Src家族激酶在脑缺血海人藻酸受体功能调控中的作用及分子机制

Ischemic stroke is a common disease with high risk of recurrence, disability and mortality in China but there is no ideal clinical method for treatment of ischemic brain damage. Our previous studies have demonstrated that kainate(KA) receptors contribute to ischemic neuronal death, which involves GluK2-PSD-95-MLK3 signaling complex and its downstream MLK3-JNKs signaling cascades. However, the molecular mechanism underlying the regulation of GluK2-KA receptors after cerebral ischemia is still unknown. Here，using the four-vessel occlusion global ischemia model and oxygen/glucose deprivation (OGD) model in cultured hippocamal neurons, whole-cell patch-damp recording, cofocal imaging, in vitro kinase assay, molecular biology and other technologies, we try to investigate the changes of GluK2 tyrosine phosphorylation, the effect of Src family of protein tyrosine kinases (SrcPTKs) on GluK2 tyrosine phosphorylation, the role of SrcPTKs-mediated GluK2 tyrosine phosphorylation in the regulation of KA receptor function and in the activation of downstream MLK3-JNKs singnaling. The results will reveal the pathological significance of postischemic GluK2 tyrosine phosphorylation. Our data will help to find potential drug targets for treatment of ischemic stroke.

缺血性脑血管病是我国常见病,复发率、致残率、致死率均较高，但临床上还没有有效的治疗措施。本室研究证实海人藻酸（kainate, KA）受体参与缺血性脑损伤，其机制主要与GluK2-PSD-95-MLK3信号复合体组装及下游MLK3-JNKs通路激活有关。但含GluK2亚基的KA受体（GluK2-KA）在脑缺血/复灌中的调控机制尚未阐明。本项目拟采用大鼠四动脉结扎全脑缺血模型、原代培养海马神经元缺氧缺糖（OGD）模型，以全细胞膜片钳、激光共聚焦、体外激酶反应、分子生物学等技术，观察脑缺血/复灌时GluK2酪氨酸磷酸化的变化规律；鉴定Src家族激酶对GluK2的磷酸化作用；研究GluK2酪氨酸磷酸化在GluK2-KA受体功能调节中的作用及其对下游MLK3-JNKs信号通路的影响，从而揭示脑缺血中GluK2酪氨酸磷酸化的病理意义，为开发脑缺血治疗药物提供潜在的靶点。

缺血性脑血管病是我国常见病和高发病，目前NMDA受体介导的兴奋毒学说不能完全解释其发病机制，NMDA受体拮抗剂在临床上也未取得理想的诊疗效果。本室前期研究证实海人藻酸（kainate, KA）受体参与缺血性神经元损伤，其机制主要与GluK2-PSD9-MLK3信号复合体组装及其下游MLK3-JNKs信号通路的激活有关。但含有GluK2亚基的KA受体（GluK2-KA）在脑缺血/复灌中的调控方式及分子机制至今不清。本项目研究显示Src家族激酶参与了KA受体功能的调节。大鼠脑缺血复灌诱导海马CA1区Src激酶介导的KA受体GluK2亚基酪氨酸磷酸化。提示在缺血性脑损伤中Src激酶介导的GluK2酪氨酸磷酸化具有重要作用。GluK2结合Src，并被Src激酶磷酸化修饰的主要位点在GluK2的Y590位酪氨酸残基上。GluK2的Y590位酪氨酸磷酸化是KA刺激后全细胞电流增加的主要原因。KA处理的细胞引起GluK2的Y590位酪氨酸磷酸化促进了GluK2亚基的内吞，并激活了JNK3及其底物c-Jun。综上所述，脑缺血复灌诱导的Src磷酸化修饰GluK2在KA受体通道开放和下游促凋亡信号通路激活中起重要作用。研究结果提示Src家族激酶在GluK2-KA受体功能调控中的病理意义，为开发基于KA受体GluK2亚基的缺血性脑血管病的防治和药物研发提供重要的靶标。