MOLECULAR MECHANISMS OF NEURONAL DIFFERENTIATION

拟议研究的总体目标是实现 对神经生长因子分子基础的认识 行为。这些行动包括建立、维护和 神经元特性的再生，包括形态(轴突 和树突)，动作电位机制，神经递质 储存和释放，以及突触发生。一个这样的增长因素， 神经生长因子(NGF)是最有说服力的 在体内扮演这样的角色。NGF是生存和生存所必需的 大交感神经和感觉神经周围神经的分化 神经元。包括那些受家族性自主神经障碍影响的人，因为 以及中枢神经系统神经元，包括 胆碱能神经元在阿尔茨海默病中的影响。 基于之前对神经元样细胞系PC12的研究结果，我们 建立了NGF的信号转导模型，并对NGF的信号转导机制进行了研究 产生生长因子作用的第二信使。在 模型，细胞光癌基因src和ras，转导神经生长因子 信号进入刺激两个第二信使系统，cAMP 和磷脂酰肌醇及其相关蛋白的周转 激活剂。本研究将通过以下方式直接测试该模型 对第二信使和蛋白质的特异性抑制 然后评估所产生的抑制作用 它们在NGF活动中的预测功能作用。一套化验方法 涉及酪氨酸羟基酶磷酸化的分析(通过 磷酸肽定位)和激活(通过原位分析)，以及 导致儿茶酚胺增加的事件 神经递质生物合成。另一种化验涉及分析 另一种光癌基因fos被认为是转录激活的 通过基因表达的变化，在 NGF的短期和长期作用。的产生和发展 将讨论由NGF引起的神经过程，以便 考察不同的第二信使在这一重要的 事件。Ras和src在NGF信号中的具体作用 转导也将通过其特定的 抑制和(通过基因转染)它们的引入 致癌的“激活”形式转化为细胞，然后评估 NGF的作用如上所述。 这些方法预计将提供对可能的 关键神经属性丢失的原因以及 帕金森氏症、阿尔茨海默氏症的合理治疗方法 家族性自主神经紊乱及其他相关的中枢和外周神经 退化的疾病。

The overall aim of the proposed research is to achieve an understanding of the molecular basis of neuronal growth factor actions. These actions include the establishment, maintenance and regeneration of neuronal properties including morphology (axons and dendrites), the action potential mechanism, neurotransmitter storage and release, and synaptogenesis. One such growth factor, the Nerve Growth Factor (NGF), is the one best established to have such roles in vivo. NGF is required for the survival and differentiation of the major sympathetic and sensory peripheral neurons. Including those affected in familial dysautonomia, as well as of central nervous system neurons including those cholinergic neurons affected in Alzheimer's disease. Based on previous results with the neuron-like cell line, PC12, we have established a model for the signal transduction of NGF and the second messengers which generate the growth factor actions. In the model, the cellular photo-oncogenes src and ras, transduce the NGF signal into the stimulation of two second messenger systems, cAMP and phosphatidylinositol turnover, and of their associated protein kinases. The present research will test the model directly by specific inhibition of the second messengers and of the protein kinases followed by an assessment of the resulting inhibition of their predicted functional roles in NGF actions. One set of assays involves the analysis of tyrosine hydroxylase phosphorylation (by phosphopeptide mapping) and activation (by an in situ assay), an event which results in the enhancement of catecholamine neurotransmitter biosynthesis. Another assay involves analysis of transcriptional activation of another photo-oncogene, fos, believed to provide one link, through changes in gene expression, between short and long term NGF actions. The generation and growth of neural processes, caused by NGF, will be addressed in order to examine the role of the various second messengers in this important event. The specific roles of ras and src in NGF signal transduction will also be determined through their specific inhibition and by the introduction (by gene transfection) of their oncogenic "activated" forms into cells, followed by assessment of NGF actions as described above. These approaches are expected to provide insights into the possible causes of the loss of critical neural properties as well as rational approaches to treatment in Parkinson's, Alzheimer's, familial dysautonomic and other related central and peripheral degenerating diseases.