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Background: Allergic rhinitis (AR) is caused by allergic reaction to allergens such as pollen. Galanin (GAL), a neuropeptide that regulates inflammatory processes, is widely expressed in the central and peripheral nervous systems. Although neuropeptides are implicated in arthritis and chemically induced ileitis, their roles in AR remain unclear. Methods: We developed a murine model of AR and generated control, systemic sensitization, mild AR, and severe AR groups. We examined GAL and GAL receptor (GALR) mRNA and protein levels and localization patterns in each group using reverse transcription PCR, western blotting, and immunohistochemical analyses. Additionally, we evaluated the effects of M871, a GALR2 antagonist, on mice with severe AR. Results: Gal and Galr2 are expressed in nasal mucosa and brain (control) samples from control and AR mice. GAL and GALR2 were expressed at similar levels and localized to ciliated epithelial and submucosal gland cells of the nasal mucosa in all four groups. Intranasal M871 administration significantly reduced the incidence of nose rubbing behaviors and sneezing (p

Our and other’s studies showed that administration of neuropeptide galanin may mitigate insulin resistance via promoting glucose transporter 4 (GLUT4) expression and translocation in rats. The objective of this study is to investigate whether galanin receptor 2 (GAL2-R) in brain mediates the ameliorative effect of galanin on insulin resistance in adipose tissues of type 2 diabetic rats. In this study galanin, GAL2-R agonist M1145 and GAL2-R antagonist M871 were respectively or cooperatively injected into intracerebroventricles of type 2 diabetic rats once a day for successive fifteen days. Then the plasma and fat tissues of rats were used to estimate the alterations of insulin resistance indexes. The central administration of galanin enhanced 2-deoxy-[3H]-d-glucose, peroxisome proliferator-activated receptor γ and adiponectin levels, food intake and body weight, GLUT4 mRNA expression and GLUT4 concentration in plasma membranes, as well as homeostasis model assessment-insulin resistance index. Those effects of galanin may be blocked by M817, and imitated by M1145 except for food intake and body weight. Those results suggest that central GAL2-R mediates the beneficial effects of galanin on insulin sensitivity in type 2 diabetic rats. GAL2-R agonist may be taken as a potential antidiabetic agent to treat insulin resistance and type 2 diabetes.

Background: Cognitive disorders associated with schizophrenia are closely linked to prefrontal cortex (PFC) dysfunction. Administration of the non-competitive NMDA receptor antagonist ketamine (KET) induces cognitive impairment in animals, producing effects similar to those observed in schizophrenic patients. In a previous study, we showed that KET (20 mg/kg) induces cognitive deficits in mice and that administration of clozapine (CLZ) reverses this effect. To identify biochemical mechanisms related to CLZ actions in the context of KET-induced impairment, we performed a biochemical analysis using the same experimental paradigm—acute and sub-chronic administration of these drugs (0.3 and 1 mg/kg). Methods: Since the effect of CLZ mainly depends on G-protein-related receptors, we used the Signaling PathwayFinder Kit to identify 84 genes involved in GPCR-related signal transduction and then verified the genes that were statistically significantly different on a larger group of mice using RT-PCR and Western blot analyses after the administration of acute and sub-chronic drugs. Results: Of the 84 genes involved in GPCR-related signal transduction, the expression of six, βarrestin1, βarrestin2, galanin receptor 2 (GalR2), dopamine receptor 2 (DRD2), metabotropic glutamate receptor 1 (mGluR1), and metabotropic glutamate receptor 5 (mGluR5), was significantly altered. Since these genes affect the levels of other signaling proteins, e.g., extracellular signal-regulated kinase 1/2 (ERK1/2), G protein-coupled receptor kinase 2 (Grk2), and G protein-gated inwardly rectifying potassium 3 (Girk3), we determined their levels in PFC using Western blot. Most of the observed changes occurred after acute treatment with 0.3 mg/kg CLZ. We showed that acute treatment with CLZ at a lower dose significantly increased βarrestin1 and ERK1/2. KET treatment induced the upregulation of βarrestin1. Joint administration of these drugs had no effect on the βarrestin1 level. Conclusion: The screening kit we used to study the expression of GPCR-related signal transduction allowed us to select several important genes affected by CLZ. However, the obtained data do not explain the mechanism of action of CLZ that is responsible for reversing KET-induced cognitive impairment.

The regulatory (neuro)peptide galanin is widely distributed in the central and peripheral nervous systems, where it mediates its effects via three G protein-coupled receptors (GAL1-3R). Galanin has a vast diversity of biological functions, including modulation of feeding behavior. However, the clinical application of natural galanin is not practicable due to its rapid in vivo breakdown by peptidases and lack of receptor subtype specificity. Much effort has been put into the development of receptor-selective agonists and antagonists, and while receptor selectivity has been attained to some degree, most ligands show overlapping affinity. Therefore, we aimed to develop a novel ligand with specificity to a single galanin receptor subtype and increased stability. To achieve this, a lanthionine amino acid was enzymatically introduced into a galanin-related peptide. The residue’s subsequent cyclization created a conformational constraint which increased the peptide’s receptor specificity and proteolytic resistance. Further exchange of certain other amino acids resulted in a novel methyllanthionine-stabilized galanin receptor agonist, a G1pE-T3N-S6A-G12A-methyllanthionine[13–16]-galanin-(1–17) variant, termed M89b. M89b has exclusive specificity for GAL2R and a prolonged half-life in serum. Intranasal application of M89b to unfasted rats significantly reduced acute 24 h food intake inducing a drop in body weight. Combined administration of M89b and M871, a selective GAL2R antagonist, abolished the anorexigenic effect of M89b, indicating that the effect of M89b on food intake is indeed mediated by GAL2R. This is the first demonstration of in vivo activity of an intranasally administered lanthipeptide. Consequently, M89b is a promising candidate for clinical application as a galanin-related peptide-based therapeutic.

