TSPO Ligands Research Articles

125 I]IodoDPA-713 Binding to 18 kDa Translocator Protein (TSPO) in a Mouse Model of Intracerebral Hemorrhage: Implications for Neuroimaging.

Intracerebral hemorrhage (ICH) is a fatal stroke subtype with significant public health impact. Although neuroinflammation is a leading cause of neurological deficits after ICH, no imaging tool is currently available to monitor brain inflammation in ICH patients. Given the role of TSPO in neuroinflammation, herein we investigate whether a second-generation TSPO ligand, [125 I]IodoDPA-713 can be used to monitor the changes in TSPO expression in a preclinical model of intracerebral hemorrhage. Male CD1 mice were subjected to ICH/Sham. The brain sections, collected at different time points were incubated with [125 I]IodoDPA-713 and the brain uptake of [125 I]IodoDPA-713 was estimated using autoradiography. The specificity of [125 I]IodoDPA-713 binding was confirmed by a competitive displacement study with an unlabeled TSPO ligand, PK11195. [125 I]IodoDPA-713 binding was higher in the ipsilateral striatum with an enhanced binding observed in the peri-hematomal brain region after ICH, whereas the brain sections from sham as well as contralateral brain areas of ICH exhibited marginal binding of [125 I]IodoDPA-713. PK11195 completely reversed the [125 I] IodoDPA-713 binding to brain sections suggesting a specific TSPO-dependent binding of [125 I]IodoDPA-713 after ICH. This was further confirmed with immunohistochemistry analysis of adjacent sections, which revealed a remarkable expression of TSPO in the areas of high [125 I]IodoDPA-713 binding after ICH. The specific as well as enhanced binding of [125 I]IodoDPA-713 to the ipsilateral brain areas after ICH as assessed by autoradiography analysis provides a strong rationale for testing the applicability of [125 I]IodoDPA-713 for non-invasive neuroimaging in preclinical models of ICH.

The translocator protein ligand XBD173 improves clinical symptoms and neuropathological markers in the SJL/J mouse model of multiple sclerosis

Multiple sclerosis (MS) is a severe autoimmune disease characterized by inflammatory, demyelinating and neurodegenerative components causing motor, sensory, visual and/or cognitive symptoms. The relapsing-remitting MS affecting 85% of patients is reliably mimicked by the proteolipid-protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE) SJL/J-mouse model. Significant progress was made for MS treatment but the development of effective therapies devoid of severe side-effects remains a great challenge. Here, we combine clinical, behavioral, histopathological, biochemical and molecular approaches to demonstrate that low and well tolerated doses (10–20mg/kg) of TSPO ligand XBD173 (Emapunil) efficiently ameliorate clinical signs and neuropathology of PLP-EAE mice. In addition to the conventional clinical scoring of symptoms, we applied the robust behavioral Catwalk-method to confirm that XBD173 (10mg/kg) increases the maximum contact area parameter at EAE-disease peak, indicating an improvement/recovery of motor functions. Consistently, histopathological studies coupled with microscope-cellSens quantification and RT-qPCR analyzes showed that XBD173 prevented demyelination by restoring normal protein and mRNA levels of myelin basic protein that was significantly repressed in PLP-EAE mice spinal cord and brain. Interestingly, ELISA-based measurement revealed that XBD173 increased allopregnanolone concentrations in PLP-EAE mice spinal and brain tissues. Furthermore, flow cytometry assessment demonstrated that XBD173 therapy decreased serum level of pro-inflammatory cytokines, including interleukin-17A, Interleukin-6 and tumor-necrosis-factor alpha in PLP-EAE mice. As the optimal XBD173 dosing exerting the maximal beneficial action in EAE mice is the lower 10mg/kg dose, the paper opens interesting perspectives for the development of efficient and safe therapies against MS with slight or no side-effects.

The Low-Affinity Binding of Second Generation Radiotracers Targeting TSPO is Associated with a Unique Allosteric Binding Site.

