Design and synthesis of novel orexin 2 receptor agonists with a 1,3,5­trioxazatriquinane skeleton.

A novel series of 1,3,5­trioxazatriquinane with multiple effective residues (TriMER) derivatives with amino-methylene side chains was designed and synthesized based on the docking-simulation results between orexin receptors (OXRs) and TriMER-type OXR antagonists. In vitro screening against orexin receptors identified six TriMER derivatives with a cis side-chain configuration, and, among these, 20d and 28d showed full agonist activity against OX2R at a concentration of 10 µM. To determine the absolute stereochemistry of these hit compounds, we also conducted the first asymmetric synthesis of a 1,3,5­trioxazatriquinane skeleton using a Katsuki-Sharpless asymmetric epoxidation as the key reaction and obtained a set of the individual stereoisomers. After evaluating their activity, (+)-20d (EC50 = 3.87 µM for OX2R) and (+)-28d (EC50 = 1.62 µM for OX2R) were determined as eutomers for OX2R agonist activity. Our results provide a new class of skeleton consisting of an (R)-1,3,5­trioxazatriquinane core with flexible methylene linkers and hydrophobic substituents at the terminals of the side chains via carbamates/sulfonamides as OX2R agonists.

Discovery of orexin 2 receptor selective and dual orexin receptor agonists based on the tetralin structure: Switching of receptor selectivity by chirality on the tetralin ring.

A novel series of 1-amino-tetralin derivatives were designed and synthesized based on the putative binding mode of the naphthalene-type orexin receptor agonist 5 and their agonist activities against orexin receptors were evaluated. The introduction of N-methyl-(3-methoxyphenyl)acetamide unit onto the 1-amino-tetralin skeleton remarkably enhanced the potency of the agonist. The asymmetric synthesis of 6 revealed that (-)-6 having a (S)-1-amino-tetralin skeleton showed a OX2R selective agonist activity (EC50 = 2.69 nM for OX2R, OX1R/OX2R = 461) yet its enantiomer (R)-(+)-6 showed a potent OX1/2R dual agonist activity (EC50 = 13.5 nM for OX1R, 0.579 nM for OX2R, OX1R/OX2R = 23.3). These results suggested that upward orientation of the amide side chain against the tetralin scaffold (S-configuration) would be selective for OX2R activation, and the downward orientation (R-configuration) would be significant for dual agonist activity. To our best knowledge, there have been no reports thus far that the stereochemistry of one carbon center on the agonist structure regulates the orexin receptor selectivity. Our results would provide important information for the development of OX1R selective agonists.

Design and synthesis of novel orexin 2 receptor agonists based on naphthalene skeleton.

A novel series of naphthalene derivatives were designed and synthesized based on the strategy focusing on the restriction of the flexible bond rotation of OX2R selective agonist YNT-185 (1) and their agonist activities against orexin receptors were evaluated. The 1,7-naphthalene derivatives showed superior agonist activity than 2,7-naphthalene derivatives, suggesting that the bent form of 1 would be favorable for the agonist activity. The conformational analysis of 1,7-naphthalene derivatives indicated that the twisting of the amide unit out from the naphthalene plane is important for the enhancement of activity. The introduction of a methyl group on the 2-position of 1,7-naphthalene ring effectively increased the activity, which led to the discovery of the potent OX2R agonist 28c (EC50 = 9.21 nM for OX2R, 148 nM for OX1R). The structure-activity relationship results were well supported by a comparison of the docking simulation results of the most potent derivative 28c with an active state of agonist-bound OX2R cryo-EM SPA structure. These results suggested important information for understanding the active conformation and orientation of pharmacophores in the orexin receptor agonists, which is expected as a chemotherapeutic agent for the treatment of narcolepsy.

Brain site-specific regulation of hedonic intake by orexin and DYN peptides: role of the PVN and obesity.

