Membrane Expression Of Receptors Research Articles

A transient blood IL-17 increase triggers neuroinflammation in cerebellum and motor incoordination in hyperammonemic rats

Patients with liver cirrhosis may show minimal hepatic encephalopathy (MHE) with motor incoordination which is reproduced in hyperammonemic rats. Hyperammonemia induces peripheral inflammation which triggers neuroinflammation and enhanced GABAergic neurotransmission in cerebellum and motor incoordination. The mechanisms involved remain unknown. The aims were to assess if the early increase of peripheral IL-17 triggers motor incoordination in hyperammonemic rats and to identify some underlying mechanisms. We assessed if blocking peripheral IL-17 with anti-IL-17 at 2–4 days of hyperammonemia prevents motor incoordination and analyzed underlying mechanisms. Hyperammonemia induces a transient blood IL-17 increase at days 3–4. This is associated with increased IL-17 receptor membrane expression and activation in cerebellum, leading to NADPH oxidase activation, increased superoxide production and MLCK that induce blood–brain barrier (BBB) permeabilization by reducing occludin and ZO-1. BBB permeabilization facilitates the entry of IL-17, which increases in cerebellum and activates microglia. This increases TNFα and the TNFR1-S1PR2-CCL2-BDNF-TrkB pathway. This enhances GABAergic neurotransmission which impairs motor coordination. Blocking peripheral IL-17 with anti-IL-17 prevents all the above process and prevents motor incoordination. Early treatment to reduce blood IL-17 may be a useful treatment to reverse motor incoordination in patients with MHE.Graphical abstract

Insulin Signaling Differentially Regulates the Trafficking of Insulin and Amyloid Beta Peptides at the Blood-Brain Barrier.

The blood-brain barrier (BBB) is instrumental in clearing toxic metabolites from the brain, such as amyloid-β (Aβ) peptides, and in delivering essential nutrients to the brain, like insulin. In Alzheimer's disease (AD) brain, increased Aβ levels are paralleled by decreased insulin levels, which are accompanied by insulin signaling deficits at the BBB. Thus, we investigated the impact of insulin-like growth factor and insulin receptor (IGF1R and IR) signaling on Aβ and insulin trafficking at the BBB. Following intravenous infusion of an IGF1R/IR kinase inhibitor (AG1024) in wild-type mice, the BBB trafficking of 125I radiolabeled Aβ peptides and insulin was assessed by dynamic SPECT/CT imaging. The brain efflux of [125I]iodo-Aβ42 decreased upon AG1024 treatment. Additionally, the brain influx of [125I]iodoinsulin, [125I]iodo-Aβ42, [125I]iodo-Aβ40, and [125I]iodo-BSA (BBB integrity marker) was decreased, increased, unchanged, and unchanged, respectively, upon AG1024 treatment. Subsequent mechanistic studies were performed using an in vitro BBB cell model. The cell uptake of [125I]iodoinsulin, [125I]iodo-Aβ42, and [125I]iodo-Aβ40 was decreased, increased, and unchanged, respectively, upon AG1024 treatment. Further, AG1024 reduced the phosphorylation of insulin signaling kinases (Akt and Erk) and the membrane expression of Aβ and insulin trafficking receptors (LRP-1 and IR-β). These findings reveal that insulin signaling differentially regulates the BBB trafficking of Aβ peptides and insulin. Moreover, deficits in IGF1R and IR signaling, as observed in the brains of type II diabetes and AD patients, are expected to increase Aβ accumulation while decreasing insulin delivery to the brain, which has been linked to the progression of cognitive decline in AD.

KCTD13-mediated ubiquitination and degradation of GluN1 regulates excitatory synaptic transmission and seizure susceptibility.

