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Stroke is the leading cause of death and disability worldwide. Novel and effective therapies for ischemic stroke are urgently needed. Here, we report that melatonin receptor 1A (MT1) agonist ramelteon is a neuroprotective drug candidate as demonstrated by comprehensive experimental models of ischemic stroke, including a middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia in vivo, organotypic hippocampal slice cultures ex vivo, and cultured neurons in vitro; the neuroprotective effects of ramelteon are diminished in MT1-knockout (KO) mice and MT1-KO cultured neurons. For the first time, we report that the MT1 receptor is significantly depleted in the brain of MCAO mice, and ramelteon treatment significantly recovers the brain MT1 losses in MCAO mice, which is further explained by the Connectivity Map L1000 bioinformatic analysis that shows gene-expression signatures of MCAO mice are negatively connected to melatonin receptor agonist like Ramelteon. We demonstrate that ramelteon improves the cerebral blood flow signals in ischemic stroke that is potentially mediated, at least, partly by mechanisms of activating endothelial nitric oxide synthase. Our results also show that the neuroprotection of ramelteon counteracts reactive oxygen species-induced oxidative stress and activates the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 pathway. Ramelteon inhibits the mitochondrial and autophagic death pathways in MCAO mice and cultured neurons, consistent with gene set enrichment analysis from a bioinformatics perspective angle. Our data suggest that Ramelteon is a potential neuroprotective drug candidate, and MT1 is the neuroprotective target for ischemic stroke, which provides new insights into stroke therapy. MT1-KO mice and cultured neurons may provide animal and cellular models of accelerated ischemic damage and neuronal cell death., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Polycystic ovary syndrome (PCOS) is an endocrine disorder seen in women of reproductive age and has been gradually increasing over the years. The mechanism of the syndrome has still not been clearly understood. In this study, the possible effects of exogenously administrated melatonin on melatonin (MT1) receptor, Growth Differentiation Factor-9 (GDF9), and Bone Morphogenetic Protein-15 (BMP15) in experimental PCOS were investigated. Thirty-two 6-8-week-old Sprague-Dawley rats were randomly divided into four groups (n = 8 in each) as Sham control (Group 1), Melatonin (Group 2), PCOS (Group 3), and PCOS + Melatonin (Group 4) groups. At the end of the 21st day, the experiment was terminated, the ovary tissues were taken, and Hematoxylin-Eosin staining, MT1, GDF9, BMP15 immunohistochemical labeling, western blot, and quantitative real-time polymerase chain reaction (qPCR) analyses were performed. Serum Luteinizing Hormone (LH)/Follicle Stimulating Hormone (FSH) levels and colpo-cytological examinations were also carried out. The results revealed that melatonin administration increased the expression levels of the MT1 receptor, GDF9, and BMP15 in PCOS at protein and mRNA levels. It was determined that melatonin administration reduced the microscopic symptoms of PCOS. Melatonin was found to be effective via the MT1 receptor in the pathogenesis of PCOS, and it suppressed the transport pathways of GDF9 to granulosa cells in antral follicles., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

This study evaluated the protective effect of melatonin before cyclophosphamide administration on ovarian function and its potential mechanism in a mouse model. Two studies were performed. In the first, mice were pretreated with melatonin (10, 20, or 30 mg/kg body weight, i.p.) once daily for 3 days, followed by injection with a single dose of cyclophosphamide (200 mg/kg body weight, i.p.) 30 min after the last melatonin injection. The second study analyzed whether melatonin type 1 and/or 2 receptors mediate the effects of melatonin on the ovary through administration of non-selective MT 1 /MT 2 antagonist (luzindole) or selective MT 2 antagonist (4-PPDOT) before the treatment with melatonin plus cyclophosphamide. After treatment groups, the ovaries were harvested and destined to histology, immunohistochemistry, and fluorescence analyses. Lastly, we examined the p-PTEN, p-Akt, and p-FOXO3a participation in the protective effect of melatonin in cyclophosphamide-induced ovarian damage. Results demonstrated that pretreatment with 20 mg/kg melatonin before cyclophosphamide administration showed more morphologically normal follicles, attenuated primordial follicle loss, decreased growing follicle atresia and mitochondrial damage, and increased GSH concentrations. Furthermore, treatment with luzindole blocked the protective effects of melatonin against the damage caused by cyclophosphamide. Additionally, pretreatment with 20 mg/kg melatonin regulated the PTEN/Akt/FOXO3a signaling pathway components after cyclophosphamide treatment. In conclusion, pretreatment with 20 mg/kg melatonin prevented primordial follicle loss and reduced apoptosis and oxidative damage in the mouse ovary during experimental chemotherapy with cyclophosphamide. Furthermore, the MT 1 receptor and PTEN/Akt/FOXO3a proteins mediated these cytoprotective effects., (© 2021. Society for Reproductive Investigation.)

Advanced prostate cancer often develops into bone metastasis, which is characterized by aberrant bone formation with chronic pain and lower chances of survival. No treatment exists as yet for osteoblastic bone metastasis in prostate cancer. The indolamine melatonin (N-acetyl-5-methoxytryptamine) is a major regulator of the circadian rhythm. Melatonin has shown antiproliferative and antimetastatic activities but has not yet been shown to be active in osteoblastic bone lesions of prostate cancer. Our study investigations reveal that melatonin concentration-dependently decreases the migratory and invasive abilities of two osteoblastic prostate cancer cell lines by inhibiting FAK, c-Src, and NF-κB transcriptional activity via the melatonin MT 1 receptor, which effectively inhibits integrin α 2 β 1 expression. Melatonin therapy appears to offer therapeutic possibilities for reducing osteoblastic bone lesions in prostate cancer., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

