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

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.

Surface translocator protein 18 kDa (TSPO) localization on immune cells upon stimulation with LPS and in ART-treated HIV+ subjects

“…Given our findings of TSPO surface detection in the absence of permeabilization and the distinct staining patterns between surface and intracellular TSPO, we conclude that TSPO is found on the surface of immune cells and is not an artifact of antibody intercalation inside the cell. This novel finding in cells of myeloid lineage has recently been confirmed in primary microglia as shown by Loth and coworkers using flow cytometry 43 . Furthermore, another study in BV2 cells, a microglia cell line, has also shown that BV2 cells express plasma membrane TSPO that can be increased by LPS activation 44 …”

Oxytocin, Dopamine, and Opioid Interactions Underlying Pair Bonding: Highlighting a Potential Role for Microglia

“…LPS is a known endotoxin that induces a proinflammatory cascade by binding to Toll-like receptor 4 (TLR4), predominantly expressed in microglia in the central nervous system ( 137 ). LPS has been used in several models to induce an acute immune response within an animal ( 135 ) and to activate microglia in cell culture studies ( 111 , 138 , 139 ) ( Fig. 3B ).…”

Peroxisomal Metabolite and Cofactor Transport in Humans

“…The majority of heme-dependent proteins bind heme in the cytosol. A few heme-dependent proteins are folded inside the ER lumen where they subsequently bind heme; it has been suggested that heme is transported into the ER through a mitochondria-associated endoplasmic reticulum membrane (MAM) ( Asagami et al, 1994 ; Loth et al, 2020 ). So far there is no indication for free heme transport into peroxisomes, but there has been a few reports that suggest that catalase with complexed heme is assembled prior to peroxisomal import ( Koepke et al, 2007 ; Otera and Fujiki, 2012 ; Okumoto et al, 2020 ) and thus imported via the peroxisomal oligomeric protein import route ( Léon et al, 2006 ; Wolf et al, 2010 ).…”