Temporal lobe epilepsy (TLE) is the most common and severe form of epilepsy in adults; however, its underlying pathomechanisms remain elusive. Dysregulation of ubiquitination is increasingly recognized to contribute to the development and maintenance of epilepsy. Herein, we observed for the first time that potassium channel tetramerization domain containing 13 (KCTD13) protein, a substrate-specific adapter for cullin3-based E3 ubiquitin ligase, was markedly down-regulated in the brain tissue of patients with TLE. In a TLE mouse model, the protein expression of KCTD13 dynamically changed during epileptogenesis. Knockdown of KCTD13 in the mouse hippocampus significantly enhanced seizure susceptibility and severity, whereas overexpression of KCTD13 showed the opposite effect. Mechanistically, GluN1, an obligatory subunit of N-methyl-D-aspartic acid receptors (NMDARs), was identified as a potential substrate protein of KCTD13. Further investigation revealed that KCTD13 facilitates lysine-48-linked polyubiquitination of GluN1 and its degradation through the ubiquitin-proteasome pathway. Besides, the lysine residue 860 of GluN1 is the main ubiquitin site. Importantly, dysregulation of KCTD13 affected membrane expression of glutamate receptors and impaired glutamate synaptic transmission. Systemic administration of the NMDAR inhibitor memantine significantly rescued the epileptic phenotype aggravated by KCTD13 knockdown. In conclusion, our results demonstrated an unrecognized pathway of KCTD13-GluN1 in epilepsy, suggesting KCTD13 as a potential neuroprotective therapeutic target for epilepsy.

Extracellular vesicles from mesenchymal stem cells reduce neuroinflammation in hippocampus and restore cognitive function in hyperammonemic rats

Chronic hyperammonemia, a main contributor to hepatic encephalopathy (HE), leads to neuroinflammation which alters neurotransmission leading to cognitive impairment. There are no specific treatments for the neurological alterations in HE. Extracellular vesicles (EVs) from mesenchymal stem cells (MSCs) reduce neuroinflammation in some pathological conditions. The aims were to assess if treatment of hyperammonemic rats with EVs from MSCs restores cognitive function and analyze the underlying mechanisms. EVs injected in vivo reach the hippocampus and restore performance of hyperammonemic rats in object location, object recognition, short-term memory in the Y-maze and reference memory in the radial maze. Hyperammonemic rats show reduced TGFβ levels and membrane expression of TGFβ receptors in hippocampus. This leads to microglia activation and reduced Smad7–IkB pathway, which induces NF-κB nuclear translocation in neurons, increasing IL-1β which alters AMPA and NMDA receptors membrane expression, leading to cognitive impairment. These effects are reversed by TGFβ in the EVs from MSCs, which activates TGFβ receptors, reducing microglia activation and NF-κB nuclear translocation in neurons by normalizing the Smad7–IkB pathway. This normalizes IL-1β, AMPA and NMDA receptors membrane expression and, therefore, cognitive function. EVs from MSCs may be useful to improve cognitive function in patients with hyperammonemia and minimal HE.

Effect of D4 Dopamine Receptor on Na+-K+-ATPase Activity in Renal Proximal Tubule Cells.

NKA activity, nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) levels were measured in RPT cells treated with the D4 receptor agonist PD168077 and/or the D4 receptor antagonist L745870, the NO synthase inhibitor NG-nitro-L-arginine-methyl ester (L-NAME) or the soluble guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo-[4,3-a] quinoxalin-1-one (ODQ). Total D4 receptor expression and its expression in the plasma membrane were investigated by immunoblotting in RPT cells from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). Activation of D4 receptors with PD168077, inhibited NKA activity in RPT cells from WKY rats in a concentration- and time-dependent manner. The inhibitory effect of PD168077 on NKA activity was prevented by the addition of the D4 receptor antagonist L745870, which by itself had no effect. The NO synthase inhibitor L-NAME and the soluble guanylyl cyclase inhibitor ODQ, which by themselves had no effect on NKA activity, eliminated the inhibitory effect of PD168077 on NKA activity. Activation of D4 receptors also increased NO levels in the culture medium and cGMP levels in RPT cells. However, the inhibitory effect of D4 receptors on NKA activity was absent in RPT cells from SHRs, which could be related to decreased plasma membrane expression of D4 receptors in SHR RPT cells. Activation of D4 receptors directly inhibits NKA activity via the NO/cGMP signaling pathway in RPT cells from WKY rats but not SHRs. Aberrant regulation of NKA activity in RPT cells may be involved in the pathogenesis of hypertension.

