Membrane Expression Of Receptors Research Articles

Functional dissection of the N-terminal extracellular domains of Frizzled 6 reveals their roles for receptor localization and Dishevelled recruitment

The Frizzled (FZD) proteins belong to class F of G protein-coupled receptors (GPCRs) and are essential for various pathways involving the secreted lipoglycoproteins of the wingless/int-1 (WNT) family. A WNT-binding cysteine-rich domain (CRD) in FZDs is N-terminally located and connected to the seven transmembrane domain-spanning receptor core by a linker domain that has a variable length in different FZD homologs. However, the function and importance of this linker domain are poorly understood. Here we used systematic mutagenesis of FZD6 to define the minimal N-terminal domain sufficient for receptor surface expression and recruitment of the intracellular scaffold protein Dishevelled (DVL). Further, we identified a triad of evolutionarily conserved cysteines in the FZD linker domain that is crucial for receptor membrane expression and recruitment of DVL. Our results are in agreement with the concept that the conserved cysteines in the linker domain of FZDs assist with the formation of a common secondary structure in this region. We propose that this structure could be involved in agonist binding and receptor activation mechanisms that are similar to the binding and activation mechanisms known for other GPCRs.

Role of NMDA and AMPA glutamatergic receptors in the effects of social defeat on the rewarding properties of MDMA in mice.

Exposure to social stress alters the response to drugs of abuse of experimental animals. Changes in the glutamatergic system seem to play a role in the effects of social defeat stress on the rewarding properties of cocaine and amphetamine. The aim of the present study was to evaluate the involvement of N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors in the effects of social defeat on the conditioned place preference induced by 3,4-methylenedioxymethamphetamine (MDMA). Our hypothesis was that changes in these receptors could mediate the effects of social defeat on MDMA reward. Young adult male mice were exposed to an episode of social defeat with an aggressive conspecific immediately before each conditioning session with MDMA (1.25mg/kg, four sessions on alternating days). According to the treatment received before defeats, six groups were used: saline, 5 or 10mg/kg of memantine (NMDA antagonist) and 0.25, 1 or 5mg/kg of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (AMPA antagonist). One control group was exposed to exploration before place conditioning. In two additional defeated and control groups, the membrane expression of NMDA and AMPA receptors was determined in the striatum and the hippocampus. Control and memantine-treated groups developed place preference, but not defeated mice treated with saline or CNQX, suggesting that the blockade of NMDA receptors reversed the effects of social defeat. Social defeat decreased the expression of several subunits of NMDA and AMPA receptors, mainly GluN1 and GluA1. These results demonstrated that glutamatergic plasticity is involved in the effects of social defeat stress on MDMA reward.

Extracellular cGMP Reverses Altered Membrane Expression of AMPA Receptors in Hippocampus of Hyperammonemic Rats: Underlying Mechanisms.

Chronic hyperammonemia impairs spatial memory by altering membrane expression of GluA1 and GluA2 subunits of AMPA receptors in hippocampus. Intracerebral administration of extracellular cGMP to hyperammonemic rats restores spatial memory and membrane expression of AMPA receptors. The underlying molecular mechanisms remain unknown and cannot be analyzed in vivo. The aims of the present work were to (1) assess whether extracellular cGMP reverses the alterations in membrane expression of GluA1 and GluA2 in hippocampus of hyperammonemic rats ex vivo and (2) identify the underlying mechanisms. To reach these aims, we used freshly isolated hippocampal slices from control and hyperammonemic rats and treated them ex vivo with extracellular cGMP. Extracellular cGMP normalizes membrane expression of GluA2 restoring its phosphorylation in Ser880 because it restores PKCζ activation by Thr560 auto-phosphorylation, which is a consequence of normalization by extracellular cGMP of phosphorylation and activity of p38 which was increased in hyperammonemic rats. Normalization of p38 is a consequence of normalization of membrane expression of the GluN2B subunit of NMDA receptor, mediated by a reduction in its phosphorylation in Tyr1472 due to reduction of Src activation, which was over-activated in hyperammonemic rats. Extracellular cGMP also restores membrane expression of GluA1 increasing its phosphorylation at Ser831 because it restores CaMKII membrane association and phosphorylation in Thr286. All these effects of extracellular cGMP are due to a reduction of hippocampal IL-1β levels in hyperammonemic rats, which reduces IL-1 receptor-mediated Src over-activation. Reduction in IL-1β levels is due to the reduction of microglia activation in hippocampus of hyperammonemic rats.

