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Hepatic encephalopathy (HE) is a neuropsychiatric syndrome caused by liver insufficiency and/or portosystemic shunting. HE is mostly episodic and as such reversible. Hyperammonemia clearly plays a key role in the pathophysiology, but the precise detrimental events in the brain leading to HE remain equivocal. Several pathogenic models have been proposed, but few have been linked to clinical studies and observations. Decreased oxygen metabolism is observed in both type A and C HE and in this review, we advocate that this reflects an actual reduced oxygen demand and not a primary cause of HE. As driving force, we propose that the hyperammonemia via astrocytic glutamine synthetase causes an increased γ-aminobutyric acid (GABA) mediated neuro-inhibition which subsequently leads to an overall decreased energy demand of the brain, something that can be enhanced by concomitant neuroinflammation. This also explains the reversibility of the condition., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Ammonia levels are orchestrated by a series of complex interrelated pathways in which the urea cycle has a central role. Liver dysfunction leads to an accumulation of ammonia, which is toxic and is strongly associated with disruption of potassium homeostasis, mitochondrial dysfunction, oxidative stress, inflammation, hypoxaemia and dysregulation of neurotransmission. Hyperammonaemia is a hallmark of hepatic encephalopathy and has been strongly associated with liver-related outcomes in patients with cirrhosis and liver failure. In addition to the established role of ammonia as a neurotoxin in the pathogenesis of hepatic encephalopathy, an increasing number of studies suggest that it can lead to hepatic fibrosis progression, sarcopenia, immune dysfunction and cancer. However, elevated systemic ammonia levels are uncommon in patients with metabolic dysfunction-associated steatotic liver disease. A clear causal relationship between ammonia-induced immune dysfunction and risk of infection has not yet been definitively proven. In this Review, we discuss the mechanisms by which ammonia produces its diverse deleterious effects and their clinical relevance in liver diseases, the importance of measuring ammonia levels for the diagnosis of hepatic encephalopathy, the prognosis of patients with cirrhosis and liver failure, and how our knowledge of inter-organ ammonia metabolism is leading to the development of novel therapeutic approaches., (© 2024. Springer Nature Limited.)

Cirrhotic patients may show minimal hepatic encephalopathy (MHE), with mild cognitive impairment and motor deficits. Hyperammonemia and inflammation are the main contributors to the cognitive and motor alterations of MHE. Hyperammonemic rats reproduce these alterations. There are no specific treatments for the neurological alterations of MHE. Extracellular vesicles from mesenchymal stem cells (MSC-EVs) are promising to treat inflammatory and immune diseases. We aimed to assess whether treatment of hyperammonemic rats with MSC-EVs reduced neuroinflammation in cerebellum and restored motor coordination and to study the mechanisms involved. The effects of MSC-EVs were studied in vivo by intravenous injection to hyperammonemic rats and ex vivo in cerebellar slices. Motor coordination was analyzed using the beam walking test. Effects on neuroinflammation were assessed by immunohistochemistry, immunofluorescence and Western blot. Injection of MSC-EVs reduced microglia and astrocytes activation in cerebellum and restored motor coordination in hyperammonemic rats. Ex vivo experiments show that MSC-EVs normalize pro-inflammatory factors, including TNFα, NF-kB activation and the activation of two key pathways leading to motor incoordination (TNFR1-NF-kB-glutaminase-GAT3 and TNFR1-CCL2-BDNF-TrkB-KCC2). TGFβ in the EVs was necessary for these beneficial effects. MSC-EVs treatment reverse neuroinflammation in the cerebellum of hyperammonemic rats and the underlying mechanisms leading to motor incoordination. Therapy with MSC-EVs may be useful to improve motor function in patients with MHE., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome that develops in patients with severe liver dysfunction and/or portocaval shunting. Despite more than a century of research into the relationship between liver damage and development of encephalopathy, pathogenetic mechanisms of hepatic encephalopathy have not yet been fully elucidated. It is generally recognized, however, that the main trigger of neurologic complications in hepatic encephalopathy is the neurotoxin ammonia/ammonium, concentration of which in the blood increases to toxic levels (hyperammonemia), when detoxification function of the liver is impaired. Freely penetrating into brain cells and affecting NMDA-receptor-mediated signaling, ammonia triggers a pathological cascade leading to the sharp inhibition of aerobic glucose metabolism, oxidative stress, brain hypoperfusion, nerve cell damage, and formation of neurological deficits. Brain hypoperfusion, in turn, could be due to the impaired oxygen transport function of erythrocytes, because of the disturbed energy metabolism that occurs in the membranes and inside erythrocytes and controls affinity of hemoglobin for oxygen, which determines the degree of oxygenation of blood and tissues. In our recent study, this causal relationship was confirmed and novel ammonium-induced pro-oxidant effect mediated by excessive activation of NMDA receptors leading to impaired oxygen transport function of erythrocytes was revealed. For a more complete evaluation of "erythrocytic" factors that diminish brain oxygenation and lead to encephalopathy, in this study, activity of the enzymes and concentration of metabolites of glycolysis and Rapoport-Lubering shunt, as well as morphological characteristics of erythrocytes from the rats with acute hyperammoniemia were determined. To elucidate the role of NMDA receptors in the above processes, MK-801, a non-competitive receptor antagonist, was used. Based on the obtained results it can be concluded that it is necessary to consider ammonium-induced morphofunctional disorders of erythrocytes and hemoglobinemia which can occur as a result of alterations in highly integrated networks of metabolic pathways may act as an additional systemic "erythrocytic" pathogenetic factor to prevent the onset and progression of cerebral hypoperfusion in hepatic encephalopathy accompanied by hyperammonemia.

Transceptors, solute transporters that facilitate intracellular entry of molecules and also initiate intracellular signaling events, have been primarily studied in lower-order species. Ammonia, a cytotoxic endogenous metabolite, is converted to urea in hepatocytes for urinary excretion in mammals. During hyperammonemia, when hepatic metabolism is impaired, nonureagenic ammonia disposal occurs primarily in skeletal muscle. Increased ammonia uptake in skeletal muscle is mediated by a membrane-bound, 12 transmembrane domain solute transporter, Rhesus blood group-associated B glycoprotein (RhBG). We show that in addition to its transport function, RhBG interacts with myeloid differentiation primary response-88 (MyD88) to initiate an intracellular signaling cascade that culminates in activation of NFκB. We also show that ammonia-induced MyD88 signaling is independent of the canonical toll-like receptor-initiated mechanism of MyD88-dependent NFκB activation. In silico, in vitro, and in situ experiments show that the conserved cytosolic J-domain of the RhBG protein interacts with the Toll-interleukin-1 receptor (TIR) domain of MyD88. In skeletal muscle from human patients, human-induced pluripotent stem cell-derived myotubes, and myobundles show an interaction of RhBG-MyD88 during hyperammonemia. Using complementary experimental and multiomics analyses in murine myotubes and mice with muscle-specific RhBG or MyD88 deletion, we show that the RhBG-MyD88 interaction is essential for the activation of NFkB but not ammonia transport. Our studies show a paradigm of substrate-dependent regulation of transceptor function with the potential for modulation of cellular responses in mammalian systems by decoupling transport and signaling functions of transceptors., Competing Interests: Competing interests statement:The authors declare no competing interest.

Sepsis-associated encephalopathy (SAE) is a significant cause of mortality in patients with sepsis. Despite extensive research, its exact cause remains unclear. Our previous research indicated a relationship between non-hepatic hyperammonemia (NHH) and SAE. This study aimed to investigate the relationship between NHH and SAE and the potential mechanisms causing cognitive impairment. In the in vivo experimental results, there were no significant abnormalities in the livers of mice with moderate cecal ligation and perforation (CLP); however, ammonia levels were elevated in the hippocampal tissue and serum. The ELISA study suggest that fecal microbiota transplantation in CLP mice can reduce ammonia levels. Reduction in ammonia levels improved cognitive dysfunction and neurological impairment in CLP mice through behavioral, neuroimaging, and molecular biology studies. Further studies have shown that ammonia enters the brain to regulate the expression of aquaporins-4 (AQP4) in astrocytes, which may be the mechanism underlying brain dysfunction in CLP mice. The results of the in vitro experiments showed that ammonia up-regulated AQP4 expression in astrocytes, resulting in astrocyte damage. The results of this study suggest that ammonia up-regulates astrocyte AQP4 expression through the gut-brain axis, which may be a potential mechanism for the occurrence of SAE., (© 2024. The Author(s).)

Background: Patients with liver cirrhosis may show minimal hepatic encephalopathy (MHE) with mild cognitive impairment and motor incoordination. Rats with chronic hyperammonemia reproduce these alterations. Motor incoordination in hyperammonemic rats is due to increased GABAergic neurotransmission in cerebellum, induced by neuroinflammation, which enhances TNFα-TNFR1-S1PR2-CCL2-BDNF-TrkB pathway activation. The initial events by which hyperammonemia triggers activation of this pathway remain unclear. MHE in cirrhotic patients is triggered by a shift in inflammation with increased IL-17. The aims of this work were: (1) assess if hyperammonemia increases IL-17 content and membrane expression of its receptor in cerebellum of hyperammonemic rats; (2) identify the cell types in which IL-17 receptor is expressed and IL-17 increases in hyperammonemia; (3) assess if blocking IL-17 signaling with anti-IL-17 ex-vivo reverses activation of glia and of the TNFα-TNFR1-S1PR2-CCL2-BDNF-TrkB pathway., Results: IL-17 levels and membrane expression of the IL-17 receptor are increased in cerebellum of rats with hyperammonemia and MHE, leading to increased activation of IL-17 receptor in microglia, which triggers activation of STAT3 and NF-kB, increasing IL-17 and TNFα levels, respectively. TNFα released from microglia activates TNFR1 in Purkinje neurons, leading to activation of NF-kB and increased IL-17 and TNFα also in these cells. Enhanced TNFR1 activation also enhances activation of the TNFR1-S1PR2-CCL2-BDNF-TrkB pathway which mediates microglia and astrocytes activation., Conclusions: All these steps are triggered by enhanced activation of IL-17 receptor in microglia and are prevented by ex-vivo treatment with anti-IL-17. IL-17 and IL-17 receptor in microglia would be therapeutic targets to treat neurological impairment in patients with MHE., (© 2024. The Author(s).)