A way to study G protein-coupled receptors in a minimal system is to reconstruct artificial membrane mimics, made of detergent and/or of lipids in which the purified receptor is maintained. In particular, it is now possible to generate lipid nanoparticles, such as nanodiscs, in which a single receptor molecule is included. Such objects offer the invaluable potential of studying an isolated receptor stabilized in a finely controlled membrane-like environment to evaluate its pharmacology, its function, and its structure at the molecular level. In this chapter, we detail the different steps from the extraction and isolation of a recombinant MT1 melatonin receptor in detergent, down to its reconstitution into nanodiscs. A G protein activation test is further described in order to exemplify how the functionality of such particles may be investigated., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Prostate cancer has high metastatic potential. Men with higher urinary levels of the sleep hormone melatonin are much less likely to develop advanced prostate cancer compared with men with lower levels of melatonin. Melatonin has shown anticancer activity in experimental investigations. Nevertheless, the therapeutic effect of melatonin in metastatic prostate cancer has largely remained a mystery. Analyses of Gene Expression Omnibus data and human tissue samples indicated that levels of matrix metallopeptidase 13 (MMP-13) expression are higher in prostate cancer patients than in healthy cancer-free individuals. Mechanistic investigations revealed that melatonin inhibits MMP-13 expression and the migratory and invasive capacities of prostate cancer cells via the MT 1 receptor and the phospholipase C, p38, and c-Jun signaling cascades. Importantly, tumor growth rate and metastasis to distant organs were suppressed by melatonin in an orthotopic prostate cancer model. This is the first demonstration showing that melatonin impedes metastasis of prostate cancer by suppressing MMP-13 expression in both in vitro and in vivo models. Thus, melatonin is promising in the management of prostate cancer metastasis and deserves to undergo clinical investigations., (© 2020 Wiley Periodicals LLC.)

Melatonin, a neurohormone that binds to two G protein-coupled receptors MT 1 and MT 2, is involved in pain regulation, but the distinct role of each receptor has yet to be defined. We characterized the nociceptive responses of mice with genetic inactivation of melatonin MT 1 (MT 1 -/- ), or MT 2 (MT 2 -/- ), or both MT 1 /MT 2 (MT 1 -/- /MT 2 -/- ) receptors in the hot plate test (HPT), and the formalin test (FT). In HPT and FT, MT 1 -/- display no differences compared to their wild-type littermates (CTL), whereas both MT 2 -/- and MT 1 -/- mice. Our results show that the genetic inactivation of melatonin MT 2 -/- receptors, produces a distinct effect on nociceptive threshold, suggesting that the melatonin MT 2 partial agonist UCM924 induced an antinociceptive effect in MT 1 -/- but not in MT 2 -/- and MT 1 -/- /MT 2 -/- mice. Also, the competitive opioid antagonist naloxone had no effects in CTL, whereas it induced a decrease of nociceptive thresholds in MT 2 -/- mice. Our results show that the genetic inactivation of melatonin MT 2 , but not MT 1 receptors, produces a distinct effect on nociceptive threshold, suggesting that the melatonin MT 2 receptor subtype is selectively involved in the regulation of pain responses., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The pharmacological potential of targeting selectively melatonin MT1 or MT2 receptors has not yet been exploited in medicine. Research using selective MT1/MT2 receptor ligands and MT1/MT2 receptor knockout mice has indicated that the activation of MT2 receptors selectively increases non-rapid eye movement (NREM) sleep whereas MT1 receptors seem mostly implicated in the regulation of REM sleep. Moreover, MT1 knockout mice show an increase in NREM sleep, while MT2 knockout a decrease, suggesting an opposite role of these two receptors. A recent paper in mice by Sharma et al (J Pineal Res, 2018, e12498) found that MT1 but not MT2 receptors are expressed on orexin neurons in the perifornical lateral hypothalamus (PFH). Moreover, after injecting melatonin or luzindole into the mouse PFH, the authors suggest that melatonin promotes NREM sleep because activates PFH MT1 receptors, which in turn inhibit orexin neurons that are important in promoting arousal and maintaining wakefulness. In this commentary, we have critically commented on some of these findings on the bases of previous literature. In addition, we highlighted the fact that no conclusions could be drawn on the melatonin receptor subtype mediating the effects of melatonin on sleep because the authors used the non-selective MT1/MT2 receptors antagonist luzindole. More solid research should further characterize the pharmacological function of these two melatonin receptors in sleep., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Melatonin promotes sleep. However, the underlying mechanisms are unknown. Orexin neurons in the perifornical lateral hypothalamus (PFH) are pivotal for wake promotion. Does melatonin promote sleep by inhibiting orexin neurons? We used C57BL/6J mice and designed 4 experiments to address this question. Experiment 1 used double-labeled immunofluorescence and examined the presence of melatonin receptors on orexin neurons. Second, mice, implanted with bilateral guides targeted toward PFH and sleep-recording electrodes, were infused with melatonin (500 pmole/50 nL/side) at dark onset (onset of active period), and spontaneous bouts of sleep-wakefulness were examined. Third, mice, implanted with bilateral guides into the PFH, were infused with melatonin (500 pmole/50 nL/side) at dark onset and euthanized 2 hours later, to examine the activation of orexin neurons using c-Fos expression in orexin neurons. Fourth, mice, implanted with PFH bilateral guides and sleep-recording electrodes, were infused with melatonin receptor antagonist, luzindole (10 pmol/50 nL/side), at light onset (onset of sleep period), and spontaneous bouts of sleep-wakefulness were examined. Our results suggest that orexin neurons express MT1, but not MT2 receptors. Melatonin infusion into the PFH, at dark onset, site-specifically and significantly increased NREM sleep (43.7%, P = .003) and reduced wakefulness (12.3%, P = .013). Local melatonin infusion at dark onset inhibited orexin neurons as evident by a significant reduction (66%, P = .0004) in the number of orexin neurons expressing c-Fos. Finally, luzindole infusion-induced blockade of melatonin receptors in PFH at sleep onset significantly increased wakefulness (44.1%, P = .015). Based on these results, we suggest that melatonin may act via the MT1 receptors to inhibit orexin neurons and promote sleep., (Published 2018. This article is a U.S. Government work and is in the public domain in the USA.)

Introduction: Melatonin is a neurohormone involved in the regulation of circadian rhythms, with potent antioxidant activity. It has a wide functional repertoire, with effects almost on all tissues and organs. It is mainly used as a dietary supplement for sleep regulation and re-synchronization of disrupted circadian rhythms. Melatonin has very low toxicity, but some pharmacokinetic issues, such as limited oral bioavailability and short half-life, limit its tissue availability., Areas Covered: Patents and patent applications from 2012 to September 2014 in which melatonin or synthetic analogues are claimed for the prevention or treatment of pathological conditions., Expert Opinion: Melatonin is considered a valuable substance that can be safely administered for the prevention and treatment of many diverse diseases. A major trend in 2012 - 2014 patents is the co-administration of melatonin with other drugs to increase the efficacy of the treatment and reduce side-effects. Two different actions have been combined in hybrid ligands (e.g., melatonin-tamoxifen and melatonin-tacrine derivatives). Further experimental evidence is needed to support the usefulness of these approaches. The number of new melatonin analogues has shown a marked decrease in the past 3 years, with claimed applications mainly as hypnotic or antioxidant agents.

