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Impaired amino acid metabolism in cells affected by the T8993G (NARP) mtDNA mutation: A potential novel therapeutic target
652
Abstracts
the deficit of amino acid substitution is observed. This implies that the mutations are removed after passing through the mtDNA genetic bottleneck in early embryo development or during the proliferation of mtDNA up to differentiation of the primary oocytes. We are seeking to refine the timing of this selection by utilizing mtDNA mutator mice that also express GFP markers of primordial germs cells. Once the selective window is detected, exact molecular mechanisms can be thoroughly investigated. This data is consistent with observations in human patients, where there are relatively few disease-causing mutations in mtDNA protein genes, which occupy most of the mtDNA chromosome. By contrast, tRNA mutations appear to be under very little or no selective constraint in the mice, and are far more common in human patients, despite their occupation of only 10% of the mtDNA chromosome. This work will allow more accurate models of mtDNA transmission to be developed, and allow for more accurate genetic counseling of families that carry mitochondrial DNA mutations. doi:10.1016/j.mito.2011.03.052
40 Impaired amino acid metabolism in cells affected by the T8993G (NARP) mtDNA mutation: A potential novel therapeutic target Marilena D'Aurelio a,⁎, Lichuan Yang a, Qiuying Chen b, Flint Beal a, Steven Gross b, Giovanni Manfredi a a
Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, NY 10065, United States b Department of Pharmacology, Weill Medical College of Cornell University, New York, NY 10065, United States Mutations in the mitochondrial DNA (mtDNA) result in defects of energy metabolism. Although the genetic defects are known, many aspects of mitochondrial disease pathogenesis are yet to be elucidated. Attempts to treat mitochondrial diseases have been disappointing thus far, mostly due to the lack of defined targets. We have utilized a compelling new mass spectrometry-based “metabolomics” technology to identify differences in the levels of thousands of functionally relevant metabolites in controls and in cells harboring the T8993G mtDNA mutation in the gene encoding subunit 6 of ATPase, associated with ATP synthesis defects and the NARP syndrome. In the cybrid system utilized, mutant and wild type cells have a similar nuclear genome, so any metabolic change detected must be linked to the effects of the mtDNA mutation. We found unbalanced levels of the “glutamate family” of amino acids, of metabolites involved in the urea cycle for ammonia detoxification and disposal, and of end products of amino acid catabolism in NARP cells. In particular, levels of glutamine, glutamate, glutathione, citrulline, aspartate and proline were decreased while arginine, Nacetyl glutamate and taurine levels were increased. Our results support the novel hypothesis that redox and pH changes, due to forced aerobic glycolytic metabolism in NARP cells, are directly responsible for decreased glutamine uptake and glutamate availability. Glutamate is the most versatile amino acid and plays important roles in all the tissues. Glutamate is at the center of all the metabolic changes found in our mutants: glutamate is required for the synthesis of glutathione, it is involved in the aspartate shuttle, plays key role in transaminations, it can be converted in α-ketoglutarate serving anaplerotic function for the Krebs cycle, and is the substrate for the synthesis of N-acetyl glutamate an essential activator of the urea cycle. The mutant cells' ability to survive and to improve replication in oxidative metabolic conditions (galactose medium) was tested upon supplementation with specific metabolites. Quantification of metabolic substrates, following enzymatic and growth condition modula-
tions, obtained by high-performance liquid chromatography, was also used for rescue evaluation. We identified conditions that significantly improved growth of NARP cells in galactose, suggesting that metabolic supplementation can bypass the defective metabolic steps and rescue the defective phenotype. These findings provide missing links between genetic defects and their fatal metabolic consequences in the blockage of crucial steps of the glutamate intermediate metabolism, and suggest this blockage as a novel therapeutic target.
