Transcriptional Dynamics of Neuronal Survival

描述（由申请人提供）：这个项目的主要目的是剖析连接生存途径和分化程序的转录网络的潜在机制。这项工作的重点是由神经元特异性的基本Helix-Loop-Helix （bHLH）转录因子Nex1/Math-2控制的转录动力学。在左右脑发育过程中，以及与认知功能相关的其他区域，它在未来的语言中都有独特的表达。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ngf诱导的PC12细胞分化途径的执行至关重要。其过表达诱导自发神经细胞发生，加速神经生长因子诱导的分化和神经突再生。最重要的是，我们的研究首次揭示了Nex1具有神经保护特性的证据，因为它：1)防止营养因子剥夺后PC12-Nex1细胞的凋亡；2)同时调节关键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存活，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，我们计划实现以下三个目标：目的1将研究Nex1在E2F-Rb通路中的作用，E2F-Rb通路是NGF通路的关键组成部分，将细胞周期阻滞与生存联系起来。E2F-Rb成员的功能参与将通过基于sirna的基因敲除结合细胞周期和细胞凋亡的流式细胞术检测进行测试。Aim II将通过对细胞周期、细胞凋亡和神经元相关通路进行基因组分析，阐明特定的nex1介导的转录网络。nex1调控基因的功能作用将通过基于sirna的沉默和基于流式细胞术的功能细胞周期/凋亡测定来评估，在nex1介导的生存的不同阶段。进而确定Nex1的直接靶基因。在来自Aim I和II的PC12细胞系统中获得的综合结果将在皮层和小脑颗粒神经元培养中验证其各自的细胞死亡模式。Aim III将剖析Bcl-w基因的转录调控，我们已经确定并表征了该基因是Nex1靶基因。关键调控元件的身份和功能将在神经元分化和生存过程中分析，使用DNasel足迹，EMSA和荧光素酶测定。因此，Aim III解决了NIH ENCODE倡议，以揭示人类基因组中的关键功能元件，并将关键调控元件中的snp定位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治疗方法具有广泛的意义。

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.