Background Galanin-like peptide (Galp) and alarin (Ala) are 2 new members of the galanin peptide family. Galanin (Gal), the "parental" peptide of the entire family, is known to regulate numerous physiological processes, including energy and osmotic homeostasis, reproduction, food intake, and secretion of adrenocortical hormones. Galp and Ala are known to regulate food intake. In the rat, Galp mRNA has been found in the brain, exclusively in the hypothalamic arcuate nucleus (ARC) and median eminence, which are involved in the regulation of energy homeostasis. Alarin-like immunoreactivity is present in the locus coeruleus (LC) and the ARC of rats and mice. Objectives The aim of the study was to investigate the expression of Ala, Galp and their receptors in the organs of the hypothalamo-pituitary-adrenal (HPA) axis of the rat. Material and methods The expression of the examined genes was measured in different models of adrenal growth of the rat in vivo (postnatal ontogenesis, compensatory adrenal growth, adrenocortical regeneration, adrenocorticotropic hormone (ACTH) administration). The expression was evaluated using the Affymetrix® microarray system or quantitative polymerase chain reaction (qPCR). Results The expression of Ala gene was observed in each organ of the HPA axis (the hypothalamus, hypophysis and adrenal gland). The elevated level of expression of this gene was observed in the pituitary of 2-day rats, while very low levels of Ala mRNA were observed in the adrenals. Galp mRNA expression was observed only in the hypothalamus and the hypophysis during postnatal ontogenesis. The expression of Gal receptors was demonstrated in the hypothalamus, the hypophysis and the adrenal gland. In different compartments of the adrenal glands of adult, intact male and female rats, the expression of Ala, Galp and galanin receptor 1 (Galr1) genes was negligible, but the expression of galanin receptor 2 (Galr2), galanin receptor 3 (Galr3) and neurotrophic receptor tyrosine kinase 2 (Ntrk2) genes was noticeable. Conclusions The examined genes showed different expression levels within the studied HPA axis; some of them were neither expressed in the hypothalamus or the pituitary gland, nor in the adrenal gland.

This study declared effect of spexin (SPX) on renal dysfunction in obese rats and its potential mitigating mechanisms which could mediated via galanin receptor-2 (GALR-2). Thirty two 32 Wistar male rats were arranged into four groups: control, high fat/fructose diet (HFFD), HFFD + SPX and HFFD + M871 (galanin receptor 2 antagonist)+SPX. At the termination of the experiment, urine volume, body mass index, Lee index and mean arterial blood pressure were assessed. Renal function was evaluated. Lipid profile, fasting glucose, insulin, insulin resistance and SPX levels were estimated. Also, renal histopathological, immunohistochemical and relative gene expression of renal tissue were done. Also, renal protein carbonyl, reduced glutathione, interferon gamma, monocyte chemoattractant protein-1, interleukin-10 and hydroxyproline were determined.Our results explored that SPX treatment prominently mitigated the metabolic changes and renal dysfunction induced by HFFD via GALR-2. SPX improved insulin resistance, dyslipidemia, renal oxidative stress, inflammation, apoptosis, and fibrosis. So, SPX can be considered as prospective therapeutic agent for treating renal dysfunction.

Our previous research has shown that galanin plays an antinociceptive effect via binding to galanin receptors (GalRs) in nucleus accumbens (NAc). This study focused on the involvement of GalR2 in galanin-induced antinociceptive effect in NAc of neuropathic pain rats. The chronic constriction injury of sciatic nerve (CCI) was used to mimic neuropathic pain model. The hind paw withdrawal latency (HWL) to thermal stimulation and hind paw withdrawal threshold (HWT) to mechanical stimulation were measured as the indicators of pain threshold. The results showed that 14 and 28 days after CCI, the expression of GalR2 was up-regulated in bilateral NAc of rats, and intra-NAc injection of GalR2 antagonist M871 reversed galanin-induced increases in HWL and HWT of CCI rats. Furthermore, intra-NAc injection of GalR2 agonist M1145 induced increases in HWL and HWT at day 14 and day 28 after CCI, which could also be reversed by M871. Finally, we found that M1145-induced antinociceptive effect in NAc of CCI rats was stronger than that in intact rats. These results imply that the GalR2 is activated in the NAc from day 14 to day 28 after CCI and GalR2 is involved in the galanin-induced antinociceptive effect in NAc of CCI rats.

It has been reported that galanin has an analgesic effect via activating galanin receptors (GALRs). This study focused on the involvement of GALR2 in the galanin-induced analgesic effect and its signaling mechanism in the nucleus accumbens (NAc) of inflammatory rats. Animal models were established through injecting carrageenan into the plantar of rats’ left hind paw. The results showed that GALR2 antagonist M871 weakened partially the galanin-induced increases in hind paw withdrawal latency (HWL) to thermal stimulation and hind paw withdrawal threshold (HWT) to mechanical stimulation in NAc of inflammatory rats. Moreover, the GALR2 agonist M1145 prolonged the HWL and HWT, while M871 blocked the M1145-induced increases in HWL and HWT. Western blotting showed that the phosphorylation of calcium/calmodulin-dependent protein kinase II (p-CaMKII) and protein kinase C (p-PKC) in NAc were upregulated after carrageenan injection, while p-PKC and p-CaMKII were downregulated after intra-NAc administration of M871. Furthermore, the CaMKII inhibitor KN93 and PKC inhibitor GO6983 attenuated M1145-induced increases in HWL and HWT in NAc of rats with inflammatory pain. These results prove that GALR2 is involved in the galanin-induced analgesic effect by activating CaMKII and PKC in NAc of inflammatory pain rats, implying that GALR2 agonists probably are potent therapeutic options for inflammatory pain.

