PICK1在脑内氧化应激损伤中的作用及其机制研究

The oxidative stress caused neuron apoptosis is an important factor in several neurologic disorders. Studies show that glutathione (GSH) plays a pivotal role in maintaining the balance of oxidation-reduction equilibrium in brain, and EAAC1 plays a decisive role in GSH synthesis by uptake cysteine. The study on the mechanism of EAAC1 is rare. The supporting protein PICK1 has multiple biological functions and associated with many major diseases. However, the role of PICK1 in oxidative stress is unclear. Our preliminary data first showed that PICK1 knockout could lead to oxidative stress by decreasing the anti-oxidative injury marker GSH level and increasing the oxidative injury marker Nitrotyrosine and 4-HNE levels indicating that PICK1 knockout could cause the oxidative stress. PICK1 could coimmunoprecipitated with EAAC1 indicating that PICK1 might regulate EAAC1 in the oxidative stress. In this project, we will continue to study how PICK1 regulates the oxidative stress in CNS using combined techniques from both in vivo and in vitro, including gene knockout mice model and oxidative injury mice model , western blot, immunocytochemistry, neuron primary culture. This project will reveal the regulatory mechanisms of PICK1 on the oxidative stress and provide new potential treatment target for the oxidative stress related diseases.

氧化应激损伤造成的神经细胞凋亡是导致神经系统疾病的重要因素。谷胱甘肽GSH对维持脑内氧化还原平衡至关重要，而谷氨酸转运体EAAC1对神经元摄取半胱氨酸从而合成GSH又起着决定性作用，但是EAAC1的调节机制研究还非常欠缺。支架蛋白PICK1具有多种生物学功能，在突触可塑性和一些疾病中发挥着重要作用，但未见其在氧化应激中作用的报道。我们首次发现，PICK1敲除小鼠脑内GSH减少，氧化损伤指标nitrotyrosine和4-HNE显著升高，提示PICK1缺失导致了氧化应激反应。PICK1可以和EAAC1免疫共沉淀，提示在抗氧化应激反应中PICK1可能起到调控EAAC1的作用。本项目将在前期基础上，利用PICK1基因敲除模型和氧化损伤小鼠模型，结合western blot、细胞免疫荧光、神经元和脑片原代培养、电生理等技术，研究PICK1调节神经元氧化损伤的机制，为氧化应激相关疾病的治疗提供新靶点

氧化应激损伤造成的神经细胞凋亡是导致神经系统疾病的重要因素。谷胱甘肽GSH对维持脑内氧化还原平衡至关重要，而谷氨酸转运体EAAC1对神经元摄取半胱氨酸从而合成GSH又起着决定性作用，但是EAAC1的调节机制研究还非常欠缺。支架蛋白PICK1具有多种生物学功能，在突触可塑性和一些疾病中发挥着重要作用，但未见其在氧化应激中作用的报道。我们利用PICK1基因敲除模型和氧化损伤小鼠模型，结合western blot、细胞免疫荧光、神经元和脑片原代培养、电生理等技术，首次发现，PICK1敲除小鼠脑内GSH减少，氧化损伤指标nitrotyrosine和4-HNE显著升高，提示PICK1缺失导致了氧化应激反应。PICK 敲除鼠中氧化应激反应的增强具有年龄依赖性。我们进一步确定了PICK1缺失导致的氧化应激反应增强主要在神经元，同时伴随着小胶质细胞的激活，其机制可能是由于神经元中ROS水平升高，对氧化应激更敏感。PICK1敲除小鼠体内的还原性谷胱甘肽GSH相比氧化型谷胱甘肽GSSG明显减少。BSO可抑制GSK的形成，既然敲除鼠中GSK减少，那么给予BSO时，细胞氧化应该更强。结果证明PICK1敲除鼠神经元GSK减少是由细胞表面的EAAC1显著降低导致的。本项目证明PICK1在抑制氧化应激中起着重要的作用，这将为氧化应激相关疾病的治疗提供新靶点。