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Hepatic encephalopathy (HE) is a clinical manifestation of a low grade cerebral edema with a mutual interrelationship between osmotic- and oxidative stress. This leads to RNA oxidation and posttranslational protein modifications such as protein tyrosine nitration with pathophysiological relevance. Here, we report on O -GlcNAcylation as another ammonia-induced posttranslational protein modification in cultured rat astrocytes. NH 4 Cl induced O -GlcNAcylation of distinct proteins (25–250 kDa) in astrocytes in a dose- and time-dependent manner. Exposure of astrocytes to NH 4 Cl (5 mmol/l) for 48 h and 72 h significantly increased protein O -GlcNAcylation by about 2-fold and 4-fold, respectively. NH 4 Cl at a concentration of 1 mmol/l was sufficient to double protein O -GlcNAcylation in astrocytes after 72 h as compared to untreated controls. Ammonia-induced protein O -GlcNAcylation was sensitive towards glutamine-synthetase inhibition by methionine sulfoximine (MSO), but was not induced by hypoosmolarity (205 mosmol/l) or CH 3 NH 3 Cl (5 mmol/l). Increased protein O -GlcNAcylation in NH 4 Cl (5 mmol/l, 48 h)-treated astrocytes was fully reversible within 24 h after withdrawal of NH 4 Cl from culture medium. Amongst the proteins which are O -GlcNAcylated in response to ammonia, GAPDH was identified. It is concluded that ammonia induces reversible protein O -GlcNAcylation in astrocytes that depends on glutamine synthesis but not on astrocyte swelling per se or ammonia-induced pH-changes. In view of the complex involvement of O -GlcNAcylation in cell regulation, such as energy metabolism, apoptosis and circadian rhythmicity and in pathologies, such as neurodegenerative diseases, O -GlcNAcylation might contribute to the pathophysiology of hepatic encephalopathy.

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/β, IL-6 and TNF-α. NH 4 Cl (5 mmol/l) had no effect on LPS-induced upregulation of CD14, IL-1α/β 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/β, TNF-α, IL-6, CD14 but not of IL-10, IL-12A/B or TGFβ 1−3 mRNA was attenuated by NH 4 Cl. LPS-induced upregulation of IL-1α/β, IL-6 and TNF-α was also diminished by the TGR5-ligands allopregnanolone and taurolithocholic acid in mono-cultured microglia. NH 4 Cl 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.

Hepatic encephalopathy is a neuropsychiatric syndrome evolving from cerebral osmotic disturbances and oxidative/nitrosative stress. Ammonia, the main toxin of hepatic encephalopathy, triggers astrocyte senescence in an oxidative stress-dependent way. As miRNAs are critically involved in cell cycle regulation and their expression may be regulated by oxidative stress, we analysed, whether astrocyte senescence is a consequence of ammonia-induced miRNA expression changes. Using a combined miRNA and gene microarray approach, 43 miRNA species which were downregulated and 142 genes which were upregulated by NH 4 Cl (5 mmol/l, 48 h) in cultured rat astrocytes were found. Ammonia-induced miRNA and gene expression changes were validated by qPCR and 43 potential miRNA target genes, including HO-1, were identified by matching upregulated mRNA species with predicted targets of miRNA species downregulated by ammonia. Inhibition of HO-1 targeting miRNAs which were downregulated by NH 4 Cl strongly upregulated HO-1 mRNA and protein levels and inhibited astrocyte proliferation in a HO-1-dependent way. Preventing ammonia-induced upregulation of HO-1 by taurine (5 mmol/l) as well as blocking HO-1 activity by tin-protoporphyrine IX fully prevented ammonia-induced proliferation inhibition and senescence. The data suggest that ammonia induces astrocyte senescence through NADPH oxidase-dependent downregulation of HO-1 targeting miRNAs and concomitant upregulation of HO-1 at both mRNA and protein level.

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. NH 4 Cl 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 NH 4 Cl-treated astrocytes. NH 4 Cl-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 NH 4 Cl or tunicamycin abolished fibroblast growth factor-induced and EphR A4-dependent astrocyte proliferation. NH 4 Cl-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.

Objective: This study aimed to assess the relationship between Type 2 Diabetes Mellitus (DM) and its complica­tions with the severity of depression. Methods: Eighty diabetics under the age of 65 using insulin were enrolled and clinical and biochemical data were recorded. The severity of neuropathy was deter­mined by the “United Kingdom neuropathy screening score” (UKNS) whereas the severity of depression was determined by the “Beck depression scale” (BDS). Ne­phropathy and retinopathy were assessed by the micro­albumin-creatinine ratio in spot urine samples and by de­tailed examination of the retina with an ophthalmoscope, respectively. Results: There were more patients who has high neurop­athy severity in the group with the highest Beck depres­sion scale score (p<0.001). In the correlation analyses, positive correlation was found between the severity of depression determined by the BDS and UKNS (r=0.231, p=0.039). No relationship was found between nephropa­thy, retinopathy and severity of depression. Conclusion: There was significant relationship between the severity of neuropathy and that of depression. Hence, cases with a diagnosis of Type 2 DM require screening in terms of neuropathy and the presence of depression. Ap­propriate approach towards such conditions associated with each other may be crucial in the context of increasing the quality of life.