The orexin peptides promote hedonic intake and other reward behaviors through different brain sites. The opioid dynorphin peptides are co-released with orexin peptides but block their effects on reward in the ventral tegmental area (VTA). We previously showed that in the paraventricular hypothalamic nucleus (PVN), dynorphin and not orexin peptides enhance hedonic intake, suggesting they have brain-site-specific effects. Obesity alters the expression of orexin and dynorphin receptors, but whether their expression across different brain sites is important to hedonic intake is unclear. We hypothesized that hedonic intake is regulated by orexin and dynorphin peptides in PVN and that hedonic intake in obesity correlates with expression of their receptors. Here we show that in mice, injection of DYN-A1-13 (an opioid dynorphin peptide) in the PVN enhanced hedonic intake, whereas in the VTA, injection of OXA (orexin-A, an orexin peptide) enhanced hedonic intake. In PVN, OXA blunted the increase in hedonic intake caused by DYN-A1-13. In PVN, injection of norBNI (opioid receptor antagonist) reduced hedonic intake but a subsequent OXA injection failed to increase hedonic intake, suggesting that OXA activity in PVN is not influenced by endogenous opioid activity. In the PVN, DYN-A1-13 increased the intake of the less-preferred food in a two-food choice task. In obese mice fed a cafeteria diet, orexin 1 receptor mRNA across brain sites involved in hedonic intake correlated with fat preference but not caloric intake. Together, these data support that orexin and dynorphin peptides regulate hedonic intake in an opposing manner with brain-site-specific effects.

Synthesis of [(11)C]MK-1064 as a new PET radioligand for imaging of orexin-2 receptor.

The reference standard MK-1064 {5″-chloro-N-((5,6-dimethoxypyridin-2-yl)methyl)-[2,2':5',3″-terpyridine]-3'-carboxamide} was synthesized from methyl 2-chloro-5-iodonicotinate and 5-(chloropyridin-3-yl)boronic acid in 4 steps with 33% overall chemical yield. The precursor desmethyl-MK-1064 {5″-chloro-N-((5-hydroxy-6-methoxypyridin-2-yl)methyl)-[2,2':5',3″-terpyridine]-3'-carboxamide} for radiolabeling was synthesized from 2-bromopyridin-3-ol and 5″-chloro-[2,2':5',3″-terpyridine]-3'-carboxylic acid in 6 steps with 17% overall chemical yield. The target tracer [(11)C]MK-1064 {5″-chloro-N-((5-[(11)C]methoxy-6-methoxypyridin-2-yl)methyl)-[2,2':5',3″-terpyridine]-3'-carboxamide} was prepared by O-[(11)C]methylation of its corresponding precursor desmethyl-MK-1064 with [(11)C]CH3OTf under basic condition and isolated by a simplified solid-phase extraction (SPE) method in 50-60% decay corrected radiochemical yields based on [(11)C]CO2 at end of bombardment (EOB). The overall synthesis time from EOB was 23min, the radiochemical purity was >99%, and the specific activity at end of synthesis (EOS) was 185-555GBq/µmol.

Orexin/hypocretin activates mTOR complex 1 (mTORC1) via an Erk/Akt-independent and calcium-stimulated lysosome v-ATPase pathway.

The lack of the neuropeptide orexin, also known as hypocretin, results in narcolepsy, a chronic sleep disorder characterized by frequent sleep/cataplexy attacks and rapid eye movement sleep abnormalities. However, the downstream pathways of orexin signaling are not clearly understood. Here, we show that orexin activates the mTOR pathway, a central regulator of cell growth and metabolism, in the mouse brain and multiple recombinant cell lines that express the G protein-coupled receptors (GPCRs), orexin 1 receptor (OX1R) or orexin 2 receptor (OX2R). This orexin/GPCR-stimulated mTOR activation is sensitive to rapamycin, an inhibitor of mTOR complex 1 (mTORC1) but is independent of two well known mTORC1 activators, Erk and Akt. Rather, our studies indicate that orexin activates mTORC1 via extracellular calcium influx and the lysosome pathway involving v-ATPase and Rag GTPases. Moreover, a cytoplasmic calcium transient is sufficient to mimic orexin/GPCR signaling to mTORC1 activation in a v-ATPase-dependent manner. Together, our studies suggest that the mTORC1 pathway functions downstream of orexin/GPCR signaling, which plays a crucial role in many physiological and metabolic processes.

