基于 CaV1.2和CaM/CaMKII通路探讨神经肽Y对癫痫神经保护作用与机制

The massive influx of Ca2+ through calcium channels may result in abnormal neuronal firing and induce onset of epileptic seizure. Neuropeptide Y (NPY), as an endogenous neuroprotective substance, has been closely related to epileptogenesis, but the mechanism is not yet clear. Previous works of our group on genetic epileptic rats (tremor rat, TRM) has revealed that the concentration of Ca2+ in hippocampal neurons was increased, accompanied with altered expressions of CaV1.2, calmodulin (CaM) and CaMKII. Intraventricular administration of NPY could reduce spike waves in EEG. Therefore, we propose the hypothesis that NPY might influence the modulation of calcium current by CaV1.2 located on neuronal membrane and thus change the concentration of Ca2+ in the neuron. Then the synaptic plasticity could be changed by activating CaM/CaMKII pathway as well. In the present study, we will focus on the neuroprotection against epilepsy by NPY, especially the role of CaM/CaMKII pathway in this effect, by using in-vivo (TRM and Trimethyltin chloride injected rat ) and in-vitro (Mg++-free cultured neuron) models of epilepsy. Our study will provide experimental and theoretical basis for elucidating epileptic pathogenesis, developing specific medicine and gene therapy for seizures.

Ca2+通过细胞膜上的钙离子通道大量内流导致神经元突发性异常放电是癫痫发作的主要原因之一。神经肽Y（NPY）作为具有神经保护功能的内源性物质与癫痫密切相关，但作用机制尚未阐明。课题组前期研究发现遗传性癫痫大鼠模型TRM海马神经元内Ca2+浓度升高，L型钙通道亚型CaV1.2、钙调蛋白CaM和钙调蛋白依赖激酶CaMKII表达发生变化；侧脑室注射NPY能够降低TRM癫痫发作频率。因此，我们提出NPY可能通过位于神经元胞膜上的CaV1.2调控钙电流而改变胞内Ca2+浓度，并通过CaM/CaMKII通路影响神经元突触可塑性发挥神经保护作用的假说。本研究拟应用TRM大鼠、急性癫痫模型三甲基氯化锡注射大鼠和无镁处理海马神经元模型研究NPY对癫痫的保护作用，并重点针对其与CaV1.2及CaM/CaMKII通路之间的关系揭示其作用机制。为探究癫痫发病机理、开发特异性药物以及开展基因治疗提供科学依据。

癫痫(Epilepsy)是一种以短暂的脑功能失常为特征的慢性脑部疾病，其发病机制很复杂，至今仍未完全阐明，特别是遗传性癫痫，目前尚缺乏副作用少的有效治疗药物。我们的前期研究结果发现神经肽Y（Neuropeptide Y, NPY）与遗传性癫痫大鼠TRM癫痫发作有关，因此本课题拟进一步探讨其分子机制。我们通过脑电图记录发现侧脑室注射NPY能够明显降低TRM癫痫发作的次数和持续时间，水迷宫结果提示NPY能够提升TRM学习记忆能力，说明NPY对TRM具有一定的神经保护作用。免疫印迹结果显示TRM海马内CaM和p-CaMKII蛋白表达在给药前后显著改变，提示NPY可能通过CaM/CaMKII通路发挥保护作用。我们利用体外模型发现在CaMKII阻断剂KN93的作用下，NPY对于细胞的保护作用显著降低，并且与未阻断组相比，CaV1.2与CaM的结合增多，提示NPY可能通过CaMKII调节CaV1.2通道的敏感性。利用TRM的实验结果也验证了这一推断：KN93处理后，NPY没有明显降低TRM癫痫发作次数或改善学习记忆能力。综上，本研究结合分子生物学、电生理技术、组织化学和细胞培养等方法探究NPY对遗传性癫痫的神经保护作用及分子机制。揭示了NPY可能通过调节CaM/CaMKII通路影响CaV1.2通道蛋白的敏感性，从而抵御癫痫造成的神经元损伤，本项目为开发治疗遗传性癫痫的特异性多肽药物以及未来的基因治疗提供了更有价值的理论依据。