Detection of recombinant and endogenous mouse melatonin receptors by monoclonal antibodies targeting the C‐terminal domain

Melatonin receptors play important roles in the regulation of circadian and seasonal rhythms, sleep, retinal functions, the immune system, depression, and type 2 diabetes development. Melatonin receptors are approved drug targets for insomnia, non‐24‐hour sleep‐wake disorders, and major depressive disorders. In mammals, two melatonin receptors (MTRs) exist, MT₁ and MT₂, belonging to the G protein‐coupled receptor (GPCR) superfamily. Similar to most other GPCRs, reliable antibodies recognizing melatonin receptors proved to be difficult to obtain. Here, we describe the development of the first monoclonal antibodies (mABs) for mouse MT₁ and MT₂. Purified antibodies were extensively characterized for specific reactivity with mouse, rat, and human MT₁ and MT₂ by Western blot, immunoprecipitation, immunofluorescence, and proximity ligation assay. Several mABs were specific for either mouse MT₁ or MT₂. None of the mABs cross‐reacted with rat MTRs, and some were able to react with human MTRs. The specificity of the selected mABs was validated by immunofluorescence microscopy in three established locations (retina, suprachiasmatic nuclei, pituitary gland) for MTR expression in mice using MTR‐KO mice as control. MT₂ expression was not detected in mouse insulinoma MIN6 cells or pancreatic beta‐cells. Collectively, we report the first monoclonal antibodies recognizing recombinant and native mouse melatonin receptors that will be valuable tools for future studies.     

Melatonin‐mediated inhibition of Cav3.2 T‐type Ca2+ channels induces sensory neuronal hypoexcitability through the novel protein kinase C‐eta isoform

Recent studies implicate melatonin in the antinociceptive activity of sensory neurons. However, the underlying mechanisms are still largely unknown. Here, we identify a critical role of melatonin in functionally regulating Cav3.2 T‐type Ca²⁺ channels (T‐type channel) in trigeminal ganglion (TG) neurons. Melatonin inhibited T‐type channels in small TG neurons via the melatonin receptor 2 (MT₂ receptor) and a pertussis toxin‐sensitive G‐protein pathway. Immunoprecipitation analyses revealed that the intracellular subunit of the MT₂ receptor coprecipitated with Gα_o. Both shRNA‐mediated knockdown of Gα_o and intracellular application of QEHA peptide abolished the inhibitory effects of melatonin. Protein kinase C (PKC) antagonists abolished the melatonin‐induced T‐type channel response, whereas inhibition of conventional PKC isoforms elicited no effect. Furthermore, application of melatonin increased membrane abundance of PKC‐eta (PKC_η) while antagonism of PKC_η or shRNA targeting PKC_η prevented the melatonin‐mediated effects. In a heterologous expression system, activation of MT₂ receptor strongly inhibited Cav3.2 T‐type channel currents but had no effect on Cav3.1 and Cav3.3 current amplitudes. The selective Cav3.2 response was PKC_η dependent and was accompanied by a negative shift in the steady‐state inactivation curve. Furthermore, melatonin decreased the action potential firing rate of small TG neurons and attenuated the mechanical hypersensitivity in a mouse model of complete Freund's adjuvant‐induced inflammatory pain. These actions were inhibited by T‐type channel blockade. Together, our results demonstrated that melatonin inhibits Cav3.2 T‐type channel activity through the MT₂ receptor coupled to novel G_βγ‐mediated PKC_η signaling, subsequently decreasing the membrane excitability of TG neurons and pain hypersensitivity in mice.     

