Transcriptional Dynamics of Neuronal Differentiation

神经元分化的转录动力学

• 批准号: 6318508
• 负责人: Anne Eliane CHIARAMELLO
• 金额: $ 33.23万
• 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 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6318508
• 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（摘自申请人的摘要）： 该项目的主要目的是剖析 控制神经元分化，维护的转录网络 和生存。该应用程序专门关注转录 由神经元特异性基本螺旋 - 环螺旋（BHLH）控制的动力学 转录因子nexl/Math-2，它是神经轨道亚科的成员。 我们的研究表明，NEX 1是神经元的关键调节剂 差异化程序作为其表达对于执行很重要 NGF诱导的分化途径。它在PC12细胞中的过表达可诱导 神经元标记GAP-43的神经突产物和表达 独立于NGF的方式。最重要的是，我们的研究揭示了第一个证据 该NEX 1可防止NGF剥夺PC12细胞的凋亡。因此，BHLH NEX 1 可能是将终端区分与维护和维护和 分化神经元的生存。测试我们的假设并阐明 NEX1在神经元分化和存活过程中的转录功能， 我们计划执行以下三个目标中概述的实验。我们的 策略将采用大鼠嗜铬细胞瘤细胞系PC 12，因为它是一个 良好的系统来研究神经元分化，维护和 生存。目的我将重点放在NEX 1调节的转录途径上 导致末端神经元分化。最初，我们将生成一个 双重稳定NEX 1诱导PC 12单元线，该系列将允许操纵 Nex I表达的时间和水平。我们将确定程度 通过使用A评估神经元标记水平的神经元分化 蛋白质印迹和免疫细胞化学的结合。最后，我们将执行 在NEX1诱导的不同阶段，动态基因分析通过 差分显示和cDNA表达微阵列。 AIM II将解决 NEX1促进神经元存活的分子机制。我们将衡量 RT-PCR以及抗凋亡和促凋亡基因的表达以及 使用我们稳定的细胞系的神经元标记的时间表达。 最后，AIM III将探索控制NEX1的转录机制 结合使用NGF诱导的分化途径 DNase I足迹，EMSA和CAT分析分析。 NEX 1的事实 展示神经保护特性使其成为增强的有希望的目标 神经元生存和设计治疗的新型治疗方法 神经退行性疾病，与年龄相关的神经元疾病和中枢神经系统损伤。