Our laboratory has recently demonstrated a melatonin MT1 receptor-mediated antiproliferative signaling mechanism in androgen receptor (AR)-positive prostate epithelial cells which involves up-regulation of p27(Kip1) through dual activation of Gα(s)/protein kinase A (PKA) and Gα(q)/protein kinase C (PKC) in parallel, and down-regulation of activated AR signaling via PKC stimulation. The aim of the present investigation was to identify the transcription factor that mediates melatonin's up-regulatory effect on p27(Kip1) in LNCaP and 22Rv1 prostate cancer cells. Deletion mapping and reporter assays of the p27(Kip1) promoter revealed that the putative melatonin-responsive transcription factor binds to a 116 base-pair region of the promoter sequence, which contains a potential nuclear factor kappa B (NF-κB) binding site. When the NF-κB binding site was abolished by site-directed mutagenesis, the stimulatory effect of melatonin on p27(Kip1) promoter activity was mitigated. Notably, melatonin inhibited the DNA binding of activated NF-κB via MT1 receptor-induced PKA and PKC stimulation. Furthermore, melatonin's up-regulatory effect on p27(Kip1) transcription and consequent cell antiproliferation were abrogated by NF-κB activator but mimicked by NF-κB inhibitor. The results indicate that inhibition of constitutively active NF-κB via melatonin MT1 receptor-induced dual activation of (Gα(s)) PKA and (Gα(q)) PKC can de-repress the p27(Kip1) promoter leading to transcriptional up-regulation of p27(Kip1). MT1 receptor-mediated inhibition of activated NF-κB signaling provides a novel mechanism supporting the use of melatonin in prostate cancer chemoprevention and therapy., (© 2012 John Wiley & Sons A/S.)

The treatment of bipolar disorder (BD) still remains a challenge. Melatonin (MLT), acting through its two receptors MT1 and MT2, plays a key role in regulating circadian rhythms which are dysfunctional in BD. Using a translational approach, we examined the implication and potential of MT1 receptors in the pathophysiology and psychopharmacology of BD. We employed a murine model of the manic phase of BD (Clock mutant (ClockΔ19) mice) to study the activation of MT1 receptors by UCM871, a selective partial agonist, in behavioral pharmacology tests and in-vivo electrophysiology. We then performed a high-resolution Nuclear Magnetic Resonance study on isolated membranes to characterize the molecular mechanism of interaction of UCM871. Finally, in a cohort of BD patients, we investigated the link between clinical measures of BD and genetic variants located in the MT1 receptor and CLOCK genes. We demonstrated that: 1) UCM871 can revert behavioral and electrophysiological abnormalities of ClockΔ19 mice; 2) UCM871 promotes the activation state of MT1 receptors; 3) there is a significant association between the number of severe manic episodes and MLT levels, depending on the genetic configuration of the MT1 rs2165666 variant. Overall, this work lends support to the potentiality of MT1 receptors as target for the treatment of BD.

Advanced prostate cancer often develops into bone metastasis, which is characterized by aberrant bone formation with chronic pain and lower chances of survival. No treatment exists as yet for osteoblastic bone metastasis in prostate cancer. The indolamine melatonin (N‐acetyl‐5‐methoxytryptamine) is a major regulator of the circadian rhythm. Melatonin has shown antiproliferative and antimetastatic activities but has not yet been shown to be active in osteoblastic bone lesions of prostate cancer. Our study investigations reveal that melatonin concentration‐dependently decreases the migratory and invasive abilities of two osteoblastic prostate cancer cell lines by inhibiting FAK, c‐Src, and NF‐κB transcriptional activity via the melatonin MT1 receptor, which effectively inhibits integrin α2β1 expression. Melatonin therapy appears to offer therapeutic possibilities for reducing osteoblastic bone lesions in prostate cancer. [ABSTRACT FROM AUTHOR]

Melatonin is a hormone that produces behavioral, pharmacological, and physiological effects through the activation of MT1 and MT2 melatonin receptors. Melatonin receptors participate in the modulation of the reinforcing effects of cocaine. Some studies report that dosing of melatonin decreases cocaine-induced locomotor activity and cocaine self-administration and that luzindole, an MT1, and MT2 melatonin receptor antagonist, blocks the melatonin-dependent decrease in cocaine-induced locomotor activity. The objective of this study was to evaluate the effect of acute or chronic dosing of melatonin on the induction and expression of cocaine-induced locomotor sensitization and cocaine-CPP in rats. Male Wistar rats received cocaine during the induction and expression of locomotor sensitization. Melatonin was administered 30 min before cocaine. After each treatment, locomotor activity was recorded for 30 min. Additionally, dopamine levels were determined in the ventral striatum, the prefrontal cortex (PFc), and the ventral tegmental area (VTA) by HPLC in animals treated with melatonin and cocaine. Melatonin decreased cocaine-induced locomotor sensitization and intracellular dopamine levels, as well as cocaine-CPP. Luzindole blocked the melatonin-induced decrease in the expression of locomotor sensitization in rats. These data suggest that melatonin may be a useful therapeutic agent to reduce cocaine abuse; additionally, they suggest that MT1 and MT2 receptors could be therapeutic targets, useful for the treatment of drug abuse disorder. [ABSTRACT FROM AUTHOR]

