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Hepatic Encephalopathy and Astrocyte Senescence

  • Boris Görg
    Affiliations
    Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Düsseldorf, Germany
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  • Ayşe Karababa
    Affiliations
    Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Düsseldorf, Germany
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  • Dieter Häussinger
    Correspondence
    Address for correspondence: Dieter Häussinger, Universitätsklinikum Düsseldorf, Klinik für Gastroenterologie, Hepatologie und Infektiologie, Moorenstrasse 5, 40225 Düsseldorf, Germany. Tel.: +49 211 811 7569; fax: +49 211 811 8838.
    Affiliations
    Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Düsseldorf, Germany
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      Hepatic Encephalopathy (HE) is a severe complication of acute or chronic liver diseases with a broad spectrum of neurological symptoms including motor disturbances and cognitive impairment of different severity. Contrary to former beliefs, a growing number of studies suggest that cognitive impairment may not fully reverse after an acute episode of overt HE in patients with liver cirrhosis. The reasons for persistent cognitive impairment in HE are currently unknown but recent observations raise the possibility that astrocyte senescence may play a role here. Astrocyte senescence is closely related to oxidative stress and correlate with irreversible cognitive decline in aging and neurodegenerative diseases. In line with this, surrogate marker for oxidative stress and senescence were upregulated in ammonia-exposed cultured astrocytes and in post mortem brain tissue from patients with liver cirrhosis with but not without HE. Ammonia-induced senescence in astrocytes involves glutamine synthesis-dependent formation of reactive oxygen species (ROS), p53 activation and upregulation of cell cycle inhibitory factors p21 and GADD45α. More recent studies also suggest a role of ROS-induced downregulation of Heme Oxygenase (HO)1-targeting micro RNAs and upregulation of HO1 for ammonia-induced proliferation inhibition in cultured astrocytes.
      Further studies are required to identify the precise sequence of events that lead to astrocyte senescence and to elucidate functional implications of senescence for cognitive performance in patients with liver cirrhosis and HE.

      Abbreviations:

      ARE (Antioxidant Response Elements), BDNF (Brain-Derived Neurotrophic Factor), Eph (Ephrine), EphR (Ephrine Receptor), GADD45α (Growth Arrest and DNA Damage Inducible 45α), GS (Glutamine Synthetase), HE (Hepatic Encephalopathy), HO1 (Heme Oxygenase 1), LOLA (l-Ornithine-l-Aspartate), MAP (Mitogen Activated Protein Kinases), mPT (Mitochondrial Permeability Transition), NAPDH (Reduced Form of Nicotinamide Adenine Dinucleotide Phosphate), nNOS (Neuronal-Type Nitric-Oxide Synthase), Nox (NADPH Oxidase), Nrf2 (Nuclear Factor-Like 2), PBR (Peripheral-Type Benzodiazepine Receptor), PTN (Protein Tyrosine Nitration), RNOS (Reactive Nitrogen and Oxygen Species), ROS (Reactive Oxygen Species), SA-β-Gal (Senescence-Associated β-d-Galactosidase), TrkBT (Truncated Tyrosine Receptor Kinase B), TSP (Trombospondin), ZnPP (Zinc Protoporphyrin)

      Keywords

      Hepatic Encephalopathy (HE) is a frequent neuropsychiatric complication of acute and chronic liver diseases. Prominent symptoms of HE include motor disturbances such as ataxia and asterixis as well as intellectual deficits reflected by impaired learning ability and memory formation.
      • Häussinger D.
      • Sies H.
      Hepatic encephalopathy: clinical aspects and pathogenetic concept.
      In chronic liver disease clinical symptoms of HE are caused by a low grade cerebral edema which is accompanied by oxidative stress.
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      • Laubenberger J.
      • vom Dahl S.
      • et al.
      Proton magnetic resonance spectroscopy studies on human brain myo-inositol in hypo-osmolarity and hepatic encephalopathy.
      • Häussinger D.
      • Kircheis G.
      • Fischer R.
      • Schliess F.
      • vom Dahl S.
      Hepatic encephalopathy in chronic liver disease: a clinical manifestation of astrocyte swelling and low-grade cerebral edema?.
      Osmotic and oxidative stress trigger multiple functional consequences in astrocytes which are suggested to disturb the communication between glia and neurons and oscillatory networks in the brain and thereby account for symptoms of HE.
      • Görg B.
      • Schliess F.
      • Häussinger D.
      Osmotic and oxidative/nitrosative stress in ammonia toxicity and hepatic encephalopathy.
      A very recent study identified premature senescence as another downstream consequence of ammonia-induced oxidative stress in astrocytes.
      • Görg B.
      • Karababa A.
      • Shafigullina A.
      • Bidmon H.J.
      • Häussinger D.
      Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      Importantly, surrogate markers for both, oxidative stress and senescence were also upregulated in post mortem brain tissue from patients with liver cirrhosis with but not without HE.
      • Görg B.
      • Karababa A.
      • Shafigullina A.
      • Bidmon H.J.
      • Häussinger D.
      Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      • Görg B.
      • Qvartskhava N.
      • Bidmon H.J.
      • et al.
      Oxidative stress markers in the brain of patients with cirrhosis and hepatic encephalopathy.
      As astrocyte senescence strongly correlates with irreversible cognitive decline in aging and neurodegenerative diseases, it was suggested that persistent cognitive impairment in patients with liver cirrhosis after resolution of an episode of overt HE
      • Bajaj J.S.
      • Schubert C.M.
      • Heuman D.M.
      • et al.
      Persistence of cognitive impairment after resolution of overt hepatic encephalopathy.
      may result from astrocyte senescence.
      • Görg B.
      • Karababa A.
      • Shafigullina A.
      • Bidmon H.J.
      • Häussinger D.
      Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      The present review summarizes our current view on the role of senescence for the pathogenesis of HE in chronic liver failure.