Ethnopharmacological Relevance: Tinospora cordifolia (TC) a potential medicinal herb, has been ethnobotanically used as an eco-friendly supplement to manage various diseases, including cerebral fever. Earlier studies have shown that TC exhibits diverse beneficial effects, including hepatoprotective and neuroprotective effects. However, the effects of TC remain unexplored in animal models of encephalopathy including hepatic encephalopathy (HE)., Aim of the Study: To evaluate the effects of TC stem extract against thioacetamide (TAA)-induced behavioural and molecular alterations in HE rats., Methods and Materials: The extract was preliminarily screened through phytochemical and HR-LC/MS analysis. Animals were pre-treated with TC extract at doses 30 and 100 mg/kg, orally. Following 7 days of TC pre-treatment, HE was induced by administering TAA (300 mg/kg, i. p. thrice). Behavioural assessments were performed after 56 h of TAA first dose. The animals were then sacrificed to assess biochemical parameters in serum, liver and brain. Liver tissue was used for immunoblotting and histological studies to evaluate inflammatory and fibrotic signalling. Moreover, brain tissue was used to evaluate brain edema, activation of glial cells (GFAP, IBA-1) and NF-κB/NLRP3 downstream signalling via immunoblotting and immunohistochemical analysis in cortex and hippocampus., Results: The pre-treatment with TC extract effective mitigated TAA-induced behavioural alterations, lowered serum LFT (AST, ALT, ALP, bilirubin) and oxidative stress markers in liver and brain. TC treatment significantly modulated hyperammonemia, cerebral edema and preserved the integrity of BBB proteins in HE animals. TC treatment attenuated TAA-induced histological changes, tissue inflammation (pNF-κB (p65), TNF-α, NLRP3) and fibrosis (collagen, α-SMA) in liver. In addition, immunoblotting analysis revealed TC pre-treatment inhibited fibrotic proteins such as vimentin, TGF-β1 and pSmad2/3 in the liver. Our study further showed that TC treatment downregulated the expression of MAPK/NF-κB inflammatory signalling, as well as GFAP and IBA-1 (glial cell markers) in cortex and hippocampus of TAA-intoxicated rats. Additionally, TC-treated animals exhibited reduced expression of caspase3/9 and BAX induced by TAA., Conclusion: This study revealed promising insights on the protective effects of TC against HE. The findings clearly demonstrated that the significant inhibition of MAPK/NF-κB signalling and glial cell activation could be responsible for the observed beneficial effects of TC in TAA-induced HE rats., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The urea cycle (UC) is a critically important metabolic process for the disposal of nitrogen (ammonia) produced by amino acids catabolism. The impairment of this liver-specific pathway induced either by primary genetic defects or by secondary causes, namely those associated with hepatic disease or drug administration, may result in serious clinical consequences. Urea cycle disorders (UCD) and certain organic acidurias are the major groups of inherited rare diseases manifested with hyperammonemia (HA) with UC dysregulation. Importantly, several commonly prescribed drugs, including antiepileptics in monotherapy or polytherapy from carbamazepine to valproic acid or specific antineoplastic agents such as asparaginase or 5-fluorouracil may be associated with HA by mechanisms not fully elucidated. HA, disclosing an imbalance between ammoniagenesis and ammonia disposal via the UC, can evolve to encephalopathy which may lead to significant morbidity and central nervous system damage. This review will focus on biochemical mechanisms related with HA emphasizing some poorly understood perspectives behind the disruption of the UC and mitochondrial energy metabolism, namely: i) changes in acetyl-CoA or NAD + levels in subcellular compartments; ii) post-translational modifications of key UC-related enzymes, namely acetylation, potentially affecting their catalytic activity; iii) the mitochondrial sirtuins-mediated role in ureagenesis. Moreover, the main UCD associated with HA will be summarized to highlight the relevance of investigating possible genetic mutations to account for unexpected HA during certain pharmacological therapies. The ammonia-induced effects should be avoided or overcome as part of safer therapeutic strategies to protect patients under treatment with drugs that may be potentially associated with HA., Competing Interests: Declaration of competing interest All authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

(1) Background: Valproic acid (VPA) is one of the frequently prescribed antiepileptic drugs and is generally considered well tolerated. However, VPA neurologic adverse effects in the absence of liver failure are fairly common, suggesting that in the mechanism for the development of VPA-induced encephalopathy, much more is involved than merely the exposure to hyperammonemia (HA) caused by liver insufficiency to perform detoxification. Taking into account the importance of the relationship between an impaired brain energy metabolism and elevated ammonia production, and based on the ability of VPA to interfere with neuronal oxidative pathways, the current study intended to investigate a potential regional ammoniagenic effect of VPA on rats' brains by determining activities of the enzymes responsible for ammonia production and neutralization. (2) Methods: Rats received a single intraperitoneal injection of VPA (50, 100, 250, 500 mg/kg). Plasma, the neocortex, the cerebellum, and the hippocampus were collected at 30 min after injection. The levels of ammonia, urea, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured in blood plasma. The activities of glutaminase and glutamate dehydrogenase (GDH) in mitochondria and the activities of AMP deaminase (AMPD), adenosine deaminase (ADA), and glutamine synthetase (GS) in cytosolic fractions isolated from rat brain regions were measured. Ammonia, ALT, and AST values were determined in the mitochondrial and cytosolic fractions. (3) Results: Multi-dose VPA treatment did not significantly affect the plasma levels of ammonia and urea or the ALT and AST liver enzymes. Significant dose-independent increases in the accumulation of ammonia were found only in the cytosol from the cerebellum and there was a strong correlation between the ammonia level and the ADA activity in this brain structure. A significant decrease in the AMPD and AST activities was observed, while the ALT activity was unaffected. Only the highest VPA dose (500 mg/kg) was associated with significantly less activity of GS compared to the control in all studied brain structures. In the mitochondria of all studied brain structures, VPA caused a dose-independent increases in ammonia levels, a high concentration of which was strongly and positively correlated with the increased GDH and ALT activity, while glutaminase activity remained unchanged, and AST activity significantly decreased compared to the control in all studied brain structures. (4) Conclusions: This study highlights the rat brain region-specific ammoniagenic effects of VPA, which may manifest themselves in the absence of hyperammonemia. Further research should analyze how the responsiveness of the different brain regions may vary in VPA-treated animals that exhibit compromised energy metabolism, leading to increased ammoniagenesis.

