Targeting translocator protein (18 kDa) (TSPO) dampens pro-inflammatory microglia reactivity in the retina and protects from degeneration
© Scholz et al. 2015
Received: 23 September 2015
Accepted: 26 October 2015
Published: 2 November 2015
Reactive microglia are commonly seen in retinal degenerative diseases, and neurotoxic microglia responses can contribute to photoreceptor cell death. We and others have previously shown that translocator protein (18 kDa) (TSPO) is highly induced in retinal degenerations and that the selective TSPO ligand XBD173 (AC-5216, emapunil) exerts strong anti-inflammatory effects on microglia in vitro and ex vivo. Here, we investigated whether targeting TSPO with XBD173 has immuno-modulatory and neuroprotective functions in two mouse models of acute retinal degeneration using bright white light exposure.
BALB/cJ and Cx3cr1 GFP/+ mice received intraperitoneal injections of 10 mg/kg XBD173 or vehicle for five consecutive days, starting 1 day prior to exposure to either 15,000 lux white light for 1 h or 50,000 lux focal light for 10 min, respectively. The effects of XBD173 treatment on microglia and Müller cell reactivity were analyzed by immuno-stainings of retinal sections and flat mounts, fluorescence-activated cell sorting (FACS) analysis, and mRNA expression of microglia markers using quantitative real-time PCR (qRT-PCR). Optical coherence tomography (OCT), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stainings, and morphometric analyses were used to quantify the extent of retinal degeneration and photoreceptor apoptosis.
Four days after the mice were challenged with bright white light, a large number of amoeboid-shaped alerted microglia appeared in the degenerating outer retina, which was nearly completely prevented by treatment with XBD173. This treatment also down-regulated the expression of TSPO protein in microglia but did not change the TSPO levels in the retinal pigment epithelium (RPE). RT-PCR analysis showed that the microglia/macrophage markers Cd68 and activated microglia/macrophage whey acidic protein (Amwap) as well as the pro-inflammatory genes Ccl2 and Il6 were reduced after XBD173 treatment. Light-induced degeneration of the outer retina was nearly fully blocked by XBD173 treatment. We further confirmed these findings in an independent mouse model of focal light damage. Retinas of animals receiving XBD173 therapy displayed significantly more ramified non-reactive microglia and more viable arrestin-positive cone photoreceptors than vehicle controls.
Targeting TSPO with XBD173 effectively counter-regulates microgliosis and ameliorates light-induced retinal damage, highlighting a new pharmacological concept for the treatment of retinal degenerations.
Microglial cells are the resident tissue macrophages of the central nervous system (CNS), including the retina. In the healthy retina, they are located in the plexiform layers from where they permanently scan the retinal environment with their motile protrusions . Several receptors that are specific for the binding of chemokines, cytokines, complement factors, antibodies, or damage-associated molecular patterns enable these cells to recognize and immediately respond to pathological changes of their environment [2–4]. Beside their homeostatic function in the healthy retina, microglia reactivity and age-related changes of microglia physiology contribute to degenerative pathologies of the retina and the entire CNS [3, 5–14]. Together with monocytes and macrophages, microglial cells are major players in chronic immune processes including parainflammation . Reactive microglia are detectable in the damaged photoreceptor layers of patients with degenerative retinal diseases such as retinitis pigmentosa and age-related macular degeneration (AMD) . Microglia not only phagocytose dead cells but also take up living rods in a mouse model for retinitis pigmentosa . Therefore, microglial activation cannot be just regarded as a bystander effect but rather actively contributes to photoreceptor cell death during retinal degeneration.
In attempts to better characterize retinal microglia reactivity and find novel markers, we and others have previously identified very high expression of translocator protein (18 kDa) in reactive retinal microglia [16, 17]. Translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, likely mediates the transport of cholesterol into the inner mitochondrial membrane, where it serves as a precursor for steroids and neurosteroids . The protein is constitutively expressed in steroidogenic tissues and up-regulated in activated glial cells [19–21]. Glial up-regulation of TSPO is a major hallmark of neurodegenerative diseases , and various TSPO ligands have been developed as molecular markers to detect gliosis by means of PET imaging .
