- Email: [email protected]
31. Molecular Dissection of Mitochondrial Complex I Assembly In a Genetically Tractable Model System
68
Abstracts / Mitochondrion 9 (2009) 61–77
decreasing the threshold for the induction of beta cell apoptisis and increasing susceptibility to T1D. doi:10.1016/j.mito.2008.12.024
28. Neurological phenotype is the key predictor of long-term outcome in mitochondrial DNA depletion resulting from deoxyguanosine kinase deficiency David P. Dimmock a, J. Kay Dunn b, Annette Feigenbaum c, Tony Rupar d, Rita Horvath e, Peter Freisinger f, Be´ne´dicte Mousson de Camaret g, Lee-Jun Wong a, Fernando Scaglia a,* a Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA; b School of Public Health, University of Texas Health Science Center at Houston; c Division of Genetics, Hospital for Sick Children, Toronto, Ontario, Canada; d Departments of Biochemistry and Pediatrics, University of Western Ontario, London, Ontario, Canada; e Mitochondrial Research Group, Department of Neurology, University of Newcastle, Newcastle upon Tyne, UK; f Metabolic Disease Center, Department of Pediatrics, University of Technology, Mu¨nchen, Germany; g Service des Maladies He´re´ditaires du Me´tabolisme, Centre de Biologie et de Pathologie Est, 69677 Bron, France Deoxyguanosine kinase (DGUOK) deficiency is the commonest type of mitochondrial DNA depletion associated with a hepatocerebral phenotype. The objective of this analysis was to evaluate predictors of survival and therapeutic options in patients with deoxyguanosine kinase deficiency. A systematic search of MEDLINE, LILAC & SCIELO was carried out to identify peer-reviewed clinical trials, randomized controlled trials, metaanalyses, and other studies with clinical pertinence. DGUOK deficiency was searched using the terms dGK, DGUOK, mitochondrial DNA depletion, mtDNA, and hepatocerebral. Bibliographies of identified articles were reviewed for additional references. Thirteen identified studies met inclusion criteria and were used in this study. Results of the study revealed that DGUOK deficiency is associated with a variable clinical phenotype. Long-term survival is best predicted by the presence or absence of neurological features. In the presence of neurological features, there is increased mortality and liver transplantation does not confer increased survival. Conversely, in the absence of neurological disease, liver transplantation may be considered as a potential treatment. doi:10.1016/j.mito.2008.12.025
29. Assembly of mitochondrial c-type cytochromes: Emergence of novel redox components Delphine Bernard b, Mazakazu Hirasawa c, David Knaff c, Bernard Guiard d, Sabeeha Merchant e, Patrice Hamel a, Vincent Corvest a,b,c,d,e,* a Department of Plant Cellular and Molecular Biology and Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH 43210, USA; b Institut Jacques Monod-CNRS, Universite´ Paris VI and Paris VII, Paris, France; c Department of Chemistry and Biochemistry, Center for Biotechnology and Genomics, Texas Tech University, Lubbock, TX 79409, USA; d Centre de Ge´ne´tique Mole´culaire, CNRS, 91198 Gif-surYvette, France; e Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA 90095, USA The c-type cytochromes are universal metalloproteins defined by the covalent attachment of heme. Besides their role as electron shuttle in energy-transducing systems, c-types cytochromes can also be recruited in signaling in the cell death pathways. The study of their biogenesis is crucial since it is well documented that many mitochondrial diseases are directly related to defects in respiratory chain or apoptosis. The covalent binding of the heme can occur via three pathways (systems I, II, III). With only two factors, CCHL and CC1HL (cytochrome c and c1 heme lyases), system III, which operates in animal and fungal mitochondria, appears deceptively simple in contrast to the complexity
of systems I and II. This strongly suggests that additional players are implicated in mitochondrial c-type cytochromes assembly. Recently, a new component Cyc2p has been discovered. In vivo studies suggest that this mitochondrial flavoprotein could be involved in the redox chemistry of c-type cytochromes biogenesis by controlling the reactivity of the cysteines at the heme binding site of apocytochrome c1. In order to test our model, we undertook a biochemical approach by measuring the midpoint redox potential of the different partners (Cyc2p, apocytochromes c1) and demonstrating the direct reduction of apocytochromes c1 by Cyc2p. The absence of structural homolog of Cyc2p in animal mitochondria (including humans) is unexpected as it is very likely that the redox function of Cyc2p is conserved in all mitochondria. In order to identify a functional homolog of Cyc2p in mammals, we chose a heterologous complementation approach and discovered two human cDNAs, LDHA and LDHB encoding the L-lactate dehydrogenase subunits M and H respectively, able to substitute for the function of Cyc2p in yeast. LDH activity in mitochondrial c-type cytochromes assembly is still obscure and we are currently attempting to better define its implication. doi:10.1016/j.mito.2008.12.026