Hyperammonemia Enhances GABAergic Neurotransmission in Hippocampus: Underlying Mechanisms and Modulation by Extracellular cGMP.

Rats with chronic hyperammonemia reproduce the cognitive and motor impairment present in patients with hepatic encephalopathy. It has been proposed that enhanced GABAergic neurotransmission in hippocampus may contribute to impaired learning and memory in hyperammonemic rats. However, there are no direct evidences of the effects of hyperammonemia on GABAergic neurotransmission in hippocampus or on the underlying mechanisms. The aims of this work were to assess if chronic hyperammonemia enhances the function of GABAA receptors in hippocampus and to identify the underlying mechanisms. Activation of GABAA receptors is enhanced in hippocampus of hyperammonemic rats, as analyzed in a multielectrode array system. Hyperammonemia reduces membrane expression of the GABA transporters GAT1 and GAT3, which is associated with increased extracellular GABA concentration. Hyperammonemia also increases gephyrin levels and phosphorylation of the β3 subunit of GABAA receptor, which are associated with increased membrane expression of the GABAA receptor subunits α1, α2, γ2, β3, and δ. Enhanced levels of extracellular GABA and increased membrane expression of GABAA receptors would be responsible for the enhanced GABAergic neurotransmission in hippocampus of hyperammonemic rats. Increasing extracellular cGMP reverses the increase in GABAA receptors activation by normalizing the membrane expression of GABA transporters and GABAA receptors. The increased GABAergic neurotransmission in hippocampus would contribute to cognitive impairment in hyperammonemic rats. The results reported suggest that reducing GABAergic tone in hippocampus by increasing extracellular cGMP or by other means may be useful to improve cognitive function in hyperammonemia and in cirrhotic patients with minimal or clinical hepatic encephalopathy.

KIF2C regulates synaptic plasticity and cognition in mice through dynamic microtubule depolymerization.

Dynamic microtubules play a critical role in cell structure and function. In nervous system, microtubules are the major route for cargo protein trafficking and they specially extend into and out of synapses to regulate synaptic development and plasticity. However, the detailed depolymerization mechanism that regulates dynamic microtubules in synapses and dendrites is still unclear. In this study, we find that KIF2C, a dynamic microtubule depolymerization protein without known function in the nervous system, plays a pivotal role in the structural and functional plasticity of synapses and regulates cognitive function in mice. Through its microtubule depolymerization capability, KIF2C regulates microtubule dynamics in dendrites, and regulates microtubule invasion of spines in neurons in a neuronal activity-dependent manner. Using RNAi knockdown and conditional knockout approaches, we showed that KIF2C regulates spine morphology and synaptic membrane expression of AMPA receptors. Moreover, KIF2C deficiency leads to impaired excitatory transmission, long-term potentiation, and altered cognitive behaviors in mice. Collectively, our study explores a novel function of KIF2C in the nervous system and provides an important regulatory mechanism on how activity-dependent microtubule dynamic regulates synaptic plasticity and cognition behaviors.

GABA-B receptors enhance GABA-A receptor currents by modulation of membrane trafficking in dentate gyrus granule cells

Activation of postsynaptic GABA-B receptors enhances tonic inhibition mediated by high-affinity extrasynaptic GABAA receptors in dentate gyrus granule cells (DGGCs), thalamocortical neurons, and cerebellar granule cells. We investigated the mechanism(s) of GABA current modulation by GABAB receptors in DGGCs using a combination of electrophysiological and biochemical approaches. In acute hippocampal brain slices the GABAB receptor agonist baclofen increased GABA-evoked currents in ∼2/3rds of DGGCs, significantly increasing GABAA currents by 41% on average. Nonstationary noise analysis was performed to estimate the effects of baclofen on single channel conductance, mean open time, and channel number; these estimates suggest that GABAB receptor activation increases receptor number but does not modify single channel properties of GABAA receptors. To directly assess baclofen-induced changes in plasma membrane expression of GABAA receptors, biotinylated western blots were performed. Treatment of hippocampal slices with baclofen significantly increased the surface expression of GABAA receptor subunits (both δ and γ2 subunits) and this effect was inhibited by the GABAB receptor antagonist CGP55845. These data indicate that changes in membrane trafficking and increased number of GABAA receptors in plasma membrane contribute to the enhancement of GABA currents produced by GABAB receptor activation in DGGCs.