The present study aimed to characterize the role of spexin (SPX) in maintaining glucose and lipid homeostasis in vivo in rats with diet-induced obesity. The in vitro effect of spexin on metabolic and endocrine functions of adipocytes isolated from obese rats was also investigated. The in vivo experiment was conducted on rats with diet-induced obesity and administered with SPX for 7 days. Lipid and carbohydrate parameters, liver markers, and hormonal profile were measured. In in vitro studies, adipocytes isolated from obese rats were used. The effect of SPX on lipolysis, lipogenesis, and leptin secretion from fat cells was assessed. The results showed that short-term administration of SPX causes weight loss, increases insulin sensitivity, and improves the metabolic state of obese rats. The in vitro experiments showed that spexin and its receptors, namely galanin receptor 2 (GALR2) and galanin receptor 3 (GALR3), were expressed in various fat depots and in adipocytes from obese rats. We also found that the addition of spexin increased the basal and isoproterenol-stimulated lipolysis and reduced the basal and insulin-stimulated lipogenesis in adipocytes isolated from obese rats. Molecular analysis showed that SPX activated hormone-sensitive lipase (HSL) phosphorylation and upregulated perilipin and HSL mRNA expression. These results suggest that SPX regulates metabolism of obese rats by affecting lipolysis and lipogenesis in adipocytes. Moreover, the present study for the first time demonstrates that SPX modulates leptin synthesis and secretion from isolated adipocytes.

The aim of this study was to assess gene expression of neurotrophins and their receptors in keloids. Skin samples of normal skin and keloids were obtained from patients in the control (n = 12) and keloid (n = 12) groups, respectively. Ribonucleic acid was extracted from the skin specimens, purified, evaluated by spectrophotometry, and used to synthesize complementary DNA. Real-time quantitative polymerase chain reaction analysis of 84 human neurotrophin genes and their receptors was performed. Twelve genes, including heat shock 27-kDa protein 1, gastrin-releasing peptide receptor, corticotropin-releasing hormone receptor 2, neuropeptide Y Y2 receptor, interleukin 6 signal transducer, nerve growth factor, metallothionein 3, B-cell chronic lymphocytic leukemia/lymphoma 2, cholecystokinin A receptor, persephin, galanin receptor 2, and fibroblast growth factor receptor 3, were down-regulated in keloid tissue compared with normal skin. The genes 27-kDa heat shock protein 1, gastrin-releasing peptide receptor, corticotropin-releasing hormone receptor 2, nerve growth factor, metallothionein 3, B-cell chronic lymphocytic leukemia/lymphoma 2, and persephin protein were considered priority genes associated with keloid formation.

Summary There exists solid evidence that endogenous galanin and galanin agonists exert anticonvulsive actions mediated both by galanin 1 receptor (GAL1‐R) and galanin 2 receptor (GAL2‐R). We have now investigated whether depletion of the recently identified third galanin receptor, GAL3‐R, and that of GAL2‐R, alters the threshold to the systemically applied γ‐aminobutyric acid (GABA) antagonist pentylenetetrazole (PTZ) or to intrahippocampally administered kainic acid (KA). In neither model, GAL3‐KO mice differed in their latency to the first seizure, mean seizure duration, total number of seizures, or time spent in seizures compared to wild‐type controls. In addition, consistent with previous data, the response to PTZ was not altered in GAL2‐KO mice. In contrast, intrahippocampal KA resulted in a significantly increased number of seizures and time spent in seizures in GAL2‐KO mice, although the latency to the first seizure and the duration of individual seizures was not altered. These results are consistent with the previous data showing that GAL2‐R knockdown does not affect the number of perforant path stimulations required for initiating status epilepticus but significantly increases the seizure severity during the ongoing status. In conclusion, our data support a specific role of GAL2‐R but not of GAL3‐R in mediating the anticonvulsive actions of endogenous galanin.

The results of our and other's studies showed that activation of galanin receptor 1 could mitigate insulin resistance via promoting glucose transporter 4 (GLUT4) expression and translocation in the skeletal muscle of rats. But no literature are available regarding the effect of galanin receptor 2 (GALR2) on insulin resistance in skeletal muscle of type 2 diabetes. Herein, in this study we intended to survey the effect of GALR2 and its signal mechanisms in the mice with high fat diet-induced obese. The mice were intraperitoneally injected with vehicle, GALR2 agonist M1145 and antagonist M871 respectively once a day for continuous 21 days. The skeletal muscles were processed for determination of glucose uptake, and GLUT4 mRNA and protein expression levels. The PGC-1α, AKT, p38MAPK, AS160, pAKT, pP38MAPK and pAS160 expression levels were quantitatively assessed too. We found that pharmacological activation of GALR2 enhanced energy expenditure, and increased GLUT4 expression and translocation in skeletal muscle of mice during high-fat diet regimens. Activation of GALR2 alleviated insulin resistance through P38MAPK/PGC-1α/GLUT4 and AKT/AS160/GLUT4 pathway in the skeletal muscle of mice. Overall, these results identify that GALR2 is a regulator of insulin resistance and activation of GALR2 represents a promising strategy against obesity-induced insulin resistance.

