Transcriptional Dynamics of Neuronal Differentiation

描述(摘自申请人的摘要)： 这个项目的主要目标是剖析 控制神经元分化、维持的转录网络 和生存。这个应用程序特别关注转录 神经元特异性基本螺旋-环-螺旋(BHLH)控制的动力学 转录因子Nex1/Math-2，它是神经D亚家族的成员。 我们的研究表明，Nex1是神经元的关键调节因子 区分程序作为它的表达方式，对于执行 NGF诱导分化途径。其在PC12细胞中的过表达诱导 神经细胞突起生长及神经元标记物GAP-43的表达 不依赖于NGF的方式。最重要的是，我们的研究揭示了第一个证据 Nex1可以阻止NGF剥夺的PC12细胞的凋亡。因此，bHLHNex1 可能是连接终端分化和维护的关键因素 分化的神经元存活。来检验我们的假设并阐明 Nex1在神经元分化和存活过程中的转录功能 我们计划进行以下三个目标中概述的实验。我们的 战略将使用大鼠嗜铬细胞瘤细胞系PC 12，因为它是一种 建立完善的研究神经元分化、维持和 生死存亡。目的我将重点介绍Nex1调控的转录途径 导致终末神经元分化。最初，我们将生成一个 双稳定的Nex1可诱导的PC 12细胞系将允许操纵 Nex I表达的时间和水平。我们将确定其程度 通过评估神经元标志物的水平来研究神经元分化 免疫印迹和免疫细胞化学相结合。最后，我们将表演一个 Nex1诱导分化不同阶段的动态基因图谱 差异显示和cDNA微阵列。AIM II将针对 Nex1促进神经元存活的分子机制。我们将衡量 逆转录-聚合酶链式反应检测抗凋亡和促凋亡基因的表达 使用我们稳定的细胞系，神经元标志物的时间表达。 最后，Aim III将探索控制Nex1的转录机制 在NGF诱导的分化途径中的表达，使用联合 DNase I足迹、EMSA和CAT分析。事实是Nex1 显示出神经保护特性，使其成为有望增强的目标 神经元存活和设计新的治疗方法 神经退行性疾病、与年龄相关的神经元障碍和中枢神经系统损伤。

DESCRIPTION (From the Applicant's Abstract): The main objective of this project is to dissect the underlying mechanisms of the transcriptional network controlling neuronal differentiation, maintenance and survival. This application specifically focuses on the transcriptional dynamics controlled by the neuronal-specific basic Helix-Loop-Helix (bHLH) transcription factor Nexl/Math-2, which is a member of the NeuroD subfamily. Our studies suggest that Nex 1 is a key regulator of the neuronal differentiation program as its expression is important to the execution of the NGF-induced differentiation pathway. Its overexpression in PC12 cells induces neurite outgrowth and expression of the neuronal marker GAP-43 in an NGF-independent manner. Most importantly, our studies reveal the first evidence that Nex 1 prevents apoptosis of NGF-deprived PC12 cells. Thus, the bHLH Nex 1 may be a key factor linking terminal differentiation to maintenance and survival of differentiated neurons. To test our hypotheses and to elucidate the transcriptional functions of Nex1 during neuronal differentiation and survival, we plan to execute the experiments outlined in the three following aims. Our strategy will employ the rat pheochromocytoma cell line, PC 12, as it is a well-established system to study neuronal differentiation, maintenance, and survival. Aim I will focus on the Nex 1-modulated transcriptional pathways leading to terminal neuronal differentiation. Initially, we will generate a double stable Nex 1 inducible PC 12 cell line that will permit the manipulation of both the timing and levels of Nex I expression. We will determine the extent of neuronal differentiation by assessing the level of neuronal markers using a combination of western blot and immunocytochemistry. Finally, we will perform a dynamic gene profiling at different stages of Nex1-induced differentiation by differential display and cDNA expression microarray. Aim II will address the molecular mechanisms by which Nex1 promotes neuronal survival. We will measure the expression of anti-apoptotic and pro-apoptotic genes by RT-PCR, as well as the temporal expression of neuronal markers, using our stable cell lines. Finally, aim III will explore the transcriptional mechanisms controlling Nex1 expression in the NGF-induced differentiation pathway, using a combination of DNase I footprinting, EMSA, and CAT assay analyses. The fact that Nex 1 exhibits neuroprotective properties makes it a promising target to enhance neuronal survival and design novel therapeutical approaches to treat neurodegenerative diseases, age-related neuronal disorders, and CNS injuries.