Intracellular and extracelluar cyclic GMP in the brain and the hippocampus.

Cyclic Guanosine-Monophosphate (cGMP) is implicated as second messenger in a plethora of pathways and its effects are executed mainly by cGMP-dependent protein kinases (PKG). It is involved in both peripheral (cardiovascular regulation, intestinal secretion, phototransduction, etc.) and brain (hippocampal synaptic plasticity, neuroinflammation, cognitive function, etc.) processes. Stimulation of hippocampal cGMP signaling have been proved to be beneficial in animal models of aging, Alzheimer's disease or hepatic encephalopathy, restoring different cognitive functions such as passive avoidance, object recognition or spatial memory. However, even when some inhibitors of cGMP-degrading enzymes (PDEs) are already used against peripheral pathologies, their utility as neurological treatments is still under clinical investigation. Additionally, it has been demonstrated a list of cGMP roles as not second but first messenger. The role of extracellular cGMP has been specially studied in hippocampal function and cognitive impairment in animal models and it has emerged as an important modulator of neuroinflammation-mediated cognitive alterations and hippocampal synaptic plasticity malfunction. Specifically, it has been demonstrated that extracellular cGMP decreases hippocampal IL-1β levels restoring membrane expression of glutamate receptors in the hippocampus and cognitive function in hyperammonemic rats. The mechanisms implicated are still unclear and might involve complex interactions between hippocampal neurons, astrocytes and microglia. Membrane targets for extracellular cGMP are still poorly understood and must be addressed in future studies.

Deorphanization of novel biogenic amine-gated ion channels identifies a new serotonin receptor for learning

SummaryPentameric ligand-gated ion channels (LGICs) play conserved, critical roles in both excitatory and inhibitory synaptic transmission and can be activated by diverse neurochemical ligands. We have performed a characterization of orphan channels from the nematode C. elegans, identifying five new monoamine-gated LGICs with diverse functional properties and expression postsynaptic to aminergic neurons. These include polymodal anion channels activated by both dopamine and tyramine, which may mediate inhibitory transmission by both molecules in vivo. Intriguingly, we also find that a novel serotonin-gated cation channel, LGC-50, is essential for aversive olfactory learning of pathogenic bacteria, a process known to depend on serotonergic neurotransmission. Remarkably, the redistribution of LGC-50 to neuronal processes is modulated by olfactory conditioning, and lgc-50 point mutations that cause misregulation of receptor membrane expression interfere with olfactory learning. Thus, the intracellular trafficking and localization of these receptors at synapses may represent a molecular cornerstone of the learning mechanism.

Kv7 channel trafficking by the microtubule network in vascular smooth muscle.

In arterial smooth muscle cells, changes in availability of integral membrane proteins influence the regulation of blood flow and blood pressure, which is critical for human health. However, the mechanisms that coordinate the trafficking and membrane expression of specific receptors and ion channels in vascular smooth muscle are poorly understood. In the vasculature, very little is known about microtubules, which form a road network upon which proteins can be transported to and from the cell membrane. This review article summarizes the impact of the microtubule network on arterial contractility, highlighting the importance of the network, with an emphasis on our recent findings regarding the trafficking of the voltage‐dependent Kv7 channels.