[11C]-PK11195 (PK11195) has been widely used with positron emission tomography (PET) to assess levels of the translocator protein 18kDa (TSPO) as a marker of neuroinflammation. Recent ligands, such as [11C]-PBR28 and [11C]-DPA713, have improved signal-to-noise ratio and specificity for TSPO over PK11195. However, these second generation radiotracers exhibit binding differences due to a single polymorphism (rs6971) that leads to three genotypes: C/C, C/T and T/T associated with high, mixed and low binding affinities, respectively. Here we report that [3H]-DPA-713 in the presence of cholesterol or PK11195 has an accelerated dissociation rate from TSPO in platelets isolated from individuals with the T/T genotype. This allosteric interaction was not observed in platelets isolated from individuals with the C/C or C/T genotype. The results provide a molecular rationale for low binding affinity of T/T TSPO and further support the exclusion of these subjects from PET imaging studies using second generation TSPO ligands.

A TSPO ligand prevents mitochondrial sterol accumulation and dysfunction during myocardial ischemia-reperfusion in hypercholesterolemic rats

A major cause of cell death during myocardial ischemia-reperfusion is mitochondrial dysfunction. We previously showed that the reperfusion of an ischemic myocardium was associated with an accumulation of cholesterol into mitochondria and a concomitant strong generation of auto-oxidized oxysterols. The inhibition of mitochondrial accumulation of cholesterol abolished the formation of oxysterols and prevented mitochondrial injury at reperfusion. The aim of this study was to investigate the impact of hypercholesterolemia on sterol and oxysterol accumulation in rat cardiac cytosols and mitochondria and to analyse the effect of the translocator protein ligand 4′-chlorodiazepam on this accumulation and mitochondrial function. Hypercholesterolemic ZDF fa/fa rats or normocholesterolemic lean rats were submitted to 30min of coronary artery occlusion followed by 15min reperfusion where cardiac cytosols and mitochondria were isolated. Hypercholesterolemia increased the cellular cardiac concentrations of cholesterol, cholesterol precursors and oxysterols both in cytosol and mitochondria in non-ischemic conditions. It also amplified the accumulation of all these compounds in cardiac cells and the alteration of mitochondrial function with ischemia-reperfusion. Administration of 4′-chlorodiazepam to ZDF fa/fa rats had no effect on the enhancement of sterols and oxysterols observed in the cytosols but inhibited cholesterol transfer to the mitochondria. It also alleviated the mitochondrial accumulation of all the investigated sterols and oxysterols. This was associated with a restoration of oxidative phosphorylation and a prevention of mitochondrial transition pore opening.The inhibition of cholesterol accumulation with TSPO ligands represents an interesting strategy to protect the mitochondria during ischemia-reperfusion in hypercholesterolemic conditions.

Modified benzoxazolone derivative as 18-kDa TSPO ligand.

We have synthesized six new congeners of acetamidobenzoxazolone for Translocator Protein [18 kDa, TSPO] imaging. The best in vitro binding affinity (10.8±1.2nm) for TSPO was found for N-methyl-2-(5-(naphthalen-1-yl)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-phenylacetamide, (NBMP). NBMP was synthesised by Suzuki coupling reaction between 2-(5-bromo-2-oxo-1,3-benzoxazol-3(2H)-yl)-N-phenylacetamide and napthalene-1-boronic acid. Computational docking and simulation studies showed not much impact of intersubject variability on binding which is one of the major drawbacks of several TSPO ligands. These findings suggested that NBMP may become a promising marker for visualization of neuroinflammation via TSPO targeting.

GQSAR modeling and combinatorial library generation of 4-phenylquinazoline-2-carboxamide derivatives as antiproliferative agents in human Glioblastoma tumors

