Mitochondrial Translocator Protein Research Articles

11C]PK11195-PET Brain Imaging of the Mitochondrial Translocator Protein in Mitochondrial Disease.

ObjectiveTo explore the possibilities of radioligands against the mitochondrial outer membrane translocator protein (TSPO) as biomarkers for mitochondrial disease, we performed brain PET-MRI with [11C]PK11195 in 14 patients with genetically confirmed mitochondrial disease and 33 matched controls.MethodsCase–control study of brain PET-MRI with the TSPO radioligand [11C]PK11195.ResultsForty-six percent of symptomatic patients had volumes of abnormal radiotracer binding greater than the 95th percentile in controls. [11C]PK11195 binding was generally greater in gray matter and significantly decreased in white matter. This was most striking in patients with nuclear TYMP or mitochondrial m.3243A>G MT-TL1 mutations, in keeping with differences in mitochondrial density seen postmortem. Some regional binding patterns corresponded to clinical presentation and underlying mutation, even in the absence of structural changes on MRI. This was most obvious for the cerebellum, where patients with ataxia had decreased binding in the cerebellar cortex, but not necessarily volume loss. Overall, there was a positive correlation between aberrant [11C]PK11195 binding and clinical severity.ConclusionThese findings endorse the use of PET imaging with TSPO radioligands as a noninvasive in vivo biomarker of mitochondrial pathology.Classification of EvidenceThis study provides Class III evidence that brain PET-MRI with TSPO radioligands identifies mitochondrial pathology.

Specific and non-specific binding of a tracer for the translocator-specific protein in schizophrenia: an [11C]-PBR28 blocking study

ObjectiveThe mitochondrial 18-kDa translocator protein (TSPO) is expressed by activated microglia and positron emission tomography enables the measurement of TSPO levels in the brain. Findings in schizophrenia have shown to vary depending on the outcome measure used and this discrepancy in TSPO results could be explained by lower non-displaceable binding (VND) in schizophrenia, which could obscure increases in specific binding. In this study, we have used the TSPO ligand XBD173 to block the TSPO radioligand [11C]-PBR28 and used an occupancy plot to quantify VND in patients with schizophrenia.MethodsA total of 7 patients with a diagnosis of schizophrenia were recruited for this study. Each patient received two separate PET scans with [11C]PBR28, one at baseline and one after the administration of the TSPO ligand XBD173. All patients were high-affinity binders (HABs) for the TSPO gene. We used an occupancy plot to quantify the non-displaceable component (VND) using 2TCM kinetic estimates with and without vascular correction. Finally we computed the VND at a single subject level using the SIME method.ResultsAll patients showed a global and generalized reduction in [11C]PBR28 uptake after the administration of XBD173. Constraining the VND to be equal for all patients, the population VND was estimated to be 1.99 mL/cm3 (95% CI 1.90 to 2.08). When we used vascular correction, the fractional TSPO occupancy remained similar.ConclusionsIn schizophrenia patients, a substantial component of the [11C]PBR28 signal represents specific binding to TSPO. Furthermore, the VND in patients with schizophrenia is similar to that previously reported in healthy controls. These results suggest that changes in non-specific binding between schizophrenia patients and healthy controls do not account for discrepant PET findings in this disorder.

Direct and specific binding of cholesterol to the mitochondrial translocator protein (TSPO) using PhotoClick cholesterol analogue.

The translocator protein (TSPO) is a five-helix transmembrane protein localized to the outer mitochondria membrane. Radioligand binding assays and chemical crosslinking showed TSPO to be a high affinity cholesterol-binding protein. In this report, we show that TSPO in mitochondrial fractions from MA-10 mouse tumour Leydig cells can interact directly and competitively with the clickable photoreactive cholesterol analogue. PhotoClick cholesterol showed saturable photoaffinity labelling of TSPO that could be specifically immunoprecipitated with anti-TSPO antibody, following the click reaction with the fluorescent-azide probe, tetramethylrhodamine (TAMRA)-azide. Moreover, excess cholesterol reduced the photolabelling of both total mitochondrial proteins and TSPO. Together, the results of this study demonstrated direct binding of PhotoClick cholesterol to TSPO and that this interaction occurs at physiologically relevant site(s).

