The sphingolipid, ceramide-1-phosphate (C1P), has been shown to promote the inflammatory phase and inhibit the proliferation and remodeling stages of wound repair via direct interaction with group ...IVA cytosolic phospholipase A2, a regulator of eicosanoid biosynthesis that fine-tunes the behaviors of various cell types during wound healing. However, the anabolic enzyme responsible for the production of C1P that suppresses wound healing as well as bioactive eicosanoids and target receptors that drive enhanced wound remodeling have not been characterized. Herein, we determined that decreasing C1P activity via inhibitors or genetic ablation of the anabolic enzyme ceramide kinase (CERK) significantly enhanced wound healing phenotypes. Importantly, postwounding inhibition of CERK enhanced the closure rate of acute wounds, improved the quality of healing, and increased fibroblast migration via a “class switch” in the eicosanoid profile. This switch reduced pro-inflammatory prostaglandins (e.g., prostaglandin E2) and increased levels of 5-hydroxyeicosatetraenoic acid and the downstream metabolite 5-oxo-eicosatetraenoic acid (5-oxo-ETE). Moreover, dermal fibroblasts from mice with genetically ablated CERK showed enhanced wound healing markers, while blockage of the murine 5-oxo-ETE receptor (oxoeicosanoid receptor 1) inhibited the enhanced migration phenotype of these cell models. Together, these studies reinforce the vital roles eicosanoids play in the wound healing process and demonstrate a novel role for CERK-derived C1P as a negative regulator of 5-oxo-ETE biosynthesis and the activation of oxoeicosanoid receptor 1 in wound healing. These findings provide foundational preclinical results for the use of CERK inhibitors to shift the balance from inflammation to resolution and increase the wound healing rate.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Systemic injection of lipopolysaccharide (LPS) is a widely used model of immune/inflammatory challenge, which can invoke a host of CNS responses, including activation of the ...hypothalamic-pituitary-adrenal (HPA) axis. Inducible vascular prostaglandin E(2) (PGE(2)) synthesis by endothelial (ECs) and/or perivascular cells (PVCs) (a macrophage-derived vascular cell type) is implicated in the engagement of HPA and other CNS responses, by virtue of their capacity to express cyclooxygenase-2 (COX-2) and microsomal PGE(2) synthase-1. Evidence from genetic and pharmacologic studies also supports a role for the constitutively expressed COX-1 in inflammation-induced activation of the HPA axis, although histochemical evidence to support relevant localization(s) and regulation of COX-1 expression is lacking. The present experiments fill this void in showing that COX-1 immunoreactivity (IR) and mRNA are detectable in identified PVCs and parenchymal microglia under basal conditions and is robustly expressed in these and ECs 1-3 h after intravenous injection of LPS (2 microg/kg). Confocal and electron microscopic analyses indicate distinct cellular/subcellular localizations of COX-1-IR in the three cell types. Interestingly, COX-1 expression is enhanced in ECs of brain PVC-depleted rats, supporting an anti-inflammatory role of the latter cell type. Functional involvement of COX-1 is indicated by the observation that central, but not systemic, pretreatment with the selective COX-1 inhibitor SC-560 attenuated the early phase of LPS-induced increases in adrenocorticotropin and corticosterone secretion. These findings support an involvement of COX-1 in bidirectional interplay between ECs and PVCs in initiating vascular PGE(2) and downstream HPA response to proinflammatory challenges.