30. Structure-based Drug Design in the Search for New Treatment of Mitochondrial Diseases Carolyn P. Ojano-Dirain a,*, Andrew Magis b, David A. Ostrov b, Peter W. Stacpoole a,c,d a Department of Medicine, Division of Endocrinology and Metabolism, University of Florida, Gainesville, FL 32610, USA; b Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 32610, USA; c Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610, USA; d General Clinical Research Center, University of Florida, Gainesville, FL 32610, USA In structure-based drug design (SBDD), the atomic structure of proteins is used to precisely examine the interactions between atoms in the protein target and the potential drug compounds. SBDD allows researchers to design a more optimal drug or identify new drug molecules. Dichloroacetate (DCA) is an investigational compound used as a therapy in patients with inborn errors of mitochondrial metabolism such as pyruvate dehydrogenase complex (PDC) deficiency. The primary action of DCA is activation of PDC by inhibiting the activity of pyruvate dehydrogenase kinase (PDK) that inhibits PDC. To search for new compounds that may be more potent than DCA, we used a structure-based high-throughput molecular docking method to identify novel drug-like small molecules with high affinity for the binding pocket of DCA in the PDK2 isozyme. We screened 140,000 compounds in a large chemical library. Fifteen of the top-scoring compounds were tested in human fibroblast cultures from three healthy subjects for their effect on PDC activity. DCA and dimethyl sulfoxide (DMSO) were used as positive and vehicle controls, respectively. PDC activity, measured by the rate of 14 CO2 formation from ½1 14 Clabeled pyruvate, was run in triplicate per cell line. Data were the mean SE of the 3 cell lines. DCA increased PDC enzymatic activity by 18629 to 28430% compared to the DMSO control (p60.05). Five of the drugs that we tested also significantly increased PDC activity compared to DMSO control (18810, 16930, 17126, 18940, and 17924%). In this study, we identified 5 compounds that appear to increase PDC activity in fibroblasts in a manner comparable to DCA. Experiments to define the utility of these drug-like small molecules as potential therapy for mitochondrial diseases are currently under way. doi:10.1016/j.mito.2008.12.027
31. Molecular Dissection of Mitochondrial Complex I Assembly In a Genetically Tractable Model System M. Rosario Barbieri a,*, Ve´ronique Larosa b, Pierre Cardol b, Claire Remacle a,b, Patrice Hamel a
Abstracts / Mitochondrion 9 (2009) 61–77 a Department of Plant Cellular and Molecular Biology and Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH 43210, USA; b Ge´ne´tique des Microorganismes, De´partement des Sciences de la Vie, Institut de Botanique, Universite´, France
Deficiencies in assembly of respiratory complexes cause severe mitochondrial diseases. NADH:ubiquinone oxidoreductase (complex I) is the largest respiratory complex in the mitochondrial inner membrane with around 45 subunits in eukaryotes, of which 14 are of prokaryotic origin (core catalytic subunits). The emaining ‘‘accessory” subunits are also generally conserved. 60% of human patients with complex I defects carry no mutations in the structural genes, suggesting that mutations in yet-to-be discovered assembly factors of complex I as important causes of disease. We are using Chlamydomonas reinhardtii, a unicellular green alga as a model system to study complex I assembly. In Chlamydomonas, 33 out the 45 human complex I subunits, as well as the complex I assembly factor CIA30 are conserved, suggesting that the assembly pathway may also be generally conserved. In contrast to human and other model systems, Chlamydomonas mutants lacking complex I are viable. Chlamydomonas can be grown either autotrophically in the light, or heterotrophically on acetate in the dark and complex I-less mutants display a characteristic slow growth in the dark phenotype. To identify complex I assembly factors, we generated an insertional mutant library and specifically screened for the mutant phenotype. Two non-allelic nuclear mutants, amc1 and amc2 (assembly of mitochondrial complex I), obtained by insertional and chemical mutagenesis, respectively, lack a functional, fully assembled complex I, but accumulate a membrane-bound subcomplex with NADH dehydrogenase activity, indicating a defect in the assembly of the membrane domain of complex I. Preliminary results including sequencing and expression level analyses show that neither the nuclear genes encoding for the membrane domain subunits nor CIA30 are affected at the molecular level in the amc2 strain. Therefore, amc2 is likely mutated in a complex I assembly gene. To identify the AMC1 and AMC2 genes, we are currently performing a map-based cloning approach. doi:10.1016/j.mito.2008.12.028
