Sublethal necroptosis signaling promotes inflammation and liver cancer.

It is currently not well known how necroptosis and necroptosis responses manifest in vivo. Here, we uncovered a molecular switch facilitating reprogramming between two alternative modes of necroptosis signaling in hepatocytes, fundamentally affecting immune responses and hepatocarcinogenesis. Concomitant necrosome and NF-κB activation in hepatocytes, which physiologically express low concentrations of receptor-interacting kinase 3 (RIPK3), did not lead to immediate cell death but forced them into a prolonged "sublethal" state with leaky membranes, functioning as secretory cells that released specific chemokines including CCL20 and MCP-1. This triggered hepatic cell proliferation as well as activation of procarcinogenic monocyte-derived macrophage cell clusters, contributing to hepatocarcinogenesis. In contrast, necrosome activation in hepatocytes with inactive NF-κB-signaling caused an accelerated execution of necroptosis, limiting alarmin release, and thereby preventing inflammation and hepatocarcinogenesis. Consistently, intratumoral NF-κB-necroptosis signatures were associated with poor prognosis in human hepatocarcinogenesis. Therefore, pharmacological reprogramming between these distinct forms of necroptosis may represent a promising strategy against hepatocellular carcinoma.

Hyperammonemia-induced changes in the cerebral transcriptome and proteome.

Molecular alterations underlying cerebral impairment in hyperammonemic disorders such as in hepatic encephalopathy (HE) are only poorly understood. Using transcriptomics and proteomics on brains of mice with systemic hyperammonemia resulting from knockout of hepatic glutamine synthetase (LGS-KO) we identified up to 214 genes and 34 proteins whose expressions were altered in brains of LGS-KO mice in a brain region-specific way. Differentially expressed genes were enriched for those related to oxidative stress, cell proliferation, heme metabolism and others. Due to their particularly high expression changes, coactivator associated arginine methyltransferase 1 (CARM1), TROVE2 and Lipocalin-2 (LCN2) were selected for further analyses. All selected candidates were expressed by astrocytes in rodent brain and challenging cultured astrocytes with NH4Cl changed their protein and mRNA levels similar to what was found in brains of LGS-KO mice. Further functional analyses suggested a role of CARM1 for senescence, TROVE2 for RNA quality control and LCN2 for disturbed iron homeostasis in ammonia-exposed astrocytes. LCN2 protein and Trove2 mRNA were also elevated in cerebral cortex of ammonium acetate-challenged rats and in post mortem brain tissue from patients with liver cirrhosis and HE, respectively. This study identified new molecular players potentially relevant for cerebral dysfunction in HE.

Taurine transporter (TauT) deficiency impairs ammonia detoxification in mouse liver.

Hepatic ammonia handling was analyzed in taurine transporter (TauT) KO mice. Surprisingly, hyperammonemia was present at an age of 3 and 12 months despite normal tissue integrity. This was accompanied by cerebral RNA oxidation. As shown in liver perfusion experiments, glutamine production from ammonia was diminished in TauT KO mice, whereas urea production was not affected. In livers from 3-month-old TauT KO mice protein expression and activity of glutamine synthetase (GS) were unaffected, whereas the ammonia-transporting RhBG protein was down-regulated by about 50%. Double reciprocal plot analysis of glutamine synthesis versus perivenous ammonia concentration revealed that TauT KO had no effect on the capacity of glutamine formation in 3-month-old mice, but doubled the ammonia concentration required for half-maximal glutamine synthesis. Since hepatic RhBG expression is restricted to GS-expressing hepatocytes, the findings suggest that an impaired ammonia transport into these cells impairs glutamine synthesis. In livers from 12-, but not 3-month-old TauT KO mice, RhBG expression was not affected, surrogate markers for oxidative stress were strongly up-regulated, and GS activity was decreased by 40% due to an inactivating tyrosine nitration. This was also reflected by kinetic analyses in perfused liver, which showed a decreased glutamine synthesizing capacity by 43% and a largely unaffected ammonia concentration dependence. It is concluded that TauT deficiency triggers hyperammonemia through impaired hepatic glutamine synthesis due to an impaired ammonia transport via RhBG at 3 months and a tyrosine nitration-dependent inactivation of GS in 12-month-old TauT KO mice.

Glutamine synthetase as a central element in hepatic glutamine and ammonia metabolism: novel aspects.

