NMDA受体GRIN1基因新生突变p.L655Q在癫痫发病中的作用及其机制

The N-methyl-D-aspartate receptors (NMDARs) are ligand-gated ionotropic glutamate receptors that play important roles in synaptic plasticity and normal brain development. Recent advances in next-generation whole exome sequencing technologies show NMDAR mutations are responsible for a significant number of epilepsy cases while it stays unknown how the mutations contribute to the patients’ phenotypes and the mechanism of the disease development. We report a de novo GRIN1 missense mutation (p. L655Q) in a patient with refractory epilepsy. Previous study has shown the mutant L655Q enhanced agonist potency (glutamate and glycine) and prolonged synaptic-like response time course. We intent to study the effects of L655Q mutation on protein stability with computational technology and use various methods to evaluate the receptor gating, trafficking, neurotoxicity and synaptic plasticity, such as molecular biology techniques, electrophysiologic recordings and so on. Our study aims to assess the genotype-phenotype relationships and find out the the possible mechanism of the epileptic seizure development with in vivo and in vitro experiments. Moreover, a number of NMDAR-targeted drugs including FDA-approved NMDAR channel blockers will be evaluated for their ability to inhibit receptors containing L655Q as a first step to exploring the potential for rescue pharmacology and personalized medicine.

N-甲基-D-门冬氨酸（NMDA）受体是由配体门控的离子型谷氨酸能受体，在突触可塑性及中枢神经系统发育过程中具有关键作用。随着全外显子测序技术的发展，临床上大量癫痫相关病例被证明与NMDA受体基因突变相关，但这些突变通过何种机制影响患者的临床表现并使疾病发生发展尚不明确。我们在难治性癫痫患儿上发现了NMDA受体GRIN1基因新生突变p.L655Q。前期研究显示L655Q突变使NMDA受体对激动剂谷氨酸和甘氨酸的效能增强，并延长其类突触反应时程。本课题拟进一步用计算机分析L655Q突变受体蛋白结构稳定性的变化，通过分子生物学、电生理学等方法测定突变对NMDA受体功能、蛋白膜表达、细胞形态学、突触可塑性等方面的影响，从体内和体外实验来探究L655Q突变与癫痫之间的关系与机制。此外，我们尝试评估FDA批准的NMDA受体拮抗药物的疗效与作用，为探寻潜在的个体化治疗药物提供线索和理论依据。

N-甲基-D-门冬氨酸（NMDA）受体是由配体门控的离子型谷氨酸能受体，在突触可塑性及中枢神经系统发育过程中具有关键作用。临床上大量癫痫相关病例被证明与NMDA受体基因突变相关，但这些突变通过何种机制影响患者的临床表现并使疾病发生发展尚不明确。我们在难治性癫痫患儿身上发现了NMDA受体GRIN1基因新生突变p.L655Q，并首次对其进行功能学评估。电生理学记录提示含有GluN1-L655Q突变的NMDA受体对激动剂（谷氨酸和甘氨酸）的效能增强、通道去活化时程延长，这说明L655Q为功能增强性突变，可使NMDA受体过度活化、促进神经兴奋性毒性，导致患者出现癫痫症状。分子动力学模拟结果显示L655Q突变使受体中GluN1亚基的稳定性降低，使GluN2A亚基的pre-M1结构域原子活动度增强，提示这可能是导致NMDA受体功能增强的机制。GRIN1-L655Q knock-in小鼠的纯合子存在胚胎致死效应，杂合子小鼠海马区神经活动增强、对药物致痫更易感（PTZ，NMDA，kainic acid）并存在焦虑样行为，进一步证实GluN1-L655Q突变是导致患者出现癫痫的原因。分子生物学实验提示杂合子小鼠海马区组织NMDA各亚基（GluN1、GluN2A、GluN2B）的表达均增高，这可能是导致其药物致痫敏感和焦虑样表现的原因。我们评估了大量NMDA受体拮抗剂对GluN1-L655Q突变受体的抑制效果，并最终选择美金刚尝试治疗小鼠。经美金刚长期治疗，杂合子小鼠的药物致痫易感性和焦虑样行为得到显著缓解，为探寻潜在的个体化治疗药物提供线索和理论依据。

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