Hyperammonemia refers to elevated levels of ammonia in the blood, which is an important pathological feature of liver cirrhosis and hepatic failure. Preclinical studies suggest tropifexor (TXR), a novel non-bile acid agonist of Farnesoid X Receptor (FXR), has shown promising effects on reducing hepatic steatosis, inflammation, and fibrosis. This study evaluates the impact of TXR on hyperammonemia in a piglet model of cholestasis. We here observed blood ammonia significantly elevated in patients with biliary atresia (BA) and was positively correlated with liver injury. Targeted metabolomics and immunblotting showed glutamine metabolism and urea cycles were impaired in BA patients. Next, we observed that TXR potently suppresses bile duct ligation (BDL)-induced injuries in liver and brain with improving the glutamine metabolism and urea cycles. Within the liver, TXR enhances glutamine metabolism and urea cycles by up-regulation of key regulatory enzymes, including glutamine synthetase (GS), carbamoyl-phosphate synthetase 1 (CPS1), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase 1 (ARG1). In primary mice hepatocytes, TXR detoxified ammonia via increasing ureagenesis. Mechanically, TXR activating FXR to increase express enzymes that regulating ureagenesis and glutamine synthesis through a transcriptional approach. Together, these results suggest that TXR may have therapeutic implications for hyperammonemic conditions in cholestatic livers., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to disclose., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background & Aims: Dysbiosis contributes to alcohol-associated liver disease (ALD); however, the precise mechanisms remain elusive. Given the critical role of the gut microbiota in ammonia production, we herein aim to investigate whether and how gut-derived ammonia contributes to ALD., Methods: Blood samples were collected from human subjects with/without alcohol drinking. Mice were exposed to the Lieber-DeCarli isocaloric control or ethanol-containing diets with and without rifaximin (a nonabsorbable antibiotic clinically used for lowering gut ammonia production) supplementation for five weeks. Both in vitro (NH 4 Cl exposure of AML12 hepatocytes) and in vivo (urease administration for 5 days in mice) hyperammonemia models were employed. RNA sequencing and fecal amplicon sequencing were performed. Ammonia and triglyceride concentrations were measured. The gene and protein expression of enzymes involved in multiple pathways were measured., Results: Chronic alcohol consumption causes hyperammonemia in both mice and human subjects. In healthy livers and hepatocytes, ammonia exposure upregulates the expression of urea cycle genes, elevates hepatic de novo lipogenesis (DNL), and increases fat accumulation. Intriguingly, ammonia promotes ethanol catabolism and acetyl-CoA formation, which, together with ammonia, synergistically facilitates intracellular fat accumulation in hepatocytes. Mechanistic investigations uncovered that ATF4 activation, as a result of ER stress induction and general control nonderepressible 2 activation, plays a central role in ammonia-provoked DNL elevation. Rifaximin ameliorates ALD pathologies in mice, concomitant with blunted hepatic ER stress induction, ATF4 activation, and DNL activation., Conclusions: An overproduction of ammonia by gut microbiota, synergistically interacting with ethanol, is a significant contributor to ALD pathologies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Perturbations in the metabolism of ammonia, a cytotoxic endogenous metabolite, occur in a number of chronic diseases, with consequent hyperammonemia. Increased skeletal muscle ammonia uptake causes metabolic, molecular, and phenotype alterations including cataplerosis of (loss of tricarboxylic acid cycle (TCA) cycle intermediate) α-ketoglutarate (αKG), mitochondrial oxidative dysfunction, and senescence-associated molecular phenotype (SAMP). L-Isoleucine (Ile) is an essential, branched-chain amino acid (BCAA) that simultaneously provides acetyl-CoA as an oxidative substrate and succinyl-CoA for anaplerosis (providing TCA cycle intermediates). Our multiomics analyses in myotubes and skeletal muscle from hyperammonemic mice and human patients with cirrhosis showed perturbations in BCAA transporters and catabolism. We, therefore, determined if Ile reverses hyperammonemia-induced impaired mitochondrial oxidative function and SAMP. Studies were performed in differentiated murine C2C12 myotubes that were early passage, late passage (senescent), or those depleted of LAT1/SLC7A5 and human induced pluripotent stem cell-derived myotubes (hiPSCM). Ile reverses hyperammonemia-induced reduction in the maximum respiratory capacity, complex I, II, and III functions in early passage murine myotubes and hiPSCM. Consistently, low ATP content and impaired global protein synthesis (high energy requiring cellular process) during hyperammonemia are reversed by Ile in murine myotubes and hiPSCM. Lower abundance of critical regulators of protein synthesis in mTORC1 signaling, and increased phosphorylation of eukaryotic initiation factor 2α are also reversed by Ile. Genetic depletion studies showed that Ile responses are independent of the amino acid transporter LAT1/SLC7A5. Our studies show that Ile reverses the hyperammonemia-induced impaired mitochondrial oxidative function, cataplerosis, and SAMP in a LAT1/SLC7A5 transporter-independent manner., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Hepatic encephalopathy (HE) symptoms associated with liver insufficiency are linked to the neurotoxic effects of ammonia and other toxic metabolites reaching the brain via the blood-brain barrier (BBB), further aggravated by the inflammatory response. Cumulative evidence documents that the non-coding single-stranded RNAs, micro RNAs (miRs) control the BBB functioning. However, miRs' involvement in BBB breakdown in HE is still underexplored. Here, we hypothesized that in rats with acute liver failure (ALF) or rats subjected to hyperammonemia, altered circulating miRs affect BBB composing proteins., Methods: Transmission electron microscopy was employed to delineate structural alterations of the BBB in rats with ALF (thioacetamide (TAA) intraperitoneal (ip.) administration) or hyperammonemia (ammonium acetate (OA) ip. administration). The BBB permeability was determined with Evans blue dye and sodium fluorescein assay. Plasma MiRs were profiled by Next Generation Sequencing (NGS), followed by in silico analysis. Selected miRs, verified by qRT-PCR, were examined in cultured rat brain endothelial cells. Targeted protein alterations were elucidated with immunofluorescence, western blotting, and, after selected miR mimics transfection, through an in vitro resistance measurement., Results: Changes in BBB structure and increased permeability were observed in the prefrontal cortex of TAA rats but not in the brains of OA rats. The NGS results revealed divergently changed miRNA-ome in the plasma of both rat models. The in silico analysis led to the selection of miR-122-5p and miR-183-5p with their target genes occludin and integrin β1, respectively, as potential contributors to BBB alterations. Both proteins were reduced in isolated brain vessels and cortical homogenates in TAA rats. We documented in cultured primary brain endothelial cells that ammonia alone and, in combination with TNFα increases the relative expression of NGS-selected miRs with a less pronounced effect of TNFα when added alone. The in vitro study also confirmed miR-122-5p-dependent decrease in occludin and miR-183-5p-related reduction in integrin β1 expression., Conclusion: This work identified, to our knowledge for the first time, potential functional links between alterations in miRs residing in brain endothelium and BBB dysfunction in ALF., (© 2023. The Author(s).)

Hyperammonemia contributes to hepatic encephalopathy. In hyperammonemic rats, cognitive function is impaired by altered glutamatergic neurotransmission induced by neuroinflammation. The underlying mechanisms remain unclear. Enhanced sphingosine-1-phosphate receptor 2 (S1PR2) activation in the cerebellum of hyperammonemic rats contributes to neuroinflammation. in In hyperammonemic rats, we assessed if blocking S1PR2 reduced hippocampal neuroinflammation and reversed cognitive impairment and if the signaling pathways were involved. S1PR2 was blocked with intracerebral JTE-013, and cognitive function was evaluated. The signaling pathways inducing neuroinflammation and altered glutamate receptors were analyzed in hippocampal slices. JTE-013 improved cognitive function in the hyperammonemic rats, and hyperammonemia increased S1P. This increased IL-1β, which enhanced Src activity, increased CCL2, activated microglia and increased the membrane expression of the NMDA receptor subunit GLUN2B. This increased p38-MAPK activity, which altered the membrane expression of AMPA receptor subunits and increased BDNF, which activated the TrkB → PI3K → Akt → CREB pathway, inducing sustained neuroinflammation. This report unveils key pathways involved in the induction and maintenance of neuroinflammation in the hippocampus of hyperammonemic rats and supports S1PR2 as a therapeutic target for cognitive impairment.

Background: The role of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in the pathogenesis of hepatic encephalopathy (HE) is unclear. Mitochondrial reactive oxygen species (mtROS) is a signal for NLRP3 inflammasome activation. Therefore, we aimed to determine whether mtROS-dependent NLRP3 inflammasome activation is involved in HE, using in vivo and in vitro models., Methods: Bile duct ligation (BDL) in C57/BL6 mice was used as an in vivo HE model. NLRP3 activation was assessed in the hippocampus. Immunofluorescence staining was performed to determine the cellular source of NLRP3 in the hippocampal tissue. For the in vitro experiment, BV-2 microglial cells were primed with lipopolysaccharide (LPS), followed by ammonia treatment. NLRP3 activation and mitochondrial dysfunction were measured. Mito-TEMPO was used to suppress mtROS production., Results: BDL mice showed cognitive impairment with hyperammonemia. Both the priming and activation steps of NLRP3 inflammasome activation were processed in the hippocampus of BDL mice. Moreover, intracellular ROS levels increased in the hippocampus, and NLRP3 was mainly expressed in the microglia of the hippocampus. In LPS-primed BV-2 cells, ammonia treatment induced NLRP3 inflammasome activation and pyroptosis, with elevation of mtROS and altered mitochondrial membrane potential. Pretreatment with Mito-TEMPO suppressed mtROS production and the subsequent NLRP3 inflammasome activation and pyroptosis under LPS and ammonia treatment in BV-2 cells., Conclusions: Hyperammonemia in HE may be involved in mtROS overproduction and subsequent NLRP3 inflammasome activation. Further studies using NLRP3-specific inhibitor or NLRP3 knockout mice are needed to elucidate the important role of NLRP3 inflammasome in HE development., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