Esmethadone (REL-1017) Restores NMDA Receptor 1 Subunit Expression in an In Vitro Model of Glutamatergic Excitotoxicity

Esmethadone (dextromethadone; REL-1017) is a novel NMDA channel blocker currently in development for major depressive disorder (MDD). NMDA receptors are known to play a primary role in neural plasticity. An excess of glutamate in the synaptic cleft and impaired neural plasticity have been implicated in the pathogenesis of MDD. The NMDA receptor 1 subunit (NMDAR1) is essential for NMDA receptor membrane expression. To gain insight into the mechanism of action of REL-1017, we examined the effect of glutamate and REL-1017 on the expression of NMDAR1 in ARPE-19 cells.

Conserved C-terminal motifs in odorant receptors instruct their cell surface expression and cAMP signaling.

The highly individual plasma membrane expression and cAMP signaling of odorant receptors have hampered their ligand assignment and functional characterization in test cell systems. Chaperones have been identified to support the cell surface expression of only a portion of odorant receptors, with mechanisms remaining unclear. The presence of amino acid motifs that might be responsible for odorant receptors' individual intracellular retention or cell surface expression, and thus, for cAMP signaling, is under debate: so far, no such protein motifs have been suggested. Here, we demonstrate the existence of highly conserved C-terminal amino acid motifs, which discriminate at least between class-I and class-II odorant receptors, with their numbers of motifs increasing during evolution, by comparing C-terminal protein sequences from 4808 receptors across eight species. Truncation experiments and mutation analysis of C-terminal motifs, largely overlapping with helix 8, revealed single amino acids and their combinations to have differential impact on the cell surface expression and on stimulus-dependent cAMP signaling of odorant receptors in NxG108CC15 cells. Our results demonstrate class-specific and individual C-terminal motif equipment of odorant receptors, which instruct their functional expression in a test cell system, and in situ may regulate their individual cell surface expression and intracellular cAMP signaling.

Role of Conserved Residues and F322 in the Extracellular Vestibule of the Rat P2X7 Receptor in Its Expression, Function and Dye Uptake Ability.

Activation of the P2X7 receptor results in the opening of a large pore that plays a role in immune responses, apoptosis, and many other physiological and pathological processes. Here, we investigated the role of conserved and unique residues in the extracellular vestibule connecting the agonist-binding domain with the transmembrane domain of rat P2X7 receptor. We found that all residues that are conserved among the P2X receptor subtypes respond to alanine mutagenesis with an inhibition (Y51, Q52, and G323) or a significant decrease (K49, G326, K327, and F328) of 2′,3′-O-(benzoyl-4-benzoyl)-ATP (BzATP)-induced current and permeability to ethidium bromide, while the nonconserved residue (F322), which is also present in P2X4 receptor, responds with a 10-fold higher sensitivity to BzATP, much slower deactivation kinetics, and a higher propensity to form the large dye-permeable pore. We examined the membrane expression of conserved mutants and found that Y51, Q52, G323, and F328 play a role in the trafficking of the receptor to the plasma membrane, while K49 controls receptor responsiveness to agonists. Finally, we studied the importance of the physicochemical properties of these residues and observed that the K49R, F322Y, F322W, and F322L mutants significantly reversed the receptor function, indicating that positively charged and large hydrophobic residues are important at positions 49 and 322, respectively. These results show that clusters of conserved residues above the transmembrane domain 1 (K49–Y51–Q52) and transmembrane domain 2 (G326–K327–F328) are important for receptor structure, membrane expression, and channel gating and that the nonconserved residue (F322) at the top of the extracellular vestibule is involved in hydrophobic inter-subunit interaction which stabilizes the closed state of the P2X7 receptor channel.

Hyperammonemia alters the mismatch negativity in the auditory evoked potential by altering functional connectivity and neurotransmission.