Ionotropic Glutamate Receptors in the Paraventricular Nucleus (PVN) are Required to Trigger, but not Sustain, Sympathoexcitation by TNF alpha

In the hypothalamic PVN, pro‐inflammatory cytokines (PIC) have been implicated in driving sympathetic nerve activity (SNA) in support of neurogenic hypertension. In other brain regions, tumor necrosis factor alpha (TNF‐a), a prototypical early onset PIC, has been reported to enhance glutamatergic neurotransmission by increasing membrane trafficking and expression of postsynaptic AMPA receptors leading to synaptic long‐term potentiation (LTP). Here, we tested if TNFa triggers such a response in the PVN to increase SNA. In anesthetized male rats (n=5), unilateral PVN injection (50 nl) of aCSF vehicle had no effect on splanchnic SNA (+2 ± 0.6 %), but rat recombinant TNFa (0.6 pmol) injected 10 minutes later elicited a progressive, ramp increase that averaged 52 ± 2 % (p<0.001, n=5). In separate rats (n=3), injection of TNFa after an Emax dose of the AMPA receptor antagonist NBQX (5.2 nmol) increased SNA by only 11 ± 1 %, which was significantly less than the control response (p<0.001). Because AMPA receptor‐mediated membrane potential depolarization can relieve Mg2+ block of NMDA receptors to potentiate postsynaptic responses to AMPA receptor activation, the effect of NMDA receptor blockade with an Emax dose of AP5 (24 nmol) was tested in another group of rats (n=3). TNFα after AP5 increased SNA by 19 ± 3 %, which was significantly less than the response following vehicle and not different from that following NBQX. Results indicate that AMPA and NMDA receptors in the PVN each participate in the TNFα‐induced increase of splanchnic SNA. Of note, neither NBQX or AP5 injection 30 minutes after TNFα altered the ramp‐like increase of SNA. Mechanisms that sustain progressive sympathoexcitation in response to TNFα in the PVN remain to be determined.Support or Funding InformationCapes (AAM, GRP), NIH HL088052 & AHA 25710176 (GMT)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Variations in Cellular Responses of Mouse T Cells to Adenosine-5'-Triphosphate Stimulation Do Not Depend on P2X7 Receptor Expression Levels but on Their Activation and Differentiation Stage.

A previous report has shown that regulatory T cells (Treg) were markedly more sensitive to adenosine-5′-triphosphate (ATP) than conventional T cells (Tconv). Another one has shown that Tregs and CD45RBlow Tconvs, but not CD45RBhigh Tconvs, displayed similar high sensitivity to ATP. We have previously reported that CD45RBlow Tconvs expressing B220/CD45RABC molecules in a pre-apoptotic stage are resistant to ATP stimulation due to the loss of P2X7 receptor (P2X7R) membrane expression. To gain a clearer picture on T-cell sensitivity to ATP, we have quantified four different cellular activities triggered by ATP in mouse T cells at different stages of activation/differentiation, in correlation with levels of P2X7R membrane expression. P2X7R expression significantly increases on Tconvs during differentiation from naive CD45RBhighCD44low to effector/memory CD45RBlowCD44high stage. Maximum levels of upregulation are reached on recently activated CD69+ naive and memory Tconvs. Ectonucleotidases CD39 and CD73 expression levels increase in parallel with those of P2X7R. Recently activated CD69+ CD45RBhighCD44low Tconvs, although expressing high levels of P2X7R, fail to cleave homing receptor CD62L after ATP treatment, but efficiently form pores and externalize phosphatidylserine (PS). In contrast, naive CD45RBhighCD44low Tconvs cleave CD62L with high efficiency although they express a lower level of P2X7, thus suggesting that P2X7R levels are not a limiting factor for signaling ATP-induced cellular responses. Contrary to common assumption, P2X7R-mediated cellular activities in mouse Tconvs are not triggered in an all-or-none manner, but depend on their stage of activation/differentiation. Compared to CD45RBlow Tconvs, CD45RBlowFoxp3+ Tregs show significantly higher levels of P2X7R membrane expression and of sensitivity to ATP as evidenced by their high levels of CD62L shedding, pore formation and PS externalization observed after ATP treatment. In summary, the different abilities of ATP-treated Tconvs to form pore or cleave CD62L depending on their activation and differentiation state suggests that P2X7R signaling varies according to the physiological role of T convs during antigen activation in secondary lymphoid organs or trafficking to inflammatory sites.

