Mfn2调控的线粒体相关内质网膜结构功能变化在癫痫神经损伤中的作用及机制

Seizure-induced mitochondrial damage makes epileptic neurons more sensitive to injury. Therefore, normal mitochondrial function is important for neuronal survival in epilepsy. Mitochondria-associated endoplasmic reticulum membranes(MAMs) relates to mitochondria closely. However, its participation in seizure-induced neuronal injury and the function and mechanism of MAMs in seizure-induced mitochondrial damage is still unknown. Recent studies have revealed that Mitofusin-2（Mfn2）plays an important role in regulating MAMs. Our previous study also found that mitochondrial damage as well as the expression of Mfn2 was significantly decreased in hippocampus of epileptic rats. So we speculate Mfn2 may affect the development of epilepsy by regulation of MAMs. Using in vitro primary cultured hippocampal neuron epileptic discharge and in vivo PILO (pilocarpine, PILO)-induced rat model of temporal lobe epilepsy, we will detect the effect of Mfn2 on MAMs, mitochondrial autophagy and apoptosis by transmission electron microscope, electron tomography, immuno-histochemistry technology and by constructing lentiviral vectors to regulate the expression of Mfn2. In addition, we will further explore the mechanism of Mfn2 regulation of MAMs by regulation mitochondrial calcium uniporter (MCU inhibitor or agonist). Taken together, this study will clarify the molecular mechanism of epileptic neuronal injury through new viewpoint and provide new targets for treatment of epilepsy.

癫痫发作后线粒体受损导致神经元对痫性损伤更敏感，因此维持线粒体正常功能至关重要。线粒体相关内质网膜（MAMs）与线粒体关系密切，但其是否参与癫痫神经损伤，以及在癫痫神经损伤中发挥何种作用目前并不清楚。最近研究发现Mfn2与MAMs结构功能关系密切，我们前期研究也发现癫痫海马神经元线粒体损伤及Mfn2水平明显降低，因此推测Mfn2可能通过调控MAMs结构功能变化影响线粒体功能进而影响癫痫发生发展。为证明该假说，我们将利用体外培养海马神经元癫痫放电及PILO诱导的大鼠颞叶癫痫模型，构建慢病毒载体干预Mfn2表达，采用电镜、电子断层成像、免疫组化等技术观察对MAMs结构功能、线粒体钙浓度、ROS生成、线粒体自噬和细胞凋亡等影响；再给予调控线粒体钙通道（MCU抑制剂或激动剂），进一步探讨MAMs影响线粒体结构功能的机制。本课题将从新的视觉阐明癫痫神经损伤分子机制，为癫痫治疗提供新靶点。

癫痫发作后线粒体受损导致神经元对痫性损伤更敏感，线粒体相关内质网膜（MAMs）在清除受损线粒体和维持线粒体正常功能中发挥重要作用。最近研究发现Mfn2在调控MAMs结构功能变化中发挥重要作用。本研究利用体外培养海马神经元癫痫放电及PILO诱导的大鼠颞叶癫痫模型，通过构建慢病毒载体调控Mfn2表达，采用分子生物学、免疫组化及激光共聚焦等方法观察对MAMs结构功能、线粒体钙离子浓度、线粒体功能、氧化应激和细胞凋亡等影响，深入探讨Mfn2调控的MAMs在癫痫神经损伤中的作用及机制。课题进展顺利，相关研究成果已以通讯作者发表SCI文章18篇，中文核心文章5篇。结果如下：.一、体外原代培养新生SD大鼠海马神经元，在无镁诱导海马神经元癫痫模型中，通过构建慢病毒干预Mfn2表达发现：.1.海马神经元Mfn2表达一过性下降，SOD水平下降，Bcl-2/Bax下降，Cyt-C从线粒体释放到细胞质。.2.上调Mfn2表达能够明显增加海马神经元SOD表达水平、升高Bcl-2/Bax水平和抑制线粒体释放Cyt-C，并明显减少海马神经元凋亡。.3.下调Mfn2表达能够明显降低海马神经元SOD表达水平、降低Bcl-2/Bax水平和增加线粒体释放Cyt-C，并明显增加海马神经元细胞凋亡。.二、在PILO诱导急性颞叶癫痫大鼠模型中，构建慢病毒干预Mfn2表达发现：.1.与PILO组比较，lenti-Mfn2组海马神经元线粒体钙超载明显减轻、ROS生成明显减少，而线粒体膜电位水平明显升高。.2.与PILO组比较，lenti-Mfn2-shRNA组海马神经元线粒体钙超载明显加重、ROS生成明显增多，而线粒体膜电位水平明显降低。.3.与PILO组比较，lenti-Mfn2组海马神经元Bax表达水平、线粒体Cyt-C释放及Caspase-3激活明显减少，而Bcl-2表达水平明显增多，海马神经元凋亡明显减少。.4.与PILO组比较，lenti-Mfn2-shRNA组海马神经元Bax表达水平、线粒体Cyt-C释放及Caspase-3激活明显增多，而Bcl-2表达水平明显减少，海马神经元凋亡明显增加。.综上所述，本课题深入研究了Mfn2调控的MAMs在癫痫神经损伤中的作用及机制，结果提示Mfn2可通过减少线粒体氧化应激，抑制线粒体凋亡途径而发挥癫痫神经保护作用。我们的研究结果为抗癫痫治疗提供了可能的新的靶点。