Neuroprotective effect of mitochondrial translocator protein ligand in a mouse model of tauopathy

BackgroundThe translocator protein (TSPO) has been identified as a positron emission tomography (PET)-visible biomarker of inflammation and promising immunotherapeutic target for the treatment of Alzheimer’s disease (AD). While TSPO ligands have been shown to reduce the accumulation of the toxic Alzheimer’s beta-amyloid peptide, their effect on tau pathology has not yet been investigated. To address this, we analyzed the effects of TSPO ligand, Ro5-4864, on the progression of neuropathology in rTg4510 tau transgenic mice (TauTg).MethodsBrain atrophy, tau accumulation, and neuroinflammation were assessed longitudinally using volumetric magnetic resonance imaging, tau-PET, and TSPO-PET, respectively. In vivo neuroimaging results were confirmed by immunohistochemistry for markers of neuronal survival (NeuN), tauopathy (AT8), and inflammation (TSPO, ionized calcium-binding adaptor molecule 1 or IBA-1, and complement component 1q or C1q) in brain sections from scanned mice.ResultsTSPO ligand treatment attenuated brain atrophy and hippocampal neuronal loss in the absence of any detected effect on tau depositions. Atrophy and neuronal loss were strongly associated with in vivo inflammatory signals measured by TSPO-PET, IBA-1, and levels of C1q, a regulator of the complement cascade. In vitro studies confirmed that the TSPO ligand Ro5-4864 reduces C1q expression in a microglial cell line in response to inflammation, reduction of which has been shown in previous studies to protect synapses and neurons in models of tauopathy.ConclusionsThese findings support a protective role for TSPO ligands in tauopathy, reducing neuroinflammation, neurodegeneration, and brain atrophy.

The translocator protein ligands as mitochondrial functional modulators for the potential anti-Alzheimer agents

Small molecule modulators of mitochondrial function have been attracted much attention in recent years due to their potential therapeutic applications for neurodegenerative diseases. The mitochondrial translocator protein (TSPO) is a promising target for such compounds, given its involvement in the formation of the mitochondrial permeability transition pore in response to mitochondrial stress. In this study, we performed a ligand-based pharmacophore design and virtual screening, and identified a potent hit compound, 7 (VH34) as a TSPO ligand. After validating its biological activity against amyloid-β (Aβ) induced mitochondrial dysfunction and in acute and transgenic Alzheimer’s disease (AD) model mice, we developed a library of analogs, and we found two most active compounds, 31 and 44, which restored the mitochondrial membrane potential, ATP production, and cell viability under Aβ-induced mitochondrial toxicity. These compounds recovered learning and memory function in acute AD model mice with improved pharmacokinetic properties.

Mitochondrial TSPO Deficiency Triggers Retrograde Signaling in MA-10 Mouse Tumor Leydig Cells.

The mitochondrial translocator protein (TSPO) has been shown to bind cholesterol with high affinity and is involved in mediating its availability for steroidogenesis. We recently reported that targeted Tspo gene deletion in MA-10 mouse tumor Leydig cells resulted in reduced cAMP-stimulated steroid formation and significant reduction in the mitochondrial membrane potential (ΔΨm) compared to control cells. We hypothesized that ΔΨm reduction in the absence of TSPO probably reflects the dysregulation and/or maintenance failure of some basic mitochondrial function(s). To explore the consequences of TSPO depletion via CRISPR-Cas9-mediated deletion (indel) mutation in MA-10 cells, we assessed the transcriptome changes in TSPO-mutant versus wild-type (Wt) cells using RNA-seq. Gene expression profiles were validated using real-time PCR. We report herein that there are significant changes in nuclear gene expression in Tspo mutant versus Wt cells. The identified transcriptome changes were mapped to several signaling pathways including the regulation of membrane potential, calcium signaling, extracellular matrix, and phagocytosis. This is a retrograde signaling pathway from the mitochondria to the nucleus and is probably the result of changes in expression of several transcription factors, including key members of the NF-κB pathway. In conclusion, TSPO regulates nuclear gene expression through intracellular signaling. This is the first evidence of a compensatory response to the loss of TSPO with transcriptome changes at the cellular level.

Genome-wide expression analysis of a new class of lncRNAs driven by SINE B2.

