MOLECULAR MECHANISMS OF NEURONAL DIFFERENTIATION

大鼠嗜铬细胞瘤细胞系PC 12是最好的细胞培养物 神经营养因子、神经生长因子 （NGF）。我们提出了一个统一的机制来解释神经生长因子的作用， PC12细胞。神经生长因子介导的形态分化是由一个原细胞介导的， 通过依次激活Trk，Src， Ras和Raf蛋白。这个线性途径的分支点介导 磷酸化和遗传调节导致其他神经元 性质，如神经递质的合成和获得 电兴奋性在下一个补助期，我们将测试具体的 该模型的预测，并确定关键的生化中间体 在神经生长因子途径中的作用我们的具体目标是： l）检验Src、Raf和一种新的原癌蛋白Shc， 2）检验Ras-和Ras-介导的神经生长因子诱导的分化的假设， Raf-dependent蛋白激酶是介导 刺激神经递质的合成，和3）检查的机制， Ras和Raf介导两个神经特异性基因VGF的NGF诱导， 和GAP-43，它们在转录和后 转录水平。所有具体目标都取决于 瞬时和稳定的PC12亚系的产生， Src、Ras、Raf、Shc和MAP激酶的显性干扰形式。在 用于产生新的PC12系的载体，癌基因或激酶cDNA 将置于组成型和/或诱导型启动子的控制下。 在特定的目标l，要求和排序的癌基因 活性将通过测定神经元分化来确定， 表达激活和显性干扰形式组合的细胞 癌基因的基因。在具体目标2中，酪氨酸的磷酸化 羟化酶（TH）将用于测定NGF中激活的激酶 信号通路在PC12亚系中，表达激活的或显性的 Ras、Raf或MAP激酶的干扰形式，Th磷酸化将被抑制。 通过磷酸肽图谱分析。将采用FPLC技术进行纯化 一种新的Raf-dependent激酶活性， TH在丝氨酸31上的磷酸化。在具体目标3中，PC12亚线 表达Ras和Raf的活化或显性干扰形式， 用含有调节片段的质粒转染的宿主细胞 VGF和GAP-43基因。突变分析将用于确定 那些赋予调控的基因序列。拟议的研究将 与整个神经营养素家族有直接关系， 调节体内神经元的存活和分化。这些方法 预计将深入了解损失的可能原因， 关键的神经元特性，以及合理的治疗方法 在帕金森氏症、阿尔茨海默氏症、家族性自主神经功能障碍和其他相关疾病中 中枢和外周神经元变性疾病。

The rat pheochromocytoma cell line, PC12, is the premier cell culture model for the differentiating actions neurotrophin, Nerve Growth Factor (NGF). We have proposed a unifying mechanism to explain NGF actions on PC12 cells. Morphological differentiation by NGF is mediated by a proto- oncogene signaling pathway through the sequential activation of Trk, Src, Ras and Raf proteins. Branchpoints off this linear pathway mediate phosphorylation and genetic regulation leading to other neuronal properties, such as neurotransmitter synthesis and the acquisition of electrical excitability. In the next grant period we will test specific predictions of this model and identify critical biochemical intermediates in the NGF pathway to neuronal differentiation. Our specific aims are to: l) test the prediction that Src, Raf and a novel protooncoprotein Shc, mediate NGF-induced differentiation, 2) test the hypothesis that Ras- and Raf-dependent protein kinases are intermediaries in the pathway to stimulation of neurotransmitter synthesis, and 3) examine the mechanism by which Ras and Raf mediate NGF-induction of two neural specific genes, VGF and GAP-43, which are regulated at the transcriptional and post- transcriptional levels, respectively. All of the specific aims rely on the generation of transient and stable PC12 sublines expressing activated or dominant interfering forms of Src, Ras, Raf,, Shc, and MAP kinase. In the vectors used to generate the new PC12 lines, the oncogene or kinase cDNAs will be placed under control of constitutive and/or inducible promoters. In specific aim l, the requirements for and ordering of oncogene activities will be determined by assaying neuronal differentiation in cells expressing combinations of activated and dominant interfering forms of the oncogenes. In specific aim 2, phosphorylation of tyrosine hydroxylase (TH) will be used to assay for kinases activated in the NGF signaling pathway. In PC12 sublines expressing activated or dominant interfering forms of Ras, Raf, or MAP kinase, Th phosphorylation will be analyzed by phosphopeptide mapping. FPLC technology will be used to purify a novel Raf-dependent kinase activity which mediates long term phosphorylation of TH on serine 31. In specific aim 3, the PC12 sublines expressing activated or dominant interfering forms of Ras and Raf, will be host cells for transfections by plasmids containing regulatory fragments of the VGF and GAP-43 genes. Mutational analysis will be used to determine those gene sequences which confer regulation. The proposed studies will have direct relevance to the entire family of neurotrophins, which regulate neuronal survival and differentiation in vivo. These approaches are expected to yield insights into the possible causes of the loss of critical neuronal properties, as well as rational approaches to treatment in Parkinson's, Alzheimer's, familial dysautonomic, and other related central and peripheral neuron degenerating diseases.