The neuropeptide galanin is distributed in the central and peripheral nervous systems and in non-neuronal peripheral organs, including the skin. Galanin acts via three G protein-coupled receptors which, except galanin receptor 1, are expressed in various skin structures. The galanin system has been associated with inflammatory processes of the skin and of several other organs. Psoriasis is an inflammatory skin disease with increased neovascularization, keratinocyte hyperproliferation, a proinflammatory cytokine milieu, and immune cell infiltration. In this study, we showed that galanin receptor 3 is present in endothelial cells in human and murine dermal vessels and is co-expressed with nestin in neo-vessels of psoriatic patients. Moreover, in a murine psoriasis model, we showed that C57/BL6 mice lacking galanin receptor 3 display a milder course of psoriasis upon imiquimod treatment, leading to decreased disease severity, delayed neo-vascularization, reduced infiltration of neutrophils, and significantly lower levels of proinflammatory cytokines compared with wild-type mice. In contrast, galanin receptor 2-knockout animals did not differ significantly from wild type mice at both the macroscopic and molecular levels in their inflammatory response to imiquimod treatment. Our data indicate that galanin receptor 3, but not galanin receptor 2, plays an important role in psoriasis-like skin inflammation.

Spexin (SPX) is a recently discovered peptide that binds to galanin receptors 2 (GalR2) and 3 (GalR3). Emerging evidence implicates SPX as a modulator of satiety and energy balance as circulating SPX levels are dysregulated in obesity. However, it is currently unknown how SPX and its receptors are regulated in the hypothalamus, a brain region critical for energy homeostasis. As protein kinase signalling is involved in the regulation of many hypothalamic neuropeptides and receptors, we hypothesized that SPX and its receptors would be regulated by at least one of the protein kinases: PKA, PKC or PKG. We therefore examined hypothalamic Spx, GalR2 and GalR3 mRNA expression in response to several protein kinase activators in a clonal adult-derived hypothalamic cell model, mHypoA-59, using quantitative reverse transcription PCR (qRT-PCR). Treatments over 24 hours revealed that Spx, GalR2 and GalR3 mRNA were all induced with 100 µM sodium nitroprusside (SNP), a nitric oxide donor that can activate the PKG pathway. However, direct stimulation of PKG with increasing concentrations of 8-Bromo-cGMP failed to induce Spx, GalR2 or GalR3 suggesting that the regulation of these genes by nitric oxide was independent of the canonical cGMP/PKG pathway. Characterization of the 5’ regulatory region of Spx led to identification of putative response elements for C/EBP-β and OCT-1. Interestingly, treatment with 100 µM SNP resulted in the induction of C/ebp-β, but not Oct-1 mRNA. Subsequent chromatin immunoprecipitation studies revealed that 100 µM SNP enhanced binding of C/EBP-β, but not OCT-1 to the 5’ regulatory region of Spx, suggesting that increased binding of C/EBP-β is involved in the induction of Spx mRNA in the response to SNP. The results thus far suggest that nitric oxide induces Spx, GalR2 and GalR3 mRNA independently of PKG and that C/EBP-β may be involved in the induction of Spx. Elucidating the pathways that regulate gene expression of SPX and its receptors in the hypothalamus will contribute to current models of energy balance and provide insight into how components of this regulatory circuit may be dysregulated in obesity.

Since foods with high hedonic value are often consumed in excess of energetic needs, this study was designed to identify the mechanisms that may counter anorexigenic signalling in the presence of hedonic foods in lean animals.Mice, in different states of satiety (fed/fasted, or fed/fasted and treated with ghrelin or leptin, respectively), were allowed to choose between high-fat/high-sucrose and standard foods. Intake of each food type and the activity of hypothalamic neuropetidergic neurons that regulate appetite were monitored. In some cases, food choice was monitored in leptin-injected fasted mice that received microinjections of galanin receptor agonists into the lateral hypothalamus.Appetite-stimulating orexin neurons in the lateral hypothalamus are rapidly activated when lean, satiated mice consume a highly palatable food (PF); such activation (upregulated c-Fos expression) occurred even after administration of the anorexigenic hormone leptin and despite intact leptin signalling in the hypothalamus. The ability of leptin to restrain PF eating is restored when a galanin receptor 2 (Gal2R) agonist is injected into the lateral hypothalamus.Hedonically-loaded foods interrupt the inhibitory actions of leptin on orexin neurons and interfere with the homeostatic control of feeding. Overeating of palatable foods can be curtailed in lean animals by activating Gal2R in the lateral hypothalamus. This article is protected by copyright. All rights reserved.

Galanin is an endogenous factor involved in the negative regulation of the biological effects of leptin in bioenergetic metabolism. Leptin promotes fibrogenic effects in hepatic stellate cells (HSCs), however, little is known about the effects of galanin on HSCs. In the present study, the biological functions of galanin and its receptors (GalRs) in HSCs were investigated using cell culture in vitro. It was found that galanin and GalR3 mRNA are expressed in quiescent and activated HSCs. GalR2 expression was undetectable in quiescent HSCs but was markedly induced in activated HSCs. In the HSC-T6 cell line, which is an activated rat HSC cell line, treatment with 100 nmol/l galanin significantly inhibited cell proliferation. It also inhibited transforming growth factor (TGF)-β1 and α-smooth muscle actin (SMA) expression and upregulated peroxisome proliferator-activated receptor (PPAR)-γ expression. Following the knockdown of GalR2 by specific small interfering RNA, the activation of GalR3 by galanin does not influence these effects of galanin on HSCs. However, activation of GalR2 alone by galanin following the knockdown of GalR3 inhibits HSC proliferation and TGF-β1 and α-SMA expression, in addition to inducing PPAR-γ expression. These data suggest that galanin inhibits HSC activation and suppresses the profibrogenic features of these cells, and these effects might be mediated by GalR2. Thus, galanin is a potential endogenous factor in the inhibition of liver fibrosis.