      Osmotic and Oxidative Stress in HE

      By using 1H Magnetic Resonance Spectroscopy (MRS) pioneering work by Häussinger
      • Häussinger D.
      • Laubenberger J.
      • vom Dahl S.
      • et al.
      Proton magnetic resonance spectroscopy studies on human brain myo-inositol in hypo-osmolarity and hepatic encephalopathy.
      and confirmed by others
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      • Alonso J.
      • Rovira A.
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      The development of low-grade cerebral edema in cirrhosis is supported by the evolution of (1)H-magnetic resonance abnormalities after liver transplantation.
      • Lodi R.
      • Tonon C.
      • Stracciari A.
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      Diffusion MRI shows increased water apparent diffusion coefficient in the brains of cirrhotics.
      • Kale R.A.
      • Gupta R.K.
      • Saraswat V.A.
      • et al.
      Demonstration of interstitial cerebral edema with diffusion tensor MR imaging in type C hepatic encephalopathy.
      consistently showed that the generation of a low-grade cerebral edema in patients with liver cirrhosis is a key event in the pathogenesis of HE. A more recent study confirmed these findings by directly quantifying the tissue water content in brain using magnetic resonance imaging.
      • Shah N.J.
      • Neeb H.
      • Kircheis G.
      • Engels P.
      • Haussinger D.
      • Zilles K.
      Quantitative cerebral water content mapping in hepatic encephalopathy.
      This study revealed brain region-specific changes in tissue water content which were significantly correlated with the severity of HE.
      • Shah N.J.
      • Neeb H.
      • Kircheis G.
      • Engels P.
      • Haussinger D.
      • Zilles K.
      Quantitative cerebral water content mapping in hepatic encephalopathy.
      However, a recent study found that treating patients with liver cirrhosis and minimal HE with l-Ornithine-l-Aspartate (LOLA) improved cognitive performance but had no effect on regional brain volume nor on cerebral osmolyte and glutamine levels.
      • McPhail M.J.
      • Leech R.
      • Grover V.P.
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      Modulation of neural activation following treatment of hepatic encephalopathy.
      From these findings it was concluded that astrocyte swelling and brain volume changes are of no relevance for cognitive impairment in HE in chronic liver failure.
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      • Leech R.
      • Grover V.P.
      • et al.
      Modulation of neural activation following treatment of hepatic encephalopathy.
      Unfortunately, blood ammonia levels were not assessed in the study and the number of patients included in the study was relatively low. Therefore, the power of the study may be too low to detect significant changes.
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      • Leech R.
      • Grover V.P.
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      Modulation of neural activation following treatment of hepatic encephalopathy.
      Astrocyte swelling per se triggers RNOS formation
      • Kruczek C.
      • Görg B.
      • Keitel V.
      • et al.
      Hypoosmotic swelling affects zinc homeostasis in cultured rat astrocytes.
      • Schliess F.
      • Foster N.
      • Görg B.
      • Reinehr R.
      • Häussinger D.
      Hypoosmotic swelling increases protein tyrosine nitration in cultured rat astrocytes.
      and RNOS in turn induce astrocyte swelling.
      • Lachmann V.
      • Görg B.
      • Bidmon H.J.
      • Keitel V.
      • Häussinger D.
      Precipitants of hepatic encephalopathy induce rapid astrocyte swelling in an oxidative stress dependent manner.
      In line with this, HE-relevant factors such as ammonia, benzodiazepines and pro-inflammatory cytokines induce astrocyte swelling as well as the formation of Reactive Nitrogen and Oxygen Species (RNOS) in astrocytes.
      • Lachmann V.
      • Görg B.
      • Bidmon H.J.
      • Keitel V.
      • Häussinger D.
      Precipitants of hepatic encephalopathy induce rapid astrocyte swelling in an oxidative stress dependent manner.
      • Schliess F.
      • Görg B.
      • Fischer R.
      • et al.
      Ammonia induces MK-801-sensitive nitration and phosphorylation of protein tyrosine residues in rat astrocytes.
      • Görg B.
      • Foster N.
      • Reinehr R.
      • et al.
      Benzodiazepine-induced protein tyrosine nitration in rat astrocytes.
      • Görg B.
      • Bidmon H.J.
      • Keitel V.
      • et al.
      Inflammatory cytokines induce protein tyrosine nitration in rat astrocytes.
      • Reinehr R.
      • Görg B.
      • Becker S.
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      Hypoosmotic swelling and ammonia increase oxidative stress by NADPH oxidase in cultured astrocytes and vital brain slices.
      • Murthy C.R.
      • Rama Rao K.V.
      • Bai G.
      • Norenberg M.D.
      Ammonia-induced production of free radicals in primary cultures of rat astrocytes.
      Therefore it was postulated that HE-relevant factors engage a vicious cycle between osmotic and oxidative stress in astrocytes.
      • Schliess F.
      • Görg B.
      • Häussinger D.
      Pathogenetic interplay between osmotic and oxidative stress: the hepatic encephalopathy paradigm.
      RNOS sources in astrocytes include neuronal
      • Kruczek C.
      • Görg B.
      • Keitel V.
      • et al.
      Hypoosmotic swelling affects zinc homeostasis in cultured rat astrocytes.
      • Görg B.
      • Foster N.
      • Reinehr R.
      • et al.
      Benzodiazepine-induced protein tyrosine nitration in rat astrocytes.
      and inducible
      • Schliess F.
      • Görg B.
      • Fischer R.
      • et al.
      Ammonia induces MK-801-sensitive nitration and phosphorylation of protein tyrosine residues in rat astrocytes.
      • Chastre A.
      • Jiang W.
      • Desjardins P.
      • Butterworth R.F.
      Ammonia and proinflammatory cytokines modify expression of genes coding for astrocytic proteins implicated in brain edema in acute liver failure.
      type nitric oxide synthases, NADPH Oxidase (Nox) 2