Minimal hepatic encephalopathy (MHE) is a neuropsychiatric syndrome produced by central nervous system dysfunction subsequent to liver disease. Hyperammonemia and inflammation act synergistically to alter neurotransmission, leading to the cognitive and motor alterations in MHE, which are reproduced in rat models of chronic hyperammonemia. Patients with MHE show altered functional connectivity in different neural networks and a reduced response in the cognitive potential mismatch negativity (MMN), which correlates with attention deficits. The mechanisms by which MMN is altered in MHE remain unknown. The objectives of this work are as follows: To assess if rats with chronic hyperammonemia reproduce the reduced response in the MMN found in patients with MHE. Analyze the functional connectivity between the areas (CA1 area of the dorsal hippocampus, prelimbic cortex, primary auditory cortex, and central inferior colliculus) involved in the generation of the MMN and its possible alterations in hyperammonemia. Granger causality analysis has been applied to detect the net flow of information between the population neuronal activities recorded from a local field potential approach. Analyze if altered MMN response in hyperammonemia is associated with alterations in glutamatergic and GABAergic neurotransmission. Extracellular levels of the neurotransmitters and/or membrane expression of their receptors have been analyzed after the tissue isolation of the four target sites. The results show that rats with chronic hyperammonemia show reduced MMN response in hippocampus, mimicking the reduced MMN response of patients with MHE. This is associated with altered functional connectivity between the areas involved in the generation of the MMN. Hyperammonemia also alters membrane expression of glutamate and GABA receptors in hippocampus and reduces the changes in extracellular GABA and glutamate induced by the MMN paradigm of auditory stimulus in hippocampus of control rats. The changes in glutamatergic and GABAergic neurotransmission and in functional connectivity between the brain areas analyzed would contribute to the impairment of the MMN response in rats with hyperammonemia and, likely, also in patients with MHE.

Extracellular Nucleotides and P2 Receptors in Renal Function.

The understanding of the nucleotide/P2 receptor system in the regulation of renal hemodynamics and transport function has grown exponentially over the last 20 yr. This review attempts to integrate the available data while also identifying areas of missing information. First, the determinants of nucleotide concentrations in the interstitial and tubular fluids of the kidney are described, including mechanisms of cellular release of nucleotides and their extracellular breakdown. Then the renal cell membrane expression of P2X and P2Y receptors is discussed in the context of their effects on renal vascular and tubular functions. Attention is paid to effects on the cortical vasculature and intraglomerular structures, autoregulation of renal blood flow, tubuloglomerular feedback, and the control of medullary blood flow. The role of the nucleotide/P2 receptor system in the autocrine/paracrine regulation of sodium and fluid transport in the tubular and collecting duct system is outlined together with its role in integrative sodium and fluid homeostasis and blood pressure control. The final section summarizes the rapidly growing evidence indicating a prominent role of the extracellular nucleotide/P2 receptor system in the pathophysiology of the kidney and aims to identify potential therapeutic opportunities, including hypertension, lithium-induced nephropathy, polycystic kidney disease, and kidney inflammation. We are only beginning to unravel the distinct physiological and pathophysiological influences of the extracellular nucleotide/P2 receptor system and the associated therapeutic perspectives.