Melatonin‐mediated inhibition of Purkinje neuron P‐type Ca2+ channels in vitro induces neuronal hyperexcitability through the phosphatidylinositol 3‐kinase‐dependent protein kinase C delta pathway

Although melatonin receptors are widely expressed in the mammalian central nervous system and peripheral tissues, there are limited data regarding the functions of melatonin in cerebellar Purkinje cells. Here, we identified a novel functional role of melatonin in modulating P‐type Ca²⁺ channels and action‐potential firing in rat Purkinje neurons. Melatonin at 0.1 μm reversibly decreased peak currents (I_Ba) by 32.9%. This effect was melatonin receptor 1 (MT_R1) dependent and was associated with a hyperpolarizing shift in the voltage dependence of inactivation. Pertussis toxin pretreatment, intracellular application of QEHA peptide, and a selective antibody raised against the G_β subunit prevented the inhibitory effects of melatonin. Pretreatment with phosphatidylinositol 3‐kinase (PI3K) inhibitors abolished the melatonin‐induced decrease in I_Ba. Surprisingly, melatonin responses were not regulated by Akt, a common downstream target of PI3K. Melatonin treatment significantly increased protein kinase C (PKC) activity 2.1‐fold. Antagonists of PKC, but not of protein kinase A, abolished the melatonin‐induced decrease in I_Ba. Melatonin application increased the membrane abundance of PKC_δ, and PKC_δ inhibition (either pharmacologically or genetically) abolished the melatonin‐induced I_Ba response. Functionally, melatonin increased spontaneous action‐potential firing by 53.0%; knockdown of MT_R1 and blockade of P‐type channels abolished this effect. Thus, our results suggest that melatonin inhibits P‐type channels through MT_R1 activation, which is coupled sequentially to the βγ subunits of G_i/o‐protein and to downstream PI3K‐dependent PKC_δ signaling. This likely contributes to its physiological functions, including spontaneous firing of cerebellar Purkinje neurons.     

Protein interactome mining defines melatonin MT1 receptors as integral component of presynaptic protein complexes of neurons

In mammals, the hormone melatonin is mainly produced by the pineal gland with nocturnal peak levels. Its peripheral and central actions rely either on its intrinsic antioxidant properties or on binding to melatonin MT₁ and MT₂ receptors, belonging to the G protein‐coupled receptor (GPCR) super‐family. Melatonin has been reported to be involved in many functions of the central nervous system such as circadian rhythm regulation, neurotransmission, synaptic plasticity, memory, sleep, and also in Alzheimer's disease and depression. However, little is known about the subcellular localization of melatonin receptors and the molecular aspects involved in neuronal functions of melatonin. Identification of protein complexes associated with GPCRs has been shown to be a valid approach to improve our understanding of their function. By combining proteomic and genomic approaches we built an interactome of MT₁ and MT₂ receptors, which comprises 378 individual proteins. Among the proteins interacting with MT₁, but not with MT₂, we identified several presynaptic proteins, suggesting a potential role of MT₁ in neurotransmission. Presynaptic localization of MT₁ receptors in the hypothalamus, striatum, and cortex was confirmed by subcellular fractionation experiments and immunofluorescence microscopy. MT₁ physically interacts with the voltage‐gated calcium channel Ca_v2.2 and inhibits Ca_v2.2‐promoted Ca²⁺ entry in an agonist‐independent manner. In conclusion, we show that MT₁ is part of the presynaptic protein network and negatively regulates Ca_v2.2 activity, providing a first hint for potential synaptic functions of MT₁.     

Genetic deletion of MT1 and MT2 melatonin receptors differentially abrogates the development and expression of methamphetamine‐induced locomotor sensitization during the day and the night in C3H/HeN mice

Abstract: This study explored the role of the melatonin receptors in methamphetamine (METH)‐induced locomotor sensitization during the light and dark phases in C3H/HeN mice with genetic deletion of the MT₁ and/or MT₂ melatonin receptors. Six daily treatments with METH (1.2 mg/kg, i.p.) in a novel environment during the light phase led to the development of locomotor sensitization in wild‐type (WT), MT₁KO and MT₂KO mice. Following four full days of abstinence, METH challenge (1.2 mg/kg, i.p.) triggered the expression of locomotor sensitization in METH‐pretreated but not in vehicle (VEH)‐pretreated mice. In MT₁/MT₂KO mice, the development of sensitization during the light phase was significantly reduced and the expression of sensitization was completely abrogated upon METH challenge. During the dark phase the development of locomotor sensitization in METH‐pretreated WT, MT₁KO and MT₂KO mice was statistically different from VEH‐treated controls. However, WT and MT₂KO, but not MT₁KO mice receiving repeated VEH pretreatments during the dark phase expressed a sensitized response to METH challenge that is of an identical magnitude to that observed upon 6 days of METH pretreatment. We conclude that exposure to a novel environment during the dark phase, but not during the light phase, facilitated the expression of sensitization to a METH challenge in a manner dependent on MT₁ melatonin receptor activation by endogenous melatonin. We suggest that MT₁ and MT₂ melatonin receptors are potential targets for pharmacotherapeutic intervention in METH abusers.     

