Small-molecule non-peptide antagonists of the PACAP receptor attenuate acute restraint stress-induced anxiety-like behaviors in mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a highly conserved pleiotropic neuropeptide, implicated in emotional stress responses and anxiety-related disorders. Here, we examined whether our recently developed small-molecule non-peptide PACAP receptor antagonists could ameliorate anxiety-like behaviors induced by acute restraint stress in mice. The antagonists PA-9 and its derivative PA-915 improved anxiety-like behaviors in mice subjected to restraint stress. An anxiolytic effect was observed with single acute dose, suggesting their fast-acting properties. PA-915 demonstrated a statistically significant anxiolytic effect whereas fluoxetine did not. These results indicate the potential of PAC1 antagonists as a novel treatment for anxiety.

The dorsal hippocampal protein targeting to glycogen maintains ionotropic glutamate receptor subunits expression and contributes to working and short-term memories in mice.

Brain glycogen metabolism is known to be involved in the learning and memory processes. Protein targeting to glycogen (PTG) is a crucial molecule for glycogenesis, and its expression level is shown to be increased in the dorsal hippocampus during fear memory acquisition and recall, suggesting that PTG may contribute to the memory process. However, its detailed role in the dorsal hippocampus remains unclear. Therefore, we knocked down the expression of PTG in the dorsal hippocampus and attempted to analyze its function behaviorally. PTG expression was found to be enriched in astrocytes. Furthermore, short hairpin RNA against PTG suppressed the expression of PTG in astrocytes. Mice with knockdown of PTG in the dorsal hippocampus showed suppressed alternation behavior in the Y-maze test and reduced memory recall at the first hour after acquisition in the passive avoidance test. Knockdown of mouse dorsal hippocampal astrocyte-specific PTG also impaired working memory in the Y-maze test. GluR1, GluR2, and NR2a subunits expressions were significantly down-regulated in the dorsal hippocampus of mice in which PTG was knocked down. These results indicate that PTG in the dorsal hippocampal astrocytes may contribute to working and short-term memories by maintaining the expression of glutamate receptor subunits.

The novel small-molecule antagonist of PAC1 receptor attenuates formalin-induced inflammatory pain behaviors in mice.

We recently developed PA-8, a novel small-molecule antagonist of PACAP type 1 (PAC1) receptor. In the present study, we examined whether PA-8 was effective against formalin-induced inflammatory pain in mice. Both intrathecal and oral administration of PA-8 resulted in the dose-dependent attenuation of the second phase of formalin-induced nociceptive responses. PA-8 also inhibited c-fos upregulation in the ipsilateral dorsal horn of the spinal cord. The results suggested that PACAP-PAC1 receptor signaling system in the spinal cord were primarily involved in the transmission of inflammatory pain, and PA-8 could be useful for the development of novel analgesics for treating inflammatory pain.

In Silico Screening Identified Novel Small-molecule Antagonists of PAC1 Receptor.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are present in the spinal dorsal horn and dorsal root ganglia, suggesting an important role of PACAP signaling systems in the modulation of spinal nociceptive transmission. Previously, we found that intrathecal injection of PACAP or maxadilan, a selective PACAP type I (PAC1) receptor agonist, induced transient aversive responses followed by a long-lasting mechanical allodynia in mice, suggesting that PACAP-PAC1 receptor systems are involved in chronic pain and that selective PAC1 antagonists may become a new class of analgesics. Although several PAC1 antagonists, such as PACAP 6-38, have been reported, all of them are peptide compounds. In the present study, we identified new small-molecule antagonists of the PAC1 receptor using in silico screening and in vitro/vivo pharmacological assays. The identified small-molecule compounds, named PA-8 and PA-9, dose dependently inhibited the phosphorylation of CREB induced by PACAP in PAC1-, but not VPAC1- or VPAC2-receptor-expressing CHO cells. PA-8 and PA-9 also dose dependently inhibited PACAP-induced cAMP elevation with an IC50 of 2.0 and 5.6 nM, respectively. In vivo pharmacological assays showed that intrathecal injection of these compounds blocked the induction of PACAP-induced aversive responses and mechanical allodynia in mice. In contrast, the compounds when administered alone exerted neither agonistic nor algesic actions in the in vitro/vivo assays. The compounds identified in the present study are new and the first small-molecule antagonists of the PAC1 receptor; they may become seed compounds for developing novel analgesics.