Hyperammonemia alters membrane expression of GluA1 and GluA2 subunits of AMPA receptors in hippocampus by enhancing activation of the IL-1 receptor: underlying mechanisms

BackgroundHyperammonemic rats reproduce the cognitive alterations of patients with hepatic encephalopathy, including altered spatial memory, attributed to altered membrane expression of AMPA receptor subunits in hippocampus. Neuroinflammation mediates these cognitive alterations. We hypothesized that hyperammonemia-induced increase in IL-1β in hippocampus would be responsible for the altered GluA1 and GluA2 membrane expression. The aims of this work were to (1) assess if increased IL-1β levels and activation of its receptor are responsible for the changes in GluA1 and/or GluA2 membrane expression in hyperammonemia and (2) identify the mechanisms by which activation of IL-1 receptor leads to altered membrane expression of GluA1 and GluA2.MethodsWe analyzed in hippocampal slices from control and hyperammonemic rat membrane expression of AMPA receptors using the BS3 cross-linker and phosphorylation of the GluA1 and GluA2 subunits using phosphor-specific antibodies. The IL-1 receptor was blocked with IL-Ra, and the signal transduction pathways involved in modulation of membrane expression of GluA1 and GluA2 were analyzed using inhibitors of key steps.ResultsHyperammonemia reduces GluA1 and increases GluA2 membrane expression and reduces phosphorylation of GluA1 at Ser831 and of GluA2 at Ser880. Hyperammonemia increases IL-1β, enhancing activation of IL-1 receptor. This leads to activation of Src. The changes in membrane expression of GluA1 and GluA2 are reversed by blocking the IL-1 receptor with IL-1Ra or by inhibiting Src with PP2.After Src activation, the pathways for GluA2 and GluA1 diverge. Src increases phosphorylation of GluN2B at Tyr14721 and membrane expression of GluN2B in hyperammonemic rats, leading to activation of MAP kinase p38, which binds to and reduces phosphorylation at Thr560 and activity of PKCζ, resulting in reduced phosphorylation at Ser880 and enhanced membrane expression of GluA2.Increased Src activity in hyperammonemic rats also activates PKCδ which enhances phosphorylation of GluN2B at Ser1303, reducing membrane expression of CaMKII and phosphorylation at Ser831 and membrane expression of GluA1.ConclusionsThis work identifies two pathways by which neuroinflammation alters glutamatergic neurotransmission in hippocampus. The steps of the pathways identified could be targets to normalize neurotransmission in hyperammonemia and other pathologies associated with increased IL-1β by acting, for example, on p38 or PKCδ.Graphical abstractIL-1β alters membrane expression of GluA1 and GluA2 AMPA receptor subunits by two difrerent mechanisms in the hippocampus of hyperammonemic rats.

Basophil Membrane Expression of Epithelial Cytokine Receptors in Patients with Severe Asthma

Background: Severe asthma is a heterogeneous disease, which is characterized by airway damage and remodeling. All triggers of asthma, such as allergens, bacteria, viruses, and pollutants, interact with the airway epithelial cells, which drive the airway inflammatory response through the release of cytokines, particularly IL-25, IL-33, and thymic stromal lymphopoietin (TSLP). Objectives and Methods: To investigate whether the expression of the IL-25, IL-33, and TSLP receptors on the basophil membrane are associated with asthma severity. Twenty-six patients with asthma (11 severe and 15 moderate/mild) and 10 healthy subjects (controls) were enrolled in the study. The results of the basophil activation test and flow cytometry analysis were assessed to investigate basophil membrane expression of IL-25, TSLP, and IL-33 receptors before and after IgE stimulation. Results: IL-25 and IL-33 receptor expression on the basophil membrane at baseline were significantly higher in patients with severe asthma than in those with mild/moderate asthma or healthy subjects, independent of atopy, eosinophilia, asthma control, and exacerbation frequency. Following IgE stimulation, a significantly higher increase in the IL-25 and IL-33 receptors was observed in mild/moderate versus severe asthma. Conclusions: The high expression of the IL-25 and IL-33 receptors on the basophil membrane of patients with severe asthma indicates an overstimulation of basophils by these cytokines in severe asthma. This finding can possibly be used as a biomarker of asthma severity.