BackgroundTSPO translocator protein, encoded in humans by the Tspo gene plays a crucial role in mitochondria mediated apoptosis and necrotic cell death through its association with Mitochondrial Permeability Transition pore (MPTP). It has been shown that this function can be exploited as a potential treatment for human Glioblastoma Multiforme. In this study, a novel robust fragment based QSAR model has been developed for a series of 4-phenylquinazoline-2-carboxamides experimentally known to be ligands for TSPO, thus triggering apoptotic mechanism cascade. ResultsModel developed showed satisfactory statistical parameters for the experimentally reported dataset (r2=0.8259, q2=0.6788, pred_r2=0.8237 and F-test=37.9). Low standard error values (r2_se=0.253, q2_se=0.34, pred_r2_se=0.14) confirmed the accuracy of the generated model. The model obtained had 4 descriptors, namely, R1-Volume, R2-SsCH3E-index, R3-SsCH3count and R5-EpsilonR. Two of them had positive contribution while the other two had negative correlation. ConclusionThe high binding affinity and the presence of essential structural features in these compounds make them an ideal choice for the consideration as potent anti-GBM drugs. Activity predicted by GQSAR model reinforces their potential as worthy candidates for drugs against GBM. The detailed analysis carried out in this study provides a substantial basis for the prospective design and development of novel 4-phenylquinazoline-2-carboxamide compounds as TSPO ligands capable of inducing apoptosis in cancer cells.

Translational evaluation of translocator protein as a marker of neuroinflammation in schizophrenia.

Positron emission tomography (PET) imaging with radiotracers that target translocator protein 18 kDa (TSPO) has become a popular approach to assess putative neuroinflammatory processes and associated microglia activation in psychotic illnesses. It remains unclear, however, whether TSPO imaging can accurately capture low-grade inflammatory processes such as those present in schizophrenia and related disorders. Therefore, we evaluated the validity of TSPO as a disease-relevant marker of inflammation using a translational approach, which combined neurodevelopmental and neurodegenerative mouse models with PET imaging in patients with recent-onset schizophrenia and matched controls. Using an infection-mediated neurodevelopmental mouse model, we show that schizophrenia-relevant behavioral abnormalities and increased inflammatory cytokine expression are associated with reduced prefrontal TSPO levels. On the other hand, TSPO was markedly upregulated in a mouse model of acute neurodegeneration and reactive gliosis, which was induced by intrahippocampal injection of kainic acid. In both models, the changes in TSPO levels were not restricted to microglia but emerged in various cell types, including microglia, astrocytes and vascular endothelial cells. Human PET imaging using the second-generation TSPO radiotracer [11C]DPA-713 revealed a strong trend towards reduced TSPO binding in the middle frontal gyrus of patients with recent-onset schizophrenia, who were previously shown to display increased levels of inflammatory cytokines in peripheral and central tissues. Together, our findings challenge the common assumption that central low-grade inflammation in schizophrenia is mirrored by increased TSPO expression or ligand binding. Our study further underscores the need to interpret altered TSPO binding in schizophrenia with caution, especially when measures of TSPO are not complemented with other markers of inflammation. Unless more selective microglial markers are available for PET imaging, quantification of cytokines and other inflammatory biomarkers, along with their molecular signaling pathways, may be more accurate in attempts to characterize inflammatory profiles in schizophrenia and other mental disorders that lack robust reactive gliosis.

4′-Chlorodiazepam modulates the development of primary hippocampal neurons in a sex-dependent manner

The translocator protein 18kDa (TSPO) is located in the outer mitochondrial membrane and is involved in the cholesterol transport into the mitochondria and in the regulation of steroidogenesis and other mitochondrial functions. It is known that steroid hormones, such as estradiol, testosterone and dihydrotestosterone are neuroprotective and regulate neuritogenesis in the CNS by different mechanisms. However, the developmental effects of TSPO ligands in the CNS are not known. Therefore, the aim of this study was to identify the developmental effects of 4′-chlorodiazepam (4′-CD), a TSPO ligand, in primary cultures of male and female mouse hippocampal neurons. We observed that female neurons showed an advanced neuritogenesis compared to male neurons after 2days in vitro. Moreover, it was shown that 4′-CD administration accelerated the maturation of male hippocampal neurons, without changing the development of female neurons. These findings, showing that 4′-CD modulates the development of hippocampal neurons in a sex-dependent manner, suggest that TSPO may be involved in the regulation of neuritogenesis.

