nitrosative stress and hepatic encephalopathy

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Oxidative/nitrosative stress and hepatic encephalopathy

32 Dieter Häussinger, Boris Görg

Clinic for Gastroenterology, Hepatology, and Infectious Diseases, Heinrich-Heine-University, Düsseldorf, Germany

Abstract Hepatic encephalopathy (HE) is a neuropsychiatric syndrome that frequently occurs in the course of acute or chronic liver diseases. HE is triggered by a heterogeneous group of factors such as ammonia, which is considered a main toxin in HE; hyponatremia; proinflammatory cytokines; and benzodiazepines. Symptoms of HE mainly comprise disturbances of cognitive and motoric function. HE in patients with liver cirrhosis is seen as the clinical manifestation of a low-grade cerebral edema and accompanying cerebral oxidative/nitrosative stress, which trigger a variety of functional consequences. These include posttranslational protein modifications such as tyrosine nitration and O-GlcNAcylation of proteins, oxidation of RNA, gene and protein expression changes, and senescence. It is assumed that these alterations impair the functions of astrocytes and neurons leading to disturbances of glio-neuronal communication in the brain with consequences for neurotransmission and oscillatory networks in the brain. ­Keywords: Astrocytes, Ammonia, Glutamine, Protein tyrosine nitration, RNA oxidation, MicroRNA, Senescence

­Introduction Oxidative stress plays an important role in health and disease (for a review, see Sies, Berndt, & Jones, 2017, Sies, 2017). On the one hand, it serves as a physiological regulator of diverse cell functions through redox signaling (called oxidative eustress), but on the other hand, it can give rise to oxidative damage of organs due to an overshooting imbalance between oxidant generation and removal (called oxidative distress) (Sies, 2017; Sies, 2018). Deregulated oxidative eustress and oxidative distress are involved in the pathogenesis of a variety of disorders such as Alzheimer’s disease, diabetes, and coronary heart disease and drug toxicity. Here, we summarize our current knowledge on the role of oxidative stress in the pathogenesis of hepatic encephalopathy and ammonia toxicity. Hepatic encephalopathy (HE) is a potentially life-threatening neuropsychiatric syndrome that frequently arises in the course of acute or chronic liver disease. Mild Oxidative Stress. https://doi.org/10.1016/B978-0-12-818606-0.00032-8 © 2020 Elsevier Inc. All rights reserved.

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forms of HE were reported to occur in up to 80% of patients with liver cirrhosis. HE in chronic liver disease comprises a broad spectrum of symptoms of varying severity with disturbances of cognitive and fine motor functions being the most prominent (Häussinger & Blei, 2007; Häussinger & Schliess, 2008; Häussinger & Sies, 2013a). While symptoms of HE are in principle reversible, cognitive disturbances may persist after an acute episode of HE (Bajaj et  al., 2010; Riggio et  al., 2011). HE in chronic liver disease is a consequence of a low-grade cerebral edema (Cordoba et al., 2001; Häussinger et al., 1994; Häussinger, Kircheis, Fischer, Schliess, & Vom Dahl, 2000; Shah et al., 2008) and cerebral oxidative/nitrosative stress (Görg et al., 2010; Görg, Bidmon, & Häussinger, 2013; Häussinger & Schliess, 2008), which trigger a variety of functional disturbances including gene and protein expression changes (Görg et  al., 2010; Jördens et  al., 2015; Schliess et  al., 2002; Sobczyk, Jördens, Karababa, Görg, & Häussinger, 2015; Warskulat et al., 2002; Zemtsova et al., 2011; Zhou & Norenberg, 1999), posttranslational protein modifications (Görg et al., 2010; Karababa, Görg, Schliess, & Häussinger, 2014; Schliess et al., 2002), oxidation of RNA, and senescence (Görg et al., 2010; Görg, Karababa, Shafigullina, Bidmon, & Häussinger, 2015) (for reviews see Häussinger & Schliess, 2008, Görg, Schliess, & Häussinger, 2013). As a result, communication between astrocytes and neurons and synaptic plasticity becomes impaired, and oscillatory networks are disturbed, thereby triggering symptoms of HE (Fig. 1). Most importantly, pathogenetic key observations that were derived from animal or cell culture experiments have also been confirmed in the human brain.

­Astrocyte swelling in HE There is general agreement that astrocytes play a key role and ammonia is a major toxin in the pathogenesis of HE (Häussinger & Blei, 2007; Häussinger & Schliess, 2008; Häussinger & Sies, 2013b; Norenberg, 1987). In the brain, ammonia is detoxified in the astrocytes through glutamine synthesis (Norenberg & Martinez-Hernandez, 1979). 1H-MR spectroscopic studies showed that glutamine levels are increased, whereas the levels of the astrocytic organic osmolyte myoinositol are decreased in brains of patients with liver cirrhosis and HE (Cordoba et al., 2001; Häussinger et al., 1994). From these findings, it was proposed that glutamine accumulation triggers in patients with liver cirrhosis the development of a low-grade cerebral edema with impaired volume-regulatory capacity due to a depletion of the myo-inositol pool in astrocytes (Cordoba et al., 2001; Häussinger et al., 1994; Häussinger et al., 2000) (Fig. 2). Direct evidence for an increased brain water content in patients with liver cirrhosis and HE was also provided by quantitative water mapping by magnetic resonance imaging (MRI) of human brains (Shah et al., 2003; Shah et al., 2008). These studies showed that changes in brain water content in patients with liver cirrhosis are brain region-specific and correlate with the severity of HE (Shah et al., 2003, Shah et al., 2008). The most prominent increase in brain water content was observed within the white matter,

