Molecular control of neurogenesis in the adult subventricular zone

描述（由申请人提供）：新神经元和神经胶质的活跃产生在成年哺乳动物大脑的有限区域中继续进行，包括侧脑室内衬的室下区（SVZ）。在这些所谓的神经源性小生境中，神经干细胞（NSC）和其他祖细胞（统称为NPC）在整个生命中持续存在，并作为新神经元和神经胶质细胞的来源。最近的研究表明，这种连续的神经发生在成年人有助于大脑的生理和病理。此外，通过操纵内源性NPC再生损伤后的特定类型的神经元是恢复神经学的重要目标之一。然而，我们对这一重要现象背后的机制的理解仍然非常有限，进一步的研究势在必行。在这个提议中，我们将通过研究同源域转录因子Gsx 1和Gsx 2在成人神经发生中的作用来解决这个问题。Gsx 1和Gsx 2控制端脑发育的许多方面，包括NPC的区域特异性和增殖以及神经元分化。然而，它们在成年人中的功能尚未被研究。我们的初步数据表明，Gsx 1和Gsx 2的表达在特定的亚群的NPC，居住在解剖学上离散的亚域的成人SVZ。我们假设Gsx 1和Gsx 2在控制成人SVZ中NPC离散亚群的维持和/或分化中起着至关重要的作用，从而有助于特定OB中间神经元亚型的产生。我们还假设，Gsx 1+和Gsx 2+祖细胞有助于损伤诱导的神经发生在成年人的大脑。 我们将通过以下四个具体目标中概述的实验来测试这些假设：目标1：揭示成人SVZ中Gsx 1+和Gsx 2+祖细胞的身份。我们的初步数据表明，Gsx 1和Gsx 2的表达在NPC居住在成人SVZ的离散子域。我们将通过以下方法揭示这些细胞的特性：1）使用Gsx 2flox/+和Gsx 1：：GFP小鼠进行详细的分子标记物分析，2）使用有丝分裂标记物5-溴-2 '脱氧尿苷（BrdU）进行短期和长期标记，并结合使用抗有丝分裂剂b-D-阿拉伯呋喃糖苷（Ara-C）和各种生长因子进行处理，以及3）克隆细胞培养测定。目的2：探讨Gsx 1和Gsx 2在成年SVZ神经发生和胶质形成中的作用。为了揭示Gsx 1和Gsx 2的成人特异性功能，我们将在体内进行条件性功能获得（GOF）和功能丧失（LOF）分析。携带Gsx 1和Gsx 2的floxed等位基因的小鼠将用于成年脑中的祖细胞亚型特异性敲除。我们还将使用携带tet-O启动子驱动的Gsx 1和Gsx 2转基因的小鼠进行条件GOF实验。将平行进行病毒介导的Gsx 1和Gsx 2过表达和敲减。目的3：利用体外培养的方法研究Gsx 1和Gsx 2的功能。为了更好地理解Gsx 1和Gsx 2的作用机制，我们将使用NPC的克隆培养进行GOF和LOF分析。我们还将通过全基因组染色质免疫沉淀（ChIP）和转录组分析确定Gsx 1和Gsx 2的转录靶点。目标4：揭示Gsx 1和Gsx 2在成人脑损伤诱导的神经发生中的作用。成人脑损伤诱导的神经发生的机制在很大程度上仍然未知。我们将研究Gsx 1+和Gsx 2+祖细胞是否参与喹啉酸（QA）诱导的纹状体兴奋性毒性损伤后的异位神经发生。我们还将使用上述转基因和病毒介导的方法，通过GOF和LOF分析，确定Gsx 1和Gsx 2是否在SVZ祖细胞和神经发生的损伤依赖性控制中发挥任何作用。 揭示Gsx 1/2在成人脑中的作用将为成人神经发生机制提供新的见解，特别是成人NPC特异性的分子基础。因此，这项研究将显着推进我们对成人神经发生和NPC生物学的理解。鉴于成人神经发生在大脑生理和病理学中的重要性，本研究的结果将对促进人类健康和福利做出重大贡献。

DESCRIPTION (provided by applicant): Active production of new neurons and glia continues in restricted regions of the adult mammalian brain, including the subventricular zone (SVZ) lining the lateral ventricle. In these so-called neurogenic niches, neural stem cells (NSCs) and other progenitors (collectively called NPCs) persist throughout life and serve as the source of new neurons and glia. Recent studies have revealed that this continuous neurogenesis in adults contribute to physiology and pathology of the brain. Moreover, regenerating specific types of neurons after damage by manipulating endogenous NPCs is one of the important goals in restorative neurology. Yet, our understanding of the mechanisms underlying this important phenomenon is still very limited, and further research is imperative. In this proposal, we will address this issue by studying the roles for the homeodomain transcription factors Gsx1 and Gsx2 in adult neurogenesis. Gsx1 and Gsx2 control many aspects of telencephalic development, including regional specification and proliferation of NPCs, and neuronal differentiation. Their function in adults, however, has not yet been investigated. Our preliminary data show that Gsx1 and Gsx2 are expressed in specific subpopulations of NPCs that reside in anatomically discrete subdomains of the adult SVZ. We hypothesize that Gsx1 and Gsx2 play crucial roles in controlling