ANALYSIS OF GENES ESSENTIAL FOR NEURONAL DIFFERENTIATION

拟议的实验将阐明两个PAN的功能- 神经表达蛋白ELAV和APPL在分化中的作用 以及所有神经元的维护。研究的长期目标 与ELAV相关的是了解神经元特异性的机制 另一种拼接。神经元特异性剪接的机制 使用ELAV进行研究，这是一种神经元特异性RNA结合 蛋白质，以及它~S下游确定的靶标，调控的内含子 神经胶质细胞的基因。具体来说，这些研究将阐明 神经元特异性NRG选择性剪接的机制 通过定义参与调控的顺式元件来实现Pre-mRNA 结合ELAV和其他核因子的内含子，并评估它们的 活体意义。其他神经元特异性因子调节 这一过程将被确定。这些研究将使用记者 体内和细胞培养中的转基因检测以评估剪接， 研究RNA-蛋白质相互作用的生物化学方法，以及 用遗传学方法来识别其他因素。果蝇的应用 蛋白质是β-淀粉样前体蛋白(App)的一员。 蛋白质家族。这个家庭中第一个被确认的成员， APP，被指在阿尔茨海默病~S病中有作用。 尽管这一蛋白质家族广泛表达于 神经系统，这些蛋白质的正常生理作用 都没有被很好地理解。假设APPL蛋白存在于 果蝇神经元功能受细胞表面受体的调节 将对神经元突起和突触进行测试。其他蛋白质 将确定哪些人参与了APPL介导的过程。 实验设计将广泛使用分子和 果蝇可能的遗传途径：使用野生型和 体外诱变转基因和转基因动物，并有针对性 表达，以检测体内功能。免疫细胞化学 技术和共聚焦显微镜将被用于研究神经元 乔木和神经生理学研究将进行研究 突触功能。由于ELAV和APPL都属于 进化上保守的蛋白质家族这一分析将 有助于从机械的角度理解 人类体内的相关蛋白质。

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.