      • Schliess F.
      • Foster N.
      • Görg B.
      • Reinehr R.
      • Häussinger D.
      Hypoosmotic swelling increases protein tyrosine nitration in cultured rat astrocytes.
      and mitochondria.
      • Görg B.
      • Karababa A.
      • Shafigullina A.
      • Bidmon H.J.
      • Häussinger D.
      Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      Meanwhile, multiple functional consequences arising from the osmotic/oxidative stress response have been identified: Protein tyrosine residues become nitrated,
      • Schliess F.
      • Foster N.
      • Görg B.
      • Reinehr R.
      • Häussinger D.
      Hypoosmotic swelling increases protein tyrosine nitration in cultured rat astrocytes.
      • Schliess F.
      • Görg B.
      • Fischer R.
      • et al.
      Ammonia induces MK-801-sensitive nitration and phosphorylation of protein tyrosine residues in rat astrocytes.
      RNA is oxidized
      • Görg B.
      • Qvartskhava N.
      • Keitel V.
      • et al.
      Ammonia induces RNA oxidation in cultured astrocytes and brain in vivo.
      and gene expression and signaling are altered.
      • Kruczek C.
      • Görg B.
      • Keitel V.
      • et al.
      Hypoosmotic swelling affects zinc homeostasis in cultured rat astrocytes.
      • Schliess F.
      • Görg B.
      • Fischer R.
      • et al.
      Ammonia induces MK-801-sensitive nitration and phosphorylation of protein tyrosine residues in rat astrocytes.
      • Kruczek C.
      • Görg B.
      • Keitel V.
      • Bidmon H.J.
      • Schliess F.
      • Häussinger D.
      Ammonia increases nitric oxide, free Zn(2+), and metallothionein mRNA expression in cultured rat astrocytes.
      • Görg B.
      • Bidmon H.J.
      • Häussinger D.
      Gene expression profiling in the cerebral cortex of patients with cirrhosis with and without hepatic encephalopathy.
      • Moriyama M.
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      • Tong X.Y.
      • Norenberg M.D.
      Role of mitogen-activated protein kinases in the mechanism of oxidant-induced cell swelling in cultured astrocytes.
      Importantly, biomarkers for oxidative stress were also upregulated in post mortem brain samples from patients with liver cirrhosis and HE, but not those without HE.
      • Görg B.
      • Qvartskhava N.
      • Bidmon H.J.
      • et al.
      Oxidative stress markers in the brain of patients with cirrhosis and hepatic encephalopathy.
      • Görg B.
      • Bidmon H.J.
      • Häussinger D.
      Gene expression profiling in the cerebral cortex of patients with cirrhosis with and without hepatic encephalopathy.
      Furthermore, the astrocytic protein glutamine synthetase was identified among the proteins that were nitrated in brain of patients with liver cirrhosis and HE.
      • Görg B.
      • Qvartskhava N.
      • Bidmon H.J.
      • et al.
      Oxidative stress markers in the brain of patients with cirrhosis and hepatic encephalopathy.
      While this finding clearly indicates the presence of oxidative/nitrosative stress in astrocytes in brain of patients with liver cirrhosis and HE, it concurrently does not rule out, that also other cell types produce RNOS or are target of the deleterious actions of RNOS.
      • Görg B.
      • Qvartskhava N.
      • Bidmon H.J.
      • et al.
      Oxidative stress markers in the brain of patients with cirrhosis and hepatic encephalopathy.
      This speculation is supported by studies on cultured brain cells showing that ammonia induces RNOS formation not only in astrocytes, but also in microglia,
      • Zemtsova I.
      • Görg B.
      • Keitel V.
      • Bidmon H.J.
      • Schrör K.
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      Microglia activation in hepatic encephalopathy in rats and humans.
      neurons
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      • Görg B.
      • Keitel V.
      • Bidmon H.J.
      • Schliess F.
      • Häussinger D.
      Ammonia increases nitric oxide, free Zn(2+), and metallothionein mRNA expression in cultured rat astrocytes.
      and endothelial cells.
      • Liu X.M.
      • Peyton K.J.
      • Durante W.
      Ammonia promotes endothelial cell survival via the heme oxygenase-1-mediated release of carbon monoxide.
      • Jayakumar A.R.
      • Tong X.Y.
      • Ospel J.
      • Norenberg M.D.
      Role of cerebral endothelial cells in the astrocyte swelling and brain edema associated with acute hepatic encephalopathy.
      However, further research is required to decipher the functional consequences of RNOS formation in these cell types. Collectively, these studies univocally demonstrated that osmotic and oxidative stress are key factors in the pathogenesis of HE.
      Most recent studies identified premature senescence as a novel functional consequence of ammonia-induced Reactive Oxygen Species (ROS) formation in astrocytes.
      • Görg B.
      • Karababa A.
      • Shafigullina A.
      • Bidmon H.J.
      • Häussinger D.
      Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      Importantly, enhanced expression levels of senescence biomarkers were also found in post mortem brain samples from patients with liver cirrhosis and HE but not in those without HE
      • Görg B.
      • Karababa A.
      • Shafigullina A.
      • Bidmon H.J.
      • Häussinger D.
      Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      (Figure 1).
      Figure thumbnail gr1
      Figure 1Pathogenetic model of hepatic encephalopathy. Ammonia and other HE precipitating factors induce astrocyte swelling and oxidative stress which mutually amplify each other. This vicious cycle between osmotic and oxidative stress triggers various functional consequences such as nitration of tyrosine residues in proteins, oxidation of RNA, alterations of intracellular signaling pathways and gene expression and senescence. These changes impair the astrocytic/neuronal communication, neurotransmission and synaptic plasticity and disturb oscillatory networks in brain which is reflected by respective motor and cognitive symptoms (modified from
      [
      • Häussinger D.
      • Sies H.
      Hepatic encephalopathy: clinical aspects and pathogenetic concept.
      ]
      ).