Melatonin, a neurohormone that binds to two G protein‐coupled receptors MT1 and MT2, is involved in pain regulation, but the distinct role of each receptor has yet to be defined. We characterized the nociceptive responses of mice with genetic inactivation of melatonin MT1 (MT1−/−), or MT2 (MT2−/−), or both MT1/MT2 (MT1−/−/MT2−/−) receptors in the hot plate test (HPT), and the formalin test (FT). In HPT and FT, MT1−/− display no differences compared to their wild‐type littermates (CTL), whereas both MT2−/− and MT1−/−/MT2−/− mice showed a reduced thermal sensitivity and a decreased tonic nocifensive behavior during phase 2 of the FT in the light phase. The MT2 partial agonist UCM924 induced an antinociceptive effect in MT1−/− but not in MT2−/− and MT1−/−/MT2−/− mice. Also, the competitive opioid antagonist naloxone had no effects in CTL, whereas it induced a decrease of nociceptive thresholds in MT2−/− mice. Our results show that the genetic inactivation of melatonin MT2, but not MT1 receptors, produces a distinct effect on nociceptive threshold, suggesting that the melatonin MT2 receptor subtype is selectively involved in the regulation of pain responses. [ABSTRACT FROM AUTHOR]

The treatment of bipolar disorder (BD) still remains a challenge. Melatonin (MLT), acting through its two receptors MT 1 and MT 2 , plays a key role in regulating circadian rhythms which are dysfunctional in BD. Using a translational approach, we examined the implication and potential of MT 1 receptors in the pathophysiology and psychopharmacology of BD. We employed a murine model of the manic phase of BD (Clock mutant (ClockΔ19) mice) to study the activation of MT 1 receptors by UCM871, a selective partial agonist, in behavioral pharmacology tests and in-vivo electrophysiology. We then performed a high-resolution Nuclear Magnetic Resonance study on isolated membranes to characterize the molecular mechanism of interaction of UCM871. Finally, in a cohort of BD patients, we investigated the link between clinical measures of BD and genetic variants located in the MT 1 receptor and CLOCK genes. We demonstrated that: 1) UCM871 can revert behavioral and electrophysiological abnormalities of ClockΔ19 mice; 2) UCM871 promotes the activation state of MT 1 receptors; 3) there is a significant association between the number of severe manic episodes and MLT levels, depending on the genetic configuration of the MT 1 rs2165666 variant. Overall, this work lends support to the potentiality of MT 1 receptors as target for the treatment of BD. [Display omitted] [ABSTRACT FROM AUTHOR]

The pathophysiological function of the G-protein coupled melatonin MT1 and MT2 receptors has not yet been well-clarified. Recent advancements using selective MT1/ MT2 receptor ligands and MT1/MT2 receptor knockout mice have suggested that the activation of the MT1 receptors are mainly implicated in the regulation of rapid eye movement (REM) sleep, whereas the MT2 receptors selectively increase non-REM (NREM) sleep. Studies in mutant mice show that MT1 knockout mice have an increase in NREM sleep and a decrease in REM sleep, while MT2 knockout mice a decrease in NREM sleep. The localization of MT1 receptors is also distinct from MT2 receptors; for example, MT2 receptors are located in the reticular thalamus (NREM area), while the MT1 receptors in the Locus Coeruleus and lateral hypothalamus (REM areas). Altogether, these findings suggest that these two receptors not only have a very specialized function in sleep, but that they may also modulate opposing effects. These data also suggest that mixed MT1-MT2 receptors ligands are not clinically recommended given their opposite roles in physiological functions, confirmed by the modest effects of melatonin or MT1/MT2 non-selective agonists when used in both preclinical and clinical studies as hypnotic drugs. In sum, MT1 and MT2 receptors have specific roles in the modulation of sleep, and consequently, selective ligands with agonist, antagonist, or partial agonist properties could have therapeutic potential for sleep; while the MT2 agonists or partial agonists might be indicated for NREM-related sleep and/or anxiety disorders, the MT1 agonists or partial agonists might be so for REM-related sleep disorders. Furthermore, MT1 but not MT2 receptors seem involved in the regulation of the circadian rhythm. Future research will help further develop MT1 and/or MT2 receptors as targets for neuropsychopharmacology drug development.

Melatonin (MLT), a neuromodulator mainly acting through two G-protein coupled receptors MT 1 and MT 2 , regulates many brain functions, including circadian rhythms, mood, pain and sleep. MLT and non-selective MT 1 /MT 2 receptor agonists are clinically used in neuropsychiatric and/or sleep disorders. However, the selective roles of the MT 1 and MT 2 receptors need to be clarified. Here, we review the role of the MT 1 receptor in neuropsychopharmacology, describe the anatomical localization of MT 1 receptors in the brain, discuss the medicinal chemistry, biochemistry and molecular aspects of the receptor, and explore the findings linking MT 1 receptors to psychiatric and neurological disorders. MT 1 receptors are localized in brain regions which regulate circadian rhythms, sleep, and mood, such as the suprachiasmatic nucleus, cortex, hippocampus, dorsal raphe nucleus and lateral hypothalamus. Their activation modulates intracellular signaling pathways also targeted by psychoactive drugs, including antidepressants and mood stabilizers. MT 1 receptor knockout mice display increased anxiety, a depressive-like phenotype, increased propensity to reward and addiction, and reduced Rapid-Eye-Movement sleep. These behavioral dysfunctions are associated with altered serotonergic and noradrenergic neurotransmissions. Several studies indicate that the MT 1 , rather than MT 2 , receptor is implicated in circadian rhythm regulation. The involvement of MT 1 receptors in Alzheimer's and Huntington diseases has also been proposed. Postmortem studies in depressed patients have further confirmed the possible involvement of MT 1 receptors in depression. Overall, there is substantial evidence indicating a role for MT 1 receptor in modulating brain function and mood. Consequently, this MLT receptor subtype deserves to be further examined as a novel target for neuropsychopharmacological drug development. [ABSTRACT FROM AUTHOR]

The pathophysiological function of the G-protein coupled melatonin MT1 and MT2 receptors has not yet been well-clarified. Recent advancements using selective MT1/ MT2 receptor ligands and MT1/MT2 receptor knockout mice have suggested that the activation of the MT1 receptors are mainly implicated in the regulation of rapid eye movement (REM) sleep, whereas the MT2 receptors selectively increase non-REM (NREM) sleep. Studies in mutant mice show that MT1 knockout mice have an increase in NREM sleep and a decrease in REM sleep, while MT2 knockout mice a decrease in NREM sleep. The localization of MT1 receptors is also distinct from MT2 receptors; for example, MT2 receptors are located in the reticular thalamus (NREM area), while the MT1 receptors in the Locus Coeruleus and lateral hypothalamus (REM areas). Altogether, these findings suggest that these two receptors not only have a very specialized function in sleep, but that they may also modulate opposing effects. These data also suggest that mixed MT1-MT2 receptors ligands are not clinically recommended given their opposite roles in physiological functions, confirmed by the modest effects of melatonin or MT1/MT2 non-selective agonists when used in both preclinical and clinical studies as hypnotic drugs. In sum, MT1 and MT2 receptors have specific roles in the modulation of sleep, and consequently, selective ligands with agonist, antagonist, or partial agonist properties could have therapeutic potential for sleep; while the MT2 agonists or partial agonists might be indicated for NREM-related sleep and/or anxiety disorders, the MT1 agonists or partial agonists might be so for REM-related sleep disorders. Furthermore, MT1 but not MT2 receptors seem involved in the regulation of the circadian rhythm. Future research will help further develop MT1 and/or MT2 receptors as targets for neuropsychopharmacology drug development. [ABSTRACT FROM AUTHOR]

