质膜完整性对发育期惊厥海马苔藓纤维再生性发芽的影响及调节

We have recently discovered that high-fat ketogenic diet inhibits the upregulation of hippocampal autophagy marker molecular beclin-1 as well as regenerative mossy fiber (MF) sprouting following develpmental seizures (supported by the National Natural Science Foundation of China, 30870808). Interestingly,the latest study has also demonstrated that beclin-1 is a novel membrane protein, mainly located in the phospholipid-rich lipid membrane.These data strongly suggest that plasma membrane integrity might play a key role in the pathophysiology of developmental seizure-induced hippocampal MF sprouting. In the present project using in vivo and in vitro developmental seizure models, we sought to determine the role of membrane lipid metabolism molecules (PRGs,Cyp46,cPLA,ACAT1,SMase) in the pathophysiology of developmental seizure-induced hippocampal plasma membrane damage/repair and regenerative MF sprouting. We will also explore the intervention effects of S1P/PI3K/mTOR pathway blockers on the expression of membrane lipid metabolism molecules. Furthermore, we will determine whether ketogenic diet would have therapy effects on developmental seizure-induced brain damage. After the experiments we might propose a new point of view that plasma membrane repairing is the therapeutic target by which to inhibit developmental seizure-induce hippocampal mossy fiber regenerative sprouting and functional impairment.

我们刚刚结题的国家自然基金项目（30870808）发现高脂性生酮饮食能够显著抑制发育期惊厥后海马自噬标记分子beclin-1表达的上调和苔藓纤维（MF）再生性发芽。而最新研究证明beclin-1是一种新型的膜蛋白，主要位于富含磷脂的脂质膜，因而提示质膜完整性在发育期惊厥致海马MF发芽中具有重要意义。本项目拟通过体内（in vivo）和体外（in vitro）发育期惊厥模型进一步验证膜脂质代谢分子（PRGs、Cyp46、cPLA2、ACAT1、SMase）与发育期惊厥后海马神经质膜损伤修复和MF再生性发芽有关；作用于S1P/P13K/mTOR通路阻滞剂能够通过调节膜脂质代谢分子表达阻止发育期惊厥后海马神经质膜完整性破坏，抑制海马异常发芽；并采用生酮饮食治疗发育期惊厥性脑损伤，籍此证明修复损伤的神经细胞质膜是抑制发育期惊厥后海马轴突异常再生和功能损害的治疗靶点。

发育期惊厥性脑损伤可造成严重的神经后遗症。海马齿状回内分子层去神经支配（denervation）和苔藓纤维（MF）再生性发芽为主要神经解剖学基础。我们的前期工作（国家自然科学基金项目30470555）发现Zn2+转运体（ZnT）参与再生性发芽的分子机制。进一步研究发现（国家自然科学基金项目30870808）生酮饮食能够显著抑制发育期惊厥后海马自噬标记分子beclin-1 表达的上调和MF再生性发芽。而beclin-1 是一种新型的膜蛋白，主要位于富含磷脂的脂质膜，因而推测质膜完整性在发育期惊厥致海马MF 发芽中具有重要意义。本项目应用动物整体的发育期惊厥模型，采用分子生物学、病理学和行为学结合的技术手段证明：1. 发育期惊厥后远期海马组织存在质膜损伤相关性脂质代谢分子（PRGs、Cyp46a1、cPLA2、ACAT1、nSMase、Kcnj11、Lepr、Drd2、Mc4r、Apoa1、Oprk1、Pdk4、ApoE）表达改变，并可能与神经行为、认知损伤和海马MF再生性发芽有关；2. 生酮饮食、质膜氧化损伤保护剂melatonin、脂质代谢调节分子leptin以及作用于P13K/mTOR/autophagy 通路阻滞剂E-64d能够抑制膜脂质代谢分子的异常表达使之恢复正常，并减轻海马MF异常发芽和神经行为、认知改变；3.生酮饮食及其它神经保护剂可能通过cPLA2/drp1/autophygy通路修复损伤的神经细胞质膜，进而调节Zinc/CaMK II信号分子的表达，从而实现抑制发育期惊厥后海马轴突异常再生和功能损害的治疗作用。这些结果为今后在分子水平寻找抑制发育期惊厥性脑损伤的新的靶分子提供了新思路。