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向其最终位置的协调迁移，以及随后神经突向其目标的投射。神经元迁移和形态发生的缺陷可导致发育性神经病变，如自闭症、无脑畸形、智力迟钝和精神分裂症。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。为此，我将研究在神经元分化过程中，狭缝机器人GTPase激活蛋白2 （srGAP2）的作用和调控。最近，Polleux实验室发现srGAP2作为皮层神经元迁移的负调节因子和同一神经元中神经突起始和分支的正调节因子的功能。此外，我们发现srGAP2通过形成富含肌动蛋白、丝状足样膜突起的能力，增加皮层神经元的神经突起始和分支。我们的初步结果表明，这一功能主要是通过其N端，膜变形的F-BAR结构域来实现的。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断了srGAP2在神经突分支和神经元迁移中的功能。相反，缺少c末端的srGAP2的截断形式，包括SH3结构域，增加了神经突分支并阻碍了神经元迁移，其效果与全长srGAP2一样。为了解释这些结果，我们假设srGAP2的c端结构域与F-BAR结构域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SH3结构域的结合通过释放F-BAR结构域激活srGAP2，使其二聚化并使膜变形。为了验证这一假设，我将把srGAP2的突变形式转染到皮质神经元中，以便进行结构/功能分析，旨在确定srGAP2的自抑制和激活的残基（目的1）。此外，我将使用这种技术，连同共免疫沉淀，以确定神经元特异性，sh3相互作用的蛋白，并确定这些候选蛋白是否参与激活srGAP2在皮质神经元形态发生（目的2）。最后，我们获得了srGAP2敲除小鼠来帮助确定srGAP2在体内的作用（目的3）。加强我们对srGAP2在神经突形成和神经元迁移中的作用机制的理解，将有助于我们对皮层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.