Specific TSPO ligands are also under investigation as treatment options for neurological disorders including Alzheimer’s disease , multiple sclerosis , neuropathic pain , peripheral nerve injury , and anxiety disorders . Classical synthetic TSPO ligands such as the benzodiazepine derivative 4′-chlorodiazepam (Ro5-4864) and the isoquinoline carboxamide PK11195 directly enhance GABAergic neurotransmission . In contrast, structurally different synthetic TSPO ligands such as etifoxine (Stresam) and XBD173 (AC-5216, emapunil) also stimulate the synthesis of neurosteroids and exert potent anti-inflammatory and neuroprotective effects . XBD173 is a very selective and high-affinity phenylpurine ligand for TSPO whereas the benzoxazine etifoxine additionally binds GABAA receptors . Since XBD173 has a high and specific affinity for TSPO with a more beneficial side-effect profile than benzodiazepine derivatives, a precise and potentially long-term application to limit neuroinflammation seems feasible.
In a previous report, we have comprehensively characterized the anti-inflammatory effects of the TSPO ligand XBD173 using murine and human microglial cells as well as cultured mouse retinal explants . XBD173 strongly suppressed pro-inflammatory gene expression in LPS-challenged microglia and diminished their neurotoxic potential on photoreceptor cell cultures, indicating that targeting TSPO with XBD173 is a promising approach to control microglial reactivity . In this study, we addressed the questions whether XBD173 influences microglial reactivity in vitro and protects from acute white light-induced retinal degeneration in two different mouse models. We selected white light exposure as it is an environmental risk factor and mimics several features of human retinal degenerative diseases in rodents [31–33]. This model is also very useful for a quantitative correlation of microglial responses with processes of retinal degeneration [34–36].
XBD173 (emapunil) was obtained by custom synthesis from APAC Pharmaceuticals, Ellicott City, MD21042. XBD173 was dissolved in DMSO.
RNA isolation and reverse transcription
Total RNA was extracted from murine retinas using the NucleoSpin® RNA Mini Kit (Macherey-Nagel, Dueren, Germany). RNA was quantified spectrophotometrically with a NanoDrop 2000 (Thermo Scientific). First-strand cDNA synthesis was carried out with the Revert Aid H Minus First-strand cDNA Synthesis Kit (Fermentas, K1632).
cDNA (25 ng) were amplified in 10 μl reaction mixture consisting of 5 μl Fast Start Universal Probe Master (Rox) (Roche), 2 μl of primers (10 μM), 0.375 μl purified water, and 0.125 μl of dual-labeled UPL probe (Roche Applied Science, Basel, Switzerland) with an Applied Biosystems 7900 HT Fast Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA). The following reaction parameters were used: 10 min 95 °C hold, followed by 40 cycles of 15 s 95 °C melt, and 1 min 60 °C anneal/extension. Primer sequences and UPL probe numbers were as follows: Cd68, forward primer 5′-ctctctaaggctacaggctgct-3′, reverse primer 5′-tcacggttgcaagagaaaca-3′, probe #27; Amwap, forward primer 5′-tttgatcactgtggggatga-3′, reverse primer 5′-acactttctggtgaaggcttg-3′, probe #1; Tspo, forward primer 5′-actgtattcagccatggggta-3′, reverse primer 5′-accatagcgtcctctgtgaaa-3′, probe #33; Il6, forward primer 5′-gatggatgctaccaaactggat-3′, reverse primer 5′-ccaggtagctatggtactccaga-3′, probe #6; iNos, forward primer 5′-ctttgccacggacgagac-3′, reverse primer 5′-tcattgtactctgagggctga-3′, probe #13; Ccl2, forward primer 5′-catccacgtgttggctca-3′, reverse primer 5′-gatcatcttgctggtgaatgagt-3′, probe #62; Casp8, forward primer 5′-tgaacaatgagatccccaaat-3′, reverse primer 5′-caaaaatttcaagcaggctca-3′, probe #11; and Atp5b, forward primer 5′-ggcacaatgcaggaaagg-3′, reverse primer 5′-tcagcaggcacatagatagcc-3′, probe #77. Measurements were performed in triplicates. Atp5b expression was used as reference gene, and results were analyzed with the ABI sequence detector software version 2.4 using the ΔΔCt method for relative quantification.
