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.

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.