Increased levels and activation of the IL-17 receptor in microglia contribute to enhanced neuroinflammation in cerebellum of hyperammonemic rats.

BACKGROUND: Patients with liver cirrhosis may show minimal hepatic encephalopathy (MHE) with mild cognitive impairment and motor incoordination. Rats with chronic hyperammonemia reproduce these alterations. Motor incoordination in hyperammonemic rats is due to increased GABAergic neurotransmission in cerebellum, induced by neuroinflammation, which enhances TNFα-TNFR1-S1PR2-CCL2-BDNF-TrkB pathway activation. The initial events by which hyperammonemia triggers activation of this pathway remain unclear. MHE in cirrhotic patients is triggered by a shift in inflammation with increased IL-17. The aims of this work were: (1) assess if hyperammonemia increases IL-17 content and membrane expression of its receptor in cerebellum of hyperammonemic rats; (2) identify the cell types in which IL-17 receptor is expressed and IL-17 increases in hyperammonemia; (3) assess if blocking IL-17 signaling with anti-IL-17 ex-vivo reverses activation of glia and of the TNFα-TNFR1-S1PR2-CCL2-BDNF-TrkB pathway. RESULTS: IL-17 levels and membrane expression of the IL-17 receptor are increased in cerebellum of rats with hyperammonemia and MHE, leading to increased activation of IL-17 receptor in microglia, which triggers activation of STAT3 and NF-kB, increasing IL-17 and TNFα levels, respectively. TNFα released from microglia activates TNFR1 in Purkinje neurons, leading to activation of NF-kB and increased IL-17 and TNFα also in these cells. Enhanced TNFR1 activation also enhances activation of the TNFR1-S1PR2-CCL2-BDNF-TrkB pathway which mediates microglia and astrocytes activation. CONCLUSIONS: All these steps are triggered by enhanced activation of IL-17 receptor in microglia and are prevented by ex-vivo treatment with anti-IL-17. IL-17 and IL-17 receptor in microglia would be therapeutic targets to treat neurological impairment in patients with MHE.

Neuroinflammation alters GABAergic neurotransmission in hyperammonemia and hepatic encephalopathy, leading to motor incoordination. Mechanisms and therapeutic implications.

Enhanced GABAergic neurotransmission contributes to impairment of motor coordination and gait and of cognitive function in different pathologies, including hyperammonemia and hepatic encephalopathy. Neuroinflammation is a main contributor to enhancement of GABAergic neurotransmission through increased activation of different pathways. For example, enhanced activation of the TNFα-TNFR1-NF-κB-glutaminase-GAT3 pathway and the TNFα-TNFR1-S1PR2-CCL2-BDNF-TrkB pathway in cerebellum of hyperammonemic rats enhances GABAergic neurotransmission. This is mediated by mechanisms affecting GABA synthesizing enzymes GAD67 and GAD65, total and extracellular GABA levels, membrane expression of GABAA receptor subunits, of GABA transporters GAT1 and GAT three and of chloride co-transporters. Reducing neuroinflammation reverses these changes, normalizes GABAergic neurotransmission and restores motor coordination. There is an interplay between GABAergic neurotransmission and neuroinflammation, which modulate each other and altogether modulate motor coordination and cognitive function. In this way, neuroinflammation may be also reduced by reducing GABAergic neurotransmission, which may also improve cognitive and motor function in pathologies associated to neuroinflammation and enhanced GABAergic neurotransmission such as hyperammonemia, hepatic encephalopathy or Parkinson's disease. This provides therapeutic targets that may be modulated to improve cognitive and motor function and other alterations such as fatigue in a wide range of pathologies. As a proof of concept it has been shown that antagonists of GABAA receptors such as bicuculline reduces neuroinflammation and improves cognitive and motor function impairment in rat models of hyperammonemia and hepatic encephalopathy. Antagonists of GABAA receptors are not ideal therapeutic tools because they can induce secondary effects. As a more effective treatment to reduce GABAergic neurotransmission new compounds modulating it by other mechanisms are being developed. Golexanolone reduces GABAergic neurotransmission by reducing the potentiation of GABAA receptor activation by neurosteroids such as allopregnanolone. Golexanolone reduces neuroinflammation and GABAergic neurotransmission in animal models of hyperammonemia, hepatic encephalopathy and cholestasis and this is associated with improvement of fatigue, cognitive impairment and motor incoordination. This type of compounds may be useful therapeutic tools to improve cognitive and motor function in different pathologies associated with neuroinflammation and increased GABAergic neurotransmission.

