Doublecortin in Neuronal Migration

双皮质素在神经元迁移中的作用

• 批准号: 7252002
• 负责人: JOSEPH G GLEESON
• 金额: $ 33.75万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7252002
• 关键词: Actin-Binding Protein; Actins; Address; Biochemical Genetics; Brain; Cell Nucleus; Cells; Centrosome; Cerebral Palsy; Condition; Cortical Malformation; Coupling; Cyclin-Dependent Kinase 5; Cytoskeleton; Data; Defect; Development; Disruption; Epilepsy; Family; Female; Gene Family; Gene Silencing; Generations; Genes; Goals; Growth Cones; Homologous Gene; Human; Imaging Techniques; Immigration; Knock-out; Knockout Mice; Lead; Life; Link; Masks; Mediating; Mental Retardation; Microtubule Stabilization; Microtubule-Associated Proteins; Microtubules; Miller-Dieker Syndrome; Molecular; Movement; Mus; Mutation; Neurocognitive Deficit; Neuronal Migration Disorder; Neurons; Nuclear; Nuclear Translocation; Pathway interactions; Patients; Phase; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physical condensation; Placement; Process; Protein Dephosphorylation; Protein phosphatase; Proteins; Reagent; Regulation; Reporter; Reporting; Role; Signal Pathway; Site; Small Interfering RNA; Specificity; Syndrome; Testing; Transgenic Organisms; Work; cellular imaging; in vivo; link protein; male; migration; nervous system disorder; neuronal cell body; neuronal growth; novel; relating to nervous system; spinophilin

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.

描述（由申请人提供）：由于神经元迁移障碍导致的皮质发育畸形越来越被认为是癫痫、智力低下和脑瘫的常见原因。双皮质素（DCX）基因对人类神经元迁移至关重要，因为突变导致男性X连锁无脑畸形和女性皮质下带状异位，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，并通过磷酸化依赖性机制参与几个信号通路。我们还确定了一个潜在的作用Dcx耦合核的中心体在微管依赖性的方式在迁移的核易位阶段。Dcx是Dck 1和Dck 2基因家族的一部分，Dck 1和Dck 2各自编码一种强脑表达蛋白，具有紧密匹配的Dcx结构域和激酶结构域。这项更新申请的总体目标是阐明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，这将协同提供一个强大的方法来实现这一目标。目标1.测试Dcx同源物Dck 1和Dck 2在神经元迁移和大脑发育中的功能冗余程度。Dcx在小鼠迁移中的功能可能与Dck 1和Dck 2基因是冗余的。我们将通过分析Dck 1和Dck 2单基因敲除小鼠以及双基因敲除小鼠和三基因敲除小鼠的表型来分析功能冗余的程度，并将这些结果与siRNA介导的基因敲除方法进行比较。目标2.检测Dcx家族基因失活中MT稳定性和核-中心体偶联的缺陷。利用Aim 1的方法和先进的活细胞成像技术，我们将测试Dcx基因家族是否是神经元迁移中MT依赖性核运动所必需的。目标3.检测Dcx基因家族MT效应的磷酸化和磷酸酶依赖性调节。我们以前的数据表明，Dcx功能通过磷酸化进行强烈的负调控。我们现在有遗传和生化数据，肌动蛋白连接的蛋白磷酸酶I和MT连接的DCK 1/2提供额外的磷酸化依赖性调节水平。我们将测试这些相互作用的特异性，使用这里生成的试剂，并测试它们在整合微管和肌动蛋白细胞骨架所需的稳定神经生长锥的作用。摘要：doublecortin的突变导致人类严重的神经系统疾病，这是由于通过未知的机制改变了大脑发育。本研究旨在利用先进的分子和细胞方法来确定doublecortin基因家族的功能。