      Oxidative Stress and Ammonia-Induced Astrocyte Senescence

      The term senescence originally referred to the process of biological aging when it was discovered that aged cells lose the ability to replicate due to an arrest of the cell cycle. Therefore, senescence is a characteristic feature of normal brain aging
      • Nagelhus E.A.
      • Amiry-Moghaddam M.
      • Bergersen L.H.
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      The glia doctrine: addressing the role of glial cells in healthy brain ageing.
      but it is also observed in many neurodegenerative diseases.
      • Chinta S.J.
      • Woods G.
      • Rane A.
      • Demaria M.
      • Campisi J.
      • Andersen J.K.
      Cellular senescence and the aging brain.
      Importantly, senescence in brain and in particular in astrocytes strongly correlates with cerebral oxidative stress and a decline of cognitive functions.
      • Nagelhus E.A.
      • Amiry-Moghaddam M.
      • Bergersen L.H.
      • et al.
      The glia doctrine: addressing the role of glial cells in healthy brain ageing.
      • Chinta S.J.
      • Woods G.
      • Rane A.
      • Demaria M.
      • Campisi J.
      • Andersen J.K.
      Cellular senescence and the aging brain.
      Apart from an age-associated irreversible exhaustion of the replicatory potential, senescence may also be triggered by a variety of stressors such as RNOS, toxins or DNA-damage.
      • Chen Q.M.
      Replicative senescence and oxidant-induced premature senescence. Beyond the control of cell cycle checkpoints.
      This so-called “premature senescence” is in principle reversible upon removal of the triggering event.
      • Kuilman T.
      • Michaloglou C.
      • Mooi W.J.
      • Peeper D.S.
      The essence of senescence.
      Recent studies showed, that ammonia, a main toxin in HE, inhibits the proliferation of cultured astrocytes in a dose-dependent
      • Görg B.
      • Karababa A.
      • Shafigullina A.
      • Bidmon H.J.
      • Häussinger D.
      Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      • Bodega G.
      • Segura B.
      • Ciordia S.
      • et al.
      Ammonia affects astroglial proliferation in culture.
      and reversible way
      • Görg B.
      • Karababa A.
      • Shafigullina A.
      • Bidmon H.J.
      • Häussinger D.
      Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      probably due to an arrest in S-phase of the cell cycle.
      • Bodega G.
      • Segura B.
      • Ciordia S.
      • et al.
      Ammonia affects astroglial proliferation in culture.
      Importantly, ammonia also upregulates senescence-associated β-d-galactosidase (SA-β-Gal),
      • Görg B.
      • Karababa A.
      • Shafigullina A.
      • Bidmon H.J.
      • Häussinger D.
      Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      which represents an established biomarker for senescence.
      • Kuilman T.
      • Michaloglou C.
      • Mooi W.J.
      • Peeper D.S.
      The essence of senescence.
      Both, upregulation of SA-β-Gal and growth inhibition by ammonia are sensitive to inhibitors of glutamine synthetase and Nox which block ammonia-induced ROS formation in astrocytes.
      • Görg B.
      • Karababa A.
      • Shafigullina A.
      • Bidmon H.J.
      • Häussinger D.
      Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      • Murthy C.R.
      • Rama Rao K.V.
      • Bai G.
      • Norenberg M.D.
      Ammonia-induced production of free radicals in primary cultures of rat astrocytes.
      The ammonia-induced ROS formation triggers phosphorylation of p53 at serine392 via mitogen activated protein kinases such as p38 (p38MAPK)
      • Panickar K.S.
      • Jayakumar A.R.
      • Rao K.V.
      • Norenberg M.D.
      Ammonia-induced activation of p53 in cultured astrocytes: role in cell swelling and glutamate uptake.
      and its nuclear accumulation.
      • Görg B.
      • Karababa A.
      • Shafigullina A.
      • Bidmon H.J.
      • Häussinger D.
      Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      p53 is considered a master regulator of the cell cycle that can stall proliferation by activating the transcription of cell cycle inhibitory factors such as p21.
      • Thornton T.M.
      • Rincon M.
      Non-classical p38 map kinase functions: cell cycle checkpoints and survival.
      In line with this, enhanced transcription of p21 and GADD45α as well as enhanced translation and nuclear accumulation of p21 were observed in ammonia-exposed astrocytes.
      • Görg B.
      • Karababa A.
      • Shafigullina A.
      • Bidmon H.J.
      • Häussinger D.
      Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      However, whether further cell cycle inhibitory factors such as p15 or p16
      • Kuilman T.
      • Michaloglou C.
      • Mooi W.J.
      • Peeper D.S.
      The essence of senescence.
      are also involved in ammonia-induced proliferation inhibition in astrocytes is currently unknown.
      In line with the tight interdependence between osmotic and oxidative stress in astrocytes, also hypoosmolarity-induced astrocyte swelling triggers ROS formation
      • Schliess F.
      • Foster N.
      • Görg B.
      • Reinehr R.
      • Häussinger D.
      Hypoosmotic swelling increases protein tyrosine nitration in cultured rat astrocytes.
      and induces senescence
      • Oenarto J.
      • Karababa A.
      • Castoldi M.
      • Bidmon H.J.
      • Görg B.
      • Häussinger D.
      Ammonia-induced miRNA expression changes in cultured rat astrocytes.
      in astrocytes. Currently, it remains unclear, whether apart from astrocytes, ammonia also induces senescence in other brain cells in HE.
      Taken together, these studies established premature senescence in astrocytes as a new functional consequence of osmotic and oxidative stress in the pathogenesis of HE.