Repetitive short interspersed elements B2 (SINE B2) have been shown to possess two promoters: polymerase III promoter for producing short B2-S RNAs and polymerase II promoter for driving the expression of long non-coding RNA (B2-AS lncRNAs). Using a B2-antisense (B2-AS) transcript sequence from the SINE B2 resident in mitochondrial translocator protein gene (Tspo) locus, we constructed a B2-AS specific RNA library and identified 96,862 sequences encoding potential B2-mediated lncRNAs, of which 55,592 lncRNAs with more than 390 nt in length possess a feature of potential genomic locus-specific effect. In addition, small RNA-Northern hybridization showed that the new B2-AS lncRNAs are constantly degraded by the Dicer1 enzyme, a finding further confirmed by in vitro Dicer1 enzyme digestion. B2-AS lncRNAs regulate the expression of target genes in a different fashion than B2-S RNAs. Genome-wide cross-comparison with mRNA mapping showed a total of 904 mRNA loci directly targeted by B2-AS lncRNAs, suggesting a locus-specific effect of the B2-AS lncRNAs and a general effect of B2-S RNAs.

Mitochondrial translocator protein (TSPO) ligand attenuates neuroinflammation and reduces complement component C1Q in a mouse model of tauopathy

AbstractBackgroundThe mitochondrial translocator protein (TSPO) has been widely investigated as a positron emission tomography (PET) detectable biomarker of neuroinflammation, as well as a potential therapeutic target for the treatment of neurodegenerative disease. In transgenic mouse models of AD, we and others have shown that TSPO ligands attenuate inflammation and beta amyloid burden (Barron et al., 2013). However, the effect of TSPO ligands on tauopathy‐induced inflammation has not yet been investigated. Here, we analysed the effects of the prototypic TSPO ligand, Ro5‐4864, on the progression of tauopathy, gliosis and atrophy in the rTg4510 tau transgenic mouse model (TauTg).MethodTauTg and WT control mice were treated with Ro5‐4864 or vehicle for 4 months. Brain atrophy was assessed using volumetric magnetic resonance imaging, while tauopathy and neuroinflammation were assessed by PET using 11C‐PBB3 and 18F‐FEBMP tracers, respectively. To corroborate in vivo findings, immunohistochemistry was performed in sections from scanned mice to measure phosphorylated tau (AT8), inflammation (IBA1, complement component 1q, C1q) and neuronal loss (NeuN).ResultRo5‐4864 treatment reduced markers of neuroinflammation, as well as expression of the neuronal ‘eat me’ signal, C1q in rTg4510 mice. In vitro experiments using the immortalized microglial cell line, BV2, confirmed the TSPO ligands, Ro5‐4864, reduced expression of C1q in response to inflammatory stimuli.ConclusionThese findings support a protective role for TSPO ligands in reducing tauopathy‐induced neuroinflammation.

Cholesterol transport between red blood cells and lipoproteins contributes to cholesterol metabolism in blood

Lipoproteins play a key role in transport of cholesterol to and from tissues. Recent studies have also demonstrated that red blood cells (RBCs), which carry large quantities of free cholesterol in their membrane, play an important role in reverse cholesterol transport. However, the exact role of RBCs in systemic cholesterol metabolism is poorly understood. RBCs were incubated with autologous plasma or isolated lipoproteins resulting in a significant net amount of cholesterol moved from RBCs to HDL, while cholesterol from LDL moved in the opposite direction. Furthermore, the bi-directional cholesterol transport between RBCs and plasma lipoproteins was saturable and temperature-, energy-, and time-dependent, consistent with an active process. We did not find LDLR, ABCG1, or scavenger receptor class B type 1 in RBCs but found a substantial amount of ABCA1 mRNA and protein. However, specific cholesterol efflux from RBCs to isolated apoA-I was negligible, and ABCA1 silencing with siRNA or inhibition with vanadate and Probucol did not inhibit the efflux to apoA-I, HDL, or plasma. Cholesterol efflux from and cholesterol uptake by RBCs from Abca1+/+ and Abca1-/- mice were similar, arguing against the role of ABCA1 in cholesterol flux between RBCs and lipoproteins. Bioinformatics analysis identified ABCA7, ABCG5, lipoprotein lipase, and mitochondrial translocator protein as possible candidates that may mediate the cholesterol flux. Together, these results suggest that RBCs actively participate in cholesterol transport in the blood, but the role of cholesterol transporters in RBCs remains uncertain.