­Astrocyte swelling in HE

FIG. 1 Pathogenetic model of hepatic encephalopathy. Factors that trigger episodes of hepatic encephalopathy (HE-precipitating factors) induce astrocyte swelling and the formation of reactive nitrogen and oxygen species (RONS) in astrocytes. Astrocyte swelling and RONS formation are mutually interrelated and self-amplify each other, thereby triggering a variety of functional consequences. These are suggested to induce astrocytic/neuronal dysfunction and thereby to disturb oscillatory networks in the brain (Butz et al., 2010; Butz, May, Häussinger, & Schnitzler, 2013), which is reflected by the symptoms. Modified from Görg, B., Karababa, A., Häussinger, D. (2018). Hepatic encephalopathy and astrocyte senescence. Journal of Clinical and Experimental Hepatology 8, 294–300, Häussinger, D. & Schliess, F. (2008). Pathogenetic mechanisms of hepatic encephalopathy. Gut 57, 1156–65, Häussinger, D. & Sies, H. (2013b). Hepatic encephalopathy: Clinical aspects and pathogenetic concept. Archives of Biochemistry and Biophysics 536, 97–100.

which consists mainly of axon-myelinating astrocytes, thereby strengthening a role of astrocyte swelling in patients with liver cirrhosis and HE (Shah et  al., 2003, Shah et al., 2008). Not only ammonia but also other conditions known to precipitate or to worsen HE episodes in patients with liver cirrhosis, such as proinflammatory cytokines, hyponatremia, and sedatives of the benzodiazepine-type induce astrocyte swelling (Bender & Norenberg, 1998; Lachmann, Görg, Bidmon, Keitel, & Häussinger, 2013; Norenberg et al., 1991; Rama Rao, Jayakumar, Tong, Alvarez, & Norenberg, 2010;

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FIG. 2 Development of a low-grade cerebral edema in HE. Hyperammonemia due to chronic liver dysfunction triggers osmotic stress by elevating the synthesis of glutamine in cerebral astrocytes. This is compensated by the release of other organic osmolytes such as myoinositol. The resulting depletion of the astrocytic osmolyte pool diminishes the volumeregulatory capacity and renders the astrocyte vulnerable for the swelling-inducing effects of HE-precipitating factors, which then exacerbate the low-grade cerebral edema. Modified from Häussinger, D. & Sies, H. (2013b). Hepatic encephalopathy: Clinical aspects and pathogenetic concept. Archives of Biochemistry and Biophysics 536, 97–100.

Reinehr et al., 2007). Astrocyte swelling in response to the HE-precipitating factors such as ammonia and TNFα is suggested to result from an activation and/or upregulation of the Na+-K+-2Cl− cotransporter 1 (NKCC1), respectively (Jayakumar et al., 2008; Pozdeev et al., 2017). Moreover, aquaporin 4, which in the brain is expressed by astrocytes, was suggested as a water entry route in animal models of acute liver failure induced by thioacetamide or acetaminophen (Rama Rao, Verkman, Curtis, & Norenberg, 2014) and in ammonia-exposed cultured astrocytes (Bodega et al., 2012; Rama Rao, Chen, Simard, & Norenberg, 2003). Thus, astrocyte swelling represents a point of convergence of the actions of the remarkably heterogeneous group of HEprecipitating factors. These findings established the paradigm that HE in chronic liver disease is a clinical manifestation of a low-grade cerebral edema that exacerbates in response to HEprecipitating factors after an ammonia-induced exhaustion of the volume-regulatory capacity of the astrocyte (Häussinger et al., 2000) (Fig. 2).

­Astrocyte swelling and oxidative/nitrosative stress in astrocytes in HE

­ strocyte swelling and oxidative/nitrosative stress in A astrocytes in HE As in many cell types also in astrocytes, changes in cell hydration or cell volume were shown to affect metabolic cell function and gene expression (for review, see Häussinger & Lang, 1991, Lang et  al., 1998). Swelling of the astrocytes, either induced by hypoosmotic cell culture medium (Schliess, Foster, Görg, Reinehr, & Häussinger, 2004), ammonia (Schliess et al., 2002), diazepam (Görg et al., 2003), or TNFα (Görg et al., 2006), elevates intracellular Ca2+ levels [Ca2+]i in a N-methyld-aspartate receptor (NMDAR)-dependent way. While the initial channel opening of the ionotropic NMDAR is suggested to be a consequence of plasma membrane stretch (Kloda, Lua, Hall, Adams, & Martinac, 2007) and a depolarization-induced removal of the Mg2+ blockade (Mayer, Westbrook, & Guthrie, 1984), a subsequent prostanoid-dependent release of vesicular L-glutamate amplifies the NMDARdependent elevation of [Ca2+]i. This elevation of [Ca2+]i by HE-precipitating factors rapidly triggers the formation of RONS through protein kinase Cζ-dependent serine phosphorylation of the NAPDH oxidase (Nox) subunit p47phox (Reinehr et al., 2007) and activation of the neuronal nitric oxide synthase (nNOS) (Kruczek et al., 2009; Reinehr et al., 2007; Schliess et al., 2004). Another source for nitric oxide (NO) in astrocytes is the inducible nitric oxide synthase (iNOS), which becomes upregulated by ammonia in a NFκB-dependent way (Chastre, Jiang, Desjardins, & Butterworth, 2010; Schliess et al., 2002; Sinke et al., 2008). Upregulation of nNOS and iNOS was also observed in rat astrocytes and pyramidal neurons in the cerebral cortex (Suarez, Bodega, Arilla, Felipo, & Fernandez, 2006) and in cerebellar Bergmann glia and Purkinje cells (Suarez, Bodega, Rubio, Felipo, & Fernandez, 2005) after portacaval anastomosis, which is frequently used as an animal model for HE. However, no upregulation of iNOS was found in the cerebral cortex after acute ammonia intoxication or after portal vein ligation (Brück et al., 2011). Likewise, iNOS and nNOS protein and mRNA levels were unaffected in postmortem brain samples from the cerebral cortex of patients with liver cirrhosis and HE (Görg et  al., 2010; Görg, Bidmon, & Häussinger, 2013; Zemtsova et  al., 2011). These contradictory findings may reflect species differences or relate to the animal model and/or to an insufficient detection sensitivity when analyzing protein and mRNA changes in brain homogenates by Western blot and qPCR as compared with immunofluorescence analysis. Apart from p47phox, nNOS, and iNOS, the Nox isoform 4 becomes upregulated by ammonia in cultured astrocytes (Görg et al., 2019) in a glutamine synthesis-­dependent way (Häussinger and Görg, unpublished). Of particular interest, Nox4 is constitutively active, and in contrast to other known Nox isoforms such as O2−-producing Nox2, Nox4 is the only isoform that produces H2O2 (Takac et al., 2011) (for review, see Nayernia, Jaquet, & Krause, 2014, Bedard & Krause, 2007, Brandes & Schröder, 2008). Depending on the cell type, Nox4 was found to be expressed in different subcellular compartments such as in the mitochondria (Block, Gorin, & Abboud, 2009), the nucleus (Hilenski, Clempus, Quinn, Lambeth, & Griendling, 2004), and the