Glutamine synthetase (GS) in the liver is expressed in a small perivenous, highly specialized hepatocyte population and is essential for the maintenance of low, non-toxic ammonia levels in the organism. However, GS activity can be impaired by tyrosine nitration of the enzyme in response to oxidative/nitrosative stress in a pH-sensitive way. The underlying molecular mechanism as investigated by combined molecular simulations and in vitro experiments indicates that tyrosine nitration can lead to a fully reversible and pH-sensitive regulation of protein function. This approach was also used to understand the functional consequences of several recently described point mutations of human GS with clinical relevance and to suggest an approach to restore impaired GS activity.

Pathomechanisms in hepatic encephalopathy.

Hepatic encephalopathy (HE) is a frequent neuropsychiatric complication in patients with acute or chronic liver failure. Symptoms of HE in particular include disturbances of sensory and motor functions and cognition. HE is triggered by heterogeneous factors such as ammonia being a main toxin, benzodiazepines, proinflammatory cytokines and hyponatremia. HE in patients with liver cirrhosis is triggered by a low-grade cerebral edema and cerebral oxidative/nitrosative stress which bring about a number of functionally relevant alterations including posttranslational protein modifications, oxidation of RNA, gene expression changes and senescence. These alterations are suggested to impair astrocyte/neuronal functions and communication. On the system level, a global slowing of oscillatory brain activity and networks can be observed paralleling behavioral perceptual and motor impairments. Moreover, these changes are related to increased cerebral ammonia, alterations in neurometabolite and neurotransmitter concentrations and cortical excitability in HE patients.

Characterization of the scavenger cell proteome in mouse and rat liver.

The structural-functional organization of ammonia and glutamine metabolism in the liver acinus involves highly specialized hepatocyte subpopulations like glutamine synthetase (GS) expressing perivenous hepatocytes (scavenger cells). However, this cell population has not yet been characterized extensively regarding expression of other genes and potential subpopulations. This was investigated in the present study by proteome profiling of periportal GS-negative and perivenous GS-expressing hepatocytes from mouse and rat. Apart from established markers of GS+ hepatocytes such as glutamate/aspartate transporter II (GLT1) or ammonium transporter Rh type B (RhBG), we identified novel scavenger cell-specific proteins like basal transcription factor 3 (BTF3) and heat-shock protein 25 (HSP25). Interestingly, BTF3 and HSP25 were heterogeneously distributed among GS+ hepatocytes in mouse liver slices. Feeding experiments showed that RhBG expression was increased in livers from mice fed with high protein diet compared to standard chow. While spatial distributions of GS and carbamoylphosphate synthetase 1 (CPS1) were unaffected, periportal areas constituted by glutaminase 2 (GLS2)-positive hepatocytes were enlarged or reduced in response to high or low protein diet, respectively. The data suggest that the population of perivenous GS+ scavenger cells is heterogeneous and not uniform as previously suggested which may reflect a functional heterogeneity, possibly relevant for liver regeneration.

Biallelic mutation of human SLC6A6 encoding the taurine transporter TAUT is linked to early retinal degeneration.

We previously reported that inactivation of the transmembrane taurine transporter (TauT or solute carrier 6a6) causes early retinal degeneration in mice. Compatible with taurine's indispensability for cell volume homeostasis, protein stabilization, cytoprotection, antioxidation, and immuno- and neuromodulation, mice develop multisystemic dysfunctions (hearing loss; liver fibrosis; and behavioral, heart, and skeletal muscle abnormalities) later on. Here, by genetic, cell biologic, in vivo1H-magnetic resonance spectroscopy and molecular dynamics simulation studies, we conducted in-depth characterization of a novel disorder: human TAUT deficiency. Loss of TAUT function due to a homozygous missense mutation caused panretinal degeneration in 2 brothers. TAUTp.A78E still localized in the plasma membrane but is predicted to impact structural stabilization. 3H-taurine uptake by peripheral blood mononuclear cells was reduced by 95%, and taurine levels were severely reduced in plasma, skeletal muscle, and brain. Extraocular dysfunctions were not yet detected, but significantly increased urinary excretion of 8-oxo-7,8-dihydroguanosine indicated generally enhanced (yet clinically unapparent) oxidative stress and RNA oxidation, warranting continuous broad surveillance.-Preising, M. N., Görg, B., Friedburg, C., Qvartskhava, N., Budde, B. S., Bonus, M., Toliat, M. R., Pfleger, C., Altmüller, J., Herebian, D., Beyer, M., Zöllner, H. J., Wittsack, H.-J., Schaper, J., Klee, D., Zechner, U., Nürnberg, P., Schipper, J., Schnitzler, A., Gohlke, H., Lorenz, B., Häussinger, D., Bolz, H. J. Biallelic mutation of human SLC6A6 encoding the taurine transporter TAUT is linked to early retinal degeneration.