Galanin receptor 2 (GAL2R) is a G protein-coupled receptor for the neuropeptide galanin that regulates many important physiological functions and pathological processes. To investigate the molecular mechanism governing GAL2R gene transcription, the rat GAL2R promoter was isolated and analyzed. We found that the region from -320 to -300 of the GAL2R promoter contains two putative ETS-1 elements and plays an important role in regulating GAL2R promoter activity. We also showed that transcription factor ETS-1 bound to this region in vitro and in vivo. Overexpression of ETS-1 significantly increased GAL2R promoter activity and transcription of the GAL2R gene, whereas knockdown of ETS-1 produced the opposite effects. In addition, we showed that ETS-1 recruited co-activator p300 to the GAL2R promoter. These data indicate a role for ETS-1 in the control of the GAL2R gene expression and provide a basis for understanding the transcriptional regulation of the GAL2R gene.

The function of the gonadotropin-releasing hormone (GnRH) neuron is critical to maintain reproductive function and a significant decrease in GnRH can lead to disorders affecting fertility, including hypogonadotropic hypogonadism. Spexin (SPX) is a novel hypothalamic neuropeptide that exerts inhibitory effects on reproduction and feeding by acting through galanin receptor 2 (GALR2) and galanin receptor 3 (GALR3). Fatty acids can act as nutritional signals that regulate the hypothalamic-pituitary-gonadal (HPG) axis, and elevated levels of circulating saturated fatty acids associated with high fat diet (HFD)-feeding have been shown to induce neuroinflammation, endoplasmic reticulum stress and hormonal resistance in the hypothalamus, as well as alter neuropeptide expression. We previously demonstrated that palmitate, the most common saturated fatty acid in a HFD, elevates the expression of Spx, Galr2 and Galr3 mRNA in a model of appetite-regulating neuropeptide Y hypothalamic neurons. Here, we found that Spx, Galr2 and Galr3 mRNA were also significantly induced by palmitate in a model of reproductive GnRH neurons, mHypoA-GnRH/GFP. As a follow-up to our previous report, we examined the molecular pathways by which Spx and galanin receptor mRNA was regulated in this cell line. Furthermore, we performed inhibitor studies, which revealed that the effect of palmitate on Spx and Galr3 mRNA involved activation of the innate immune receptor TLR4, and we detected differential regulation of the three genes by the protein kinases PKC, JNK, ERK, and p38. However, the intracellular metabolism of palmitate to ceramide did not appear to be involved in the palmitate-mediated gene regulation. Overall, this suggests that SPX may play a role in reproduction at the level of the hypothalamus and the pathways by which Spx, Galr2 and Galr3 are altered by fatty acids could provide insight into the mechanisms underlying reproductive dysfunction in obesity.

The neuropeptide galanin functions via three G-protein coupled receptors, Gal1-3-R. Both Gal1-R and 2-R are involved in pain signaling at the spinal level. Here a Gal2-R-EGFP transgenic (TG) mouse was generated and studied in pain tests and by characterizing Gal2-R expression in both sensory ganglia and spinal cord. After peripheral spared nerve injury, mechanical allodynia developed and was ipsilaterally similar between wild type (WT) and TG mice. A Gal2-R-EGFP-positive signal was primarily observed in small and medium-sized dorsal root ganglion (DRG) neurons and in spinal interneurons and processes. No significant difference in size distribution of DRG neuronal profiles was found between TG and WT mice. Both percentage and fluorescence intensity of Gal2-R-EGFP-positive neuronal profiles were overall significantly upregulated in ipsilateral DRGs as compared to contralateral DRGs. There was an ipsilateral reduction in substance P-positive and calcitonin gene-related peptide (CGRP)-positive neuronal profiles, and this reduction was more pronounced in TG as compared to WT mice. Moreover, Gal2-R-EGFP partly co-localized with three pain-related neuropeptides, CGRP, neuropeptide Y and galanin, both in intact and injured DRGs, and with galanin also in local neurons in the superficial dorsal horn. Taken together, the present results provide novel information on the localization and phenotype of DRG and spinal neurons expressing the second galanin receptor, Gal2-R, and on phenotypic changes following peripheral nerve injury. Gal2-R may also be involved in autoreceptor signaling.

ETS-1 is a transcription factor that is a member of the E26 transformation-specific (ETS) family. Galanin receptor 2 (GalR2), a subtype of receptors of the neuropeptide galanin, has been shown to have an antidepressant-like effect after activation in rodents. Our previous study has shown that overexpression of ETS-1 increases the expression of GalR2 in PC12 phaeochromocytoma cells. However, whether ETS-1 has an antidepressant-like effect is still unclear. In this study, we found that chronic mild stress (CMS) decreased the expression of both ETS-1 and GalR2 in the ventral hippocampus of rats. Meanwhile, we demonstrated that overexpression of ETS-1 increased the expression of GalR2 in primary hippocampal neurons. Importantly, we showed that overexpression of ETS-1 in the ventral hippocampus counteracted the depression-like behaviors of CMS rats. Furthermore, we found that overexpression of ETS-1 increased the level of downstream phosphorylated extracellular signal-regulated protein kinases 1 and 2 (p-ERK1/2) of GalR2 in the ventral hippocampus of CMS rats. Taken together, our findings suggest that ETS-1 has an antidepressant-like effect in rats, which might be mediated by increasing the level of GalR2 and its downstream p-ERK1/2 in the ventral hippocampus. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12264-019-00412-6) contains supplementary material, which is available to authorized users.

