Identification of hyperammonemia-induced changes in the cerebral transcriptome and proteome

Question The molecular changes underlying neurological impairment in hyperammonemic disorders such as in hepatic encephalopathy (HE) are only incompletely understood. In the present study we investigated gene and protein alterations in different brain regions of mice with systemic hyperammonemia resulting from knockout of hepatic glutamine synthetase (LGS-KO) and analyzed potential functional consequences in astrocytes in vitro.

Methods Proteome and transcriptome analyses were performed by mass spectrometry and gene array, respectively. mRNA and protein levels were analyzed by qPCR, Western blot and immunofluorescence analysis.

Results Using transcriptomics and proteomics we identified 214,44 and 163 mRNA species and 4, 36 and 15 proteins with altered levels in the cerebral cortex, the hippocampus and the cerebellum of LGS-KO mice, respectively. Differentially-expressed genes related to oxidative and endoplasmic reticulum stress, energy metabolism and cell proliferation and others. For selected candidates CARM1, TROVE2, LCN2 and ASCT2 we found, that all of them are expressed by astrocytes in mouse and rat brain as shown by immunofluorescence analyses and that NH₄Cl (5mM, 72h) changed their protein and mRNA levels in rat astrocytes in vitro similar to what was found in brains of LGS-KO mice. Functional consequences of the protein level changes of the selected candidates were investigated in cultured rat astrocytes using pharmacological inhibitors of CARM1, ASCT2, iron chelators or siRNA-mediated knockdown of TROVE2. The results suggest a role of CARM1 and ASCT2 for senescence, of LCN2 for disturbed iron homeostasis and of TROVE2 for RNA quality control in NH₄Cl-exposed astrocytes. Challenging rats with ammonia acetate (4.5mmol/kg BW, 24h) also elevated ASCT2 and LCN2 protein levels in cerebral cortex and ASCT2 and TROVE2 mRNA levels were also elevated in post mortem brain tissue from patients with liver cirrhosis with HE.

Conclusions In the present study we identified previously unknown changes in the cerebral transcriptome and proteome potentially relevant for hyperammonemia-induced neurological dysfunction in HE. The findings suggest an important role of the methyltransferase CARM1, the glutamine transporter ASCT2, the iron chelator LCN2 and the RNA binding protein TROVE2 for ammonia toxicity and the pathogenesis of HE.

Supported by DFG through SFB974 “Communication and Systems Relevance in Liver Injury and Regeneration”.

Publication History

Article published online:
04 January 2021