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e­ ndoplasmic reticulum (Chen, Kirber, Xiao, Yang, & Keaney Jr, 2008). However, the intracellular localization of Nox4 in the astrocytes remains to be established. While astrocyte swelling is sufficient to trigger RONS formation, H2O2 and the nitric oxide donor spermine-NONOate in turn induce astrocyte swelling (Lachmann et al., 2013; Moriyama, Jayakumar, Tong, & Norenberg, 2010). In view of this mutual interrelationship between osmotic and oxidative/nitrosative stress, it was proposed that HE-precipitating factors engage a self-amplifying cycle in the astrocytes (Schliess, Görg, & Häussinger, 2006). This cycle may further be augmented by the synergistic actions of HE-precipitating factors as demonstrated for proinflammatory cytokines and ammonia, both being triggers for astrocyte swelling (Rama Rao et al., 2010) and formation of oxidative stress (Häussinger & Schliess, 2008) (Fig. 1). Currently, only little is known about the effects of ammonia and other HEprecipitating factors on the formation of reactive oxygen species (ROS) and NO in brain cell types other than astrocytes. Ammonia was shown to trigger ROS formation in cortical neurons (Kruczek et  al., 2011) and cerebellar granule cells (Bobermin et  al., 2015) and to elevate ROS and NO in microglia (Rao, Brahmbhatt, & Norenberg, 2013; Zemtsova et al., 2011) and in endothelial cells in vitro (Jayakumar, Tong, Ospel, & Norenberg, 2012), but the underlying mechanisms are unknown. Interestingly, in vitro studies suggest that RONS derived from ammonia-exposed microglia and endothelial cells may also contribute to the ammonia-induced astrocyte swelling (Jayakumar et al., 2012; Rao et al., 2013) and therefore may further amplify osmotic and oxidative/nitrosative stress in astrocytes. Whether a mutual interdependence between the formation of reactive nitrogen and oxygen species (RONS) and cell swelling, similar to what is observed in astrocytes, also exists in other brain cell types is currently unknown. However, ammonia does not trigger cell swelling in cultured neurons (Lachmann et al., 2013). Ammonia induces swelling of microglia in culture in a glutamine synthesis-dependent way; however, expression of glutamine synthetase under these conditions may represent a cell culture artifact (Lachmann et al., 2013).

­Mitochondria and oxidative stress in HE Studies on ammonia-exposed isolated mitochondria (Niknahad, Jamshidzadeh, Heidari, Zarei, & Ommati, 2017), ammonia-exposed astrocytes (Görg et al., 2015), and animal models for HE (Chadipiralla, Reddanna, Chinta, & Reddy, 2012; Dhanda, Sunkaria, Halder, & Sandhir, 2018; Jamshidzadeh et  al., 2017; Kosenko et  al., 2017) identified mitochondria as another site of ammonia-induced ROS formation (ROSmito), which may due to an induction of mitochondrial permeability transition (mPT) (Rama Rao, Jayakumar, & Norenberg, 2005). Furthermore, ammonia dissipates the mitochondrial membrane potential (ΔΨ), decreases the synthesis of ATP, and triggers swelling and fragmentation of mitochondria in astrocytes (Bai et  al., 2001; Görg et  al., 2015; Pichili, Rao, Jayakumar, & Norenberg, 2007; Rama Rao & Norenberg, 2014). Swelling of mitochondria, ROSmito formation, and impaired