This study was to investigate the effects of ammonia and manganese in the metabolism of minimal hepatic encephalopathy (MHE). A total of 32 Sprague-Dawley rats were divided into four subgroups: chronic hyperammonemia (CHA), chronic hypermanganese (CHM), MHE and control group (CON). 1H-NMR-based metabolomics was used to detect the metabolic changes. Sparse projection to latent structures discriminant analysis was used for identifying and comparing the key metabolites. Significant elevated blood ammonia were shown in the CHA, CHM, and MHE rats. Significant elevated brain manganese (Mn) were shown in the CHM, and MHE rats, but not in the CHA rats. The concentrations of γ-amino butyric acid (GABA), lactate, alanine, glutamate, glutamine, threonine, and phosphocholine were significantly increased, and that of myo-inositol, taurine, leucine, isoleucine, arginine, and citrulline were significantly decreased in the MHE rats. Of all these 13 key metabolites, 10 of them were affected by ammonia (including lactate, alanine, glutamate, glutamine, myo-inositol, taurine, leucine, isoleucine, arginine, and citrulline) and 5 of them were affected by manganese (including GABA, lactate, myo-inositol, taurine, and leucine). Enrichment analysis indicated that abnormal metabolism of glutamine and TCA circle in MHE might be affected by the ammonia, and abnormal metabolism of GABA might be affected by the Mn, and abnormal metabolism of glycolysis and branched chain amino acids metabolism might be affected by both ammonia and Mn. Both ammonia and Mn play roles in the abnormal metabolism of MHE. Chronic hypermanganese could lead to elevated blood ammonia. However, chronic hyperammonemia could not lead to brain Mn deposition., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Perturbed metabolism of ammonia, an endogenous cytotoxin, causes mitochondrial dysfunction, reduced NAD + /NADH (redox) ratio, and postmitotic senescence. Sirtuins are NAD + -dependent deacetylases that delay senescence. In multiomics analyses, NAD metabolism and sirtuin pathways are enriched during hyperammonemia. Consistently, NAD + -dependent Sirtuin3 (Sirt3) expression and deacetylase activity were decreased, and protein acetylation was increased in human and murine skeletal muscle/myotubes. Global acetylomics and subcellular fractions from myotubes showed hyperammonemia-induced hyperacetylation of cellular signaling and mitochondrial proteins. We dissected the mechanisms and consequences of hyperammonemia-induced NAD metabolism by complementary genetic and chemical approaches. Hyperammonemia inhibited electron transport chain components, specifically complex I that oxidizes NADH to NAD + , that resulted in lower redox ratio. Ammonia also caused mitochondrial oxidative dysfunction, lower mitochondrial NAD + -sensor Sirt3, protein hyperacetylation, and postmitotic senescence. Mitochondrial-targeted Lactobacillus brevis NADH oxidase (MitoLbNOX), but not NAD+ precursor nicotinamide riboside, reversed ammonia-induced oxidative dysfunction, electron transport chain supercomplex disassembly, lower ATP and NAD + content, protein hyperacetylation, Sirt3 dysfunction and postmitotic senescence in myotubes. Even though Sirt3 overexpression reversed ammonia-induced hyperacetylation, lower redox status or mitochondrial oxidative dysfunction were not reversed. These data show that acetylation is a consequence of, but is not the mechanism of, lower redox status or oxidative dysfunction during hyperammonemia. Targeting NADH oxidation is a potential approach to reverse and potentially prevent ammonia-induced postmitotic senescence in skeletal muscle. Since dysregulated ammonia metabolism occurs with aging, and NAD + biosynthesis is reduced in sarcopenia, our studies provide a biochemical basis for cellular senescence and have relevance in multiple tissues., (© 2023 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Hepatic encephalopathy (HE) is a neurological disease occurring in patients with hepatic insufficiency and/or portal-systemic blood shunting based on cirrhosis. The pathogenesis is not completely clear till now, but it is believed that hyperammonemia is the core of HE. Hyperammonemia caused by increased sources of ammonia and decreased metabolism further causes mental problems through the gut-liver-brain axis. The vagal pathway also plays a bidirectional role in the axis. Intestinal microorganisms play an important role in the pathogenesis of HE through the gut-liver-brain axis. With the progression of cirrhosis to HE, intestinal microbial composition changes gradually. It shows the decrease of potential beneficial taxa and the overgrowth of potential pathogenic taxa. Changes in gut microbiota may lead to a variety of effects, such as reduced production of short-chain fatty acids (SCFAs), reduced production of bile acids, increased intestinal barrier permeability, and bacterial translocation. The treatment aim of HE is to decrease intestinal ammonia production and intestinal absorption of ammonia. Prebiotics, probiotics, antibiotics, and fecal microbiota transplantation (FMT) can be used to manipulate the gut microbiome to improve hyperammonemia and endotoxemia. Especially the application of FMT, it has become a new treated approach to target microbial composition and function. Therefore, restoring intestinal microbial homeostasis can improve the cognitive impairment of HE, which is a potential treatment method., (© 2023 The Author(s).)

Objectives: To determine whether hyperammonemia has a direct impact on steatohepatitis in mice fed with a high-fat diet (HFD)., Methods: Male C57BL/6 mice were divided into two groups receiving either chow diet or HFD. After 12-week NASH modeling, hyperammonemia was induced by intragastric administration of ammonium chloride solution (NH 4 Cl) or liver-specific carbamoyl phosphate synthetase 1 (Cps1) knockdown. In vitro experiments were performed in HepG2 cells induced by free fatty acid (FFA) and NH 4 Cl., Results: NH 4 Cl administration led to increased levels of plasma and hepatic ammonia in NASH mice. NH 4 Cl-induced hyperammonemia did not influence liver histological changes in mice fed with HFD; however, elevated plasma cholesterol level, and an increasing trend of liver lipid content were observed. No significant effect of hyperammonemia on hepatic inflammation and fibrosis in NASH mice was found. In vitro cell experiments showed that NH 4 Cl treatment failed to increase the lipid droplet content and the expressions of de novo lipogenesis genes in HepG2 cells induced by FFA. The knockdown of Cps1 in HFD-fed mice resulted in elevated plasma ammonia levels but did not cause histological change in the liver., Conclusions: Our study revealed a limited role of ammonia in aggravating the progression of NASH. Further studies are needed to clarify the role and mechanism of ammonia in NASH development., (© 2023 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.)

Patients with cirrhosis are prone to electrolyte disorders, including hypokalaemia. The available evidence suggests that hypokalaemia facilitates hyperammonaemia and thus increases the risk for hepatic encephalopathy (HE). In case studies, plasma potassium decrements were followed by plasma ammonia increments and HE progression, which was reversed by potassium supplementation. The explanation to the hyperammonaemia may be that hypokalaemia both stimulates renal ammonia production and reduces hepatic ammonia elimination by urea synthesis. Further, hypokalaemia eases the entrance of the increased ammonia into the central nervous system because the lower potassium ion concentration favours the competition of NH 4 + ions for potassium transporters across the blood brain barrier, and because hypokalaemia-induced metabolic alkalosis increases the amount of gaseous ammonia, which freely passes the barrier. Potassium depletion thus seems to be a mechanistic contributor to HE, supporting the clinical notion of routinely correcting low potassium in patients with cirrhosis., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Chronic hyperammonemia is a main contributor to the cognitive and motor impairment in patients with hepatic encephalopathy. Sustained hyperammonemia induces the TNFα expression in Purkinje neurons, mediated by NF-κB activation. The aims were the following: (1) to assess if enhanced TrkB activation by BDNF is responsible for enhanced NF-κB activation in Purkinje neurons in hyperammonemic rats, (2) to assess if this is associated with increased content of NF-κB modulated proteins such as TNFα, HMGB1, or glutaminase I, (3) to assess if these changes are due to enhanced activation of the TNFR1-S1PR2-CCR2-BDNF-TrkB pathway, (4) to analyze if increased activation of NF-κB is mediated by the PI3K-AKT pathway. It is shown that, in the cerebellum of hyperammonemic rats, increased BDNF levels enhance TrkB activation in Purkinje neurons leading to activation of PI3K, which enhances phosphorylation of AKT and of IκB, leading to increased nuclear translocation of NF-κB which enhances TNFα, HMGB1, and glutaminase I content. To assess if the changes are due to enhanced activation of the TNFR1-S1PR2-CCR2 pathway, we blocked TNFR1 with R7050, S1PR2 with JTE-013, and CCR2 with RS504393. These changes are reversed by blocking TrkB, PI3K, or the TNFR1-SP1PR2-CCL2-CCR2-BDNF-TrkB pathway at any step. In hyperammonemic rats, increased levels of BDNF enhance TrkB activation in Purkinje neurons, leading to activation of the PI3K-AKT-IκB-NF-κB pathway which increased the content of glutaminase I, HMGB1, and TNFα. Enhanced activation of this TrkB-PI3K-AKT-NF-κB pathway would contribute to impairing the function of Purkinje neurons and motor function in hyperammonemic rats and likely in cirrhotic patients with minimal or clinical hepatic encephalopathy., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Patients with liver cirrhosis show hyperammonemia and peripheral inflammation and may show hepatic encephalopathy with cognitive impairment, reproduced by rats with chronic hyperammonemia. Peripheral inflammation induces neuroinflammation in hippocampus of hyperammonemic rats, altering neurotransmission and leading to cognitive impairment. Extracellular vesicles (EVs) may transmit pathological effects from the periphery to the brain. We hypothesized that EVs from peripheral blood would contribute to cognitive alterations in hyperammonemic rats. The aims were to assess whether EVs from plasma of hyperammonemic rats (HA-EVs) induce cognitive impairment and to identify the underlying mechanisms. Injection of HA-EVs impaired learning and memory, induced microglia and astrocytes activation and increased TNFα and IL-1β. Ex vivo incubation of hippocampal slices from control rats with HA-EVs reproduced these alterations. HA-EVs increased membrane expression of TNFR1, reduced membrane expression of TGFβR2 and Smad7 and IκBα levels and increased IκBα phosphorylation. This led to increased activation of NF-κB and IL-1β production, altering membrane expression of NR2B, GluA1 and GluA2 subunits, which would be responsible for cognitive impairment. All these effects of HA-EVs were prevented by blocking TNFα, indicating that they were mediated by enhanced activation of TNFR1 by TNFα. We show that these mechanisms are very different from those leading to motor incoordination, which is due to altered GABAergic neurotransmission in cerebellum. This demonstrates that peripheral EVs play a key role in the transmission of peripheral alterations to the brain in hyperammonemia and hepatic encephalopathy, inducing neuroinflammation and altering neurotransmission in hippocampus, which in turn is responsible for the cognitive deficits., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome derived from metabolic disorders due to various liver failures. Clinically, HE is characterized by hyperammonemia, EEG abnormalities, and different degrees of disturbance in sensory, motor, and cognitive functions. The molecular mechanism of HE has not been fully elucidated, although it is generally accepted that HE occurs under the influence of miscellaneous factors, especially the synergistic effect of toxin accumulation and severe metabolism disturbance. This review summarizes the recently discovered cellular mechanisms involved in the pathogenesis of HE. Among the existing hypotheses, ammonia poisoning and the subsequent oxidative/nitrosative stress remain the mainstream theories, and reducing blood ammonia is thus the main strategy for the treatment of HE. Other pathological mechanisms mainly include manganese toxicity, autophagy inhibition, mitochondrial damage, inflammation, and senescence, proposing new avenues for future therapeutic interventions.

