The Mitochondrial Complex I Inhibitor Rotenone Induces Endoplasmic Reticulum Stress and Activation of GSK-3β in Cultured Rat Retinal Cells
The Mitochondrial Complex I Inhibitor Rotenone Induces Endoplasmic Reticulum Stress and Activation of GSK-3β in Cultured Rat Retinal Cells
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DOI：
10.1167/iovs.14-14371
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发表时间：
2014-09-01
影响因子：
4.4
通讯作者：
Wood, John P. M.
中科院分区：
文献类型：
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作者：
Han, Guoge;Casson, Robert J.;Wood, John P. M.
目的.线粒体功能障碍和内质网（ER）应激都与神经退行性疾病的发病机制有关。本研究的目的是探讨培养的视网膜细胞线粒体功能障碍引起的细胞损伤的机制，特别是ER应激是否在这种反应中发挥作用.从新生大鼠制备含有神经元和神经胶质的混合视网膜细胞培养物。使用复合物I抑制剂鱼藤酮来诱导线粒体功能障碍。免疫细胞化学，蛋白质印迹，活性氧（ROS），三磷酸腺苷（ATP）和TdT-dUTP末端缺口末端标记（TUNEL）的测定被用作标准，以阐明细胞反应。神经元更迅速地受到鱼藤酮（1 μ M）比神经胶质细胞，与显着的损失，这些细胞出现6小时后应用。胶质细胞死亡在24小时时是明显的，并且与TUNEL的阳性核标记相关。所有细胞损失与ROS产生增加有关，但神经元损失也与细胞ATP的显著消耗同时发生。在鱼藤酮处理的神经胶质细胞培养物中，6小时后，特征性ER应激组分免疫球蛋白重链结合蛋白（BiP）、ATF-4（转录激活因子4）、磷酸化PERK（胰腺内质网激酶/PKR样内质网激酶）和生长停滞和DNA损伤诱导蛋白/C/EBP同源蛋白（CHOP）的表达增加是明显的。此外，用LiCl抑制糖原合成酶激酶-3 β（GSK-3 β）能够保护神经胶质细胞，而用钙蛋白酶抑制剂III抑制钙蛋白酶仅保护神经元。这些数据共同表明，视网膜细胞中诱导的线粒体功能障碍可通过多种机制引起病理学，包括ATP耗竭、ROS升高、ER应激或GSK-3b或钙蛋白酶的活化。这种机制主要取决于线粒体挑战的浓度和持续时间以及受影响的细胞类型。
PURPOSE. Both mitochondrial dysfunction and endoplasmic reticulum (ER) stress have been implicated in the pathogenesis of neurodegenerative disorders. The purpose of the present study was to investigate the mechanisms of cellular damage resulting from mitochondrial dysfunction induced in cultured retinal cells and, in particular, whether ER stress plays a role in this response.METHODS. Mixed retinal cell cultures containing neurons and glia were prepared from neonatal rats. The complex I inhibitor rotenone was employed to induce mitochondrial dysfunction. Immunocytochemistry, Western blotting, and assays for reactive oxygen species (ROS), adenosine triphosphate (ATP), and TdT-dUTP terminal nick-end labeling (TUNEL) were used as standard to elucidate cellular responses.RESULTS. Neurons were more rapidly affected by rotenone (1 mu M) than glial cells, with significant loss of these cells appearing by 6 hours after application. Glial death was apparent by 24 hours and was associated with positive nuclear labeling by TUNEL. All cell loss was associated with increased ROS production, but neuronal loss was also concurrent with a significant depletion in cellular ATP. Increased expression of the characteristic ER stress components immunoglobulin heavy-chain binding protein (BiP), ATF-4 (activating transcription factor 4), phospho-PERK (pancreatic endoplasmic reticulum kinase/PKR-like endoplasmic reticulum kinase), and growth arrest and DNA damage-inducible protein/C/EBP homologous protein (CHOP) was evident in the rotenone-treated cultures after 6 hours in glial cells. Furthermore, inhibition of glycogen synthase kinase-3 beta (GSK-3 beta) with LiCl was able to protect glia cells, whereas inhibition of the calpain with calpain inhibitor III protected only neurons.CONCLUSIONS. These data together demonstrate that a mitochondrial dysfunction induced in retinal cells can give rise to pathology via a variety of mechanisms including ATP depletion, ROS elevation, ER stress, or activation of GSK-3b or calpain. Such mechanisms predominantly depend upon the concentration and duration of mitochondrial challenge and the type of cell affected.