Molecular control of neurogenesis in the adult subventricular zone

描述（由申请人提供）：在成年哺乳动物大脑的受限区域，包括侧脑室内衬的室下区（SVZ），新神经元和胶质细胞继续活跃地产生。在这些所谓的神经源性壁龛中，神经干细胞（NSCs）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的来源。最近的研究表明，这种持续的成人神经发生有助于大脑的生理和病理。此外，通过操纵内源性神经细胞再生特定类型的损伤神经元是恢复性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，进一步的研究是必要的。在本文中，我们将通过研究同源结构域转录因子Gsx1和Gsx2在成人神经发生中的作用来解决这一问题。Gsx1和Gsx2控制着远脑发育的许多方面，包括NPCs的区域规范和增殖以及神经元分化。然而，它们在成人中的功能尚未被研究过。我们的初步数据显示，Gsx1和Gsx2在成人SVZ解剖学上离散的亚域的npc的特定亚群中表达。我们假设Gsx1和Gsx2在控制成年SVZ中NPCs离散亚群的维持和/或分化中发挥关键作用，从而促进特定OB中间神经元亚型的产生。我们还假设Gsx1+和Gsx2+祖细胞参与了成人脑损伤诱导的神经发生。我们将通过以下四个具体目标来验证这些假设：目的1：揭示成人SVZ中Gsx1+和Gsx2+祖细胞的身份。我们的初步数据显示，Gsx1和Gsx2在位于成人SVZ离散子域的npc中表达。我们将通过以下方法揭示这些细胞的特性：1)使用Gsx2flox/+和Gsx1::GFP小鼠进行详细的分子标记分析；2)使用有丝分裂标记物5-溴-2'脱氧脲嘧啶（BrdU）进行短期和长期标记，联合使用抗有丝分裂剂b- d -阿拉伯糖醛酸苷（Ara-C）和各种生长因子进行处理；3)克隆细胞培养试验。目的2：揭示Gsx1和Gsx2在成人SVZ神经发生和胶质瘤发生中的作用。为了揭示Gsx1和Gsx2的成人特异性功能，我们将在体内进行条件功能获得（GOF）和功能丧失（LOF）分析。携带Gsx1和Gsx2固定等位基因的小鼠将用于在成年大脑中进行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Gsx1和Gsx2转基因的小鼠进行条件GOF实验。病毒介导的Gsx1和Gsx2的过表达和敲低将并行进行。目的3：通过体外培养揭示Gsx1和Gsx2的功能。为了更好地了解Gsx1和Gsx2的作用机制，我们将使用NPCs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Gsx1和Gsx2的转录靶点。目的4：揭示Gsx1和Gsx2在成人脑损伤性神经发生中的作用。成人脑损伤性神经发生的机制仍不清楚。我们将研究Gsx1+和Gsx2+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Gsx1和Gsx2是否在SVZ祖细胞的损伤依赖性控制和神经发生中发挥任何作用。揭示Gsx1/2在成人大脑中的作用将为成人神经发生机制，特别是成人npc特异性的分子基础提供新的见解。因此，这项研究将显著促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中的重要性，本研究的结果将对促进人类健康和福祉作出重大贡献。

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. PUBLIC HEALTH RELEVANCE: Continuous production of new neurons in the adult brain (adult neurogenesis) has been shown to play an important role in higher brain functions as well as repair of damaged brains. Yet, our understanding of the mechanisms underlying this important phenomenon is still limited. This study will address this issue by studying the roles of the transcription factors Gsx1 and Gsx2 in adult neurogenesis. Thus, the outcomes of the proposed research will have significant contribution to the promotion of human health and welfare.