周期蛋白依赖性蛋白激酶5调控神经突触发育的机制研究

The objectives of this proposal are to understand the interaction and regulation of cyclin-dependent kinase 5 (Cdk5) and its upstream cell surface receptor tyrosine kinase EphB2, and to unravel the novel mechanisms by which Cdk5 regulates EphB2-dependent synapse development in hippocampal pyramidal neurons. Through combinatory use of multiple tools in molecular and cellular biology, immunocytochemistry, electrophysiology etc., we will investigate (1) the in vivo and in vitro interaction between Cdk5 and EphB2, as well as the dependency on their kinase activity; (2) the mutual regulation of their kinase activity between Cdk5 and EphB2 and the key phosphorylation sites; (3) the role of EphB2 activation on the dendritic spine morphogenesis, synaptic protein trafficking, and synaptic transmission; (4) the regulation of EphB2 function at synapse by Cdk5 activity; and (5) the molecular mechanisms by which Cdk5 regulates EphB2-dependent synapse development, with special focus on the phosphorylation sites, activity modulation and action of Cdk5 substrates. The findings from this study will not only advance our understanding of the spatiotemporal mechanisms regulating synapse development and function, especially how cell surface receptors and their downstream kinase receive and process neuronal signals, but also shed new light on the development of therapeutic tools for repairing synapse.

本项研究旨在解析蛋白激酶Cdk5与上游受体酪氨酸激酶EphB2的相互作用与调控机制，明确Cdk5在EphB2依赖性的突触发育中的作用。本项目以海马锥体神经元为研究对象，综合运用分子细胞生物学、免疫细胞化学、电生理学等方法，研究（1）Cdk5与EphB2在体内、体外的相互作用，及其相互作用对激酶活性的依赖性；（2）Cdk5与EphB2对相互之间激酶活性的调控，及其关键磷酸化位点；（3）EphB2激活对神经元树突棘发育、突触蛋白转运、突触传递的调控作用；（4）调控Cdk5活性对于EphB2突触功能的影响；（5）Cdk5参与EphB2依赖性突触调控的机制，尤其是Cdk5底物的磷酸化位点、活性改变和作用机理。 通过本项目的研究，我们能够加深对突触发育和功能的时空调控机制的理解，进一步明确神经元通过细胞表面受体和下游蛋白激酶对神经信号的接收、加工和处理机制，也能为修复突触障碍的手段研发提供新的思路。

本项目解析了蛋白激酶Cdk5与上游受体酪氨酸激酶EphB2的相互作用与调控机制，明确Cdk5在EphB2依赖性的突触发育中的作用。取得了以下主要研究发现：（1）明确EphB2与Cdk5/p35复合物的相互作用，及其相互作用对激酶活性的依赖性；（2）揭示了EphB2激活对神经元树突棘发育、突触蛋白转运、突触传递的调控作用；（3）论证了Cdk5/p35复合物参与EphB2依赖性突触调控的机制。这些研究发现，不但能够加深我们对突触发育和功能的时空调控机制的理解，尤其是神经元通过细胞表面受体和下游蛋白激酶对神经信号的接收、加工和处理机制，也能为修复突触障碍的手段研发提供新的思路。