Mitochondrial Translocator Protein (TSPO) Prevents Heart Failure by Increasing Cardiac Utilization of Fatty Acids

Introduction: For more than two decades, the peripheral mitochondrial translocator protein (TSPO) was considered to play a major role in mPTP formation, cholesterol transport, and steroidogenesis. However, recent genetic studies have questioned these previously established functions of TSPO, and therefore, the exact molecular mechanism of TSPO action remains unclear. We have previously demonstrated that TSPO expression was significantly upregulated in heart failure (HF) induced by transverse aortic constriction (TAC) in mice, and cardiac-specific conditional knockout (KO) of TSPO limited the development of pressure overload induced HF (Thai, Sci Reports, 2018, 8(1):16213). However, the exact mechanism of this protection has not been elucidated. Objective: Determine the mechanism of TSPO KO protection against pressure-overload HF. Methods and Results: We examined protein profiles in hearts from 4 experimental groups (WT sham, KO sham, WT TAC and KO TAC) using liquid chromatography-mass spectrophotometry. Our data revealed major changes in NADH:ubiquinone oxidoreductase subunit AB1 of the mitochondrial complex I (NDUFAB1), mitochondrial acyl-CoA binding protein (ACBP), mitochondrial long-chain specific acyl-CoA dehydrogenase (ACADL), and mitochondrial D-2-hydroxyglutarate dehydrogenase between KO and WT animals, with more modest changes in cytoskeletal and collagen associated proteins. Since NDUFAB1 and ACBP participate in free fatty acids (FFA) synthesis and ACADL is an important enzyme in FFA oxidation (FAO) pathway, we tested whether TSPO modulates FFA biosynthesis and oxidation.We measured changes in FAO in WT and TSPO KO mice under sham and TAC conditions and found that TSPO KO significantly increased fatty acid consumption and oxidation. Conclusion: Since HF is characterized by a shift in metabolism from FFA to less efficient glycolysis, thereby resulting in energetic failure, contractile dysfunction and cell death, the recovery of FFA metabolism by TSPO inhibition may be an effective drug target for HF.

Glioblastoma Exhibits Inter-Individual Heterogeneity of TSPO and LAT1 Expression in Neoplastic and Parenchymal Cells.

Molecular imaging is essential for diagnosis and treatment planning for glioblastoma patients. Positron emission tomography (PET) with tracers for the detection of the solute carrier family 7 member 5 (SLC7A5; also known as the amino acid transporter light chain L system, LAT1) and for the mitochondrial translocator protein (TSPO) is successfully used to provide additional information on tumor volume and prognosis. The current approaches for TSPO-PET and the visualization of tracer ([18F] Fluoroethyltyrosine, FET) uptake by LAT1 (FET-PET) do not yet exploit the full diagnostic potential of these molecular imaging techniques. Therefore, we investigated the expression of TSPO and LAT1 in patient glioblastoma (GBM) samples, as well as in various GBM mouse models representing patient GBMs of different genetic subtypes. By immunohistochemistry, we found that TSPO and LAT1 are upregulated in human GBM samples compared to normal brain tissue. Next, we orthotopically implanted patient-derived GBM cells, as well as genetically engineered murine GBM cells, representing different genetic subtypes of the disease. To determine TSPO and LAT1 expression, we performed immunofluorescence staining. We found that both TSPO and LAT1 expression was increased in tumor regions of the implanted human or murine GBM cells when compared to the neighboring mouse brain tissue. While LAT1 was largely restricted to tumor cells, we found that TSPO was also expressed by microglia, tumor-associated macrophages, endothelial cells, and pericytes. The Cancer Genome Atlas (TCGA)-data analysis corroborates the upregulation of TSPO in a bigger cohort of GBM patient samples compared to tumor-free brain tissue. In addition, AIF1 (the gene encoding for the myeloid cell marker Iba1) was also upregulated in GBM compared to the control. Interestingly, TSPO, as well as AIF1, showed significantly different expression levels depending on the GBM genetic subtype, with the highest expression being exhibited in the mesenchymal subtype. High TSPO and AIF1 expression also correlated with a significant decrease in patient survival compared to low expression. In line with this finding, the expression levels for TSPO and AIF1 were also significantly higher in (isocitrate-dehydrogenase wild-type) IDHWT compared to IDH mutant (IDHMUT) GBM. LAT1 expression, on the other hand, was not different among the individual GBM subtypes. Therefore, we could conclude that FET- and TSPO-PET confer different information on pathological features based on different genetic GBM subtypes and may thus help in planning individualized strategies for brain tumor therapy in the future. A combination of TSPO-PET and FET-PET could be a promising way to visualize tumor-associated myeloid cells and select patients for treatment strategies targeting the myeloid compartment.

