Glial Cell Biology Laboratory Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150-180 Porto, Portugal.
Glial Cell Biology Laboratory Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150-180 Porto, Portugal.
Free Radic Biol Med. 2015 Feb;79:45-55. doi: 10.1016/j.freeradbiomed.2014.11.019. Epub 2014 Dec 5.
3-O-caffeoylquinic acid (3-CQA) is an isomer of chlorogenic acid, which has been shown to regulate lipopolysaccharide-induced tumor necrosis factor production in microglia. Whereas overactivation of microglia is associated with neuronal loss in brain diseases via reactive oxygen species (ROS) production and glutamate excitotoxicity, naïve (nonactivated) microglia are believed to generate little ROS under basal conditions, contributing to the modulation of synaptic activity and nerve tissue repair. However, the signaling pathways controlling basal ROS homeostasis in microglial cells are still poorly understood. Here we used time-lapse microscopy coupled with highly sensitive FRET biosensors (for detecting c-Src activation, ROS generation, and glutamate release) and lentivirus-mediated shRNA delivery to study the pathways involved in antioxidant-regulated ROS generation and how this associates with microglia-induced neuronal cell death. We report that 3-CQA abrogates the acquisition of an amoeboid morphology in microglia triggered by Aβ oligomers or the HIV Tat peptide. Moreover, 3-CQA deactivates c-Src tyrosine kinase and abrogates c-Src activation during proinflammatory microglia stimulation, which shuts off ROS production in these cells. Moreover, forced increment of c-Src catalytic activity by overexpressing an inducible c-Src heteromerization construct in microglia increases ROS production, abrogating the 3-CQA effects. Whereas oxidant (hydrogen peroxide) stimulation dramatically enhances glutamate release from microglia, such release is diminished by the 3-CQA inhibition of c-Src/ROS generation, significantly alleviating cell death in cultures from embryonic neurons. Overall, we provide further mechanistic insight into the modulation of ROS production in cortical microglia, indicating antioxidant-regulated c-Src function as a pathway for controlling microglia-triggered oxidative damage.
3-O-咖啡酰奎宁酸（3-CQA）是绿原酸的一种异构体，已被证明可调节小胶质细胞中脂多糖诱导的肿瘤坏死因子产生。虽然小胶质细胞的过度激活会通过活性氧（ROS）产生和谷氨酸兴奋性毒性导致脑疾病中的神经元丧失，但在基础条件下，未激活的小胶质细胞产生的 ROS 很少，有助于调节突触活性和神经组织修复。然而，控制小胶质细胞中基础 ROS 动态平衡的信号通路仍知之甚少。在这里，我们使用延时显微镜结合高灵敏度 FRET 生物传感器（用于检测 c-Src 激活、ROS 生成和谷氨酸释放）和慢病毒介导的 shRNA 传递来研究参与抗氧化剂调节的 ROS 生成的途径，以及这与小胶质细胞诱导的神经元细胞死亡如何相关。我们报告 3-CQA 可阻止 Aβ 寡聚体或 HIV Tat 肽触发的小胶质细胞获得阿米巴样形态。此外，3-CQA 可使 c-Src 酪氨酸激酶失活并阻止促炎小胶质细胞刺激过程中的 c-Src 激活，从而阻止这些细胞中的 ROS 生成。此外，通过在小胶质细胞中过表达可诱导的 c-Src 异源二聚体构建体强制增加 c-Src 催化活性，会增加 ROS 生成，从而消除 3-CQA 的作用。虽然氧化剂（过氧化氢）刺激可显著增强小胶质细胞中谷氨酸的释放，但 3-CQA 抑制 c-Src/ROS 生成会减少这种释放，从而显著减轻胚胎神经元培养物中的细胞死亡。总的来说，我们为皮质小胶质细胞中 ROS 产生的调节提供了进一步的机制见解，表明抗氧化剂调节的 c-Src 功能作为控制小胶质细胞触发的氧化损伤的途径。
