Transcriptional Dynamics of Neuronal Survival

描述（由申请人提供）：该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。这项工作的重点是转录动力学控制的神经元特异性的基本螺旋环受阻（bHLH）转录因子，Nex 1/数学2。它在左右脑发育过程中以及与认知功能相关的其他区域中独特地表达在未来语言中。我们的研究表明，Nex 1是神经元分化程序的关键调节因子，因为它的表达对PC 12细胞中NGF诱导的分化途径的执行至关重要。其过度表达诱导自发性神经突发生、加速的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Nex 1显示神经保护特性的第一个证据，因为它：1）在营养因子剥夺后防止PC 12-Nex 1细胞凋亡; 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Nex 1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Nex 1的转录网络，我们计划执行以下三个目标：目的我将调查Nex 1在E2 F-Rb通路，神经生长因子通路的关键组成部分，连接细胞周期阻滞生存的作用。E2 F-Rb成员的功能参与将使用基于siRNA的基因敲除结合基于细胞周期和凋亡的流式细胞术测定来测试。目的II将阐明特定的Nex 1介导的转录网络进行基因组分析限制在细胞周期，凋亡和神经元相关的途径。在Nex 1介导的存活的不同阶段，将通过基于siRNA的沉默和基于功能性流式细胞术的细胞周期/凋亡测定来评估Nex 1调节基因的功能作用。此外，还将鉴定Nex 1的直接靶基因。将在皮质和小脑颗粒神经元培养物（具有各自的细胞死亡范例）中验证在目标I和II的PC 12细胞系统中获得的组合结果。目的III将剖析Bcl-w基因的转录调控，我们已经确定并表征为Nex 1靶基因。关键调控元件的身份和功能将在神经元分化和存活期间使用DNase 1足迹法、EMSA和荧光素酶测定法进行分析。因此，目标III提出了NIH ENCODE倡议，以解开人类基因组中的关键功能元件，并将关键调控元件中的SNP图谱与神经元相关疾病联系起来。最终，Nex 1的组合分化和神经保护特性可能对治疗神经退行性疾病和CNS损伤的新型治疗方法的设计具有广泛的意义。

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.