      Mitochondrial Dysfunction in Ammonia-Induced Astrocyte Senescence

      Impaired energy metabolism and enhanced ROS formation in mitochondria strongly contribute to cerebral aging and senescence.
      • Kuilman T.
      • Michaloglou C.
      • Mooi W.J.
      • Peeper D.S.
      The essence of senescence.
      In line with a role of mitochondrial dysfunction for senescence in HE, cell culture and animal studies suggest that ammonia impairs mitochondrial energy metabolism (for a review see [
      • Rama Rao K.V.
      • Norenberg M.D.
      Brain energy metabolism and mitochondrial dysfunction in acute and chronic hepatic encephalopathy.
      ]) and triggers ROS formation in mitochondria.
      • Görg B.
      • Karababa A.
      • Shafigullina A.
      • Bidmon H.J.
      • Häussinger D.
      Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      The ammonia-induced mitochondrial dysfunction is also reflected at the structural level by fragmentation of the mitochondrial network and mitochondrial swelling.
      • Görg B.
      • Karababa A.
      • Shafigullina A.
      • Bidmon H.J.
      • Häussinger D.
      Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      • Gregorios J.B.
      • Mozes L.W.
      • Norenberg M.D.
      Morphologic effects of ammonia on primary astrocyte cultures. II. Electron microscopic studies.
      Importantly, swelling of mitochondria in brain was also observed in animal models for chronic liver failure
      • Dhanda S.
      • Sunkaria A.
      • Halder A.
      • Sandhir R.
      Mitochondrial dysfunctions contribute to energy deficits in rodent model of hepatic encephalopathy.
      • Laursen H.
      • Diemer N.H.
      Morphometry of astrocyte and oligodendrocyte ultrastructure after portocaval anastomosis in the rat.
      • Jamshidzadeh A.
      • Heidari R.
      • Abasvali M.
      • et al.
      Taurine treatment preserves brain and liver mitochondrial function in a rat model of fulminant hepatic failure and hyperammonemia.
      as well as in post mortem brain samples from patients with acute liver failure.
      • Kato M.
      • Hughes R.D.
      • Keays R.T.
      • Williams R.
      Electron microscopic study of brain capillaries in cerebral edema from fulminant hepatic failure.
      Only little is known on the mechanisms that underlie mitochondrial dysfunction in HE. A number of studies suggested that glutamine mediates ammonia toxicity in mitochondria through a ROS-dependent and non-specific pore opening of the inner mitochondrial membrane (mitochondrial permeability transition, mPT).
      • Rama Rao K.V.
      • Norenberg M.D.
      Brain energy metabolism and mitochondrial dysfunction in acute and chronic hepatic encephalopathy.
      • Albrecht J.
      • Norenberg M.D.
      Glutamine: a Trojan horse in ammonia neurotoxicity.
      Functional consequences of the mPT are a dissipation of the mitochondrial membrane potential, mitochondrial swelling and apoptotic or necrotic cell death.
      • Oenarto J.
      • Karababa A.
      • Castoldi M.
      • Bidmon H.J.
      • Görg B.
      • Häussinger D.
      Ammonia-induced miRNA expression changes in cultured rat astrocytes.
      Interestingly, ammonia induces the mPT
      • Butterworth R.F.
      Neurosteroids in hepatic encephalopathy: novel insights and new therapeutic opportunities.
      and mitochondrial swelling
      • Görg B.
      • Karababa A.
      • Shafigullina A.
      • Bidmon H.J.
      • Häussinger D.
      Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      • Gregorios J.B.
      • Mozes L.W.
      • Norenberg M.D.
      Morphologic effects of ammonia on primary astrocyte cultures. II. Electron microscopic studies.
      but does affect the vitality of astrocytes.
      • Görg B.
      • Karababa A.
      • Shafigullina A.
      • Bidmon H.J.
      • Häussinger D.
      Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      • Oenarto J.
      • Karababa A.
      • Castoldi M.
      • Bidmon H.J.
      • Görg B.
      • Häussinger D.
      Ammonia-induced miRNA expression changes in cultured rat astrocytes.
      However, it remains to be established whether ammonia-induced swelling and fragmentation of mitochondria in astrocytes is a consequence of glutamine-mediated induction of the mPT. In this regard, results from studies on isolated brain mitochondria are inconsistent as to whether glutamine
      • Zieminska E.
      • Dolinska M.
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      Induction of permeability transition and swelling of rat brain mitochondria by glutamine.
      or ammonia per se
      • Niknahad H.
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      induce mitochondrial swelling.
      Besides ammonia, also ligands of the Peripheral-Type Benzodiazepine Receptor (PBR) induce the mPT and swelling of mitochondria.
      • Chelli B.
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      Peripheral-type benzodiazepine receptor ligands: mitochondrial permeability transition induction in rat cardiac tissue.
      These findings raise the possibility that enhanced cerebral neurosteroid levels and increased benzodiazepine binding sites in brain of patients with liver cirrhosis and HE
      • Butterworth R.F.
      Neurosteroids in hepatic encephalopathy: novel insights and new therapeutic opportunities.
      may further aggravate mitochondrial dysfunction and swelling.