Molecular and cellular pharmacological properties of 5‐methoxycarbonylamino‐N‐acetyltryptamine (MCA‐NAT): a nonspecific MT3 ligand

Abstract: 5‐Methoxycarbonylamino‐N‐acetyltryptamine (MCA‐NAT) has been initially described as a ligand at non MT₁, non MT₂ melatonin binding site (MT3) selective versus MT₁ and MT₂, two membrane melatonin receptors. MCA‐NAT activity has been reported by others in different models, in vivo, particularly in the intra‐ocular pressure (IOP) models in rabbits and monkeys. Its activity was systematically linked to either MT3 or to a new, yet unknown, melatonin receptor. In this article, the melatonin receptor pharmacology of MCA‐NAT is described. MCA‐NAT has micromolar range affinities at the melatonin receptors MT₁ and MT₂, while in functional studies, MCA‐NAT proved to be a powerful MT₁/MT₂ partial agonist in the sub‐micromolar range. These data strongly suggest that MCA‐NAT actions might be mediated by these receptors in vivo. Finally, as described by others, we show that MCA‐NAT is unable to elicit any type of receptor‐like functional responses from Chinese hamster ovary cells over‐expressing quinone reductase 2, the MT3.     

Melatonin protects chronic kidney disease mesenchymal stem cells against senescence via PrPC‐dependent enhancement of the mitochondrial function

Although mesenchymal stem cell (MSC)‐based therapy is a treatment strategy for ischemic diseases associated with chronic kidney disease (CKD), MSCs of CKD patients undergo accelerated senescence, with decreased viability and proliferation upon uremic toxin exposure, inhibiting their utility as a potent stem cell source for transplantation therapy. We investigated the effects of melatonin administration in protecting against cell senescence and decreased viability induced by pathophysiological conditions near the engraftment site. MSCs harvested from CKD mouse models were treated with H₂O₂ to induce oxidative stress. CKD‐derived MSCs exhibited greater oxidative stress‐induced senescence than normal‐mMSCs, while melatonin protected CKD‐mMSCs from H₂O₂ and associated excessive senescence. The latter was mediated by PrP^C‐dependent mitochondrial functional enhancement; melatonin upregulated PrP^C, which bound PINK1, thus promoting mitochondrial dynamics and metabolism. In vivo, melatonin‐treated CKD‐mMSCs survived longer, with increased secretion of angiogenic cytokines in ischemic disease engraftment sites. CKD‐mMSCs are more susceptible to H₂O₂‐induced senescence than normal‐mMSCs, and melatonin administration protects CKD‐mMSCs from excessive senescence by upregulating PrP^C and enhancing mitochondrial function. Melatonin showed favorable therapeutic effects by successfully protecting CKD‐mMSCs from related ischemic conditions, thereby enhancing angiogenesis and survival. These results elucidate the mechanism underlying senescence inhibition by melatonin in stem cell‐based therapies using mouse‐derived CKD‐mMSCs.     

Melatonin receptor structures shed new light on melatonin research

The tryptophan derivative melatonin is an evolutionary old molecule that is involved in a pleiotropy of physiological functions. In humans, age‐related decline of circulating melatonin levels and/or dysregulation of its circadian synthesis pattern have been associated with several disorders and disease states. Several molecular targets have been proposed for melatonin since its discovery, in 1959. Among them, melatonin MT₁ and MT₂ receptors are the best characterized melatonin targets, mediating melatonin effects in a variety of tissues. They belong to the superfamily of G protein‐coupled receptors. Two back‐to‐back articles published in the “Nature” Journal earlier this year present the first crystal structures of the human MT₁ and MT₂ in its inactive states. Here, we will briefly outline the discovery path of melatonin receptors until their structural elucidation and discuss how these new findings will guide future research toward a better understanding of their function and rational drug design.     