33. Therapeutic potential of pyruvate for mitochondrial diseases Masashi Tanaka*, Yutaka Nishigaki, Noriyuki Fuku, Tohru Ibi, Ko Sahashi, Yasutoshi Koga Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, Japan Rationale 1. Pyruvate can eliminate hydrogen peroxide to form acetate, carbon dioxide, and water. Pyruvate prevents programmed cell death induced by hydrogen peroxide. The antioxidant activity of pyruvate may be also beneficial for patients with mitochondrial disorders. 2. Pyruvate has been used in culturing rho-0 cell. Pyruvate is preferentially taken up by the cell via the monocarboxylate transporter in exchange for lactate. Pyruvate oxidizes NADH to NAD+ via LDH reaction, which restores the ATP production by glycolysis. 3. Pyruvate activates PDHC through inhibition of pyruvate dehydrogenase kinases. Dichloroacetate (DCA) inhibits pyruvate dehydrogenase kinase and thereby activates PDHC. Although DCA has been used in treatment for lactic acidosis, it causes peripheral neuropathy. Because pyruvate is a physiological inhibitor of PDK, it should have the same lactate-lowering effect as DCA without any toxicity. Design and Results 1. We administered 5 g of sodium pyruvate to a patient with CPEO carrying deleted mtDNA. At 30 min after the administration, the L/P ratio decreased from 25.7 to 16.3. The decreased L/P ratio suggests that the intracellular redox state was improved and that ATP production by the glycolytic pathway was restored by the administration of pyruvate. 2. Adult-onset type II citrullinemia (CTLN2) is caused by a mutation in the gene encoding citrin, mitochondrial aspartate glutamate carrier. This defect impairs the import of NADH from the cytosol into mitochondria, resulting in fatty liver and liver cirrhosis. We demonstrated that perfusion of the liver of Citrin-/- mice with sodium pyruvate improved the formation of urea from ammonia.
69
Conclusion 1. Pyruvate administration would ameliorate the bioenergetic defects of paitients with mitochondrial disorders. 2. Pyruvate would prevent severe hepatic failure that necessitates liver transplantation in patients with citrin deficiency. 3. We have started an open clinical study on the effects of sodium pyruvate on mitochondrial diseases and type II citrullinemia. doi:10.1016/j.mito.2008.12.029
34. A transcriptional signature of primary mitochondrial disease is present across evolution Marni J. Falk*, Zhe Zhang, David L. Gasser, Marc Yudkoff The Children’s Hospital of Philadelphia and University of Pennsylvania School of Medicine, USA Mitochondrial disease diagnosis is complicated by the absence of a biomarker that sufficiently divulges all cases. Significant bioinformatic advances have made it feasible to consider a novel approach to address this diagnostic challenge. Specifically, applying metabolic pathway clustering to global genome transcriptional profiling presents a powerful opportunity to simultaneously survey very large datasets while reducing the inherent complexity of describing metabolic regulation. We have utilized this approach in two evolutionarily divergent animal models to identify cellular adaptations to single gene defects causing primary mitochondrial dysfunction. Results in C. elegans mutants for nuclear-encoded subunits of complexes I, II, and III are highly similar to those obtained in M. musculus mutants in the Coenzyme Q biosynthetic enzyme Pdss2. Specifically, primary mitochondrial mutants across evolution concordantly upregulate 15 basic cellular metabolic pathways involving carbohydrate, amino acid, and fatty acid metabolism, as well as cellular defenses. Unique transcriptional changes were also observed, as evidenced by only the mouse mutant having upregulation of the CoQ biosynthesis pathway (likely in compensation for their CoQ deficiency); biosynthetic pathways of cholesterol, bile acids, and steroids (as substrates immediately prior to the Pdss2 enzymatic block appear to get funneled toward alternative biochemical pathways); and regulation of autophagy (supported by histologic findings of mitophagy in these animals). Expression studies in CoQ mutant mice whose lethal kidney disease can be prevented with anti-oxidants are underway to determine if normalization occurs in previously upregulated metabolic pathways and to specify which metabolic alterations may be contributing to the disease phenotype. Validation of suggested metabolic alterations was performed through quantitative amino acid profiling in both species. Importantly, a ‘signature’ of nuclear mutations affecting respiratory chain function is emerging that appears to span evolution. This work suggests that identification of a similar systems biology-based ‘biomarker’ may be possible in human patients with primary mitochondrial disease. doi:10.1016/j.mito.2008.12.030