doi:10.1016/j.mito.2011.03.053
41 Combined treatment with oral metronidazole and N-acetylcysteine is effective in ethylmalonic encephalopathy Carlo Viscomi a,⁎, Alberto Burlina b, Imad Dweikat c, Mario Savoiardo d, Valeria Tiranti a, Massimo Zeviani a a Unit of Molecular Neurogenetics, The Foundation “Carlo Besta” Institute of Neurology, Milan, Italy b Clinic of Paediatrics, University of Padua School of Mediciene, Padua, Italy c Makassed Hospital, Metabolic Disease Unit, Jerusalem, Israel d Unit of Neuroradiology, The Foundation “Carlo Besta” Institute of Neurology, Milan, Italy
Ethylmalonic Encephalopathy (EE) is an autosomal recessive, invariably fatal disorder (OMIM#602473), clinically hallmarked by (i) progressive encephalopathy; (ii) chronic hemorrhagic diarrhea; (iii) showers of petechial purpura and severe orthostatic acrocyanosis. Biochemically, EE is a peculiar entity, since it combines severe deficiency of cytochrome c oxidase (COX), the last component of the mitochondrial respiratory chain (MRC), in both muscle and brain, with blood and urinary accumulation of ethyl-malonic acid (EMA), a di-carboxylic derivative of butyrate which is produced in short-chain acyl CoA dehydrogenase (SCAD) deficiency and other rare defects of betaoxidation. EE is caused by failure to detoxify sulfide, a mitochondrial poison for both COX and SCAD, produced by intestinal anaerobes and, in trace amount, by tissues. Absence of a mitochondrial matrix sulfur dioxygenase, encoded by the ETHE1 gene, is responsible for EE. Metronidazole, a bactericidal agent specific for anaerobes, or Nacetylcysteine, a precursor of sulfide-buffering glutathione, significantly prolonged the lifetime of ETHE1-less mice, the combined treatment being additive. We then applied the same treatment to four EE children for 3–8 months. All patients showed remarkable amelioration of the main disease features, including disappearance of diarrhea and skin lesions, and improvement of neurological symptoms, with hardly any collateral effect. The remarkable prolongation of the lifespan in Ethe1−/− mice, and the evidence-based clinical improvement in the patients, prompt us to propose it as the most effective treatment nowadays available for this devastating mitochondrial disease. Since neurological symptoms in EE do not appear before 2–4 months after birth, and more slowly progressive cases, albeit however severe, have been recently described, early diagnosis by either prenatal analysis of the ETHE1 gene in at-risk fetuses, or mass-mass spectrometry screening of EMA aciduria in neonates could prompt to pre-symptomatic treatment with the aim of preventing irreversible brain damage. The results presented here warrant the development of future investigation, including a larger clinical trial with the same or similar compounds, and alternative experimental therapies that will first be attempted in the mouse model, such as gene or bone marrow cell replacement.
doi:10.1016/j.mito.2011.03.054
Abstracts
the deficit of amino acid substitution is observed. This implies that the mutations are removed after passing through the mtDNA genetic bottleneck in early embryo development or during the proliferation of mtDNA up to differentiation of the primary oocytes. We are seeking to refine the timing of this selection by utilizing mtDNA mutator mice that also express GFP markers of primordial germs cells. Once the selective window is detected, exact molecular mechanisms can be thoroughly investigated. This data is consistent with observations in human patients, where there are relatively few disease-causing mutations in mtDNA protein genes, which occupy most of the mtDNA chromosome. By contrast, tRNA mutations appear to be under very little or no selective constraint in the mice, and are far more common in human patients, despite their occupation of only 10% of the mtDNA chromosome. This work will allow more accurate models of mtDNA transmission to be developed, and allow for more accurate genetic counseling of families that carry mitochondrial DNA mutations. doi:10.1016/j.mito.2011.03.052
40 Impaired amino acid metabolism in cells affected by the T8993G (NARP) mtDNA mutation: A potential novel therapeutic target Marilena D'Aurelio a,⁎, Lichuan Yang a, Qiuying Chen b, Flint Beal a, Steven Gross b, Giovanni Manfredi a a
Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, NY 10065, United States b Department of Pharmacology, Weill Medical College of Cornell University, New York, NY 10065, United States Mutations in the mitochondrial DNA (mtDNA) result in defects of energy metabolism. Although the genetic defects are known, many aspects of mitochondrial disease pathogenesis are yet to be elucidated. Attempts to treat mitochondrial diseases have been disappointing thus far, mostly due to the lack of defined targets. We have utilized a compelling new mass spectrometry-based “metabolomics” technology to identify differences in the levels of thousands of functionally relevant metabolites in controls and in cells harboring the T8993G mtDNA mutation in the gene encoding subunit 6 of