Experiments were performed with 10–14-week-old albino BALB/cJ mice and Cx3cr1 GFP/+ mice on C57BL/6J background of both sexes. Animals were housed in an air-conditioned environment with 12-h light-dark schedule and had free access to water and food. All experimental procedures complied with the German law on animal protection and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The animal protocols used in this study were reviewed and approved by the governmental body responsible for animal welfare in the state of Nordrhein-Westfalen (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Germany) (reference number 84-02.04.2015-A039) and by the Animal Welfare and Ethical Review Board of Queen’s University Belfast under the regulation of the UK Home Office Animal (Scientific Procedures) Act 1986.
The mice received intraperitoneal injections of XBD173 at a dose of 10 mg/kg body weight, dissolved in DMSO or DMSO vehicle control twice daily for the first 2 days, starting 1 day before the light exposure and once daily for the remaining 3 days.
Light exposure regimens
BALB/cJ and Cx3cr1 GFP/+ mice were dark-adapted for 16 h before light exposure. After pupil dilatation with 1 % phenylephrine and 2.5 % tropicamide under dim red light, the mice were exposed to bright white light with an intensity of 15,000 lux for 1 h or focal white light with an intensity of 50,000 lux delivered by an otoscope (1218AA, Karl Storz, Tuttlingen, Germany) for 10 min, respectively. After light exposure, the animals were housed in dark-reared conditions overnight and then maintained under normal light conditions for the remaining experimental period.
Eyes were harvested for immunohistochemical analysis 4 days after light exposure. After fixation with 4 % paraformaldehyde, eyes were embedded in optimal cutting temperature compound or dissected for retinal flat mount analysis. Sixteen-micrometer sections were rehydrated with phosphate-buffered saline (PBS) and blocked with 1 % dried milk solution containing 0.01 % Triton X-100. Flat mounts were incubated with 5 % Tween and 5 % Triton X-100 in PBS overnight, and non-specific binding was blocked by incubation with dried milk solution. Subsequently, retinal sections and flat mounts were incubated with primary antibodies at 4 °C overnight. Primary antibodies targeting the following proteins were used: rabbit anti-Iba1 antibody (dilution 1:500; Wako Chemicals, Neuss, Germany), rabbit anti-TSPO antibody (dilution 1:250; Abcam, Cambridge, UK), rabbit anti-glial fibrillary acidic protein (dilution 1:200; G9269, Sigma, USA), mouse anti-glutamine synthetase (dilution 1:200; MAB302, Millipore, Darmstadt, Germany), and rabbit anti-cone arrestin (Millipore, Darmstadt, Germany). After a washing step, the sections and flat mounts were incubated with a secondary antibody either conjugated to Alexa488 (green; dilution 1:1000) or Alexa594 (red; dilution 1:800) (Jackson Immuno-Research, West Grove, PA, USA) for 1 h. After counterstaining with 4′,6-Diamidin-2-phenylindol (DAPI) in some instances, the samples were mounted in DAKO fluorescent mounting medium (Dako Deutschland GmbH, Hamburg, Germany) and analyzed with an Axioskop2 MOT Plus Apotome microscope (Carl Zeiss) or an Eclipse TE200-U confocal microscope (Nikon).
Optical coherence tomography
Animals were anesthetized by intraperitoneal injection of Rompun (10 mg/kg body weight)-Ketavet (100 mg/kg body weight), and their pupils were dilated with phenylephrine HCl (0.25 %)-tropicamide (0.05 %) before image acquisition. Spectral domain optical coherence tomography (SD-OCT) was performed on both eyes with a Spectralis™ HRA + OCT device (Heidelberg Engineering) to investigate structural changes in the retina after light exposure and XBD173 administration. Thickness measurements were performed using the Heidelberg Eye Explorer Software using a circular ring scan (circle diameters 3 and 6 mm), centered on the optic nerve head, which represents the average retinal thickness (μm) in a certain field.
