TRPM2通道调控海马神经元锌离子内流在癫痫发生中的作用及机制研究

Epilepsy is a major cause of chronic neurological disabilities including cognitive dysfunctions in children. The underlying mechanisms are complicated and multiple, and remain not fully understood. TRPM2 forms non-selective cationic channels. It induces Ca2+ and Zn2+ influx. Cytosolic Zn2+ accumulation induced by TRPM2 have a critical role in driving delayed pyramidal neuronal death, but the underlying mechanisms are not fully understood. In this study, we investigate the TRPM2 channel promotes epileptogenesis via regulating Zn2+ influx in hippocampal neurons. Using the MES, PTZ and KA kindling epilepsy models, we study the hipocampal pathological changes, the cognitive dysfunction, the cytosolic Zn2+ and Ca2+ levels ([Zn2+]i, [Ca2+]i) in hippocampal neurons and neural excities. The data will verify the important relationship between TRPM2 and epileptogenesis. The mechanisms of TRPM2 channel mediates Zn2+ influx in epileptogenesis will be validated by inhibitors of Zn2+(PTEN) and Ca2+(EDTA).To address the cell specificity in epileptogenesis, we generate TRPM2 conditional knockout (cKO) mice using a set of universal Cre-driver mouse lines. Selective deletion of TRPM2 was achieved in hippocampal CA3 pyramidal neurons (GRIK4-TRPM2 cKO), or in gamma amino butyric acidergic (GABAergic) neurons (Vgat- TRPM2 cKO). The data from in vivo and in vitro will verify the cell specificity of TRPM2 in epileptogenesis. The specific neuron TRPM2 channel activation can be a crucial mechanism responsible for the Zn2+ influx in epileptogenesis. Our study will reveal a new endogenous regulator of TRPM2 and uncover a novel mechanism underlying TRPM2-induced Zn2+ influx in epileptogenesis. TRPM2 inhibition may be a promising strategy of developing novel therapeutic treatments to control epileptic seizures, mitigate brain injury and improve cognitive dysfunction.

癫痫是临床常见的脑功能障碍性疾病，其发生机制尚未完全阐明。TRPM2通道能介导Zn2+内流，加剧细胞钙超载，增加神经元兴奋性，但与癫痫发生的相关性及介导机制等仍不明确。我们先期研究发现TRPM2基因敲除能抑制癫痫发生。在此基础上，本项目拟利用TRPM2-/-小鼠制备癫痫模型，检测神经元[Zn2+]i和[Ca2+]i、细胞电流及海马神经元形态学变化，体内体外实验记录癫痫波发放，明确TRPM2促进癫痫发生的作用；然后运用Zn2+和Ca2+螯合剂，通过癫痫行为及神经元兴奋性检测，明确TRPM2介导Zn2+内流促进癫痫发生；最后应用Cre-loxp基因重组技术，获得GRIK4-TRPM2 cKO小鼠和Vgat-TRPM2 cKO小鼠，通过体内体外实验进一步明确TRPM2在癫痫发生中的神经元特异性。实验结果将明确TRPM2在癫痫发生中的作用及相关机制，将为儿童癫痫的防治提供新思路和新靶点。

癫痫发作是大脑皮层神经元突发性异常放电，伴随大量离子通道开放，如钠通道、钾通道、钙通道等。钙离子（Ca2+）作为重要的细胞内信使，它对于神经元之间信息传递、神经递质释放和神经元存活等都具有重要意义。癫痫发作中存在神经元内Ca2+超载现象，异常的Ca2+内流是神经元同步化放电的先决条件。近年来研究发现细胞内Ca2+超载可能与锌离子（Zn2+）内流密切相关。本项目旨在阐明TRPM2通道调控海马神经元Zn2+内流在癫痫发生中的作用及机制。我们利用TRPM2基因敲除小鼠，通过戊四氮点燃癫痫模型，观察动物癫痫行为及海马神经元形态学变化，检测神经元[Zn2+]i和[Ca2+]i，膜片钳检测神经元电流，体内体外实验记录癫痫波发放，明确TRPM2促进癫痫发生的作用；然后运用Zn2+和Ca2+螯合剂，通过癫痫行为及神经元兴奋性检测，明确TRPM2介导Zn2+内流促进癫痫发生；最后应用Cre-loxp基因重组技术，获得GRIK4-TRPM2 cKO小鼠和Vgat-TRPM2 cKO小鼠，通过体内体外实验进一步明确TRPM2在癫痫发生中的神经元特异性。本研究重要结果：1. TRPM2基因敲除能够减轻癫痫所致的海马病理损害及降低星形胶质细胞的活化。2.TRPM2基因沉默可一定程度上抑制癫痫所致的焦虑行为及短期空间记忆损害，改善由癫痫所致的自主运动活性下降。3．TRPM2基因敲除可能通过下调PARP1及其下游的信号通路起到保护作用。4.TRPM2基因敲除后海马CA1锥体神经元兴奋性增加可能与Kv7相关。本项目将为探索TRPM2与癫痫发生提供实验依据，为难治性癫痫控制发作提供药物治疗新靶点，对改善癫痫患儿的生活质量具有重要的临床意义。