Ovarian cancer is a highly aggressive disease that is frequently diagnosed in advanced stages. Melatonin, with its numerous antitumor properties, holds great promise in cancer treatment. Herein, we investigated the effects of melatonin on apoptosis, cell migration, and kinase levels in human ovarian carcinoma SKOV-3 cells and determined whether these effects are mediated by the activation of the MT1 receptor. SKOV-3 cells were exposed to different concentrations of melatonin based on the presence of MT1 receptor, and we also performed specific silencing of the melatonin receptor gene MTNR1A. Our findings revealed that melatonin reduced cell viability as shown by the MTT assay, and flow cytometry analysis showed increased rates of apoptosis and necrosis in all melatonin-treated cells. Melatonin significantly decreased the migratory and invasive capacities of the cells. Propidium iodide labeling indicated that melatonin induced cell cycle arrest by reducing DNA content in the S and G2/M phases in SKOV-3 cells. Additionally, the levels of AKT, ERK1/2, JNK, CREB, p70S6K, STAT3/5, and p38 MAP kinase involved in cell survival, proliferation, motility, and stress responses were depressed by melatonin and further reduced after MT1 knockdown. These molecules were found to be associated with lower overall survival in ovarian cancer patients. Melatonin had obvious oncostatic actions on ovarian cancer cells, and MT1 receptor knockdown intensified its antitumor effect. The inhibition of the MT1 receptor resulted in a substantial reduction in the migratory and invasive capacities of the cells, suggesting its potential as a therapeutic target for the treatment of ovarian cancer. • Melatonin abolishes ovarian cancer cell advantages. • Melatonin reduces growth and migratory potential of cells. • The melatonin's inhibitory effects involve kinase signaling in ovarian cancer cells. • MT1 receptor silencing seems to potentiate the pharmacological effect of melatonin. [ABSTRACT FROM AUTHOR]

The present study analysed the involvement of melatonin, acting via its receptors (MT1 and MT2), in ram sperm functionality. Indirect immunofluorescence assays revealed no changes in the distribution or intensity of MT1 receptors, whereas different subpopulations were established for MT2 receptors in control, in vitro capacitated and acrosome-reacted ram spermatozoa. Chlortetracycline staining revealed the following correlations between the pattern of staining for MT2 receptors in: (1) non-capacitated (NC) sperm rate and the proportion of spermatozoa with equal immunostaining intensity in the acrosome and post-acrosome (r=0.59, P<0.001); (2) in capacitated (C) sperm rate and the proportion of spermatozoa with stronger reactivity in the acrosome (r=0.60, P<0.001); and (3) in acrosome-reacted (AR) sperm rate and the proportion of spermatozoa with more intense staining on the post-acrosome (r=0.67, P<0.001). Incubation of swim-up-selected samples with either 1 μM melatonin or MT1 and MT2 receptor agonists (2-phenylmelatonin 1 μM and 8-Methoxy-2-propionamidotetralin (8M-PDOT) 1 μM and 10 nM) at 39°C and 5% CO2 for 3 h resulted in a higher proportion of the NC pattern compared with the control group (P<0.05), whereas treatment with MT1 and MT2 receptor antagonists (luzindole 1 μM and 4-phenyl-2-propionamidotetralin (4P-PDOT) 1 μM and 10 nM) decreased the proportion of spermatozoa exhibiting the NC pattern (P<0.001) concomitant with an increase in those exhibiting the C pattern (P<0.01). In conclusion, melatonin exerts a modulating effect on ram sperm functionality, primarily via activation of the MT2 receptor. [ABSTRACT FROM AUTHOR]

Highlights: [•] Melatonin decreases the latency to NREM sleep. [•] Melatonin does not alter the duration of sleep during the 24-h light-dark cycle. [•] The non-selective MT1/MT2 receptor agonist UCM793 affects the number of episodes of wakefulness but not sleep. [•] MLT and the non-selective MT1/MT2 receptor agonist UCM793 differently influence quality of the three vigilance states. [•] Pharmacological activation of MT2 receptors increases NREMS. [ABSTRACT FROM AUTHOR]

Highlights: [•] Short Day (SD) enhanced the proliferation of splenocytes, PBMC and GM-CFU. [•] SD induced circulatory melatonin increased the IL-2 and anti-KLH IgG production. [•] Melatonin acts as an immunostimulator via increasing expression of receptor MT1. [Copyright &y& Elsevier]

Abstract: The neurohormone melatonin activates two G-protein coupled receptors, MT1 and MT2. Melatonin is implicated in circadian rhythms and sleep regulation, but the role of its receptors remains to be defined. We have therefore characterized the spontaneous vigilance states in wild-type (WT) mice and in three different types of transgenic mice: mice with genetic inactivation of MT1 (MT1 −/−), MT2 (MT2 −/−) and both MT1/MT2 (MT1 −/−/MT2 −/−) receptors. Electroencephalographic (EEG) and electromyographic sleep–wake patterns were recorded across the 24-h light–dark cycle. MT1 −/−mice displayed a decrease (−37.3%) of the 24-h rapid eye movement sleep (REMS) time whereas MT2 −/−mice showed a decrease (−17.3%) of the 24-h non rapid eye movement sleep (NREMS) time and an increase in wakefulness time (14.8%). These differences were the result of changes occurring in particular during the light/inactive phase. Surprisingly, MT1 −/−/MT2 −/− mice showed only an increase (8.9%) of the time spent awake during the 24-h. These changes were correlated to a decrease of the REMS EEG theta power in MT1 −/−mice, of the NREMS EEG delta power in MT2 −/−mice, and an increase of the REMS and wakefulness EEG theta power in MT1 −/−/MT2 −/− mice. Our results show that the genetic inactivation of both MT1 and MT2 receptors produces an increase of wakefulness, likely as a result of reduced NREMS due to the lack of MT2 receptors, and reduced REMS induced by the lack of MT1 receptors. Therefore, each melatonin receptor subtype differently regulates the vigilance states: MT2 receptors mainly NREMS, whereas MT1 receptors REMS. [Copyright &y& Elsevier]

