Fig 1: Immunocytochemistry and confocal imaging of TSPO colocalization with gp91phox, p22phox, VDAC, and LAMP-2 and effect of LPS activation in primary microglia. a Triple-label immunofluorescent confocal images of TSPO colocalization with gp91phox, p22phox, or VDAC in primary microglia (Mac-1 labeled) cells. Confocal images show that TSPO colocalizes with gp91phox, p22phox, and VDAC in primary microglia. Images in upper panels are at a low magnification: scale bar = 40 µm. Cells in white boxes were selected to be expressed at a higher magnification: scale bar = 10 µm (lower panel). b Analyses of protein pair signal colocalization revealed that TSPO has a high degree of colocalization with gp91phox (64.68%), p22phox (72.82%), and VDAC (81.48%) and a lower level of colocalization (20.13%) with the lysosomal marker, LAMP-2 in vehicle-treated microglia. Analysis of variance with Tukey’s post hoc tests shows a significant effect of protein colocalization with TSPO (F3,6 = 9.8; p = 0.0006) where TSPO colocalization with LAMP-2 is significantly lower than with gp91phox, p22phox, and VDAC (* = p < 0.01). c The percentage of gp91phox, p22phox, and VDAC that colocalized with TSPO decreased when microglia were activated with 100 ng/mL of LPS for 18 h relative to vehicle conditions (% gp91 with TSPO: p = 0.004; %p22 with TSPO: p = 0.002; % VDAC with TSPO: p = 0.003). These results indicate that microglia activation disrupts TSPO’s association with gp91phox, p22phox, and VDAC. d Further analysis of signal colocalization indicates that under LPS-stimulated conditions, TSPO associated with gp91 and TSPO associated with p22, exhibit significantly decreased colocalization with VDAC suggesting a movement from the mitochondria to other cellular compartments (% (TSPO with gp91)/ VDAC: p < 0.001. % (TSPO with p22) / VDAC: p = 0.004). Data are expressed as mean ± s.e.m. n = 5–7 independent experiments with > 35 microglia counted per treatment condition per experiment. n = 3 independent experiments for TSPO/LAMP-2 labeling with > 35 microglia counted per treatment condition per experiment. Student’s paired t test was performed; *p < 0.05 compared to vehicle-treated microglia
Fig 2: Reactive oxygen species (ROS) modulates surface expression of TSPO in primary microglia cells. a Representative flow cytometry dot plots of surface TSPO and CellROX® fluorescence in live primary microglia cells that were treated with and without N-acetyl cysteine (NAC), a ROS scavenger followed by tert-butyl hydroperoxide (TBHP), an ROS inducer. Percentage of surface TSPO expression (b) and mean fluorescence intensity (MFI) of ROS levels (c) with TBHP, ± NAC treatment. Surface TSPO expression and ROS levels increase with TBHP, while NAC treatment inhibited ROS and surface TSPO caused by TBHP. Data are expressed as mean ± sem n = 3 independent experiments: One-way ANOVA was performed; *p = 0.0003, F2,6 = 44.2 compared to basal levels; **p = 0.0046, F2,6 = 15.1 compared to basal levels
Fig 3: CCI Results in an Increase in the Number of GFAP Positive Cells in Injury Penumbra 5 Days After Injury. A: Whole brain photomicrographs (1.25×) of Sham and CCI with GFAP antibody. Note the increase in fluorescence in the CCI photomicrograph in comparison to Sham. Scale bar 1 mm. B: There is an overall lack of co-staining of PBR with GFAP positive cells in either groups (Sham or CCI). Scale bar: 25 µm.
Fig 4: CCI Results in an Increase in the Number of Amoeboid IBA1 Positive Cells in the Ipsilateral Injury Penumbra 5 Days After Injury. A: Whole brain photomicrographs (1.25×) of Sham and CCI with IBA1 antibody. Note the increase in fluorescence in the CCI photomicrograph in comparison to Sham. Scale bar 1 mm. B: There is a lack of co-staining of PBR with ramified (sham) IBA1 positive cells (merged) in comparison to amoeboid (CCI) IBA1 positive cells (merged). Note the increase in PBR (red) in CCI in comparison to Sham. Scale bar 25 µm. C: Ipsilateral to the injury, CCI significantly increased the number of amoeboid IBA1 positive cells in comparison to sham and significantly decreased the ramified cells in CCI (*: p < 0.05) by unpaired non parametric Mann-Whitney test. D: CCI significantly increased the number of amoeboid IBA1/PBR positive cells in comparison to sham.
Fig 5: Bioinformatics-based modeling of the hTSPO monomer and putative heme-binding capabilities. (a) A ribbon diagram of hTSPO mapped to mouse TSPO structure (PDB: 2mgy.1) with a-helices color coded (blue: Helix 1, blue-green: Helix 2, green: Helix 3, green-yellow: Helix 4, orange-red: Helix 5). The N-terminus (N') is labeled along with the C-terminus (C') and conserved tryptophan-33 (W33) in the cytosolic a-loop. (b) immediately below W33 is the proposed porphyrin-binding site identified in crystal structures of Rhodobacter sphaeroides TSPO illustrated by the appearance of protoporphyrin IX (PP9). The conserved a-loop motif of WYXXLXKP, wherein W33, Y34, and L37 of hTSPO were identified as potential heme-interacting residues. Using the HemeBind software and the sequence and modeled structure of hTSPO, we were able to identify amino acids with the potential for heme binding that were not implicated in porphyrin-binding in previous models. Including a motif (WXXLYXXM) in the intermembrane space of the mitochondria that could be the site of heme loading at the close of biosynthesis typified by the presence of W68 in hTSPO at the bottom of helix 2 (c). (d) An examination of the predicted protein surface topology using the same colors as the ribbon structure reveal a groove or trough between helices 1 and 3 (i). This trough has stretches of hydrophobicity (ii, red) and aromaticity (iii, blue) that resemble traits of other heme binding proteins without conserved or strong heme-binding motifs
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