谷氨酸AMPA受体GluR2/GAPDH干扰肽在颞叶癫痫中的作用

Over 50 million people suffer from epilepsy in the world and about 40% of patients exhibiting epilepsy have chronic temporal lobe epilepsy (TLE)， which is characterized by a progressive expansion of complex partial seizures arising from the limbic system regions such as the hippocampus. Hippocampal sclerosis is one of the most prevailing pathological marker of TLE and shows widespread neuronal loss in the dentate hilus and the CA1 and CA3 subfields, accompanied with learning and memory impairments. 35% TLE are resistant to antiepileptic drugs. Alternative antiepileptic drugs approaches are needed to influencing the course of the disease rather than merely provide symptomatic treatment. AMPA receptor (AMPAR) antagonists have been investigated for antiseizure activity both preclinically and clinically, and have the potential to reduce excessive excitatory responses providing neuroprotection and seizure suppression. These effects are mainly attributed to their relieving of glutamate induced excitotoxic injury. However, AMPA receptors in most excitatory synapses contain the GluR2 subunit. The exact antiepileptic mechanisms of AMPAR antagonists are still unknown. We have recently found that GluR2 forms a protein complex with GAPDH, which is critical in mediating glutamate induced cell apoptosis. Disruption of this complex with a competitive protein peptide (TAT-G-Gpep) could rescue glutamate induced excitatory neuronal death in primary neuronal culture and animal models of ischemic stroke. The present study aims to investigate the potential protective effects of this peptide in pilocarpine model of temporal lobe epilepsy. We will study in this model: GluR2/GAPDH levels in this model and whether TAT-G-Gpep could disrupts this complex in vivo; effects of TAT-G-Gpep in epilepsy induced neuronal degeneration and cell death; whether TAT-G-Gpep could reverse epilepsy induced cognitive deficits; influence of TAT-G-Gpep on the pathological course of epilepsy。This study will not only elucidate the molecular mechanisms of GluR2-AMPAR in epilepsy but also provide novel therapeutic alternative to TLE.

约40%的成人癫痫患者为颞叶癫痫，多为药物难治性，并常伴有海马神经元丢失和认知功能障碍。近年来发现谷氨酸AMPA受体（AMPAR）拮抗剂是一类极具潜力的新型抗癫痫药物，但其作用机制尚不清楚。我们发现AMPAR的GluR2亚基可以和甘油醛-3-磷酸脱氢酶（GAPDH）组成蛋白复合物，并参与细胞凋亡过程，而破坏GluR2/GAPDH的蛋白肽（G-Gpep）能够挽救原代海马培养神经元和缺血性中风动物模型中谷氨酸诱导的兴奋性细胞死亡。本项目拟研究干扰肽在匹罗卡品诱发颞叶癫痫动物模型中的保护作用，验证GluR2/GAPDH在癫痫动物模型中的耦合及干扰肽的打断耦合的活性；证明具有穿膜能力的TAT-G-Gpep在致痫大鼠中防止海马神经元变性和丢失的能力；评估TAT-G-Gpep肽对癫痫发作诱导的海马依赖性记忆损害的影响；研究TAT-G-Gpep肽对癫痫动物模型病程发生发展的影响；为癫痫提供新的治疗策略。

GluR2/GAPDH蛋白复合体参与细胞凋亡和谷氨酸介导的兴奋性神经毒性，而后者已证明为癫痫发病机制和病理过程之一。已有研究表明，AMPA受体亚基GluR2干扰肽（TAT-GluR2NT1-3-2）通过干扰GluR2/GAPDH的耦合作用在缺血脑损伤中起保护作用，提示它可能应用于癫痫的治疗。本课题通过Western Blot、免疫共沉淀、细胞组织化学检测到干扰肽可阻断癫痫模型大鼠脑内神经元胞质中GluR2/GAPDH复合体的形成，并已测定干扰肽的最佳脑内给药时间窗为2小时、剂量为50 nmol/kg，细胞组织化学也提示干扰肽对动物模型中海马神经元变性和凋亡有保护作用；通过在诱导癫痫之前预给药干扰肽发现干扰肽可降低癫痫诱导成功率，延长潜伏期；利用Y迷宫、水迷宫、逃避实验等行为学实验证实干扰肽对癫痫大鼠急性期海马依赖的认知功能障碍有改善作用；在体脑电监测评价干扰肽对癫痫慢性病程中癫痫发作的影响仍在进行当中。综合上述结果，本课题对包含GluR2的AMPA受体在癫痫发病中的作用进行了研究，阐述了特异性干扰肽TAT-GluR2NT1-3-2对颞叶癫痫病理生理机制的影响，为以AMPA受体相关蛋白为靶点的癫痫治疗提供了新策略。