Our laboratory has recently demonstrated a melatonin MT1 receptor-mediated antiproliferative signaling mechanism in androgen receptor (AR)-positive prostate epithelial cells which involves up-regulation of p27Kip1 through dual activation of Gαs/protein kinase A (PKA) and Gαq/protein kinase C (PKC) in parallel, and down-regulation of activated AR signaling via PKC stimulation. The aim of the present investigation was to identify the transcription factor that mediates melatonin's up-regulatory effect on p27Kip1 in LNCaP and 22Rv1 prostate cancer cells. Deletion mapping and reporter assays of the p27Kip1 promoter revealed that the putative melatonin-responsive transcription factor binds to a 116 base-pair region of the promoter sequence, which contains a potential nuclear factor kappa B (NF-κB) binding site. When the NF-κB binding site was abolished by site-directed mutagenesis, the stimulatory effect of melatonin on p27Kip1 promoter activity was mitigated. Notably, melatonin inhibited the DNA binding of activated NF-κB via MT1 receptor-induced PKA and PKC stimulation. Furthermore, melatonin's up-regulatory effect on p27Kip1 transcription and consequent cell antiproliferation were abrogated by NF-κB activator but mimicked by NF-κB inhibitor. The results indicate that inhibition of constitutively active NF-κB via melatonin MT1 receptor-induced dual activation of (Gαs) PKA and (Gαq) PKC can de-repress the p27Kip1 promoter leading to transcriptional up-regulation of p27Kip1. MT1 receptor-mediated inhibition of activated NF-κB signaling provides a novel mechanism supporting the use of melatonin in prostate cancer chemoprevention and therapy. [ABSTRACT FROM AUTHOR]

To determine whether melatonin, via its MT1 G protein-coupled receptor, impacts mouse mammary gland development, we generated a mouse mammary tumor virus (MMTV)-MT1-Flag-mammary gland over-expressing (MT1-mOE) transgenic mouse. Increased expression of the MT1-Flag transgene was observed in the mammary glands of pubescent MT1-mOE transgenic female mice, with further significant increases during pregnancy and lactation. Mammary gland whole mounts from MT1-mOE mice showed significant reductions in ductal growth, ductal branching, and terminal end bud formation. Elevated MT1 receptor expression in pregnant and lactating female MT1-mOE mice was associated with reduced lobulo-alveolar development, inhibition of mammary epithelial cell proliferation, and significant reductions in body weights of suckling pups. Elevated MT1 expression in pregnant and lactating MT1-mOE mice correlated with reduced mammary gland expression of Akt1, phospho-Stat5, Wnt4, estrogen receptor alpha, progesterone receptors A and B, and milk proteins β-casein and whey acidic protein. Estrogen- and progesterone-stimulated mammary gland development was repressed by elevated MT1 receptor expression and exogenous melatonin administration. These studies demonstrate that the MT1 melatonin receptor and its ligand melatonin play an important regulatory role in mammary gland development and lactation in mice through both growth suppression and alteration of developmental paradigms. [ABSTRACT FROM AUTHOR]

Purpose: To investigate (i) the effect of monochromatic light on inhibiting induction of light-induced melatonin and (ii) the roles of melanopsin and MT1 receptor in light-induced myopia in the guinea pig. Methods: Forty-eight guinea pigs were randomly distributed into three treatment groups: white-light (control), green-light (530 nm), and blue-light (480 nm) groups. Levels of pineal gland melatonin were measured twice daily--10:00 a.m. and 10:00 p.m.--10 days after initial light treatment. Thirty additional guinea pigs were also assigned to these groups and treated similarly. For these latter animals, refractive status, ocular length, and vitreous depth were measured before and after light treatment. Eight weeks after light treatment, retinal and sceral levels of melanopsin, melatonin receptor type (MT) 1, and mRNA protein were determined by Western blotting, real-time polymerase chain reaction (RT-PCR), and immunohistochemistry. Results: The level of pineal gland melatonin in the green-light group was significantly higher than that in the blue-light group. Biometric measurements showed that guinea pigs in the green-light group had a somewhat myopic refractive status. Expressions of retinal melanopsin mRNA and protein were significantly higher in the blue-light group and lower in the green-light group when compared to controls. Conversely, expressions of MT1 receptor mRNA and protein in retina and sclera were significantly higher in the green-light group and lower in the blue-light group when compared to controls. Conclusions: Green light appears to suppress induction of melatonin production. In addition, 530 nm of green light is involved in the development of myopia. In the guinea pig, MT1 receptor and melanopsin appear to play roles in the development of myopia induced by 530 nm of light. [ABSTRACT FROM AUTHOR]

The MT1 melatonin receptor is bound and activated by the pineal hormone melatonin. This G protein-coupled melatonin receptor is expressed in human breast tumor cell lines, and when activated, mediates the growth-suppressive and steroid hormone/nuclear receptor modulatory actions of melatonin on breast tumor cells. In the current studies, we have examined the expression of the MT1 receptor in breast cancer cell lines and primary human breast tumors and correlated MT1 receptor expression with the deletion, rearrangement and amplification of the MT1 gene and established markers of breast cancer such as tumor size, stage, estrogen receptor alpha (ERα) and progesterone receptor (PR) expression. Theses studies suggest amplification of the MT1 gene in some breast tumors and an inverse correlation with ERα, PR and MT1 protein expression. Furthermore, these approaches employing immunohistochemical and immunofluorescent/confocal microscopic studies demonstrate that the MT1 receptor is localized to the caveoli and that MT1 expression in MCF-7 breast cancer cells can be repressed by estradiol and melatonin. [ABSTRACT FROM AUTHOR]

