Abstract
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 gp91phox and p22phox, the principal subunits of NOX2 in primary murine microglia. The association of TSPO with gp91phox and p22phox was observed using co-immunoprecipitation, confocal immunofluorescence imaging, and proximity ligation assay. We found that besides gp91phox and p22phox, 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 gp91phox and p22phox 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 gp91phox and p22phox since the MAM is a point of communication between outer mitochondria membrane proteins (TSPO) and ER proteins (gp91phox and p22phox) 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 gp91phox and p22phox. We hypothesize that this interaction may regulate Cytb558 formation and modulate NOX2 levels, ROS production, and redox homeostasis in microglia.
Highlights
Translocator protein 18 kDa (TSPO) is a widely used preclinical and clinical biomarker of brain injury and neuroinflammation that is able to detect diverse brain pathologies [1,2,3]
Co-immunoprecipitation of Translocator protein kDa (TSPO) with gp91phox, p22phox, and voltage-dependent anion channel (VDAC) in Microglia To explore the putative association between TSPO and the NADPH oxidase 2 (NOX2) principal subunits gp91phox and p22phox, we performed TSPO immunoprecipitation experiments of primary mouse microglia using antibodies independently validated in our lab (Supplementary Fig. S1) under both LPSactivated and vehicle conditions
Primary murine microglia cultures were greater than 94% pure as determined by macrophage integrin 1 (Mac-1 called CD11b) immunostaining (Supplementary Fig. S2)
Summary
Translocator protein 18 kDa (TSPO) is a widely used preclinical and clinical biomarker of brain injury and neuroinflammation that is able to detect diverse brain pathologies [1,2,3]. During the last several decades, a number of diverse functions have been attributed to TSPO, including cholesterol transport into the mitochondria for steroidogenesis; regulation of the mitochondrial permeability transition pore (mPTP); reactive oxygen species (ROS) production; and porphyrin/heme transport, among several other putative functions [1, 19,20,21,22]. Most of these studies were performed in steroidogenic primary cultures or cell lines, with limited studies in primary glial cells. Elucidating the functional significance of TSPO upregulation in glial cells under conditions of diverse neuropathology is important in order to advance the understanding of TSPO and glial cell biology
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