Establishment of disseminated intravascular coagulation (DIC) model by a single iv administration of Escherichia coli-derived lipopolysaccharide (LPS) to cynomolgus monkeys and evaluation of its pathophysiological status.

We prepared a DIC model by administrating LPS to cynomolgus monkeys, and investigated its potential for evaluations of new medicines for DIC therapy. Peripheral blood mononuclear cells (PBMC) collected from cynomolgus monkeys were incubated with LPS (8 types), and TNF-α levels in the media were measured. LPS from Escherichia coli (K-235) was most appropriate in terms of larger increases and smaller variation in TNF-α levels. PBMC from rats, cynomolgus monkeys or humans were incubated with LPS (K-235), and the TNF-α response to LPS was investigated. The response was comparable between cynomolgus monkeys and humans but small in rats. In an in vivo experiment, LPS (K-235) was administered once intravenously to cynomolgus monkeys with or without recombinant human thrombomodulin (rhTM) to investigate any changes in coagulation and fibrinolysis biomarkers and the suppressive effect of rhTM. The liver, kidney, and lung were examined histopathologically. Almost all of the changes resembled the pathophysiological status of human DIC and were suppressed by co-administration of rhTM. The DIC model resembling human DIC was established by LPS (K-235) treatment in cynomolgus monkeys, and therapeutic effect of rhTM was noted, suggesting that this model is useful in evaluations of the efficacy of new medicines for DIC therapy.

The Deletion of GPR40/FFAR1 Signaling Damages Maternal Care and Emotional Function in Female Mice.

The free fatty acid receptor 1 (GPR40/FFAR1) is activated by polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acids (DHA). This receptor has been the focus of many studies regarding physiological functions of the central nervous system. PUFAs are essential for neuronal development and maintenance of neuronal function; thus, the decrease of PUFAs in the brain is closely related to the induction of psychiatric diseases associated with emotional disorder, such as anxiety, depression, and schizophrenia. However, details of the mechanisms remain unclear. In this study, we investigated changes of maternal and/or emotional behavior caused by a deficiency of GPR40/FFAR1 signaling. GPR40/FFAR1 deficient (FFAR1-/-) female mice exhibited impaired maternal care such as retrieving behaviors and an increased rate of neglect and infanticide when compared to wild type (WT) female mice. Furthermore, FFAR1-/- female mice showed increased time spent in the open arms in an elevated plus maze test, reduction of locomotor activity and social interaction behavior, and decreased sucrose intake, when compared to WT female mice. In conclusion, these findings suggest that PUFAs-GPR40/FFAR1 signaling might function, at least in part, as a regulatory factor of emotional and maternal behavior in mice.

Spinal astrocytic activation contributes to both induction and maintenance of pituitary adenylate cyclase-activating polypeptide type 1 receptor-induced long-lasting mechanical allodynia in mice.

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are present in the spinal dorsal horn and dorsal root ganglia, suggesting an important role of PACAP-PACAP receptors signaling system in the modulation of spinal nociceptive transmission. We have previously reported that a single intrathecal injection of PACAP or a PACAP specific (PAC1) receptor selective agonist, maxadilan, in mice induced dose-dependent aversive behaviors, which lasted more than 30 min, and suggested that the maintenance of the nociceptive behaviors was associated with the spinal astrocytic activation. RESULTS: We found that a single intrathecal administration of PACAP or maxadilan also produced long-lasting hind paw mechanical allodynia, which persisted at least 84 days without affecting thermal nociceptive threshold. In contrast, intrathecal application of vasoactive intestinal polypeptide did not change mechanical threshold, and substance P, calcitonin gene-related peptide, or N-methyl-D-aspartate induced only transient mechanical allodynia, which disappeared within 21 days. Western blot and immunohistochemical analyses with an astrocytic marker, glial fibrillary acidic protein, revealed that the spinal PAC1 receptor stimulation caused sustained astrocytic activation, which also lasted more than 84 days. Intrathecal co-administration of L-α-aminoadipate, an astroglial toxin, with PACAP or maxadilan almost completely prevented the induction of the mechanical allodynia. Furthermore, intrathecal treatment of L-α-aminoadipate at 84 days after the PAC1 stimulation transiently reversed the mechanical allodynia accompanied by the reduction of glial fibrillary acidic protein expression level. CONCLUSION: Our data suggest that spinal astrocytic activation triggered by the PAC1 receptor stimulation contributes to both induction and maintenance of the long-term mechanical allodynia.