O-GlcNAcylation-dependent upregulation of HO1 triggers ammonia-induced oxidative stress and senescence in hepatic encephalopathy.

BACKGROUND & AIMS: Cerebral oxidative stress plays an important role in the pathogenesis of hepatic encephalopathy (HE), but the underlying mechanisms are incompletely understood. Herein, we analyzed a role of heme oxygenase (HO)1, iron and NADPH oxidase 4 (Nox4) for the induction of oxidative stress and senescence in HE. METHODS: Gene and protein expression in human post-mortem brain samples was analyzed by gene array and western blot analysis. Mechanisms and functional consequences of HO1 upregulation were studied in NH4Cl-exposed astrocytes in vitro by western blot, qPCR and super-resolution microscopy. RESULTS: HO1 and the endoplasmic reticulum (ER) stress marker grp78 were upregulated, together with changes in the expression of multiple iron metabolism-related genes, in post-mortem brain samples from patients with liver cirrhosis and HE. NH4Cl elevated HO1 protein and mRNA in cultured astrocytes through glutamine synthetase (GS)-dependent upregulation of glutamine/fructose amidotransferases 1/2 (GFAT1/2), which blocked the transcription of the HO1-targeting miR326-3p in a O-GlcNAcylation dependent manner. Upregulation of HO1 by NH4Cl triggered ER stress and was associated with elevated levels of free ferrous iron and expression changes in iron metabolism-related genes, which were largely abolished after knockdown or inhibition of GS, GFAT1/2, HO1 or iron chelation. NH4Cl, glucosamine (GlcN) and inhibition of miR326-3p upregulated Nox4, while knockdown of Nox4, GS, GFAT1/2, HO1 or iron chelation prevented NH4Cl-induced RNA oxidation and astrocyte senescence. Elevated levels of grp78 and O-GlcNAcylated proteins were also found in brain samples from patients with liver cirrhosis and HE. CONCLUSION: The present study identified glucosamine synthesis-dependent protein O-GlcNAcylation as a novel mechanism in the pathogenesis of HE that triggers oxidative and ER stress, as well as senescence, through upregulation of HO1 and Nox4. LAY SUMMARY: Patients with liver cirrhosis frequently exhibit hyperammonemia and suffer from cognitive and motoric dysfunctions, which at least in part involve premature ageing of the astrocytes in the brain. This study identifies glucosamine and an O-GlcNAcylation-dependent disruption of iron homeostasis as novel triggers of oxidative stress, thereby mediating ammonia toxicity in the brain.

Regulation of Plasma Membrane Localization of the Na⁺-Taurocholate Co-Transporting Polypeptide by Glycochenodeoxycholate and Tauroursodeoxycholate.

