Role of Cdc42 and Par6 Polarity Complex in CNS Neuronal Migration

描述(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的迁移，它们在那里产生，并组装成神经层，在那里形成突触连接。由于中枢神经系统移行障碍与多种皮质畸形相关，是人类发育过程中智力低下和癫痫等疾病的主要原因，因此，对中枢神经系统神经元移行的分子调控机制的进一步了解将有助于人类发育性脑疾病的诊断和治疗。神经元的迁移在很大程度上取决于神经元在力矩方向上的极化，在这一资助的支持下，我们先前证实，在迁移的小脑颗粒神经元中，保守的mPar61极性复合体定位于中心体，并通过激活前导突近端区域的肌球蛋白收缩马达来协调中心体和胞体的向前运动。这项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，包括高极化主导过程的形成和维持，并确定它们在神经元黏附蛋白受体运输中的作用(S)。尽管CDC42在许多非神经细胞的迁移、树突分枝和轴突引导中的作用已经明确，但在活的、迁移的CNS神经元的高分辨率时间推移成像中，CDC42的作用和Par6亚型的相对作用还没有被分析。鉴于胶质引导的迁移对大脑皮层神经元层形成的重要性，我们将以小脑颗粒神经元在胶质细胞上迁移为模型系统，重点研究这一高度专业化的迁移系统。在目标1中，我们将研究CDC42功能的条件性丧失；在目标3中，将对颗粒细胞前体(GCP)中表达的三种Par6亚型中的每一种实施类似的计划。作为补充，在AIMS 2和3中，我们将使用siRNAs和shRNAs敲除CDC42和Par6亚型水平，并比较敲除表型和条件性功能丧失表型。如果CDC42与任何或所有Par6亚型处于相同的遗传途径，我们预计会看到CDC42和Par6亚型功能丧失的类似表型。我们还将使用松田美树博士提供的Raichu探针来检测cdc42，这是一种基于FRET的探针，可以监测细胞局部区域的cdc42激活情况。这些探针将使我们能够评估CDC42激活相对于Par6亚型在迁移的GCP中的时空定位。Par6在小脑发育过程中表达变化模式的发现为了解它们对神经元迁移的相对贡献以及它们是否在CDC42信号通路中发挥作用提供了一个令人兴奋的机会。

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.