Studies on Neuronal Development

神经元发育研究

• 批准号: 7231594
• 负责人: TADMIRI Rangachar VENKATESH
• 金额: $ 19.52万
• 依托单位: CITY COLLEGE OF NEW YORK
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7231594
• 关键词: 26S proteasome; Address; Axon; Binding; Biochemical; Biochemical Genetics; Biological Assay; Blood - brain barrier anatomy; Brain; Catalytic Domain; Cell Cycle Progression; Cell Extracts; Cell physiology; Cells; Clone Cells; Co-Immunoprecipitations; Complex; Confocal Microscopy; Development; Disease; Drosophila genus; ETS Family Protein; Equilibrium; Eye; Genes; Genetic; Goals; Histocompatibility Testing; Human; Immunohistochemistry; In Vitro; Lead; Localized; Mediating; Modeling; Molecular; Molecular Genetics; Morula; Mutation; Nervous system structure; Neuraxis; Neurobiology; Neurodegenerative Disorders; Neuroglia; Neuronal Plasticity; Neurons; Numbers; Pattern; Positioning Attribute; Proteins; RNA Interference; Regulation; Role; Signal Pathway; Structure; Supporting Cell; System; Techniques; Testing; Thinking; Time; Tissue Extracts; Tissues; Two-Hybrid System Techniques; Ubiquitination; Work; Yeasts; anaphase-promoting complex; base; gain of function; genetic manipulation; in vivo; insight; loss of function; mutant; nervous system development; nervous system disorder; novel; protein degradation; research study; response; synaptogenesis; tissue culture; transcription factor; ubiquitin ligase

SPECIFIC AIMS A primary goal of this proposal is to understand the molecular genetic mechanisms regulating glia differentiation. Traditionally, glia were thought of mainly as support cells for neuronal function. In recent years, however, it has become increasingly clear that glia are pivotal for proper neuronal development and function. Glia mediate a remarkable array of cellular functions including axon ensheathment, establishment of blood brain barrier, trophic response, ionic equilibrium, synaptogenesis, axon pruning, engulfment and neuronal plasticity. To carry out these important functions, glia must themselves differentiate and function properly. Indeed, glia malfunction often precedes neuronal/axonal degeneration in many human neurodegenerative diseases (BAUMANN and PHAM-DINH 2001; BENARROCH 2005; FREEMAN 2005b; FREEMAN 2005c; KIM and DE VELLIS 2005; SCHWABE et a/. 2005; SHAHAM 2005; WYSS-CORAY and MUCKE 2002). Despite their immense importance in neurobiology, glia are remarkably understudied and the molecular genetic mechanisms that direct the differentiation of glia are poorly understood. The developing nervous system of Drosophila offers a superb experimental system in which to understand these mechanisms. Drosophila glia are remarkably similar to the mammalian glia in their development, structure and function (FREEMAN and DOHERTY 2005). In Drosophila, molecular and genetic manipulations are readily feasible and the cellular signaling pathways that regulate nervous system development are highly conserved between Drosophila and mammalian systems. Thus an understanding of the regulatory mechanisms that direct glia differentiation in Drosophila will provide important insights into mammalian glia differentiation and will be invaluable in our understanding of human neurological disorders. The focus of our proposal is on the role of ubiquitination as a regulatory mechanism during glia differentiation. Tagging of specific proteins for degradation by ubiquitination has emerged as an important regulatory mechanism in nervous system development and disease. We previously identified and molecularly characterized Rap/Fzr, an activator of the multi-subunit ubiquitin ligase, APC (Anaphase promoting complex). Our work has shown that Rap/Fzr regulates cell cycle progression and is required for proper neuronal patterning in the developing eye (JACOBS etal. 2002; KARPILOW et a/. 