Extracellular Cyclic GMP Modulates Membrane Expression of The GluA1 and GluA2 Subunits of AMPA Receptor in Cerebellum: Molecular Mechanisms Involved

There is increasing evidence that extracellular cGMP modulates glutamatergic neurotransmission and some forms of learning. However, the underlying mechanisms remain unknown. We proposed the hypotheses that extracellular cGMP may regulate membrane expression of AMPA receptors. To do this extracellular cGMP should act on a membrane protein and activate signal transduction pathways modulating phosphorylation of the GluA1 and/or GluA2 subunits. It has been shown that extracellular cGMP modulates glycine receptors. The aims of this work were to assess: 1) whether extracellular cGMP modulates membrane expression of GluA1 and GluA2 subunits of AMPA receptors in cerebellum in vivo; 2) whether this is mediated by glycine receptors; 3) the role of GluA1 and GluA2 phosphorylation and 4) identify steps of the intracellular pathways involved. We show that extracellular cGMP modulates membrane expression of GluA1 and GluA2 in cerebellum in vivo and unveil the mechanisms involved. Extracellular cGMP reduced glycine receptor activation, modulating cAMP, protein kinases and phosphatases, and GluA1 and GluA2 phosphorylation, resulting in increased GluA1 and reduced GluA2 membrane expression. Extracellular cGMP therefore modulates membrane expression of AMPA receptors and glutamatergic neurotransmission. The steps identified may be therapeutic targets to improve neurotransmission and neurological function in pathological situations with abnormal glutamatergic neurotransmission.

Kainate receptor mediated presynaptic LTP in agranular insular cortex contributes to fear and anxiety in mice

Anxiety disorders represent serious social problems worldwide. Recent neuroimaging studies have found that elevated activity and altered connectivity of the insular cortex might account for the negative emotional states in highly anxious individuals. However, the exact synaptic mechanisms of specific insular subregions have yet to be studied in detail. To assess the electrophysiological properties of agranular insular cortex (AIC) neurons, basic synaptic transmission was recorded and different protocols were used to induce presynaptic and postsynaptic long-term potentiation in mice with anxiety-related behaviors. The presynaptic membrane expression of kainate receptors (KARs) and pharmacologic manipulations were quantified to examine the role of Gluk1 subtype in anxiety-like behaviors. Fear conditioning occludes electrically induced postsynaptic-LTP in the AIC. Quantal analysis of LTP expression in this region revealed a significant presynaptic component reflected by an increase in the probability of transmitter release. A form of presynaptic-LTP that requires KARs has been characterized. Interestingly, a simple emotional anxiety stimulus resulted in selective occlusion of presynaptic-LTP, but not of postsynaptic-LTP. Finally, injecting GluK1-specific antagonists into the AIC reduced behavioral responses to fear or anxiety stimuli in the mouse. These findings suggest that activity-dependent synaptic plasticity takes place in the AIC due to exposure to fear or anxiety, and inhibiting the presynaptic KAR function may help to prevent or treat anxiety disorder.

Histone deacetylase inhibition induces odor preference memory extension and maintains enhanced AMPA receptor expression in the rat pup model.

Histone deacetylase (HDAC) plays a role in synaptic plasticity and long-term memory formation. We hypothesized that trichostatin-A (TSA), an HDAC inhibitor, would promote long-term odor preference memory and maintain enhanced GluA1 receptor levels that have been hypothesized to support memory. We used an early odor preference learning model in neonate rat pups that normally produces only 24-h memory to test behavior and examine receptor protein expression. Our behavioral studies showed that intrabulbar infusion of TSA, prior to pairing of the conditioned stimulus (peppermint odor) with the unconditioned stimulus (tactile stimulation), prolonged 24-h odor preference memory for at least 9 d. The prolonged odor preference memory was selective for the paired odor and was also observed using a specific HDAC6 inhibitor, tubacin, supporting a role for histone acetylation in associative memory. Immunoblot analysis showed that GluA1 receptor membrane expression in the olfactory bulbs of the TSA-treated group was significantly increased at 48 h unlike control rats without TSA. Immunohistochemistry revealed significant increase of GluA1 expression in olfactory bulb glomeruli 5 d after training. These results extend previous evidence for a close relationship between enhanced GluA1 receptor membrane expression and memory expression. Together, these findings provide a new single-trial appetitive model for understanding the support and maintenance of memories of varying duration.

Differential molecular regulation of processing and membrane expression of Type-I BMP receptors: implications for signaling.

The Type-I bone morphogenetic protein receptors (BMPRs), BMPR1A and BMPR1B, present the highest sequence homology among BMPRs, suggestive of functional similitude. However, sequence elements within their extracellular domain, such as signal sequence or N-glycosylation motifs, may result in differential regulation of biosynthetic processing and trafficking and in alterations to receptor function. We show that (i) BMPR1A and the ubiquitous isoform of BMPR1B differed in mode of translocation into the endoplasmic reticulum; and (ii) BMPR1A was N-glycosylated while BMPR1B was not, resulting in greater efficiency of processing and plasma membrane expression of BMPR1A. We further demonstrated the importance of BMPR1A expression and glycosylation in ES-2 ovarian cancer cells, where (i) CRISPR/Cas9-mediated knockout of BMPR1A abrogated BMP2-induced Smad1/5/8 phosphorylation and reduced proliferation of ES-2 cells and (ii) inhibition of N-glycosylation by site-directed mutagenesis, or by tunicamycin or 2-deoxy-D-glucose treatments, reduced biosynthetic processing and plasma membrane expression of BMPR1A and BMP2-induced Smad1/5/8 phosphorylation.

