Molecular Determinants of Mossy Fiber Presynaptic Channelopathies in Epilepsy

描述(申请人提供)：癫痫是一组不同的发作性疾病，与遗传或获得性离子通道和/或神经递质受体功能障碍有关，即所谓的“通道病”。在人类和实验性内侧颞叶癫痫(MTLE)中，海马苔藓纤维经历了分子和结构重排。最近的研究表明，癫痫大鼠苔藓纤维中的代谢性谷氨酸受体(MGluR II)和大电导钙激活钾通道(BK通道)均显著下调。在正常大脑中，突触前自身受体mGluR II与其他离子通道(即BK通道)的“反馈”激活可以减弱过度的兴奋性传递。因此，突触前缺失的mGluR II和BK通道可能在癫痫的发生中起重要作用。本项目的具体目的是：(1)验证癫痫大鼠苔藓纤维中mGluR II和BK通道下调与类似的mRNA转录缺陷和颗粒细胞剪接变体异常表达相关的假说；(2)测试癫痫发作诱导的mGluR II和BK通道下调影响突触前功能并加剧实验性MTLE苔藓纤维-CA3锥体细胞突触的兴奋性传递的假说；以及(3)确定mGluR II和BK通道能否减弱MTLE大鼠新的“复发”苔藓纤维颗粒细胞突触的兴奋性传递。现代技术的结合将被用来研究对照组和慢性癫痫大鼠的突触前机制。这些方法将包括组织和癫痫监测、单细胞实时定量聚合酶链式反应、颗粒细胞的可视化膜片钳记录，以及通过荧光苯乙烯染料FM1-43直接显示突触前功能。长期的目标是破译“获得性突触前通道病”的分子机制，并研究这种突触前功能障碍在MTLE发病机制中的后果。这项建议的数据可能为癫痫的新治疗干预措施提供基础。

DESCRIPTION (provided by applicant): Epilepsies are a diverse group of paroxysmal disorders that have been linked to genetic or acquired ion channel and/or neurotransmitter receptor dysfunctions, the so-called "channelopathies." The hippocampal mossy fibers undergo molecular and structural rearrangement in human and experimental mesial temporal lobe epilepsy (MTLE). Recent data revealed a robust down-regulation of both group II metabotropic glutamate receptors (mGluR II) and the large conductance Ca2+activated potassium channel (BK channels) in mossy fibers of epileptic rats. In the normal brain, "feedback" activation of presynaptic autoreceptor mGluR II in concert with other ion channels (i.e., BK channels) attenuates excessive excitatory transmission. Hence, presynaptic deficit in mGluR II and BK channels may play a major role in epileptogenesis. The specific aims of this project are: (1) to test the hypothesis that down-regulation of mGluR II and BK channels in mossy fiber of epileptic rats correlates with similar deficit in mRNA transcripts and abnormal expression of splice variants in granule cells; (2) to test the hypothesis that seizure-induced down-regulation of mGluR II and BK channels affects presynaptic function and exacerbates excitatory transmission at mossy fiber-CA3 pyramidal cell synapses in an experimental MTLE; and (3) to determine whether mGluR II and BK channels can attenuate excessive excitatory transmission at newly "recurrent" mossy fiber-granule cell synapses in a rat model of MTLE. A combination of modern techniques will be used to study presynaptic mechanisms in control versus chronically epileptic rats. Such approaches will include tissue and seizure monitoring, single-cell quantitative real-time PCR, visualized patch-clamp recordings from granule cells and direct visualization of the presynaptic function via the fluorescent styryl dye FM1-43. The long-term goal is to decipher the molecular mechanisms determining "acquired presynaptic channelopathies" and to investigate the consequences of such presynaptic dysfunction in the pathogenesis of MTLE. Data from this proposal may provide the foundation for novel therapeutic interventions for epilepsy.