Inflammation is associated with increased sympathetic drive in cardiovascular diseases. Blood-borne proinflammatory cytokines, markers of inflammation, induce cyclooxygenase 2 (COX-2) activity in ...perivascular macrophages of the blood-brain barrier. COX-2 generates prostaglandin E2, which may enter the brain and increase sympathetic nerve activity. We examined the contribution of this mechanism to augmented sympathetic drive in rats after myocardial infarction (MI). Approximately 24 hours after acute MI, rats received an intracerebroventricular injection (1 μL/min over 40 minutes) of clodronate liposomes (MI+CLOD) to eliminate brain perivascular macrophages, liposomes alone, or artificial cerebrospinal fluid. A week later, COX-2 immunoreactivity in perivascular macrophages and COX-2 mRNA and protein had increased in hypothalamic paraventricular nucleus of MI rats treated with artificial cerebrospinal fluid or liposomes alone compared with sham-operated rats. In MI+CLOD rats, neither perivascular macrophages nor COX-2 immunoreactivity was seen in the paraventricular nucleus, and COX-2 mRNA and protein levels were similar to those in sham-operated rats. Prostaglandin E2 in cerebrospinal fluid, paraventricular nucleus neuronal excitation, and plasma norepinephrine were less in MI+CLOD rats than in MI rats treated with artificial cerebrospinal fluid or liposomes alone but more than in sham-operated rats. Intracerebroventricular CLOD had no effect on interleukin 1β and tumor necrosis factor-α mRNA and protein in the paraventricular nucleus or plasma interleukin-1β and tumor necrosis factor-α, which were increased in MI compared with sham-operated rats. In normal rats, pretreatment with intracerebroventricular CLOD reduced (P<0.05) the renal sympathetic, blood pressure, and heart rate responses to intracarotid artery injection of tumor necrosis factor-α (0.5 μg/kg); intracerebroventricular liposomes had no effect. The results suggest that proinflammatory cytokines stimulate sympathetic excitation after MI by inducing COX-2 activity and prostaglandin E2 production in perivascular macrophages of the blood-brain barrier.
We have used double in situ hybridization to examine the cellular localization of 5-HT2C receptor mRNA in relation to serotonergic and GABAergic neurons in the anterior raphe nuclei of the rat. In ...the dorsal and median raphe nuclei 5-HT2C receptor mRNA was not detected in serotonergic cells identified as those expressing serotonin (5-HT) transporter mRNA. In contrast, 5-HT2C receptor mRNA was found in most GABAergic cells, recognized by the presence of glutamic acid decarboxylase mRNA. Such 5-HT2C receptor-positive GABAergic neurons were mainly located in the intermediolateral and lateral portions of the dorsal raphe and lateral part of the median raphe. The present data give anatomical support to a previous hypothesis that proposed a negative-feedback loop involving reciprocal connections between GABAergic interneurons bearing 5-HT2A/2C receptors and 5-HT neurons in the dorsal raphe and surrounding areas. According to this model, the excitation of GABAergic interneurons through these 5-HT2C (and also 5-HT2A) receptors would result in the suppression of 5-HT cell firing.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
All phases of lipopolysaccharide (LPS)-induced fever are mediated by prostaglandin (PG) E2. It is known that the second febrile phase (which starts at approximately 1.5 h post-LPS) and subsequent ...phases are mediated by PGE2 that originated in endotheliocytes and perivascular cells of the brain. However, the location and phenotypes of the cells that produce PGE2 triggering the first febrile phase (which starts at approximately 0.5 h) remain unknown. By studying PGE2 synthesis at the enzymatic level, we found that it was activated in the lung and liver, but not in the brain, at the onset of the first phase of LPS fever in rats. This activation involved phosphorylation of cytosolic phospholipase A2 (cPLA2) and transcriptional up-regulation of cyclooxygenase (COX)-2. The number of cells displaying COX-2 immunoreactivity surged in the lung and liver (but not in the brain) at the onset of fever, and the majority of these cells were identified as macrophages. When PGE2 synthesis in the periphery was activated, the concentration of PGE2 increased both in the venous blood (which collects PGE2 from tissues) and arterial blood (which delivers PGE2 to the brain). Most importantly, neutralization of circulating PGE2 with an anti-PGE2 antibody both delayed and attenuated LPS fever. It is concluded that fever is initiated by circulating PGE2 synthesized by macrophages of the LPS-processing organs (lung and liver) via phosphorylation of cPLA2 and transcriptional up-regulation of COX-2. Whether PGE2 produced at the level of the blood-brain barrier also contributes to the development of the first phase remains to be clarified.