GABA-A receptor and mitochondrial TSPO signaling act in parallel to regulate melanocyte stem cell quiescence in larval zebrafish.

Tissue regeneration and homeostasis often require recruitment of undifferentiated precursors (adult stem cells; ASCs). While many ASCs continuously proliferate throughout the lifetime of an organism, others are recruited from a quiescent state to replenish their target tissue. A long-standing question in stem cell biology concerns how long-lived, non-dividing ASCs regulate the transition between quiescence and proliferation. We study the melanocyte stem cell (MSC) to investigate the molecular pathways that regulate ASC quiescence. Our prior work indicated that GABA-A receptor activation promotes MSC quiescence in larval zebrafish. Here, through pharmacological and genetic approaches we show that GABA-A acts through calcium signaling to maintain MSC quiescence. Unexpectedly, we identified translocator protein (TSPO), a mitochondrial membrane-associated protein that regulates mitochondrial function and metabolic homeostasis, as a parallel regulator of MSC quiescence. We found that both TSPO-specific ligands and induction of gluconeogenesis likely act in the same pathway to promote MSC activation and melanocyte production in larval zebrafish. In contrast, TSPO and gluconeogenesis appear to act in parallel to GABA-A receptor signaling to regulate MSC quiescence and vertebrate pigment patterning.

2163Molecular imaging of cardiac and neuroinflammation early after myocardial infarction and in progressive heart failure

Abstract Background/Introduction Myocardial infarction (MI) triggers local inflammation to support endogenous healing and repair. Recent imaging studies of the macrophage- and microglia-expressed mitochondrial translocator protein (TSPO) identified concurrent neuroinflammation after acute MI and in chronic heart failure. The source of this neuroinflammation and its relationship to cardiac function early and late after MI are unknown. Purpose We aimed to characterize the cellular basis of the TSPO PET signal by modulating early inflammation via clodronate-mediated macrophage depletion, and modifying late mitochondrial function using the TSPO inhibitor PK11195. Methods C57BL/6 mice underwent permanent coronary artery ligation (n=47) or sham surgery (n=9). Subgroups were treated 24h prior surgery with clodronate liposomes (n=18) to deplete peripheral macrophages or continuously with the cardioprotective TSPO inhibitor PK11195 (n=13). Cardiac and neuroinflammation were evaluated by whole-body PET using the TSPO ligand 18F-GE180 at 1wk, 4wk and 8wk after surgery. Cardiac function and perfusion were assessed by ECG-gated 99mTc-sestamibi SPECT. Results Untreated MI mice showed elevated TSPO signal in the infarct territory compared to sham at 1wk post-MI (ID/g, 10.5±2.9 vs 7.2±1.6, p<0.001), and elevated remote myocardial TSPO signal at 8wk (ID/g, 9±1.9 vs 7±1.6, p=0.003). TSPO signal in brain of MI mice was also increased compared to sham at 1wk (ID/g, 2.1±0.3 vs 1.8±0.2, p=0.006) and 8wk (ID/g, 2.0±0.3 vs 1.8±0.2, p=0.033), reflecting neuroinflammation. Clodronate macrophage depletion lowered the infarct territory TSPO signal at 1wk compared to untreated (ID/g, 4.9±1 vs 10.5±3, p<0.001), consistent with lack of peripheral macrophage recruitment. Conversely, brain TSPO remained elevated (ID/g, 2.7±0.3 vs 2.2±0.3, p<0.001), suggesting resident microglial activation as the source of cerebral PET signal. Late signal at 8wk was comparable between clodronate and untreated (p=NS). TSPO inhibition by PK11195 treatment did not affect acute TSPO signal in heart or brain compared to untreated (p=NS). At 8wk, remote myocardial signal was reduced (ID/g, 7.4±1 vs 9.0±2, p=0.040) in parallel with attenuated cardiac dysfunction in PK11195 treated mice (%EF, 49.8±6 vs 37.3±5, p<0.001). Late brain TSPO signal at 8wk was comparable between PK11195 treatment and untreated (p=NS). Consistently, cardiac and brain TSPO signal were proportional (r=0.637, p<0.001), and neuroinflammation was correlated to cardiac function at 8wk after MI (r=−0.345, p=0.005). Conclusions Cardiac TSPO signal reflects acute macrophage activity and chronic mitochondrial dysfunction in heart failure. Neuroinflammation derives from resident microglia, and is proportional to cardiac function at late stages. As such, TSPO PET provides insight into inflammation and mitochondrial dysfunction in progressive heart failure, and may guide novel therapies such as cardioprotection via TSPO inhibition.