BACKGROUND/AIMS: Hydrophobic bile salts, such as glycochenodeoxycholate (GCDC) can trigger hepatocyte apoptosis, which is prevented by tauroursodesoxycholate (TUDC), but the effects of GCDC and TUDC on sinusoidal bile salt uptake via the Na⁺-taurocholate transporting polypeptide (Ntcp) are unclear. METHODS: The effects of GCDC and TUDC on the plasma membrane localization of Ntcp were studied in perfused rat liver by means of immunofluorescence analysis and super-resolution microscopy. The underlying signaling events were investigated by Western blotting and inhibitor studies. RESULTS: GCDC (20 µmol/l) induced within 60 min a retrieval of Ntcp from the basolateral membrane into the cytosol, which was accompanied by an activating phosphorylation of the Src kinases Fyn and Yes. Both, Fyn activation and the GCDC-induced Ntcp retrieval from the plasma membrane were sensitive to the NADPH oxidase inhibitor apocynin, the antioxidant N-acetylcysteine and the Src family kinase inhibitors SU6656 and PP-2, whereas PP-2 did not inhibit GCDC-induced Yes activation. Internalization of Ntcp by GCDC was also prevented by the protein kinase C (PKC) inhibitor Gö6850. TUDC (20 µmol/l) reversed the GCDC-induced retrieval of Ntcp from the plasma membrane and prevented the activation of Fyn and Yes in GCDC-perfused rat livers. Reinsertion of Ntcp into the basolateral membrane in GCDC-perfused livers by TUDC was sensitive to the protein kinase A (PKA) inhibitor H89 and the integrin-inhibitory peptide GRGDSP, whereas the control peptide GRADSP was ineffective. Ex posure of cultured rat hepatocytes to GCDC (50 µmol/l, 15min) increased the fluorescence intensity of the reactive oxygen fluorescent indicator DCF to about 1.6-fold of untreated controls in a TUDC (50 µmol/l)-sensitive way. GCDC caused a TUDC-sensitive canalicular dilatation without evidence for Bsep retrieval from the canalicular membrane. CONCLUSION: The present study suggests that GCDC triggers the retrieval of Ntcp from the basolateral membrane into the cytosol through an oxidative stress-dependent activation of Fyn. TUDC prevents the GCDC-induced Fyn activation and Ntcp retrieval through integrin-dependent activation of PKA.

Tauroursodeoxycholate protects from glycochenodeoxycholate-induced gene expression changes in perfused rat liver.

Tauroursodeoxycholate (TUDC) is well known to protect against glycochenodeoxycholate (GCDC)-induced apoptosis in rat hepatocytes. In the present study, we analyzed whether TUDC also exerts protective effects by modulating GCDC-induced gene expression changes. For this, gene array-based transcriptome analysis and quantitative polymerase chain reaction (qPCR) were performed on RNA isolated from rat livers perfused with GCDC, TUDC or a combination of both (each 20 µm for 2 h). GCDC led to a significant increase of lactate dehydrogenase (LDH) into the effluent perfusate, which was prevented by TUDC. GCDC, TUDC and co-perfusion induced distinct gene expression changes. While GCDC upregulated the expression of several pro-inflammatory genes, co-perfusion with TUDC increased the expression of pro-proliferative and anti-apoptotic p53 target genes. In line with this, levels of serine20-phosphorylated p53 and of its target gene p21 were elevated by GCDC in a TUDC-sensitive way. GCDC upregulated the oxidative stress surrogate marker 8OH(d)G and the pro-apoptotic microRNAs miR-15b/16 and these effects were prevented by TUDC. The upregulation of miR-15b and miR-16 in GCDC-perfused livers was accompanied by a downregulation of several potential miR-15b and miR-16 target genes. The present study identified changes in the transcriptome of the rat liver which suggest, that TUDC is hepatoprotective by counteracting GCDC-induced gene expression changes.

Hyperammonemia in gene-targeted mice lacking functional hepatic glutamine synthetase.

Urea cycle defects and acute or chronic liver failure are linked to systemic hyperammonemia and often result in cerebral dysfunction and encephalopathy. Although an important role of the liver in ammonia metabolism is widely accepted, the role of ammonia metabolizing pathways in the liver for maintenance of whole-body ammonia homeostasis in vivo remains ill-defined. Here, we show by generation of liver-specific Gln synthetase (GS)-deficient mice that GS in the liver is critically involved in systemic ammonia homeostasis in vivo. Hepatic deletion of GS triggered systemic hyperammonemia, which was associated with cerebral oxidative stress as indicated by increased levels of oxidized RNA and enhanced protein Tyr nitration. Liver-specific GS-deficient mice showed increased locomotion, impaired fear memory, and a slightly reduced life span. In conclusion, the present observations highlight the importance of hepatic GS for maintenance of ammonia homeostasis and establish the liver-specific GS KO mouse as a model with which to study effects of chronic hyperammonemia.

Ammonia Attenuates LPS-Induced Upregulation of Pro-Inflammatory Cytokine mRNA in Co-Cultured Astrocytes and Microglia.

