Role of Cdc42 and Par6 Polarity Complex in CNS Neuronal Migration

描述（由申请人提供）：哺乳动物大脑的发育依赖于神经元前体从生发区（它们产生的地方）迁移到神经元层（突触连接形成的地方）。由于中枢神经系统迁移障碍与许多皮质畸形有关，并且是发育中的人类大脑疾病的主要原因，包括智力迟钝和癫痫，因此更清楚地了解中枢神经系统神经元迁移的分子控制可能与人类发育性大脑疾病的诊断和治疗有关。神经元迁移严重依赖于神经元在瞬间方向上的极化，在此资助的支持下，我们之前建立了保守的mPar61极性复合物定位于中心体，并通过一种机制协调中心体和体的向前运动，该机制包括在迁移小脑颗粒神经元的前导过程的近端区域激活肌球蛋白收缩马达。本研究的总体目标是确定主极性调节因子Cdc42和三种Par6亚型在中枢神经系统迁移的关键步骤中的相对贡献，包括高度极化先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。尽管Cdc42在许多非神经元细胞的迁移、树突树突和轴突引导中发挥了明确的作用，但在活体迁移的CNS神经元的高分辨率时移成像中，尚未分析Cdc42的作用以及Par6同种异构体的相对作用。鉴于神经胶质引导的迁移对大脑皮层神经元层形成的重要性，我们将重点研究这种高度专业化的迁移系统，使用小脑颗粒神经元在神经胶质上迁移作为我们的模型系统。在Aim 1中，我们将研究Cdc42的条件功能丧失；在Aim 3中，将对颗粒细胞祖细胞（gcp）中表达的三种Par6亚型中的每一种实施类似的计划。作为一种补充方法，在Aims 2和3中，我们将使用sirna和shrna敲低Cdc42和Par6亚型水平，并将敲低表型与条件功能缺失表型进行比较。如果Cdc42与任何或所有Par6亚型处于相同的遗传途径中，我们将期望看到Cdc42和Par6亚型功能丧失的相似表型。我们还将使用Miki Matsuda博士提供的Raichu Cdc42探针，这是一种基于fret的探针，用于监测细胞局部区域的Cdc42激活。这些探针将使我们能够评估迁移gcp中Cdc42激活相对于Par6亚型的时空定位。发现小脑发育过程中Par6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Cdc42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.