TSPO deficiency induces mitochondrial dysfunction, leading to hypoxia, angiogenesis, and a growth-promoting metabolic shift toward glycolysis in glioblastoma.

The ligands of mitochondrial translocator protein (TSPO) have been widely used as diagnostic biomarkers for glioma. However, the true biological actions of TSPO in vivo and its role in glioma tumorigenesis remain elusive. TSPO knockout xenograft and spontaneous mouse glioma models were employed to assess the roles of TSPO in the pathogenesis of glioma. A Seahorse Extracellular Flux Analyzer was used to evaluate mitochondrial oxidative phosphorylation and glycolysis in TSPO knockout and wild-type glioma cells. TSPO deficiency promoted glioma cell proliferation in vitro in mouse GL261 cells and patient-derived stem cell-like GBM1B cells. TSPO knockout increased glioma growth and angiogenesis in intracranial xenografts and a mouse spontaneous glioma model. Loss of TSPO resulted in a greater number of fragmented mitochondria, increased glucose uptake and lactic acid conversion, decreased oxidative phosphorylation, and increased glycolysis. TSPO serves as a key regulator of glioma growth and malignancy by controlling the metabolic balance between mitochondrial oxidative phosphorylation and glycolysis.1. TSPO deficiency promotes glioma growth and angiogenesis.2. TSPO regulates the balance between mitochondrial oxidative phosphorylation and glycolysis.

Microglial Pro-Inflammatory and Anti-Inflammatory Phenotypes Are Modulated by Translocator Protein Activation

A key role of the mitochondrial Translocator Protein 18 KDa (TSPO) in neuroinflammation has been recently proposed. However, little is known about TSPO-activated pathways underlying the modulation of reactive microglia. In the present work, the TSPO activation was explored in an in vitro human primary microglia model (immortalized C20 cells) under inflammatory stimulus. Two different approaches were used with the aim to (i) pharmacologically amplify or (ii) silence, by the lentiviral short hairpin RNA, the TSPO physiological function. In the TSPO pharmacological stimulation model, the synthetic steroidogenic selective ligand XBD-173 attenuated the activation of microglia. Indeed, it reduces and increases the release of pro-inflammatory and anti-inflammatory cytokines, respectively. Such ligand-induced effects were abolished when C20 cells were treated with the steroidogenesis inhibitor aminoglutethimide. This suggests a role for neurosteroids in modulating the interleukin production. The highly steroidogenic ligand XBD-173 attenuated the neuroinflammatory response more effectively than the poorly steroidogenic ones, which suggests that the observed modulation on the cytokine release may be influenced by the levels of produced neurosteroids. In the TSPO silencing model, the reduction of TSPO caused a more inflamed phenotype with respect to scrambled cells. Similarly, during the inflammatory response, the TSPO silencing increased and reduced the release of pro-inflammatory and anti-inflammatory cytokines, respectively. In conclusion, the obtained results are in favor of a homeostatic role for TSPO in the context of dynamic balance between anti-inflammatory and pro-inflammatory mediators in the human microglia-mediated inflammatory response. Interestingly, our preliminary results propose that the TSPO expression could be stimulated by NF-κB during activation of the inflammatory response.

