receptor activation in the developing SCG raises the question of the extent to which caspase-8 and caspase-9 contribute to apoptosis during PCD, and which death receptors are driving this process. These questions will be the subject of Aim 2. Collectively the experiments proposed here will define the molecular mechanisms and magnitude of the role played by death receptor pathways, such as Npn-1/PA3 and p75, in PCD.