ANALYSIS OF GENES ESSENTIAL FOR NEURONAL DIFFERENTIATION

所提出的实验将阐明两个泛的功能 分化中的神经表达蛋白 ELAV 和 APPL 和所有神经元的维护。学习的长期目标 与 ELAV 相关的是了解神经元特异性的机制 选择性拼接。 神经元特异性剪接机制 使用 ELAV（一种神经元特异性 RNA 结合）进行研究 蛋白质及其下游确定的目标，即受调控的内含子 神经胶质细胞基因。 具体来说，这些研究将阐明 nrg神经元特异性选择性剪接机制 pre-mRNA 通过定义参与调节的顺式元件 结合 ELAV 和其他核因子的内含子，并评估它们的 体内意义。 其他调节神经元特异性的因素 这个过程将被识别。 这些研究将使用记者 体内和细胞培养测定中的转基因以评估剪接， 研究 RNA-蛋白质相互作用的生化方法，以及 遗传方法来识别其他因素。 果蝇APPL 蛋白质是 β 淀粉样前体蛋白 (APP) 的成员 蛋白质家族。 这个家族中第一个被确认的成员， APP 被认为与阿尔茨海默病有关。 尽管该蛋白家族广泛表达于 神经系统，这些蛋白质的正常生理作用 没有被很好地理解。 假设APPL蛋白在 果蝇神经元作为细胞表面受体发挥调节作用 将测试神经元过程和突触。 其他蛋白质 参与 APPL 介导过程的那些将被识别。 实验设计将广泛利用分子和 果蝇中可能的遗传方法：使用野生型和 体外诱变转基因和转基因动物，并靶向 表达，以测定体内功能。 免疫细胞化学 技术和共聚焦显微镜将用于研究神经元 将进行乔木和神经生理学研究 突触功能。 由于 ELAV 和 APPL 都属于 该分析将进化上保守的蛋白质家族 有助于对功能的机械理解 人类的相关蛋白质。

The proposed experiments will elucidate functions of two pan- neurally expressed proteins, ELAV and APPL, in differentiation and maintenance of all neurons. The long term objective of studies related to ELAV is to understand mechanism of neuron-specific alternative splicing. Mechanisms of neuron-specific splicing will be investigated using ELAV, a neuron-specific RNA binding protein, and it~s downstream identified target, the regulated intron of the gene neuroglian. Specifically, these studies will elucidate the mechanism of neuron-specific alternative splicing of the nrg pre-mRNA by defining cis-elements involved in the regulated intron that bind ELAV and other nuclear factors, and assess their in vivo significance. Other neuron-specific factors that regulate this process will be identified. These studies will use reporter transgenes in vivo and in cell culture assays to assess splicing, biochemical approaches to study RNA-protein interactions, and genetic methods to identify other factors. The Drosophila APPL protein is a member of the beta amyloid precursor protein (APP) family of proteins. The first identified member of this family, APP, is implicated to have a role in Alzheimer~s disease. Although this family of proteins is widely expressed within the nervous system, the normal physiological roles of these proteins are not well understood. The hypothesis that the APPL protein in Drosophila neuron function sas a cell surface receptor to regulate neuronal processes and synapses will be tested. Other proteins that are involved in APPL-mediated processes will be identified. The experimental design will make extensive use of molecular and genetic approaches possible in Drosophila: use of wild type and in vitro mutagenized transgenes and transgenic animals, and targeted expression, to assay in vivo function. Immunocytochemical techniques and confocal microscopy will be used to study neuronal arbors and neurophysiological studies will be conducted study synaptic function. Since ELAV and APPL both belong to evolutionarily conserved protein families this analysis will contribute to the mechanistic understanding of the function of related proteins in humans.