Involvement of the GABAA receptor α subunit in the mode of action of etifoxine

Etifoxine (EFX) is a non-benzodiazepine psychoactive drug which exhibits anxiolytic effects through a dual mechanism, by directly binding to GABAA receptors (GABAARs) and to the mitochondrial 18-kDa translocator protein, resulting in the potentiation of the GABAergic function. The β subunit subtype plays a key role in the EFX-GABAAR interaction, however this does not explain the anxiolytic effects of this drug. Here, we combined behavioral and electrophysiological experiments to challenge the role of the GABAAR α subunit in the EFX mode of action. After single administrations of anxiolytic doses (25–50 mg/kg, intraperitoneal), EFX did not induce any neurological nor locomotor impairments, unlike the benzodiazepine bromazepam (0.5–1 mg/kg, intraperitoneal). We established the EFX pharmacological profile on heteropentameric GABAARs constructed with α1 to α6 subunit expressed in Xenopus oocyte. Unlike what is known for benzodiazepines, neither the γ nor δ subunits influenced EFX-mediated potentiation of GABA-evoked currents. EFX acted first as a partial agonist on α2β3γ2S, α3β3γ2S, α6β3γ2S and α6β3δ GABAARs, but not on α1β3γ2S, α4β3γ2S, α4β3δ nor α5β3γ2S GABAARs. Moreover, EFX exhibited much higher positive allosteric modulation towards α2β3γ2S, α3β3γ2S and α6β3γ2S than for α1β3γ2S, α4β3γ2S and α5β3γ2S GABAARs. At 20 μM, corresponding to brain concentration at anxiolytic doses, EFX increased GABA potency to the highest extent for α3β3γ2S GABAARs. We built a docking model of EFX on α3β3γ2S GABAARs, which is consistent with a binding site located between α and β subunits in the extracellular domain. In conclusion, EFX preferentially potentiates α2β3γ2S and α3β3γ2S GABAARs, which might support its advantageous anxiolytic/sedative balance.

024 The mitochondrial translocator protein single nucleotide polymorphism rs6971 may influence pain and fatigue in rheumatoid arthritis: results of a preliminary study

024 The mitochondrial translocator protein single nucleotide polymorphism rs6971 may influence pain and fatigue in rheumatoid arthritis: results of a preliminary study

The agonistic TSPO ligand XBD173 attenuates the glial response thereby protecting inner retinal neurons in a murine model of retinal ischemia

BackgroundLigand-driven modulation of the mitochondrial translocator protein 18 kDa (TSPO) was recently described to dampen the neuroinflammatory response of microglia in a retinal light damage model resulting in protective effects on photoreceptors. We characterized the effects of the TSPO ligand XBD173 in the postischemic retina focusing on changes in the response pattern of the major glial cell types of the retina—microglia and Müller cells.MethodsRetinal ischemia was induced by increasing the intraocular pressure for 60 min followed by reperfusion of the tissue in mice. On retinal cell types enriched via immunomagnetic separation expression analysis of TSPO, its ligand diazepam-binding inhibitor (DBI) and markers of glial activation were performed at transcript and protein level using RNA sequencing, qRT-PCR, lipid chromatography-mass spectrometry, and immunofluorescent labeling. Data on cell morphology and numbers were assessed in retinal slice and flatmount preparations. The retinal functional integrity was determined by electroretinogram recordings.ResultsWe demonstrate that TSPO is expressed by Müller cells, microglia, vascular cells, retinal pigment epithelium (RPE) of the healthy and postischemic retina, but only at low levels in retinal neurons. While an alleviated neurodegeneration upon XBD173 treatment was found in postischemic retinae as compared to vehicle controls, this neuroprotective effect of XBD173 is mediated putatively by its action on retinal glia. After transient ischemia, TSPO as a marker of activation was upregulated to similar levels in microglia as compared to their counterparts in healthy retinae irrespective of the treatment regimen. However, less microglia were found in XBD173-treated postischemic retinae at 3 days post-surgery (dps) which displayed a more ramified morphology than in retinae of vehicle-treated mice indicating a dampened microglia activation. Müller cells, the major retinal macroglia, show upregulation of the typical gliosis marker GFAP. Importantly, glutamine synthetase was more stably expressed in Müller glia of XBD173-treated postischemic retinae and homeostatic functions such as cellular volume regulation typically diminished in gliotic Müller cells remained functional.ConclusionsIn sum, our data imply that beneficial effects of XBD173 treatment on the postischemic survival of inner retinal neurons were primarily mediated by stabilizing neurosupportive functions of glial cells.

A natural anticancer pigment,Pheophytin a,from a seagrass acts as a high affinity human mitochondrial translocator protein (TSPO) ligand, in silico, to reduce mitochondrial membrane Potential (∆ψmit) in adenocarcinomic A549 cells.

