Ammonia triggers astrocyte senescence through glucosamine synthesis-dependent upregulation of ASCT2 and KGA

Question Recent studies point to an important role of glucosamine synthesis-dependent astrocyte senescence for persistent cognitive impairment in patients with liver cirrhosis and hepatic encephalopathy (HE) (Görg et al. GLIA 2015, 107: 1475-1480 and J Hepatol 2019, 71: 930-941). In the present study we investigated a potential role of the glutamine transporter ASCT2 and the kidney-type glutaminase (KGA) for ammonia-induced astrocyte senescence.

Methods KGA and ASCT2 mRNA and protein levels were analyzed by qPCR, Western blot and immunofluorescence analysis, respectively. ASCT2, KGA, GFAT1/2 proteins were downregulated in rat astrocytes in vitro using specific siRNAs. The activity of senescence-associated β-galactosidase (SAβGal) was measured using the SAβGal substrate C₁₂FDG and fluorescence microscopy.

Results Immunofluorescence analysis, confocal laserscanning and superresolution microscopy showed that KGA and glutamine synthetase (GS) are present in one and the same astrocyte in culture and in rat brain and that ASCT2 and KGA are both found in mitochondria. NH₄Cl (5mM, 72h) upregulated KGA protein and ASCT2 protein and mRNA levels in cultured astrocytes. Upregulation of KGA protein by NH₄Cl was completely prevented by the GS inhibitors methionine-sulfoximine (3mM) and phosphinothricin (100µM). siRNA-mediated knockdown of GFAT1/2 inhibited the NH₄Cl-induced upregulation of KGA protein and ASCT2 mRNA and protein. Knockdown of KGA or ASCT2 by specific siRNAs inhibited the upregulation of surrogate markers for oxidative and endoplasmic reticulum stress (heme oxygenase 1, glucose regulated protein 78) and senescence (growth arrest and DNA damage inducible 45α) in NH₄Cl-exposed astrocytes. Knockdown of KGA also prevented the enhanced SAβGal activity and astrocyte proliferation inhibition in NH₄Cl-exposed astrocytes. KGA protein levels were also elevated in rat cerebral cortex after inducing hyperammonemia through intraperitoneal injection of ammonia acetate.

Conclusion Our findings suggest that ammonia triggers astrocyte senescence through glucosamine synthesis-dependent upregulation of ASCT2 and KGA and ASCT2-dependent glutamine import into mitochondria and subsequent KGA-dependent glutaminolysis. These findings may have important implications for senescence-induced persistent cognitive impairment in patients with liver cirrhosis and HE.

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

Publikationsverlauf

Artikel online veröffentlicht:
04. Januar 2021