Golexanolone improves fatigue, motor incoordination and gait and memory in rats with bile duct ligation.

BACKGROUND AND AIMS: Many patients with the chronic cholestatic liver disease primary biliary cholangitis (PBC) show fatigue and cognitive impairment that reduces their quality of life. Likewise, rats with bile duct ligation (BDL) are a model of cholestatic liver disease. Current PBC treatments do not improve symptomatic alterations such as fatigue or cognitive impairment and new, more effective treatments are therefore required. Golexanolone reduces the potentiation of GABAA receptors activation by neurosteroids. Golexanolone reduces peripheral inflammation and neuroinflammation and improves cognitive and motor function in rats with chronic hyperammonemia. The aims of the present study were to assess if golexanolone treatment improves fatigue and cognitive and motor function in cholestatic BDL rats and if this is associated with improvement of peripheral inflammation, neuroinflammation, and GABAergic neurotransmission in the cerebellum. METHODS: Rats were subjected to bile duct ligation. One week after surgery, oral golexanolone was administered daily to BDL and sham-operated controls. Fatigue was analysed in the treadmill, motor coordination in the motorater, locomotor gait in the Catwalk, and short-term memory in the Y-maze. We also analysed peripheral inflammation, neuroinflammation, and GABAergic neurotransmission markers by immunohistochemistry and Western blot. RESULTS: BDL induces fatigue, impairs memory and motor coordination, and alters locomotor gait in cholestatic rats. Golexanolone improves these alterations, and this was associated with improvement of peripheral inflammation, neuroinflammation, and GABAergic neurotransmission in the cerebellum. CONCLUSION: Golexanolone may have beneficial effects to treat fatigue, and motor and cognitive impairment in patients with the chronic cholestatic liver disease PBC.

Enhanced Activation of the S1PR2-IL-1ß-Src-BDNF-TrkB Pathway Mediates Neuroinflammation in the Hippocampus and Cognitive Impairment in Hyperammonemic Rats.

Hyperammonemia contributes to hepatic encephalopathy. In hyperammonemic rats, cognitive function is impaired by altered glutamatergic neurotransmission induced by neuroinflammation. The underlying mechanisms remain unclear. Enhanced sphingosine-1-phosphate receptor 2 (S1PR2) activation in the cerebellum of hyperammonemic rats contributes to neuroinflammation. in In hyperammonemic rats, we assessed if blocking S1PR2 reduced hippocampal neuroinflammation and reversed cognitive impairment and if the signaling pathways were involved. S1PR2 was blocked with intracerebral JTE-013, and cognitive function was evaluated. The signaling pathways inducing neuroinflammation and altered glutamate receptors were analyzed in hippocampal slices. JTE-013 improved cognitive function in the hyperammonemic rats, and hyperammonemia increased S1P. This increased IL-1ß, which enhanced Src activity, increased CCL2, activated microglia and increased the membrane expression of the NMDA receptor subunit GLUN2B. This increased p38-MAPK activity, which altered the membrane expression of AMPA receptor subunits and increased BDNF, which activated the TrkB → PI3K → Akt → CREB pathway, inducing sustained neuroinflammation. This report unveils key pathways involved in the induction and maintenance of neuroinflammation in the hippocampus of hyperammonemic rats and supports S1PR2 as a therapeutic target for cognitive impairment.

Hepatic encephalopathy: investigational drugs in preclinical and early phase development.

INTRODUCTION: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, in patients with liver disease, which affects life quality and span. Current treatments are lactulose or rifaximin, acting on gut microbiota. Treatments aiming ammonia levels reduction have been tested with little success. AREAS COVERED: Pre-clinical research shows that the process inducing HE involves sequentially: liver failure, altered microbiome, hyperammonemia, peripheral inflammation, changes in immunophenotype and extracellular vesicles and neuroinflammation, which alters neurotransmission impairing cognitive and motor function. HE may be reversed using drugs acting at any step: modulating microbiota with probiotics or fecal transplantation; reducing peripheral inflammation with anti-TNFα, autotaxin inhibitors or silymarin; reducing neuroinflammation with sulforaphane, p38 MAP kinase or phosphodiesteras 5 inhibitors, antagonists of sphingosine-1-phosphate receptor 2, enhancing meningeal lymphatic drainage or with extracellular vesicles from mesenchymal stem cells; reducing GABAergic neurotransmission with indomethacin or golexanolone. EXPERT OPINION: A factor limiting the progress of HE treatment is the lack of translation of research advances into clinical trials. Only drugs acting on microbiota or ammonia reduction have been tested in patients. It is urgent to change the mentality on how to approach HE treatment to develop clinical trials to assess drugs acting on the immune system/peripheral inflammation, neuroinflammation or neurotransmission to improve HE.