GPR40/FFAR1 deficient mice increase noradrenaline levels in the brain and exhibit abnormal behavior.

The free fatty acid receptor 1 (GPR40/FFAR1) is a G protein-coupled receptor, which is activated by long chain fatty acids. We have previously demonstrated that activation of brain GPR40/FFAR1 exerts an antinociceptive effect that is mediated by the modulation of the descending pain control system. However, it is unclear whether brain GPR40/FFAR1 contributes to emotional function. In this study, we investigated the involvement of GPR40/FFAR1 in emotional behavior using GPR40/FFAR1 deficient (knockout, KO) mice. The emotional behavior in wild and KO male mice was evaluated at 9-10 weeks of age by the elevated plus-maze test, open field test, social interaction test, and sucrose preference test. Brain monoamines levels were measured using LC-MS/MS. The elevated plus-maze test and open field tests revealed that the KO mice reduced anxiety-like behavior. There were no differences in locomotor activity or social behavior between the wild and KO mice. In the sucrose preference test, the KO mice showed reduction in sucrose preference and intake. The level of noradrenaline was higher in the hippocampus, medulla oblongata, hypothalamus and midbrain of KO mice. Therefore, these results suggest that brain GPR40/FFAR1 is associated with anxiety- and depression-related behavior regulated by the increment of noradrenaline in the brain.

Pituitary adenylate cyclase-activating polypeptide type 1 receptor signaling evokes long-lasting nociceptive behaviors through the activation of spinal astrocytes in mice.

Intrathecal (i.t.) administration of pituitary adenylate cyclase-activating polypeptide (PACAP) induces long-lasting nociceptive behaviors for more than 60 min in mice, while the involvement of PACAP type1 receptor (PAC1-R) has not been clarified yet. The present study investigated signaling mechanisms of the PACAP-induced prolonged nociceptive behaviors. Single i.t. injection of a selective PAC1-R agonist, maxadilan (Max), mimicked nociceptive behaviors in a dose-dependent manner similar to PACAP. Pre- or post-treatment of a selective PAC1-R antagonist, max.d.4, significantly inhibited the nociceptive behaviors by PACAP or Max. Coadministration of a protein kinase A inhibitor, Rp-8-Br-cAMPS, a mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase inhibitor, PD98059 or a c-Jun N-terminal kinase (JNK) inhibitor, SP600125, significantly inhibited the nociceptive behaviors by Max. Immunohistochemistry and immunoblotting analysis revealed that spinal administration of Max-induced ERK phosphorylation and JNK phosphorylation, and also augmented an astrocyte marker, glial fibrillary acidic protein in mouse spinal cord. Furthermore, an astroglial toxin, l-α-aminoadipate, significantly attenuated the development of the nociceptive behaviors and ERK phosphorylation by Max. These results suggest that the activation of spinal PAC1-R induces long-lasting nociception through the interaction of neurons and astrocytes.

Attenuation of inflammatory and neuropathic pain behaviors in mice through activation of free fatty acid receptor GPR40.

