analysis of srGAP2 in cortical development in vitro and in vivo

描述（由申请人提供）：大脑皮层中功能性神经元回路的形成涉及锥体神经元协调迁移至其最终位置，以及神经突随后投射至其靶点。神经元迁移和形态发生的缺陷可导致发育性神经病理学，如自闭症、无脑回畸形、精神发育迟滞和精神分裂症。通过阐明调节这些过程的机制，我们可以提高我们对这些社会破坏性疾病的发展机制的理解。为了这个目的，我将检查的作用和调节的蛋白质，slit- robo GTP酶激活蛋白2（srGAP 2），在神经元分化。最近，Polleux实验室已经发现srGAP 2作为皮质神经元迁移的负调节剂和相同神经元中神经突起始和分支的正调节剂的功能。此外，我们发现srGAP 2通过其形成富含肌动蛋白的丝状伪足样膜突起的能力增加皮质神经元中的神经突起始和分支。我们的初步结果表明，这一功能主要是由其N-末端，膜变形F-BAR结构域。有趣的是，阻止其Src同源3（SH 3）结构域与其结合伴侣相互作用的突变阻断了srGAP 2在神经突分支和神经元迁移中的功能。相反，缺乏C-末端的srGAP 2的截短形式，包括SH 3结构域，增加神经突分支并阻断神经元迁移，与全长srGAP 2一样有效。为了解释这些结果，我们假设srGAP 2的C-末端结构域与其F-BAR结构域的突出活性相互作用并抑制其突出活性。在这种“自抑制”模型中，特定相互作用物与SH 3结构域的结合通过释放F-BAR结构域激活srGAP 2，使其二聚化并使膜变形。为了验证这一假设，我将把srGAP 2的突变形式导入皮层神经元，以进行结构/功能分析，目的是鉴定参与srGAP 2自抑制和激活的残基（目的1）。此外，我将使用这种技术，沿着与免疫共沉淀，以确定神经元特异性，SH 3相互作用的蛋白质，并确定这些候选蛋白质是否参与激活srGAP 2在皮层神经元形态发生（目的2）。最后，我们获得了靶向srGAP 2敲除小鼠，以帮助建立srGAP 2在体内的作用（目的3）。增强我们对srGAP 2在神经突形成和神经元迁移中的功能机制的理解将提高我们对皮质发育的分子机制的理解。

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.