Function of GFL-Dependent & Independent RET Activation

描述（由申请人提供）：神经营养因子支持中枢和周围神经元的存活、生长和分化。神经营养因子的作用具有很大的特异性和保真度，部分原因是它们激活了高亲和力的细胞表面受体。神经营养因子是第一个确定的神经营养因子家族，以神经生长因子（NGF）为例，通过激活其受体酪氨酸激酶（rtk） （Trks）发挥作用。第二种营养因子家族是神经胶质细胞系衍生的神经营养因子（GDNF）家族配体（GFLs），其功能是通过激活它们的RTK （Ret）来实现的。我们最近发现了一种RTK间的信号传导机制，通过该机制，NGF受体（TrkA）的激活在体外和体内导致GFLs缺失时的成熟依赖性激活Ret。ngf介导的Ret激活增强了成熟而非未成熟交感神经元的营养状态。为了确定NGF促进Ret激活的机制，利用体外维持的成熟交感神经元，提出了旨在系统测试最可能假说的生化实验。确定NGF促进Ret激活的机制对于揭示哪些受体参与rtk间信号传导以及该过程具有哪些发育功能的长期项目的形成至关重要。为了在体内鉴定gfl依赖性和gfl非依赖性（即ngf依赖性）Ret功能，将产生缺乏所有Ret功能或仅缺乏gfl依赖性Ret功能的转基因动物。对这些动物的中枢和周围神经系统的详细检查将首次揭示Ret的产后功能，因为Ret缺乏的动物的围产期死亡率不允许检查产后发育。作为主要研究者，我将学习一系列重要的实验技能，例如在解剖、生理和行为水平上生产和分析转基因动物，以及生产用于在初级神经元中表达外源蛋白的慢病毒载体。这些拟议实验的结果，以及我将学习的技术，将为我追求我的职业目标——作为学术实验室的主要研究者建立一个独立的研究项目——提供坚实的基础。

DESCRIPTION (provided by applicant): 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.