TUNEL assay and morphometric analyses
Retinal sections were labeled with an in situ cell death detection kit, fluorescein (Roche), to detect the amount of apoptotic cells 4 days after light exposure. For a better overview, the sections were also counterstained with DAPI for 10 min. After a washing procedure, sections were mounted in DAKO fluorescent mounting medium (Dako Deutschland GmbH, Hamburg, Germany) and analyzed with an Axioskop2 MOT Plus Apotome microscope (Carl Zeiss). Quantitative morphometric analyses were performed by counting the number of rows of photoreceptor nuclei along the nasal/temporal axis.
Mouse retinas were dissected 4 days after light exposure and dissociated using the Neuronal Tissue Dissociation Kit-Postnatal Neurons (MACS, Miltenyi, Bergisch Gladbach, Germany). To identify microglia cells and macrophages, the single-cell suspension was stained with anti-mouse/human CD11b-APC antibody (MACS, Miltenyi, Bergisch Gladbach, Germany) at a dilution of 1:10 for 15 min in the dark at 4 °C. After a washing step, cells were fixed with FluoroFix Buffer (Biolegend, San Diego, CA, USA) in the dark for 30 min at room temperature. Afterwards, the cells were washed and resuspended in PBS solution (pH 7.2, 0.5 % containing bovine serum albumin (BSA), and 2 mM EDTA) until flow cytometric analyses with a fluorescence-activated cell sorting (FACS) Canto II (Becton Dickinson, Heidelberg, Germany). The number of CD11b+ cells was determined using FlowJo software (Treestar, Ashland, USA).
The differences between control mice and animals after light exposure that either received sham injections or XBD173 injections were analyzed using a one-way ANOVA and Dunnett’s multiple comparison test. p < 0.05 was considered statistically significant.
The TSPO ligand XBD173 prevents microglia reactivity and gliosis in murine retinas exposed to acute white light
We have previously identified that Tspo mRNA and protein are highly induced in genetic models of retinal degeneration and that its specific ligand XBD173 has potent anti-inflammatory activity on microglia in vitro . Based on these data, we hypothesized that XBD173 could also modulate microglial responses in the damaged retina in vivo. We have chosen acute light-induced degeneration as it is a fast and reproducible mouse model that mimics several features of human retinal degenerative diseases including innate immune activation and selective cell death of photoreceptor cells .
XBD173 reduces pro-inflammatory gene expression in retinal degeneration
XBD173 treatment prevents light-induced retinal degeneration
XBD173 prevents focal light-induced retinal damage
In the last set of experiments, we determined the number of cone photoreceptors in the focal light damage model using confocal microscopy of retinal flat mounts that were stained with anti-cone arrestin antibodies. The areas of focal light damage showed a much weaker arrestin staining compared to controls and compared to the retinas of XBD173-treated animals, respectively (Fig. 6g–i). The quantitative analysis of 6–8 independent retinas revealed a more than 8-fold reduction in the number of cone photoreceptors upon light damage and a 4.8-fold increase in the XBD173 therapy group, indicating a prominent effect on photoreceptor survival (Fig. 6j).
Due to its high expression in activated glia, TSPO is a marker for brain gliosis and TSPO ligands are commonly used for in vivo imaging in humans and mice [41, 42]. We have previously identified high TSPO levels in retinal microglia of murine models of inherited retinal degeneration [16, 37]. In the present study, we now show that TSPO is also strongly induced in retinal microglia after light exposure and that the TSPO ligand XBD173 has potent microglia-modulatory and neuroprotective functions in the retina in vivo. To our knowledge, this is the first report of TSPO-directed pharmacological targeting of the diseased retina.