Macrophage Polarization Modulates FcγR- and CD13-Mediated Phagocytosis and Reactive Oxygen Species Production, Independently of Receptor Membrane Expression.

In response to microenvironmental cues, macrophages undergo a profound phenotypic transformation acquiring distinct activation phenotypes ranging from pro-inflammatory (M1) to anti-inflammatory (M2). To study how activation phenotype influences phagocytosis and production of reactive oxygen species (ROS) mediated by receptors for IgG antibodies (Fcγ receptors) and by CD13, human monocyte-derived macrophages were polarized to distinct phenotypes using IFN-γ (Mϕ-IFN-γ), IL-4 (Mϕ-IL-4), or IL-10 (Mϕ-IL-10). Phenotypically, Mϕ-IFN-γ were characterized as CD14+CD80+CD86+ cells, Mϕ-IL-4 as CD209highCD206+CD11b+CD14low, and Mϕ-IL-10 as CD16+CD163+ cells. Compared to non-polarized macrophages, FcγRI expression increased in Mϕ-IFN-γ and Mϕ-IL-10 and FcγRIII expression increased in Mϕ-IL-10. None of the polarizing cytokines modified FcγRII or CD13 expression. Functionally, we found that cytokine-mediated activation significantly and distinctively affected FcγR- and CD13-mediated phagocytosis and ROS generation. Compared to non-polarized macrophages, FcγRI-, FcγRII-, and CD13-mediated phagocytosis was significantly increased in Mϕ-IL-10 and decreased in Mϕ-IFN-γ, although both cytokines significantly upregulated FcγRI expression. IL-10 also increased phagocytosis of Escherichia coli, showing that the effect of IL-10 on macrophage phagocytosis is not specific for a particular receptor. Interestingly, Mϕ-IL-4, which showed poor FcγR- and CD13-mediated phagocytosis, showed very high phagocytosis of E. coli and zymosan. Coupled with phagocytosis, macrophages produce ROS that contribute to microbial killing. As expected, Mϕ-IFN-γ showed significant production of ROS after FcγRI-, FcγRII-, or CD13-mediated phagocytosis. Unexpectedly, we found that Mϕ-IL-10 can also produce ROS after simultaneous stimulation through several phagocytic receptors, as coaggregation of FcγRI/FcγRII/CD13 induced a belated but significant ROS production. Together, these results demonstrate that activation of macrophages by each cytokine distinctly modulates expression of phagocytic receptors, FcγR- and CD13-mediated phagocytosis, and ROS production.

Probing the Allosteric Role of the α5 Subunit of α3β4α5 Nicotinic Acetylcholine Receptors by Functionally Selective Modulators and Ligands.

Nicotinic acetylcholine receptors regulate the nicotine dependence encountered with cigarette smoking, and this has stimulated a search for drugs binding the responsible receptor subtypes. Studies link a gene cluster encoding for α3β4α5-D398N nicotinic acetylcholine receptors to lung cancer risk as well as link a second mutation in this cluster to an increased risk for nicotine dependence. However, there are currently no recognized drugs for discriminating α3β4α5 signaling. In this study, we describe the development of homogeneous HEK-293 cell clones of α3β4 and α3β4α5 receptors appropriate for drug screening and characterizing biochemical and pharmacological properties of incorporated α5 subunits. Clones were assessed for plasma membrane expression of the individual receptor subunits by mass spectrometry and immunochemistry, and their calcium flux was measured in the presence of a library of kinase inhibitors and a focused library of acetylcholine receptor ligands. We demonstrated an incorporation of two α3 subunits in approximately 98% of plasma membrane receptor pentamers, indicating a 2/3 subunit expression ratio of α3 to β4 alone or to coexpressed β4 and α5. With prolonged nicotine exposure, the plasma membrane expression of receptors with and without incorporated α5 increased. Whereas α5 subunit expression decreased the cell calcium response to nicotine and reduced plasma membrane receptor number, it partially protected receptors from nicotine mediated desensitization. Hit compounds from both libraries suggest the α5 and α5-D398N subunits allosterically modify the behavior of nicotine at the parent α3β4 nicotinic acetylcholine receptor. These studies identify pharmacological tools from two distinct classes of drugs, antagonists and modifiers that are α5 and α5-D398N subtype selective that provide a means to characterize the role of the CHRNA5/A3/B4 gene cluster in smoking and cancer.