Hepatic encephalopathy (HE) is associated with cerebral microglia activation. Ammonia, a major toxin of HE, activates microglia in vitro but does not trigger pro-inflammatory cytokine synthesis. In the present study we analysed effects of ammonia on lipopolysaccharide (LPS)-induced upregulation of microglia activation and cytokine mRNA as well as on cytokine secretion in mono-cultured microglia and co-cultured astrocytes and microglia. In mono-cultured microglia LPS (100 ng/ml, 18 h) strongly elevated mRNA levels of the microglia activation marker CD14 and the pro-inflammatory cytokines IL-1α/ß, IL-6 and TNF-α. NH4Cl (5 mmol/l) had no effect on LPS-induced upregulation of CD14, IL-1α/ß and IL-6 mRNA but enhanced LPS-induced upregulation of TNF-α mRNA in mono-cultured microglia. In co-cultured astrocytes and microglia, however, LPS-induced upregulation of IL-1α/ß, TNF-α, IL-6, CD14 but not of IL-10, IL-12A/B or TGFß1-3 mRNA was attenuated by NH4Cl. LPS-induced upregulation of IL-1α/ß, IL-6 and TNF-α was also diminished by the TGR5-ligands allopregnanolone and taurolithocholic acid in mono-cultured microglia. NH4Cl also attenuated LPS-induced release of MCP-1, IL-6 and IL-10 in mono-cultured microglia. mRNA level of surrogate marker for microglia activation (CD14) and for the anti-inflammatory M2-type microglia (CD163, CXCL1, CXCL2) were also elevated in post mortem brain tissue taken from the fusiforme gyrus of patients with liver cirrhosis and HE. The findings suggest that ammonia attenuates LPS-induced microglia reactivity in an astrocyte-dependent way. One may speculate that these anti-inflammatory effects of ammonia may be triggered by neurosteroids derived from astrocytes and may account for absence of microglia reactivity in cerebral cortex of cirrhotic patients with HE.

Molecular Mechanisms of Glutamine Synthetase Mutations that Lead to Clinically Relevant Pathologies.

Glutamine synthetase (GS) catalyzes ATP-dependent ligation of ammonia and glutamate to glutamine. Two mutations of human GS (R324C and R341C) were connected to congenital glutamine deficiency with severe brain malformations resulting in neonatal death. Another GS mutation (R324S) was identified in a neurologically compromised patient. However, the molecular mechanisms underlying the impairment of GS activity by these mutations have remained elusive. Molecular dynamics simulations, free energy calculations, and rigidity analyses suggest that all three mutations influence the first step of GS catalytic cycle. The R324S and R324C mutations deteriorate GS catalytic activity due to loss of direct interactions with ATP. As to R324S, indirect, water-mediated interactions reduce this effect, which may explain the suggested higher GS residual activity. The R341C mutation weakens ATP binding by destabilizing the interacting residue R340 in the apo state of GS. Additionally, the mutation is predicted to result in a significant destabilization of helix H8, which should negatively affect glutamate binding. This prediction was tested in HEK293 cells overexpressing GS by dot-blot analysis: Structural stability of H8 was impaired through mutation of amino acids interacting with R341, as indicated by a loss of masking of an epitope in the glutamate binding pocket for a monoclonal anti-GS antibody by L-methionine-S-sulfoximine; in contrast, cells transfected with wild type GS showed the masking. Our analyses reveal complex molecular effects underlying impaired GS catalytic activity in three clinically relevant mutants. Our findings could stimulate the development of ATP binding-enhancing molecules by which the R324S mutant can be repaired extrinsically.

Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy.

Hepatic encephalopathy (HE) is a frequent complication of liver cirrhosis and is due to a low-grade cerebral edema associated with oxidative/nitrosative stress. Recent reports suggest that cognitive impairment in cirrhotic patients may not resolve completely after an attack of manifest HE. As astrocyte dysfunction is central to the pathogenesis of HE and astrocytes are critically involved in synaptic plasticity, we tested for sustained impairment of astrocyte function by analyzing expression levels of senescence biomarkers in ammonia-treated cultured rat astrocytes and in postmortem brain samples from cirrhotic patients with or without HE. NH4 Cl time- and dose-dependently inhibited proliferation of cultured astrocytes by up to 45% (5 mmol/L, 72 h) and strongly increased senescence-associated ß-galactosidase activity. Inhibition of astrocyte proliferation by ammonia was mediated by a l-methionine sulfoximine-, oxidative stress-, and p38(MAPK) -dependent activation of p53 associated with enhanced transcription of cell cycle inhibitory genes GADD45α and p21. Mitochondria and the nucleus were identified as sources of oxygen radical formation after prolonged NH4 Cl exposure. Concurrently, NH4 Cl (5 mmol/L) treatment inhibited both epidermal growth factor- and brain-derived neurotrophic factor (BDNF)-induced proliferation as well as BDNF-mediated astrocyte morphology changes through downregulation of the respective growth factor receptors epidermal growth factor receptor and truncated tyrosine receptor kinase B. Increased mRNA expression levels of senescence-associated genes were also found in post mortem brain samples from patients with liver cirrhosis with HE, but not in those without HE. The data suggest that ammonia toxicity and HE are associated with premature astrocyte senescence, which may impair neurotransmission and contribute to persistence of cognitive disturbances after resolution of episodes of overt HE.

