Pharmacologic rescue of hyperammonemia-induced toxicity in zebrafish by inhibition of ornithine aminotransferase

PLOS ONE, Sep 2018

Hyperammonemia is the common biochemical hallmark of urea cycle disorders, activating neurotoxic pathways. If untreated, affected individuals have a high risk of irreversible brain damage and mortality. Here we show that acute hyperammonemia strongly enhances transamination-dependent formation of osmolytic glutamine and excitatory glutamate, thereby inducing neurotoxicity and death in ammoniotelic zebrafish larvae via synergistically acting overactivation of NMDA receptors and bioenergetic impairment induced by depletion of 2-oxoglutarate. Intriguingly, specific and irreversible inhibition of ornithine aminotransferase (OAT) by 5-fluoromethylornithine rescues zebrafish from lethal concentrations of ammonium acetate and corrects hyperammonemia-induced biochemical alterations. Thus, OAT inhibition is a promising and effective therapeutic approach for preventing neurotoxicity and mortality in acute hyperammonemia.

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Pharmacologic rescue of hyperammonemia-induced toxicity in zebrafish by inhibition of ornithine aminotransferase

September Pharmacologic rescue of hyperammonemia- induced toxicity in zebrafish by inhibition of ornithine aminotransferase Matthias Zielonka 0 1 Maximilian Breuer 0 1 JuÈ rgen GuÈ nther Okun 0 1 Matthias Carl 1 Georg Friedrich Hoffmann 0 1 Stefan KoÈ lker 0 1 0 University Hospital Heidelberg, Center for Child and Adolescent Medicine, Division for Pediatric Neurology and Metabolic Medicine , Heidelberg, Germany , 2 Heidelberg Research Center for Molecular Medicine (HRCMM) , Heidelberg, Germany, 3 Heidelberg University , Medical Faculty Mannheim, Department of Cell and Molecular Biology, Mannheim, Germany, 4 University of Trento, Center for Integrative Biology (CIBIO), Laboratory of Translational Neurogenetics , Trento , Italy 1 Editor: Jyotshna Kanungo, National Center for Toxicological Research , UNITED STATES Hyperammonemia is the common biochemical hallmark of urea cycle disorders, activating neurotoxic pathways. If untreated, affected individuals have a high risk of irreversible brain damage and mortality. Here we show that acute hyperammonemia strongly enhances transamination-dependent formation of osmolytic glutamine and excitatory glutamate, thereby inducing neurotoxicity and death in ammoniotelic zebrafish larvae via synergistically acting overactivation of NMDA receptors and bioenergetic impairment induced by depletion of 2oxoglutarate. Intriguingly, specific and irreversible inhibition of ornithine aminotransferase (OAT) by 5-fluoromethylornithine rescues zebrafish from lethal concentrations of ammonium acetate and corrects hyperammonemia-induced biochemical alterations. Thus, OAT inhibition is a promising and effective therapeutic approach for preventing neurotoxicity and mortality in acute hyperammonemia. - Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: Matthias Zielonka was supported by the Physician-Scientist Program at Ruprecht-KarlsUniversity Heidelberg Faculty of Medicine and a Career Development Fellowship provided by the Heidelberg Research Center for Molecular Medicine (HRCMM) in the framework of Excellence Initiative II of the German Research Foundation. The funders had no role in study design, data Introduction Nitrogen is an essential building block of amino and nucleic acids in all living organisms. Protein ingested by food or derived from the body is the major source of excess nitrogen once nitrogen-containing compounds are used to build energy substrates. Deamination of amino acids liberates ammonium (NH4+). If present in increased concentrations, NH4+ is highly toxic to living organisms. Species have developed different strategies to dispose excess NH4+. While fishes (ammoniotelic organisms) excrete up to 90% of their nitrogenous waste directly into their aqueous environment, reptiles and birds save water by excreting uric acid (uricotelic organisms) [1]. Humans and terrestrial animals (ureotelic organisms) are unable to excrete NH4+ directly or to package large amounts of NH4+ into uric acid and metabolize NH4+ to water-soluble urea, an energy-dependent mechanism requiring three moles of ATP for each mole of urea. collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. The urea cycle contains five enzymes and two transporters that are involved in the irreversible detoxification of NH4+ to urea including carbamoylphosphate synthetase 1 (CPS1), ornithine transcarbamylase (OTC), argininosuccinate synthetase 1 (ASS1), argininosuccinate lyase (ASL), arginase 1 (ARG1), citrin or aspartate/glutamate carrier and the mitochondrial ornithine transporter 1 [2]. Furthermore, carbonic anhydrase VA and N-acetylglutamate synthase (NAGS) are required to form bicarbonate and N-acetylglutamate for the first enzymatic step, the formation of carbamoylphosphate. Urea cycle disorders (UCDs) are caused by inherited deficiencies of the NH4+-detoxifying ureagenetic machinery. Estimated cumulative incidence of UCDs is between 1:35,000 to 1:50,000 newborns [3, 4]. UCD patients, except for those with citrin and ARG1 deficiency, characteristically present with hyperammonemic encephalopathy (HE), which often manifests as early as the first days of life, but can present with first symptoms at any age afterwards. Onset type and the peak plasma ammonium concentration of the initial manifestation correlate with clinical severity and outcome. The most severe manifestation is neonatal HE with coma; it is associated with a high risk of mortality and, in survivors, of severe neurologic dysfunction and intellectual disability [3, 5±11]. Maintenance treatment of UCDs is based on a low protein diet, supplementation with essential amino acids, citrulline and/or arginine, and application of nitrogen scavenging drugs (sodium benzoate, sodium and glycerol phenylbutyrate) as well as carglumic acid (for NAGS deficiency, partially responsiv (...truncated)


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Matthias Zielonka, Maximilian Breuer, Jürgen Günther Okun, Matthias Carl, Georg Friedrich Hoffmann, Stefan Kölker. Pharmacologic rescue of hyperammonemia-induced toxicity in zebrafish by inhibition of ornithine aminotransferase, PLOS ONE, 2018, Volume 13, Issue 9, DOI: 10.1371/journal.pone.0203707