Hyperammonemia‐induced DNA‐damage Response Leads to the Acquisition of Cellular Senescence in Neurons after Chronic Liver Failure

Abstract

BackgroundHepatic encephalopathy (HE) is a serious neuropsychiatric complication of chronic liver disease (Type C HE) that affects health‐related quality of life, clinical management, and patient survival. The current understanding of HE pathophysiology has centered around the synergistic actions of hyperammonemia with peripheral and central inflammation to precipitate HE. Furthermore, HE is associated with oxidative stress with consequences on lipid, protein and RNA structure, but the impact of these reactive species on DNA structure and function has not been analyzed and the implications of oxidative DNA lesions and the DNA damage response (DDR) has not been appreciated. In other organs, when DNA repair mechanisms fail, activation of the DDR may result in cellular senescence, a permanent cell cycle arrest with epigenetic alterations that promotes the production of a senescence‐associated phenotype (SASP) leading to the secretion of many factors including cytokines, chemokines, growth factors, proteases and insoluble extracellular matrix components13, In HE, senescent markers have been found in astrocytes, but research into neuronal senescence has been limited. The aim of this study is to assess the oxidative DNA lesions and DDR in HE.MethodsSD rats underwent bile duct ligation (BDL) surgery and C57BL/6 mice were treated with CCl4 for 16 wks to induce chronic liver failure. In parallel, human autopsy brain samples from patients with HE due to liver cirrhosis and age‐ and sex‐matched controls were used. In vitro, hippocampal neurons were treated with various concentrations of ammonium chloride (up to 5 mM) in the presence of the ATM inhibitor KU55933. Oxidative DNA lesions and double‐stranded DNA breaks were visualized by DNA comet assay (in vitro only), 8‐oxo‐2‐deoxyguanosinie (8‐oxoG) and g‐H2AX Immunofluorescence respectively. The DDR (phosphoATM, GADD45b) and markers of senescence (p16, p21, b‐Gal and SASPs; CCL2, TGFb1, IGFBP3, PAI‐1 and IL‐6) were assessed by IHC and qPCR.ResultsIn rodent models of HE and in human autopsy samples, oxidative DNA lesions and double‐stranded DNA breaks were evident in neurons of the CA1 and dentate gyrus in the hippocampus and in the purkinje cell layer in the cerebellum, regions known for their involvement in learning and memory and motor control. Evidence of these DNA lesions correlated with increased expression of markers of the DDR and cellular senescence in the same neuronal cell population. Treatment of hippocampal neurons with ammonia resulted in oxidative DNA lesions, double stranded DNA breaks, DDR activation and the acquisition of a senescent‐like phenotype. Inhibition of the DDR, in vitro attenuated the expression of markers of senescence.ConclusionHyperammonemia induces oxidative DNA damage and the subsequent activation of a DDR in neurons during Type C HE. Aberrant activation of the DDR promotes the hyperammonemia‐induced acquisition of a senescent‐like phenotype in neurons that may contribute to pathogenesis of Type C HE.