Knockdown of type 2 orexin receptor in adult mouse testis potentiates testosterone production and germ cell proliferation.

Orexins (OXs) are neuropeptides which regulate various physiological processes. OXs exist in two different forms, mainly orexin A (OXA) and orexin B (OXB) and their effects are mediated via OX1R and OX2R. Presence of OXB and OX2R in mouse testis is also reported. However, the role of OXB/OX2R in the male gonad remains unexplored. Herein we investigated the role of OXB/OX2R system in testicular physiology under in vivo and ex vivo conditions. Adult mice were given a single dose of bilateral intratesticular injection of siRNA targeting OX2R and were sacrificed 96 h post-injection. OX2R-knockdown potentiated serum and intratesticular testosterone levels with up-regulation in the expressions of major steroidogenic proteins. Germ cell proliferation also increased in siRNA-treated mice. Results of the ex vivo experiment also supported the findings of the in vivo study. In conclusion, OX2R may regulate testosterone production and thereby control the fine-tuning between steroidogenesis and germ cell dynamics.

The orexin receptor 2 (OX2R)-selective agonist TAK-994 increases wakefulness without affecting cerebrospinal fluid orexin levels in cynomolgus monkeys.

Orexin A (OX-A) and orexin B are neuropeptides produced in orexin neurons located in the lateral hypothalamus that exert multiple biological functions through the activation of orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R) throughout the central nervous system. OX1R and OX2R have distinct functions: OX1R is involved in reward seeking, whereas OX2R has a pivotal role in sleep/wake regulation. OX2R-selective agonists are in development as novel therapeutic agents for the treatment of hypersomnia. However, their potential to induce orexin release, which may indirectly stimulate both OX1R and OX2R in vivo, is unclear. Herein, we assessed the effects of the OX2R-selective agonist TAK-994 on wakefulness and orexin release in monkeys. Oral administration of TAK-994 at 10 mg/kg in the beginning of the sleep phase (zeitgeber time [ZT] 12) significantly increased wakefulness time in monkeys but did not increase OX-A levels in monkey cisternal cerebrospinal fluid (CSF). Moreover, oral administration of TAK-994 (10 mg/kg) during the active phase (ZT1) did not increase OX-A levels in monkey CSF. These findings indicate that the OX2R agonist TAK-994 selectively activates OX2R in vivo and would not robustly induce spontaneous orexin release during the daytime or nighttime in monkeys.

Jingqianshu granules mitigates premenstrual depression by regulating orexin signaling.

Introduction: Premenstrual dysphoric disorder (PMDD), a severe form of premenstrual syndrome (PMS), is a serious health disorder that affects patient moods. It is caused by cyclic psychological symptoms and its pathogenesis is still unclear. Abnormalities in the basolateral amygdala (BLA) orexin system, which are important causes of the development of depressive mood, have not been reported in PMDD, so exploring its intrinsic mechanisms is meaningful for enriching the pathomechanisms of PMDD. Methods: High performance liquid chromatography was used for the determination of the active ingredients of Jingqianshu granules. Developing a rat model of premenstrual depression using the forced swimming test (FST). The experiment consisted of two parts. In Part 1, the rats were divided into the control group, the model group, the model + Jingqianshu group, and the model + fluoxetine group. The FST, open field test, and elevated plus maze test, were used to assess the behavior of the rats as well as to evaluate the effect of drug intervention. Immunofluorescence and RT-qPCR were used to detect the expression of orexin and its receptors OX1R and OX2R genes and proteins. The expression of Toll-like receptor 4, nuclear factor kappa-B, tumor necrosis factor-α, interleukin 6, and interleukin-1ß in the BLA brain region was detected by Western-Blot. In part 2, the rats were injected intracerebrally with orexin-A. Observe the behavioral activities of rats in the control group, model group, and model+orexin-A group. Immunofluorescence was used to detect microglia in the BLA area of rats, and the expression levels of the above inflammatory factors were detected by Western-Blot. Results: The five components of Jingqianshu granules are: paeoniflorin, erulic acid, liquiritin, hesperidin, and paeonol. During the estrous cycle, rats exhibited depressive-like behavior during the non-receptive phase of the behavioral test, which disappeared during the receptive phase. Immunofluorescence and RT-qPCR showed reduced gene and protein expression of orexin, OX1R, and OX2R in the BLA region of rats in the model group.WB showed elevated levels of inflammatory factors. All returned to control levels after drug treatment. In part 2, injection of orexin-A into the BLA brain region of model rats resulted in reduced immunoreactivity of microglia and decreased expression levels of inflammatory factors. Discussion: Jianqianshu granules can achieve the purpose of treating premenstrual depression by regulating orexin-mediated inflammatory factors, which provides a new idea for further research on the pathogenesis of PMDD. However, the current study is still preliminary and the pathogenesis of PMDD is complex. Therefore, more in-depth exploration is needed.