To mimic exposure to bright daylight, which is a well-known environmental risk factor for retinal degenerations, we exposed the mice to 15,000 lux UV-free white light for 1 h in the first set of experiments and to 50,000 lux focal cold white light for 10 min in the validation studies. White light has an emission spectrum relatively similar to that of daylight, and it is less artificial than the light of a particular wavelength [43, 44]. Furthermore, white light contains a significant fraction of short-wavelength blue light (403 nm) which is thought to have a higher damaging potential than the light of longer wavelength including green light (490–580 nm), which was used in some studies to mimic retinal degeneration . The higher damaging potential of blue light is due to a process called photo-reversal, the regeneration of rhodopsin from bleaching intermediates that results in a higher number of photon absorption in a certain time span .
Very early microglial activation was observed already 1 day after light challenge in both models (data not shown), indicating that microglia sense subtle disturbances in the light-damaged retina before overt cell death occurs. TSPO expression was present in these reactive microglia, and our analyses towards the phenotype and behavior of microglia together with previous in vitro data  suggest that XBD173 exerts its neuroprotective function at least partially via modulation of microglia. Likewise, another specific TSPO ligand, etifoxine, also potently diminished inflammatory pathology and thereby attenuated the clinical score of experimental autoimmune encephalomyelitis, an experimental mouse model for multiple sclerosis . The tight correlation of microglia reactivity with TSPO expression was also identified in the genetic mouse model of myosin VII deficiency that mimics both features of retinal dystrophies and glaucoma . Therefore, TSPO induction in retinal microglia may be an early and reliable marker for changes in the microglia phenotype in different types of retinal degenerative diseases .
Our mRNA expression data of XBD173-treated retinas showed not only decreased transcript levels of Cd68, Amwap, and Tspo but also reduced Ccl2 and Il6 expression. The CCL2/CCR2 axis is crucial in subretinal macrophage and microglia accumulation in retinal degeneration models and human AMD, and these findings implicate that CCL2/CCR2 inhibition may be a novel tool to limit inflammation and neurodegeneration in the retina [48, 49]. Similarly, human reactive microglia express IL6, which in turn prevents retinal regeneration and promotes subretinal immune cell survival [50, 51]. Thus, the XBD173/TSPO axis seems to target two key pathways of chronic microglia reactivity in the retina.
As we have shown previously in microglial cell cultures , one potential mechanism of the anti-inflammatory effects of XBD173 could be the local synthesis of pregnenolone, as has been also demonstrated for brain astrocytes . Pregnenolone can be further metabolized to progesterone and allopregnanolone that are both potent neuroprotective and anti-inflammatory molecules [53–55]. When orally applied to rd1 mice with inherited retinal degeneration, progesterone potently reduced oxidative stress levels, diminished gliosis, and provided a temporal improvement in photoreceptor function .
We have demonstrated constitutive mitochondrial TSPO expression in the RPE that was unaffected by light exposure or XBD173 therapy. Therefore, TSPO in RPE mitochondria could potentially fulfill a housekeeping function. Of note, increased damage of mitochondrial DNA specifically in the RPE has been recently implicated as a risk factor for AMD and mutations of electron transport chain components could potentially limit energy production . Thus, in addition to its anti-inflammatory effects on microglia, TSPO may sustain mitochondrial homeostasis and integrity by regulating the oxygen consumption rate. However, this function and the previously identified physiological roles of TSPO remain to be characterized with novel cell-type specific knockout mouse models [58, 59].
We have shown that modulation of microglia with the synthetic TSPO ligand XBD173 preserved the retinal structure by counter-regulation of microglial pro-inflammatory responses during light exposure. Our data suggest that targeting TSPO in the retina may be a novel promising approach for anti-inflammatory and neuroprotective therapies in retinal degenerative disorders.
This work was supported by the DFG (LA1203/6-2, LA1203/9-1, LA1203/10-1, and FOR2240), the Velux Foundation, the Hans und Marlies Stock-Foundation, the Pro Retina Foundation, the Bayer Graduate Program in Pharmacology, and Fight for Sight (1425/1426). We thank the members of the Brachvogel Laboratory, Institute of Biochemistry, University of Cologne, for their help with the FACS analyses.
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