Antidepressant-like effects of YL-IPA08, a potent ligand for the translocator protein (18 kDa) in chronically stressed rats

The present study aimed to examine the molecular and cellular mechanisms underlying the antidepressant-like effect of YL-IPA08, a novel TSPO ligand designed and synthesized at our institute. We firstly used the chronic unpredictable stress (CUS) procedure of rats, a well validated stress-related animal model of depression, to further determine the antidepressant-like of YL-IPA08. And we found that YL-IPA08 caused significant suppression of inhibiting of locomotor activity, reducing the sucrose preference and increasing the latency to eat induced by CUS. In addition, YL-IPA08 treatment increased the levels of progesterone and allopregnanolone in the hippocampus and prefrontal cortex of post- CUS rats. Furthermore, long-term YL-IPA08 administration reversed dendritic shrinkage, down-regulation of neurotrophic signaling pathway within the hippocampus, as well as HPA dysfunctions simultaneously observed in the CUS rats. Collectively, the evidence presented above supports the notion that binding to TSPO and the subsequent synthesis of neurosteroid, maintenance of hippocampal morphologic and functional plasticity, and preventing HPA axis dysfunction, may account for the profound molecular and cellular mechanism underlying the antidepressant-like effect of YL-IPA08.

Systematic Analysis of Translocator Protein 18 kDa (TSPO) Ligands on Toll-like Receptors-mediated Pro-inflammatory Responses in Microglia and Astrocytes.

Translocator protein 18 kDa (TSPO) is a mitochondrial protein highly expressed on reactive microglia and astrocytes, and is considered as a biomarker for neurodegeneration and brain damage, especially neuroinflammation. Toll-like receptors (TLRs) are closely related with inflammatory responses of microglia and astrocytes and these signaling pathways regulate neuroinflammation. Previous reports have identified the anti-inflammatory effects of TSPO ligands, however study of their effects in relation to the TLR signaling was limited. Here, we investigated the effects of five representative TSPO ligands on microglia and astrocytes following activation by various TLR ligands. Our results show that TSPO ligands reduce the pro-inflammatory response elicited by the TLR ligands with more profound effects on microglia than astrocytes.

Synthesis and in vitro characterization of novel fluorinated derivatives of the translocator protein 18 kDa ligand CfO-DPA-714

The translocator protein 18 kDa (TSPO) is today a validated target for a number of therapeutic applications, but also a well-recognized diagnostic/imaging biomarker for the evaluation of inflammatory related-disease state and progression, prompting the development of specific and dedicated TSPO ligands worldwide. For this purpose, pyrazolo[1,5-a]pyrimidine acetamides constitute a unique class of high affinity and selectivity TSPO ligands; it includes DPA-714, a fluorine-containing derivative that has also been labelled with the positron-emitter fluorine-18, and is nowadays widely used as a Positron Emission Tomography imaging probe. Recently, to prevent defluorination issues encountered in vivo with this tracer, a first series of analogues was reported where the oxygen atom bridging the phenyl ring of the core structure and the fluorinated moiety was replaced with a more robust linkage. Among this new series, CfO-DPA-714 was discovered as a highly promising TSPO ligand. Herein, a novel series of fluorinated analogues of the latter molecule were synthesized and in vitro characterized, where the pharmacomodulation at the amide position of the molecule was explored. Thirteen compounds were thus prepared from a common key-ester intermediate (synthesized in 7 steps from 4-iodobenzoate – 11% overall yield) and a set of commercially available amines and obtained with moderate to good yields (23–81%) and high purities (>95%). With one exception, all derivatives displayed nanomolar to subnanomolar affinity for the TSPO and also high selectivity versus the CBR (Ki (CBR)/Ki (TSPO) > 103). Within this series, three compounds showed better Ki values (0.25, 0.26 and 0.30 nM) than that of DPA-714 (0.91 nM) and CfO-DPA-714 (0.37 nM), and favorable lipophilicity for brain penetration (3.6 < logD7.4 < 4.4). Among these three compounds, the N-methyl-N-propyl amide analogue (9) exhibited similar metabolic stability when compared to CfO-DPA-714 in mouse, rat and human microsomes. Therefore, the latter compound stands out as a promising candidate for drug development or for use as a PET probe, once fluorine-18-labelled, for in vivo neuroinflammation imaging.

Regulation of Translocator Protein 18 kDa (TSPO) Expression in Rat and Human Male Germ Cells.

