Transcriptional Dynamics of Neuronal Survival

神经元存活的转录动力学

• 批准号: 7659004
• 负责人: Anne Eliane CHIARAMELLO
• 金额: $ 6.02万
• 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: 2001-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7659004
• 关键词: Address; Agreement; Apoptosis; Apoptotic; Area; BHLH Protein; Biological; Biological Assay; Boxing; Brain; Cell Cycle; Cell Cycle Arrest; Cell Cycle Regulation; Cell Death; Cell Survival; Cells; Cessation of life; Cytoplasmic Granules; Development; Disease; E2F1 gene; EMSA; Elements; Excision; Flow Cytometry; Future; G1 Phase; Gene Targeting; Genes; Genomics; Grant; Growth; Helix-Turn-Helix Motifs; Human Genome; Indium; Knowledge; Language; Lead; Left; Link; Luciferases; Mediating; Metabolism; Natural regeneration; Nerve Growth Factor 1; Nerve Growth Factor Pathway; Neurites; Neurodegenerative Disorders; Neuronal Differentiation; Neurons; PC12 Cells; Pathway interactions; Phase; Phosphorylation; Phylogenetic Analysis; Play; Process; Property; Protein Overexpression; Proteins; RBL2 gene; Regulation; Regulatory Element; Regulatory Pathway; Reporter; Repression; Research Personnel; Role; Serum; Signal Transduction; Single Nucleotide Polymorphism Map; Site-Directed Mutagenesis; Small Interfering RNA; Staging; Switching Complex; System; Testing; Transactivation; Transcription Initiation Site; Transcriptional Regulation; United States National Institutes of Health; Withdrawal; Work; base; central nervous system injury; cognitive function; deprivation; design; gain of function; gliogenesis; human RBL2 protein; member; neurogenesis; neuron apoptosis; neuronal survival; novel therapeutics; prevent; programs; promoter; protein protein interaction

DESCRIPTION (provided by applicant): The main objective of this project is to dissect the underlying mechanisms of the transcriptional network linking the survival pathway to the differentiation program. This work focuses on the transcriptional dynamics controlled by the neuronal-specific basic Helix-Loop-Helix (bHLH) transcription factor, Nex1/Math-2. It is uniquely expressed in the future language during the left-right brain development as well as other areas associated with cognitive functions. Our studies show that Nex1 is a key regulator of the neuronal differentiation program, as its expression is critical to the execution of the NGF-induced differentiation pathway in PC12 cells. Its overexpression induces spontaneous neuritogenesis, accelerated NGF-induced differentiation, and neurite regeneration. Most importantly, our studies reveal the first evidence that Nex1 displays neuro-protective properties since it: 1) prevents apoptosis of PC12-Nex1 cells upon trophic factor deprivation; and 2) concomitantly modulates the expression of key anti-apoptotic and cell cycle regulators. Accordingly, we hypothesize that Nex1 promotes survival by orchestrating several distinct but interconnected programs, linking neuronal differentiation to cell cycle withdrawal and the anti-apoptotic pathway. To test our overarching hypothesis and elucidate the transcriptional network of Nex1, we plan to execute the three following aims: Aim I will investigate the role of Nex1 in the E2F-Rb pathway, a critical component of the NGF pathway, linking cell cycle arrest to survival. The functional participation of E2F-Rb members will be tested using siRNA-based gene knockdown combined with cell cycle and apoptosis-based flow cytometry assays. Aim II will elucidate the specific Nex1-mediated transcriptional network by performing genomic analyses restricted to the cell cycle, apoptosis, and neuronal-related pathways. The functional roles of Nex1-regulated genes will be assessed by siRNA-based silencing and functional flow cytometry-based cell cycle/apoptosis assays, at different phases of Nex1-mediated survival. Furthermore, direct target genes of Nex1 will be identified. The combined results obtained in the PC12 cell system from Aim I and II will be validated in cortical and cerebellar granule neurons cultures with their respective cell death paradigms. Aim III will dissect the transcriptional regulation of the Bcl-w gene, which we have identified and characterized as being a Nex1 target gene. The identity and functions of the critical regulatory elements will be analyzed during neuronal differentiation and survival, using DNasel footprinting, EMSA, and luciferase assays. Accordingly, Aim III addresses the NIH ENCODE initiative to unravel critical functional elements in the human genome, and to link SNPs mapping in key regulatory elements with neuronal-related diseases. Ultimately, the combined differentiation and neuro-protective properties of Nex1 may have broad implications for the design of novel therapeutic approaches to treat neurodegenerative diseases and CNS injuries.

描述(由申请人提供)：这个项目的主要目标是剖析将生存途径与分化程序联系起来的转录网络的潜在机制。本工作主要研究神经元特异性碱性螺旋-环-螺旋(BHLH)转录因子Nex1/Math-2控制的转录动力学。它在未来的语言中有独特的表达，在左右脑发育过程中以及其他与认知功能相关的区域。我们的研究表明，Nex1是神经元分化程序的关键调节因子，因为它的表达对于执行NGF诱导的PC12细胞分化途径至关重要。其过表达可诱导自发神经发生、加速NGF诱导分化和轴突再生。最重要的是，我们的研究揭示了Nex1表现出神经保护特性的第一个证据，因为它：1)阻止营养因子剥夺时PC12-Nex1细胞的凋亡；2)同时调节关键的抗凋亡和细胞周期调节因子的表达。因此，我们假设Nex1通过协调几个不同但相互关联的程序来促进生存，将神经元分化与细胞周期退出和抗凋亡途径联系起来。为了验证我们的总体假设并阐明Nex1的转录网络，我们计划执行以下三个目标：目的I将研究Nex1在E2F-Rb途径中的作用，E2F-Rb途径是NGF途径的关键组成部分，将细胞周期停滞与生存联系在一起。E2F-Rb成员的功能参与将使用基于siRNA的基因敲除结合细胞周期和基于凋亡的流式细胞术分析来测试。目的II将通过对细胞周期、细胞凋亡和神经元相关通路的基因组分析，阐明Nex1介导的特定转录网络。Nex1调控基因的功能作用将通过基于siRNA的沉默和基于功能流式细胞仪的细胞周期/凋亡分析来评估，在Nex1介导的生存的不同阶段。此外，还将确定Nex1的直接靶基因。AIM I和AIM II在PC12细胞系统中获得的综合结果将在大脑皮层和小脑颗粒神经元培养中得到验证，它们各自的细胞死亡模式。目的分析Bclw基因的转录调控，我们已鉴定该基因为Nex1靶基因。在神经元分化和存活过程中，将使用DNasel足迹、EMSA和荧光素酶分析来分析关键调控元件的身份和功能。因此，AIM III解决了NIH ENCODE倡议，以解开人类基因组中的关键功能元件，并将关键调节元件中的SNPs映射与神经元相关疾病联系起来。最终，Nex1的联合分化和神经保护特性可能对设计治疗神经退行性疾病和中枢神经系统损伤的新疗法具有广泛的意义。