Patients who died with steatohepatitis or liver cirrhosis show neuroinflammation and neuronal loss in hippocampus.

BACKGROUND: Neuroinflammation in the cerebral cortex of patients who died with liver cirrhosis and neuroinflammation, and neuronal death in the cerebellum of patients who died with steatohepatitis or cirrhosis, were reported. Hippocampal neuroinflammation could contribute to cognitive decline in patients with liver disease, but this has yet to be studied. The study aims were to assess if hippocampus from patients who died with steatohepatitis or cirrhosis showed: (i) glial activation, (ii) altered cytokine content, (iii) immune cell infiltration, (iv) neuronal apoptosis and (v) neuronal loss. METHODS: Post-mortem hippocampus was obtained from 6 controls, 19 patients with steatohepatitis (SH) and 4 patients with liver cirrhosis. SH patients were divided into SH1 (n = 9), SH2 (n = 6) and SH3 (n = 4) groups depending on disease severity. Glial activation, IL-1ß and TNFα content, CD4 lymphocyte and monocyte infiltration, neuronal apoptosis and neuronal loss were analyzed by immunohistochemistry. RESULTS: Patients who died in SH1 showed astrocyte activation, whereas those who died in SH2 also showed microglial activation, CD4 lymphocyte and monocyte infiltration, neuronal apoptosis and neuronal loss. These changes remained in patients in SH3, who also showed increased IL-1ß and TNFα. Patients who died of liver cirrhosis did not show CD4 lymphocyte infiltration, neuronal apoptosis or increase in TNFα, but still showed glial activation, increased IL-1ß and neuronal loss. CONCLUSIONS: Patients with steatohepatitis showed glial activation, immune cell infiltration, apoptosis and neuronal loss. Glial activation and neuronal loss remained in cirrhotic patients. This may explain the irreversibility of some cognitive alterations in hepatic encephalopathy. Cognitive reserve may contribute to different grades of cognitive impairment despite similar neuronal loss.

Sustained Hyperammonemia Activates NF-κB in Purkinje Neurons Through Activation of the TrkB-PI3K-AKT Pathway by Microglia-Derived BDNF in a Rat Model of Minimal Hepatic Encephalopathy.

Chronic hyperammonemia is a main contributor to the cognitive and motor impairment in patients with hepatic encephalopathy. Sustained hyperammonemia induces the TNFα expression in Purkinje neurons, mediated by NF-κB activation. The aims were the following: (1) to assess if enhanced TrkB activation by BDNF is responsible for enhanced NF-κB activation in Purkinje neurons in hyperammonemic rats, (2) to assess if this is associated with increased content of NF-κB modulated proteins such as TNFα, HMGB1, or glutaminase I, (3) to assess if these changes are due to enhanced activation of the TNFR1-S1PR2-CCR2-BDNF-TrkB pathway, (4) to analyze if increased activation of NF-κB is mediated by the PI3K-AKT pathway. It is shown that, in the cerebellum of hyperammonemic rats, increased BDNF levels enhance TrkB activation in Purkinje neurons leading to activation of PI3K, which enhances phosphorylation of AKT and of IκB, leading to increased nuclear translocation of NF-κB which enhances TNFα, HMGB1, and glutaminase I content. To assess if the changes are due to enhanced activation of the TNFR1-S1PR2-CCR2 pathway, we blocked TNFR1 with R7050, S1PR2 with JTE-013, and CCR2 with RS504393. These changes are reversed by blocking TrkB, PI3K, or the TNFR1-SP1PR2-CCL2-CCR2-BDNF-TrkB pathway at any step. In hyperammonemic rats, increased levels of BDNF enhance TrkB activation in Purkinje neurons, leading to activation of the PI3K-AKT-IκB-NF-κB pathway which increased the content of glutaminase I, HMGB1, and TNFα. Enhanced activation of this TrkB-PI3K-AKT-NF-κB pathway would contribute to impairing the function of Purkinje neurons and motor function in hyperammonemic rats and likely in cirrhotic patients with minimal or clinical hepatic encephalopathy.

Enhanced BDNF and TrkB Activation Enhance GABA Neurotransmission in Cerebellum in Hyperammonemia.