­Mitochondria and oxidative stress in HE

ATP synthesis were also observed in ammonia-exposed isolated brain mitochondria (Niknahad et al., 2017), in animal models for HE (Dhanda et al., 2018; Jamshidzadeh et al., 2017; Laursen & Diemer, 1980; Reddy, Murthy Ch, & Reddanna, 2004) and in astrocytes in postmortem brain samples from patients with acute liver failure (Kato, Hughes, Keays, & Williams, 1992). Interestingly, in thioacetamide-induced liver failure, feeding of the animals with a diet containing the antioxidant and osmolyte taurine fully prevented mitochondrial swelling and ROSmito formation and preserved mitochondrial ATP levels (Jamshidzadeh et al., 2017; Niknahad et al., 2017). While this suggests a close relationship between osmotic and oxidative/nitrosative stress in mitochondria, it remains to be established whether the ammonia-induced swelling of mitochondria is a consequence of ROSmito formation or vice versa and whether ROSmito and mitochondrial swelling mutually depend on each other. Although the ammonia-induced structural and molecular changes in mitochondria are clearly indicative for mitochondrial dysfunction, ammonia does not impair the viability of the astrocytes (Oenarto et al., 2016; Schliess et al., 2002). This may be explained by ammonia-induced mitophagy, which serves as a quality control and degrades defective mitochondria (Polletta et al., 2015). Interestingly, ammonia-induced ROSmito formation in astrocytes in  vitro depends on the synthesis and the import of glutamine into mitochondria (Görg et al., 2015; Pichili et al., 2007). According to the so-called Trojan horse hypothesis, the ­glutaminase-dependent hydrolysis of glutamine will raise intramitochondrial ammonia levels that then trigger ROSmito formation via MPT and the dissipation of ΔΨ and decrease the synthesis of ATP (Rama Rao & Norenberg, 2014). However, it remains to be determined, whether either the glutaminolysis-triggered elevation of intramitochondrial ammonia or glutamate levels or both account for ROSmito formation. In line with this, knockdown of glutaminase in astrocytes prevented ammonia-induced RNA oxidation (Görg and Häussinger, unpublished result). Ammonia and benzodiazepines of the diazepam type, which precipitate HE episodes, trigger ROSmito formation in astrocytes (Görg et al., 2003; Jayakumar, Panickar, & Norenberg, 2002) due to upregulation or activation of the peripheral-type benzodiazepine receptor (PBR) (Kruczek et al., 2011), which is located in the outer mitochondrial membrane. Activation of the PBR may trigger the formation of neurosteroids, which are elevated in brains from patients with liver cirrhosis and HE (Ahboucha, Pomier-Layrargues, Mamer, & Butterworth, 2006; Zaman, 1990). Whether these observations relate to MPT pore opening is yet unclear. Neurosteroids can also activate the membrane-bound bile acid receptor TGR5 (G protein-coupled bile acid receptor, Gpbar-1), which is expressed in astrocytes and neurons and whose activation is coupled to adenylate cyclase activation, elevation of [Ca2+]i, and ROS formation (Keitel et al., 2010). However, TGR5 is downregulated not only in astrocytes by ammonia but also in cerebral cortex from patients with liver cirrhosis and HE (Keitel et al., 2010). Therefore, a significant contribution of TGR5 to ammonia-induced ROS formation is questionable and needs to be established. Fig. 3 summarizes the currently discussed mechanisms triggering ammonia-induced RONS formation.

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FIG. 3 Mechanisms and sources of the ammonia-induced RONS formation in astrocytes. Ammonia induces the formation of RONS in astrocytes through an NMDA receptordependent elevation of the intracellular calcium concentration [Ca2+]i, which is initiated by a depolarization-induced removal of the Mg2+ blockade and membrane stretch and which is amplified by prostanoids and exocytosis of glutamate-containing intracellular vesicles. The elevation of [Ca2+]i stimulates the formation of NO through the activation of nNOS and by upregulating iNOS via degradation of IκBα and activation of Nox2, which generates the synthesis of O2−. Ammonia also triggers the synthesis of H2O2 by increasing the expression of Nox4. Upregulation of the benzodiazepine receptor (PBR) by ammonia stimulates the synthesis of neurosteroids, which are exported from the cell via Mrp4 and which then can activate the GS-coupled bile acid receptor TGR5 in an autocrine and paracrine fashion. TGR5-induced cAMP synthesis contributes to the elevation of [Ca2+]i and RONS formation. Glutaminase-dependent hydrolysis of glutamine inside the mitochondria triggers mitochondrial O2− formation. NO and O2− also combine to form ONOO−, which can trigger protein tyrosine nitration. Modified from Görg, B., Schliess, F., Häussinger, D. (2013). Osmotic and oxidative/nitrosative stress in ammonia toxicity and hepatic encephalopathy. Archives of Biochemistry and Biophysics 536, 158–163.

­Oxidative/nitrosative stress and protein tyrosine nitration in HE

F­ unctional consequences of osmotic and oxidative/ nitrosative stress in HE Since the first reports on cerebral nitric oxide, superoxide, and peroxynitrite formation in animal models for HE (Kosenko et al., 1997; Kosenko, Kaminski, Lopata, Muravyov, & Felipo, 1999; Larsen, Gottstein, & Blei, 2001; Master, Gottstein, & Blei, 1999; Schliess et al., 2002) and in ammonia-exposed astrocytes in vitro (Murthy, Rama Rao, Bai, & Norenberg, 2001; Schliess et al., 2002), several functional consequences were identified. These include covalent protein modifications (Jayakumar et al., 2008; Schliess et al., 2002; Widmer, Kaiser, Engels, Jung, & Grune, 2007), RNA oxidation (Görg et al., 2008), altered protein and gene expression (Jayakumar, Panickar, Murthy Ch, & Norenberg, 2006; Jördens et  al., 2015), signal transduction (Moriyama et al., 2010; Schliess et al., 2002), disturbances of zinc homeostasis (Kruczek et al., 2009; Kruczek et al., 2011), inhibition of proliferation, and induction of senescence (Bodega et  al., 2015; Görg et  al., 2015; Görg et  al., 2018; Oenarto et al., 2016) (Fig. 1). Most importantly, such alterations were also identified in postmortem brain samples from patients with liver cirrhosis and HE, but not those with liver cirrhosis without HE. The first evidence for ongoing oxidative stress in the brain of HE patients was significantly increased levels of heat shock protein 27, a surrogate marker for oxidative stress in the cerebral cortex of patients with liver cirrhosis and HE when compared with controls without liver disease or patients with liver cirrhosis without HE (Görg et al., 2010).