the maintenance and/or differentiation of discrete subpopulations of NPCs in the adult SVZ, thereby contributing to the generation of specific OB interneuron subtypes. We also hypothesize that Gsx1+ and Gsx2+ progenitors contribute to injury-induced neurogenesis in the adult brain. We will test these hypotheses by experiments outlined in the following four specific aims: Aim 1: To reveal the identity of Gsx1+ and Gsx2+ progenitors in the adult SVZ. Our preliminary data show that Gsx1 and Gsx2 are expressed in NPCs that reside in discrete subdomains of the adult SVZ. We will reveal the properties of these cells by 1) detailed molecular marker analysis using Gsx2flox/+ and Gsx1::GFP mice, 2) short-term and long-term labeling with the mitotic marker 5-bromo-2'deoxyuridine (BrdU) combined with treatment with the anti-mitotic agent b-D-arabinofuranoside (Ara-C) and various growth factors, and 3) clonal cell culture assays. Aim 2: To reveal the roles for Gsx1 and Gsx2 in neurogenesis and gliogenesis in the adult SVZ. To reveal the adult-specific function of Gsx1 and Gsx2, we will perform conditional gain-of-function (GOF) and loss-of-function (LOF) analyses in vivo. Mice carrying floxed alleles of Gsx1 and Gsx2 will be used for progenitor subtype-specific knockout in the adult brain. We will also perform conditional GOF experiments using mice carrying tet-O promoter-driven Gsx1 and Gsx2 transgenes. Virus-mediated overexpression and knockdown of Gsx1 and Gsx2 will be performed in parallel. Aim 3: To reveal the function of Gsx1 and Gsx2 using in vitro culture. To better understand the mechanisms of action of Gsx1 and Gsx2, we will perform GOF and LOF analyses using clonal culture of NPCs. We will also identify the transcriptional targets for Gsx1 and Gsx2 by genome-wide chromatin-immunoprecipitation (ChIP) and transcriptome assays. Aim 4: To reveal the roles for Gsx1 and Gsx2 in injury-induced neurogenesis in the adult brain The mechanisms underlying injury-induced neurogenesis in the adult brain remain largely unknown. We will examine if Gsx1+ and Gsx2+ progenitors participate in ectopic neurogenesis following quinolinic acid (QA)-induced excitotoxic injury in the striatum. We will also determine if Gsx1 and Gsx2 play any role in injury-dependent control of SVZ progenitors and neurogenesis through GOF and LOF analyses using the aforementioned transgenic and virus-mediated approaches. Revealing the roles of Gsx1/2 in the adult brain will provide novel insights into the mechanisms for adult neurogenesis, in particular, the molecular basis for the specificity of adult NPCs. Thus, this study will significantly advance our understanding of adult neurogenesis and the biology of NPCs. Given the importance of adult neurogenesis in physiology and pathology of the brain, the outcomes of this study will have significant contribution to the promotion of human health and welfare.