Interaction modes of human orexin 2 receptor with selective and nonselective antagonists studied by NMR spectroscopy.

Orexin neuropeptides have many physiological roles in the sleep-wake cycle, feeding behavior, reward demands, and stress responses by activating cognitive receptors, the orexin receptors (OX1R and OX2R), distributed in the brain. There are only subtle differences between OX1R and OX2R in the orthosteric site, which has hindered the rational development of subtype-selective antagonists. In this study, we utilized solution-state NMR to capture the structural plasticity of OX2R labeled with 13CH3-ε-methionine in complex with antagonists. Mutations in the orthosteric site allosterically affected the intracellular tip of TM6. Ligand exchange experiments with the subtype-selective EMPA and the nonselective suvorexant identified three methionine residues that were substantially perturbed. The NMR spectra suggested that the suvorexant-bound state exhibited more structural plasticity than the EMPA-bound state, which has not been foreseen from the close similarity of their crystal structures, providing insights into dynamic features to be considered in understanding the ligand recognition mode.

Localization and expression of Orexin B (OXB) and its type 2 receptor (OX2R) in mouse testis during postnatal development.

The orexins (OXs) were first reported in hypothalamus of rat, and they play an important role in diverse physiological actions. The OXs consist of orexin A (OXA) and orexin B (OXB) peptides and their actions are mediated via two G-protein-coupled receptors, orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R), respectively. Presence of OXA and OX1R has been also reported in peripheral organs like reproductive tissues. These findings, therefore, highlight a possible role of OXs and their receptors in male reproductive health. Though, expression and localization of OXB and OX2R in the testis and their role in spermatogenesis are not finally clarified. Herein, we elucidated the localization and the patterns of expression of OXB and OX2R in Parkes mice testes during postnatal development. Results suggest that the precursor prepro-orexin (PPO), OXB and OX2R are expressed at the transcript and protein levels in mouse testis throughout the postnatal development. Immunostaining further showed the localization of OXB and OX2R both in interstitium and tubular compartments of the testis. On 7 day postpartum (7 dpp), only spermatogonia showed immunoreactivity of OXB and OX2R, while at 14, 28, 42 and 90 dpp, immunolocalization of OXB and OX2R were noted in the seminiferous tubules, especially in leptotene, pachytene spermatocytes, round and elongating spermatids, and in Leydig cells and Sertoli cells. The immunoreactivity of OXB and OX2R appeared to be stage-specific in adult mouse testis. The results suggest the expression of OXB and OX2R in mouse testis and their possible regulatory role in spermatogenesis and steroidogenesis.

The present and future of synthetic orexin receptor agonists.

The neuropeptide orexin/hypocretin plays a crucial role in various physiological processes, including the regulation of sleep/wakefulness, appetite, emotion and the reward system. Dysregulation of orexin signaling has been implicated in hypersomnia, especially in narcolepsy, which is a chronic neurological disorder characterized by excessive daytime sleepiness (EDS), sudden loss of muscle tone while awake (cataplexy), sleep paralysis, and hallucinations. Small-molecule orexin receptor agonists have emerged as promising therapeutics for these disorders, and significant progress has been made in this field in the past decade. This review summarizes recent advances in the design and synthesis of orexin receptor agonists, with a focus on peptidic and small-molecule OX2R-selective, dual, and OX1R-selective agonists. The review discusses the key structural features and pharmacological properties of these agonists, as well as their potential therapeutic applications.

