Parvalbumin介导的Glu-GABA代谢途径在癫痫发作终止中的作用及分子机制

The mechanism of controlling seizure termination arouses wide concerns as patients who develop status epilepticus often have unfavourable prognosis. Previous studies have indicated that Parvalbumin(PV) might play crucial roles in termination of seizure via Glu-GABA pathway, but the mechanism remains largely unclear so far. Thus, in this study, we will employ biochemical assays to investigate the changes in the expression levels of PV in hippocampus of mice with status epilepticus. Subsequently, we will construct lenti-virus expressing hM3DGq, and infect the in vitro cultured neurons derived from PV-Cre mouse hippocampus, the change of PV level when PV-positive neurons are activated is evaluated through DREADD technique; lenti-virus expressing PV-shRNA and PV-overexpresion are applied to detect the relationship between PV, Glutamate decarboxylase, Glutamic acid, and GABA. Finally, we will infect the hippocampus of PV-Cre mice in vivo by the above lenti-viruses, and then investigate the changes in behavioral phenotypes and the biochemical markers in kainic acid models treated with DREADD strategy. We plan to clarify the specific role of PV and PV-positive interneurons in seizure termination and dissect out the underlying molecular mechanisms. This work will greatly help us to understand the novel regulatory mechanisms of termination of epilepsy, and provide new pharmacological targets and therapeutic strategies against epilepsy.

癫痫持续状态可对患者造成严重危害，尽快终止发作因关系到患者预后而备受关注。研究表明，微清蛋白PV可能通过Glu-GABA代谢途径，介导癫痫发作的终止，但具体机制目前尚不明确。因此我们拟用分子生物学手段，探究PV在癫痫持续状态小鼠脑内海马组织表达水平变化，与癫痫终止建立关联性；利用DREADD技术，构建hM3DGq慢病毒载体，体外感染培养的PV-Cre小鼠海马神经元，给予CNO激活，联合PV基因沉默及过表达技术，探究PV阳性神经元激活后相关蛋白变化及机制；运用立体定位技术，实现hM3DGq慢病毒载体在体感染PV-Cre小鼠海马神经元，后采用海人酸造模，使用CNO干预，通过行为评估及分子生物学方法，探索小鼠海马PV阳性神经元激活对小鼠痫性发作的影响及相关机制。这将帮助我们增进对癫痫终止内在分子调控机制的理解，并为癫痫防治提供新的分子靶点和科学理论依据。

癫痫是一组由大脑神经元异常放电引起的短暂的中枢神经系功能失常为特征的慢性脑部疾病。癫痫若得不到及时、正确的控制，有发展成为癫痫持续状态或难治性癫痫的可能，严重影响患者及其家庭的生活，加重社会的负担。研究表明，小清蛋白（Parvalbumin,PV）可通过多条途径影响癫痫的发生、发展及终止。研究发现，PV表达增多时伴有谷氨酸脱羧酶（Glutamic acid decarboxylase，GAD）水平的升高，而GAD是谷氨酸（Glutamic acid,Glu）向γ-氨基丁酸（Gamma Aminobutyric,GABA）转化的关键酶，但PV参与癫痫发作终止的调节机制仍未明确。本研究通过构建PV过表达及PV沉默慢病毒载体体外感染PC12细胞，并加入神经生长因子（Nerve growth factor，NGF）诱导分化处理，利用免疫荧光、RT-PCR、Western blotting、高效液相色谱法，在细胞层面上明确了PV通过影响GAD65及GAD67的表达，参与了Glu-GABA代谢途径；通过构建PV沉默及PV过表达腺相关病毒载体在体感染PV-Cre小鼠模型，予海人酸（Kainic acid， KA）海马注射造癫痫持续状态（Epileptic state，SE）模型，在动物层面上研究PV对GAD、Glu、GABA的影响。本课题通过探讨PV介导的Glu-GABA代谢途径在癫痫发作终止中的作用及分子机制，发现PV可能通过提高GABA水平和降低Glu水平，减轻兴奋性，从而对癫痫发作终止产生有益的影响，可能作为治疗癫痫发作的潜在靶点。