The clinical laboratory criteria of infectious complications in primary patients with acute leucosis.

The study was carried out with the purpose of investigating clinical laboratory features of immune reaction in primary patients with acute leucosis and their significance in development of infectious complications. The analysis was applied to indices of immunograms, main anti-inflammatory cytokines - IL-1β, IL-6, IL-8 and FNOα, molecule of inter-cellular adhesion sICAM-1 and also expression of receptors to IgG (CD16, CD64) on cells of macrophage phagocyte component and intracellular content of myeloperoxidase. It is established that development of infectious complications is connected to imbalance of congenital immunity and is characterized by involvement into systemic inflammation anti-inflammatory cytokines FNOα, IL-6, molecule of inter-cellular adhesion sICAM-1 and reduced capacity of neutrophils to phagocytosis conditioned by insufficiency of membrane expression of receptors to IgG - CD16 combined with enzymatic insufficiency.

Reducing Peripheral Inflammation with Infliximab Reduces Neuroinflammation and Improves Cognition in Rats with Hepatic Encephalopathy.

Inflammation contributes to cognitive impairment in patients with hepatic encephalopathy (HE). However, the process by which peripheral inflammation results in cognitive impairment remains unclear. In animal models, neuroinflammation and altered neurotransmission mediate cognitive impairment. Taking into account these data, we hypothesized that in rats with HE: (1) peripheral inflammation is a main contributor to neuroinflammation; (2) neuroinflammation in hippocampus impairs spatial learning by altering AMPA and/or NMDA receptors membrane expression; (3) reducing peripheral inflammation with infliximab (anti-TNF-a) would improve spatial learning; (4) this would be associated with reduced neuroinflammation and normalization of the membrane expression of glutamate receptors. The aims of this work were to assess these hypotheses. We analyzed in rats with portacaval shunt (PCS) and control rats, treated or not with infliximab: (a) peripheral inflammation by measuring prostaglandin E2, IL10, IL-17, and IL-6; (b) neuroinflammation in hippocampus by analyzing microglial activation and the content of TNF-a and IL-1b; (c) AMPA and NMDA receptors membrane expression in hippocampus; and (d) spatial learning in the Radial and Morris water mazes. We assessed the effects of treatment with infliximab on peripheral inflammation, on neuroinflammation and AMPA and NMDA receptors membrane expression in hippocampus and on spatial learning and memory. PCS rats show increased serum prostaglandin E2, IL-17, and IL-6 and reduced IL-10 levels, indicating increased peripheral inflammation. PCS rats also show microglial activation and increased nuclear NF-kB and expression of TNF-a and IL-1b in hippocampus. This was associated with altered AMPA and NMDA receptors membrane expression in hippocampus and impaired spatial learning and memory in the radial and Morris water maze. Treatment with infliximab reduces peripheral inflammation in PCS rats, normalizing prostaglandin E2, IL-17, IL-6, and IL-10 levels in serum. Infliximab also prevents neuroinflammation, reduces microglial activation, translocates NF-kB into nucleoli and normalizes TNF-a and IL-1b content in hippocampus. This was associated with normalization of AMPA receptors membrane expression in hippocampus and of spatial learning and memory. The results suggest that peripheral inflammation contributes to spatial learning impairment in PCS rats. Treatment with anti-TNF-a could be a new therapeutic approach to improve cognitive function in patients with HE.

In vivo administration of extracellular cGMP normalizes TNF-α and membrane expression of AMPA receptors in hippocampus and spatial reference memory but not IL-1β, NMDA receptors in membrane and working memory in hyperammonemic rats

