Role of Cdc42 and Par6 Polarity Complex in CNS Neuronal Migration

描述（由申请人提供）：哺乳动物脑的发育取决于神经元前体从它们产生的生发区迁移，以及它们组装成神经元板层，在那里形成突触连接。由于CNS迁移障碍与许多皮质畸形相关，并且是发育中的人脑中疾病的主要原因，包括精神发育迟滞和癫痫，因此更清楚地了解CNS神经元迁移的分子控制可能与人类发育性脑障碍的诊断和治疗相关。神经元迁移严重依赖于极化的神经元的方向的时刻，并支持从这个补助金，我们以前建立了保守的mPar 61极性复合物定位到中心体和协调的中心体和索马的向前运动，通过一种机制，包括激活肌动蛋白收缩马达在近端区域的领先过程中，在迁移小脑颗粒神经元。拟议研究的总体目标是确定主极性调节剂Cdc 42和三种Par 6亚型对CNS迁移关键步骤的相对贡献，包括高度极化的前导过程的形成和维持，并确定它们在神经元粘附蛋白受体运输中的作用。虽然Cdc 42在许多非神经元细胞的迁移中，以及在树突状分支和轴突引导中已经确定了明确的作用，但Cdc 42的作用和Par 6同种型的相对作用尚未在活体迁移的CNS神经元的高分辨率延时成像中进行分析。鉴于胶质细胞引导的迁移在大脑皮层区域神经元层形成中的重要性，我们将专注于这个高度专业化的迁移系统，使用小脑颗粒神经元迁移胶质细胞作为我们的模型系统。在目标1中，我们将研究Cdc 42功能的条件性丧失;在目标3中，将对颗粒细胞祖细胞（GCPs）中表达的三种Par 6亚型中的每一种实施类似的计划。作为补充方法，在目标2和3中，我们将使用siRNA和shRNA敲低Cdc 42和Par 6同种型水平，并比较敲低表型与功能表型的条件性丧失。如果Cdc 42与任何或所有Par 6同种型处于相同的遗传途径，我们将预期看到Cdc 42和Par 6同种型功能丧失的相似表型。我们还将使用三木松田博士提供的Cdc 42的Raichu探针，这是基于FRET的探针，可监测细胞局部区域的Cdc 42激活。这些探针将使我们能够评估Cdc 42激活相对于Par 6亚型迁移GCP的时空定位。在小脑发育过程中发现Par 6表达模式的变化是一个令人兴奋的机会，可以了解它们对神经元迁移的相对贡献以及它们是否在Cdc 42信号通路中起作用。

DESCRIPTION (provided by applicant): The development of the mammalian brain depends on the migrations of neuronal precursors from germinal zones, where they are generated, and their assembly into neuronal laminae, where synaptic connections form. Since CNS migration disorders are associated with a number of cortical malformations, and are a major cause of disease in the developing human brain, including mental retardation and epilepsy, a clearer understanding of the molecular control of CNS neuronal migration could be relevant to the diagnosis and treatment of human developmental brain disorders. Neuronal migration critically depends on the polarization of the neuron in the direction of moment, and with support from this grant, we previously established that the conserved mPar61 polarity complex localizes to the centrosome and coordinates the forward movement of the centrosome and soma, by a mechanism that includes activation of acto-myosin contractile motors in the proximal region of the leading process, in migrating cerebellar granule neurons. The overall goal of the proposed research is to define the relative contributions of the master polarity regulator Cdc42 and the three Par6 isoforms to critical steps in CNS migration, including the formation and maintenance of a highly polarized leading process, and to define their role(s) in receptor trafficking of neuronal adhesion proteins. Although a clear role for Cdc42 has been established in the migration of many non-neuronal cells, and in dendritic arborization and axon guidance, the roles of Cdc42 and the relative role of the Par6 isoforms have not been analyzed in high-resolution time-lapse imaging of live, migrating CNS neurons. Given the importance of glial-guided migration to the formation of neuronal layers in cortical regions of brain, we will focus on this highly specialized migration system, using cerebellar granule neurons migrating on glia as our model system. In Aim 1, we will study a conditional loss of function of Cdc42; a similar plan will be implemented in Aim 3 for each of the three Par6 isoforms expressed in granule cell progenitors (GCPs). As a complementary approach, in Aims 2 & 3, we will use siRNAs and shRNAs to knockdown Cdc42 and Par6 isoform levels and compare knockdown phenotypes with conditional loss of function phenotypes. If Cdc42 is in the same genetic pathway with any or all of the Par6 isoforms, we would expect to see similar phenotypes for Cdc42 and Par6 isoform loss of function. We will also use Raichu probes for Cdc42, kindly provided by Dr. Miki Matsuda, which are FRET-based probes that monitor Cdc42 activation in localized regions of a cell. These probes will enable us to evaluate the spatiotemporal localization of Cdc42 activation relative to the Par6 isoforms in migrating GCPs. The discovery of changing patterns of Par6 expression during cerebellar development is an exciting opportunity to understand their relative contributions to neuronal migration and whether they act within a Cdc42 signaling pathway.