­ xidative/nitrosative stress and protein tyrosine O nitration in HE One consequence of ammonia-induced oxidative-nitrosative stress is protein tyrosine nitration (PTN). In cultured astrocytes, PTN is induced not only by ammonia but also by benzodiazepines and inflammatory cytokines such as TNFα. These effectors act synergistically with regard to PTN. PTN is also induced by hypoosmotic exposure of astrocytes, indicating that astrocyte swelling is sufficient to trigger PTN (Görg et al., 2003; Görg et al., 2006; Schliess et al., 2002; Schliess et al., 2004). Whereas PTN under the influence of ammonia, hypoosmolarity, and TNFα involves the formation of peroxynitrite, benzodiazepine-induced PTN involves activation of the PBR. PTN in the brain was shown to occur in vivo in ammonia- and LPS-intoxicated or ­portacaval-shunted rats (Brück et al., 2011; Görg et al., 2006; Schliess et al., 2002), in hyperammonemic mice due to taurine transporter knockout (Qvartskhava et al., 2019) or liver-specific knockdown of glutamine synthetase (Qvartskhava et  al., 2015). Astrocytes located near the blood-brain barrier show particularly high levels of PTN with potential consequences for blood-barrier permeability and transastrocytic substrate transport (Brück et al., 2011, Görg et al., 2006, Schliess et al., 2002). Tyrosine nitration is a selective process and involves distinct proteins only. Among these, the PBR, glyceraldehyde-3-phosphate, Erk-1, glutamine synthetase,

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and NKCC1 have been identified (Brück et al., 2011; Görg et al., 2006; Jayakumar et al., 2008; Schliess et al., 2002). Whereas nitration of the NKCC1 was suggested to enhance its activity (Jayakumar et al., 2008) and thereby augment astrocyte swelling, PTN of GS inactivates the enzyme and may counteract ammonia-induced astrocyte swelling (Brück et al., 2011; Görg et al., 2006; Görg et al., 2007; Schliess et al., 2002). Knockdown of GS in astrocytes prevents ammonia-induced mitochondrial swelling (own unpublished result) and RNA oxidation (Görg et  al., 2019). Therefore, inactivation of GS by tyrosine nitration may counteract ammonia toxicity. Although PTN interferes with signaling elements and enzyme activities, its exact role in the pathogenesis of HE remains to be defined. However, increased levels of tyrosine-nitrated proteins and glutamine synthetase in particular are also found in postmortem cerebrocortical brain samples from patients with liver cirrhosis and HE, but not from those without HE (Görg et al., 2010).

­RNA oxidation in HE A novel aspect on the pathogenesis of HE arose from the finding that ammonia, TNFα, benzodiazepines, and hypoosmotic swelling can induce RNA oxidation in cultured astrocytes, in the brains from ammonia acetate-treated rats and in ammoniaexposed brain slices (Görg et al., 2008). Here, ROS oxidize guanosine to produce 8-oxo-7,8-dihydro-2′-guanosine (8OHG; also called 8-oxoguanosine), which is detected in astrocytes and in the cytosol of neurons from ammonia-intoxicated rats (Görg et al., 2008). Likewise, RNA oxidation is also found in brains from mice with liver-specific glutamine synthetase knockout (Qvartskhava et  al., 2015) or taurine transporter knockout (Qvartskhava et al., 2019), that is, conditions associated with systemic hyperammonemia. Most importantly, increased RNA oxidation was also found in the brain of patients with liver cirrhosis with HE. Patients with liver cirrhosis but without HE showed levels of RNA oxidation in the brain similar to control patients without liver disease (Görg et al., 2010). 8OHG immunoreactivity was also found in granular structures along the dendrites and in postsynaptic dendritic regions in association with the RNA-binding splicing protein neuro-oncological ventral antigen 2 (NOVA2). Thus, HE-associated oxidative stress apparently modifies RNA species, which participate in the granular RNA transport along the dendrites. Such neuronal RNA granules can contain all elements required for local postsynaptic protein synthesis, which is controlled by synaptic signals and plays a major role for synaptic plasticity, as reflected by late-phase long-term potentiation (L-LTP) (Schuman, Dynes, & Steward, 2006). Postsynaptic protein synthesis is required for learning and the formation of long-term memory (for reviews, see Schuman et al., 2006, Sutton & Schuman, 2005, Martin, Barad, & Kandel, 2000). RNA oxidation in response to HE-relevant conditions (e.g., ammonia, TNFα, benzodiazepines, and hyponatraemia) is a selective process. Among the oxidized RNA species, the mRNA for the glutamate uptake system GLAST and ribosomal (r) RNA were identified (Görg

