ApoE介导的Cdc42通路参与难治性癫痫异常神经网络苔藓纤维芽生的机制研究

Aberrant neural network formation is an important cause of refractory epilepsy, which regulated by a variety of factors. Our preliminary studies demonstrated that Cdc42 pathway regulating abnormal neural network formation is an important mechanism of refractory epilepsy, but its upstream regulatory factor is not clear. To furthur investigate the regulatory mechanism will provide us with a better understanding of the pathogenesis of epilepsy. In previous studies on cerebrospinal fluid (CSF) of patients with refractory epilepsy, applicants found that ApoE significantly altered. In addition, ApoE level was found obviously higher in brain tissues of patients with refractory epilepsy than in controls in preliminary experiments. It has been reported that ApoE can give rise to aberrant mossy fiber sprouting in central nervous system. Therefore, the applicant put forward the scientific hypothesis that Cdc42 pathway mediated by ApoE is involved in abnormal neural network formation of refractory epilepsy. We intend to use brain tissues of patients with refractory epilepsy after surgical treatment, ApoE knockout mice and cell model of epilepsy to carry out research, including multiplex PCR genotyping of the ApoE allele, sprouting related cytoskeletal proteins signaling pathways, as well as patch-clamp recording. We will try to clarify the relations of ApoE and its different alleles with mossy fiber sprouting, and to explore the possible mechanisms of ApoE mediating Cdc42 pathway.

异常神经网络形成是导致癫痫反复发作的重要原因，而异常神经网络系统受到多种因素的调节已受到广泛认同。课题组前期的研究已发现Cdc42是调节难治性癫痫异常神经网络的关键蛋白，但其上游调控因子尚不清楚，继续研究其调控机制将加深对癫痫发病机制的了解。申请者在前期研究中发现难治性癫痫患者脑脊液及脑组织中ApoE明显增高，文献表明ApoE可以引起中枢神经系统病理性神经纤维芽生。为此，申请人提出ApoE介导的Cdc42通路参与难治性癫痫异常网络苔藓纤维芽生这一科学假说。基于此假说，申请者拟采用难治性癫痫患者术后脑组织、ApoE基因敲除小鼠及AAV-ApoE-eGFP转染小鼠作为研究对象，进行ApoE等位基因分型多重PCR、芽生相关蛋白信号通路及膜片钳电生理检测等多方位研究，力图阐明ApoE介导Cdc42通路参与癫痫发生发展的机制，并探讨ApoE不同等位基因与癫痫苔藓纤维芽生的关系。

项目背景：.异常神经网络形成是导致癫痫反复发作的重要原因，课题组前期的研究已发现Cdc42是调节难治性癫痫异常神经网络的关键蛋白，且在前期研究中发现难治性癫痫患者脑脊液及脑组织中ApoE明显增高，文献表明ApoE可以引起中枢神经系统病理性神经纤维芽生。为此，申请人提出ApoE介导的Cdc42通路参与难治性癫痫异常网络苔藓纤维芽生这一科学假说，并通过实验进行验证。.主要研究内容：.研究者对癫痫患者脑组织进行ApoE等位基因检测，并检测了ApoE不同等位基因的蛋白编码产物ApoE2、ApoE3以及ApoE4的纯化蛋白对癫痫模型小鼠苔藓纤维芽生的影响。分别构建了ApoE过表达和抑制表达载体，注入癫痫模型小鼠海马组织，观察模型鼠癫痫发作，膜片钳检测电生理改变，分子生物学检测下游α-SNAP、Fibronectin，Cdc42 下游的tyr-Tubulin的表达，力图阐明ApoE介导Cdc42通路参与癫痫发生发展的机制。.重要结果及关键数据：.证实ApoE在难治性癫痫组患者脑组织中的含量明显增高，ApoE等位基因ε4等位基因频率在癫痫组中增加；epsilon4纯化蛋白使苔藓纤维芽生更加显著，发作也更加频繁。ApoE过表达鼠建立模型的后放电阈值、痫样放电和发作强度较野生鼠和ApoE沉默表达鼠明显增高；ApoE过表达鼠齿状回苔藓纤维芽生较野生鼠和ApoE沉默表达鼠明显增加。给予Cdc42的抑制剂ML141后，癫痫模型鼠发作及mIPSCs波幅增加。ApoE下游的Fibronectin蛋白在癫痫患者血清和脑脊液中含量较对照组明显升高，ApoE下游，Cdc42上游分子α-SNAP在癫痫鼠脑组织中明显减低，低水平的α-SNAP增加了癫痫发作的易感性。Cdc42下游的tyr-Tubulin在癫痫患者脑组织和动物模型中明显增加，参与癫痫的发病机制。.科学意义：.ApoE调控下的Cdc42蛋白信号通路可能是介导难治性癫痫异常网络形成的重要机制。具体的机制可能是ApoE通过下调α-SNAP，上调Fibronectin，对下游Cdc42发挥调控作用，进而进一步调控下游tyr-Tubulin对微管聚合与解聚的动力学发挥调控作用，介导难治性癫痫异常网络形成。