35. Blood-feeding reduces mitochondrial function in the flight muscle of Aedes aegypti mosquito A. Galina, C.P. Figueira, M.A. Vannier-Santos, P.L. Oliveira, M.F. Oliveira, R.L.S. Goncßalves* Federal University of Rio de Janeiro, Brazil Hematophagy poses a challenge to blood-feeding organisms since products of blood digestion, such as heme and iron, exert cellular deleterious effects [1] and, to overcome their toxicity, they evolved efficient adaptive mechanisms [2]. A hypothesis raised by our group [3] suggested that hematophagous organisms would shift their energy metabolism, from aerobic to fermentative, during blood digestion, as a preventive antioxidant defense, reducing both oxygen utilization and reactive oxygen species (ROS) generation in mitochondria. Here, we tested this hypothesis by investigating mitochondrial function obtained from flight muscles (FM)
Abstracts / Mitochondrion 9 (2009) 61–77
decreasing the threshold for the induction of beta cell apoptisis and increasing susceptibility to T1D. doi:10.1016/j.mito.2008.12.024
28. Neurological phenotype is the key predictor of long-term outcome in mitochondrial DNA depletion resulting from deoxyguanosine kinase deficiency David P. Dimmock a, J. Kay Dunn b, Annette Feigenbaum c, Tony Rupar d, Rita Horvath e, Peter Freisinger f, Be´ne´dicte Mousson de Camaret g, Lee-Jun Wong a, Fernando Scaglia a,* a Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA; b School of Public Health, University of Texas Health Science Center at Houston; c Division of Genetics, Hospital for Sick Children, Toronto, Ontario, Canada; d Departments of Biochemistry and Pediatrics, University of Western Ontario, London, Ontario, Canada; e Mitochondrial Research Group, Department of Neurology, University of Newcastle, Newcastle upon Tyne, UK; f Metabolic Disease Center, Department of Pediatrics, University of Technology, Mu¨nchen, Germany; g Service des Maladies He´re´ditaires du Me´tabolisme, Centre de Biologie et de Pathologie Est, 69677 Bron, France Deoxyguanosine kinase (DGUOK) deficiency is the commonest type of mitochondrial DNA depletion associated with a hepatocerebral phenotype. The objective of this analysis was to evaluate predictors of survival and therapeutic options in patients with deoxyguanosine kinase deficiency. A systematic search of MEDLINE, LILAC & SCIELO was carried out to identify peer-reviewed clinical trials, randomized controlled trials, metaanalyses, and other studies with clinical pertinence. DGUOK deficiency was searched using the terms dGK, DGUOK, mitochondrial DNA depletion, mtDNA, and hepatocerebral. Bibliographies of identified articles were reviewed for additional references. Thirteen identified studies met inclusion criteria and were used in this study. Results of the study revealed that DGUOK deficiency is associated with a variable clinical phenotype. Long-term survival is best predicted by the presence or absence of neurological features. In the presence of neurological features, there is increased mortality and liver transplantation does not confer increased survival. Conversely, in the absence of neurological disease, liver transplantation may be considered as a potential treatment. doi:10.1016/j.mito.2008.12.025
29. Assembly of mitochondrial c-type cytochromes: Emergence of novel redox components Delphine Bernard b, Mazakazu Hirasawa c, David Knaff c, Bernard Guiard d, Sabeeha Merchant e, Patrice Hamel a, Vincent Corvest a,b,c,d,e,* a Department of Plant Cellular and Molecular Biology and Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH 43210, USA; b Institut Jacques Monod-CNRS, Universite´ Paris VI and Paris VII, Paris, France; c Department of Chemistry and Biochemistry, Center for Biotechnology and Genomics, Texas Tech University, Lubbock, TX 79409, USA; d Centre de Ge´ne´tique Mole´culaire, CNRS, 91198 Gif-surYvette, France; e Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA 90095, USA The c-type cytochromes are universal metalloproteins defined by the covalent attachment of heme. Besides their role as electron shuttle in energy-transducing systems, c-types cytochromes can also be recruited in signaling in the cell death pathways. The study of their biogenesis is crucial since it is well documented that many mitochondrial diseases are directly related to defects in respiratory chain or apoptosis. The covalent binding of the heme can occur via three pathways (systems I, II, III). With only two factors, CCHL and CC1HL (cytochrome c and c1 heme lyases), system III, which operates in animal and fungal mitochondria, appears deceptively simple in contrast to the complexity