Nociceptive responses in melatonin MT2 receptor knockout mice compared to MT1 and double MT1/MT2 receptor knockout mice

Melatonin, a neurohormone that binds to two G protein-coupled receptors MT₁ 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 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.     

Melatonin receptor ligands: A pharmaco-chemical perspective

Melatonin MT₁ and MT₂ receptor ligands have been vigorously explored for the last 4 decades. Inspection of approximately 80 publications in the field revealed that most melatonergic ligands were structural analogues of melatonin combining three essential features of the parent compound: an aromatic ring bearing a methoxy group and an amide side chain in a relative arrangement similar to that present in melatonin. While several series of MT₂-selective agents—agonists, antagonists, or partial agonists—were reported, the field was lacking MT₁-selective agents. Herein, we describe various approaches toward the development of melatonergic ligands, keeping in mind that most of the molecules/pharmacophores obtained were essentially melatonin copies, even though diverse tri- or tetra-cyclic compounds were explored. In addition to lack of structural diversity, only few studies examined the activity of the reported melatonergic ligands in vivo. Moreover, an extensive pharmacological characterization including biopharmaceutical stability, pharmacokinetic properties, specificity toward other major receptors to name a few remained scarce. For example, many of the antagonists described were not stable in vivo, were not selective for the melatonin receptor subtype of interest, and were not fully characterized from a pharmacological standpoint. Indeed, virtual screening of large compound libraries has led to the recent discovery of potent and selective melatonin receptor agonists and partial agonists of new chemotypes. Having said this, the melatonergic field is still lacking subtype-selective melatonin receptor antagonists “active” in vivo, which are critical to our understanding of melatonin and melatonin receptors’ role in basic physiology and disease.     

Anatomical and cellular localization of melatonin MT1 and MT2 receptors in the adult rat brain

The involvement of melatonin in mammalian brain pathophysiology has received growing interest, but information about the anatomical distribution of its two G‐protein‐coupled receptors, MT₁ and MT₂, remains elusive. In this study, using specific antibodies, we examined the precise distribution of both melatonin receptors immunoreactivity across the adult rat brain using light, confocal, and electron microscopy. Our results demonstrate a selective MT₁ and MT₂ localization on neuronal cell bodies and dendrites in numerous regions of the rat telencephalon, diencephalon, and mesencephalon. Confocal and ultrastructural examination confirmed the somatodendritic nature of MT₁ and MT₂ receptors, both being localized on neuronal membranes. Overall, striking differences were observed in the anatomical distribution pattern of MT₁ and MT₂ proteins, and the labeling often appeared complementary in regions displaying both receptors. Somadendrites labeled for MT₁ were observed for instance in the retrosplenial cortex, the dentate gyrus of the hippocampus, the islands of Calleja, the medial habenula, the suprachiasmatic nucleus, the superior colliculus, the substantia nigra pars compacta, the dorsal raphe nucleus, and the pars tuberalis of the pituitary gland. Somadendrites endowed with MT₂ receptors were mostly observed in the CA3 field of the hippocampus, the reticular thalamic nucleus, the supraoptic nucleus, the inferior colliculus, the substantia nigra pars reticulata, and the ventrolateral periaqueductal gray. Together, these data provide the first detailed neurocytological mapping of melatonin receptors in the adult rat brain, an essential prerequisite for a better understanding of melatonin distinct receptor function and neurophysiology.     