BACKGROUND: The G-protein-coupled receptor 40 (GPR40) is suggested to function as a transmembrane receptor for medium- to long-chain free fatty acids and is implicated to play a role in free fatty acids-mediated enhancement of glucose-stimulated insulin secretion from pancreas. However, the functional role of GPR40 in nervous system including somatosensory pain signaling has not been fully examined yet. RESULTS: Intrathecal injection of GPR40 agonist (MEDICA16 or GW9508) dose-dependently reduced ipsilateral mechanical allodynia in CFA and SNL models and thermal hyperalgesia in carrageenan model. These anti-allodynic and anti-hyperalgesic effects were almost completely reversed by a GPR40 antagonist, GW1100. Immunohistochemical analysis revealed that GPR40 is expressed in spinal dorsal horn and dorsal root ganglion neurons, and immunoblot analysis showed that carrageenan or CFA inflammation or spinal nerve injury resulted in increased expression of GPR40 in these areas. Patch-clamp recordings from spinal cord slices exhibited that bath-application of either MEDICA16 or GW9508 significantly decreased the frequency of spontaneous excitatory postsynaptic currents in the substantia gelatinosa neurons of the three pain models. CONCLUSIONS: Our results indicate that GPR40 signaling pathway plays an important suppressive role in spinal nociceptive processing after inflammation or nerve injury, and that GPR40 agonists might serve as a new class of analgesics for treating inflammatory and neuropathic pain.

Pituitary adenylate cyclase-activating polypeptide type 1 receptor (PAC1) gene is suppressed by transglutaminase 2 activation.

Pituitary adenylate cyclase-activating polypeptide (PACAP) functions as a neuroprotective factor through the PACAP type 1 receptor, PAC1. In a previous work, we demonstrated that nerve growth factor augmented PAC1 gene expression through the activation of Sp1 via the Ras/MAPK pathway. We also observed that PAC1 expression in Neuro2a cells was transiently suppressed during in vitro ischemic conditions, oxygen-glucose deprivation (OGD). Because endoplasmic reticulum (ER) stress is induced by ischemia, we attempted to clarify how ER stress affects the expression of PAC1. Tunicamycin, which induces ER stress, significantly suppressed PAC1 gene expression, and salubrinal, a selective inhibitor of the protein kinase RNA-like endoplasmic reticulum kinase signaling pathway of ER stress, blocked the suppression. In luciferase reporter assay, we found that two Sp1 sites were involved in suppression of PAC1 gene expression due to tunicamycin or OGD. Immunocytochemical staining demonstrated that OGD-induced transglutaminase 2 (TG2) expression was suppressed by salubrinal or cystamine, a TG activity inhibitor. Further, the OGD-induced accumulation of cross-linked Sp1 in nuclei was suppressed by cystamine or salubrinal. Together with cystamine, R283, TG2-specific inhibitor, and siRNA specific for TG2 also ameliorated OGD-induced attenuation of PAC1 gene expression. These results suggest that Sp1 cross-linking might be crucial in negative regulation of PAC1 gene expression due to TG2 in OGD-induced ER stress.

Alleviation of behavioral hypersensitivity in mouse models of inflammatory pain with two structurally different casein kinase 1 (CK1) inhibitors.

BACKGROUND: The phylogenetically highly conserved CK1 protein kinases consisting of at least seven isoforms form a distinct family within the eukaryotic protein kinases. CK1 family members play crucial roles in a wide range of signaling activities. However, the functional role of CK1 in somatosensory pain signaling has not yet been fully understood. The aim of this study was to clarify the role of CK1 in the regulation of inflammatory pain in mouse carrageenan and complete Freund's adjuvant (CFA) models. RESULTS: We have used two structurally different CK1 inhibitors, TG003 and IC261. TG003, which was originally identified as a cdc2-like kinase inhibitor, had potent inhibitory effects on CK1 isoforms in vitro and in cultured cells. Intrathecal injection of either TG003 (1-100 pmol) or IC261 (0.1-1 nmol) dose-dependently decreased mechanical allodynia and thermal hyperalgesia induced by carrageenan or CFA. Bath-application of either TG003 (1 µM) or IC261 (1 µM) had only marginal effects on spontaneous excitatory postsynaptic currents (sEPSCs) recorded in the substantia gelatinosa neurons of control mice. However, both compounds decreased the frequency of sEPSCs in both inflammatory pain models. CONCLUSIONS: These results suggest that CK1 plays an important pathophysiological role in spinal inflammatory pain transmission, and that inhibition of the CK1 activity may provide a novel strategy for the treatment of inflammatory pain.

A combination of in vitro comet assay and micronucleus test using human lymphoblastoid TK6 cells.