Internal and external modulation factors of the orexin system (REVIEW).

Orexin-A and -B (identical to hypocretin-1 and -2) are neuropeptides synthesized in the lateral hypothalamus and perifornical area, and orexin neurons project their axon terminals broadly throughout the entire central nervous system (CNS). The activity of orexins is mediated by two specific G protein-coupled receptors (GPCRs), termed orexin type1 receptor (OX1R) and orexin type2 receptor (OX2R). The orexin system plays a relevant role in various physiological functions, including arousal, feeding, reward, and thermogenesis, and is key to human health. Orexin neurons receive various signals related to environmental, physiological, and emotional stimuli. Previous studies have reported that several neurotransmitters and neuromodulators influence the activation or inhibition of orexin neuron activity. In this review, we summarize the modulating factors of orexin neurons in the sleep/wake rhythm and feeding behavior, particularly in the context of the modulation of appetite, body fluids, and circadian signaling. We also describe the effects of life activity, behavior, and diet on the orexin system. Some studies have observed phenomena that have been verified in animal experiments, revealing the detailed mechanism and neural pathway, while their applications to humans is expected in future research.

Relationship between APOE, PER2, PER3 and OX2R Genetic Variants and Neuropsychiatric Symptoms in Patients with Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by the presence of neuropsychiatric or behavioral and psychological symptoms of dementia (BPSD). BPSD have been associated with the APOE_ε4 allele, which is also the major genetic AD risk factor. Although the involvement of some circadian genes and orexin receptors in sleep and behavioral disorders has been studied in some psychiatric pathologies, including AD, there are no studies considering gene-gene interactions. The associations of one variant in PER2, two in PER3, two in OX2R and two in APOE were evaluated in 31 AD patients and 31 cognitively healthy subjects. Genotyping was performed using real-time PCR and capillary electrophoresis from blood samples. The allelic-genotypic frequencies of variants were calculated for the sample study. We explored associations between allelic variants with BPSD in AD patients based on the NPI, PHQ-9 and sleeping disorders questionnaires. Our results showed that the APOE_ε4 allele is an AD risk variant (p = 0.03). The remaining genetic variants did not reveal significant differences between patients and controls. The PER3_rs228697 variant showed a nine-fold increased risk for circadian rhythm sleep-wake disorders in Mexican AD patients, and our gene-gene interaction analysis identified a novel interaction between PERIOD and APOE gene variants. These findings need to be further confirmed in larger samples.

Characteristic structural difference between inactive and active states of orexin 2 receptor determined using molecular dynamics simulations.

To investigate the dynamics of the orexin 2 receptor, which is a class A G protein-coupled receptor, we recently performed several microsecond-scale molecular dynamics simulations of the wild-type protein, of a mutant that stabilizes the inactive state, and of constitutively active mutants of the class A G protein-coupled receptors. Herein, we review the results of these molecular dynamics simulations of the orexin 2 receptor. In these simulations, characteristic conformational changes were observed in the V3096.40Y mutant. The conformational changes were related to the outward movement of the transmembrane helix 6 and the inward movement of the transmembrane helix 7, which are common structural changes in the activation of G protein-coupled receptors. The index for the quantitative evaluation of the active and inactive states of class A G protein-coupled receptors and the mechanism of the inward movement of the transmembrane helix 7 were examined. In this review, we also discuss the activation mechanism by comparing the structures obtained from the molecular dynamics simulations with the structure of the active state of the orexin 2 receptor clarified by cryo-electron microscopy in the recent years.

[Increase in the level of orexin receptor 1 (OX1R) mRNA in the brain structures of rats prone to impulsivity in behavior]. / Povyshenie urovnia mRNK retseptora oreksina pervogo tipa (OX1R) v strukturakh golovnogo mozga u krys, sklonnykh k impul'sivnosti v povedenii.