Multidrug resistance-associated protein 4 expression in ammonia-treated cultured rat astrocytes and cerebral cortex of cirrhotic patients with hepatic encephalopathy.

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome frequently accompanying liver cirrhosis and reflects the clinical manifestation of a low grade cerebral edema associated with cerebral oxidative/nitrosative stress. The multidrug resistance-associated protein (Mrp) 4 is an export pump which transports metabolites that were recently suggested to play a major role in the pathogenesis of HE such as neurosteroids and cyclic nucleotides. We therefore studied Mrp4 expression changes in ammonia-exposed cultured astrocytes and postmortem human brain samples of cirrhotic patients with HE. NH4 Cl increased Mrp4 mRNA and protein levels in astrocytes in a dose- and time-dependent manner up to threefold after 72 h of exposure and concurrently inhibited N-glycosylation of Mrp4 protein. Upregulation of Mrp4 mRNA and protein as well as impaired N-glycosylation of Mrp4 protein by ammonia were sensitive towards the glutamine-synthetase inhibitor l-methionine-S-sulfoximine and were not induced by CH3 NH3 Cl (5 mmol/L). Upregulation of Mrp4 mRNA required ammonia-induced activation of nitric oxide synthases or NADPH oxidase and p38MAPK -dependent activation of PPARα. Inhibition of Mrp4 by ceefourin 1 synergistically enhanced both, inhibition of astrocyte proliferation as well as transcription of the oxidative stress surrogate marker heme oxygenase 1 by forskolin (10 µmol/L, 72 h) or NH4 Cl (5 mmol/L, 72 h) in cultured rat astrocytes. Increased Mrp4 mRNA and protein levels were also found in postmortem brain samples from patients with liver cirrhosis with HE but not in those without HE. The data show that Mrp4 is upregulated in HE, which may be relevant for the handling of neurosteroids and cyclic nucleotides in response to ammonia. GLIA 2015;63:2092-2105.

IFN-γ licenses CD11b(+) cells to induce progression of systemic lupus erythematosus.

Autoantibodies are a hallmark of autoimmune diseases, such as rheumatoid arthritis, autoimmune hepatitis, and systemic lupus erythematosus (SLE). High titers of anti-nuclear antibodies are used as surrogate marker for SLE, however their contribution to pathogenesis remains unclear. Using murine model of SLE and human samples, we studied the effect of immune stimulation on relapsing of SLE. Although autoantibodies bound to target cells in vivo, only additional activation of CD8(+) T cells converted this silent autoimmunity into overt disease. In mice as well as in humans CD8(+) T cells derived IFN-γ enhanced expression of Fc-receptors on CD11b(+) cells. High expression of Fc-receptors allowed CD11b(+) cells to bind to antibody covered target cells and to destroy them in vivo. We found that autoantibodies induce clinically relevant disease when adaptive immunity, specific for disease non-related antigen, is activated.

Ephrin/Ephrin receptor expression in ammonia-treated rat astrocytes and in human cerebral cortex in hepatic encephalopathy.

