analysis of srGAP2 in cortical development in vitro and in vivo

srGAP2 在体外和体内皮质发育中的分析

• 批准号: 8213741
• 负责人: Jaeda Coutinho-Budd
• 金额: $ 1.41万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8213741
• 关键词: Actins; Amino Acids; Autistic Disorder; Axon; Binding; Biochemical; Brain; C-terminal; Cell Culture Techniques; Cell physiology; Cells; Cerebral cortex; Charge; Co-Immunoprecipitations; Code; Coiled-Coil Domain; Complex; Cues; Cytoskeleton; Defect; Dendrites; Dependence; Development; Disease; Drosophila sli protein; Electroporation; Endocytosis; Filopodia; Functional disorder; GTPase-Activating Proteins; Genes; Genetic; Glutamates; Human; In Vitro; Introns; Knockout Mice; Lead; Length; Location; Membrane; Mental Retardation; Microtubules; Modeling; Molecular; Morphogenesis; Morphology; Mus; Mutation; N-terminal; Neurites; Neuronal Differentiation; Neurons; Newborn Infant; Pathology; Play; Point Mutation; Process; Protein Conformation; Proteins; Proteomics; Regulation; Role; Schizophrenia; Signal Pathway; Signal Transduction; Structure; T-Lymphocyte; Techniques; Testing; extracellular; hippocampal pyramidal neuron; improved; in vivo; in vivo Model; lissencephaly; loss of function; migration; mutant; neurite growth; neuropathology; novel; overexpression; prevent; response; rho

DESCRIPTION (provided by applicant): Formation of functional neuronal circuits in the cerebral cortex involves coordinated migration of pyramidal neurons to their final location, as well as subsequent projection of neurites to their targets. Defects in both neuronal migration and morphogenesis can lead to developmental neuropathologies such as autism, lissencephaly, mental retardation, and schizophrenia. By elucidating the mechanisms that regulate these processes, we can improve our understanding of the developmental mechanisms underlying some of these socially-devastating diseases. Towards this aim, I will examine the role and regulation of the protein, slit- robo GTPase Activating Protein 2 (srGAP2), during neuronal differentiation. Recently, the Polleux lab has uncovered the function of srGAP2 as a negative regulator of cortical neuron migration and a positive regulator of neurite initiation and branching in the same neurons. Moreover, we found that srGAP2 increases neurite initiation and branching in cortical neurons through its ability to form actin-rich, filopodia- like membrane protrusions. Our preliminary results show that this function is primarily carried out by its N- terminal, membrane-deforming F-BAR domain. Interestingly, a mutation preventing its Src Homology 3 (SH3) domain to interact with its binding partners blocks the function of srGAP2 in neurite branching and neuronal migration. Conversely, a truncated form of srGAP2 lacking the C-terminal end, including the SH3 domain, increases neurite branching and blocks neuronal migration as potently as full-length srGAP2. In order to explain these results, we hypothesize that the C-terminal domain of srGAP2 interacts with, and inhibits, the protruding activity of its F-BAR domain. In this 'auto-inhibitory' model, binding of specific interactors to the SH3 domain activates srGAP2 by releasing the F-BAR domain, allowing it to dimerize and deform membrane. To test this hypothesis, I will transfect mutant forms of srGAP2 into cortical neurons in order to perform a structure/function analysis aimed at identifying the residues involved in the auto-inhibition and activation of srGAP2 (Aim 1). Additionally, I will use this technique, along with co-immunoprecipitation, to identify neuron-specific, SH3-interacting proteins and determine if these candidate proteins are involved in activating srGAP2 during cortical neuron morphogenesis (Aim 2). Finally, we have acquired a targeted srGAP2 knockout mouse to help establish the role of srGAP2 in vivo (Aim 3). Enhancing our understanding of the mechanisms underlying srGAP2 function in neurite formation and neuronal migration will improve our understanding of the molecular mechanisms underlying cortical development.

描述(申请人提供)：大脑皮层功能神经元回路的形成涉及锥体神经元向其最终位置的协调迁移，以及随后神经突起向其目标的投射。神经元迁移和形态发生的缺陷都可能导致发育神经病理，如自闭症、无脑畸形、智力低下和精神分裂症。通过阐明调控这些过程的机制，我们可以更好地理解这些具有社会破坏性的疾病背后的发育机制。为此，我将研究Sit-Robo GTP酶激活蛋白2(SRGAP2)在神经元分化过程中的作用和调控。最近，Polleux实验室发现了SRGAP2作为皮质神经元迁移的负调节因子和在相同神经元中的轴突起始和分支的正调节因子的功能。此外，我们还发现，SRGAP2通过形成富含肌动蛋白的丝状足状突起，增加了皮质神经元中轴突的起始和分支。我们的初步结果表明，这一功能主要是通过其N端、膜变形的F-bar结构域实现的。有趣的是，阻止其Src同源3(SH3)结构域与其结合伙伴相互作用的突变阻止了SRGAP2在轴突分支和神经元迁移中的功能。相反，缺少C-末端的截短形式的SRGAP2，包括SH3结构域，增加了轴突分支，并像全长SRGAP2一样有效地阻止神经元迁移。为了解释这些结果，我们假设SRGAP2的C-末端结构域与其F-bar结构域相互作用并抑制其突出活性。在这个“自动抑制”模型中，特定的相互作用体与SH3结构域的结合通过释放F-bar结构域来激活SRGAP2，使其二聚化并使膜变形。为了验证这一假设，我将把SRGAP2的突变形式导入皮质神经元，以便进行结构/功能分析，目的是确定参与SRGAP2自身抑制和激活的残基(目标1)。此外，我将使用这项技术，结合免疫共沉淀，鉴定神经元特异性的SH3相互作用蛋白，并确定这些候选蛋白是否参与激活皮质神经元形态发生过程中的SRGAP2(目标2)。最后，我们获得了一只有针对性的SRGAP2基因敲除小鼠，以帮助建立SRGAP2在体内的作用(目标3)。加强对SRGAP2在轴突形成和神经元迁移中作用机制的了解，将有助于我们更好地理解皮质发育的分子机制。

项目成果

Inhibition of SRGAP2 function by its human-specific paralogs induces neoteny during spine maturation.

SRGAP2 人类特异性旁系同源物对 SRGAP2 功能的抑制可在脊柱成熟过程中诱导幼态持续

• DOI: 10.1016/j.cell.2012.03.034
• 发表时间: 2012-05-11
• 期刊: Cell
• 影响因子: 64.5
• 作者: Charrier C;Joshi K;Coutinho-Budd J;Kim JE;Lambert N;de Marchena J;Jin WL;Vanderhaeghen P;Ghosh A;Sassa T;Polleux F
• 通讯作者: Polleux F