Glucocorticoids (GCs) can affect hippocampal structure and function in animals and humans. This study was designed to investigate the possible functional molecules and mechanisms involved in the action of GCs on hippocampal neurons. Rat primary hippocampal neurons were cultured and treated with glucocorticoids at a low concentration (LC, 10-8 mol/L), a middle concentration (MC, 10-7 mol/L) and a high concentration (HC, 10-6 mol/L). The results indicate that GCs do not change the viability of hippocampal neurons but do change the catalase (CAT) activity and malondialdehyde (MDA) content. The transcription expression levels of brain derived neurotrophic factor (BDNF), galanin (GAL), galanin receptor-2 (GALR2), and neuropeptide Y receptor-5 (NPYR5) genes in the HC group were significantly higher than those in the control group (p less than 0.05). These results suggest that hippocampal neurons launch the neuron protection pathways mediated by GAL, GALR2 and BDNF molecules when encountering an experimentally high concentration of corticosteroids.

Discovery of biased ligands and receptor mutants allows characterization of G-protein- and β-arrestin-mediated signaling mechanisms of G-protein-coupled receptors (GPCRs). However, the structural mechanisms underlying biased agonism remain unclear for many GPCRs. We show that while Galanin induces the activation of the galanin receptor 2 (Galr2) that leads to a robust stimulation toward Gαq-protein and β-arrestin1/2, an alternative ligand Spexin and its analog have biased agonism toward G-protein signaling relative to Galanin. We used intramolecular fluorescein arsenical hairpin bioluminescence resonance energy transfer-based biosensors of β-arrestin2 combined with NanoBit technology to measure β-arrestin2–Galr2 interactions in real-time living systems. We found that Spexin and Galanin induce specific active conformations of Galr2, which may lead to different internalization rates of the receptor as well as different signaling outputs. This work represents an additional pharmacological evidence of endogenous G-protein-biased agonism at a GPCR.

Spexin is a novel neuropeptide discovered by bioinformatics and has been proposed as a regulator of energy balance. However, its precise role and underlying mechanisms remain unclear. In this study, we investigated the role of spexin and its receptor, galanin receptor 2 (GALR2), in regulation of energy balance in the mediobasal hypothalamus of mice. Administration of spexin into the third ventricle near the hypothalamus (I3V) significantly decreased food intake and body weight with no change in energy expenditure in mice. The central spexin-induced anorexia and weight loss were reinstated by pre-I3V administration of GALR2 antagonist, M871, indicating hypothalamic GALR2-mediated action of spexin in the regulation of energy balance. In addition, we showed that I3V administration of spexin-based selective GALR2 agonist significantly reduced food intake and body weight in a dose-dependent manner with no signs of illness assessed by a conditioned taste aversion test, suggesting a novel role of hypothalamic GALR2 in the regulation of energy balance. Furthermore, we found that activation of GALR2 induced by spexin increased phosphorylation of CREB in the mediobasal hypothalamus of mice and cells overexpressing GALR2 different from galanin, suggesting a ligand-dependent switch of G protein coupled with GALR2. Taken together, these results suggested a critical role of hypothalamic spexin and GALR2 in the regulation of energy balance in mice. Disclosure N. Ha: None. N. Kim: None. C. Kim: None. M. Song: None. H. Lee: None. B. Jin: None. M. Park: None. J. Seong: None. S. Kim: None. D. Kim: None.

Although recent results of our and other studies have showed that galanin (GAL) is an antidiabetic and anti-inflammatory neuropeptide, the molecular mechanism how central GAL regulates energy homeostasis and insulin sensitivity is still not fully understood. The aim of this study was to investigate whether central type 2 of GAL receptors (GALR2) are involved in the regulation of systemic glucose metabolism and its underlying mechanisms. In the present study, type 2 diabetic rats were intracerebroventricularly (i.c.v.) given 100 nM/kg/d GALR2 agonist M1145 or GALR2 antagonist M871 in 5 μl artificial cerebrospinal fluid once a day for consecutive 21 days. Then insulin resistance indexes, inflammatory factor and many genes associated with the function of glucose metabolism were examined in peripheral tissues. The present findings showed that the intracerebroventricular injection of M1145 or M871 respectively increased or decreased glucose infusion rates in hyperinsulinemic euglycemic clamp tests, but attenuated or enhanced the plasma inflammatory factors and glucose concentration in type 2 diabetic rats. Moreover, administration of M1145 markedly increased PGC-1α and GLUT4 expression in skeletal muscles and adipocytes of type 2 diabetic rats. In conclusion, activation of central GALR2 promotes glucose metabolism and ameliorates insulin resistance mainly through the PGC-1α/GLUT4 pathways. The central GALR2 is crucial to whole-body insulin sensitivity and energy homeostasis.

Galanin (GAL) and the NPYY1 agonist play a role in mood regulation and both neuropeptides interact in several central functions. The present study examined the interaction between Galanin receptor 2 (GALR2) and Neuropeptide Y Y1 receptor (NPYY1R) in the dentate gyrus (DG) of the Hippocampus in relation to depression-like behavior. Using receptor autoradiography, in situ hybridization and in situ proximity ligation assay an interaction between GALR and NPYY1R was demonstrated in the DG probably involving the formation of GALR2-NPYY1R heteroreceptor complexes. These complexes were specifically observed in the polymorphic and subgranular subregions of the DG, where both receptors were found to colocalize. Moreover, this GALR2/NPYY1R interaction was linked to an enhancement of the antidepressive-like behavior mediated by NPYY1R in the forced swimming test. Specific cells populations within DG subregions may be involved in this behavioral effect since the coactivation of GALR2 and NPYY1R enhances the NPYY1R-mediated reduction in the number of c-Fos immunoreactive nuclei in the polymorphic region. These results indicate that GALR2/NPYY1R interactions can provide a novel integrative mechanism in DG in depression-related behavior and may give the basis for the development of drugs targeting GALR2/NPYY1R heteroreceptor complexes in the DG of the hippocampus for the treatment of depression.