Patients with hepatic encephalopathy (HE) show working memory and visuo-spatial orientation deficits. Hyperammonemia is a main contributor to cognitive impairment in HE. Hyperammonemic rats show impaired spatial learning and learning ability in the Y maze. Intracerebral administration of extracellular cGMP restores learning in the Y-maze. The underlying mechanisms remain unknown. It also remains unknown whether extracellular cGMP improves neuroinflammation or restores spatial learning in hyperammonemic rats and if it affects differently reference and working memory. The aims of this work were:a)assess whether treatment with extracellular cGMP reduces hippocampal neuroinflammation and restores spatial learning in hyperammonemic rats.b)analyze the underlying mechanisms, including changes in membrane expression of NMDA and AMPA receptors.Spatial working and reference memory were assessed using the radial and Morris water mazes and neuroinflammation by immunohistochemistry and Western blot. Membrane expression of NMDA and AMPA receptor subunits was analyzed using the BS3 crosslinker. Extracellular cGMP was administered intracerebrally using osmotic minipumps.Chronic hyperammonemia induces neuroinflammation in hippocampus, with astrocytes activation and increased IL-1β, which are associated with increased NMDA receptors membrane expression and impaired working memory. This process is not affected by extracellular cGMP. Hyperammonemia also activates microglia and increases TNF-α, alters membrane expression of AMPA receptor subunits (increased GluA1 and reduced GluA2) and impairs reference memory. All these changes are reversed by extracellular cGMP.These results show that extracellular cGMP modulates spatial reference memory but not working memory. This would be mediated by modulation of TNF-α levels and of membrane expression of GluA1 and GluA2 subunits of AMPA receptors.

Hyperammonemia alters glycinergic neurotransmission and modulation of the glutamate-nitric oxide-cGMP pathway by extracellular glycine in cerebellum in vivo

The glutamate-nitric oxide (NO)-cGMP pathway modulates some forms of learning. How glycine modulates this pathway is unclear. Glycine could modulate the pathway biphasically, enhancing its function through NMDA receptor activation or reducing it through glycine receptor activation. Chronic hyperammonemia impairs the glutamate-NO-cGMP pathway in the cerebellum and induces cognitive impairment. The possible alterations in hyperammonemia of glycinergic neurotransmission and of glutamate-NO-cGMP pathway modulation by glycine remain unknown. The aims were to assess, by invivo microdialysis in cerebellum: (i) the effects of different glycine concentrations, administered through the microdialysis probe, on the glutamate-NO-cGMP pathway function; (ii) the effects of tonic glycine receptors activation on the pathway function, by blocking them with strychnine; (iii) whether hyperammonemia alters the pathway modulation by glycine; (iv) and whether hyperammonemia alters extracellular glycine concentration and/or glycine receptor membrane expression. In control rats, low glycine levels reduce the pathway function, likely by activating glycine receptors, while 20μM glycine enhances the pathway function, likely by enhancing NMDA receptor activation. In hyperammonemic rats, glycine did not reduce the pathway function, but enhanced it when administered at 1-20μM. Hyperammonemia reduces extracellular glycine concentration by approximately 50% and glycine receptor membrane expression. However, tonic glycine receptor activation seems to be enhanced in hyperammonemic rats, as indicated by the larger increase in extracellular cGMP induced by strychnine. These data show that glycine modulates the glutamate-NO-cGMP pathway biphasically and that hyperammonemia strongly alters glycinergic neurotransmission and modulation by glycine of the glutamate-NO-cGMP pathway. These alterations may contribute to the cerebellar aspects of cognitive alterations in hyperammonemia. The findings reported in this study show that hyperammonemia alters glycinergic neurotransmission and the glutamate-NO-cGMP pathway modulation by glycine. In control rats, low glycine levels reduced the pathway function, likely by activating glycine receptors, while 20μM glycine enhanced the pathway, likely by enhancing NMDA receptor activation. In hyperammonemic rats, glycine (administered at 1-20μM) enhances the pathway, likely by activating NMDA receptors.

Hyperammonemia induces glial activation, neuroinflammation and alters neurotransmitter receptors in hippocampus, impairing spatial learning: reversal by sulforaphane.

