CLU/自噬共靶向调控质膜完整性对发育期惊厥致海马苔藓纤维异常投射的影响及机制

We have recently discovered that the developmental seizure-induced long-term hippocampal plasma membrane integrity-related injury was in parallel with autophagy stress state and up-regulated expression of lipid-transport molecules clusterin (CLU) (supported by the National Natural Science Foundation of China, 81271458). Recent studies have shown that CLU is a novel molecular chaperone that binds to the autophagy marker molecule LC3, promotes LC3 lipid synthesis and autophagy synthesis, and expands protein toxic stress. Therefore, it is speculated that CLU and autophagy interaction and co-targeting can influence the membrane trafficking and signaling transduction in the hippocampus following developmental seizures, so as to repair the damage of the plasma membrane. In the present project using in vivo (CLU-/- mice) and in vitro developmental seizure models, we sought to further confirm the co-localization of CLU with the LC3 and hippocampal autophagic stress-induced CLU expression following developmental seizures; CLU silencing would weaken autophagy activation, while CLU overexpression enhance autophagic activity, which are in parallel with plasma membrane injury and repair, as well as with hippocampal mossy fiber sprouting; CLU can regulate the expression of membrane lipid metabolism molecules through autophagy-dependent pathways and repair the damaged plasma membrane of nerve cells. After the experiments we might propose a new point of view that co-targeting CLU and autophagy can be a new target for inhibiting hippocampal injury, abnormal axonal regeneration and functional damage in developing seizures.

我们课题组前期结题的国自然项目观察到发育期惊厥远期海马中质膜完整性调节分子cPLA2/clusterin（CLU）表达上调，并伴有自噬应激。新近研究也表明CLU是一种分子伴侣，可与自噬标记分子LC3共结合，促进LC3脂化和自噬体合成，扩大蛋白毒性应激。因而推测CLU和自噬相互作用和共靶向调控能够影响发育期惊厥远期海马质膜讯息传导，修复损伤质膜，发挥神经保护作用。本项目拟通过体内和体外（CLU-/-小鼠）发育期惊厥模型进一步验证发育期惊厥远期海马自噬应激触发CLU表达和与LC3的共定位；CLU沉默减弱自噬激活，CLU过表达增强自噬活性，并与神经质膜损伤修复和海马苔藓纤维（MFs）再生性发芽有关；CLU通过自噬依赖性途径调节膜脂质代谢分子表达，修复损伤的神经细胞质膜，籍此证明共靶向调节CLU和自噬通路是抑制发育期惊厥远期海马质膜损伤、轴突异常投射和功能损伤的新靶点。

我们的前期工作（国家自然科学基金项目81471337）证明线粒体呼吸、自噬和凋亡相关分子相互作用共同参与海马苔藓纤维异常发芽和行为、认知损伤。本项目继续探讨Clusterin介导的线粒体自噬在发育期惊厥后急性期和远期脑损伤中的作用，重点探讨对海马神经元线粒体功能稳态、苔藓纤维发芽以及行为和认知等方面的影响，以及线粒体保护剂melatonin、leptin和补锌饮食的干预作用，主要发现是：1.在整体模型中，惊厥后海马线粒体中clusterin、NIX、LC3II/I表达呈正相关，表达部位一致。Clusterin早期干预可通过NIX介导的线粒体自噬抑制神经元脱失、海马苔藓纤维异常发芽并减轻远期神经行为、认知的损伤。2. 在谷氨酸损伤细胞模型中，Clusterin能提早并提高NIX及LC3在线粒体上的表达，并能促进NIX与LC3的结合，促进线粒体自噬的发生。clusterin干预可通过上调NIX介导的线粒体自噬提高神经元细胞的存活。3. 在整体模型中探讨神经元质膜完整性和线粒体功能保护剂leptin对新生期惊厥发作后血清clusterin和血管内皮生长因子（VEGF）浓度、神经元质膜完整性相关蛋白以及神经行为表型的影响, 证明在新生期惊厥发作后立即用leptin治疗可以恢复血清clusterin的快速升高以及大脑皮层和海马中ATF-4/GRP78/自噬轴蛋白表达的上调，并改善远期神经元兴奋性表型，包括惊厥阈和海马苔藓纤维发芽，显示发育期惊厥急性期外周血clusterin与远期脑损伤表型之间存在内在联系，并具有潜在的生化标记物临床价值。4. 探讨了饮水补锌对BTBR小鼠自闭症样行为的影响机制，证明饮水补锌可以预防BTBR小鼠自闭症样行为，降低惊厥易感性，增加海马祖细胞的增殖，减轻苔藓纤维缠结，并一定程度上调clusterin蛋白水平，表明上调clusterin表达可能是饮水补锌纠正BTBR自闭症小鼠行为和兴奋性相关表型异常的机制之一。5. 构建Plppr5基因敲除小鼠（Plppr5-/-），探讨Plppr5敲除对发育期脑损伤及melatonin (神经质膜完整性保护剂)神经保护作用的影响，证明Plppr5对线粒体自噬介导的神经元氧化应激损伤具有重要的保护作用，可能是发育性惊厥性脑损伤的新靶点。这些结果为今后寻找新的抑制发育期惊厥性脑损伤的药物靶点子提供了新思路。