ATPase, associated with ATP synthesis defects and the NARP syndrome. In the cybrid system utilized, mutant and wild type cells have a similar nuclear genome, so any metabolic change detected must be linked to the effects of the mtDNA mutation. We found unbalanced levels of the “glutamate family” of amino acids, of metabolites involved in the urea cycle for ammonia detoxification and disposal, and of end products of amino acid catabolism in NARP cells. In particular, levels of glutamine, glutamate, glutathione, citrulline, aspartate and proline were decreased while arginine, Nacetyl glutamate and taurine levels were increased. Our results support the novel hypothesis that redox and pH changes, due to forced aerobic glycolytic metabolism in NARP cells, are directly responsible for decreased glutamine uptake and glutamate availability. Glutamate is the most versatile amino acid and plays important roles in all the tissues. Glutamate is at the center of all the metabolic changes found in our mutants: glutamate is required for the synthesis of glutathione, it is involved in the aspartate shuttle, plays key role in transaminations, it can be converted in α-ketoglutarate serving anaplerotic function for the Krebs cycle, and is the substrate for the synthesis of N-acetyl glutamate an essential activator of the urea cycle. The mutant cells' ability to survive and to improve replication in oxidative metabolic conditions (galactose medium) was tested upon supplementation with specific metabolites. Quantification of metabolic substrates, following enzymatic and growth condition modula-
tions, obtained by high-performance liquid chromatography, was also used for rescue evaluation. We identified conditions that significantly improved growth of NARP cells in galactose, suggesting that metabolic supplementation can bypass the defective metabolic steps and rescue the defective phenotype. These findings provide missing links between genetic defects and their fatal metabolic consequences in the blockage of crucial steps of the glutamate intermediate metabolism, and suggest this blockage as a novel therapeutic target.
doi:10.1016/j.mito.2011.03.053
41 Combined treatment with oral metronidazole and N-acetylcysteine is effective in ethylmalonic encephalopathy Carlo Viscomi a,⁎, Alberto Burlina b, Imad Dweikat c, Mario Savoiardo d, Valeria Tiranti a, Massimo Zeviani a a Unit of Molecular Neurogenetics, The Foundation “Carlo Besta” Institute of Neurology, Milan, Italy b Clinic of Paediatrics, University of Padua School of Mediciene, Padua, Italy c Makassed Hospital, Metabolic Disease Unit, Jerusalem, Israel d Unit of Neuroradiology, The Foundation “Carlo Besta” Institute of Neurology, Milan, Italy
Ethylmalonic Encephalopathy (EE) is an autosomal recessive, invariably fatal disorder (OMIM#602473), clinically hallmarked by (i) progressive encephalopathy; (ii) chronic hemorrhagic diarrhea; (iii) showers of petechial purpura and severe orthostatic acrocyanosis. Biochemically, EE is a peculiar entity, since it combines severe deficiency of cytochrome c oxidase (COX), the last component of the mitochondrial respiratory chain (MRC), in both muscle and brain, with blood and urinary accumulation of ethyl-malonic acid (EMA), a di-carboxylic derivative of butyrate which is produced in short-chain acyl CoA dehydrogenase (SCAD) deficiency and other rare defects of betaoxidation. EE is caused by failure to detoxify sulfide, a mitochondrial poison for both COX and SCAD, produced by intestinal anaerobes and, in trace amount, by tissues. Absence of a mitochondrial matrix sulfur dioxygenase, encoded by the ETHE1 gene, is responsible for EE. Metronidazole, a bactericidal agent specific for anaerobes, or Nacetylcysteine, a precursor of sulfide-buffering glutathione, significantly prolonged the lifetime of ETHE1-less mice, the combined treatment being additive. We then applied the same treatment to four EE children for 3–8 months. All patients showed remarkable amelioration of the main disease features, including disappearance of diarrhea and skin lesions, and improvement of neurological symptoms, with hardly any collateral effect. The remarkable prolongation of the lifespan in Ethe1−/− mice, and the evidence-based clinical improvement in the patients, prompt us to propose it as the most effective treatment nowadays available for this devastating mitochondrial disease. Since neurological symptoms in EE do not appear before 2–4 months after birth, and more slowly progressive cases, albeit however severe, have been recently described, early diagnosis by either prenatal analysis of the ETHE1 gene in at-risk fetuses, or mass-mass spectrometry screening of EMA aciduria in neonates could prompt to pre-symptomatic treatment with the aim of preventing irreversible brain damage. The results presented here warrant the development of future investigation, including a larger clinical trial with the same or similar compounds, and alternative experimental therapies that will first be attempted in the mouse model, such as gene or bone marrow cell replacement.
doi:10.1016/j.mito.2011.03.054