­Oxidative/nitrosative stress and gene expression changes in HE

et al., 2008). The role of RNA oxidation in ammonia neurotoxicity and HE is not yet clear; however, there is good evidence that rRNA and mRNA oxidation may compromise translation accuracy and efficacy, thereby resulting in the formation of defective or unstable proteins. Oxidation of astroglial GLAST mRNA by ammonia may partly explain the long-known ammonia-induced decrease of GLAST expression and glutamate uptake in cultured astrocytes (Chan, Hazell, Desjardins, & Butterworth, 2000; Jayakumar et  al., 2006) and contribute to the known disturbances in glutamatergic neurotransmission in HE (Vaquero & Butterworth, 2006). To what extent RNA oxidation contributes to the multiple derangements of neurotransmitter receptor systems in HE (for a review, see Häussinger & Blei, 2007) and to HE-associated alterations of gene expression (Görg, Bidmon, & Häussinger, 2013; Oenarto et al., 2016; Sobczyk et al., 2015; Song, Dhodda, Blei, Dempsey, & Rao, 2002) is currently unclear. Neuronal RNA oxidation was associated with mild cognitive impairment in early stages of Alzheimer’s disease (Nunomura et al., 2009; Nunomura et al., 2012). Cognitive impairment without neuronal degeneration is also a hallmark of HE and may involve a disturbed protein synthesis-dependent late-phase long-term potentiation (L-LTP), learning, and memory consolidation due to the oxidation of postsynaptically translated mRNA species. In line with this, learning ability is disturbed in animal models for HE such as bile duct-ligated rats or rats fed with a hyperammonemic diet (Rodrigo et al., 2010). LTP is impaired in mouse brain slices exposed to ammonia or TNFα (Swain, Blei, Butterworth, & Kraig, 1991). However, to what extent the oxidation of locally translated mRNA species contributes to the L-LTP impairment under these conditions remains to be established. Nonetheless, RNA oxidation in response to the oxidative stress as induced by HE-relevant neurotoxins can provide a mechanistic link between cell and mitochondrial swelling and oxidative stress on the one hand and alterations of synaptic plasticity on the other hand. In line with this, portal vein ligation (PVL) in rats triggered hyperammonemia, PTN, RNA oxidation, and impairment of locomotor activity. Indomethacin had no effect on the PVL-induced hyperammonemia, but prevented PTN and RNA oxidation and the disturbances of locomotor disturbances (Brück et al., 2011). These findings underline the importance of oxidative/nitrosative stress in the pathogenesis of HE.

­ xidative/nitrosative stress and gene expression O changes in HE The ammonia-induced RONS formation activates a number of transcription factors, thereby affecting the expression of several genes that are suggested to play a role in the pathogenesis of HE (Fig. 4). In this regard, ammonia- and hypoosmolarity-­ induced nitrosative stress in astrocytes activate the transcription factors specificity protein 1 (SP1) and the metal-responsive transcription factor (MTF)-1 (Kruczek et al., 2009; Kruczek et al., 2011). This was shown to be a consequence of a NOinduced liberation of zinc from Zn2+-thiolate clusters in proteins and the resulting increase of intracellular levels of free zinc ions (Kruczek et al., 2009, Kruczek et al.,

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FIG. 4 Mechanisms of RONS-induced gene expression changes in ammonia-exposed cultured rat astrocytes. Ammonia triggers expression changes of genes relevant for the pathogenesis of HE in astrocytes through ROS- and RNS-dependent activation of the transcription factors NFκB, p53, PPARα, MTF1, and SP1.

2011). As elevated levels of free zinc ions are highly toxic to cells and a MTF-1dependent upregulation of the zinc-chelating metallothioneins MT-1 and MT-2 by ammonia or hypoosmotic media or in the brain in ammonium acetate-treated rats may reflect a protective response (Kruczek et al., 2009, Kruczek et al., 2011). Gene expression levels of six MT isoforms were also significantly upregulated in postmortem brain tissue from patients with liver cirrhosis with, but not in those without HE, and here, expression levels of some MT isoforms positively correlated with the peripheral blood ammonia concentration. These results suggest an important role of ammonia as a trigger for deranged Zn2+ homeostasis in the brain of patients with liver cirrhosis and HE (Görg, Bidmon, & Häussinger, 2013). Apart from MT-1 and MT-2, also SP1 becomes activated by ammonia or hypoosmotic cell swelling in a zinc-dependent way, and the latter was shown to upregulate the mRNA levels of the peripheral-type benzodiazepine receptor (PBR) (Kruczek et al., 2009). In line with an ammonia-induced upregulation of PBR protein in astrocytes (Kruczek et al., 2011), PBR binding sites were found to be elevated in the brain from animal models for HE (Agusti, Dziedzic, Hernandez-Rabaza, Guilarte, & Felipo, 2014; Rao, Audet, Therrien, & Butterworth, 1994) and in brains from patients