Multiple Sclerosis (MS) is an inflammatory neurodegenerative disease that affects approximately 2.5 million people globally. Even though the etiology of MS remains unknown, it is accepted that it involves a combination of genetic alterations and environmental factors. Here, after performing whole exome sequencing, we found a MS patient harboring a rare and homozygous single nucleotide variant (SNV; rs61745847) of the G-protein coupled receptor (GPCR) galanin-receptor 2 (GALR2) that alters an important amino acid in the TM6 molecular toggle switch region (W249L). Nuclear magnetic resonance imaging showed that the hypothalamus (an area rich in GALR2) of this patient exhibited an important volumetric reduction leading to an enlarged third ventricle. Ex vivo experiments with patient-derived blood cells (AKT phosphorylation), as well as studies in recombinant cell lines expressing the human GALR2 (calcium mobilization and NFAT mediated gene transcription), showed that galanin (GAL) was unable to stimulate cell signaling in cells expressing the variant GALR2 allele. Live cell confocal microscopy showed that the GALR2 mutant receptor was primarily localized to intracellular endosomes. We conclude that the W249L SNV is likely to abrogate GAL-mediated signaling through GALR2 due to the spontaneous internalization of this receptor in this patient. Although this homozygous SNV was rare in our MS cohort (1:262 cases), our findings raise the potential importance of impaired neuroregenerative pathways in the pathogenesis of MS, warrant future studies into the relevance of the GAL/GALR2 axis in MS and further suggest the activation of GALR2 as a potential therapeutic route for this disease.

The present study was performed to explore the role of galanin and galanin receptor 2 in nociceptive modulation in anterior cingulate cortex (ACC) of normal rats and rats with mononeuropathy. Intra-ACC injection of galanin induced significant increases in hindpaw withdrawal latencies (HWLs) to thermal and mechanical stimulations in both normal rats and rats with mononeuropathy, the increased HWLs were attenuated significantly by intra-ACC injection of galanin receptor 2 antagonist M871, indicating an involvement of galanin receptor 2 in nociceptive modulation in ACC. Interestingly, the galanin-induced HWL was significant higher in rats with mononeuropathy than that in normal rats tested by Randall Selitto test. Furthermore, both the galanin mRNA expression and galanin content increased significantly in ACC in rats with mononeuropathy than that in normal rats. Moreover, both the mRNA levels of galanin receptor 2 and the content of galanin receptor 2 in ACC increased significantly in rats with mononeuropathy than that in normal rats. These results found that galanin induced antinociception in ACC in both normal rats and rats with mononeuropathy. And there may be plastic changes in the expression of galanin and galanin receptor 2 in rats with mononeuropathy, as well as in the galanin-induced antinociception.

Previously, we have described the Galanin (GAL) and Neuropeptide Y Y1 (NPYY1) interactions through GAL receptor 2 and NPYY1 receptor 1 (GALR2/NPYY1R) heterodimers in the Dentate Gyrus (DG) of the Hippocampus, using autoradiographic, in situ hybridization and in situ proximity ligation assay(PLA) (1,2). The current work is to evaluate GALR2 and NPYY1R interactions in relation to depression-like behaviour and c-Fos expression in the Hippocampal DG. Rats (n=6-8) were forced to swim for a 15-min period (pre-test) and 24 h later were subjected to a 5-min swimming session (test) 15 min after the administration alone or in combination of GAL, the NPYY1R agonist [Leu31,Pro34]NPY and the GALR2 antagonist M871. The total duration of immobility, swimming, and climbing periods were scored during the test. For c-Fos immunohistochemistry, experimental groups of rats were anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and perfused with 4 % Paraformaldehyde 90 min after icv injections. Then, brains were coronally sliced and immunostained. As primary antibodies, an antibody against c-Fos protein (1:5000, sc- 52, Santa Cruz Biotechnology, CA, USA), revealed with 3,3´-Diaminobenzidine (DAB) plus nickel, was used as an indirect marker of neural activity. The antibody to Calbindin-D28 k (1:1000, Santa Cruz Biotecnology, CA, USA), revealed with DAB, was used to outline the granular region since it marks mainly hippocampal granule cells. Sections were analyzed using the optical fractionator stereological method. We observed that icv injection of GAL and NPYY1R agonist significantly enhanced the decrease in the immobility (p

The distribution and (patho-)physiological role of neuropeptides in the adult and aging brain have been extensively studied. Galanin is an inhibitory neuropeptide that can coexist with ã-aminobutyric acid (GABA) in the adult forebrain. However, galanin's expression sites, mode of signaling, impact on neuronal morphology, and colocalization with amino acid neurotransmitters during brain development are less well understood. Here, we show that galaninergic innervation of cholinergic projection neurons, which preferentially express galanin receptor 2 (GalR2) in the neonatal mouse basal forebrain, develops by birth. Nerve growth factor (NGF), known to modulate cholinergic morphogenesis, increases GalR2 expression. GalR2 antagonism (M871) in neonates reduces the in vivo expression and axonal targeting of the vesicular acetylcholine transporter (VAChT), indispensable for cholinergic neurotransmission. During cholinergic neuritogenesis in vitro, GalR2 can recruit Rho-family GTPases to induce the extension of a VAChT-containing primary neurite, the prospective axon. In doing so, GalR2 signaling dose-dependently modulates directional filopodial growth and antagonizes NGF-induced growth cone differentiation. Galanin accumulates in GABA-containing nerve terminals in the neonatal basal forebrain, suggesting its contribution to activity-driven cholinergic development during the perinatal period. Overall, our data define the cellular specificity and molecular complexity of galanin action in the developing basal forebrain.

