脑皮质投射神经元极性建立过程中的肌动蛋白细胞骨架调控解析

The neural circuit formation and function of cerebral cortex are determined by a highly-laminated structure that contains six layers. Different subtypes of glutamatergic projection neurons reside in each layer, forming distinct lamina-specific cortical and subcortical connections. The precise positioning of these projection neurons is directed by radial migration of newly born neurons from the ventricular zone. Importantly, a neuronal polarization process of the newly born neurons, i.e. multipolar-to-bipolar transition, is a determining step for the initiation of radial migration. The failure of the establishment of the bipolar polarity normally stalls radial migration, hence leading to misplacement of the neurons and impairment of cortical function. Disruption of neuronal migration is among the most common causes of cortical malformation disorders and is associated with neuropsychiatric diseases such as schizophrenia and autism. Nonetheless, the molecular control of multipolar-to-bipolar transition still remains to be fully understood. The applicant's research focuses on the molecular basis of neuronal polarization and radial migration, and has contributed a great deal to the understanding of the regulation of actin cytoskeleton. The proposed project aims to thoroughly analyze the dynamics and regulation of actin cytoskeleton during multipolar-to-bipolar transition. Using in utero electroporation approach to label newly born neurons, we will examine F-actin distribution and dynamics, and assess how polymerization/depolymerization of actin affect the polarization of these neurons. Furthermore, we will design an siRNA library of actin regulators (including actin-binding proteins, Rho GTPase signaling proteins and scaffold proteins), and conduct high content screening for important regulators during polarization. We will also prepare PCR array plates to analyze whether the gene transcription of these regulators is controlled during neuronal polarization or by extracellular signals. The findings of the proposed project will not only contribute to a better understanding of the molecular mechanism of critical developmental step during cortical lamination, but also provide new insights into the study of neurological diseases related to impaired corticogenesis.

大脑皮质是一个多层结构，各层有形态和功能特异的投射神经元，由胚胎不同时期脑室区新生的神经元放射性迁移而来。放射性迁移起始的一个决定步骤是新生神经元由多极向双极形态转变的极化过程。该极性建立的失败通常导致投射神经元错误定位和脑皮质功能紊乱，并与脑皮质发育疾病相关。本课题组前期对神经元极化和放射性迁移的分子机制已有一定研究积累，并揭示了数条肌动蛋白细胞骨架的调控通路。在此基础上，本项目拟通过子宫内电穿孔技术标记胎鼠脑室区的新生神经元，观察其在极性建立时肌动蛋白细胞骨架的动态变化特征，并明确肌动蛋白的聚集或解聚对极性建立的调控作用。拟进一步通过高内涵筛选及荧光定量PCR手段筛查极性建立时重要的肌动蛋白调控因子（如肌动蛋白结合蛋白或Rho 家族小G蛋白通路蛋白等），并探索不同胞外信号对这些因子的调控作用。研究结果将有助于明确脑皮质片层化构筑的重要发育步骤，并为寻找脑皮质发育疾病的分子靶点提供线索。

神经元发育的多个阶段受到肌动蛋白细胞骨架的严格调控，而Rho GTPases是肌动蛋白细胞骨架的关键调节因子。Rho GTPases活性的时空调节对神经元的发育至关重要，而其参与通路的异常改变与多种神经精神疾病密切相关。本项目研究Rho GTPases的两种鸟苷酸置换因子Arhgef1和Dock4（分别激活RhoA和Rac1），通过对大脑皮层、海马等部位的形态及体外培养的神经元展开研究，明确该两个因子在神经元发育过程中的作用及其调控Rho GTPases的分子机制。在第一部分，发现Arhgef1在脑皮层发育早期高度表达，且其表达主要集中在神经前体细胞和早期发育的神经元中。通过loss-of-function和gain-of-function方法在体外培养的皮层神经元中研究，发现Arhgef1的表达量与神经突起的生长呈负相关，说明该蛋白是神经突起发育的负调控因子。机制研究发现，Arhgef1中激活RhoA的结构域对于其抑制神经突起的作用必不可少，并通过RhoA抑制剂及过表达不同活性RhoA蛋白的方法证实RhoA激活的确是Arhgef1作用的下游机制。进一步地，发现cofilin抑制和肌动蛋白聚集是Arhgef1-RhoA的下游机制。在第二部分，研究Dock4敲除小鼠的脑皮层和海马区的结构及神经元形态及功能的改变。结果发现，敲除鼠的侧脑室空腔呈现一定程度上增大，脑皮质厚度正常，但不同皮层片层的细胞数有所改变，如Cux1 阳性的II-IV 层和Ctip2阳性的V 层细胞数有所增多。在体外培养的皮层神经前体细胞中，发现干扰Dock4后促进细胞增殖、抑制分化。在海马区的研究发现，齿状回和CA1区的神经元树突棘密度显著减少，并伴随突触可塑性下降。这些研究有助于揭示Rho GTPases在脑发育中的时空调控模式，并为进一步研究脑发育-行为关系提供证据。