­Oxidative/nitrosative stress and gene expression changes in HE

with liver cirrhosis who died in hepatic coma (Lavoie, Layrargues, & Butterworth, 1990). Although ammonia upregulates the PBR protein, the functional consequence remains unclear because ammonia simultaneously triggers PTN of the PBR. As the PBR imports cholesterol into mitochondria and thereby promotes the synthesis of neurosteroids (Papadopoulos, 2003), upregulation of the PBR may account for increased neurosteroid levels in the brain in animal models for HE (Ahboucha et al., 2006; Ahboucha, Layrargues, Mamer, & Butterworth, 2005) and in postmortem brain tissue from patients with liver cirrhosis with HE (Ahboucha et al., 2006; Zaman, 1990). Neurosteroids are substrates of the multidrug resistance protein 4 (Mrp4), which is upregulated through a RONS-induced activation of the peroxisome proliferator-activated receptor-α (PPARα) in ammonia-exposed cultured rat astrocytes (Jördens et  al., 2015). Upregulation of Mrp4 mRNA and Mrp4 protein was also observed in postmortem brain tissue from patients with liver cirrhosis with HE (Jördens et al., 2015). Therefore, increased γ-aminobutyric acid (GABA)-dependent neurotransmission in HE (Ahboucha & Butterworth, 2004; Butterworth, 2016) may be a consequence of PBR-dependent synthesis and Mrp4-mediated release of neurosteroids from astrocytes. However as already mentioned earlier, neurosteroids may also trigger RONS formation through activation of TGR5, which in the brain is strongly expressed by astrocytes and neurons (Keitel et al., 2010). The ammonia-induced RONS formation also triggers mRNA expression changes of a variety of ephrin receptor (EphR) and ephrin (Eph) isoforms, and such changes were also observed in postmortem brain tissue from patients with liver cirrhosis and HE (Sobczyk et al., 2015). Importantly, the EphR/Eph mRNA expression changes found in ammonia-exposed cultured astrocytes were sensitive toward inhibition of NADPH oxidase and nitric oxide synthase (Sobczyk et al., 2015). Interestingly, bidirectional communication between astrocytes and neurons via EphR/Eph strongly affects synaptic transmission by modulating the expression of glial glutamate transporters (Filosa et al., 2009; Yang et al., 2014). Therefore, it was suggested that RONS-induced EphR/ Eph mRNA expression changes may contribute to disturbed glutamatergic neurotransmission and impaired synaptic plasticity in HE (Sobczyk et al., 2015). Further gene expression changes were recently identified by a transcriptome analysis on postmortem brain samples from patients with liver cirrhosis (Görg, Bidmon, & Häussinger, 2013). This study revealed 616 genes whose expression were selectively changed in postmortem brain samples from patients with liver cirrhosis with, but not in those without HE. Importantly, these genes were related to biological processes that have been meanwhile established to play an important role for the pathogenesis of HE such as oxidative stress, microglia activation, and proliferation. Among the upregulated genes that relate to the oxidative stress response, not only HO1 but also selenoprotein V, peroxisome proliferator-activated receptor α, and ­peroxiredoxin-4 were identified (Görg, Bidmon, & Häussinger, 2013). However, gene expression levels of the inducible, neuronal, and endothelial nitric oxide synthase isoforms were not significantly changed. The study also unraveled a number of genes that were selectively changed in brain samples from patient with liver cirrhosis and HE that had not been recognized before to play a role for the pathogenesis of HE

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such as genes that relate to the activation of anti-inflammatory signaling pathways such as the interleukins IL-4 and IL-10β (Görg, Bidmon, & Häussinger, 2013). Due to the cellular complexity of the brain, it remains to be established, which cell types are affected by the observed gene expression changes and whether all of them are also reflected at the protein level (Görg, Schliess, & Häussinger, 2013). Apart from triggering mRNA expression changes, RONS were also shown to account for downregulation of a specific set of microRNAs (miRNA) in ammoniaexposed cultured rat astrocytes (Oenarto et al., 2016). Therefore, these miRNAs were identified as new members of a miRNA subset named “redoximiRs,” whose expression is modulated by RONS. The study further suggested that the downregulation of redoximiRs in ammonia-exposed astrocytes may account for the upregulation of mRNA species, which are predicted to be their targets (Oenarto et  al., 2016). Potential targets of these redoximiRs include mRNAs involved in glutamine transport (solute carrier family 1 member 5 [Slc1a5]), glutaminolysis (kidney-type glutaminase [Gls1]), oxidative stress, and senescence (NADPH oxidase 4 [Nox4], heme oxygenase 1 [HO1]) (Fig. 5). Importantly, miRNAs may decrease protein levels of

FIG. 5 ROS-dependent downregulation of miRNA species in ammonia-exposed astrocytes. The ammonia-induced ROS formation downregulates a subset of miRNA species targeting mRNAs coding for HO1 (gene name: Hmox1) (Oenarto et al., 2016) and predicted to target mRNAs of Nox4, Gls1, and Slc1a5.

­Oxidative stress and astrocyte senescence in HE

a respective target mRNA either by degrading the respective mRNA or by inhibiting its translation. This explains why in the case of HO1, downregulation of miR326-3p is associated with an upregulation of HO1 mRNA and protein (Oenarto et al., 2016), whereas Nox4 protein becomes upregulated despite unchanged Nox4 mRNA levels (Görg et al., 2019). These findings suggest an important role of redoximiRs for gene expression changes relevant to the pathogenesis of HE.

­Oxidative stress and astrocyte senescence in HE Oxidative stress is well-known to trigger premature senescence that arrests the cell cycle and renders cells unresponsive toward growth factors and further signals being crucial for the regulation of many cell functions (Chen, 2000). In several neurodegenerative diseases, cerebral oxidative stress strongly correlates with an upregulation of surrogate markers for senescence in the brain (Chinta et al., 2015; Nagelhus et al., 2013), and cognitive dysfunction in a mouse model for Alzheimer’s disease was recently identified as a consequence of senescence in astroglia (Bussian et al., 2018). In line with a role of oxidative stress for senescence, surrogate markers for senescence were also upregulated in postmortem brain biopsies from patients with liver cirrhosis with HE but not in those without HE (Görg et  al., 2015). As senescent astrocytes lose the ability to stabilize synaptic contacts, astrocyte senescence may disturb synaptic connectivity and thereby impair neurotransmission (Kawano et al., 2012). In this light, astrocyte senescence may explain the recent clinical observation that symptoms of HE may not fully resolve after the resolution of an acute episode of overt HE (Bajaj et al., 2010; Riggio et al., 2011). Studies on ammonia-exposed cultured rat astrocytes confirmed a central role of oxidative stress for the induction of senescence in HE. Here, it was shown that oxidative stress triggers senescence via p38MAPK activation and p53-dependent transcription of the cell cycle inhibitory genes p21 and GADD45α and nuclear accumulation of p21 protein (Görg et al., 2015). Findings from the same study further indicated that these senescent astrocytes become unresponsive toward brain-derived growth factor (BDNF)-dependent actin remodeling, which is thought to be required for synapse stabilization. Further findings indicated a role of HO1 for ammonia-induced senescence in cultured astrocytes (Oenarto et al., 2016). This study also showed that the upregulation of HO1 mRNA is due to a downregulation of the HO1-targeting miRNAs 326-3p, 221-3p, and 221-5p (Oenarto et al., 2016) (Fig. 4). It was postulated that HO1 may induce astrocyte senescence by triggering the Fenton reaction through the liberation of ferrous iron from heme (Görg et al., 2018; Oenarto et al., 2016). The H2O2 required for the Fenton reaction will be supplied by Nox4, which is a predicted target of 326-3p and which is upregulated most likely as a consequence of downregulation of miR326-3p by ammonia (Fig. 4). In line with this, knockdown of HO1 or