An estimated one-third of the general population is affected by insomnia, and this number is increasing due to more stressful working conditions and the progressive aging of society. However, current treatment of insomnia with hypnotics, gamma-aminobutyric acid A (GABAA) receptor modulators, induces various side effects, including cognitive impairment, motor disturbance, dependence, tolerance, hangover, and rebound insomnia. Ramelteon (Rozerem; Takeda Pharmaceutical Company Limited, Osaka, Japan) is an orally active, highly selective melatonin MT1/MT2 receptor agonist. Unlike the sedative hypnotics that target GABAA receptor complexes, ramelteon is a chronohypnotic that acts on the melatonin MT1 and MT2 receptors, which are primarily located in the suprachiasmatic nucleus, the body's “master clock.” As such, ramelteon possesses the first new therapeutic mechanism of action for a prescription insomnia medication in over three decades. Ramelteon has demonstrated sleep-promoting effects in clinical trials, and coupled with its favorable safety profile and lack of abuse potential or dependence, this chronohypnotic provides an important treatment option for insomnia. [ABSTRACT FROM AUTHOR]

Circannual variation in the human serum levels of prostate-specific antigen, a growth marker of the prostate gland, has been reported recently. The present study was conducted to investigate the role of the photoperiodic hormone melatonin (MLT) and its membrane receptors in the modulation of human prostate growth. Expression of MT1 and MT2 receptors was detected in benign human prostatic epithelial tissues and RWPE-1 cells. MLT and 2-iodomelatonin inhibited RWPE-1 cell proliferation and up-regulated p27 Kip1 gene and protein expression in the cells. The effects of MLT were blocked by the nonselective MT1/MT2 receptor antagonist luzindole, but were not affected by the selective MT2 receptor antagonist 4-phenyl-2-propionamidotetraline. Of note, the antiproliferative action of MLT on benign prostate epithelial RWPE-1 cells was effected via increased p27 Kip1 gene transcription through MT1 receptor-mediated activation of protein kinase A (PKA) and protein kinase C (PKC) in parallel, a signaling process which has previously been demonstrated in 22Rv1 prostate cancer cells. Taken together, the demonstration of the MT1/PKA+PKC/p27Kip1 antiproliferative pathway in benign and malignant prostate epithelial cell lines indicated the potential importance of this MLT receptor-mediated signaling mechanism in growth regulation of the human prostate gland in health and disease. Collectively, our data support the hypothesis that MLT may function as a negative mitogenic hormonal regulator of human prostate epithelial cell growth. [ABSTRACT FROM AUTHOR]

Prostate cancer is a public health problem of the elderly men. It has been estimated that one in six men will develop prostate cancer in his lifetime in the USA. There is thus a huge clinical demand for effective therapies for the prevention and treatment of the disease. Here, the scientific evidence supporting the effectiveness of melatonin in inhibiting the development and progression of prostate cancer is reviewed. The rational use of melatonin in prostate cancer prevention, stabilization of clinically localized favourable-risk prostate cancer and palliative treatment of advanced or metastatic tumour is discussed within the context of the molecular pathogenesis of the disease. [ABSTRACT FROM AUTHOR]

Abstract: A significant increase in tumor regression was induced in N-nitroso-N-methylurea-induced mammary tumors in rats treated with the combination of melatonin and 9-cis-retinoic acid (9cRA). Treatment groups included: control (ethanolic saline), 9cRA (30mg/kg chow/day), melatonin 500μg/day, melatonin 1000μg/day, melatonin 500μg/day+9cRA and melatonin 1000μg/day+9cRA. Rats treated with the lower dose of melatonin 500μg+9cRA show the greatest degree of tumor regression (78%), with 54% undergoing complete regression and a significant increase in apoptotic cells observed by TUNEL Assay. Furthermore, tumor multiplicity and burden were significantly decreased by the combination of melatonin and 9cRA. [Copyright &y& Elsevier]

Melatonin acts through several specific receptors, including membrane receptors (MT1 and MT2) and members of the RZR/ROR nuclear receptors family, which have been identified in a large variety of mammalian and nonmammalian cells types. Both membrane and nuclear melatonin receptors have been partially characterized in Harderian gland of the Syrian hamster. Nevertheless, the identities of these receptors were unknown until this study, where the coexistence of MT1 and ROR α1 in this gland was determined by nested RT-PCR followed by amplicon sequencing and Western-blot. Furthermore, the cellular localization of both receptors was determined by immunohistochemistry. Thus, MT1 receptor was localized exclusively at the basal side of the cell acini, supporting the hypothesis that this receptor is activated by the pineal-synthesized melatonin. On the contrary, although a ROR α1-immunoreactivity was observed in nuclei of epithelial cells of both sexes, an extranuclear specific staining, which was more frequently among those cells of males, was also seen. The implication of this possible nuclear exclusion of ROR α1 on the role of this indoleamine against oxidative stress is discussed. [ABSTRACT FROM AUTHOR]

Melatonin has been shown to bind to the MT1 G protein-coupled receptor (GPCR) in MCF-7 breast cancer cells to modulate the estrogen response pathway suppressing estrogen-induced estrogen receptor alpha (ERα) transcriptional activity, blunting ER/DNA binding activity and suppressing cell proliferation. In these studies we have examined the effect of melatonin on the transcriptional activity of the ERα and other members of the steroid/thyroid hormone receptor superfamily, namely, the glucocorticoid receptor (GR) and the retinoic acid receptor alpha (RARα). As with the ERα, melatonin represses ligand (dexamethasone)-induced activation of the GR. This effect of melatonin on ERα and GR is blocked by pertussis toxin (PTX) suggesting that melatonin's actions may be mediated via a PTX-sensitive G αi protein. In contrast, melatonin potentiates the action ofall-trans-retinoic acid on RARα transcriptional activation and enhances RARα/DNA binding activity, an action which is not PTX-sensitive. Expression of a dominant-positive G αi2 protein, with which the MT1 receptor has been shown to couple, is able to mimic the effect of melatonin on ERα but not RARα transcriptional activation in breast cancer cells. This demonstrates that GPCRs can modulate the transcriptional activity of various steroid receptors in response to their ligand through activation of different G protein signaling pathways. [ABSTRACT FROM AUTHOR]