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Multiple sclerosis (MS) is a devastating neurological disease that predominantly affects young adults resulting in severe personal and economic impact. The majority of therapies for this disease were ...developed in, or are beneficial in experimental autoimmune encephalomyelitis (EAE), the animal model of MS. While known to target adaptive anti-CNS immune responses, they also target, the innate immune arm. This mini-review focuses on the role of dendritic cells (DCs), the professional antigen presenting cells of the innate immune system. The evidence for a role for DCs in the appropriate regulation of anti-CNS autoimmune responses and their role in MS disease susceptibility and possible therapeutic utility are discussed. Additionally, the current controversy regarding the evidence for the presence of functional DCs in the normal CNS is reviewed. Furthermore, the role of CNS DCs and potential routes of their intercourse between the CNS and cervical lymph nodes are considered. Finally, the future role that this nexus between the CNS and the cervical lymph nodes might play in site directed molecular and cellular therapy for MS is outlined.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
In the rat, postsynaptic 5‐hydroxytryptamine2A receptors medial prefrontal cortex control the activity of the serotonergic system through changes in the activity of pyramidal neurons projecting to ...the dorsal raphe nucleus. Here we extend these observations to mouse brain. The prefrontal cortex expresses abundant 5‐ hydroxytryptamine2A receptors, as assessed by immunohistochemistry, Western blots and in situ hybridization procedures. The application of the 5‐hydroxytryptamine2A/2C agonist DOI (100 µm) by reverse dialysis in the medial prefrontal cortex doubled the local release of 5‐hydroxytryptamine. This effect was reversed by coperfusion of tetrodotoxin, and by the selective 5‐hydroxytryptamine2A receptor antagonist M100907, but not by the 5‐hydroxytryptamine2C antagonist SB‐242084. The effect of DOI was also reversed by prazosin (α1‐adrenoceptor antagonist), BAY × 3702 (5‐hydroxytryptamine1A receptor agonist), NBQX (α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐4‐propionate/kainic acid antagonist) and 1S,3S‐ACPD (mGluR II/III agonist), but not by dizocilpine (N‐methyl‐d‐aspartate antagonist). α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐4‐propionate mimicked the 5‐hydroxytryptamine elevation produced by DOI, an effect also reversed by BAY × 3702. Likewise, the coperfusion of classical (chlorpromazine, haloperidol) and atypical antipsychotic drugs (clozapine, olanzapine) fully reversed the 5‐hydroxytryptamine elevation induced by DOI. These observations suggest that DOI increases 5‐hydroxytryptamine release in the mouse medial prefrontal cortex through the activation of local 5‐hydroxytryptamine2A receptors by an impulse‐dependent mechanism that involves/requires the activation of local α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐4‐propionate receptors. This effect is reversed by ligands of receptors present in the medial prefrontal cortex, possibly in pyramidal neurons, which are involved in the action of antipsychotic drugs. In particular, the reversal by classical antipsychotics may involve blockade of α1‐adrenoceptors, whereas that of atypical antipsychotics may involve 5‐hydroxytryptamine2A receptors and α1‐adrenoceptors.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Background
TAK-831 is a potent inhibitor of the DAAO enzyme being developed for the cognitive deficits and negative symptoms of schizophrenia. Inhibition of DAAO results in an increase in ...D-serine, a co-agonist of the NMDA receptor and a full agonist of the δ2 glutamate receptor. Augmentation of NMDA and δ2 glutamate receptor function may be beneficial in schizophrenia. To determine this, rats given TAK-831 were tested in the Novel Object Recognition (NOR) test, a basic cognition test, the Attentional Set Shifting Task (ASST), an animal assay to assess executive function in rodents, and Social Interaction (SI), an animal model of negative symptoms. To determine the impact that a target localized in the cerebellum has on learning, the compound was also profiled in eyeblink conditioning (EBC), a model of cerebellar-based associative learning.