BACKGROUND: Hyperammonemia is a main contributor to minimal hepatic encephalopathy (MHE) in cirrhotic patients. Hyperammonemic rats reproduce the motor incoordination of MHE patients, which is due to enhanced GABAergic neurotransmission in the cerebellum as a consequence of neuroinflammation. In hyperammonemic rats, neuroinflammation increases BDNF by activating the TNFR1-S1PR2-CCR2 pathway. (1) Identify mechanisms enhancing GABAergic neurotransmission in hyperammonemia; (2) assess the role of enhanced activation of TrkB; and (3) assess the role of the TNFR1-S1PR2-CCR2-BDNF pathway. In the cerebellum of hyperammonemic rats, increased BDNF levels enhance TrkB activation in Purkinje neurons, leading to increased GAD65, GAD67 and GABA levels. Enhanced TrkB activation also increases the membrane expression of the γ2, α2 and ß3 subunits of GABAA receptors and of KCC2. Moreover, enhanced TrkB activation in activated astrocytes increases the membrane expression of GAT3 and NKCC1. These changes are reversed by blocking TrkB or the TNFR1-SP1PR2-CCL2-CCR2-BDNF-TrkB pathway. Hyperammonemia-induced neuroinflammation increases BDNF and TrkB activation, leading to increased synthesis and extracellular GABA, and the amount of GABAA receptors in the membrane and chloride gradient. These factors enhance GABAergic neurotransmission in the cerebellum. Blocking TrkB or the TNFR1-SP1PR2-CCL2-CCR2-BDNF-TrkB pathway would improve motor function in patients with hepatic encephalopathy and likely with other pathologies associated with neuroinflammation.

The S1PR2-CCL2-BDNF-TrkB pathway mediates neuroinflammation and motor incoordination in hyperammonaemia.

AIMS: Chronic hyperammonaemia and inflammation synergistically induce neurological impairment, including motor incoordination, in hepatic encephalopathy. Hyperammonaemic rats show neuroinflammation in the cerebellum which enhances GABAergic neurotransmission leading to motor incoordination. We aimed to identify underlying mechanisms. The aims were (1) to assess if S1PR2 is involved in microglial and astrocytic activation in the cerebellum of hyperammonaemic rats; (2) to identify pathways by which enhanced S1PR2 activation induces neuroinflammation and alters neurotransmission; (3) to assess if blocking S1PR2 reduces neuroinflammation and restores motor coordination in hyperammonaemic rats. METHODS: We performed ex vivo studies in cerebellar slices from control or hyperammonaemic rats to identify pathways by which neuroinflammation enhances GABAergic neurotransmission in hyperammonaemia. Neuroinflammation and neurotransmission were assessed by immunochemistry/immunofluorescence and western blot. S1PR2 was blocked by intracerebral treatment with JTE-013 using osmotic mini-pumps. Motor coordination was assessed by beam walking. RESULTS: Chronic hyperammonaemia enhances S1PR2 activation in the cerebellum by increasing its membrane expression. This increases CCL2, especially in Purkinje neurons. CCL2 activates CCR2 in microglia, leading to microglial activation, increased P2X4 membrane expression and BDNF in microglia. BDNF enhances TrkB activation in neurons, increasing KCC2 membrane expression. This enhances GABAergic neurotransmission, leading to motor incoordination in hyperammonaemic rats. Blocking S1PR2 in hyperammonaemic rats by intracerebral administration of JTE-013 normalises the S1PR2-CCL2-CCR2-BDNF-TrkB-KCC2 pathway, reduces glial activation and restores motor coordination in hyperammonaemic rats. CONCLUSIONS: Enhanced S1PR2-CCL2-BDNF-TrkB pathway activation mediates neuroinflammation and incoordination in hyperammonaemia. The data raise a promising therapy for patients with hepatic encephalopathy using compounds targeting this pathway.

Blocking glycine receptors reduces neuroinflammation and restores neurotransmission in cerebellum through ADAM17-TNFR1-NF-κß pathway.

