MLK3结合蛋白对脑缺血JNK通路调控的研究

【作者】 张全光；

【导师】 张光毅； 徐天乐；

【作者基本信息】 中国科学技术大学 ， 神经生物学， 2006， 博士

【摘要】 在大鼠和沙土鼠动物全脑缺血模型中，短暂的全脑缺血导致海马CA1区锥体神经元的选择性死亡，这种迟发的神经元细胞丢失只有在缺血／复灌2—4天后才能观察到。实验证据表明，这种迟发的神经元死亡的分子机制中存在凋亡过程。JNK蛋白激酶信号的激活是介导脑缺血应激引起神经细胞凋亡的重要通路；最近，一种胞内丝／苏氨酸蛋白激酶MLK3被发现，MLK3作为JNK级联上游的重要活化因子，通过直接磷酸化并激活中游MKK4／7蛋白激酶，从而激活JNK应激信号通路。MLK3介导了JNK诱导的神经元凋亡和脑缺血损伤通路，并因此受到重视。然而，关于脑缺血／复灌激活MLK3的信号调控分子机制尚不清楚。本文主要探讨了MLK3的两种直接结合蛋白POSH和Rac1对MLK3／JNK信号通路以及对脑缺血损伤的调控作用。 1．POSH通过组装POSH-MLK3／MKK4／JNKs信号模块介导JNK通路的激活和脑缺血／复灌诱导的海马CA1区神经元损伤 我们首先用免疫印迹和免疫组化的方法观察了海马CA1区POSH蛋白在SD大鼠脑缺血／复灌后的表达分布情况。结果显示，假手术组和缺血／复灌组POSH蛋白的胞浆免疫活性在海马CA1和CA3锥体细胞较强，然而在DG颗粒细胞表达较弱。免疫共沉淀实验表明，POSH能与MLK3、MKK4以及p-JNKs免疫共结合，在复灌30分钟时达最高值，在复灌1-3天时仍高于对照组。同时，在脑缺血／复灌30分钟和3天时MLK3／MKK4／JNKs信号通路存在两个活化峰。脑室注射POSH反义寡核苷酸不仅能显著降低其蛋白表达、抑制POSH与MLK3，MKK4，和p-JNKs级联组分的结合，还能够抑制MLK3／MKK4／JNKs信号通路的激活。而且，脑室注射POSH反义寡核苷酸显著增加了脑缺血／复灌5天后海马CA1区存活神经元的数量。结果提示，POSH可能作为支架蛋白介导了脑缺血／复灌后海马CA1区JNK信号通路的激活，POSH反义寡核菅酸通过抑制MLK3／MKK4／JNKs通路，并涉及c-Jun和Caspase-3的抑制，发挥对脑缺血损伤的保护作用。更多还原

【Abstract】 Transient, severe global ischemia in rats and gerbils leads to selective and delayed neuronal death of pyramidal neurons in the hippocampal CA1, which is not detected until 2-4 d after induction of ischemia. Several lines of evidence have been proposed to suggest that apoptotic process underlies the molecular mechanism of this delayed neuronal cell death. Considerable evidence suggests that c-Jun NH2-terminal kinase (JNK) is an important kinase mediating the neuronal cell death in response to cerebral ischemia. Recently, an intracellular serine/threonine kinase, mixed-lineage kinase 3 (MLK3) has been identified as a novel upstream activator of the JNK pathway. MLK3 functions as a MAPK kinase kinase (MAPKKK) of the JNK stress pathway by directly phosphorylating and activating the JNK activators SEK1/MKK4 and MKK7. MLK3 has garnered attention as an important mediator of JNK-mediated neuronal apoptosis and ischemic injury. However, the molecular mechanisms that regulate MLK3 activity in cerebral ischemic-reperfusion has not been extensively delineated. The overall goal of the present study is to understand the molecular basis by which MLK3 and its signaling pathways are negatively regulated by targeting MLK3 interacting proteins in cerebral ischemia.1. POSH is an important mediator of JNK pathway activation and ischemic injury by compositing the POSH/MKK4/JNK signaling complex in vulnerable CA1 region following transient global ischemia.We investigated the expression and subcellular localization of the multidomain protein POSH (plenty of SH3s) by immunohistochemistry and Western blot analysis in rat hippocampal CA1 region following cerebral ischemia. Our results indicated that the cytosol immunoreactivity of POSH was strong in CA1-CA3 pyramidal cell but weak in DG granule cell of rat hippocampus both in sham control and after reperfusion. Coimmunoprecipitation experiments showed that the interactions of MLK3, MKK4 and phospho-JNKs with POSH were persistently enhanced during the early (30 min) and the later reperfusion period (from 1 d to 3 d) compared with sham更多还原