Transcriptional Dynamics of Neuronal Survival

神经元存活的转录动力学

基本信息

批准号：
8055717
负责人：
Anne Eliane CHIARAMELLO
金额：
$ 7.64万
依托单位：
GEORGE WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-08-01 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8055717
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 Neurites Neurodegenerative Disorders Neuronal Differentiation Neurons PC12 Cells Pathway interactions Phase Phosphorylation Phylogenetic Analysis Play Process Property 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 member neurogenesis neuron apoptosis neuronal survival novel therapeutic intervention overexpression prevent programs promoter protein protein interaction

项目摘要

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）：该项目的主要目的是剖析连接到差异化计划的生存途径的转录网络的基本机制。这项工作着重于由神经元特异性基本螺旋 - 环螺旋（BHLH）转录因子（NEX1/MATH-2）控制的转录动力学。它在左右大脑发育期间以及与认知功能相关的其他领域中在未来的语言中唯一表达。我们的研究表明，NEX1是神经元分化程序的关键调节剂，因为它的表达对于在PC12细胞中执行NGF诱导的分化途径至关重要。它的过表达诱导自发的神经发生，加速NGF诱导的分化和神经突再生。最重要的是，我们的研究揭示了第一个证据表明，NEX1以来显示神经保护特性：1）防止在营养因子剥夺时PC12-Nex1细胞的凋亡； 2）同时调节关键抗凋亡和细胞周期调节剂的表达。因此，我们假设NEX1通过策划了几个不同但相互联系的程序来促进生存，从而将神经元分化与细胞周期戒断和抗凋亡途径联系起来。为了检验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：目标我将研究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，将细胞周期滞留与生存联系起来。 E2F-RB成员的功能参与将使用基于siRNA的基因敲低结合细胞周期和基于细胞凋亡的流式细胞术分析进行测试。 AIM II将通过限制细胞周期，凋亡和神经元相关途径的基因组分析来阐明特定的NEX1介导的转录网络。在NEX1介导的生存期的不同阶段，基于siRNA的沉默和基于siRNA的流式细胞仪周期/凋亡测定法，将评估NEX1调节基因的功能作用。此外，将确定NEX1的直接靶基因。从AIM I和II中获得的PC12细胞系统中获得的组合结果将在具有各自的细胞死亡范式的皮质和小脑颗粒神经元培养物中得到验证。 AIM III将剖析Bcl-W基因的转录调节，我们已将其识别为NEX1靶基因。使用DNASEL足迹，EMSA和荧光素酶测定，将在神经元分化和存活期间分析关键调节元件的身份和功能。因此，AIM III解决了NIH的编码计划，以揭示人类基因组中关键功能元素的问题，并将关键调节元素中的SNP与神经元相关疾病联系起来。最终，NEX1的结合分化和神经保护特性可能对治疗神经退行性疾病和中枢神经系统损伤的新型治疗方法的设计具有广泛的影响。