BackgroundThe present investigation looks at the most likely possibilities of usage of a naturally occurring photosynthetic pigment, Pheophytin a, from the seagrass, Syringodium isoetifolium, for plausible use as human TSPO ligand. MethodsPheophytin a isolated in our laboratory previously was administered to A549 cell lines in vitro to examine its effects on cell migrations, DNA, cell cycle, Mitochondrial Membrane Potential and gene expressions. In silico tools were used to predict the nature of the compound and target binding. ResultsPheophytin a hadIC50 values of 22.9 ± 5.8 µM for cancerous A549 cell lines, whilst not targeting non-cancerous vero cells [IC50: 183.6 ± 1.92 µM]. Pheophytin a hindered cellular migration, fragmented DNA, arrested cell cycle precisely at S phase, reduced ∆ψmit and directed mRNA expressions toward apoptosis. In silico tools indicate that the compound binds to TSPO with high effectiveness to collapse ∆ψmit(which is proved using wet lab experiments) to promote mitophagy. ConclusionHence Pheophytin a could be seen as a possible TSPO ligand for targeting metastatic alveolar cancers like A549 via intrinsic apoptotic pathway. General significanceGiven the inherent non-toxic nature of the compound and easy extractability from almost all autotrophic eukaryotes, one could be confident to testing in animal models.

Targeted Molecular Imaging of Translocator Protein (TSPO) Using 18FGE180-PET for the Diagnosis of Gastrointestinal Graft Versus host Disease (GI-GVHD)

Introduction:Acute Gastrointestinal Graft Versus Host Disease (GI-GVHD) is a life-threatening complication following haematopoietic progenitor cell transplantation (HPCT). Recent data suggest that the current gold standard diagnostic test (endoscopic biopsy) fails to identify 26% of those patients requiring treatment. The mitochondrial translocator protein TSPO is upregulated in activated macrophages and inflammatory bowel diseases, and thus may be a biomarker of acute GI-GVHD. 18FGE180 is a novel TSPO-targeting radioligand for use in positron emission tomography imaging (18FGE180-PET).Methods:We performed a prospective observational pilot study evaluating 18FGE180-PET in adults with clinically suspected acute GI-GVHD occurring within 100 days following HPCT. Participants underwent a baseline 18FGE180-PET within 7 days of diagnostic endoscopy, and a follow-up 18FGE180-PET 1-4 weeks later. Tissue avidity was assessed visually in four sections of the gastrointestinal tract (GIT; stomach, duodenum, sigmoid and rectum) and rated by standardised uptake variable (SUV), with “positive” defined by SUV greater than mediastinal blood pool (MBP). Imaging findings were correlated with patient demographics, symptom severity (defined by modified Glucksberg stage), endoscopic findings, and histologic diagnosis. For patients in whom GI-GVHD treatment was initiated (defined as minimum prednisone 1mg/kg or equivalent), the clinical response was correlated with GIT avidity during the second 18FGE180-PET.Results:Six (6) patients were recruited, with median age 57 years (range 53-68 years), at a median 66 days post HPCT (range 26-95 days). Median Glucksberg stage of presumptive GI-GVHD was 2 (range 1-3). The histological diagnosis was GI-GVHD in 4 (66%) patients, and drug toxicity in the remaining two patients. Concordance between histological findings and 18FGE180-PET avidity by GIT section were 50% (stomach), 100% (duodenum), 100% (sigmoid) and 80% (rectum). 18FGE180-PET was highly accurate in duodenum and sigmoid (sensitivity 100%, specificity 100%), moderately accurate in rectum (sensitivity 66%, specificity 100%), and non-specific in stomach (sensitivity 100%, specificity 25%). Five (83%) patients subsequently underwent treatment for GI-GVHD, and of these 4 (80%) underwent repeat 18FGE180-PET for response assessment, seven days after the first. In these patients, high concordance between clinical response and 18FGE180-PET avidity was demonstrated in sigmoid (100%), rectum (100%) and duodenum (75%), compared with poor concordance in stomach (25%). The final patient, whose biopsies were negative for GI-GVHD and did not undergo GVHD treatment, was non-avid at all sites on both initial and subsequent 18FGE180-PET.Conclusion:18FGE180-PET appears to show utility in the diagnosis of GI-GVHD in small and large bowel. Larger prospective studies, correlating with histological TSPO expression, are warranted. DisclosuresNo relevant conflicts of interest to declare.