FAM134B介导的内质网自噬在无镁外液致痫海马神经元线粒体钙稳态及凋亡中的作用

Role of FAM134B-mediated ER-phagy in mitochondrial calcium homeostasis and hippocampal neurons apoptosis induced by Mg^(2+)-free solution

目的探讨FAM134B介导的内质网自噬在无镁外液致痫海马神经元线粒体钙稳态及凋亡中的作用。方法原代培养新生24 h SD大鼠海马神经元,建立无镁外液诱导的体外癫痫模型,并通过慢病毒载体干预FAM134B表达。将培养10 d的海马神经元随机分为对照组(CON)、无镁诱导组(AE)、空载病毒组(NC)、FAM134B过表达组(Lenti-FAM134B)和FAM134B低表达组(Lenti-FAM134B-shRNA)。采用TUNEL法检测细胞凋亡,钙离子荧光探针Mag-Fluo-AM和Rhod-2分别检测内质网和线粒体钙离子浓度,电镜下观察线粒体超微结构,Western blotting法检测自噬相关蛋白LC3-Ⅱ/Ⅰ、线粒体-内质网结构偶联中IP3R、细胞色素C(CytC)和活化cleaved caspase-3的表达。结果(1)与CON组相比,AE组神经元亡明显增加,FAM134B过表达使无镁外液致痫海马神经元凋亡明显减少,而降低FAM134B表达发挥相反作用;(2)与CON组相比,AE组LC3-Ⅱ/Ⅰ增加,FAM134B过表达显著增加无镁外液致痫海马神经元中LC3-Ⅱ/Ⅰ,而降低FAM134B表达发挥相反作用;(3)与CON组相比,AE组IP3R表达升高,内质网Ca^(2+)浓度降低,线粒体Ca^(2+)浓度升高;FAM134B过表达使无镁外液致痫海马神经元中IP3R表达减低,内质网Ca^(2+)浓度升高,线粒体Ca^(2+)浓度降低,而降低FAM134B表达发挥相反作用;(4)与CON组相比,AE组海马神经元线粒体结构明显损伤,CytC释放和caspase-3活化增加,FAM134B过表达使无镁外液致痫海马神经元线粒体结构损伤明显减轻,CytC释放和caspase-3活化降低,而降低FAM134B表达发挥相反作用。结论FAM134B介导的内质网自噬对无镁外液致痫海马神经元线粒体钙稳态及凋亡发挥保护作用,其机制可能为通过调节线粒体-内质网结构偶联中的IP3R水平改变内质网与线粒体之间的Ca^(2+)交换,减轻线粒体损伤,减少CytC的释放,抑制线粒体凋亡通路的激活。

Objective To study the role of FAM134B-mediated endoplasmic reticulum(ER)-phagy in calcium homeostasis and hippocampal neurons apoptosis induced by Mg^(2+)-free solution.Methods Primary cultured hippocampal neurons from newborn SD rats were induced by Mg^(2+)-free solution to establish epilepsy model and change the expression of FAM134B by lentiviral vector.After 10 days culture,neurons were randomly divided into control group,AE group,Lenti-pGV group,Lenti-FAM134B group and Len⁃ti-FAM134B-shRNA group.TUNEL assay was used to measure apoptotic neurons.Calcium fluorescence probe Mag-Fluo-AM and calcium fluorescence probe Rhod-2 were respectively used to detect ER calcium concentration and mitochondrial calcium concentra⁃tion.Ultrastructure of mitochondria were observed under electron microscope.The expressions of LC3-Ⅱ/LC3-Ⅰ,IP3R,CytC and cas⁃pase-3 were detected by Western blotting.Results Compared with the control group,neuronal apoptosis was significantly increased in AE group;FAM134B overexpression attenuated AE-induced neuronal apoptosis,while FAM134B down-expression exerted the op⁃posite effect.Compared with the control group,the ratio of LC3-Ⅱ/LC3-Ⅰwas significantly increased in the AE groups;FAM134B overexpression further increased the ratio of LC3-Ⅱ/LC3-Ⅰ,while FAM134B down-expression exerted the opposite effect.Compared with the control group,the expression of IP3R and mitochondrial calcium concentration were obviously increased and ER calcium concen⁃tration was decreased in the AE groups;FAM134B overexpression decreased the expression of IP3R and mitochondrial calcium concen⁃tration and increased ER calcium concentration induced by AE,while FAM134B down-expression exerted the opposite effect.Compared with the control group,mitochondrial of hippocampal neurons were damage and the CytC release and caspase-3 activation were increased in the AE groups;FAM134B overexpression reduced the mitochondria damage,the CytC release and caspase-3 activation,while FAM134B down-expression exerted the opposite effect.Conclusion FAM134B-mediated ER-phagy can play an important role in AE-induced neuronal apoptosis,possibly by modulating the IP3R in MAMs to alter Ca^(2+)exchange between ER and mitochondria and thus reduce mitochondria damage,decrease release of CytC and suppress mitochondrial apoptosis.