A Novel Interaction of Translocator Protein 18 kDa (TSPO) with NADPH Oxidase in Microglia.

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 increased TSPO levels on the surface of microglia and this effect was abrogated by a scavenger. These results suggest that duanyu1670 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, duanyu1670 production, and redox homeostasis in microglia.

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