MOLECULAR MECHANISMS OF NEURONAL DIFFERENTIATION

获得神经元表型的机制是 在哺乳动物神经系统中的控制是知之甚少。我们 工作的目的是确定两个突出的信号通路， 神经元表型的调节因子，神经元生长因子和 兴奋性 大鼠嗜铬细胞瘤细胞系PC12是最早的 神经营养因子分化作用的细胞培养模型， 神经生长因子 NGF对PC12细胞的主要作用，包括延长 神经突起（神经突），神经递质刺激 合成，神经基因的诱导，以及预防 凋亡性细胞死亡都是通过原癌蛋白介导的 其中Ras是一个中心成分。 一系列的部分损失- Ras的无功能突变体和推定Ras效应子的突变体 蛋白，转染到PC12细胞，将用于揭示Ras 控制不同神经特性的下游效应蛋白。 去极化诱导的钙内流对心肌细胞产生长期影响， 神经元表型 Src和PYK2蛋白酪氨酸的突变形式 在PC12中表达的激酶，将用于确定相对的 这些激酶在引起下游钙介导的 Ras依赖性和Ras依赖性的磷酸化事件 独立的道路。 长期酪氨酸的参与 磷酸化事件介导神经元表型和凋亡 变化将由刺激的特定操纵来确定。 酪氨酸激酶 Rin是一种新的钙/钙调素激活蛋白 它与Ras相关，只存在于神经元细胞中。 rin 并且Rin的活化形式将在PC12细胞中表达， 确定Rin可以调节不同方面的程度， 神经元表型响应钙升高，并确定 Rin是否能与正常Ras信号效应子相互作用。 神经生长因子和钙内流控制神经元表型 和细胞死亡通过独立但收敛的信号通路。 我们的研究旨在阐明这些控制的机制， 它的破坏是各种神经系统退化的基础 紊乱

The mechanisms by which acquisition of the neuronal phenotype is controlled in the mammalian nervous system are poorly understood. Our work is aimed at defining the signaling pathways for two prominent regulators of neuronal phenotype, neuronal growth factors and excitability. The rat pheochromocytoma cell line, PC12, is the premier cell culture model for the differentiating actions of the neurotrophin, NGF. Major actions of NGF on PC12 cells, including the extension of neuronal processes (neurites), stimulation of neurotransmitter synthesis, the induction of neural genes, and the prevention of apoptotic cell death are all mediated through a proto-oncoprotein pathway in which Ras is a central component. A series of partial loss- of-function mutants of Ras and mutants of putative Ras-effector proteins, transfected into PC12 cells, will be used to reveal the Ras downstream effector proteins that control distinct neural traits. Depolarization-induced calcium influx exerts long-term effects on neuronal phenotype. Mutant forms of Src and PYK2 protein tyrosine kinases expressed in PC12, will be used to determine the relative contributions of these kinases in causing downstream calcium-mediated phosphorylation events required for both Ras-dependent and Ras- independent pathways. The involvement of the long term tyrosine phosphorylation events in mediating neuronal phenotypic and apoptotic changes will be determined by specific manipulation of the stimulated tyrosine kinase. Rin is a novel calcium/calmodulin-activated protein that is related to Ras and present exclusively in neuronal cells. Rin and an activated form of Rin will be expressed in PC12 cells to determine the extent to which Rin can regulate distinct aspects of neuronal phenotype in response to calcium elevation, and to determine if Rin can interact with the normal Ras signaling effectors. Neuronal growth factors and calcium influx control neuronal phenotype and cell death through independent but convergent signaling pathways. Our studies are aimed at elucidating mechanisms for these controls, the disruption of which underlies a variety of degenerative nervous system disorders.