      HO1 and Ammonia-Induced Astrocyte Senescence

      Heme Oxygenase (HO) is only weakly expressed in cultured brain cells and normal brain.
      • Schipper H.M.
      • Song W.
      • Zukor H.
      • Hascalovici J.R.
      • Zeligman D.
      Heme oxygenase-1 and neurodegeneration: expanding frontiers of engagement.
      However, HO1 mRNA and protein levels strongly increase in response to oxidative stress.
      • Schipper H.M.
      • Song W.
      • Zukor H.
      • Hascalovici J.R.
      • Zeligman D.
      Heme oxygenase-1 and neurodegeneration: expanding frontiers of engagement.
      In line with this, HO1 mRNA levels as well as oxidative stress markers are concordantly upregulated in ammonia-exposed astrocytes,
      • Oenarto J.
      • Karababa A.
      • Castoldi M.
      • Bidmon H.J.
      • Görg B.
      • Häussinger D.
      Ammonia-induced miRNA expression changes in cultured rat astrocytes.
      • Warskulat U.
      • Görg B.
      • Bidmon H.J.
      • Müller H.W.
      • Schliess F.
      • Häussinger D.
      Ammonia-induced heme oxygenase-1 expression in cultured rat astrocytes and rat brain in vivo.
      in brain in animal models for HE
      • Warskulat U.
      • Görg B.
      • Bidmon H.J.
      • Müller H.W.
      • Schliess F.
      • Häussinger D.
      Ammonia-induced heme oxygenase-1 expression in cultured rat astrocytes and rat brain in vivo.
      • Song G.
      • Dhodda V.K.
      • Blei A.T.
      • Dempsey R.J.
      • Rao V.L.
      GeneChip analysis shows altered mRNA expression of transcripts of neurotransmitter and signal transduction pathways in the cerebral cortex of portacaval shunted rats.
      and in post mortem brain samples of patients with liver cirrhosis with, but not without HE.
      • Görg B.
      • Bidmon H.J.
      • Häussinger D.
      Gene expression profiling in the cerebral cortex of patients with cirrhosis with and without hepatic encephalopathy.
      However, it remains to be established in which cell types HO1 becomes upregulated in brain in patients with liver cirrhosis and HE. Interestingly, ammonia does not elevate the expression of HO1 in cultured neurons
      • Warskulat U.
      • Görg B.
      • Bidmon H.J.
      • Müller H.W.
      • Schliess F.
      • Häussinger D.
      Ammonia-induced heme oxygenase-1 expression in cultured rat astrocytes and rat brain in vivo.
      but in endothelial cells.
      • Liu X.M.
      • Peyton K.J.
      • Durante W.
      Ammonia promotes endothelial cell survival via the heme oxygenase-1-mediated release of carbon monoxide.
      ROS upregulate HO1 mRNA levels through activation of the transcription factor Nuclear Factor-Like (Nrf) 2, which binds to the Antioxidant Response Element (ARE) in the promoter region of the HO1 gene.
      • Johnson J.A.
      • Johnson D.A.
      • Kraft A.D.
      • et al.
      The Nrf2-ARE pathway: an indicator and modulator of oxidative stress in neurodegeneration.
      So far, a role of Nrf2 for enhanced HO1 mRNA levels in experimental models of HE remains to be established but recent studies suggest an involvement of Micro RNAs (miRNA) in the ammonia-induced upregulation of HO1 mRNA in cultured astrocytes.
      • Oenarto J.
      • Karababa A.
      • Castoldi M.
      • Bidmon H.J.
      • Görg B.
      • Häussinger D.
      Ammonia-induced miRNA expression changes in cultured rat astrocytes.
      In this regard, it was shown that ammonia elevates HO1 mRNA levels by downregulating a subset of HO1-targeting miRNAs in a NADPH oxidase-dependent manner. These miRNA species were identified to belong to the group of so called “redoximiRs”, as their expression levels are modulated by the intracellular redox status.
      • Oenarto J.
      • Karababa A.
      • Castoldi M.
      • Bidmon H.J.
      • Görg B.
      • Häussinger D.
      Ammonia-induced miRNA expression changes in cultured rat astrocytes.
      Importantly, blocking HO1-targeting redoximiRs by selective miRNA inhibitors strongly upregulated HO1 mRNA and protein levels and concurrently inhibited astrocyte proliferation.
      • Oenarto J.
      • Karababa A.
      • Castoldi M.
      • Bidmon H.J.
      • Görg B.
      • Häussinger D.