Abstract: Ramelteon (TAK-375) is a novel melatonin receptor agonist currently under investigation for the treatment of insomnia. This study describes the neurochemical and receptor binding characteristics of ramelteon in vitro. Ramelteon showed very high affinity for human MT1 (Mel1a) and MT2 (Mel1b) receptors (expressed in Chinese hamster ovary [CHO] cells), and chick forebrain melatonin receptors (consisting of Mel1a and Mel1c receptors) with K i values of 14.0, 112, and 23.1 pM, respectively, making the affinities of ramelteon for these receptors 3–16 times higher than those of melatonin. The affinity of ramelteon for hamster brain MT3 binding sites was extremely weak (K i: 2.65 μM) compared to melatonin''s affinity for the MT3 binding site (K i: 24.1 nM). In addition, ramelteon showed no measurable affinity for a large number of ligand binding sites (including benzodiazepine receptors, dopamine receptors, opiate receptors, ion channels, and transporters) and no effect on the activity of various enzymes. Ramelteon inhibited forskolin-stimulated cAMP production in the CHO cells that express the human MT1 or MT2 receptors. Taken together, these results indicate that ramelteon is a potent and highly selective agonist of MT1/MT2 melatonin receptors. [Copyright &y& Elsevier]

Based on the fact that human bile and, particularly gallbladder bile, contains high physiological levels of the antioxidant melatonin, the aim of this study was to investigate whether the melatonin receptor MT1 is present in human gallbladder. Expression and localization of MT1 was assessed by RT-PCR, Western blotting and immunofluorescence analysis in gallbladder samples from patients with cholelithiasis and with advanced gallbladder carcinoma. Additionally, we monitored mRNA expression of the two key enzymes of melatonin synthesis, i.e. arylalkylamine- N-acetyltransferase (AANAT) and hydroxyindole- O-methyltransferase (HIOMT). MT1 mRNA and protein were present in all cholelithiasis (n = 10) and gallbladder carcinoma (n = 5) samples. As indicated from RT-PCR and Western blot studies, MT1 is located in gallbladder epithelia. Epithelial expression was further proven by immunofluorescence staining of MT1 in paraffin-embedded cholelithiasis and gallbladder carcinoma sections. Analysis of AANAT and HIOMT mRNA expression showed that HIOMT mRNA is present in gallbladder. Surprisingly, AANAT was not detectable under conditions where it was found in a human colon specimen. The absence of AANAT suggests that in human gallbladder, HIOMT might be involved in the formation of 5-hydroxytryptamine products other than melatonin. In summary, our results provide the first evidence for the presence of MT1 in human gallbladder epithelia. Therefore, in addition to its profound antioxidative effects in the biliary system, melatonin might also act through MT1-mediated signal transduction pathways. Thereby, it might be involved in the regulation of gallbladder function. [ABSTRACT FROM AUTHOR]

Overexpression of the MT1 melatonin receptor in MCF-7 human breast cancer cells significantly enhances the response of these cells to the growth-inhibitory actions of melatonin. Athymic nude mice implanted with MT1-overexpressing MCF-7 cells developed significantly fewer palpable tumors (60% reduction) compared to mice receiving vector-transfected MCF-7 cells (vt-MCF-7). In response to exogenous melatonin, tumor incidence in the mice receiving the MT1-overexpressing MCF-7 cells was decreased by 80% compared to mice receiving vt-MCF-7 cells. Interestingly, daily melatonin administration did not decrease tumor incidence in mice receiving vt-MCF-7 cells, but rather stimulated overall tumor formation. [Copyright &y& Elsevier]

Melatonin inhibits the proliferation of estrogen receptor alpha (ERα)-positive (MCF-7), but not ERα-negative (MDA-MB-231) breast cancer cells. Here, we assessed the effect of MT1 melatonin receptor stable overexpression in MCF-7 and MDA-MB-231 breast cancer cells on the growth-suppressive effects of melatonin. Parental and vector-transfected MCF-7 cells demonstrated a modest, but significant, growth-suppressive response to melatonin; however, melatonin treatment of MT1-transfected MCF-7 cells resulted in significantly enhanced growth-suppression. This response was blocked by an MT1/MT2 melatonin receptor antagonist. Interestingly, MT1-overexpression did not induce a melatonin-sensitive phenotype in melatonin-insensitive MDA-MB-231 cells. Finally, Northern blot analysis demonstrated an enhanced inhibition of ERα mRNA expression and an enhanced induction of pancreatic spasmolytic polypeptide (pS2) by melatonin in MT1-transfected MCF-7 cells relative to vector-transfected MCF-7 cells. These data suggest the involvement of the MT1 melatonin receptor in mediation of melatonin effects on growth-suppression and gene-modulation in breast cancer cells. [Copyright &y& Elsevier]

Abstract Melatonin acts via high affinity, G-protein coupled, seven transmembrane domain receptors. To precisely localize these receptors, antibodies were raised in chickens against a 15 amino acid fragment at the intracellular C-terminal region of the human melatonin receptor subtype mt1 (DSSNDVADRVKWKPS, mt1338-352). A chimeric form of the receptor with a hydrophilic Flag peptide (DYKDDDDK) in sequence with the extracellular N-terminus (Flag-mt1) was generated by polymerase chain reaction and expressed in mammalian cell lines. An IgY antibody (Y31), which gave high antibody titres by enzyme-linked immunosorbent assay, was used to localize Flag-mt1 in stably transfected cells by immunofluoresence. Flag-mt1 localization with Y31 was identical to that obtained with the M5 antibody directed against the Flag epitope and was mainly localized to the Golgi apparatus with some staining at the cell surface. No staining was seen in untransfected cells with either antibody. Y31 staining was abolished using antibody preabsorbed with peptide antigen. Y31 immunofluorescence in fetal human kidney sections was restricted to nephrogenic regions and matched that of 2-(125I)iodomelatonin binding and mt1 gene expression by in situ hybridization. Y31 was used to immunoprecipitate biotinylated membrane proteins from Flag-mt1 stably transfected and untransfected CHO cells. Western blotting of immunoprecipitated proteins revealed two major bands specific to stably transfected cells, one at 63 kDa and one at 86 kDa. The first band almost certainly corresponds to the glycosylated form of Flag-mt1 and the second band to receptor dimers. Thus, Y31 antibody is suitable for use in detecting the human mt1 receptor subtype in tissues and in transfected cells. [ABSTRACT FROM AUTHOR]