The Neuroprotective Gene Botch in Stroke-Induced Neurogenesis

描述（由申请人提供）：中风是世界范围内死亡的主要原因之一，但其病理生理机制尚不完全清楚。我们的实验室发现了一种新的基因，Botch，它可以保护缺血模型中的细胞死亡，并调节神经前体细胞的增殖和分化。Botch是从神经保护基因的功能筛选中鉴定出来的，并通过缺血预处理上调。此外，该基因的表达可以保护原代皮层神经元免受氧葡萄糖剥夺（OGD）和NMDA的兴奋性毒性。该基因在包括大脑和心血管系统在内的多个器官的不同发育阶段表达。我们实验室以前的数据显示，Botch在体内促进胚胎和成人神经发生，在体外促进神经元分化。此外，Botch可以结合Notchi和Notch3并抑制Notch信号通路，Notch信号通路参与促进神经前体存活和调节细胞分化。我们的实验室已经开发了工具来阐明Botch的功能，包括敲除（KO）小鼠，基因过表达和敲除的构建物，以及特异性单克隆和多克隆抗体。我们在免疫组织化学和逆转录病毒标记技术方面有丰富的经验来评估体内的Botch-regulated神经发生。此外，我们的实验室在采用局灶性脑缺血的小鼠中风模型方面具有专业知识。我们建议，在脑缺血的背景下，研究Botch在成人神经发生中的作用将进一步加深我们对导致发病率的潜在细胞和分子过程的理解，最终目标是开发出减少中风病理结果的治疗方法。目的1:Botch对成人神经发生的影响。两者在体内是否影响神经元的分化和整合？我们假设，Botch KO小鼠将显示出胶质细胞生成率增加和新生神经元功能整合降低。基于初步数据，我们假设Botch过表达会导致体内神经元分化增加。此外，我们假设在行为测试中，Botch KO小鼠将表现出海马体依赖空间记忆的缺陷。目的2:Botch对中风后神经发生和细胞死亡的影响是什么？我们假设Botch KO小鼠在中风后会经历更严重的损伤以及中风后神经发生和恢复的失调。

DESCRIPTION (provided by applicant): Stroke is among the leading causes of mortality worldwide, yet the mechanisms underlying its pathophysiology are not fully understood. Our laboratory has found a novel gene, Botch, which confers protection from cell death in models of ischemia, and also regulates the proliferation and differentiation of neural precursor cells. Botch was identified from a functional screen for neuroprotective genes, and is upregulated by ischemic preconditioning. Futhermore, expression of the gene confers protection from oxygen glucose deprivation (OGD) and NMDA excitotoxicity in primary cortical neuron cultures. The gene is expressed during various stages of development in multiple organs including the brain and cardiovascular system. Previous data generated in our laboratory show that Botch promotes both embryonic and adult neurogenesis in vivo and neuronal differentiation in vitro. Furthermore, Botch can bind to Notchi and Notch3 and inhibit Notch signaling, a pathway known to be involved in promoting neural precursor survival and regulating cellular differentiation. Our laboratory has developed tools to elucidate the function of Botch, including a knock-out (KO) mouse, constructs for gene overexpression and knockdown, and specific monoclonal and polyclonal antibodies. We have experience in immunohistochemisty and retrovirus labeling techniques to evaluate Botch-regulated neurogensis in vivo. Furthermore, our laboratory has expertise in a mouse model of stroke that employs focal cerebral ischemia. We propose that examining Botch's role on adult neurogenesis in the context of cerebral ischemia will further our understanding of underlying cellular and molecular processes that contribute to morbidity, with the ultimate goal of developing therapies to curtail the pathological outcome of stroke. Aim 1: The effect of Botch on adult neurogenesis. Does Botch influence neuronal differentiation and integration in vivo? We hypothesize that the Botch KO mouse will display increased rates of gliogenesis and a decreased functional integration of newborn neurons. Based on preliminary data, we hypothesize that Botch over- expression will lead to increased neuronal differentiation in vivo. Additionally, we hypothesize that in behavioral testing, the Botch KO mouse will exhibit a deficit in hippocampal-dependent spatial memory. Aim 2: What is Botch's impact on neurogenesis and cell death following stroke? We hypothesize that the Botch KO mouse will experience a greater severity of injury following stroke and disregulation of post-stroke neurogenesis and recovery.