      Ammonia-induced miRNA expression changes in cultured rat astrocytes.
      In line with an important role of HO1 for ammonia-induced senescence further studies showed that blocking HO1 activity by tin protoporphyrin as well as inhibition of HO1 transcription by taurine fully prevented ammonia-induced astrocyte senescence.
      • Oenarto J.
      • Karababa A.
      • Castoldi M.
      • Bidmon H.J.
      • Görg B.
      • Häussinger D.
      Ammonia-induced miRNA expression changes in cultured rat astrocytes.
      However, the mechanisms by which HO1 promotes astrocyte senescence are not understood and currently under investigation in our laboratories.
      HO1-catalyzed heme degradation yields on the one hand redox active ferrous iron and on the other the antioxidant biliverdin and anti-inflammatory carbon monoxide.
      • Schipper H.M.
      • Song W.
      • Zukor H.
      • Hascalovici J.R.
      • Zeligman D.
      Heme oxygenase-1 and neurodegeneration: expanding frontiers of engagement.
      Therefore, HO1 can not only promote but also counteract oxidative stress. However, the factors that decide whether HO1 promotes pro- or anti-oxidative effects remain largely obscure. There is evidence indicating that HO1 promotes ROS formation in a ferrous iron-dependent way when it is constantly overexpressed in astrocytes.
      • Schipper H.M.
      • Song W.
      • Zukor H.
      • Hascalovici J.R.
      • Zeligman D.
      Heme oxygenase-1 and neurodegeneration: expanding frontiers of engagement.
      Whether HO1 triggers senescence in ammonia-exposed astrocytes through induction of oxidative stress is currently unclear and under investigation in our laboratories. Interestingly, a recent study showed that systemic application of the HO1 inhibitor Zinc Protoporphyrin (ZnPP) diminished oxidative stress and ameliorated behavioral abnormalities in brain of rats fed with an ammonium-containing diet.
      • Wang Q.M.
      • Yin X.Y.
      • Duan Z.J.
      • Guo S.B.
      • Sun X.Y.
      Role of the heme oxygenase/carbon monoxide pathway in the pathogenesis and prevention of hepatic encephalopathy.
      However, further studies are required to exclude that side effects of ZnPP account for the observed anti-oxidative effects in brain and normalization of behavior in hyperammonemic rats.
      • Wang Q.M.
      • Yin X.Y.
      • Duan Z.J.
      • Guo S.B.
      • Sun X.Y.
      Role of the heme oxygenase/carbon monoxide pathway in the pathogenesis and prevention of hepatic encephalopathy.
      Besides ammonia, also hypoosmolarity-induced astrocyte swelling triggers ROS formation,
      • Schliess F.
      • Foster N.
      • Görg B.
      • Reinehr R.
      • Häussinger D.
      Hypoosmotic swelling increases protein tyrosine nitration in cultured rat astrocytes.
      upregulation of HO1
      • Oenarto J.
      • Karababa A.
      • Castoldi M.
      • Bidmon H.J.
      • Görg B.
      • Häussinger D.
      Ammonia-induced miRNA expression changes in cultured rat astrocytes.
      and senescence
      • Oenarto J.
      • Karababa A.
      • Castoldi M.
      • Bidmon H.J.
      • Görg B.
      • Häussinger D.
      Ammonia-induced miRNA expression changes in cultured rat astrocytes.
      in astrocytes. This again underlines the fundamental role of osmotic and oxidative stress in astrocytes for the pathogenesis of HE (Figure 2).
      Figure thumbnail gr2
      Figure 2Proposed signaling mechanisms underlying ammonia-induced senescence. Ammonia rapidly induces ROS formation and upregulates HO1 in a glutamine synthesis-dependent way. Downregulation of HO1-targeting miRNAs/redoxymiRs by ROS elevate HO1 mRNA and protein levels. HO1 may then contribute to ROS formation via promoting the Fenton reaction. ROS also upregulate the cell cycle inhibitory genes p21 and GADD45α and this involves p38-dependent phosphorylation and nuclear accumulation of p53. Subsequently, the cell cycle is arrested and astrocytes become senescent (modified from
      [
      • Görg B.
      • Karababa A.
      • Shafigullina A.
      • Bidmon H.J.
      • Häussinger D.
      Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      ]
      ).

