T-type 钙离子通道与内侧颞叶癫痫的发病机制研究

Medial temporal lobe epilepsy (MTLE) is the most common, drug resistant form of epilepsy in adults. One of the characteristic features of this syndrome is the latent period which is a delay between the precipitating insults (such as traumatic head injury, status epilepticus et al. ) and the onset of spontaneous behavioral seizures. It is during this period that the cellular and network level changes leading to chronic MTLE occur. Thus, therapeutic intervention during this period could be beneficial in delaying the onset of chronic MTLE. Entorhinal cortex (EC) and hippocampus are the principle brain regions involved in the induction and maintenance of MTLE. In human, seizure can initiate in the EC and propagate to the hippocampus. In addition, during latent period the EC in vivo and in vitro has been shown to be hyperexcitable. However, the molecular mechanism by which MTLE onset is initiated in the EC is still not clear. Low-voltage-activated T-type calcium channels open at sub-threshold membrane potentials and it is ideal for them to regulate the threshold and firing pattern of action potentials. Thus, They play an important role in regulating neuronal excitability. We have first shown that functional T-type calcium channels are present in the EC (Huang et al.(2011)Nat Neurosci 14(4):478-86). In addition,our preliminary data suggested that T-type calcium channels mediated currents (T-currents) are significantly enhanced in the EC during latent period. Therefore, we proposed that the enhancement in T-currents increases neuronal excitability in the EC,thereby facilitates the development of MTLE and leads the onset of spontaneous epilepsy. To test this hypothesis, we will use patch-clamping, two-photon calcium imaging and glutamate uncaging techniques combined with pharmacological, immunological and molecular biological tools to investigate the role of T-type calcium channels in MTLE. Besides, we will utilize the selective T-type calcium channels blockers, TTA-P2 and TTA-A2, to block T-currents in latent period to assess if we can decay or stop the onset of MTLE. This study will uncover the fundamental causes of MTLE and thus identify possible therapeutic target.

内侧颞叶癫痫是成年人最为常见的顽固型癫痫。内嗅皮层和海马体是其发病的起始部位，但发病机制不详。T-type钙离子通道在动作电位阈值下被激活,直接调节细胞的兴奋性。我们曾首次报道，内嗅皮层中大量表达功能性T-type钙离子通道。在预实验中我们还发现在癫痫潜伏期，内嗅皮层T-type 钙离子通道调节的电流（T-currents）明显增加。由此，我们假设"内嗅皮层T-currents增加，直接增加神经细胞的兴奋性，从而导致癫痫的发病"可能是颞叶癫痫发病的新机制。本研究拟采用卡英酸颞叶癫痫模型，通过应用膜片钳电生理记录、双光子钙离子成像和谷氨酸释放等方法，从分子、细胞兴奋性及整体不同层次证明T-type钙离子通道的表达与颞叶癫痫的发病之间的关系。最后，申请人还将在癫痫潜伏期应用选择性T-currents阻断剂，从而评估是否可以延缓或中止癫痫病的发病，为颞叶癫痫的预防和治疗提供新的思路和新靶点。

内侧颞叶癫痫是成年人最为常见的顽固型癫痫。内嗅皮层和海马体是其发病的起始部位，但发病机制不详。T-type钙离子通道在动作电位阈值下被激活，直接调节细胞的兴奋性。因此我们假设T-type钙离子通道可能参与颞叶癫痫的发生和发展。在研究中，我们验证了在颞叶癫痫潜伏期，T-type钙离子通道的表达明显升高。与传统的报道不同，我们发现T-type钙离子通道定位与皮层神经元的轴突起始段，其受乙酰胆碱能和多巴胺能神经元的调控，从而调节神经元的阈值，在神经元的信息处理中发挥重要作用。此外，我们还发现，CDYL是表观遗传学因子。在颞叶癫痫发病的潜伏期，CDYL的表达明显降低，从而导致其下游调控基因Nav1.6和T-type钙离子通道的表达升高，从而导致神经元兴奋性的升高。在潜伏期，特异性的升高CDYL可以抑制癫痫的发生和发展。我们的结果为颞叶癫痫的防治提供了新机制和新靶点。相关的研究成果发表学术论文6篇，其中责任作者论文两篇(Nature Communication（2017）、Journal of Physiology（2014）)，第一作者论文两篇(Scientific Reports（2017）、Journal of Physiology（2014）)，第二作者论文一篇（Neuron（2015））。申请中国专利三项。参与编著Springer论著一部。受邀参与国际会议四次，其中两次进行大会报告，两次作为分会报告。在此期间，项目负责人获得教育部新世纪人才的支持和绿叶生物医药杰出青年奖。并先后培养博士生4人，研究生8人（两人已毕业），其中一名博士人荣获国家奖学金，多人获北京大学一等奖学金。共有10余人次的学生出席不同的国内及国际学术会议。相关结果已经完成基金申请时所承诺的预期发表论文数目和人才培养目标。