The comet assay has been widely used as a genotoxicity test for detecting primary DNA damage in individual cells. The micronucleus (MN) test is also a well-established assay for detecting clastogenicity and aneugenicity. A combination of the comet assay (COM) and MN test is capable of detecting a variety of genotoxic potentials as an in vitro screening system. Although the in vitro MN test has a robust protocol and Organisation for Economic Co-operation and Development (OECD) test guideline, the in vitro COM does not. To establish a robust protocol for the COM and to compare its sensitivity with that of the MN, we conducted COM and MN concurrently for five genotoxic agents (ethyl methanesulfonate, methyl methanesulfonate, hydrogen peroxide, gamma-rays and mitomycin C) and one non-genotoxic agent (triton X-100), using human lymphoblastoid TK6 cells. Relative cell count (RCC), relative population doubling (RPD), relative increase in cell count (RICC) and relative cell viability determined by trypan blue dye-exclusion assay (TBDE) were employed as cytotoxic measurements. However, the relative cell viability determined by TBDE just after the treatment was not an appropriate parameter of cytotoxicity for the genotoxic agents because it remained constant even at the highest doses, which showed severe cytotoxicity by RCC, RPD and RICC. The results of the COM showed qualitative agreement (positive or negative) with those of the MN except for mitomycin C, which is an interstrand cross-linker. The COM always required higher doses than the MN to detect the genotoxic potential of the genotoxic agents under the test conditions applied here. The doses that induced a comet tail always yielded <50% RICC, and do not accord to the OECD test guideline for MN because of their high cytotoxicity. These results are helpful for interpreting the results of the COM and MN in in vitro genotoxic hazard assessments. Further investigation is required to standardise the COM.

Muscle wasting associated with pathologic change is a risk factor for the exacerbation of joint swelling in collagen-induced arthritis in cynomolgus monkeys.

BACKGROUND: Not only joint destruction but also muscle wasting due to rheumatoid cachexia has been problem in terms of quality of life of patients with rheumatoid arthritis (RA). In the present study, we performed histopathological examination and assessed relationships between characteristic parameters relating to muscle and joint swelling in a collagen-induced arthritis (CIA) model using cynomolgus monkeys (CMs). METHODS: Female CMs were used and CIA was induced by twice immunizations using bovine type II collagen with Freund's complete adjuvant. Arthritis level was evaluated from the degree of swelling at the peripheral joints of the fore and hind limbs. Food consumption, body weight, and serum biochemical parameters were measured sequentially. Five or 6 animals per time point were sacrificed at 2, 3, 5 and 9 weeks after the first immunization to obtain quadriceps femoris specimens for histopathology. Pimonidazole hydrochloride was intravenously administered to determine tissue hypoxia in skeletal muscle. RESULTS: Gradual joint swelling was observed and the maximum arthritis score was noted at Week 5. In histopathology, necrosis of muscle fiber in the quadriceps femoris was observed only at Week 2 and the most significant findings such as degeneration, atrophy, and regeneration of muscle fiber were mainly observed at Week 5. Food consumption was decreased up to Week 4 but recovered thereafter. Body weight decreased up to Week 5 and did not completely recover thereafter. A biphasic increase in serum cortisol was also observed at Weeks 2 and 5. Histopathology showed that muscle lesions were mainly composed of degeneration and atrophy of the muscle fibers, and ATPase staining revealed that the changes were more pronounced in type II muscle fiber than type I muscle fiber. In the pimonidazole experiment, mosaic pattern in skeletal muscle was demonstrated in the intact animal, but not the CIA animal. Increased arthritis score was accompanied by a decrease in serum creatinine, a marker that reflects muscle mass. CONCLUSIONS: Muscle wasting might exacerbate joint swelling in a collagen-induced arthritis model of cynomolgus monkeys.

The Pivotal Role of Neuropeptide Crosstalk from Ventromedial-PACAP to Dorsomedial-Galanin in the Appetite Regulation in the Mouse Hypothalamus.