      Functional Consequences of Astrocyte Senescence in HE

      Astrocytes control the maturation and the stability of synaptic connections through bidirectional communication with neurons.
      • Chung W.S.
      • Allen N.J.
      • Eroglu C.
      Astrocytes control synapse formation, function, and elimination.
      This communication is established through the release of growth factors and signaling molecules such as Brain-Derived Neurotrophic Factor (BDNF) or Thrombospondins (TSP)
      • Chung W.S.
      • Allen N.J.
      • Eroglu C.
      Astrocytes control synapse formation, function, and elimination.
      or by interaction of ephrins with corresponding ephrin receptors.
      • Klein R.
      Bidirectional modulation of synaptic functions by Eph/ephrin signaling.
      However, in brain in normal aging and in neurodegenerative diseases astrocyte senescence is strongly associated with impaired neurotransmission and cognitive decline.
      • Nagelhus E.A.
      • Amiry-Moghaddam M.
      • Bergersen L.H.
      • et al.
      The glia doctrine: addressing the role of glial cells in healthy brain ageing.
      • Chinta S.J.
      • Woods G.
      • Rane A.
      • Demaria M.
      • Campisi J.
      • Andersen J.K.
      Cellular senescence and the aging brain.
      The mechanisms by which astrocyte senescence may disturb neurotransmission are not yet fully understood, but there is reason to assume that astrocyte senescence decreases synaptic connectivity. In line with this assumption, synaptic contacts were reduced in brains from different animal models for HE, as well as in ammonia-exposed astrocyte/neuron cocultures.
      • Galland F.
      • Negri E.
      • Da Re C.
      • et al.
      Hyperammonemia compromises glutamate metabolism and reduces BDNF in the rat hippocampus.
      • Jayakumar A.R.
      • Tong X.Y.
      • Curtis K.M.
      • et al.
      Decreased astrocytic thrombospondin-1 secretion after chronic ammonia treatment reduces the level of synaptic proteins: in vitro and in vivo studies.
      Importantly, the impaired synaptic connectivity in these HE models is associated with decreased BDNF
      • Galland F.
      • Negri E.
      • Da Re C.
      • et al.
      Hyperammonemia compromises glutamate metabolism and reduces BDNF in the rat hippocampus.
      and TSP
      • Jayakumar A.R.
      • Tong X.Y.
      • Curtis K.M.
      • et al.
      Decreased astrocytic thrombospondin-1 secretion after chronic ammonia treatment reduces the level of synaptic proteins: in vitro and in vivo studies.
      levels. BDNF was shown to induce Tyrosine Receptor Kinase B (TrkBT)-dependent morphology changes in astrocytes at active synapses and this is suggested to stabilize synaptic contacts.
      • Ohira K.
      • Funatsu N.
      • Homma K.J.
      • et al.
      Truncated TrkB-T1 regulates the morphology of neocortical layer I astrocytes in adult rat brain slices.
      In line with this, BDNF rapidly triggers filopodia outgrowth as well as actin polymerization in cultured astrocytes.
      • Görg B.
      • Karababa A.
      • Shafigullina A.
      • Bidmon H.J.
      • Häussinger D.
      Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      However, BDNF-induced actin polymerization is completely abolished by ammonia in cultured astrocytes.
      • Görg B.
      • Karababa A.
      • Shafigullina A.
      • Bidmon H.J.
      • Häussinger D.
      Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      This phenomenon may be explained by the observation that ammonia inhibits TrkBT N-glycosylation which may abolish TrkBT-mediated signaling.
      • Görg B.
      • Karababa A.
      • Shafigullina A.
      • Bidmon H.J.
      • Häussinger D.
      Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      In line with this, inhibiting TrkBT N-glycosylation by tunicamycin also blocks BDNF-induced actin polymerization.
      • Görg B.
      • Karababa A.
      • Shafigullina A.
      • Bidmon H.J.
      • Häussinger D.
      Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      Besides BDNF-dependent TrkBT signaling, also the interaction of Ephrins (Eph) with Ephrin-Receptors (EphR) on astrocytes and neurons at the tripartite synapse establishes and strengthens synaptic contacts.
      • Klein R.
      Bidirectional modulation of synaptic functions by Eph/ephrin signaling.
      However, ammonia strongly changes the expression patterns of Eph and EphR isoforms and concurrently inhibits EphR4-dependent signaling in cultured astrocytes.
      • Sobczyk K.
      • Jördens M.S.
      • Karababa A.
      • Görg B.
      • Häussinger D.
      Ephrin/Ephrin receptor expression in ammonia-treated rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      Like the situation with TrkBT, impaired EphR4 signaling is associated with decreased N-glycosylation of the receptor and this may inhibit ligand-induced signaling.
      • Sobczyk K.
      • Jördens M.S.
      • Karababa A.
      • Görg B.
      • Häussinger D.
      Ephrin/Ephrin receptor expression in ammonia-treated rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      Importantly, Eph/EphR isoform expression changes were also observed in post mortem brain tissue of cirrhotic patients with HE.
      • Sobczyk K.
      • Jördens M.S.
      • Karababa A.
      • Görg B.
      • Häussinger D.
      Ephrin/Ephrin receptor expression in ammonia-treated rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      Therefore, one may speculate that ammonia-induced astrocyte senescence is associated with disturbed synaptic stability and connectivity by downregulating BDNF levels
      • Galland F.
      • Negri E.
      • Da Re C.
      • et al.
      Hyperammonemia compromises glutamate metabolism and reduces BDNF in the rat hippocampus.
      on the one hand and by blocking TrkBT-dependent BDNF-signaling on the other.
      • Görg B.
      • Karababa A.
      • Shafigullina A.
      • Bidmon H.J.
      • Häussinger D.
      Ammonia-induced senescence in cultured rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      Impaired ephrin/ephrin receptor signaling in senescent astrocytes may further decrease synaptic connectivity in brain.
      • Sobczyk K.
      • Jördens M.S.
      • Karababa A.
      • Görg B.
      • Häussinger D.
      Ephrin/Ephrin receptor expression in ammonia-treated rat astrocytes and in human cerebral cortex in hepatic encephalopathy.
      Thus, impaired astrocytic/neuronal communication and astrocyte senescence may induce permanent structural changes in brain in HE that may persist after resolution of an episode of overt HE. However, further work is needed to establish a role of astrocyte senescence for the disturbed astrocytic/neuronal communication in brain in HE.

      Perspectives

      Research over the past years univocally demonstrated a fundamental role of oxidative/nitrosative stress and related functional consequences for the pathogenesis of HE. This view was again strengthened by recent findings showing that ammonia induces astrocyte senescence in an oxidative stress dependent way. However, it remains to be established whether astrocyte senescence disturbs synaptic connectivity and accounts for persistence of cognitive impairment in cirrhotic patients after an overt episode of HE. Moreover, there is a lack of knowledge on the precise sequence of events that lead to astrocyte senescence.
      In view of the fact that RNOS are important signaling molecules, it seems likely that a broad therapeutic intervention with antioxidants will have unpredictable side effects. Given the important role of HO1 for ammonia-induced astrocyte senescence, future studies may explore whether HO1 inhibition possesses therapeutic potential against persistent cognitive decline in patients with liver cirrhosis and HE.

      Conflicts of Interest

      The authors have none to declare.

      Acknowledgements

      Our own studies reported in this article were supported by Deutsche Forschungsgemeinschaft (DFG) through Collaborative Research Centers 575 “Experimental Hepatology” and 974 “Communication and Systems Relevance in Liver Injury and Regeneration”, (Düsseldorf). The authors are greatful to the Australian Brain Donor Programs NSW Tissue Resource Centre, which is supported by the University of Sydney, National Health and Medical Research Council of Australia, Schizophrenia Research Institute, National Institute of Alcohol Abuse and Alcoholism and NSW Department of Health for tissue support.

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