Role of 5-lipoxygenase for ammonia-induced astrocyte senescence

Question Recent studies suggested an important role of 5-lipoxygenase (5-LO) for the induction of senescence in the brain during aging (Manev et al. 2000, FASEBJ 14, 1464-9) and showed that astrocytes become senescent in hepatic encephalopathy (HE) (Görg et al. 2019, J Hepatol 71, 930-941). Whether 5-LO also contributes to senescence in HE is currently unknown and was investigated in the present study.

Methods Protein levels of total and serine⁵⁰⁵-phosphorylated cytosolic phospholipase A₂ (cPLA₂), 5-LO, Coactosin-like protein 1 (COTL1), NADPH-oxidase 4 (NOX4) serine³⁹²-phosphorylated P53, P21 and growth arrest damage inducible protein 45α (GADD45α) were analyzed by immunofluorescence analyses and fluorescence microscopy. Heme oxygenase 1 (HO1), glucose regulated protein 78 (GRP78), P21 and GADD45α mRNA levels were quantified by realtime-PCR. Senescence-associated β-galactosidase (SAβGal) activity was measured using the SAβGal substrate C₁₂FDG and fluorescence microscopy.

Results Cytosolic and nuclear cPLA₂ and nuclear P^Ser505-cPLA₂, 5-LO and COTL1 immunoreactivities strongly increased 24 and 72h after incubating cultured rat astrocytes with NH₄Cl (5mM). Levels of nuclear NOX4 and p^Ser392-P53 and HO1, P21 and GADD45α mRNA increased in NH₄Cl (5mM, 72h)-exposed astrocytes. Inhibition of cPLA₂ with CAY10650 (1µM) or 5-LO with Zileuton (10µM) inhibited the upregulation of HO1, GRP78, P21 and GADD45α mRNA at 24h but not at 72h after NH₄Cl (5mM)-exposure. The NH₄Cl-induced nuclear accumulation of P21 and GADD45α and elevation of C₁₂FDG fluorescence was completely prevented by Zileuton. Transcriptome analyses data showed that mRNA levels of 5-LO, 5-LO activating protein and COTL1 were upregulated in human post mortem brain tissue from patients with liver cirrhosis with HE but not in those without HE.

Conclusions The present study suggests an important role of cPLA₂ and 5-LO for ammonia-induced transcription of surrogate markers for oxidative and ER stress and senescence and the nuclear accumulation of P21 and GADD45α and the induction of astrocyte senescence. Elevated mRNA levels of cPLA₂, 5-LO, 5-LO activating protein and COTL1 mRNAs in post mortem brain samples from patients with liver cirrhosis with HE further suggest that upregulation of 5-LO and 5-LO dependent leukotriene synthesis plays a role for senescence in HE.

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