Transcriptional Dynamics of Neuronal Differentiation

神经元分化的转录动力学

• 批准号: 7232204
• 负责人: Anne Eliane CHIARAMELLO
• 金额: $ 2.5万
• 依托单位: GEORGE WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7232204
• 关键词: DNA footprinting; PC12 cells; apoptosis; biological signal transduction; cell differentiation; gel mobility shift assay; gene expression; immunocytochemistry; microarray technology; neurogenesis; neurons; neurotrophic factors; polymerase chain reaction; protein structure; protein structure function; tissue /cell culture; transcription factor; western blottings

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.

描述（来自申请人的摘要）： 本项目的主要目标是剖析 转录网络控制神经元的分化、维持 和生存本申请特别关注转录 由神经元特异性基本螺旋环阻滞（bHLH）控制的动力学 转录因子Nexl/Math-2，其是NeuroD亚家族的成员。 我们的研究表明，Nex 1是神经元的关键调节因子， 微分程序，因为它的表达是重要的执行， 神经生长因子诱导的分化途径。它在PC 12细胞中的过表达诱导 神经突起生长和神经元标记物GAP-43的表达， 神经生长因子独立的方式。最重要的是，我们的研究揭示了 Nex 1抑制去NGF的PC 12细胞凋亡。因此，bHLH Nex 1 可能是连接终末分化和维持的关键因素， 分化的神经元的存活。为了验证我们的假设并阐明 Nex 1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 该策略将使用大鼠嗜铬细胞瘤细胞系PC 12，因为它是一种 一个完善的系统来研究神经元的分化，维持， 生存目的本论文主要研究Nex 1调控的转录途径 导致终末神经元分化。首先，我们将生成一个 双稳定的Nex 1诱导型PC 12细胞系，允许操作 Nex I表达的时间和水平。我们将确定 神经元的分化，通过评估神经元标记物的水平， 免疫印迹和免疫细胞化学相结合。最后，我们将执行一个 在Nex 1诱导分化的不同阶段的动态基因谱分析， 差异显示和cDNA表达微阵列。目标二将解决 Nex 1促进神经元存活的分子机制。我们将测量 通过RT-PCR检测抗凋亡和促凋亡基因的表达，以及 神经元标记物的时间表达，使用我们的稳定细胞系。 最后，目的III将探索控制Nex 1的转录机制 在NGF诱导的分化途径中的表达，使用 DNA酶I足迹法、EMSA和CAT测定分析。事实上，NEX 1 显示出神经保护特性，使其成为一个有前途的目标，以提高 神经元存活和设计新的治疗方法， 神经变性疾病、年龄相关的神经元病症和CNS损伤。