ADAM10起始的膜剪切调控皮层神经元迁移的上下游机制

Membrane signal paradigms are essential strategies that migrating neurons converge extracellular stimuli thereby formulating instructive orders to directing internal molecular machinery. Mutations of associated genes usually cause severe migration defects. Regulated intramembrane proteolysis (RIP) is one of the critical paradigms that regulate migration, whereas its function and underlying mechanisms remain largely unknown. We have previously identified ADAM10-initiated RIP as a critical regulator of cortical neuron migration，while the undisclosed contextual mechanism requires further investigation. In this application, we will on one hand study the crosstalk between Reelin/Dab signaling and ADAM10-initiated RIP, especially the mechanism and functional significance of the modulation of ADAM10 by Reelin. On the other hand, giving the many substrate proteins that would be cleaved by ADAM10-initiated RIP, unveiling the versatile downstream effective mechanisms is crucial to understand how RIP acts in regulating neuron migration. Our preliminary results showed that ADAM10 initiated RIP of Robo2 in cortical neurons releases the ectodomain into the extracellular space. Interestingly, the function of Robo2 in regulating neuron migration did not rely on the receptor function of Robo2, but instead depended on the soluble Robo2-ECD, which might bind to another membrane partner, thereby controlling neuron migration. This effective mechanism is different from that of ADAM10-initiated RIP of Notch, which releases NICD into the cytosol and in turn translocates into the nucleus. The work of this application will bridge ADAM10-initiated RIP and Reelin signaling, and moreover expand the conceptual frame of membrane signal network that regulates the neuron migration of cerebral cortex.

膜信号转导机制是迁移神经元整合细胞外信号并形成控制内部分子机器导向指令的核心方式。受调控的膜剪切（RIP）是重要的膜信号转导机制之一。我们前期发现ADAM10起始的RIP在皮层神经元迁移中有重要作用，但其上下游机制仍不清楚。本项目将研究Reelin/Dab1信号通路与ADAM10起始的RIP之间的Crosstalk，解析ADAM10受到Reelin调控的分子机制，并检验该调控的功能意义。再者,初步结果显示ADAM10起始的RIP对Robo2的剪切释放的Robo2胞外端，将Robo2-ECD从一个受体结构域转变成游离的配体，可能通过膜上新的分子发挥调控迁移的作用。这一机制不同于Notch剪切后NICD的入核效应机制，体现了RIP下游效应机制的复杂性和独特性。本项目的工作将弥合Reelin信号和ADAM10起始的RIP通路之间的空白，并拓展调控大脑皮层神经元迁移的膜信号网络的机制框架。

膜信号转导机制是迁移神经元整合细胞外信号并形成控制内部分子机器导向指令的核心方式。在皮层神经元放射状迁移过程中，受调控的膜剪切（RIP）是重要的膜信号转导机制之一。我们前期发现ADAM10介导的RIP通过释放Notch胞内段进而在皮层神经元迁移中发挥重要作用，但其上游的调控机制仍不清楚。本项目采用分子生化实验方法，结合多个品系的敲除和条件性敲除小鼠，以及胚胎电转，显微镜形态学等多种技术手段，研究发现Reelin/Dab1信号通路与ADAM10起始的RIP之间存在显著的Crosstalk。深入解析发现，ADAM10的表达量，特别是膜表达量受到Reelin分子的调控；其在reelin ko小鼠中表达明显下降， 而在reelin重组蛋白刺激后则有明显的暂时性上升的现象。再者，通过Co-IP等方法发现，ADAM10与Dab1存在互作，且ADAM10的表达量在dab1 KO鼠中的表达也出现下降了。并且，ADAM10小鼠中发现Dab1的tyrosine磷酸化出现改变，且Dab1的tyrosine磷酸化可以调节ADAM10与Dab1的相互作用强度。本项目的工作将弥合Reelin信号和ADAM10起始的RIP通路之间的空白，并拓展调控大脑皮层神经元迁移的膜信号网络的机制框架。