In hepatic encephalopathy, hyperammonemia (HA) causes cognitive impairment and anxiety by causing neuroinflammation. Ibuprofen and 1,8- cineol have anti-inflammatory and antioxidant properties, respectively. The aim of this study was to evaluate the effects of ibuprofen alone and in combination with 1,8- cineol on anxiety and oxidative stress in a HA rat animal model. For this purpose, 36 rats were divided into six groups (n = 6) including the HA (received intraperitoneally (IP) ammonium acetate 2.5 mg/kg for four week), ibuprofen (induced HA rats that received 15 mg/kg, IP), cineol (induced HA rats that received 5 and 10 mg/kg, IP), Ib + cineol (induced HA rats that received 15 and 10 mg/kg, respectively, IP), and the control groups (received normal saline, IP). Except the HA group, all other groups received the aforementioned treatment for two weeks.. The Morris water maze and elevated plus maze were used to assess cognitive function and anxiety in the animals, respectively. Superoxide dismutase (SOD) activity was measured to evaluate oxidative stress. The mRNA expression levels of interleukin (IL)-6 and IL-1β was assessed by real-time PCR in the animal's brain. The results showed a significant improvement in spatial memory and anxiety of the Ib group compared to the HA group (P < 0.01), but no significant change was observed in SOD activity (P > 0.05). There was a significant improvement in spatial memory and anxiety as well as a significant increase in SOD activity in the Ib + cineol group (P < 0.01) compared to the HA group. These results indicate that the Ib + cineol, not only improve cognitive function and reduce anxiety, also reduce oxidative stress, therefore, the simultaneous use of these two compounds may be useful in improving HA-induced cognitive disorders and anxiety., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Chronic hyperammonemia, a main contributor to hepatic encephalopathy (HE), leads to neuroinflammation which alters neurotransmission leading to cognitive impairment. There are no specific treatments for the neurological alterations in HE. Extracellular vesicles (EVs) from mesenchymal stem cells (MSCs) reduce neuroinflammation in some pathological conditions. The aims were to assess if treatment of hyperammonemic rats with EVs from MSCs restores cognitive function and analyze the underlying mechanisms. EVs injected in vivo reach the hippocampus and restore performance of hyperammonemic rats in object location, object recognition, short-term memory in the Y-maze and reference memory in the radial maze. Hyperammonemic rats show reduced TGFβ levels and membrane expression of TGFβ receptors in hippocampus. This leads to microglia activation and reduced Smad7-IkB pathway, which induces NF-κB nuclear translocation in neurons, increasing IL-1β which alters AMPA and NMDA receptors membrane expression, leading to cognitive impairment. These effects are reversed by TGFβ in the EVs from MSCs, which activates TGFβ receptors, reducing microglia activation and NF-κB nuclear translocation in neurons by normalizing the Smad7-IkB pathway. This normalizes IL-1β, AMPA and NMDA receptors membrane expression and, therefore, cognitive function. EVs from MSCs may be useful to improve cognitive function in patients with hyperammonemia and minimal HE., (© 2023. The Author(s).)

Hepatic encephalopathy (HE) is a neuropsychiatric hepatic‑induced syndrome in which several factors are involved in promoting brain perturbations, with ammonia being the primary factor. Motor impairment, incoordination, and gut dysbiosis are some of the well‑known symptoms of HE. Nevertheless, the link between the direct effect of hyperammonemia and associated gut dysbiosis in the pathogenesis of HE is not well established. Thus, this work aimed to assess motor function in hyperammonemia and gut dysbiosis in mice. Twenty‑eight Swiss mice were distributed into three groups: two‑week and four‑week hyperammonemia groups were fed with an ammonia‑rich diet (20% w/w), and the control group was pair‑fed with a standard diet. Motor performance in the three groups was measured through a battery of motor tests, namely the rotarod, parallel bars, beam walk, and static bars. Microbial analysis was then carried out on the intestine of the studied mice. The result showed motor impairments in both hyperammonemia groups. Qualitative and quantitative microbiological analysis revealed decreased bacterial load, diversity, and ratios of both aerobic and facultative anaerobic bacteria, following two and four weeks of ammonia supplementation. Moreover, the Shannon diversity index revealed a time‑dependent cutback of gut bacterial diversity in a treatment‑time‑dependent manner, with the presence of only Enterobacteriaceae, Streptococcaceae, and Enterococcaceaeat at four weeks. The data showed that ammonia‑induced motor coordination deficits may develop through direct and indirect pathways acting on the gut‑brain axis.

Arginase 1 Deficiency (ARG1-D) is a rare urea cycle disorder that results in persistent hyperargininemia and a distinct, progressive neurologic phenotype involving developmental delay, intellectual disability, and spasticity, predominantly affecting the lower limbs and leading to mobility impairment. Unlike the typical presentation of other urea cycle disorders, individuals with ARG1-D usually appear healthy at birth and hyperammonemia is comparatively less severe and less common. Clinical manifestations typically begin to develop in early childhood in association with high plasma arginine levels, with hyperargininemia (and not hyperammonemia) considered to be the primary driver of disease sequelae. Nearly five decades of clinical experience with ARG1-D and empirical studies in genetically manipulated models have generated a large body of evidence that, when considered in aggregate, implicates arginine directly in disease pathophysiology. Severe dietary protein restriction to minimize arginine intake and diversion of ammonia from the urea cycle are the mainstay of care. Although this approach does reduce plasma arginine and improve patients' cognitive and motor/mobility manifestations, it is inadequate to achieve and maintain sufficiently low arginine levels and prevent progression in the long term. This review presents a comprehensive discussion of the clinical and scientific literature, the effects and limitations of the current standard of care, and the authors' perspectives regarding the past, current, and future management of ARG1-D., (© 2022 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Patients with urea cycle disorders intermittently develop episodes of decompensation with hyperammonemia. Although such an episode is often associated with starvation and catabolism, its molecular basis is not fully understood. First, we attempted to elucidate the mechanism of such starvation-associated hyperammonemia. Using a mouse embryonic fibroblast (MEF) culture system, we found that glucose starvation increases ammonia production, and that this increase is associated with enhanced glutaminolysis. These results led us to focus on α-ketoglutarate (AKG), a glutamate dehydrogenase inhibitor, and a major anaplerotic metabolite. Hence, we sought to determine the effect of dimethyl α-ketoglutarate (DKG), a cell-permeable AKG analog, on MEFs and found that DKG mitigates ammonia production primarily by reducing flux through glutamate dehydrogenase. We also verified that DKG reduces ammonia in an NH 4 Cl-challenged hyperammonemia mouse model and observed that DKG administration reduces plasma ammonia concentration to 22.8% of the mean value for control mice that received only NH 4 Cl. In addition, we detected increases in ornithine concentration and in the ratio of ornithine to arginine following DKG treatment. We subsequently administered DKG intravenously to a newborn pig with hyperammonemia due to ornithine transcarbamylase deficiency and found that blood ammonia concentration declined significantly over time. We determined that this effect is associated with facilitated reductive amination and glutamine synthesis. Our present data indicate that energy starvation triggers hyperammonemia through enhanced glutaminolysis and that DKG reduces ammonia accumulation via pleiotropic mechanisms both in vitro and in vivo. Thus, cell-permeable forms of AKG are feasible candidates for a novel hyperammonemia treatment., (© 2022 SSIEM.)

Aims: Hyperammonemic rats show peripheral inflammation, increased GABAergic neurotransmission and neuroinflammation in cerebellum and hippocampus which induce motor incoordination and cognitive impairment. Neuroinflammation enhances GABAergic neurotransmission in cerebellum by enhancing the TNFR1-glutaminase-GAT3 and TNFR1-CCL2-TrkB-KCC2 pathways. Golexanolone reduces GABA A receptors potentiation by allopregnanolone. This work aimed to assess if treatment of hyperammonemic rats with golexanolone reduces peripheral inflammation and neuroinflammation and restores cognitive and motor function and to analyze underlying mechanisms., Methods: Rats were treated with golexanolone and effects on peripheral inflammation, neuroinflammation, TNFR1-glutaminase-GAT3 and TNFR1-CCL2-TrkB-KCC2 pathways, and cognitive and motor function were analyzed., Results: Hyperammonemic rats show increased TNFα and reduced IL-10 in plasma, microglia and astrocytes activation in cerebellum and hippocampus, and impaired motor coordination and spatial and short-term memories. Treating hyperammonemic rats with golexanolone reversed changes in peripheral inflammation, microglia and astrocytes activation and restored motor coordination and spatial and short-term memory. This was associated with reversal of the hyperammonemia-enhanced activation in cerebellum of the TNFR1-glutaminase-GAT3 and TNFR1-CCL2-TrkB-KCC2 pathways., Conclusion: Reducing GABA A receptors activation with golexanolone reduces peripheral inflammation and neuroinflammation and improves cognitive and motor function in hyperammonemic rats. The effects identified would also occur in patients with hepatic encephalopathy and, likely, in other pathologies associated with neuroinflammation., (© 2022 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

Hepatic encephalopathy (HE) is a frequent and devastating but generally reversible neuropsychiatric complication secondary to chronic and acute liver failure. During HE, brain energy metabolism is markedly reduced and it remains unclear whether this is due to external or internal energy supply limitations, or secondary to depressed neuronal cellular functions - and if so, which mechanisms that are in play. The extent of deteriorated cerebral function correlates to blood ammonia levels but the metabolic link to ammonia is not clear. Early studies suggested that high levels of ammonia inhibited key tricarboxylic acid (TCA) cycle enzymes thus limiting mitochondrial energy production and oxygen consumption; however, later studies by us and others showed that this is not the case in vivo. Here, based on a series of translational studies from our group, we advocate the view that the low cerebral energy metabolism of HE is likely to be caused by neuronal metabolic depression due to an elevated GABAergic tone rather than by restricted energy availability. The increased GABAergic tone seems to be secondary to synthesis of large amounts of glutamine in astrocytes for detoxification of ammonia with the glutamine acting as a precursor for elevated neuronal synthesis of vesicular GABA., (Copyright © 2022. Published by Elsevier Inc.)