Orexin and its receptors are involved in the mechanisms of pathological craving for alcohol and psychoactive drugs. The orexin system is also involved in the mechanisms of non-chemical forms of addiction: binge eating and gambling. The aim of this work was to study the level of orexin receptor mRNA in the hypothalamus, hippocampus, and prefrontal cortex of rats prone to impulsivity in behavior in a model for studying the elements of gambling addiction (a variant of the Iowa Gambling Task test). Brain structures were isolated on the 22nd day of the experiment. The expression of the OX1R gene was higher in the hypothalamus by 122% and in the hippocampus by 149% in rats that preferred to receive a high reward, but with a low probability as compared with a group of animals that preferred a low level of reinforcement, but with a 100% probability. In the prefrontal cortex, on the contrary, no significant changes were observed in the level of OX1R mRNA. The level of OX2R mRNA insignificantly changed in the hypothalamus, hippocampus, and prefrontal cortex of rats prone to impulsivity in behavior. The data indicate involvement of OX1R in the hypothalamus and hippocampus in mechanisms mediating impulsive behavior and the choice of the significance of positive reinforcement in terms of its varying strength and probability.

Soporific effect of modified Suanzaoren Decoction on mice models of insomnia by regulating Orexin-A and HPA axis homeostasis.

AIM: Modified Suanzaoren Decoction (MSZRD) is obtained by improving Suanzaoren Decoction (SZRT), a traditional Chinese herbal prescription that has been used to treat insomnia for more than thousands of years. Our previous study showed that MSZRD can improve the gastrointestinal discomfort related insomnia by regulating Orexin-A. This study is the first study to evaluate the effects and possible mechanisms of MSZRD in mice with insomnia caused by p-chlorophenylalanine (PCPA) combined with multifactor random stimulation. METHODS: After 14 days of multifactor stimulation to ICR mice, a PCPA suspension (30 mg/mL) was injected intraperitoneally for two consecutive days to establish an insomnia model. Three different doses of MSZRD (3.6, 7.2, and 14.4 g/kg/day) were given to ICR mice for 24 days. The food intake and back temperature were measured, and behavioral tests and pentobarbital sodium-induced sleep tests were conducted. The levels of Orexin-A, corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and adrenocortical hormones (CORT) in the serum and 5-hydroxytryptamine (5-HT), dopamine (DA), and norepinephrine (NE) in hypothalamus were measured using enzyme-linked immunosorbent assay (ELISA) kits. The levels of γ-aminobutyric acid (GABA) and glutamic acid (Glu) were measured by high-performance liquid chromatography (HPLC). The expression of 5HT1A receptor (5-HTRIA) and orexin receptor 2 antibody (OX2R) was measured by Western blot (WB) and immunohistochemical staining (ICH). Hematoxylin and eosin (H&E) staining and Nissl staining were used to assess the histological changes in hypothalamus tissue. RESULTS: Of note, MSZRD can shorten the sleep latency of insomnia mice (P < 0.05, 0.01), prolonged the sleep duration of mice (P < 0.05, 0.01), and improve the circadian rhythm disorder relative to placebo-treated animals. Furthermore, MSZRD effectively increased the content of 5-HT and 5-HTR1A protein in the hypothalamus of insomnia mice (P < 0.05, 0.01), while downregulated the content of DA and NE (P < 0.05, 0.01). Importantly, serum GABA concentration was increased by treatment with MSZRD (P < 0.05), as reflected by a decreased Glu/GABA ratio (P < 0.05). Moreover, MSZRD decreased the levels of CORT, ACTH, and CRH related hormones in HPA axis (P < 0.05, 0.01). At the same time, MSZRD significantly downregulated the serum Orexin-A content in insomnia mice (P < 0.05), as well as hypothalamic OX2R expression (P < 0.05). In addition, MSZRD also improved the histopathological changes in hypothalamus in insomnia mice. CONCLUSION: MSZRD has sleep-improvement effect in mice model of insomnia. The mechanism may be that regulating the expression of Orexin-A affects the homeostasis of HPA axis and the release of related neurotransmitters in mice with insomnia.

Evolution of Orexin Neuropeptide System: Structure and Function.