BackgroundPatients with liver cirrhosis and minimal hepatic encephalopathy (MHE) show mild cognitive impairment and spatial learning dysfunction. Hyperammonemia acts synergistically with inflammation to induce cognitive impairment in MHE. Hyperammonemia-induced neuroinflammation in hippocampus could contribute to spatial learning impairment in MHE. Two main aims of this work were: (1) to assess whether chronic hyperammonemia increases inflammatory factors in the hippocampus and if this is associated with microglia and/or astrocytes activation and (2) to assess whether hyperammonemia-induced neuroinflammation in the hippocampus is associated with altered membrane expression of glutamate and GABA receptors and spatial learning impairment. There are no specific treatments for cognitive alterations in patients with MHE. A third aim was to assess whether treatment with sulforaphane enhances endogenous the anti-inflammatory system, reduces neuroinflammation in the hippocampus of hyperammonemic rats, and restores spatial learning and if normalization of receptor membrane expression is associated with learning improvement.MethodsWe analyzed the following in control and hyperammonemic rats, treated or not with sulforaphane: (1) microglia and astrocytes activation by immunohistochemistry, (2) markers of pro-inflammatory (M1) (IL-1β, IL-6) and anti-inflammatory (M2) microglia (Arg1, YM-1) by Western blot, (3) membrane expression of GABA, AMPA, and NMDA receptors using the BS3 cross-linker, and (4) spatial learning using the radial maze.ResultsThe results reported show that hyperammonemia induces astrocytes and microglia activation in the hippocampus, increasing pro-inflammatory cytokines IL-1β and IL-6. This is associated with altered membrane expression of AMPA, NMDA, and GABA receptors which would be responsible for altered neurotransmission and impairment of spatial learning in the radial maze. Treatment with sulforaphane promotes microglia differentiation from pro-inflammatory M1 to anti-inflammatory M2 phenotype and reduces activation of astrocytes in hyperammonemic rats. This reduces neuroinflammation, normalizes membrane expression of glutamate and GABA receptors, and restores spatial learning in hyperammonemic rats.ConclusionsHyperammonemia-induced neuroinflammation impairs glutamatergic and GABAergic neurotransmission by altering membrane expression of glutamate and GABA receptors, resulting in impaired spatial learning. Sulforaphane reverses all these effects. Treatment with sulforaphane could be useful to improve cognitive function in cirrhotic patients with minimal or clinical hepatic encephalopathy.

Basophils Membrane Expression of Epithelial Cytokines Receptors in Eosinophilic and Non Eosinophilic Asthma

Basophils are effector cells most often associated with airway inflammation in patients with asthma and can have an important role in the initiation of Th2 inflammation, as these cells may be activated directly by tissue cytokines (IL-25, IL-33 and TSLP) released by epithelial cells in response to damage signals from allergens, bacterials, virus or pollutants. We investigated whether IL-25, IL-33 and TSLP receptors expression levels on basophil membrane are associated with asthma phenotype. 24 patients with asthma (12 eosinophilic, 12 non eosinophilic) have been enrolled in the study. Basophils membrane expression of IL25 R, TSLP R and IL33 R was analyzed before and after IgE, fMLP, LPS, LTA-SA (Lipoteicoic Acid from S. Aureus) stimulation and correlated with FEV1 of each patient. Following LPS stimulation a significant increase in IL25 R was observed in eosinophilic (p=0.001) but not in non eosinophilic asthma. IgE stimulation significantly increased IL25 R expression on basophils membrane, in both eosinophilic and non eosinophilic asthma (p= 0.001 and p= 0.001, respectevely), the percentual increase being greater in eosinophilic compared to non-eosinophilic asthma (p=0.007).

Sildenafil reduces neuroinflammation and restores spatial learning in rats with hepatic encephalopathy: underlying mechanisms.

BackgroundThere are no specific treatments for the neurological alterations of cirrhotic patients with minimal hepatic encephalopathy (MHE). Rats with MHE due to portacaval shunt (PCS) show impaired spatial learning. The underlying mechanisms remain unknown. The aims of this work were to assess: (a) whether PCS rats show neuroinflammation in hippocampus, (b) whether treatment with sildenafil reduces neuroinflammation and restores spatial learning in PCS rats, and (c) analyze the underlying mechanisms.MethodsNeuroinflammation was assessed by determining inflammatory markers by Western blot. Phosphorylation of MAP-kinase p38 was assessed by immunohistochemistry. Membrane expression of GABA and glutamate receptors was analyzed using BS3 cross-linker. Spatial learning was analyzed using the radial and Morris water mazes. To assess if sildenafil reverses the alterations, rats were treated with sildenafil in the drinking water.ResultsPCS rats show increased IL-1β and TNF-α levels and phosphorylation (activity) of p38 in hippocampus. Membrane expression of subunits α1 of GABAA receptor and GluR2 of AMPA receptor are increased in PCS rats, while subunits GluR1 of AMPA receptors and NR1 and NR2a of NMDA receptors are reduced. PCS rats show reduced spatial learning in the radial and Morris water mazes. Sildenafil treatment normalizes IL-1β and TNF-α levels, p38 phosphorylation, and membrane expression of GABAA, AMPA, and NMDA receptors and restores spatial learning.ConclusionsIncreased IL-1β alters GABAergic and glutamatergic neurotransmission in hippocampus and impairs spatial learning in rats with MHE. Sildenafil reduces neuroinflammation and restores learning. Phosphodiesterase-5 inhibitors may be useful to improve cognitive function in patients with MHE.