P2X7R异常激活介导AICWs参与皮质发育障碍致痫机制研究

Malformation of cortical development (MCD) represents a well-recognized cause of intractable epilepsy. Understanding the mechanism of epilepsy induced by MCD may provide novel therapeutic target in the management and prevention of intractable epilepsy. Recently, the astrocytes in MCD have been received great attention. It is reported that intercellular calcium waves in central neural system is mainly existed in astrocytes. There was hyperplasia of astrocytes in epileptogenic foci of MCD. The purinergic ionotropic P2X7 receptor (P2X7R) is an atypical P2X subtype that gates calcium and sodium ions. In vivo studies showed that P2X7R activation may exacerbate seizures and P2X7R agonists increased the severity of seizures during status epilepticus triggered by intra-amygdala Kainic Acid in mice. And P2X7R-deficient mice are resistant to the development of epilepsy. Our previous studies showed that P2X7R is up-regulated and primarily expressed in reactive astrocytes in patients of Focal cortical dysplasias. We hypothesize that P2X7R involved in the epileptogenesis of MCD by positively regulating AICWs to affect neuron-to-astrocyte communication. Therefore, this project will elucidate how P2X7R affect MCD by regulating AICWs. Therefore, this project will focus on the astrocytes in MCD, using clinical specimens and animal model and cultivation of brain slice, to elucidate the mechanism of how AICWs regulated by over expression of P2X7R. The results may be helpful for us to better understanding of the refractory of MCD epilepsy.

皮质发育障碍（MCD）是难治性癫痫的重要病因。已有研究证实：1）中枢神经系统钙波主要起源于星形胶质细胞；2）MCD致痫灶内星形胶质细胞增生形成“神经-胶质复合物”；3）P2X7R异常激活致细胞内Ca2+浓度增高；4）P2X7R-/-小鼠癫痫易感性显著减低。课题组前期研究发现MCD病灶中Ca2+释放事件增加；P2X7R在MCD致痫灶中尤其是星形胶质细胞表达显著增高，且其升高程度与癫痫发病频率呈正相关，因此我们推测P2X7R异常激活，调控星形胶质间钙波（AICWs），致神经元超同步化放电，诱发MCD癫痫发生。本项目拟以MCD临床标本及动物模型为研究对象，采用脑片培养、电生理、激光共聚焦钙成像等技术，结合信号通路激动剂及抑制剂，从体内外水平探讨并明确P2X7R过度激活调控AICWs致神经元超同步化放电的致痫机制，为进一步揭示MCD的发病机制提供新的线索。

皮质发育障碍(MCD)是难治性癫痫的重要病因。已有研究证实:1)中枢神经系统钙波主要起源于星形胶质细胞;2)MCD致痫灶内星形胶质细胞增生形成“神经-胶质复合物”; 3)P2X7R异常激活致细胞内Ca2+浓度增高;4)P2X7R-/-小鼠癫痫易感性显著减低。 课题组前期研究发现MCD病灶中Ca2+释放事件增加;P2X7R在MCD致痫灶中尤其是小胶质细胞、星形胶质细胞表达显著增高，且其升高程度与癫痫发病频率呈正相关，因此，我们首先通过对人MCD的重要病理亚型（结节性硬化TSC)进行研究，发现P2X7R在来自于病人手术切除的TSC病理结节标本中异常激活，高表达于小胶质细胞中，我们以及其他课题组研究表明，TSC结节有明显的星形胶质细胞增生，且为癫痫的放电起源，由此推论，p2x7R 、小胶质细胞及星形胶质细胞必然对癫痫的发生、发展及神经元损伤有一定的相关性，故本项目以MCD临床标本及转基因动物模型为研究对象，进而研究了小胶质细胞及星形胶质细胞之间的作用机制，结合信号通路激动剂、抑制剂及基因敲除工具小鼠，我们发现高表达P2x7R的小胶质细胞在在模拟癫痫病理条件下被激活后，可以通过补体C3-C3aR通路，正反馈胶质细胞增生，从而导致神经元损伤；应用维持小胶质细胞生存和增殖的关键分子-集落刺激因子1 受体抑制剂后，可明显减轻海人酸脑室注射诱导癫痫发作后的海马胶质细胞增生， 课题组进而从体内外水平，动物模型方面，探讨并明确P2X7R过度激活调控小胶质细胞激活、癫痫病灶内胶质化以及神经元损伤的重要病理机制，为进一步揭示癫痫的发病和诊治提供新的线索。