Life-threatening hyperammonemia occurs in both inherited and acquired liver diseases affecting ureagenesis, the main pathway for detoxification of neurotoxic ammonia in mammals. Protein O-GlcNAcylation is a reversible and nutrient-sensitive post-translational modification using as substrate UDP-GlcNAc, the end-product of hexosamine biosynthesis pathway. Here we show that increased liver UDP-GlcNAc during hyperammonemia increases protein O-GlcNAcylation and enhances ureagenesis. Mechanistically, O-GlcNAcylation on specific threonine residues increased the catalytic efficiency for ammonia of carbamoyl phosphate synthetase 1 (CPS1), the rate-limiting enzyme in ureagenesis. Pharmacological inhibition of O-GlcNAcase, the enzyme removing O-GlcNAc from proteins, resulted in clinically relevant reductions of systemic ammonia in both genetic (hypomorphic mouse model of propionic acidemia) and acquired (thioacetamide-induced acute liver failure) mouse models of liver diseases. In conclusion, by fine-tuned control of ammonia entry into ureagenesis, hepatic O-GlcNAcylation of CPS1 increases ammonia detoxification and is a novel target for therapy of hyperammonemia in both genetic and acquired diseases., (© 2022. The Author(s).)

Hyperammonemia plays a main role in the neurological impairment in cirrhotic patients with hepatic encephalopathy. Rats with chronic hyperammonemia reproduce the motor incoordination of patients with minimal hepatic encephalopathy, which is due to enhanced GABAergic neurotransmission in cerebellum as a consequence of neuroinflammation. Extracellular vesicles (EVs) could play a key role in the transmission of peripheral alterations to the brain to induce neuroinflammation and neurological impairment in hyperammonemia and hepatic encephalopathy. EVs from plasma of hyperammonemic rats (HA-EVs) injected to normal rats induce neuroinflammation and motor incoordination, but the underlying mechanisms remain unclear. The aim of this work was to advance in the understanding of these mechanisms. To do this we used an ex vivo system. Cerebellar slices from normal rats were treated ex vivo with HA-EVs. The aims were: 1) assess if HA-EVs induce microglia and astrocytes activation and neuroinflammation in cerebellar slices of normal rats, 2) assess if this is associated with activation of the TNFR1-NF-kB-glutaminase-GAT3 pathway, 3) assess if the TNFR1-CCL2-BDNF-TrkB pathway is activated by HA-EVs and 4) assess if the increased TNFα levels in HA-EVs are responsible for the above effects and if they are prevented by blocking the action of TNFα. Our results show that ex vivo treatment of cerebellar slices from control rats with extracellular vesicles from hyperammonemic rats induce glial activation, neuroinflammation and enhance GABAergic neurotransmission, reproducing the effects induced by hyperammonemia in vivo . Moreover, we identify in detail key underlying mechanisms. HA-EVs induce the activation of both the TNFR1-CCL2-BDNF-TrkB-KCC2 pathway and the TNFR1-NF-kB-glutaminase-GAT3 pathway. Activation of these pathways enhances GABAergic neurotransmission in cerebellum, which is responsible for the induction of motor incoordination by HA-EVs. The data also show that the increased levels of TNFα in HA-EVs are responsible for the above effects and that the activation of both pathways is prevented by blocking the action of TNFα. This opens new therapeutic options to improve motor incoordination in hyperammonemia and also in cirrhotic patients with hepatic encephalopathy and likely in other pathologies in which altered cargo of extracellular vesicles contribute to the propagation of the pathology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Izquierdo-Altarejos, Martínez-García and Felipo.)

Chronic hyperammonemia alters membrane expression of AMPA and NMDA receptors subunits in hippocampus leading to impaired memory and learning. Increasing extracellular cGMP normalizes these alterations. However, it has not been studied whether hyperammonemia alters the function of AMPA and NMDA receptors. The aims of this work were: (1) assess if hyperammonemia alters AMPA and NMDA receptors function; (2) analyze if extracellular cGMP reverses these alterations. A multielectrode array device was used to stimulate Schäffer collaterals and record postsynaptic currents in the CA1 region in hippocampal slices from control and hyperammonemic rats and analyze different features of the excitatory postsynaptic potentials. Hyperammonemia reduces the amplitude and delays appearance of AMPA EPSPs, whereas increases amplitude, hyperpolarization, depolarization and desensitization area of the NMDA EPSPs. These alterations in AMPA and NMDA function are accentuated as the stimulation intensity increases. Adding extracellular cGMP reverses the alteration in amplitude in both, AMPA and NMDA EPSPs. In control slices extracellular cGMP decreases the AMPA and NMDA EPSPs amplitude and delays the response of neurons and the return to the resting potential at all stimulation intensities. In conclusion, hyperammonemia decreases the AMPA response, whereas increases the NMDA response and extracellular cGMP reverses these alterations., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

This study was planned to assess the potential protective effects of taxifolin against thioacetamide-induced hepatic encephalopathy and subsequently to portray its behavioural results. The experimental model was induced with three doses of (200 mg/kg i.p.) thioacetamide and taxifolin (50 and 100 mg/kg, p.o.) was administered for fourteen days. Taxifolin effectively attenuated hepatic encephalopathy through decrease in AST, ALT, ALP and LDH concentrations and improvement of hyperammonemia, and increase in antioxidant capacity by decreasing MDA, ROS, and increasing CAT and GSH. In addition, the expressions of NF-κB, TNF-α, IL-1β, caspase-3 and Bax was down-regulated while IL-10 and Bcl-2 expressions were up-regulated with taxifolin treatment. The recovery was confirmed by downregulation of iNOS and 8-OHdG expressions in our immunohistochemical analysis. Taxifolin treatment reduced the disrupting role of thioacetamide as seen by corrected hyperammonemia as well as preservation of astrocyte and hepatocyte structure. Elevated plus maze and locomotor activity tests also proved that taxifolin might repeal the neurobehavioral disabilities. In conclusion, taxifolin has shown hepatoprotective and neuroprotective roles with antioxidant and anti-inflammatory effects, as well as suppressing the excessive release of ammonia, and it eventually reversed neurobehavioral impairments., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Aims: Chronic hyperammonaemia and inflammation synergistically induce neurological impairment, including motor incoordination, in hepatic encephalopathy. Hyperammonaemic rats show neuroinflammation in the cerebellum which enhances GABAergic neurotransmission leading to motor incoordination. We aimed to identify underlying mechanisms. The aims were (1) to assess if S1PR2 is involved in microglial and astrocytic activation in the cerebellum of hyperammonaemic rats; (2) to identify pathways by which enhanced S1PR2 activation induces neuroinflammation and alters neurotransmission; (3) to assess if blocking S1PR2 reduces neuroinflammation and restores motor coordination in hyperammonaemic rats., Methods: We performed ex vivo studies in cerebellar slices from control or hyperammonaemic rats to identify pathways by which neuroinflammation enhances GABAergic neurotransmission in hyperammonaemia. Neuroinflammation and neurotransmission were assessed by immunochemistry/immunofluorescence and western blot. S1PR2 was blocked by intracerebral treatment with JTE-013 using osmotic mini-pumps. Motor coordination was assessed by beam walking., Results: Chronic hyperammonaemia enhances S1PR2 activation in the cerebellum by increasing its membrane expression. This increases CCL2, especially in Purkinje neurons. CCL2 activates CCR2 in microglia, leading to microglial activation, increased P2X4 membrane expression and BDNF in microglia. BDNF enhances TrkB activation in neurons, increasing KCC2 membrane expression. This enhances GABAergic neurotransmission, leading to motor incoordination in hyperammonaemic rats. Blocking S1PR2 in hyperammonaemic rats by intracerebral administration of JTE-013 normalises the S1PR2-CCL2-CCR2-BDNF-TrkB-KCC2 pathway, reduces glial activation and restores motor coordination in hyperammonaemic rats., Conclusions: Enhanced S1PR2-CCL2-BDNF-TrkB pathway activation mediates neuroinflammation and incoordination in hyperammonaemia. The data raise a promising therapy for patients with hepatic encephalopathy using compounds targeting this pathway., (© 2022 British Neuropathological Society.)

