NWD1/GluN2B介导的胞吞再循环在海马癫痫样神经兴奋性中的作用及机制

Glutamic acid receptor subunit GluN2B plays an important role in neuroexcitability, but its distribution, location and transport are regulated by several factors; therefore, the specific mechanism of epileptogenesis in epilepsy is still unclear. NACHT and WD repeat domain-containing protein 1 (NWD1) is a member of the innate immune protein subfamily, and NWD1 is highly expressed in the brain. It has not been reported whether NWD1 is involved in the pathophysiological process of epilepsy. We found that the levels of NWD1 protein increased in hippocampal tissue obtained from a mouse model of epilepsy. The epileptiform discharges decreased in pyramidal neurons and in field potential after silencing NWD1, and there was a decrease in the GluN2B receptor-mediated excitatory postsynaptic currents. NWD1 was positively correlated with GluN2B membrane protein levels and with the phosphorylation of GluN2B-Tyr1472, and these protein were coexpressed in the postsynaptic membrane. This project aims to explore the regulation of the phosphorylation of GluN2B-Tyr1472 by NWD1, which mediates the dynamic transport of GluN2B endocytosis and recycling, both in vitro and in vivo. This study will identify and clarify the role and mechanism of NWD1/GluN2B-Tyr1472 in homeostasis and the mechanism of hippocampal neuroexcitability as well as in the occurrence and development of epilepsy. This study will also provide new clues about the pathogenesis of epilepsy and new strategies for the diagnosis and treatment of epilepsy.

谷氨酸受体亚基GluN2B在神经兴奋性中起着重要的作用，由于其分布、定位和转运受多种因素调控，其在癫痫发生发展中的机制仍不明确。NACHT和WD重复序列蛋白1（NWD1）是固有免疫蛋白亚家族成员，在大脑中高表达，目前尚未见报道其是否参与癫痫病理生理过程。我们前期在癫痫模型小鼠海马组织中发现NWD1蛋白增高，沉默NWD1减少GluN2B受体介导的海马锥体神经元及场电位癫痫样放电和兴奋性突触后电位。而NWD1与GluN2B膜蛋白水平及GluN2B-Tyr1472磷酸化程度正相关，且共表达于神经突触后膜。本项目拟通过体内和体外实验研究NWD1调控GluN2B-Tyr1472磷酸化介导GluN2B的胞吞再循环的动态转运规律，从而影响海马神经兴奋性平衡稳态及其机制，明确NWD1/GluN2B-Tyr1472在癫痫发生发展中的作用和机制。该项目将为癫痫的发病机制提供新线索，并为癫痫的诊治提供新策略。

谷氨酸受体亚基GluN2B在神经兴奋性中起着重要的作用，由于其分布、定位和转运受多种因素调控，其在癫痫发生发展中的机制仍不明确。NACHT和WD重复序列蛋白1（NWD1）是固有免疫蛋白亚家族成员，在大脑中高表达，目前尚未见报道其是否参与癫痫病理生理过程。本研究中，在颞叶癫痫患者及癫痫模型小鼠海马及皮层组织中发现NWD1蛋白水平增高，且在兴奋性神经元突触后膜表达；沉默NWD1减少GluN2B受体介导的海马锥体神经元癫痫样放电和兴奋性突触后电位；动物行为学实验证实，在急性癫痫模型中，NWD1表达较少可导致的癫痫发作潜伏期延长和发作频率减少。此外，NWD1与GluN2B膜蛋白水平及GluN2BTyr1472磷酸化程度正相关，且共表达于神经元突触后膜，沉默NWD1减少GluN2B膜蛋白水平及GluN2BTyr1472磷酸化程度。GluN2B受体首先在内质网组装合成，然后到达高尔基体，被囊泡包裹后运输到细胞膜上。为进一步验证NWD1调控GluN2B-Tyr1472磷酸化介导GluN2B的胞吞再循环的动态转运规律，在无镁脑脊液孵育后的海马神经元HT22细胞中发现沉默Nwd1后GluN2BTyr1472的内吞无明显变化，但GluN2BTyr1472内吞后的降解增加，再循环至细胞膜受体数量减少，由此证实，Nwd1 通过调控NMDA受体亚基GluN2BTyr1472再循环介导兴奋性神经突触传递，从而影响海马神经兴奋性平衡稳态及其机制。既往证据表明神经元兴奋性/抑制性失衡损害神经可塑性，从而导致认知能力下降。在APP/PS1转基因痴呆鼠中利用红藻氨酸诱导慢性自发性癫痫发现，沉默Nwd1后癫痫发作频率和严重程度明显减少，认知障碍程度明显得到改善，推测Nwd1在癫痫相关认知障碍病理生理过程中也可能起着重要作用。因此，该项目将为癫痫的发病机制提供新线索，并为癫痫的诊治提供新策略。