Translocator protein 18 kDa (TSPO) is a high affinity cholesterol- and drug-binding protein highly expressed in steroidogenic cells, such as Leydig cells, where it plays a role in cholesterol mitochondrial transport. We have previously shown that TSPO is expressed in postnatal day 3 rat gonocytes, precursors of spermatogonial stem cells. Gonocytes undergo regulated phases of proliferation and migration, followed by retinoic acid (RA)-induced differentiation. Understanding these processes is important since their disruption may lead to the formation of carcinoma in situ, a precursor of testicular germ cell tumors (TGCTs). Previously, we showed that TSPO ligands do not regulate gonocyte proliferation. In the present study, we found that TSPO expression is downregulated in differentiating gonocytes. Similarly, in F9 embryonal carcinoma cells, a mouse TGCT cell line with embryonic stem cell properties, there is a significant decrease in TSPO expression during RA-induced differentiation. Silencing TSPO expression in gonocytes increased the stimulatory effect of RA on the expression of the differentiation marker Stra8, suggesting that TSPO exerts a repressive role on differentiation. Furthermore, in normal human testes, TSPO was located not only in Leydig cells, but also in discrete spermatogenic phases such as the forming acrosome of round spermatids. By contrast, seminomas, the most common type of TGCT, presented high levels of TSPO mRNA. TSPO protein was expressed in the cytoplasmic compartment of seminoma cells, identified by their nuclear expression of the transcription factors OCT4 and AP2G. Thus, TSPO appears to be tightly regulated during germ cell differentiation, and to be deregulated in seminomas, suggesting a role in germ cell development and pathology.

TSPO activation modulates the effects of high pressure in a rat ex vivo glaucoma model

We previously reported that elevated pressure induces axonal swelling and facilitates the synthesis of the neurosteroid, allopregnanolone (AlloP), in the ex vivo rat retina. Exogenously applied AlloP attenuates the axonal swelling, suggesting that the neurosteroid plays a neuroprotective role against glaucomatous pressure-induced injuries, although mechanisms underlying neurosteroidogenesis have not been clarified. The aim of this study was to determine whether AlloP synthesis involves activation of translocator protein 18 kD (TSPO) and whether TSPO modulates pressure-induced retinal injury. Ex vivo rat retinas were exposed to various pressures (10, 35, or 75 mmHg) for 24 h. Expression of TSPO, 5α-reductase (5aRD), and AlloP was examined by quantitative real-time RT-PCR, ELISA, immunohistochemistry, and LC-MS/MS. We also examined the effects of TSPO ligands on AlloP synthesis and retinal damage. In this acute model, quantitative real-time RT-PCR and ELISA analyses revealed that elevated pressure facilitated TSPO expression. Similarly, these methods also detected enhanced 5aRD (mostly type II), which was observed in retinal ganglion cells (RGC) and the inner nuclear layer (INL). Atriol, a TSPO antagonist, suppressed pressure mediated AlloP synthesis and induced more severe histological changes in the inner retina when combined with elevated pressure. PK11195, a TSPO ligand that facilitates AlloP synthesis by itself, remarkably diminished pressure-mediated retinal degeneration. These results suggest that AlloP synthesis is induced by sequential activation of TSPO and 5aRD in an ex vivo glaucoma model, and that TSPO agonists may serve as potential therapeutic agents for the prevention of pressure-induced retinal damage.

A novel translocator protein 18kDa ligand, ZBD-2, exerts neuroprotective effects against acute spinal cord injury.

Traumatic spinal cord injury (SCI) happens accidently and often leads to motor dysfunction due to a series of biochemical and pathological events and damage, either temporarily or permanently. Translocator protein 18 (TSPO) has been found to be involved in the synthesis of endogenous neurosteroids which have multiple effects on neurons, but the internal mechanisms are not clear. N-benzyl-N-ethyl-2-(7,8-oxo-2-phenyl-9H-purin-9-yl) acetamide (ZBD-2), a newly reported ligand of TSPO, shows some neuroprotective effect against focal cerebral ischemia in vivo and NMDA-induced neurotoxicity in vitro. The present study aims to examine the role of ZBD-2 in SCI mice and elucidate the underlying molecular mechanisms. The SCI model was established by crushing spinal cord. ZBD-2 (10mg/kg) significantly enhanced the hindlimb locomotor functions after SCI and decreased the tissue damage and conserved the white matter of the spinal cord. High-dose ZBD-2 alleviated the oxidative stress induced by SCI and regulated the imbalance between NR2B-containing NMDA and GABA receptors by increasing the levels of GAD67 in the spinal cord of SCI mice. Additionally, ZBD-2 (10mg/kg) increased phosphorylated Akt (p-Akt) and decreased the ratio of Bax/Bcl-2. These results demonstrate that ZBD-2 performs neuroprotection against SCI through regulating the synaptic transmission and the PI3K/AKT signaling pathway.

