MOLECULAR MECHANISMS OF NEURONAL DIFFERENTIATION

拟议研究的总体目标是实现 了解神经生长因子的分子基础 行动 这些行动包括建立、维持和 神经元特性的再生，包括形态学（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。 一个这样的生长因子， 神经生长因子（NGF）是最好的一种 在体内的作用。 NGF是生存所必需的， 主要交感神经和感觉周围神经的分化 神经元 包括家族性自主神经功能障碍患者， 以及中枢神经系统的神经元， 胆碱能神经元在阿尔茨海默病中受到影响。 基于以前的神经元样细胞系PC12的结果，我们 建立了NGF信号转导模型， 第二信使产生的生长因子的行动。 在 模型，细胞光癌基因src和ras，抑制NGF 信号转化为两个第二信使系统的刺激，cAMP 和磷脂酰肌醇的周转，以及它们的相关蛋白质 激酶。 本研究将通过以下方法直接检验该模型： 第二信使和蛋白质的特异性抑制 激酶，然后评估所产生的抑制 它们在NGF作用中的预测功能作用。 一组试验 涉及酪氨酸羟化酶磷酸化的分析（通过 磷酸肽图谱）和活化（通过原位测定）， 导致儿茶酚胺增强的事件 神经递质生物合成 另一种测定涉及分析 另一种光致癌基因fos的转录激活， 通过基因表达的变化， 短期和长期的NGF作用。 的产生和生长 神经过程，由神经生长因子，将被解决，以 研究各种第二信使在这一重要领域的作用， 活动 ras和src在神经生长因子信号转导中的作用 转导也将通过它们的特异性 抑制和通过引入（通过基因转染）它们的 致癌"激活"形式进入细胞，然后评估 如上所述的NGF作用。 预计这些方法将提供对可能的 关键神经特性丧失的原因以及 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的中枢和外周 退化性疾病

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.