Orexins are hypothalamic neuropeptides that were initially identified in the rat brain as endogenous ligands for an (previously) orphan G-protein-coupled receptor (GPCR). They are multitasking peptides involved in many physiological functions, including regulation of feeding behavior, wakefulness and autonomic/neuroendocrine functions, and sleep/wakefulness states in mammals. There are two isopeptides of orexin, orexin A and orexin B, which are produced from a common precursor peptide, prepro-orexin. Structures of orexins, as well as orexin genes, are highly conserved throughout mammalian species, suggesting strong evolutionary pressure that maintains the structures. Their lengths and structure suggested that orexin B is the ancestral form of the orexin neuropeptide. In mammals, orexins bind to two subtypes of GPCRs, i.e., orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R). Phylogenetically, the orexin system is present exclusively in vertebrates. In genomes of species outside mammals, there is only one orexin receptor, which is similar to OX2R, suggesting that OX2R is the prototype receptor for orexins. OX1R is likely to have evolved during early mammalian evolution. Orexin-producing neurons (orexin neurons) are mainly located in the lateral hypothalamic area (LHA) in mammals and are also found in hypothalamic regions in many other vertebrates. Orexins are likely to be closely related to the regulation of active, motivated behavior in many species. The orexin system seems to have evolved as a system that supports active and purposeful behavior which is closely related with wakefulness.

Mechanisms of Memory Impairment Induced by Orexin-A via Orexin 1 and Orexin 2 Receptors in Post-traumatic Stress Disorder Rats.

Post-traumatic stress disorder (PTSD) patients exhibit abnormal learning and memory. Axons from orexin neurons in the lateral hypothalamus innervate the hippocampus, modulating learning and memory via the orexin 1 and 2 receptors (OX1R and OX2R). However, the role of the orexin system in the learning and memory dysfunction observed in PTSD is unknown. This was investigated in the present study using PTSD animal model-single prolonged stress (SPS) rats. Spatial learning and memory in the rats were evaluated with the Morris water maze (MWM) test; changes in body weight and food intake were recorded to assess changes in appetite; and the expression of orexin-A and its receptors in the hypothalamus and hippocampus was examined and quantified by immunohistochemistry, western blotting and real-time PCR. The results showed that spatial memory was impaired and food intake was decreased in SPS rats; this was accompanied by downregulation of orexin-A in the hypothalamus and upregulation of OX1R and OX2R in the hippocampus and of OX1R in the hypothalamus. Intracerebroventricular administration of orexin-A improved spatial memory and enhanced appetite in SPS rats and partly reversed the increases in OX1R and OX2R levels in the hippocampus and hypothalamus. These results suggest that the orexin system plays a critical role in the memory and appetite dysfunction observed in PTSD.

Signaling transduction regulated by 5-hydroxytryptamine 1A receptor and orexin receptor 2 heterodimers.

As G-protein-coupled receptors (GPCRs), 5-hydroxytryptamine 1A receptor (5-HT1AR) and orexin receptor 2 (OX2R) regulate the levels of the cellular downstream molecules. The heterodimers of different GPCRs play important roles in various of neurological diseases. Moreover, 5-HT1AR and OX2R are involved in the pathogenesis of neurological diseases such as depression with deficiency of hippocampus plasticity. However, the direct interaction of the two receptors remains elusive. In the present study, we firstly demonstrated the heterodimer formation of 5-HT1AR and OX2R. Exchange protein directly activated by cAMP (Epac) cAMP bioluminescence resonance energy transfer (BRET) biosensor analysis revealed that the expression levels of cellular cAMP significantly increased in HEK293T cells transfected with the two receptors compared with the 5-HT1AR group. Additionally, the cellular level of calcium was upregulated robustly in HEK293T cells co-transfected with 5-HT1AR and OX2R group after agonist treatment. Furthermore, western blotting data showed that 5-HT1AR and OX2R heterodimer decreased the levels of phosphorylation of extracellular signal-regulated kinase (ERK) and cAMP-response element-binding protein (CREB). These results not only unraveled the formation of 5-HT1AR and OX2R heterodimer but also suggested that the heterodimer affected the downstream signaling pathway, which will provide new insights into the function of the two receptors in the brain.