Rats with chronic hyperammonemia reproduce the cognitive and motor impairment present in patients with hepatic encephalopathy. It has been proposed that enhanced GABAergic neurotransmission in hippocampus may contribute to impaired learning and memory in hyperammonemic rats. However, there are no direct evidences of the effects of hyperammonemia on GABAergic neurotransmission in hippocampus or on the underlying mechanisms. The aims of this work were to assess if chronic hyperammonemia enhances the function of GABA A receptors in hippocampus and to identify the underlying mechanisms. Activation of GABA A receptors is enhanced in hippocampus of hyperammonemic rats, as analyzed in a multielectrode array system. Hyperammonemia reduces membrane expression of the GABA transporters GAT1 and GAT3, which is associated with increased extracellular GABA concentration. Hyperammonemia also increases gephyrin levels and phosphorylation of the β3 subunit of GABA A receptor, which are associated with increased membrane expression of the GABA A receptor subunits α1, α2, γ2, β3, and δ. Enhanced levels of extracellular GABA and increased membrane expression of GABA A receptors would be responsible for the enhanced GABAergic neurotransmission in hippocampus of hyperammonemic rats. Increasing extracellular cGMP reverses the increase in GABA A receptors activation by normalizing the membrane expression of GABA transporters and GABA A receptors. The increased GABAergic neurotransmission in hippocampus would contribute to cognitive impairment in hyperammonemic rats. The results reported suggest that reducing GABAergic tone in hippocampus by increasing extracellular cGMP or by other means may be useful to improve cognitive function in hyperammonemia and in cirrhotic patients with minimal or clinical hepatic encephalopathy., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Amino acids, the building blocks of proteins in the cells and tissues, are of fundamental importance for cell survival, maintenance, and proliferation. The liver plays a critical role in amino acid metabolism and detoxication of byproducts such as ammonia. Urea cycle disorders with hyperammonemia remain difficult to treat and eventually necessitate liver transplantation. In this study, ornithine transcarbamylase deficient (Otc spf-ash ) mouse model was used to test whether knockdown of a key glutamine metabolism enzyme glutaminase 2 (GLS2, gene name: Gls2) or glutamate dehydrogenase 1 (GLUD1, gene name: Glud1) could rescue the hyperammonemia and associated lethality induced by a high protein diet. We found that reduced hepatic expression of Gls2 but not Glud1 by AAV8-mediated delivery of a short hairpin RNA in Otc spf-ash mice diminished hyperammonemia and reduced lethality. Knockdown of Gls2 but not Glud1 in Otc spf-ash mice exhibited reduced body weight loss and increased plasma glutamine concentration. These data suggest that Gls2 hepatic knockdown could potentially help alleviate risk for hyperammonemia and other clinical manifestations of patients suffering from defects in the urea cycle., (© 2022 Regeneron Pharmaceuticals, Inc. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Hyperammonemia is known to cause various neurological dysfunctions such as seizures and cognitive impairment. Several studies have suggested that hyperammonemia may also be linked to the development of Alzheimer's disease (AD). However, the direct evidence for a role of ammonia in the pathophysiology of AD remains to be discovered. Herein, we report that hyperammonemia increases the amount of mature amyloid precursor protein (mAPP) in astrocytes, the largest and most prevalent type of glial cells in the central nervous system that are capable of metabolizing glutamate and ammonia, and promotes amyloid beta (Aβ) production. We demonstrate the accumulation of mAPP in astrocytes was primarily due to enhanced endocytosis of mAPP from the plasma membrane. A large proportion of internalized mAPP was targeted not to the lysosome, but to the endoplasmic reticulum, where processing enzymes β-secretase BACE1 (beta-site APP cleaving enzyme 1) and γ-secretase presenilin-1 are expressed, and mAPP is cleaved to produce Aβ. Finally, we show the ammonia-induced production of Aβ in astrocytic endoplasmic reticulum was specific to Aβ42, a principal component of senile plaques in AD patients. Our studies uncover a novel mechanism of Aβ42 production in astrocytes and also provide the first evidence that ammonia induces the pathogenesis of AD by regulating astrocyte function., Competing Interests: Conflict of interest The authors declare no conflicts of interest associated with this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Molecular alterations underlying cerebral impairment in hyperammonemic disorders such as in hepatic encephalopathy (HE) are only poorly understood. Using transcriptomics and proteomics on brains of mice with systemic hyperammonemia resulting from knockout of hepatic glutamine synthetase (LGS-KO) we identified up to 214 genes and 34 proteins whose expressions were altered in brains of LGS-KO mice in a brain region-specific way. Differentially expressed genes were enriched for those related to oxidative stress, cell proliferation, heme metabolism and others. Due to their particularly high expression changes, coactivator associated arginine methyltransferase 1 (CARM1), TROVE2 and Lipocalin-2 (LCN2) were selected for further analyses. All selected candidates were expressed by astrocytes in rodent brain and challenging cultured astrocytes with NH 4 Cl changed their protein and mRNA levels similar to what was found in brains of LGS-KO mice. Further functional analyses suggested a role of CARM1 for senescence, TROVE2 for RNA quality control and LCN2 for disturbed iron homeostasis in ammonia-exposed astrocytes. LCN2 protein and Trove2 mRNA were also elevated in cerebral cortex of ammonium acetate-challenged rats and in post mortem brain tissue from patients with liver cirrhosis and HE, respectively. This study identified new molecular players potentially relevant for cerebral dysfunction in HE., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: We found through previous research that hyperammonemia can cause secondary liver damage. However, whether hepatocytes are target cells of ammonia toxicity and whether hyperammonemia affects hepatocyte metabolism remain unknown., Aims: The purpose of the current study is to examine whether the hepatocyte is a specific target cell of ammonia toxicity and whether hyperammonemia can interfere with hepatocyte metabolism., Methods: Cell viability and apoptosis were analyzed in primary hepatocytes and other cells that had been exposed to ammonium chloride. Western blotting was adopted to examine the expression of proteins related to ammonia transport. We also established a metabolomics method based on gas chromatography-mass spectrometry to understand the characteristics of the hepatocyte metabolic spectrum in a hyperammonemia microenvironment, to screen and identify differential metabolites, and to determine the differential metabolic pathway. Different technologies were used to verify the differential metabolic pathways., Results: Hepatocytes are target cells of ammonia toxicity. The mechanism is related to the ammonia transporter. Hyperammonemia interferes with hepatocyte metabolism, which leads to TCA cycle, urea cycle, and RNA synthesis disorder., Conclusions: This study demonstrates that hepatocyte growth and metabolism are disturbed in a hyperammonemia microenvironment, which further deteriorates hepatocyte function., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cyclic Guanosine-Monophosphate (cGMP) is implicated as second messenger in a plethora of pathways and its effects are executed mainly by cGMP-dependent protein kinases (PKG). It is involved in both peripheral (cardiovascular regulation, intestinal secretion, phototransduction, etc.) and brain (hippocampal synaptic plasticity, neuroinflammation, cognitive function, etc.) processes. Stimulation of hippocampal cGMP signaling have been proved to be beneficial in animal models of aging, Alzheimer's disease or hepatic encephalopathy, restoring different cognitive functions such as passive avoidance, object recognition or spatial memory. However, even when some inhibitors of cGMP-degrading enzymes (PDEs) are already used against peripheral pathologies, their utility as neurological treatments is still under clinical investigation. Additionally, it has been demonstrated a list of cGMP roles as not second but first messenger. The role of extracellular cGMP has been specially studied in hippocampal function and cognitive impairment in animal models and it has emerged as an important modulator of neuroinflammation-mediated cognitive alterations and hippocampal synaptic plasticity malfunction. Specifically, it has been demonstrated that extracellular cGMP decreases hippocampal IL-1β levels restoring membrane expression of glutamate receptors in the hippocampus and cognitive function in hyperammonemic rats. The mechanisms implicated are still unclear and might involve complex interactions between hippocampal neurons, astrocytes and microglia. Membrane targets for extracellular cGMP are still poorly understood and must be addressed in future studies., (Copyright © 2022 Elsevier Inc. All rights reserved.)