BACKGROUND: Chronic hyperammonemia induces neuroinflammation in cerebellum, with glial activation and enhanced activation of the TNFR1-NF-kB-glutaminase-glutamate-GABA pathway. Hyperammonemia also increases glycinergic neurotransmission. These alterations contribute to cognitive and motor impairment. Activation of glycine receptors is reduced by extracellular cGMP, which levels are reduced in cerebellum of hyperammonemic rats in vivo. We hypothesized that enhanced glycinergic neurotransmission in hyperammonemic rats (1) contributes to induce neuroinflammation and glutamatergic and GABAergic neurotransmission alterations; (2) is a consequence of the reduced extracellular cGMP levels. The aims were to assess, in cerebellum of hyperammonemic rats, (a) whether blocking glycine receptors with the antagonist strychnine reduces neuroinflammation; (b) the cellular localization of glycine receptor; (c) the effects of blocking glycine receptors on the TNFR1-NF-kB-glutaminase-glutamate-GABA pathway and microglia activation; (d) whether adding extracellular cGMP reproduces the effects of strychnine. METHODS: We analyzed in freshly isolated cerebellar slices from control or hyperammonemic rats the effects of strychnine on activation of microglia and astrocytes, the content of TNFa and IL1b, the surface expression of ADAM17, TNFR1 and transporters, the phosphorylation levels of ERK, p38 and ADAM17. The cellular localization of glycine receptor was assessed by immunofluorescence. We analyzed the content of TNFa, IL1b, HMGB1, glutaminase, and the level of TNF-a mRNA and NF-κB in Purkinje neurons. Extracellular concentrations of glutamate and GABA were performed by in vivo microdialysis in cerebellum. We tested whether extracellular cGMP reproduces the effects of strychnine in ex vivo cerebellar slices. RESULTS: Glycine receptors are expressed mainly in Purkinje cells. In hyperammonemic rats, enhanced glycinergic neurotransmission leads to reduced membrane expression of ADAM17, resulting in increased surface expression and activation of TNFR1 and of the associated NF-kB pathway. This increases the expression in Purkinje neurons of TNFa, IL-1b, HMGB1, and glutaminase. Increased glutaminase activity leads to increased extracellular glutamate, which increases extracellular GABA. Increased extracellular glutamate and HMGB1 potentiate microglial activation. Blocking glycine receptors with strychnine or extracellular cGMP completely prevents the above pathway in hyperammonemic rats. CONCLUSIONS: Glycinergic neurotransmission modulates neuroinflammation. Enhanced glycinergic neurotransmission in hyperammonemia would be due to reduced extracellular cGMP. These results shed some light on possible new therapeutic target pathways for pathologies associated to neuroinflammation.

Sustained hyperammonemia induces TNF-a IN Purkinje neurons by activating the TNFR1-NF-κB pathway.

BACKGROUND: Patients with liver cirrhosis may develop hepatic encephalopathy. Rats with chronic hyperammonemia exhibit neurological alterations mediated by peripheral inflammation and neuroinflammation. Motor incoordination is due to increased TNF-a levels and activation of its receptor TNFR1 in the cerebellum. The aims were to assess (a) whether peripheral inflammation is responsible for TNF-a induction in hyperammonemic rats, (b) the cell type(s) in which TNF-a is increased, (c) whether this increase is associated with increased nuclear NF-κB and TNFR1 activation, (d) the time course of TNF-a induction, and (e) if TNF-a is induced in the Purkinje neurons of patients who die with liver cirrhosis. METHODS: We analyzed the level of TNF-a mRNA and NF-κB in microglia, astrocytes, and Purkinje neurons in the cerebellum after 1, 2, and 4 weeks of hyperammonemia. We assessed whether preventing peripheral inflammation by administering an anti-TNF-a antibody prevents TNF-a induction. We tested whether TNF-a induction is reversed by R7050, which inhibits the TNFR1-NF-κB pathway, in ex vivo cerebellar slices. RESULTS: Hyperammonemia induced microglial and astrocyte activation at 1 week. This was followed by TNF-a induction in both glial cell types at 2 weeks and in Purkinje neurons at 4 weeks. The level of TNF-a mRNA increased in parallel with the TNF-a protein level, indicating that TNF-a was synthesized in Purkinje cells. This increase was associated with increased NF-κB nuclear translocation. The nuclear translocation of NF-κB and the increase in TNF-a were reversed by R7050, indicating that they were mediated by the activation of TNFR1. Preventing peripheral inflammation with an anti-TNF-a antibody prevents TNF-a induction. CONCLUSION: Sustained (4 weeks) but not short-term hyperammonemia induces TNF-a in Purkinje neurons in rats. This is mediated by peripheral inflammation. TNF-a is also increased in the Purkinje neurons of patients who die with liver cirrhosis. The results suggest that hyperammonemia induces TNF-a in glial cells and that TNF-a released by glial cells activates TNFR1 in Purkinje neurons, leading to NF-κB nuclear translocation and the induction of TNF-a expression, which may contribute to the neurological alterations observed in hyperammonemia and hepatic encephalopathy.

