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 该提案的主要目标是了解调节神经胶质细胞的分子遗传机制 差异化。传统上，神经胶质细胞主要被认为是神经元功能的支持细胞。最近几年， 然而，越来越清楚的是，神经胶质细胞对于神经元的正常发育和功能至关重要。 神经胶质细胞介导一系列显着的细胞功能，包括轴突鞘、血液的建立 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，神经胶质细胞本身必须分化并正常发挥作用。 事实上，在许多人类神经退行性疾病中，神经胶质细胞功能障碍通常先于神经元/轴突变性 疾病（BAUMANN 和 PHAM-DINH 2001；BENARROCH 2005；FREEMAN 2005b；FREEMAN 2005c；KIM 和 DE 韦利斯2005；施瓦贝等人/。 2005年；沙汉姆 2005； WYSS-CORAY 和 MUCKE 2002）。 尽管神经胶质细胞在神经生物学中非常重要，但对其的研究却非常不足，并且分子遗传学 指导神经胶质细胞分化的机制尚不清楚。正在发育的神经系统 果蝇提供了一个极好的实验系统来理解这些机制。果蝇神经胶质细胞 在发育、结构和功能上与哺乳动物神经胶质细胞非常相似（FREEMAN 和 多尔蒂 2005）。在果蝇中，分子和遗传操作是很容易实现的，并且细胞 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，了解指导神经胶质细胞分化的调节机制 果蝇将为哺乳动物神经胶质细胞分化提供重要的见解，并且对于我们的研究具有无价的价值。 了解人类神经系统疾病。 我们提案的重点是泛素化作为神经胶质细胞分化过程中的调节机制的作用。 标记特定蛋白质以通过泛素化降解已成为一种重要的监管手段 神经系统发育和疾病的机制。我们之前鉴定并从分子角度 Rap/Fzr 是多亚基泛素连接酶 APC（后期促进复合物）的激活剂。 我们的工作表明 Rap/Fzr 调节细胞周期进程，并且是正常神经元所必需的 发育中眼睛的图案（JACOBS 等人，2002 年；KARPILOW 等人，1989 年；KARPILOW 等人，1996 年；PIMENTEL 和 VENKATESH 2005b)。 为了鉴定 Rap/Fzr 的新细胞功能，我们进行了遗传学研究，结果表明 Rap/Fzr 与神经胶质分化所需的几个基因相互作用（Kaplow 等人，2006 年提交）。我们最近有 做出了 Rap/Fzr 通过与 Ets 相互作用来调节神经胶质细胞分化的新观察 结构域转录因子，尖头，已知是神经胶质细胞分化所必需的，并且具有 Apc2。 APC 泛素连接酶的催化亚基。我们的工作假设是神经胶质细胞分化是 受涉及 Rap/Fzr、Pointed 和 Apc2 的新颖相互作用调节。 Rap/Fzr 绑定 Pointed 并将其瞄准 泛素连接酶复合物 (APC)，它被泛素化。尖头最终被26S降级 蛋白酶体。在我们的模型中，Pointed 的水平由 Rap/Fzr 调节，并且是 神经胶质分化的调节（图1）。该提案的具体目标是： 具体目的 I.测试Rap/Fzr是否负向调控胶质细胞分化。我们的初步 结果表明 Rap/Fzr 是神经胶质细胞分化的负调节因子，a) 我们将在细胞水平上进行测试 Rap/Fzr 功能丧失突变对发育中幼虫大脑神经胶质细胞分化的影响 眼睛。我们将使用 FLP-FRT 在野生型组织背景中生成纯合 rap/fzr 突变克隆 技术，b) 直接评估 Rap/Fzr 功能丧失对空间和神经胶质细胞分化的影响 在时间限制的方式下，我们将使用 RNAi 和 UAS-GAL4 技术来敲低 Rap/Fzr 功能 c) 为了测试 Rap/Fzr 功能获得对神经胶质细胞分化的影响，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 将决定 Rap/Fzr 在调节发育中的幼虫神经胶质细胞的数量和位置中的作用 系统。这些实验将解决 Rap/Fzr 功能对于神经胶质细胞分化是否是必需的。 细胞自主方式。 具体目标二．为了测试 Rap/Fzr 是否通过与 Pointed 直接相互作用来调节神经胶质细胞分化： 我们的工作假设是 Rap/Fzr 的目标是被泛素连接酶 APC 泛素化。尖 是神经胶质分化所需的 ETS 结构域转录因子，a) 我们将测试 Rap/Fzr 是否 使用体外和体内生化测定结合Pointed。我们将使用幼虫中央的组织提取物 神经系统 (CMS) 和组织培养 S2 细胞提取物以及 Rap/Fzr 之间结合相互作用的测定 和通过免疫共沉淀测定指出，b) 我们将测试 Rap/Fzr 和 Pointed 是否在神经胶质细胞中共定位 使用共聚焦显微镜与免疫组织化学相结合进行体内实验。 c) 我们将测试 Rap/Fzr 是否 使用酵母二杂交检测进行物理上的 Pointed 相互作用，d) 我们将进行体外泛素化检测 并测试Pointed是否可以作为Rap/Fzr和APC泛素化的底物。 具体目标 III.测试 Apc2/Morula 是否调节胶质细胞分化：Apc2 是催化亚基 桑葚基因编码的泛素连接酶复合物 APC。我们的初步研究表明 Apc2 是神经胶质分化的负调节因子。直接评估 Apc2 在神经胶质细胞分化中的作用 细胞水平：a) 我们将测试 APC 功能丧失对神经胶质细胞分化的影响。我们将诱导克隆 发育中的幼虫大脑和眼睛中的 Apc2-/Apc2- 组织，并确定 Apc2 是否是必需的细胞 自主进行神经胶质分化，b) 我们将测试 Apc2 功能丧失对神经胶质分化的影响 使用 RNAi 技术，c) 我们将测试 Apc2 功能获得对神经胶质细胞分化的影响 使用 UAS-GAL4 和 FLP-FRT 技术生成表达 Apc2 的细胞的随机克隆。