1989; KARPILOW etal. 1996; PIMENTEL and VENKATESH 2005b). To identify novel cellular functions of Rap/Fzr we carried out genetic studies which showed that Rap/Fzr interacts with several genes required for glia differentiation (Kaplow et al. 2006 submitted). We have recently made the novel observation that Rap/Fzr regulates glia differentiation by interacting with an Ets domain transcription factor, Pointed, already known to be required for glia differentiation and with Apc2. a catalytic subunit of the APC ubiquitin ligase. Our working hypothesis is that glia differentiation is regulated by novel interactions involving Rap/Fzr, Pointed and Apc2. Rap/Fzr binds Pointed and targets it to the ubiquitin ligase complex (APC), where it is ubiquitinated. Pointed is eventually degraded by the 26S proteosome. In our model, the level of Pointed is regulated by Rap/Fzr, and is a key determinant in the regulation of glia differentiation (Figure 1). The specific aims of this proposal are: Specific Aim I. To test whether glia differentiation is negatively regulated by Rap/Fzr. Our preliminary results suggest that Rap/Fzr is a negative regulator of glia differentiation, a) We will test at the cellular level the effects of loss-of-function Rap/Fzr mutations on glia differentiation in the developing larval brain and the eye. We will generate homozygous rap/fzr- mutant clones in a wild type tissue background using the FLP-FRT technique, b) To directly assess the effects of Rap/Fzr loss-of-function on glia differentiation in a spatially and temporally restricted manner, we will use RNAi and UAS-GAL4 techniques to knockdown Rap/Fzr function in specific tissues at specific times, c) To test the effects of gain-of-function of Rap/Fzr on glia differentiation, we will generate random clones of cells expressing Rap/Fzr, using UAS-GAL4 as well as FLP-FRT systems. We will determine the role of Rap/Fzr in regulating the number and position of glia in the developing larval nervous system. These experiments will address whether the Rap/Fzr function is necessary for glia differentiation in a cell autonomous manner. Specific aim II. To test whether Rap/Fzr regulates glia differentiation by direct interaction with Pointed: Our working hypothesis is that Rap/Fzr targets Pointed for ubiquitination by the ubiquitin ligase APC. Pointed is an ETS domain transcription factor which is required for glia differentiation, a) We will test whether Rap/Fzr binds Pointed using in vitro and in vivo biochemical assays. We will use tissue extracts from larval central nervous system (CMS) and tissue culture S2 cell extracts and assay for binding interactions between Rap/Fzr and Pointed by co-immunoprecipitation assays, b) We will test whether Rap/Fzr and Pointed co-localize in glia in vivo using confocal microscopy in combination with immunohistochemistry. c) We will test whether Rap/Fzr and Pointed interact physically using yeast two-hybrid assays, d) We will perform in vitro ubiquitination assays and test whether Pointed can serve as a substrate for ubiquitination by Rap/Fzr and APC. Specific Aim III. To test whether Apc2/Morula regulates glia differentiation: Apc2 is the catalytic subunit of the ubiquitin ligase complex, APC, encoded by the morula gene. Our preliminary studies suggest that Apc2 is a negative regulator of glia differentiation. To directly assess the role of Apc2 in glia differentiation at the cellular level: a) We will test the effects of loss-of-function of APC on glia differentiation. We will induce clones of Apc2-/Apc2- tissue in the developing larval brain and the eye and determine whether Apc2 is required cell autonomously for glia differentiation, b) We will test the effect of loss-of-function of Apc2 on glia differentiation using the RNAi technique, c) We will test the effects of the gain-of-function of Apc2 on glia differentiation by generating random clones of cells expressing Apc2 using the UAS-GAL4 and the FLP-FRT techniques.