PB2195 ASSESSMENT OF PLATELET MEMBRANE ALTERATION IN PATIENTS WITH MYELOID METAPLASIA WITH MYELOFIBROSIS

Background:Patients with Myeloid Metaplasia with Myelofibrosis (MMM) present at the onset or during evolution thrombotic complications. In the assessment of thrombosis risk are important the presence of JAK2 mutation as well as platelet function. Receptors that define the status of activated platelets (CD62P, CD36) are better expressed in chronic myeloproliferative neoplasms (MPNs) patients with thrombotic complications. Numerous studies have shown a high oxidative status in MPNs patients, a situation that correlates with a higher thrombotic risk.Aims:The aim of our study was to determine if resting membrane potential could be correlated with alterations in expression of platelet receptors and reactive species production (ROS)MethodsThis retrospective study included 35 patients with MMM (16 male and 19 female), median age 61.1 (95% CI of median 60,71‐72) as well as 15 healthy volunteers. The diagnosis of MMM was made according to World Health Organization (WHO) classification of MPNs. We evaluated the flow cytometry markers of platelet adhesion (CD42a, CD42b), aggregation (CD41, CD61) and CD36. Production of reactive species (ROS) was examined using fluorescence method with DCFDA and was assessed by aria under curve (AUC) in serial measurements during 900 sec. The determination of platelet membrane potential was done by fluorescence method using 3,3′‐dipropyl‐2,2′‐thiadicarbocyanine iodide (DiSC3‐5)Results:The ROS production is significantly higher in MMM group: median value of AUC patient group 12780882862,5000 compared with controls 11621365017,5000, p= 0.04. Patients in advanced phase (grade 5 Hackett of splenomegaly) has high level of ROS production compared with patients diagnosed in early stage (grade 1 or 2 Hackett of splenomegaly) median value: 13178014970 vs 12303298585, p = 0.001. There are no differences between groups that was splitted by type of treatment Ruxolitinib/Hydroxiureea or type of mutation diagnosed JAK2V617F/CALR/no mutation. Patients with MMM present higher resting membrane potential (RMP) compared with controls (median value ‐ 63 mV vs–57.5 mV, p = 0.05). The expression of CD36 receptor was higher in MMM group vs controls: median value 27.91 vs 20.91, p = 0.003. Number of microparticle (MP) derivated from platelet, erythrocyte and endothelial cells are no different between patient and control group. We obtain only lower receptor expression on MP surface for CD41/CD61, CD42b and GlyA in patient group compared with control group, p < 0.05. There are significant correlation between resting membrane potential and expression of CD42b (r ‐0.45, p = 0.04) and GlyA receptors (r ‐0.45, p = 0.04).Summary/Conclusion:Patients with MMM especially those with have a higher level of ROS production especially in advanced phase. The hyperpolarisation of cell macrophages membrane could be associated with high ROS production, no reports are regarding platelet membrane. In our study we did not obtain any significant correlation between RMP and ROS production. The expression of CD 36 is higher in MMM patients but was not correlated with ROS production. It was proved that generation of intracellular ROS is associated with CD36 signaling and represent one start point for thrombosis. Low expression of CD42b and GlyA of microparticle surface is associated with lower RMP that correspond to activated status of platelet membrane. We have to verify these results in higher number of patients in order to find significant correlations

Trans‐synaptic regulation of group III mGluR pharmacology by endogenous allosteric modulators implicated in neuropsychiatric disease

Functional characterization of the GPCR interactome has focused predominantly on intracellular interacting‐proteins, yet GPCRs are increasingly found in complex with extracellular proteins. Extracellular leucine rich repeat fibronectin type III domain containing 1 (ELFN1) was recently reported to physically anchor mGluR6 and mGluR7 across retinal and hippocampal synapses, respectively; however, the consequence and extent of trans‐synaptic interactions on GPCR physiology and pharmacology had been unknown. We explored the functional implications of ELFN1‐mGluR interactions by developing a novel transcellular GPCR signaling assay platform to monitor changes in mGluR activity in one cell following its exposure to separate ELFN1‐containing cells. Using this platform, we found the first evidence of allosteric modulation of GPCR activity in trans; whereby ELFN1‐expressing cells in co‐culture altered both agonist‐induced and constitutive receptor activity of group III mGluRs expressed in separate cell populations. We further adapted this platform for various readouts and determined transcellular ELFN1‐mediated alterations of group III mGluR cAMP signaling was not a consequence of receptor membrane expression or desensitization, but instead a direct consequence of altered G protein activation as seen by BRET‐based real‐time kinetic assays. Furthermore, we have identified additional trans‐synaptic binding partners of group III mGluRs with previously unknown function and demonstrate selective pharmacological consequence and relevance to neuropsychiatric disease. Our findings demonstrate that accepted principles of group III mGluR pharmacology are critically altered via extracellular interactions with endogenous trans‐synaptic allosteric modulators, and disruption of these interactions may endow susceptibilities to neuropsychiatric disease.Support or Funding InformationThis work was supported by NIH Grants EY018139 and DA026405 (to K.A.M.). H.A.D. is the recipient of a Canadian Institutes of Health Research Postdoctoral Fellowship award.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Peripheral inflammation induces neuroinflammation that alters neurotransmission and cognitive and motor function in hepatic encephalopathy: Underlying mechanisms and therapeutic implications.

