靶向海马谷氨酸AMPA受体GluA2/TARPγ-8的癫痫治疗研究

Epilepsy occurs as a result of episodic abnormal synchronous discharges in cerebral neuronal networks. It is characterized by an imbalance between excitatory and inhibitory neurotransmission. Although various non-conventional mechanisms are implicated in epileptic synchronization, glutamate excitatory neurons play an essential role. Cascading excitation within networks of synaptically connected neurons is a classical mechanism. AMPA receptors mediate fast synaptic excitation within and between brain regions relevant to epilepsy, and play a role in epileptogenesis and in seizure-induced brain damage. However, direct modulation of AMPA receptors may have undesirable consequences, given its wide expression within the central nervous system and critical roles on brain circuitry development. We have recently found that AMPAR GluA2 formed a complex with its auxiliary binding protein TARPγ-8 in the hippocampus. This binding is dramatically increased in the hippocampus of pilocarpine induced epilepsy rat model. Disruption of the AMPAR GluA2/TARPγ-8 binding not only decreased AMPA mediated mEPSC amplitude and frequency in primary cultured hippocampal neurons, but also improved epileptic behaviors and extended the latency of onset in PTZ induced mice epilepsy models. The proposed study will further define the biochemical nature of GluA2/TARPγ-8 interaction, examine its cellular roles in AMPA function and importance in epilepsy pathology. The results obtained could reveal the novel roles of GluA2/TARPγ-8 in epileptic pathology, and provide alternative targets to develop selective antiepileptic therapeutic targeting AMAP receptors.

癫痫是一种常见的慢性中枢神经系统（CNS）疾病，目前认为谷氨酸AMPA受体（AMPAR）在其发病中起着病因性作用。但由于AMPAR在CNS的广泛分布，以及在脑神经发育和通路形成中的重要作用，以其为靶点的选择性药物亟待开发。本课题组的前期研究发现，AMPAR GluA2亚基可以与跨膜AMPAR调节蛋白γ-8亚型（TARPγ-8）特异性结合，而且二者偶联在癫痫大鼠海马区显著增高。打断这一复合物可降低AMPAR介导的电活动，且降低癫痫小鼠的行为评分，延长其发作潜伏期。基于此，本项目拟从分子和亚细胞水平到整体行为，进一步深入研究GluA2/TARPγ-8偶联的生化和分子生物学机制，TARPγ-8调节GluA2的功能代谢及其介导的细胞生物学功能，探索二者偶联在癫痫大鼠发病中的作用，从而阐明GluA2/TARPγ-8在癫痫中的作用和机制，为开发选择性调节海马区AMPAR的抗癫痫药物提供新策略。

AMPAR介导的兴奋性神经毒性与癫痫导致的海马区神经损伤密切相关，已有研究表明AMPA受体GluA2亚基参与了癫痫的发病，干扰肽TAT-GluA2CT通过干扰GluA2/TARPγ-8的耦合在体外实验中展现了抗癫痫活性，提示它可能能够应用于癫痫的治疗。本课题通过Western Blot、免疫共沉淀检测到干扰肽TAT-GluA2CT可阻断癫痫持续状态模型大鼠脑内神经元胞质中GluA2/TARPγ-8复合体的形成，细胞组织化学结果表明应用干扰肽TAT-GluA2CT能够减轻大鼠匹罗卡品癫痫持续状态模型中海马CA1区神经元的丢失与凋亡，并且干扰肽最佳给药时间是给与匹罗卡品前1小时和后2小时；通过在诱导癫痫前脑内注射干扰肽，与癫痫组大鼠相比，发现应用干扰肽能延长潜伏期，降低诱导成功率和癫痫发作等级。综合上述结果，本课题对AMPA受体GluA2亚基与TARPγ-8复合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-GluA2CT通过打断GluA2与TARPγ-8的耦合对颞叶癫痫病理生理的影响，为以AMPA受体相关蛋白为靶点的癫痫治疗提供了新策略。