Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy

Abstract

Hepatic encephalopathy (HE) is a frequent complication of liver cirrhosis and is due to a low-grade cerebral edema associated with oxidative/nitrosative stress. Recent reports suggest that cognitive impairment in cirrhotic patients may not resolve completely after an attack of manifest HE. As astrocyte dysfunction is central to the pathogenesis of HE and astrocytes are critically involved in synaptic plasticity, we tested for sustained impairment of astrocyte function by analyzing expression levels of senescence biomarkers in ammonia-treated cultured rat astrocytes and in postmortem brain samples from cirrhotic patients with or without HE. NH4 Cl time- and dose-dependently inhibited proliferation of cultured astrocytes by up to 45% (5 mmol/L, 72 h) and strongly increased senescence-associated β-galactosidase activity. Inhibition of astrocyte proliferation by ammonia was mediated by a l-methionine sulfoximine-, oxidative stress-, and p38(MAPK) -dependent activation of p53 associated with enhanced transcription of cell cycle inhibitory genes GADD45α and p21. Mitochondria and the nucleus were identified as sources of oxygen radical formation after prolonged NH4 Cl exposure. Concurrently, NH4 Cl (5 mmol/L) treatment inhibited both epidermal growth factor- and brain-derived neurotrophic factor (BDNF)-induced proliferation as well as BDNF-mediated astrocyte morphology changes through downregulation of the respective growth factor receptors epidermal growth factor receptor and truncated tyrosine receptor kinase B. Increased mRNA expression levels of senescence-associated genes were also found in post mortem brain samples from patients with liver cirrhosis with HE, but not in those without HE. The data suggest that ammonia toxicity and HE are associated with premature astrocyte senescence, which may impair neurotransmission and contribute to persistence of cognitive disturbances after resolution of episodes of overt HE.

Multidrug resistance‐associated protein 4 expression in ammonia‐treated cultured rat astrocytes and cerebral cortex of cirrhotic patients with hepatic encephalopathy

“…Astrocyte proliferation was measured as described recently (Görg et al, ). In brief, proliferation was estimated by fluorimetric measurement of DNA content (Fluoscan Ascent FL, Thermo Electron Cooperation).…”

“…Currently, HE is seen as a clinical manifestation of a low‐grade cerebral edema (Häussinger et al, ) which is associated with oxidative/nitrosative stress (Görg et al, ; Häussinger and Görg, ). The increased formation of reactive oxygen and nitrogen species (RNOS) triggers a series of functional consequences such as tyrosine nitration of proteins (Schliess et al, ), oxidation of RNA (Görg et al, ), activation of Zn 2+ ‐dependent gene transcription (Kruczek et al, ) and astrocyte senescence (Görg et al, ). Such alterations may affect synaptic plasticity, impair neurotransmission and disturb oscillatory networks in the brain, which finally accounts for symptoms of HE (Görg et al, ; Timmermann et al, ).…”

Hepatic Encephalopathy Is Associated with Persistent Learning Impairments Despite Adequate Medical Treatment: A Multicenter, International Study

“…The mechanism for this change is not clear, although recent studies have demonstrated the activation of ''senescence'' genes in animal models of HE [32]. In humans, the brain MR evidence shows worsened brain reserve in prior HE subjects and on autopsy studies demonstrate astrocytosis [33].…”

Decreased STAT3 Phosphorylation Mediates Cell Swelling in Ammonia-Treated Astrocyte Cultures

“…While increased STAT3 phosphorylation was implicated in the mechanism of cell proliferation in CNS neoplasms [8,9,10,11,12,13], it is possible that decreased STAT3 phosphorylation, which was observed following exposure of astrocytes to ammonia, may have contributed to the impaired astrocyte proliferation following ammonia exposure, likely as a consequence of the increased senescence-associated genes expression levels [44]. …”