DISCOVERY OF NOVEL TSPO LIGANDS AS MODULATORS OF MITOCHONDRIAL FUNCTION: A POTENTIAL TREATMENT FOR ALZHEIMER’S DISEASE

DISCOVERY OF NOVEL TSPO LIGANDS AS MODULATORS OF MITOCHONDRIAL FUNCTION: A POTENTIAL TREATMENT FOR ALZHEIMER’S DISEASE

Dependence of anxiolytic effects of the dipeptide TSPO ligand GD-23 on neurosteroid biosynthesis.

The elevated plus maze test showed that GD-23 (N-carbobenzoxy-L-tryptophanyl-L-isoleucine amide), an original dipeptide ligand of TSPO, exerted anxiolytic effect when injected intraperitoneally at a dose of 0.5 mg/kg. This effect was completely blocked by the selective neurosteroid synthesis inhibitors, enzymes trilostane and finasteride. The same inhibitors do not prevent the anxiolytic effects of the benzodiazepine tranquillizer diazepam. The results of the study indicate the selective neurosteroidogenic mechanism of the anxiolytic action of GD-23.

Translocator Protein 18 kDa (TSPO): An Old Protein with New Functions?

Translocator protein 18 kDa (TSPO) was previously known as the peripheral benzodiazepine receptor (PBR) in eukaryotes, where it is mainly localized to the mitochondrial outer membrane. Considerable evidence indicates that it plays regulatory roles in steroidogenesis and apoptosis and is involved in various human diseases, such as metastatic cancer, Alzheimer's and Parkinson's disease, inflammation, and anxiety disorders. Ligands of TSPO are widely used as diagnostic tools and treatment options, despite there being no clear understanding of the function of TSPO. An ortholog in the photosynthetic bacterium Rhodobacter was independently discovered as the tryptophan-rich sensory protein (TspO) and found to play a role in the response to changes in oxygen and light conditions that regulate photosynthesis and respiration. As part of this highly conserved protein family found in all three kingdoms, the rat TSPO is able to rescue the knockout phenotype in Rhodobacter, indicating functional as well as structural conservation. Recently, a major breakthrough in the field was achieved: the determination of atomic-resolution structures of TSPO from different species by several independent groups. This now allows us to reexamine the function of TSPO with a molecular perspective. In this review, we focus on recently determined structures of TSPO and their implications for potential functions of this ubiquitous multifaceted protein. We suggest that TSPO is an ancient bacterial receptor/stress sensor that has developed additional interactions, partners, and roles in its mitochondrial outer membrane environment in eukaryotes.

A translocator protein 18 kDa ligand, Ro5-4864, inhibits ATP-induced NLRP3 inflammasome activation

Ro5-4864 and PK11195, prototypical synthetic ligands of translocator protein 18 kDa (TSPO), have shown anti-inflammatory effects in several models of inflammatory diseases; however, their biochemical mechanisms remain poorly understood. Nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation as a part of the innate immune system, has been implicated in a variety of inflammatory diseases. Here, we demonstrate for the first time that TSPO ligands, especially Ro5-4864, potently suppressed ATP-induced NLRP3 inflammasome activation in THP-1 and BMDM cells. Detailed action mechanism was further investigated in THP-1 cells. Ro5-4864 efficiently attenuated NLRP3 translocation to mitochondria, inflammasome assembly/oligomerization, activation of caspase-1, and subsequent secretion of the mature forms of interleukin-1β and −18. Ro5-4864 also reduced the production of mitochondrial superoxide and preserved the mitochondrial membrane potential in ATP-treated cells, suggesting that Ro5-4864 may act on mitochondria or more upstream targets in NLRP3 inflammasome signaling. We also observed the distinct effects of the TSPO ligands between THP-1 monocytes and macrophages, which suggested different NLRP3 inflammasome signaling depending on cell type. Collectively, our novel findings demonstrate that Ro5-4864 effectively inhibited ATP-induced NLRP3 inflammasome activation through the prevention of mitochondrial perturbation. Our results indicate Ro5-4864 as a promising candidate for the treatment of NLRP3 inflammasome-related diseases.