Several million patients with liver cirrhosis suffer minimal hepatic encephalopathy (MHE), with mild cognitive and coordination impairments that reduce their quality of life and life span. Hyperammonaemia and peripheral inflammation act synergistically to induce these neurological alterations. We propose that MHE appearance is because of the changes in peripheral immune system, which are transmitted to brain, leading to neuroinflammation that alters neurotransmission leading to cognitive and motor alterations. We summarize studies showing that MHE in cirrhotic patients is associated with alterations in the immune system and that patients died with HE show neuroinflammation in cerebellum, with microglial and astrocytic activation and Purkinje cell loss. We also summarize studies in animal models of MHE on the role of peripheral inflammation in neuroinflammation induction, how neuroinflammation alters neurotransmission and how this leads to cognitive and motor alterations. These studies identify therapeutic targets and treatments that improve cognitive and motor function. Rats with MHE show neuroinflammation in hippocampus and altered NMDA and AMPA receptor membrane expression, which impairs spatial learning and memory. Neuroinflammation in cerebellum is associated with altered GABA transporters and extracellular GABA, which impair motor coordination and learning in a Y maze. These alterations are reversed by treatments that reduce peripheral inflammation (anti-TNFα, ibuprofen), neuroinflammation (sulphoraphane, p38 inhibitors), GABAergic tone (bicuculline, pregnenolone sulphate) or increase extracellular cGMP (sildenafil or cGMP). The mechanisms identified would also occur in other chronic diseases associated with inflammation, aging and some mental and neurodegenerative diseases. Treatments that improve MHE may also be beneficial to treat these pathologies.

Chronic hyperammonemia induces peripheral inflammation that leads to cognitive impairment in rats: Reversed by anti-TNF-α treatment

Chronic hyperammonemia induces neuroinflammation which mediates cognitive impairment. How hyperammonemia induces neuroinflammation remains unclear. We aimed to assess whether: chronic hyperammonemia induces peripheral inflammation, and whether this then contributes to neuroinflammation, altered neurotransmission and impaired spatial learning - before assessing whether this neuroinflammation and impairment is reversible following hyperammonemia elimination or treatment of peripheral inflammation with anti-TNF-α. Chronic hyperammonemia was induced by feeding rats an ammonia-containing diet. Peripheral inflammation was analyzed by measuring PGE2, TNF-α, IL-6 and IL-10. We tested whether chronic anti-TNF-α treatment improves peripheral inflammation, neuroinflammation, membrane expression of glutamate receptors in the hippocampus and spatial learning. Hyperammonemic rats show a rapid and reversible induction of peripheral inflammation, with increased pro-inflammatory PGE2, TNF-α and IL-6, followed at around 10 days by reduced anti-inflammatory IL-10. Peripheral anti-TNF-α treatment prevents peripheral inflammation induction and the increase in IL-1b and TNF-α and microglia activation in hippocampus of the rats, which remain hyperammonemic. This is associated with prevention of the altered membrane expression of glutamate receptors and of the impairment of spatial memory assessed in the radial and Morris water mazes. This report unveils a new mechanism by which chronic hyperammonemia induces neurological alterations: induction of peripheral inflammation. This suggests that reducing peripheral inflammation by safe procedures would improve cognitive function in patients with minimal hepatic encephalopathy. This article unveils a new mechanism by which chronic hyperammonemia induces cognitive impairment in rats: chronic hyperammonemia per se induces peripheral inflammation, which mediates many of its effects on the brain, including induction of neuroinflammation, which alters neurotransmission, leading to cognitive impairment. It is also shown that reducing peripheral inflammation by treating rats with anti-TNF-α, whichdoes not cross the blood-brain barrier, prevents hyperammonemia-induced neuroinflammation, alterations in neurotransmission and cognitive impairment.