Association of disease‐predisposition polymorphisms of the melatonin receptors and sunshine duration in the global human populations

Abstract: Melatonin is predominantly involved in signaling circadian and seasonal rhythms, and its synthesis is regulated by the environmental light/dark cycle. The selection pressure by geographically different environmental light/dark cycles, which is predominantly determined by sunshine duration, on the global distribution of genetic polymorphisms in the melatonin pathway is not well understood. Recent genetic association studies identified various disease‐predisposition polymorphisms in this pathway. We investigated the correlations between the prevalence of these clinically important single nucleotide polymorphisms (SNPs) and sunshine duration among worldwide human populations from twelve regions in the CEPH‐HGDP database rs4753426, a recently reported predisposition SNP for type 2 diabetes in the promoter of the MT₂ melatonin receptor gene (MTNR1B), which was not included in the CEPH‐HGDP genotyping array, was additionally genotyped. This SNP showed a marginally significant correlation in 760 CEPH‐HGDP DNA samples (r = ?0.5346, P = 0.0733), and it showed the most prominent association among the candidate melatonin pathway SNPs examined. To control for population structure, which may lead to a false positive correlation, we genotyped this SNP in a replication set of 1792 subjects from China. The correlation was confirmed among Chinese populations (r = ?0.8694, P = 0.0002), and was also statistically significant after correction of other climatic and geographical covariants in multiple regression analysis (β = ?0.907, P = 1.94 × 10^?5). Taken together, it suggests that the human melatonin signaling pathway, particularly MT₂ melatonin receptor may have undergone a selective pressure in response to global variation in sunshine duration.     

Functional MT1 and MT2 melatonin receptors in mammals

Melatonin, dubbed the hormone of darkness, is known to regulate a wide variety of physiological processes in mammals. This review describes well-defined functional responses mediated through activation of high-affinity MT₁ and MT₂ proteinteoupled receptors viewed as potential targets for drug discovery. MT₁ melatonin receptors modulate neuronal firing, arterial vasoconstriction, cell proliferation in cancer cells, and reproductive and metabolic functions. Ativation of MT₂ melatonin receptors phase shift circadian rhythms of neuronal firing in the suprachiasmatic nucleus, inhibit dopamine release in retina, induce vasodilation and inhibition of leukocyte rolling in arterial beds, and enhance immune responses. The melatonin-mediated responses elicited by activation of MT₁ and MT₂ native melatonin receptors are dependent on circadian time, duration and mode of exposure to endogenous or exogenous melatonin, and functional receptor sensitivity. Together, these studies underscore the importance of carefully linking each melatonin receptor type to specific functional responses in target tissues to facilitate the design and development of novel therapeutic agent.     

Melatonin reverses H2O2‐induced premature senescence in mesenchymal stem cells via the SIRT1‐dependent pathway

Mesenchymal stem cells (MSCs) represent an attractive source for stem cell‐based regenerative therapy, but they are vulnerable to oxidative stress‐induced premature senescence in pathological conditions. We previously reported antioxidant and antiarthritic effects of melatonin on MSCs against proinflammatory cytokines. In this study, we hypothesized that melatonin could protect MSCs from premature senescence induced by hydrogen peroxide (H₂O₂) via the silent information regulator type 1 (SIRT1)‐dependent pathway. In response to H₂O₂ at a sublethal concentration of 200 μm , human bone marrow‐derived MSCs (BM‐MSCs) underwent growth arrest and cellular senescence. Treatment with melatonin before H₂O₂ exposure cannot significantly prevent premature senescence; however, treatment with melatonin subsequent to H₂O₂ exposure successfully reversed the senescent phenotypes of BM‐MSCs in a dose‐dependent manner. This result was made evident by improved cell proliferation, decreased senescence‐associated β‐galactosidase activity, and the improved entry of proliferating cells into the S phase. In addition, treatment with 100 μm melatonin restored the osteogenic differentiation potential of BM‐MSCs that was inhibited by H₂O₂‐induced premature senescence. We also found that melatonin attenuated the H₂O₂‐stimulated phosphorylation of p38 mitogen‐activated protein kinase, decreased expression of the senescence‐associated protein p16^INK4α, and increased SIRT1. Further molecular experiments revealed that luzindole, a nonselective antagonist of melatonin receptors, blocked melatonin‐mediated antisenescence effects. Inhibition of SIRT1 by sirtinol counteracted the protective effects of melatonin, suggesting that melatonin reversed the senescence in cells through the SIRT1‐dependent pathway. Together, these findings lay new ground for understanding oxidative stress‐induced premature senescence and open perspectives for therapeutic applications of melatonin in stem cell‐based regenerative medicine.