We have previously shown that pituitary adenylate cyclase-activating polypeptide (PACAP) in the ventromedial hypothalamus (VMH) enhances feeding during the dark cycle and after fasting, and inhibits feeding during the light cycle. On the other hand, galanin is highly expressed in the hypothalamus and has been reported to be involved in feeding regulation. In this study, we investigated the involvement of the VMH-PACAP to the dorsomedial hypothalamus (DMH)-galanin signaling in the regulation of feeding. Galanin expression in the hypothalamus was significantly increased with fasting, but this increment was canceled in PACAP-knockout (KO) mice. Furthermore, overexpression of PACAP in the VMH increased the expression of galanin, while knockdown (KD) of PACAP in the VMH decreased the expression of galanin, indicating that the expression of galanin in the hypothalamus might be regulated by PACAP in the VMH. Therefore, we expressed the synaptophysin-EGFP fusion protein (SypEGFP) in PACAP neurons in the VMH and visualized the neural projection to the hypothalamic region where galanin was highly expressed. A strong synaptophysin-EGFP signal was observed in the DMH, indicating that PACAP-expressing cells of the VMH projected to the DMH. Furthermore, galanin immunostaining in the DMH showed that galanin expression was weak in PACAP-KO mice. When galanin in the DMH was knocked down, food intake during the dark cycle and after fasting was decreased, and food intake during the light cycle was increased, as in PACAP-KO mice. These results indicated that galanin in the DMH may regulate the feeding downstream of PACAP in the VMH.

N-arachidonoyl glycine induces macrophage apoptosis via GPR18.

N-arachidonoyl glycine (NAGly), a member of lipoamino acids, was reported to exhibit anti-inflammatory effects in experimental ear edema or peritonitis. However the underlying mechanisms have not been clarified so far. In this study, we attempt to investigate the effects of NAGly on macrophages, including the relevant signaling pathways. NAGly potently induced apoptosis in mouse macrophage-derived cell line, RAW264.7. Pretreatment with inhibitors for MEK and p38 MAPK prevented the apoptosis induced by NAGly, although NAGly activated ERK1/2, p38 MAPK and JNK. Further, we focused on implication of GPR18, one of the orphan G protein-coupled receptors, because NAGly has been reported as a candidate ligand for GPR18. Pretreatment with pertussis toxin or siRNA to knock down the expression of GPR18 significantly attenuated the apoptosis induced by NAGly. In mouse peritoneal macrophages, the expression of GPR18 mRNA was elevated in proinflammatory stimulated macrophages but not in anti-inflammatory stimulated macrophages; consistently, NAGly remarkably reduced cell viability of the former, as compared to the latter. These results suggest that NAGly might be involved in function of macrophages through GPR18.

Spinal Astrocyte-Neuron Lactate Shuttle Contributes to the Pituitary Adenylate Cyclase-Activating Polypeptide/PAC1 Receptor-Induced Nociceptive Behaviors in Mice.

We have previously shown that spinal pituitary adenylate cyclase-activating polypeptide (PACAP)/PACAP type 1 (PAC1) receptor signaling triggered long-lasting nociceptive behaviors through astroglial activation in mice. Since astrocyte-neuron lactate shuttle (ANLS) could be essential for long-term synaptic facilitation, we aimed to elucidate a possible involvement of spinal ANLS in the development of the PACAP/PAC1 receptor-induced nociceptive behaviors. A single intrathecal administration of PACAP induced short-term spontaneous aversive behaviors, followed by long-lasting mechanical allodynia in mice. These nociceptive behaviors were inhibited by 1,4-dideoxy-1,4-imino-d-arabinitol (DAB), an inhibitor of glycogenolysis, and this inhibition was reversed by simultaneous L-lactate application. In the cultured spinal astrocytes, the PACAP-evoked glycogenolysis and L-lactate secretion were inhibited by DAB. In addition, a protein kinase C (PKC) inhibitor attenuated the PACAP-induced nociceptive behaviors as well as the PACAP-evoked glycogenolysis and L-lactate secretion. Finally, an inhibitor for the monocarboxylate transporters blocked the L-lactate secretion from the spinal astrocytes and inhibited the PACAP- and spinal nerve ligation-induced nociceptive behaviors. These results suggested that spinal PAC1 receptor-PKC-ANLS signaling contributed to the PACAP-induced nociceptive behaviors. This signaling system could be involved in the peripheral nerve injury-induced pain-like behaviors.

