In the brain neuropil, translocator protein 18 kDa (TSPO) is a stress response protein that is upregulated in microglia and astrocytes in diverse central nervous system pathologies. TSPO is widely used as a biomarker of neuroinflammation in preclinical and clinical neuroimaging studies. However, there is a paucity of knowledge on the function(s) of TSPO in glial cells. In this study, we explored a putative interaction between TSPO and NADPH oxidase 2 (NOX2) in microglia. We found that TSPO associates with gp91^phox and p22^phox, the principal subunits of NOX2 in primary murine microglia. The association of TSPO with gp91^phox and p22^phox was observed using co-immunoprecipitation, confocal immunofluorescence imaging, and proximity ligation assay. We found that besides gp91^phox and p22^phox, voltage-dependent anion channel (VDAC) also co-immunoprecipitated with TSPO consistent with previous reports. When we compared lipopolysaccharide (LPS) stimulated microglia to vehicle control, we found that a lower amount of gp91^phox and p22^phox protein co-immunoprecipitated with TSPO suggesting a disruption of the TSPO-NOX2 subunits association. TSPO immuno-gold electron microscopy confirmed that TSPO is present in the outer mitochondrial membrane but it is also found in the endoplasmic reticulum (ER), mitochondria-associated ER membrane (MAM), and in the plasma membrane. TSPO localization at the MAM may represent a subcellular site where TSPO interacts with gp91^phox and p22^phox since the MAM is a point of communication between outer mitochondria membrane proteins (TSPO) and ER proteins (gp91^phox and p22^phox) where they mature and form the cytochrome b₅₅₈ (Cytb₅₅₈) heterodimer. We also found that an acute burst of reactive oxygen species (ROS) increased TSPO levels on the surface of microglia and this effect was abrogated by a ROS scavenger. These results suggest that ROS production may alter the subcellular distribution of TSPO. Collectively, our findings suggest that in microglia, TSPO is associated with the major NOX2 subunits gp91^phox and p22^phox. We hypothesize that this interaction may regulate Cytb₅₅₈ formation and modulate NOX2 levels, ROS production, and redox homeostasis in microglia.

在大脑Neuropil中，易位蛋白18 kDa（TSPO）是一种应激反应蛋白，在多种中枢神经系统病理学中的小胶质细胞和星形胶质细胞中上调。TSPO在临床前和临床神经影像研究中被广泛用作神经炎症的生物标志物。然而，关于神经胶质细胞中TSPO功能的知识很少。在这项研究中，我们探索了小胶质细胞中TSPO和NADPH氧化酶2（NOX2）之间的假定相互作用。我们发现TSPO与gp91 ^phox和p22 ^phox有关，gp91 ^phox和p22 ^phox是初级鼠小胶质细胞中NOX2的主要亚基。TSPO与gp91 ^phox和p22 ^phox的关联使用共免疫沉淀，共聚焦免疫荧光成像和邻近结扎法观察到的免疫组织化学。我们发现除gp91 ^phox和p22 ^phox外，电压依赖性阴离子通道（VDAC）还与TSPO共免疫沉淀，这与以前的报道一致。当我们比较脂多糖（LPS）刺激的小胶质细胞与媒介物对照时，我们发现gp91 ^phox和p22 ^phox的含量较低与TSPO共免疫沉淀的蛋白质表明TSPO-NOX2亚基缔合受到破坏。TSPO免疫金电子显微镜检查证实，TSPO存在于线粒体外膜中，但也存在于内质网（ER），线粒体相关ER膜（MAM）和质膜中。TSPO定位在MAM上可能代表TSPO与gp91 ^phox和p22 ^phox相互作用的亚细胞位点，因为MAM是外部线粒体膜蛋白（TSPO）和ER蛋白（gp91 ^phox和p22 ^phox）之间交流的点，它们在那里成熟并形成细胞色素b ₅₅₈（Cytb ₅₅₈）异二聚体。我们还发现，活性氧（ROS）的突然爆发增加了小胶质细胞表面的TSPO水平，并且这种清除作用被ROS清除剂消除了。这些结果表明，ROS的产生可能改变TSPO的亚细胞分布。总的来说，我们的发现表明在小胶质细胞中，TSPO与主要的NOX2亚基gp91 ^phox和p22 ^phox相关。我们假设这种相互作用可能调节Cytb _558的形成并调节小胶质细胞中NOX2的水平，ROS的产生和氧化还原的稳态。