This article reviews the scientific career and accomplishments of the late Dr. Saul Brusilow, Professor of Pediatrics at Johns Hopkins. Dr. Brusilow's career was focused on diseases involving hyperammonemia. He and his colleagues developed a set of drugs that could lower ammonia levels in patients with genetic disorders of the urea cycle by providing alternative pathways for the synthesis of excretable nitrogenous molecules. Those drugs and their derivatives represent one of the earliest and most successful drug therapies for genetic diseases. Turning their attention to brain swelling caused by liver disease, Dr. Brusilow and colleagues developed the Osmotic Gliopathy Hypothesis to help explain the mechanism of ammonia toxicity, postulating that high ammonia drives glutamine synthetase in astrocytes to produce elevated levels of glutamine that act as a potent osmolyte, drawing water into the cell and causing cerebral edema. This hypothesis suggests that inhibiting glutamine synthetase with its well-characterized inhibitor, methionine sulfoximine, might prove therapeutic in cases of hepatic encephalopathy, a conclusion supported by their subsequent studies in animals. But although the drugs developed to treat hyperammonemia resulting from urea cycle disorders were successfully developed and approved by the FDA, the compound suggested as a treatment for hepatic encephalopathy was unable to attract sufficient interest and investment to be tested for use in humans., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Hepatic encephalopathy and depression share a number of clinical features, such as cognitive impairment and psychomotor retardation, and are highly prevalent in patients with chronic liver disease. Both conditions signify a poor prognosis, carry an increased mortality and are major determinants of reduced health related quality of life. The pathophysiology of hepatic encephalopathy is complex. Whilst cerebral accumulation of ammonia is well-recognised as being central to the development of hepatic encephalopathy, systemic inflammation, which acts in synergy with hyperammonaemia, is emerging as a key driver in its development. The pro-inflammatory state is also widely documented in depression, and peripheral to brain communication occurs resulting in central inflammation, behavioural changes and depressive symptoms. Gut dysbiosis, with a similar reduction in beneficial bacteria, increase in pathogens and decreased bacterial diversity, has been observed in both hepatic encephalopathy and depression, and it may be that the resultant increased bacterial translocation causes their shared inflammatory pathophysiology. Whilst the literature on a positive association between hepatic encephalopathy and depression in cirrhosis remains to be substantiated, there is evolving evidence that treatment with psychobiotics may be of dual benefit, improving cognition and mood in cirrhosis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Urea cycle disorders (UCD) are inherited diseases resulting from deficiency in one of six enzymes or two carriers that are required to remove ammonia from the body. UCD may be associated with neurological damage encompassing a spectrum from asymptomatic/mild to severe encephalopathy, which results in most cases from Hyperammonemia (HA) and elevation of other neurotoxic intermediates of metabolism. Electroencephalography (EEG), Magnetic resonance imaging (MRI) and Proton Magnetic resonance spectroscopy (MRS) are noninvasive measures of brain function and structure that can be used during HA to guide management and provide prognostic information, in addition to being research tools to understand the pathophysiology of UCD associated brain injury. The Urea Cycle Rare disorders Consortium (UCDC) has been invested in research to understand the immediate and downstream effects of hyperammonemia (HA) on brain using electroencephalogram (EEG) and multimodal brain MRI to establish early patterns of brain injury and to track recovery and prognosis. This review highlights the evolving knowledge about the impact of UCD and HA in particular on neurological injury and recovery and use of EEG and MRI to study and evaluate prognostic factors for risk and recovery. It recognizes the work of others and discusses the UCDC's prior work and future research priorities., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Propionic acidemia (PA) is a severe autosomal recessive metabolic disease caused by deficiency of propionyl-CoA carboxylase (PCC). We studied PA transgenic (Pat) mice that lack endogenous PCC but express a hypoactive human PCCA cDNA, permitting their survival. Pat cohorts followed from 3 to 20 weeks of age showed growth failure and lethal crises of lethargy and hyperammonemia, commoner in males (27/50, 54%) than in females (11/52, 21%) and occurring mainly in Pat mice with the most severe growth deficiency. Groups of Pat mice were studied under basal conditions (P-Ba mice) and during acute crises (P-Ac). Plasma acylcarnitines in P-Ba mice, compared to controls, showed markedly elevated C3- and low C2-carnitine, with a further decrease in C2-carnitine in P-Ac mice. These clinical and biochemical findings resemble those of human PA patients. Liver acyl-CoA measurements showed that propionyl-CoA was a minor species in controls (propionyl-CoA/acetyl-CoA ratio, 0.09). In contrast, in P-Ba liver the ratio was 1.4 and in P-Ac liver, 13, with concurrent reductions of the levels of acetyl-CoA and other acyl-CoAs. Plasma ammonia levels in control, P-Ba and P-Ac mice were 109 ± 10, 311 ± 48 and 551 ± 61 μmol/L respectively. Four-week administration to Pat mice, of carglumate (N-carbamyl-L-glutamic acid), an analogue of N-carbamylglutamate, the product of the only acyl-CoA-requiring reaction directly related to the urea cycle, was associated with increased food consumption, improved growth and absence of fatal crises. Pat mice showed many similarities to human PA patients and provide a useful model for studying tissue pathophysiology and treatment outcomes., (Copyright © 2021. Published by Elsevier Inc.)

The etiology of secondary 3-methylglutaconic aciduria (3-MGA-uria) is not well understood although is thought to be a marker of mitochondrial dysfunction. For this reason, suspicion for a secondary 3-MGA-uria often leads to an extensive clinical and laboratory work-up for mitochondrial disease, although in many cases evidence for mitochondrial dysfunction is never found. 3-methylglutaconic aciduria in healthy individuals without known metabolic disease has not been well described. Here, we describe clinical and biochemical features of 23 individuals evaluated at the Greenwood Genetic Center for low plasma free carnitine reported on newborn screening. Of the 23 individuals evaluated, four individuals were diagnosed with primary carnitine deficiency, 16 were identified as carriers for primary carnitine deficiency, and three individuals were determined to be unaffected non-carriers based on molecular and biochemical testing. Elevated 3-MGA (>20 mmol/mol of creatinine) was identified in nine carriers of primary carnitine deficiency, while all unaffected non carriers and all affected individuals with primary carnitine deficiency had a normal 3-MGA level (<20 mmol/mol of creatinine). Average 3-MGA among all carriers was 39.66 mmol/mol of creatinine. Average plasma free carnitine in among all carriers (n = 16) was 13.87 μm/L, and average plasma free carnitine was not significantly different between carriers with and those without elevated 3-MGA (p = 0.66). In summary, we describe elevated 3-MGA as a discriminatory feature in nine healthy carriers of primary carnitine deficiency. Our findings suggest that heterozygosity for pathogenic alterations on SLC22A5 should be considered in the differential for individuals with persistent 3-MGA-uria of unclear etiology., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Reduced muscle tone, muscle weakness, and physical fatigue can impact considerably on quality of life for children with neurofibromatosis type 1 (NF1). Human muscle biopsies and mouse models of NF1 deficiency in muscle show intramyocellular lipid accumulation, and preclinical data have indicated that L-carnitine supplementation can ameliorate this phenotype. The aim of this study is to examine whether daily L-carnitine supplementation is safe and feasible, and will improve muscle strength and reduce fatigue in children with NF1. A 12-week Phase 2a trial was conducted using 1000 mg daily oral levocarnitine tartrate supplementation. Recruited children were between 8 and 12 years old with a clinical diagnosis of NF1, history of muscle weakness and fatigue, and naïve to L-carnitine. Primary outcomes were safety (self-reporting, biochemical testing) and compliance. Secondary outcomes included plasma acylcarnitine profiles, functional measures (muscle strength, long jump, handwriting speed, 6-minute-walk test [6MWT]), and parent-reported questionnaires (PedsQL™, CBCL/6-18). Six children completed the trial with no self-reported adverse events. Biochemical tests for kidney and liver function were normal, and the average compliance was 95%. Plasma acylcarnitine levels were low, but within a range not clinically linked to carnitine deficiency. For strength measures, there was a mean 53% increase in dorsiflexion strength (95% confidence interval [CI] 8.89-60.75; p = 0.02) and mean 66% increase in plantarflexion strength (95% CI 12.99-134.1; p = 0.03). In terms of muscle performance, there was a mean 10% increase in long jump distance (95% CI 2.97-16.03; p = 0.01) and 6MWT distance (95% CI 5.88-75.45; p = 0.03). Comparison with the 1000 Norms Project data showed a significant improvement in Z-score for all of these measures. Parent reports showed no negative impact on quality of life, and the perceived benefits led to the majority of individuals remaining on L-carnitine after the study. Twelve weeks of L-carnitine supplementation is safe and feasible in children with NF1, and a Phase 3 trial should confirm the efficacy of treatment., (© 2021 The Authors. American Journal of Medical Genetics Part A published by Wiley Periodicals LLC.)

Ammonia is a cytotoxic molecule generated during normal cellular functions. Dysregulated ammonia metabolism, which is evident in many chronic diseases such as liver cirrhosis, heart failure, and chronic obstructive pulmonary disease, initiates a hyperammonemic stress response in tissues including skeletal muscle and in myotubes. Perturbations in levels of specific regulatory molecules have been reported, but the global responses to hyperammonemia are unclear. In this study, we used a multiomics approach to vertically integrate unbiased data generated using an assay for transposase-accessible chromatin with high-throughput sequencing, RNA-Seq, and proteomics. We then horizontally integrated these data across different models of hyperammonemia, including myotubes and mouse and human muscle tissues. Changes in chromatin accessibility and/or expression of genes resulted in distinct clusters of temporal molecular changes including transient, persistent, and delayed responses during hyperammonemia in myotubes. Known responses to hyperammonemia, including mitochondrial and oxidative dysfunction, protein homeostasis disruption, and oxidative stress pathway activation, were enriched in our datasets. During hyperammonemia, pathways that impact skeletal muscle structure and function that were consistently enriched were those that contribute to mitochondrial dysfunction, oxidative stress, and senescence. We made several novel observations, including an enrichment in antiapoptotic B-cell leukemia/lymphoma 2 family protein expression, increased calcium flux, and increased protein glycosylation in myotubes and muscle tissue upon hyperammonemia. Critical molecules in these pathways were validated experimentally. Human skeletal muscle from patients with cirrhosis displayed similar responses, establishing translational relevance. These data demonstrate complex molecular interactions during adaptive and maladaptive responses during the cellular stress response to hyperammonemia., Competing Interests: Conflict of interest The funders had no role in the design, performance, or interpretation of these studies. The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Hepatic encephalopathy (HE) is a well-studied, neurological syndrome caused by liver dysfunctions. Ammonia, the major toxin during HE pathogenesis, impairs many cellular processes within astrocytes. Yet, the molecular mechanisms causing HE are not fully understood. Here we will recapitulate possible underlying mechanisms with a clear focus on studies revealing a link between altered energy metabolism and HE in cellular models and in vivo . The role of the mitochondrial glutamate dehydrogenase and its role in metabolic rewiring of the TCA cycle will be discussed. We propose an updated model of ammonia-induced toxicity that may also be exploited for therapeutic strategies in the future., (© 2021 Marcel Zimmermann and Andreas S. Reichert, published by De Gruyter, Berlin/Boston.)