Galanin and its receptors, GALR1 and GALR2, are tumor suppressors and represent therapeutic targets in head and neck squamous cell carcinoma (HNSCC). In the present study, it was demonstrated that the re‑expression of GALR1 in GALR1 and GALR2‑negative HNSCC cells suppresses tumor cell proliferation. This is mediated via extracellular‑regulated protein kinase‑1/2 (ERK1/2)‑dependent effects on the cyclin‑dependent kinase inhibitors (CKI) and cyclin D1. In combination with galanin, GALR2 also suppressed proliferation by increasing CKI and decreasing cyclin D1 levels. In contrast to GALR1, overexpression of GALR2 also induced caspase‑3‑dependent apoptosis. It was identified that in GALR2‑transfected cells, galanin induced activation of ERK1/2 and suppressed cell proliferation. Galanin stimulation also decreased the expression of cyclin D1 and induced apoptotic DNA ladder formation in GALR2‑transfected cells. Pretreatment with the ERK1/2‑specific inhibitor U0126 and pertussis toxin prevented the suppression of cyclin D1 expression, however did not affect DNA ladder formation. In conclusion, GALR2 expression in the presence of galanin exerts antitumor effects via cell cycle arrest and apoptotic pathways, and reactivation of these pathways may have therapeutic benefits in HNSCC.

The large conductance Ca(2+)-activated K(+) (BK) channels are widely distributed in the brain, and act as intracellular calcium sensors in neurons. They play an important feedback role in controlling Ca(2+) flux and Ca(2+)-dependent processes, including neurotransmitter release and cellular excitability. In this study, the effects of the neuropeptide galanin on BK channels were examined by determining the whole-cell currents and single-channel activities in human embryonic kidney (HEK293) cells co-expressing GalR2 and the BK alpha subunit. Galanin enhanced the currents of BK channels, in a concentration-dependent and PTX-independent manner, with an ED50 value of 71.8±16.9 nM. This activation was mediated by GalR2, since its agonist AR-M1896 mimicked the effect of galanin, and since galanin did not facilitate BK currents in cells co-expressing cDNAs of BK and GalR1 or GalR3. The galanin-induced BK current persisted after replacement with Ca(2+)-free solution, suggesting that extracellular Ca(2+) is not essential. Chelating intracellular Ca(2+) by either the slow Ca(2+) buffer EGTA or the fast Ca(2+) buffer BAPTA abolished galanin-mediated activation of BK channels, indicating the important role of intracellular Ca(2+). The role of Ca(2+) efflux from the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) was confirmed by application of thapsigargin, an irreversible inhibitor that depletes Ca(2+) from SR/ER. Moreover, the inositol-1,4,5-triphosphate receptor (IP3R) was identified as the mediator responsible for increased intracellular Ca(2+) activating BK channels. Taken together, activation of GalR2 leads to elevation of intracellular Ca(2+) is due to Ca(2+) efflux from ER through IP3R sequentially opening BK channels.

One of the major pathological characteristics of Alzheimer's disease (AD) is the presence of enhanced deposits of beta-amyloid peptide (Aβ). The neuropeptide galanin (GAL) and its receptors are overexpressed in degenerating brain regions in AD. The functional consequences of galaninergic systems plasticity in AD are unclear. The objective of the present study was to investigate whether exogenous galanin could attenuate spatial memory impairment and hippocampal Aβ aggregation in rat model of AD. The effects of Aβ, galanin, galanin receptor 1 agonist M617 and galanin receptor 2 agonist AR-M1896 on spatial memory were tested by Morris water maze. The effects of Aβ, galanin, M617 and AR-M1896 on hippocampal Aβ protein expression were evaluated by western blot assay. The expression of galanin, galanin receptors 1 and 2 in rats' hippocampus were detected by real time PCR and western blot assay. The results showed that (1) Galanin administration was effective in improving the spatial memory and decreasing hippocampal Aβ levels after intracerebroventricular injection of Aβ; (2) AR-M1896 rather than M617 could imitate these effects of galanin; (3) GAL and GALR2 mRNA and protein levels increased significantly in hippocampus after Aβ administration, while GALR1 mRNA and protein levels did not change; (4) GAL, AR-M1896 and M617 administration did not show significant effect on GAL, GalR1 and GalR2 mRNA and protein levels in hippocampus after Aβ administration. These results implied that galanin receptor 2, but not receptor 1 was involved in the protective effects against spatial memory impairment and hippocampal Aβ aggregation.

Pain threshold may be up-regulated or down-regulated according to gender, age, race/ethnic and psychological state. Previous studies indicated that obesity may change pain threshold, both nociceptive and antinociceptive, which resulted from obesity-reduced variation of neuroendocrine. However there is a limited understanding of its molecular mechanism underlying this variation. A lot of evidence supports that galanin increases food intake and body weight to induce obesity in animals. This peptide may also modulate nociceptive susceptibility via central galanin receptor 1 and peripheral galanin receptor 2 in dorsal root ganglion. Whereas injury and obesity may up-regulate the galanin expression and stimulate its secretion to elevate the plasma levels of subjects. Pain may increase the risk of obesity through reduced physical activity. In this review, we highlighted the multiple bilateral interrelation between obesity and pain sensitivity, between galanin and obesity and between galanin and injure-induced pain. In view of the above, we reasoned that galanin receptors possibly participated in the modulation of the obesity-induced change in pain threshold, which need further direct evidence to support as yet. This review is helpful to explore the mechanism that galanin receptors regulate the obesity-induced change of pain sensitivity and to contribute to our understanding of the relation among galanin, obesity and pain threshold.