Brain fractalkine-CX3CR1 signalling is anti-obesity system as anorexigenic and anti-inflammatory actions in diet-induced obese mice.

Fractalkine is one of the CX3C chemokine family, and it is widely expressed in the brain including the hypothalamus. In the brain, fractalkine is expressed in neurons and binds to a CX3C chemokine receptor 1 (CX3CR1) in microglia. The hypothalamus regulates energy homeostasis of which dysregulation is associated with obesity. Therefore, we examined whether fractalkine-CX3CR1 signalling involved in regulating food intake and hypothalamic inflammation associated with obesity pathogenesis. In the present study, fractalkine significantly reduced food intake induced by several experimental stimuli and significantly increased brain-derived neurotrophic factor (BDNF) mRNA expression in the hypothalamus. Moreover, tyrosine receptor kinase B (TrkB) antagonist impaired fractalkine-induced anorexigenic actions. In addition, compared with wild-type mice, CX3CR1-deficient mice showed a significant increase in food intake and a significant decrease in BDNF mRNA expression in the hypothalamus. Mice fed a high-fat diet (HFD) for 16 weeks showed hypothalamic inflammation and reduced fractalkine mRNA expression in the hypothalamus. Intracerebroventricular administration of fractalkine significantly suppressed HFD-induced hypothalamic inflammation in mice. HFD intake for 4 weeks caused hypothalamic inflammation in CX3CR1-deficient mice, but not in wild-type mice. These findings suggest that fractalkine-CX3CR1 signalling induces anorexigenic actions via activation of the BDNF-TrkB pathway and suppresses HFD-induced hypothalamic inflammation in mice.

Design and synthesis of pyrido[2,3-d]pyrimidine derivatives for a novel PAC1 receptor antagonist.

Since PA-8 (5-(4-(Allyloxy)-3-methoxyphenyl)-2-amino-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione) was recently identified as a novel small-molecule antagonist of the pituitary adenylate cyclase-activating polypeptide (PACAP) type I (PAC1) receptor, a series of pyrido[2,3-d]pyrimidine derivatives have been designed, synthesized and subsequently evaluated for antagonistic activity on the PAC1 receptor. In this study, we synthesized 21 derivatives based on the PA-8 structure. Among them, the compound 2o (2-Amino-5-(3-trifluoromethoxy-phenyl)-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione) showed more potent antagonistic activities than PA-8. Intrathecal (i.t.) injection of 2o blocked the induction of PACAP-induced aversive behaviors and mechanical allodynia in mice, and the effects were more potent than those of PA-8. A single i.t. injection of 2o also inhibited spinal nerve ligation (SNL)-induced mechanical allodynia. Repeated intraperitoneal administration of 2o gradually reduced the SNL-induced mechanical allodynia, and this effect appeared earlier than for PA-8. In addition, 2o exhibited a favorable ADME and pharmacokinetics profiles. These results suggest that 2o may become an analgesic for the treatment of neuropathic pain.

Characterization of the testis-specific promoter region in the human pituitary adenylate cyclase-activating polypeptide (PACAP) gene.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide localized in the testis at concentration comparable to that found in the brain, suggesting involvement in spermatogenesis. In this study, we identified the human PACAP testis-specific exon (TSE) 10.9 kb upstream from the translational start site and found that the testis-specific transcript of the human PACAP gene was found to be spliced from the TSE into a region of intron 2 without a frameshift. The resulting PACAP precursor has no signal peptide, suggesting that PACAP functions physiologically in an intracrine manner in the testis. The 5'-flanking region of the TSE contains an 80-bp fragment with potent promoter activity in testicular F9 cell. Electrophoresis mobility shift assays showed that proteins from the F9 nuclear extract interacted specifically with the 80-bp fragment. DNA affinity chromatography allowed isolation of the specific proteins bound to the 80-bp fragment, two of which were identified as Poly (ADP-ribose) polymerase-1 (PARP-1) and TIA-1-related protein (TIAR) by mass spectrometry. By using their siRNAs, the depletion of their proteins in F9 cells affected the potent promoter activity of the 80-bp fragment, suggesting that they might be involved in the testis-specific gene expression of PACAP.