Department of Neurosurgery, First Affiliated Hospital, Hunan University of Chinese Medicine, Changsha, 410007, Hunan, China.
Neurochem Res. 2018 Oct;43(10):1963-1977. doi: 10.1007/s11064-018-2618-4. Epub 2018 Aug 22.
Mitochondrial dysfunction has been acknowledged as the key pathogenic mechanism in cerebral ischemia-reperfusion (IR) injury. Mitophagy is the protective system used to sustain mitochondrial homeostasis. However, the upstream regulator of mitophagy in response to brain IR injury is not completely understood. Nuclear receptor subfamily 4 group A member 1 (NR4A1) has been found to be associated with mitochondrial protection in a number of diseases. The aim of our study is to explore the functional role of NR4A1 in cerebral IR injury, with a particular focus on its influence on mitophagy. Wild-type mice and NR4A1-knockout mice were used to generate cerebral IR injury in vivo. Mitochondrial function and mitophagy were detected via immunofluorescence assays and western blotting. Cellular apoptosis was determined via MTT assays, caspase-3 activity and western blotting. Our data revealed that NR4A1 was significantly increased in the reperfused brain tissues. Genetic ablation of NR4A1 reduced the cerebral infarction area and repressed neuronal apoptosis. The functional study demonstrated that NR4A1 modulated cerebral IR injury by inducing mitochondrial damage. Higher NR4A1 promoted mitochondrial potential reduction, evoked cellular oxidative stress, interrupted ATP generation, and initiated caspase-9-dependent apoptosis. Mechanistically, NR4A1 induced mitochondrial damage by disrupting Mfn2-mediated mitophagy. Knockdown of NR4A1 elevated Mfn2 expression and therefore reversed mitophagic activity, sending a prosurvival signal for mitochondria in the setting of cerebral IR injury. Further, we demonstrated that NR4A1 modulated Mfn2 expression via the MAPK-ERK-CREB signaling pathway. Blockade of the ERK pathway could abrogate the permissive effect of NR4A1 deletion on mitophagic activation, contributing to neuronal mitochondrial apoptosis. Overall, our results demonstrate that the pathogenesis of cerebral IR injury is closely associated with a drop in protective mitophagy due to increased NR4A1 through the MAPK-ERK-CREB signaling pathway.
线粒体功能障碍已被认为是脑缺血再灌注（IR）损伤的关键发病机制。自噬是维持线粒体动态平衡的保护系统。然而，针对脑 IR 损伤的自噬的上游调节因子尚不完全清楚。核受体亚家族 4 组 A 成员 1（NR4A1）已被发现与许多疾病中的线粒体保护有关。我们的研究旨在探讨 NR4A1 在脑 IR 损伤中的功能作用，特别是其对自噬的影响。野生型小鼠和 NR4A1 敲除小鼠用于体内产生脑 IR 损伤。通过免疫荧光和 Western blot 检测线粒体功能和自噬。通过 MTT 测定、caspase-3 活性和 Western blot 测定细胞凋亡。我们的数据显示，NR4A1 在再灌注脑组织中显著增加。NR4A1 的基因缺失减少了脑梗死面积并抑制了神经元凋亡。功能研究表明，NR4A1 通过诱导线粒体损伤来调节脑 IR 损伤。较高的 NR4A1 导致线粒体电位降低，引发细胞氧化应激，中断 ATP 生成，并引发 caspase-9 依赖性细胞凋亡。机制上，NR4A1 通过破坏 Mfn2 介导的自噬来诱导线粒体损伤。NR4A1 的敲低增加了 Mfn2 的表达，从而逆转了自噬活性，为脑 IR 损伤中的线粒体提供了生存信号。此外，我们证明 NR4A1 通过 MAPK-ERK-CREB 信号通路调节 Mfn2 的表达。阻断 ERK 通路可以消除 NR4A1 缺失对自噬激活的许可作用，导致神经元线粒体凋亡。总的来说，我们的结果表明，脑 IR 损伤的发病机制与 MAPK-ERK-CREB 信号通路中因 NR4A1 增加而导致保护性自噬下降密切相关。