TSPO-ligands prevent oxidative damage and inflammatory response in C6 glioma cells by neurosteroid synthesis

Translocator protein 18kDa (TSPO) is predominantly located in the mitochondrial outer membrane, playing an important role in steroidogenesis, inflammation, cell survival and proliferation. Its expression in central nervous system, mainly in glial cells, has been found to be upregulated in neuropathology, and brain injury.In this study, we investigated the anti-oxidative and anti-inflammatory effects of a group of TSPO ligands from the N,N-dialkyl-2-phenylindol-3-ylglyoxylamide class (PIGAs), highlighting the involvement of neurosteroids in their pharmacological effects. To this aim we used a well-known in vitro model of neurosteroidogenesis: the astrocytic C6 glioma cell line, where TSPO expression and localization, as well as cell response to TSPO ligand treatment, have been established. All PIGAs reduced l-buthionine-(S,R)-sulfoximine (BSO)-driven cell cytotoxicity and lipid peroxidation. Moreover, an anti-inflammatory effect was observed due to the reduction of inducible nitric oxide synthase and cyclooxygenase-2 induction in LPS/IFNγ challenged cells. Both effects were blunted by aminoglutethimide (AMG), an inhibitor of pregnenolone synthesis, suggesting neurosteroids' involvement in PIGA protective mechanism. Finally, pregnenolone evaluation in PIGA exposed cells revealed an increase in its synthesis, which was prevented by AMG pre-treatment.These findings indicate that these TSPO ligands reduce oxidative stress and pro-inflammatory enzymes in glial cells through the de novo synthesis of neurosteroids, suggesting that these compounds could be potential new therapeutic tools for the treatment of inflammatory-based neuropathologies with beneficial effects possibly comparable to steroids, but potentially avoiding the negative side effects of long-term therapies with steroid hormones.

TSPO ligand PK11195 alleviates neuroinflammation and beta-amyloid generation induced by systemic LPS administration

Translocator protein 18 kDa (TSPO) is now an attractive drug target for controlling neuroinflammation. Studies applying TSPO ligands to neurodegenerative diseases, especially Alzheimer's disease (AD), were rare. Our study was aimed to evaluate the effect of PK11195, a specific TSPO ligand, in an animal model of neuroinflammation caused by systemic LPS administration. C57/BL6 mice were treated with lipopolysaccharide (LPS, 500 μg/kg, i.p.) three days after PK11195 administration (3mg/kg, i.p.). The drugs were not discontinued until the mice were sacrificed. Cognitive function was assessed by Morris water maze (MWM) seven days after LPS injection. Chronic LPS-injection in mice was characterized by cognitive dysfunction, increased expression of cyclooxygenase (COX)-2 and TSPO, elevated Aβ content with increased expression of β-site APP cleaving enzyme-1 (BACE-1) and insulin-degrading enzyme (IDE) as well as decreased brain progesterone and brain-derived neurophic factor (BDNF) level. PK11195 pretreatment protected cognitive function in LPS-injected animals and normalized the inflammatory proteins. Moreover, PK11195 pre-administration decreased elevated hippocampal Aβx-42 levels and increased brain levels of progesterone, allopregnanolone. However, LPS-induced BDNF decrease was not reversed by PK11195 administration. Our data demonstrated that PK11195 could protect cognitive deficits induced by chronic LPS administration. The underling mechanism may involve alleviated neuroinflammation, increased synthesis of neurosteroid and decreased Aβ accumulation accompanied by down-regulation of BACE-1.