膜结构性钾氯共转运体KCC2 在癫痫沉默期海马神经元兴奋性突触重构中的作用与机制研究

Epileptogenesis contents seizure induction, latent, and chronic seizure periods. Studies have demonstrated that hippocampal excitatory synaptic transmission reduced in the early phase of the latent period and later enhanced at the late phase of the latent period/chronic seizure period, which suggests that there would be excitatory synaptic reorganization during latent period of epileptogensis, but lack of studies to investigate its underlying mechanism. Membrane structural KCC2 has been shown to have important functional role in spine maturation and keeping spine stability. In our previous study, we have discovered that membrane KCC2 down regulation facilitated the epileptic seizure occurrence. In this proposed study, we tend to use the clinical surgical removed seizure patient epileptic brain tissue and mice seizure model to investigate :1) whether membrane structural KCC2 down regulation induces (a) disassemble of protein 4.1N with F-actin, and (b) activation of β-PIX–Rac1 pathway causing inactivation of cofilin due to its phosphorylation and subsequently impairment of F-actin function, which leads to the loss of spine in the early phase of the latent period; 2) whether the recovery of the membrane structural KCC2 in the late phase of the latent period would (a) over activate of cofilin-actin function and induce abnormal spine growing, (b) facilitate the KCC2-Neto2-GluK2 oligomeric to functional expressed along with the membrane structure KCC2 recovery from the early down regulation, which leads to abnormal excitatory synaptic current kanatics and induces spontaneous epileptiform activities; 3) to explore the possibility of early KCC2 down regulation and spinal loss is the major cause of the late phase synaptic reorganization during epileptogenesis. The output from this study would provide the valuable information of the underlying mechanism of epileptogenesis and the early intervention target for drug development.

癫痫病理进程分急性、沉默和慢性期。沉默期的海马兴奋性突触传递先抑制后增强，提示有兴奋性突触重构发生，然而其机制不明。神经元膜结构性KCC2在树突棘形成与结构维护及受体动态调控中起重要作用，而我们的前期研究发现膜KCC2在癫痫早期的下膜促进了癫痫大发作形成。本项目拟应用人源癫痫灶样本及小鼠癫痫模型，研究阐明1）膜结构性KCC2下调导致的（a）4.1N 蛋白去稳定化和（b）βPIX-Rac1激活后磷酸化cofilin而去活性二条通路导致沉默早期F-actin异常相关树突棘丢失；2）沉默后期KCC2恢复性上膜将（a）过度激活cofilin-actin导致树突棘新生异常和（b）促进KCC2-Neto2-GluK2复合体上膜形成异常突触电动力学的KAR而导致癫痫自发放电；3）探索沉默早期KCC2下调及树突棘丢失是慢性期异常兴奋性突触重构的必要条件。此研究成果将为探索癫痫进程早期干预靶点提供理论依据。

慢性癫痫的病理进程分急性、沉默和慢性期。慢性癫痫的形成，特别是癫痫沉默期在慢性癫痫的发生发展中的重要作用及分子机制不明，也越来越受到重视。沉默期的海马兴奋性突触传递先抑制后增强，提示有兴奋性突触重构发生，然而其机制不明。我们的前期研究发现膜KCC2在癫痫早期的下膜促进了癫痫大发作形成，而神经元膜结构性KCC2在树突棘形成与结构维护及受体动态调控中起重要作用, 但是膜结构性KCC2在慢性癫痫形成中的作用还没有被系统研究阐述。.在本项目中，我们运用经典的内侧颞叶癫痫模型—小鼠匹罗卡品模型作为对象，通过癫痫行为学、膜片钳电生理、荧光免疫组化以及分子生物学技术，研究了神经元膜结构性KCC2在癫痫发生发展的长时程周期中的动态变化在癫痫沉默期调控海马兴奋性突出结构与功能在癫痫发生发展中的作用及机制。研究发现：1. DG区膜KCC2在癫痫发生发展中的动态改变通过调控沉默期颗粒细胞树突棘形态和功能的重塑，慢性期兴奋性突触后AMPA和KA受体功能的增强，进而增强颗粒细胞兴奋性信号传递和整合，促进慢性期癫痫的自发发作；2. 癫痫沉默早期膜KCC2的下调使βPIX/Rac1/pS3Cofilin通路过度激活，颗粒细胞兴奋性突触连接重塑，为慢性期颗粒细胞突触后KA受体功能的增强，mEPSCs通道开放时长的延长提供条件，有利于兴奋性信号的传递与整合；3. 癫痫沉默后期膜KCC2的恢复通过与Neto2协同上膜相互作用激活了颗粒细胞突触后KA受体的异常功能，可能是调节慢性期颗粒细胞兴奋性信号整合能力增强在慢性癫痫形成中的新机制。.本课题研究探索并初步阐明了沉默早期KCC2下调及树突棘丢失是慢性期异常兴奋性突触重构的必要条件以及癫痫沉默后期膜KCC2回调激活颗粒细胞突触后KA受体异常功能的多重机制是慢性癫痫发生的可能诱因。此研究成果将为探索癫痫进程早期干预靶点提供了新的理论依据，为抗癫痫药物的干预提供了新机制新靶点。