Hepatic encephalopathy (HE) represents a neuropsychiatric syndrome, which evolves as a consequence of a low grade cerebral edema and a concomitant oxidative/nitrosative stress response. Ephrin receptors (EphR) and their ligands (ephrins) regulate astrocytic glutamate uptake and gliotransmitter release thereby governing neurotransmission, but their role in HE and ammonia toxicity is unclear. We therefore tested effects of ammonia on expression levels of EphR/ephrin isoforms in cultured rat astrocytes and analysed underlying mechanisms. NH4Cl induced mRNA expression changes of several EphR/ephrin isoforms in a methionine sulfoximine-, NADPH oxidase- and NO synthase-dependent manner in cultured astrocytes. A prominent upregulation was noted for EphR A4 mRNA and protein in NH4Cl-treated astrocytes. NH4Cl-treatment decreased EphR A4 molecular mass to similar extent as found in astrocytes treated with the N-glycosylation inhibitor tunicamycin. Knockdown of EphR A4 by siRNA, or treating astrocytes with NH4Cl or tunicamycin abolished fibroblast growth factor-induced and EphR A4-dependent astrocyte proliferation. NH4Cl-treatment also decreased GLAST mRNA levels in cultured astrocytes. This effect was sensitive to inhibitors of NAPDH oxidase or glutamine synthetase, but was insensitive to siRNA-mediated EphR A4 knockdown. Eph/ephrin gene expression changes were also found in post mortem brain samples of cirrhotic patients without or with HE compared to controls suggesting a potential in vivo relevance of the present findings. The present study suggests that ammonia modulates EphR/ephrin signaling in astrocytes and in the brain of cirrhotic patients with HE with potential implications for deranged neurotransmission in HE.

Secondary NAD+ deficiency in the inherited defect of glutamine synthetase.

Glutamine synthetase (GS) deficiency is an ultra-rare inborn error of amino acid metabolism that has been described in only three patients so far. The disease is characterized by neonatal onset of severe encephalopathy, low levels of glutamine in blood and cerebrospinal fluid, chronic moderate hyperammonemia, and an overall poor prognosis in the absence of an effective treatment. Recently, enteral glutamine supplementation was shown to be a safe and effective therapy for this disease but there are no data available on the long-term effects of this intervention. The amino acid glutamine, severely lacking in this disorder, is central to many metabolic pathways in the human organism and is involved in the synthesis of nicotinamide adenine dinucleotide (NAD(+)) starting from tryptophan or niacin as nicotinate, but not nicotinamide. Using fibroblasts, leukocytes, and immortalized peripheral blood stem cells (PBSC) from a patient carrying a GLUL gene point mutation associated with impaired GS activity, we tested whether glutamine deficiency in this patient results in NAD(+) depletion and whether it can be rescued by supplementation with glutamine, nicotinamide or nicotinate. The present study shows that congenital GS deficiency is associated with NAD(+) depletion in fibroblasts, leukocytes and PBSC, which may contribute to the severe clinical phenotype of the disease. Furthermore, it shows that NAD(+) depletion can be rescued by nicotinamide supplementation in fibroblasts and leukocytes, which may open up potential therapeutic options for the treatment of this disorder.

Gene expression profiling in the cerebral cortex of patients with cirrhosis with and without hepatic encephalopathy.

UNLABELLED: Hepatic encephalopathy (HE) is a frequent complication of liver cirrhosis and is seen as the clinical manifestation of a low-grade cerebral edema associated with oxidative-nitrosative stress. However, comprehensive data on HE-associated molecular derangements in the human brain are lacking. In the present study, we used a whole human genome microarray approach for gene expression profiling in post mortem brain samples from patients with cirrhosis with or without HE and controls without cirrhosis. Altered expression levels were found for a total of 1,012 genes in liver cirrhosis patients without and with HE, and HE-characteristic gene expression changes were identified. Genes with altered expression pattern in HE were related to oxidative stress, microglia activation, receptor signaling, inflammatory pathways, cell proliferation, and apoptosis. Despite an up-regulation of genes associated with microglia activation, pro-inflammatory cytokine messenger RNA profiles remained unchanged in the brains of patients with liver cirrhosis and HE compared with controls. Interestingly, many genes counteracting pro-inflammatory signaling and inflammatory cytokine expression were up-regulated in the cerebral cortex of patients with liver cirrhosis and HE. CONCLUSION: Pathogenetic mechanisms of HE deduced from cell culture and animal experiments, such as oxidative stress, altered Zn(2+) homeostasis and microglia activation also apply to human brain from patients with liver cirrhosis and HE. The study also revealed a not-yet recognized increased expression of genes antagonizing proinflammatory signaling and inflammatory cytokine expression. (HEPATOLOGY 2013;57:2436-2447).