mTOR信号通路调控癫痫的作用及新型机制

Our previous study has demonstrated that the mTOR signaling pathway mediates epileptogenesis in a rat model of temporal lobe epilepsy. In pilot studies to determine the effect of mTOR signaing pathway inhibitors on epileptogennesis in a rat model induced by kainic acid (KA), we initially observed a paradoxical phenomenom in different inhibitors. As the down-stream effector of mTOR signaling pathway, S6 phosphorylation was not so obvious in Akt inhibitor perifosine-treated rats compaired with mTOR inhibitor rapamycin-treated rats. However, it was robust on preventing spontaneous seizure and neuronal cell death, indicating novel mechamism of mTOR signaling pathway on regulating epileptogenensis other than classic mTOR pwthway. To explore the novel target of mTOR signaling pathway on regulating epileptogenesis and its significance in epileptogensis, in the present project, we will detect the change of phosphorylation of different target protein of mTOR pathway and their correlation both in a rat epilepsy model induced by KA and in conditional gene knockout mouse.Subsequently, the effects of PI3K, Akt and mTOR inhibitors on mTOR signaling target protein phosphorylation and on epileptogenesis will be evaluated. Proteomics study will be used to explore the new targets which interact with Akt/S6 protein, and their functions in epileptogenesis will be examined in cultured primary hippocampal neuron and in rats/transgenic mice. This project will raise novel mechachimsm for regulating epileptogenesis independent of classical mTOR signaling pathway. It will provide new strategy for both clinical implications for the use of mTOR inhibitors as potential antiepileptogenic agents and for the development of novel antiepileptic therapies.

本项目前期研究表明mTOR信号通路介导了红藻氨酸（KA）致痫大鼠自发性癫痫的发生。最近在细致探讨mTOR信号通路相关抑制剂的作用时，我们发现Akt抑制剂哌立福辛无明显降低mTOR通路下游靶点S6蛋白磷酸化的作用，但对神经元死亡及自发性癫痫的防治效果较mTOR抑制剂雷帕霉素更显著，提示mTOR信号通路调控癫痫可能存在不同于经典通路的新型机制。为进一步探讨mTOR信号通路调控癫痫的新靶点及其在癫痫发生时的意义，本项目拟在KA致痫大鼠及基因敲除小鼠检测mTOR信号各靶点磷酸化水平的变化及关联程度；探讨PI3K，Akt及mTOR抑制剂对各靶点蛋白磷酸化水平的改变及对自发性癫痫的效应；蛋白质组学探索Akt或S6蛋白的新型结合靶点，并在原代细胞/脑片培养及整体动物观测新靶点在癫痫发生中的作用，从而提出mTOR信号调控癫痫的新机制，并为mTOR通路抑制剂在癫痫治疗中的应用以及新型药物研发提供理论依据。

mTOR信号通路在自发性癫痫发生中的中心作用以及mTOR抑制剂对癫痫的防治作用正在受到广泛的关注，尤其是雷帕霉素作为FDA业已认证的药物，在癫痫的防治中应该有很好的应用前景。本项目观察了KA致痫大鼠及基因敲除小鼠后mTOR信号各靶点磷酸化水平的变化及关联程度，并检测了Akt抑制剂哌立福新和mTOR抑制剂雷帕霉素对各靶点蛋白磷酸化水平的改变及对自发性癫痫的效应，并进一步对雷帕霉素降低剂量后的效应及不良反应进行观测，且探讨了雷帕霉素对KA致痫后小鼠树突棘的变化进行检测；进一步制备选择性神经元敲除raptor基因小鼠，观测了其在癫痫发生后的反应，蛋白质组学探索S6蛋白的新型结合靶点Hsp90。结果发现，KA致痫后，perifosine能够作用于Akt，使其磷酸化水平降低，从而抑制其下游mTOR信号通路的激活；Perifosine能够减少KA致痫导致的神经细胞死亡，苔藓纤维发芽，这可能是其减少自发性癫痫产生的原因。制备的Nestin CKO小鼠在外观和体重上与正常小鼠没有区别，但脑重比低，且脑皮层神经元厚度变低。Nestin CKO小鼠KA致痫后，Nestin CKO小鼠由KA导致的mTOR信号通路过度激活得到抑制神经细胞死亡及苔藓纤维发芽得到改善，在一定程度能够减少KA导致的认知障碍和减少焦虑行为。降低雷帕霉素剂量能抑制癫痫所致的mTOR信号通路异常激活、逆转学习记忆功能损伤和焦虑抑郁状态，且低剂量雷帕霉素对KA致痫未成年期小鼠体质量和海马DCX表达无明显影响。雷帕霉素可显著减少KA致痫后对树突棘的减少，这可能是雷帕霉素保护学习记忆功能的机制。另外，HSP90的变化趋势与S6蛋白的磷酸化水平变化相一致，HSP90的抑制剂17-AAG能显著逆转KA致痫所致的Hsp90增加，且S6蛋白磷酸化水平亦显著下降，提示S6的活化受上游因子Hsp90的调控；大鼠在生理盐水或KA致痫后3h提取海马总蛋白，经HSP90免疫沉降，SDS-PAGE检测到p-S6的表达，提示HSp90与S6共沉淀。上述结果表明，哌立福新和雷帕霉素具有良好的抗癫痫作用，可用于癫痫的治疗，mTOR信号通路可作为新药研发的靶点。