The Neuroprotective Gene Botch in Stroke-Induced Neurogenesis

描述(申请人提供)：中风是世界范围内死亡的主要原因之一，但其病理生理学基础的机制还不完全清楚。我们的实验室已经发现了一个新的基因，Botch，它可以在缺血模型中保护细胞免于死亡，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，并被缺血预适应上调。此外，在原代培养的大脑皮层神经元中，该基因的表达提供了对氧糖剥夺(OGD)和NMDA兴奋毒性的保护。该基因在发育的不同阶段在多个器官中表达，包括大脑和心血管系统。我们实验室以前的数据表明，Botch在体内促进胚胎和成人的神经发生，在体外促进神经元分化。此外，Botch可以与Notchi和Notch3结合，并抑制Notch信号，这是一条已知参与促进神经前体存活和调节细胞分化的途径。我们的实验室已经开发了一些工具来阐明Botch的功能，包括基因敲除(KO)小鼠、基因过度表达和敲除的构建体以及特定的单抗和多克隆抗体。我们在免疫组织化学和逆转录病毒标记技术方面有经验来评估Botch调节的体内神经发生。此外，我们的实验室在使用局灶性脑缺血的中风小鼠模型方面拥有专业知识。我们认为，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，最终目标是开发治疗方法，以减少中风的病理结果。目的1：Botch对成体神经发生的影响。Botch是否影响体内神经元的分化和整合？我们假设Botch KO小鼠将表现出更高的胶质生成速率和更低的新生神经元功能整合。根据初步数据，我们假设Botch的过度表达将导致体内神经元分化的增加。此外，我们假设在行为学测试中，Botch KO小鼠将表现出海马区依赖的空间记忆缺陷。目标2：博奇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小鼠在卒中后将经历更严重的损伤以及卒中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.