Kir2.1钾通道调节miR-9/FOXP2通路参与癫痫后学习记忆障碍的机制研究

The learning and memory dysfunction is the most common symptom caused by temporal lobe epilepsy (TLE). So far, there is no effective treatment for learning and memory impairment. It has been suggested that the pathogenesis of learning and memory impairment is associated with neuronal activity and synapse reorganization after TLE, but its underlying mechanisms is still not fully elucidated. Our previous work demonstrate that the Kir2.1 inward rectifying potassium channel (kir2.1) is a key molecular mediates deficit of learning and memory in TLE mice. Overexpression of Kir2.1 channel decreased the excitability of hippocampal dentate granule cells, meanwhile, the level of activity-regulated miR-9 is increased in TLE mice. miR-9 can target to bind Forkhead box protein P2(FOXP2). We hypothesize that seizures may increases the function expression of Kir2.1 channel, which activate miR-9/FOXP2 signaling pathway and cause synaptic plasticity damage and learning and memory disorders. Based on the previous work, the proposed study will use the Kir2.1 mutant mice to construct cognitive dysfunction mice model induced by TLE, combined with applying molecular biology, immunohistochemistry, electrophysiological recording and animal behaviour detection methods, we will further explore the effects and mechanisms of Kir2.1 in earning and memory deficts induced by TLE . Our study will provide a new idea and strategy for the clinical treatment of cognitive dysfunction.

学习记忆功能障碍是颞叶癫痫（TLE）最常见的伴随症状，与海马神经元活性改变以及突触重塑有关，但具体机制一直未能阐明，缺乏有效治疗。申请人在前期工作中发现：内向整流钾离子通道Kir2.1亚基(Kir2.1)是介导癫痫后学习记忆障碍的关键分子。过表达Kir2.1降低海马齿状回颗粒细胞兴奋性，上调非编码小分子RNA miR-9水平，miR-9靶向结合核转录因子叉头框P2基因（FOXP2）。因此申请人推测：癫痫发作上调Kir2.1，激活活性依赖性miR-9/FOXP2信号通路，损伤突触可塑性，导致学习记忆功能减退。本项目拟在此基础上，构建过表达Kir2.1转基因小鼠，建立TLE慢性期小鼠模型，结合分子生物学、免疫组化、膜片钳电生理以及动物行为学技术进一步探讨癫痫发作后Kir2.1介导学习记忆障碍的作用与机制，为临床治疗TLE诱发的认知功能损伤提供新策略。

学习记忆功能障碍是颞叶癫痫(temporal lobe epilepsy，TLE)最常见的伴随症状，多数研究认为癫痫发作引起海马特定区域的神经细胞损伤，突触结构重塑，神经环路异常从而导致学习记忆功能缺陷，但学习记忆被编码或调控的机制仍然未知。本项目研究发现小鼠进入TLE慢性期后，空间学习和记忆能力减退，内向整流钾离子通道2.1(Kir2.1)在海马的表达明显增加，Kir2.1通道在与认知相关的大脑区域广泛分布，并能调节脑神经元兴奋性。我们构建成功条件性诱导Kir2.1过表达的转基因小鼠，从形态和功能上验证Kir2.1通道在小鼠海马神经元的高表达，运用RNA测序和qPCR技术检测Kir2.1(+)小鼠海马内差异表达的非编码微小RNA水平，发现miR-9水平明显升高，运用生物学信息技术筛选出miR-9下游与突触可塑性相关的叉头框P2基因（Forkhead box protein P2，FOXP2），通过荧光素酶报告基因实验、qPCR和免疫印迹检测证实：miR-9能靶向结合并通过转录后机制调控FOXP2的表达。.为了明确Kir2.1通道激活miR-9/FOXP2信号通路诱导TLE慢性期的学习和记忆缺陷，我们用Kir2.1转基因小鼠建立TLE慢性期模型，运用分子生物学、膜片钳电生理、免疫组化和动物行为学技术开展了基因过表达和基因表达抑制的研究实验。在Kir2.1(+)小鼠海马内用锁核苷酸修饰的反义寡核苷酸分子LNA-9抑制内源性miR-9和用慢病毒Lenti-FOXP2升高FOXP2水平后，我们发现：特异性抑制miR-9完全恢复了Kir2.1(+)小鼠海马内FOXP2蛋白的表达，而海马内FOXP2水平升高有效逆转了TLE慢性期小鼠的学习障碍和记忆衰退。与之相反，在Kir2.1(-)小鼠海马内用慢病毒过表达miR-9和用RNAi技术沉默FOXP2则会损伤突触形态结构可塑性和突触传递效能，引起小鼠的学习记忆功能减退。在本项目的研究中，我们发现了Kir2.1介导TLE诱发学习记忆缺陷的miR-9/FOXP2信号通路：癫痫发作激活Kir2.1通道，上调miR-9，miR-9靶向抑制核转录因子FOXP2，损伤突触可塑性，诱导学习记忆障碍，这一新发现将丰富我们对癫痫发作损伤学习记忆能力机制的认识，为临床治疗TLE诱发的认知功能衰退提供新的思路和策略。