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chelation of ferrous iron fully prevented the ammonia-induced RNA oxidation and induction of senescence (Görg et al., 2019). Furthermore, upregulation of HO1 in an animal model for HE was accompanied by oxidative stress and behavioral abnormalities, which were prevented by the HO1 inhibitor zinc protoporphyrin (Wang, Yin, Duan, Guo, & Sun, 2013). Importantly, significantly elevated HO1 mRNA levels were also found in postmortem brain tissue from patients with liver cirrhosis and HE but not in those without HE (Görg, Bidmon, & Häussinger, 2013).

­O-GlcNAcylation and oxidative stress in astrocytes Another covalent protein modification is O-GlcNAcylation, that is, the attachment of N-acetylglucosamine to serine or threonine residues of proteins (for a review, see Yang & Qian, 2017). O-GlcNAcylation is stress and nutrient triggered and a reversible protein modification, which relies on intracellular levels of glucosamine6-­phosphate (GlcN-6P) and which is synthesized from glutamine and fructose6-phosphate by the glutamine/fructose amidotransferases (GFAT) 1 and 2 within the hexosamine biosynthetic pathway (HBP) (Yang & Qian, 2017). Elevated intracellular levels of GlcN-6P (Görg et  al., 2019) and increased O-GlcNAcylation (Karababa et  al., 2014) were also observed in astrocytes in response to ammonia. Apart from other proteins, GAPDH becomes O-GlcNAcylated in ­ammonia-exposed astrocytes (Karababa et al., 2014). Interestingly, O-GlcNAcylation of GAPDH was shown to trigger a nuclear accumulation of GAPDH with so far unknown functional consequences (Park, Han, Kim, Kang, & Kim, 2009). While oxidative stress was shown to stimulate the O-GlcNAcylation of proteins in the ­neuroblastoma-derived SH-SY5Y cell line (Katai et al., 2016), our own studies with rat astrocytes indicate that the ammonia-induced O-GlcNAcylation is not a consequence, but rather a trigger for oxidative stress (Görg et al., 2019). In line with this, knockdown of GFAT 1 and 2 prevented the ammonia-induced RNA oxidation, which is triggered by an ammonia-induced upregulation of HO1 and induction of the Fenton reaction (Görg et  al., 2019). Upregulation of HO1 results from an O-GlcNAcylation-dependent transcription inhibition of the HO1targeting miR326-3p, which may also upregulate Nox4 (Görg et al., 2019). While the underlying mechanism is not yet settled, it was speculated that RNA polymerase II becomes inactivated by O-GlcNAcylation at the pri-miR326-3p transcription site (Görg et al., 2019). Consistent with the central role of HO1 and oxidative stress for the induction of astrocyte senescence by ammonia, knockdown of GFAT1 and 2 fully prevented the ammonia-induced senescence in cultured astrocytes (Görg et al., 2019) (Fig. 6). Importantly, significantly elevated levels of O-GlcNAcylated proteins were also found in postmortem brain tissue from patients with liver cirrhosis and HE but not in those without HE (Görg et al., 2019).

­O-GlcNAcylation and oxidative stress in astrocytes

FIG. 6 Proposed mechanism underlying the ammonia-induced RNA oxidation and senescence in cultured astrocytes. The expression of HO1 and Nox4 in the astrocyte is controlled by miR-326-3p (left side). Ammonia elevates intracellular glutamine and glucosamine-6-P (GlcN-6P) levels by the actions of glutamine synthetase (GS) and glutamine/fructose-6P amidotransferases 1 and 2 (GFAT1/2). Elevated intracellular GlcN-6-P levels trigger the O-GlcNAc-transferase (OGT)-dependent O-GlcNAcylation and inactivation of RNA polymerase II (RNAPII). As a consequence, miR326-p levels decrease, and HO1 and Nox4 levels concomitantly increase. HO1-dependent liberation of Fe(II) and Nox4dependent H2O2 formation triggers the oxidation of RNA and the induction of senescence through activation of the Fenton reaction. Modified from Görg, B., Karababa, A., Schütz, E., Paluschinski, M., Schrimpf, A., Shafigullina, A., Castoldi, M., Bidmon, H. J. & Häussinger, D. (2019). O-GlcNAcylation-dependent upregulation of HO1 triggers ­ammonia-induced oxidative stress and senescence in HE. Journal of Hepatology. doi:10.1016/j. jhep.2019.06.020.

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­Concluding remarks The findings summarized in the present article demonstrate a central role of oxidative/nitrosative stress in the pathogenesis of HE. However, it appears unlikely that antioxidants may represent a therapeutic option for the treatment of HE since they may interfere with physiological RONS-dependent signaling pathways and thereby may produce undesired side effects. Given the important role of HO1 for astrocyte senescence and cognitive impairment in animal models for HE, selective HO1 inhibition could be a potential approach for the treatment of HE.

­Funding Our own studies reported here were supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) through Sonderforschungsbereiche 575 “Experimental Hepatology” and SFB 974 “Communication and Systems Relevance in Liver Injury and Regeneration”, Projektnummer 190586431—(Düsseldorf, Germany).

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