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

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. LAY SUMMARY: 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, which does not cross the blood-brain barrier, prevents hyperammonemia-induced neuroinflammation, alterations in neurotransmission and cognitive impairment.

Differential role of interleukin-1ß in neuroinflammation-induced impairment of spatial and nonspatial memory in hyperammonemic rats.

Activated microglia and increased brain IL-1ß play a main role in cognitive impairment in much pathology. We studied the role of IL-1ß in neuroinflammation-induced impairment of the following different types of learning and memory: novel object recognition (NOR), novel object location (NOL), spatial learning, reference memory (RM), and working memory (WM). All these processes are impaired in hyperammonemic rats. We assessed which of these types of learning and memory are restored by blocking the IL-1 receptor in vivo in hyperammonemic rats and the possible mechanisms involved. Blocking the IL-1 receptor reversed microglial activation in the hippocampus, perirhinal cortex, and prefrontal cortex but not in the postrhinal cortex. This was associated with the restoration of NOR and WM but not of tasks involving a spatial component (NOL and RM). This suggests that IL-1ß would be involved in neuroinflammation-induced nonspatial memory impairment, whereas spatial memory impairment would be IL-1ß-independent and would be mediated by other proinflammatory factors.-Taoro-González, L., Cabrera-Pastor, A., Sancho-Alonso, M., Arenas, Y. M., Meseguer-Estornell, F., Balzano, T., ElMlili, N., Felipo, V. Differential role of interleukin-1ß in neuroinflammation-induced impairment of spatial and nonspatial memory in hyperammonemic rats.

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 alters extracellular glutamate, glutamine and GABA and membrane expression of their transporters in rat cerebellum. Modulation by extracellular cGMP.

Trafficking of glutamate, glutamine and GABA between astrocytes and neurons is essential to maintain proper neurotransmission. Chronic hyperammonemia alters neurotransmission and cognitive function. The aims of this work were to analyze in cerebellum of rats the effects of chronic hyperammonemia on: a) extracellular glutamate, glutamine and GABA concentrations; b) membrane expression of glutamate, glutamine and GABA transporters; c) how they are modulated by extracellular cGMP. Hyperammonemic rats show increased levels of extracellular glutamate, glutamine, GABA and citrulline in cerebellum in vivo. Hyperammonemic rats show: a) increased membrane expression of the astrocytic glutamine transporter SNAT3 and reduced membrane expression of the neuronal transporter SNAT1; b) reduced membrane expression of the neuronal GABA transporter GAT1 and increased membrane expression of the astrocytic GAT3 transporter; c) reduced membrane expression of the astrocytic glutamate transporters GLAST and GLT-1 and of the neuronal transporter EAAC1. Increasing extracellular cGMP normalizes membrane expression of SNAT3, GAT3, GAT1 and GLAST and extracellular glutamate, glutamine, GABA and citrulline hyperammonemic rats. Extracellular cGMP also modulates membrane expression of most transporters in control rats, reducing membrane expression of SNAT1, GLT-1 and EAAC1 and increasing that of GAT1 and GAT3. Modulation of SNAT3, SNAT1, GLT-1 and EAAC1 by extracellular cGMP would be mediated by inhibition of glycine receptors. These data suggest that, in pathological situations such as hyperammonemia, hepatic encephalopathy or Alzheimer's disease, reduced levels of extracellular cGMP contribute to alterations in membrane expression of glutamine, glutamate and GABA transporters, in the extracellular levels of glutamine, glutamate and GABA and in neurotransmission. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

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-1ß levels in hyperammonemic rats, which reduces IL-1 receptor-mediated Src over-activation. Reduction in IL-1ß levels is due to the reduction of microglia activation in hippocampus of hyperammonemic rats.

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

BACKGROUND: Hyperammonemic 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-1ß 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-1ß 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. METHODS: We 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. RESULTS: Hyperammonemia reduces GluA1 and increases GluA2 membrane expression and reduces phosphorylation of GluA1 at Ser831 and of GluA2 at Ser880. Hyperammonemia increases IL-1ß, 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. CONCLUSIONS: This 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-1ß by acting, for example, on p38 or PKCδ. IL-1ß alters membrane expression of GluA1 and GluA2 AMPA receptor subunits by two difrerent mechanisms in the hippocampus of hyperammonemic rats.