Translocator protein (18 kDa) (TSPO) is expressed in reactive retinal microglia and modulates microglial inflammation and phagocytosis

Abstract

BackgroundThe translocator protein (18 kDa) (TSPO) is a mitochondrial protein expressed on reactive glial cells and a biomarker for gliosis in the brain. TSPO ligands have been shown to reduce neuroinflammation in several mouse models of neurodegeneration. Here, we analyzed TSPO expression in mouse and human retinal microglia and studied the effects of the TSPO ligand XBD173 on microglial functions.MethodsTSPO protein analyses were performed in retinoschisin-deficient mouse retinas and human retinas. Lipopolysaccharide (LPS)-challenged BV-2 microglial cells were treated with XBD173 and TSPO shRNAs in vitro and pro-inflammatory markers were determined by qRT-PCR. The migration potential of microglia was determined with wound healing assays and the proliferation was studied with Fluorescence Activated Cell Sorting (FACS) analysis. Microglial neurotoxicity was estimated by nitrite measurement and quantification of caspase 3/7 levels in 661 W photoreceptors cultured in the presence of microglia-conditioned medium. The effects of XBD173 on filopodia formation and phagocytosis were analyzed in BV-2 cells and human induced pluripotent stem (iPS) cell-derived microglia (iPSdM). The morphology of microglia was quantified in mouse retinal explants treated with XBD173.ResultsTSPO was strongly up-regulated in microglial cells of the dystrophic mouse retina and also co-localized with microglia in human retinas. Constitutive TSPO expression was high in the early postnatal Day 3 mouse retina and declined to low levels in the adult tissue. TSPO mRNA and protein were also strongly induced in LPS-challenged BV-2 microglia while the TSPO ligand XBD173 efficiently suppressed transcription of the pro-inflammatory marker genes chemokine (C-C motif) ligand 2 (CCL2), interleukin 6 (IL6) and inducible nitric oxide (NO)-synthase (iNOS). Moreover, treatment with XBD173 significantly reduced the migratory capacity and proliferation of microglia, their level of NO secretion and their neurotoxic activity on 661 W photoreceptor cells. Furthermore, XBD173 treatment of murine and human microglial cells promoted the formation of filopodia and increased their phagocytic capacity to ingest latex beads or photoreceptor debris. Finally, treatment with XBD173 reversed the amoeboid alerted phenotype of microglial cells in explanted organotypic mouse retinal cultures after challenge with LPS.ConclusionsThese findings suggest that TSPO is highly expressed in reactive retinal microglia and a promising target to control microglial reactivity during retinal degeneration.

In VivoDetection of Age- and Disease-Related Increases in Neuroinflammation by18F-GE180 TSPO MicroPET Imaging in Wild-Type and Alzheimer's Transgenic Mice

“…Increased TSPO mRNA and protein levels have been reported in activated BV2 microglia induced by LPS, a pro-inflammatory inducer, whereas multiple pro-inflammatory marker genes, including interleukin-6 (IL-6) and inducible nitric oxide synthase, were suppressed by the TSPO ligand XBD173, implicating a possible correlation of TSPO and proinflammatory microglia (Karlstetter et al, 2014). Moreover, a PET imaging study in nonhuman primates given low-dose intravenous LPS showed increased whole-brain uptake of 11 C-PBR28 in parallel with elevated serum levels of IL-1␤ and IL-6, indicating that TSPO may be associated with pro-inflammatory microglial activation (Hannestad et al, 2012).…”

Double Application of Translocator Protein Ligands and RAW Cells Inflammatory Milieu

“…This broad spectrum of bioactivities makes it an attractive theurapeutic drug target and a number of TSPO ligands has been synthesised and evaluated for their functional effects on TSPO and development of potential therapeutic agents (Szewczyk and Wojtczak, 2002;Galiegue et al, 2003;Karlstetter et al, 2014;Selvaraj et al, 2015). In addition, the development of radiolabelled TSPO ligands as molecular markers for imaging (Katsifis et al, 2000;Fookes et al, 2008;Pulli and Chen, 2014;Katsifis et al, 2004) helped localizing and monitoring the TSPO upregulation on activated microglia and/or astrocytes which is one of the hallmarks of neuroinflammation and neurodegeneration (Wilms et al, 2003;Girard et al, 2008;Mattner et al, 2011;Daugherty et al, 2013;Mattner et al, 2013).…”

Cerebrovascular and microglial states are not altered by functional neuroinflammatory gene variant

“…It is hypothesized that the Alanine to Threonine substitution at position 147 results in a conformational change in TSPO structure that influences its interaction with ligands. 36,38,39 Differences in binding affinity may have important implications for the etiopathology of AD; TSPO ligands have been shown to ameliorate neuroinflammation in vitro, 40 reverse neuropathology and behavioral decline in AD mouse models, 41 reduce Ab 42 -induced neurodegeneration in drosophila, 42 as well as confer neuroprotective and regenerative effects in vivo and in vitro. [43][44][45][46] Despite this potential involvement of TSPO in the etiopathology of neurodegenerative disorders, and the well-known links between inflammation, cerebrovascular disease, and AD, no studies to date have investigated the effect of rs6971 on MRI-based phenotypes or inflammatory biomarkers.…”