Methods
Rats were given an acute and chronic dose of TAK-831 and cerebrospinal fluid (CSF); cerebellar tissue and plasma were harvested after 6 hours. For NOR, mice were dosed acutely and chronically (14d) with TAK-831 and assessed in their ability to differentiate between novel and previously seen objects after a 4-hour inter-trial interval (ITI). Rats treated with a subchronic phencyclidine (PCP) regimen (2 mg/kg, 7 days bid) showed a deficit in the extra-dimensional shift (EDS) in the ASST. Acute and chronic (14d) doses of TAK-831 were assessed for reversal of the PCP-induced deficit. TAK-831 was dosed acutely and chronically (0.3, 1, 3 mg/kg, po) and assessed for its ability to reverse a naturally occurring SI deficit seen in Balb/c mice. The same dose range was used when the SI deficit was induced by maternal treatment with Poly(I:C). In EBC, a periorbital electrical stimulus was paired with a corneal air puff resulting in classical conditioning. TAK-831 (0.1 and 1 mg/kg, po, 10d chronic dosing) was assessed for its ability to improve acquisition of the conditioned response in the presence and absence of scopolamine.
Results
TAK-831 produced a dose-dependent increase in D-serine levels across CSF, cerebellum, and in plasma. After a 4-hour ITI, vehicle-treated mice were unable to differentiate between a novel and familiar object in NOR. Animals were able to identify the novel object when treated with TAK-831 at 0.3, 1, 3 and 10 mg/kg acute doses and at 0.003, 0.01, 0.03, 0.1, 1.0 and 3 mg/kg chronic doses. TAK-831 was able to reverse the PCP-induced deficit in rats in the EDS in the ASST at 3, 10, and 30 mg/kg acute doses and at 0.1, 1.0 and 10 mg/kg chronic doses. TAK-831 was efficacious in the Balb/c SI model at 1 and 3 mg/kg acute doses and at 0.01, 0.03, 0.1, 1.0 and 10 mg/kg chronic doses; it was efficacious at 3 mg/kg acute dosing in the Poly(I:C) SI model. TAK-831 caused an improvement in both the acquisition of the conditioned response and in reversing the scopolamine-induced deficit at 0.1 and 1 mg/kg in the EBC paradigm.
Discussion
TAK-831 shows utility at a range of doses in animal models associated with negative symptoms and cognitive impairment in schizophrenia. Its efficacy in EBC shows the impact on cerebellar-based learning; this region of the brain is now considered to also be involved in schizophrenia. TAK-831 shows a different range of efficacies pre-clinically, depending on whether the compound is dosed acutely or chronically, indicating a potential plasticity of effect in the brain of preclinical species.
•Pre-synaptic and post-synaptic GABAB receptors differentially regulate network activity.•GABAB ligands modify cognition differently due to the receptor population they engage.•Pro-cognitive benefit ...in man requires critical assessment of GABAB receptor occupancy.
Metabotropic GABAB receptors clearly modify cognitive performance in preclinical animal models, yet translation to treating human disease has been elusive. Compared to their ionotropic GABAA receptor counterpart GABAB receptors not only regulate postsynaptic excitability but also regulate diverse synaptic inputs by presynaptically inhibiting neurotransmitter release. As such, the choice of agonist, antagonist, −ve or +ve modulator as well as CNS exposure level, timing of delivery and longevity of action strongly influence the probability of unlocking the procognitive potential of GABAB receptors in human disease. Quantitative clinical analysis of target/mechanistic engagement of GABAB receptors within cognitive circuits at the level of distinct pre-synaptic and post-synaptic subpopulations is required to determine the optimal pharmacological/dosing profile for different cognitive disorders.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
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