Doublecortin in Neuronal Migration

描述（由申请人提供）：由神经元迁移障碍引起的皮层发育畸形越来越被认为是癫痫、智力迟钝和脑瘫的常见原因。双皮质素（DCX）基因对人类神经元迁移至关重要，突变导致男性x连锁无脑畸形和女性皮质下带异位，产生严重的神经认知缺陷。我们确定了DCX基因，并在患有这种疾病的患者中发现了突变。我们确定了它作为微管（MT）相关蛋白的作用，并通过磷酸化依赖机制参与了几种信号通路。我们还确定了Dcx在细胞核迁移易位阶段以微管依赖的方式将细胞核与中心体耦合的潜在作用。Dcx是一个包含Dck1和Dck2的基因家族的一部分，每个基因都编码一种具有密切匹配的Dcx结构域和激酶结构域的强脑表达蛋白。本应用的总体目标是阐明Dcx基因家族在神经元迁移和脑功能中的分子和细胞机制。我们将利用基因敲除和转基因报告小鼠，结合先进的活细胞成像能力和体内分析，为实现这一目标提供强有力的方法。目的1。检测Dcx同源基因Dck1和Dck2在神经元迁移和大脑发育中的功能冗余程度。在小鼠中，Dcx在迁移中的功能可能与Dck1和Dck2基因是冗余的。我们将通过分析Dck1和Dck2单敲除、双敲除和三敲除小鼠的表型来分析功能冗余程度，并将这些结果与sirna介导的基因敲除方法进行比较。目标2。dcx家族基因失活中MT稳定性和核-中心体偶联缺陷的检测。利用Aim 1的方法和先进的活细胞成像技术，我们将测试Dcx基因家族在神经元迁移中是否需要mt依赖的核运动。目标3。测试磷酸化和磷酸酶依赖性调节MT对Dcx基因家族的影响。我们之前的数据表明，通过磷酸化，Dcx功能具有很强的负调控作用。我们现在有遗传和生化数据表明，肌动蛋白连接的蛋白磷酸酶I和mt连接的Dck1/2提供了额外水平的磷酸化依赖性调节。我们将使用这里生成的试剂来测试这些相互作用的特异性，并测试它们在整合微管和肌动蛋白细胞骨架方面的作用，这些微管和肌动蛋白细胞骨架是稳定神经元生长锥所需的。摘要：双皮质素突变可通过未知机制改变人类大脑发育，从而导致严重的神经系统疾病。本研究旨在利用先进的分子和细胞方法鉴定双皮质素基因家族的功能。

DESCRIPTION (provided by applicant): Malformations of cortical development due to disorders of neuronal migration are increasingly recognized as a common cause of epilepsy, mental retardation and cerebral palsy. The doublecortin (DCX) gene is critical for neuronal migration in humans, as mutations result in X-linked lissencephaly in males and subcortical band heterotopia in females, producing severe neurocognitive deficits. We identified the DCX gene, and found mutations in patients with this condition. We identified its role as a microtubule (MT)-associated protein and its involvement in several signaling pathways through phosphorylation-dependent mechanisms. We also identified a potential role for Dcx in coupling the nucleus to the centrosome in a microtubule-dependent fashion during the nuclear translocation phase of migration. Dcx is part of a gene family also containing Dck1 and Dck2, each encoding a strongly brain-expressed protein with a closely matching Dcx domain and a kinase domain. The overall goal of this renewal application is to elucidate the molecular and cellular mechanisms of the Dcx gene family in neuronal migration and brain function. We will utilize knockout and transgenic reporter mice combined with advanced live-cell imaging capabilities and in vivo analysis that will synergize to provide a powerful approach address this goal. Aim 1. Test the degree of functional redundancy of Dcx homologues Dck1 and Dck2 in neuronal migration and brain development. The function of Dcx in migration may be redundant with the Dck1 and Dck2 genes in mouse. We will analyze the degree of functional redundancy through the analysis of phenotype of Dck1 and Dck2 single as well as double and triple knockout mice and compare these results with siRNA-mediated gene knockdown approaches. Aim 2. Test for defects in MT stabilization and nuclear-centrosomal coupling in Dcx-family gene inactivation. Utilizing the approaches from Aim 1 and advanced live cell imaging techniques, we will test whether the Dcx gene family is required for MT-dependent nuclear movement in neuronal migration. Aim 3. Test for phosphorylation and phosphatase-dependent regulation of the MT effects of the Dcx gene family. Our previous data has indicated strong negative-regulation of Dcx function through phosphorylation. We now have genetic and biochemical data that actin-linked protein-phosphatase I and MT-linked Dck1/2 provide additional levels of phosphorylation-dependent regulation. We will test the specificity of these interactions using the reagents generated here and test their role in integrating the microtubule and actin cytoskeletons required for stabilization of neuronal growth cones. Lay Summary: Mutations in doublecortin lead to severe neurological disorders in humans due to altered brain development through unknown mechanisms. This study seeks to identify the function the family of doublecortin genes using advanced molecular and cellular approaches.