具体目标 该建议的主要目标是了解调节神经胶质的分子遗传机制 分化。传统上，神经胶质被认为主要是神经元功能的支持细胞。最近几年， 但是，越来越明显的是，神经胶质对于适当的神经元发育和功能至关重要。 Glia介导一系列出色的细胞功能，包括轴突宿内，建立血液 脑屏障，营养反应，离子平衡，突触发生，轴突修剪，吞噬和神经元 可塑性。为了执行这些重要功能，神经胶质本身必须自身分化和正常功能。 实际上，在许多人类神经退行性中，胶质功能通常在神经元/轴突变性之前 疾病（Baumann and Pham-Dinh 2001; Benarroch 2005; Freeman 2005b; Freeman 2005c; Kim and De Vellis 2005; Schwabe et a/。 2005; Shaham 2005; Wyss-Coray和Mucke 2002）。 尽管在神经生物学中极为重要，但神经胶质却进行了深入研究，分子遗传 指导神经胶质分化的机制知之甚少。发展的神经系统 果蝇提供了一个出色的实验系统，可以在其中了解这些机制。果蝇神经胶质 与哺乳动物的发展，结构和功能非常相似（Freeman和 Doherty 2005）。在果蝇中，分子和遗传操作很容易可行，细胞 调节神经系统发育的信号通路在果蝇和 哺乳动物系统。因此，了解指导胶质分化的调节机制 果蝇将提供有关哺乳动物胶质分化的重要见解，在我们的 了解人类神经系统疾病。 我们的提议的重点是泛素化作为在神经胶质分化过程中的调节机制的作用。 特异性蛋白质通过泛素化降解已成为重要的调节 神经系统发育和疾病的机制。我们先前鉴定并分子 表征了RAP/FZR，这是多亚基泛素连接酶的激活剂APC（后期促进复合物）。 我们的工作表明，RAP/FZR调节细胞周期的进展，是正确的神经元所必需的 发育中的眼睛中的图案（Jacobsetal。2002; Karpilow et a/。1989; Karpilowetal。1996; Pimentel 和Venkatesh 2005b）。 为了鉴定RAP/FZR的新细胞功能，我们进行了遗传研究，该研究表明RAP/FZR 与胶质分化所需的几种基因相互作用（Kaplow等，2006提交）。我们最近有 使RAP/FZR通过与ETS相互作用来调节神经胶质分化的新颖观察结果 尖头的域转录因子，已知是胶质分化所必需的，并且 APC2。 APC泛素连接酶的催化亚基。我们的工作假设是胶质分化是 由涉及RAP/FZR，尖和APC2的新型相互作用调节。 RAP/FZR绑定指向并将其靶向 泛素连接酶复合物（APC），在泛素化中。尖头最终被26年代降解 蛋白质体。在我们的模型中，指向的水平受RAP/FZR的调节，并且是关键的决定因素 胶质分化的调节（图1）。该提案的具体目的是： 特定目的I.测试胶质分化是否受RAP/FZR负面调节。我们的初步 结果表明RAP/FZR是胶质分化的负调节剂，a）我们将在细胞水平上进行测试 功能丧失RAP/FZR突变对发展中幼虫大脑和 眼睛。我们将使用flp-frt在野生型组织背景下生成纯合说唱/fzr-突变克隆 技术，b）直接评估RAP/FZR功能丧失对空间和 在时间上受到限制的方式，我们将使用RNAi和UAS-GAL4技术来敲除RAP/FZR功能 特定的组织在特定时间，c）测试说RAP/FZR功能获得对胶质分化的影响，我们 将使用UAS-GAL4以及FLP-FRT系统生成表达RAP/FZR的细胞的随机克隆。我们 将确定RAP/FZR在调节神经胶质的数量和位置中的作用 系统。这些实验将解决RAP/FZR功能是否对于A的神经胶质分化是必需的 细胞自主方式。 具体目标II。测试RAP/FZR是否通过与尖头直接相互作用来调节神经胶质分化： 我们的工作假设是，RAP/FZR靶标指出了泛素连接酶APC泛素化的指向。尖 是胶质分化所需的ETS域转录因子，a）我们将测试RAP/FZR是否 使用体外和体内生化测定指向的结合。我们将使用来自幼虫中央的组织提取物 神经系统（CMS）和组织培养S2细胞提取物和RAP/FZR之间的结合相互作用的测定 并由共免疫沉淀测定法指向，b）我们将测试RAP/FZR和尖锐的Glia中的尖锐定位 在体内使用共聚焦显微镜与免疫组织化学结合使用。 c）我们将测试说唱/fzr是否 并使用酵母双杂交测定法进行物理相互作用，d）我们将在体外泛素化测定法 并测试尖头是否可以用作RAP/FZR和APC泛素化的底物。 特定目标III。测试APC2/Morula是否调节神经胶质分化：APC2是催化亚基 泛素连接酶复合物的APC，由Morula基因编码。我们的初步研究表明APC2 是神经胶质分化的负调节剂。直接评估APC2在胶质分化中的作用 细胞水平：a）我们将测试APC功能丧失对神经胶质分化的影响。我们将诱发克隆 在发育中的幼虫大脑和眼睛中的APC2-/APC2-组织的结构，并确定是否需要APC2细胞 自主性胶质分化，b）我们将测试APC2功能丧失对胶质分化的影响 使用RNAi技术，c）我们将测试APC2功能获得对胶质分化的影响 使用UAS-GAL4和FLP-FRT技术生成表达APC2的细胞的随机克隆。