of systems I and II. This strongly suggests that additional players are implicated in mitochondrial c-type cytochromes assembly. Recently, a new component Cyc2p has been discovered. In vivo studies suggest that this mitochondrial flavoprotein could be involved in the redox chemistry of c-type cytochromes biogenesis by controlling the reactivity of the cysteines at the heme binding site of apocytochrome c1. In order to test our model, we undertook a biochemical approach by measuring the midpoint redox potential of the different partners (Cyc2p, apocytochromes c1) and demonstrating the direct reduction of apocytochromes c1 by Cyc2p. The absence of structural homolog of Cyc2p in animal mitochondria (including humans) is unexpected as it is very likely that the redox function of Cyc2p is conserved in all mitochondria. In order to identify a functional homolog of Cyc2p in mammals, we chose a heterologous complementation approach and discovered two human cDNAs, LDHA and LDHB encoding the L-lactate dehydrogenase subunits M and H respectively, able to substitute for the function of Cyc2p in yeast. LDH activity in mitochondrial c-type cytochromes assembly is still obscure and we are currently attempting to better define its implication. doi:10.1016/j.mito.2008.12.026
30. Structure-based Drug Design in the Search for New Treatment of Mitochondrial Diseases Carolyn P. Ojano-Dirain a,*, Andrew Magis b, David A. Ostrov b, Peter W. Stacpoole a,c,d a Department of Medicine, Division of Endocrinology and Metabolism, University of Florida, Gainesville, FL 32610, USA; b Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 32610, USA; c Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610, USA; d General Clinical Research Center, University of Florida, Gainesville, FL 32610, USA In structure-based drug design (SBDD), the atomic structure of proteins is used to precisely examine the interactions between atoms in the protein target and the potential drug compounds. SBDD allows researchers to design a more optimal drug or identify new drug molecules. Dichloroacetate (DCA) is an investigational compound used as a therapy in patients with inborn errors of mitochondrial metabolism such as pyruvate dehydrogenase complex (PDC) deficiency. The primary action of DCA is activation of PDC by inhibiting the activity of pyruvate dehydrogenase kinase (PDK) that inhibits PDC. To search for new compounds that may be more potent than DCA, we used a structure-based high-throughput molecular docking method to identify novel drug-like small molecules with high affinity for the binding pocket of DCA in the PDK2 isozyme. We screened 140,000 compounds in a large chemical library. Fifteen of the top-scoring compounds were tested in human fibroblast cultures from three healthy subjects for their effect on PDC activity. DCA and dimethyl sulfoxide (DMSO) were used as positive and vehicle controls, respectively. PDC activity, measured by the rate of 14 CO2 formation from ½1 14 Clabeled pyruvate, was run in triplicate per cell line. Data were the mean SE of the 3 cell lines. DCA increased PDC enzymatic activity by 18629 to 28430% compared to the DMSO control (p60.05). Five of the drugs that we tested also significantly increased PDC activity compared to DMSO control (18810, 16930, 17126, 18940, and 17924%). In this study, we identified 5 compounds that appear to increase PDC activity in fibroblasts in a manner comparable to DCA. Experiments to define the utility of these drug-like small molecules as potential therapy for mitochondrial diseases are currently under way. doi:10.1016/j.mito.2008.12.027
31. Molecular Dissection of Mitochondrial Complex I Assembly In a Genetically Tractable Model System M. Rosario Barbieri a,*, Ve´ronique Larosa b, Pierre Cardol b, Claire Remacle a,b, Patrice Hamel a
Abstracts / Mitochondrion 9 (2009) 61–77 a Department of Plant Cellular and Molecular Biology and Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH 43210, USA; b Ge´ne´tique des Microorganismes, De´partement des Sciences de la Vie, Institut de Botanique, Universite´, France
Deficiencies in assembly of respiratory complexes cause severe mitochondrial diseases. NADH:ubiquinone oxidoreductase (complex I) is the largest respiratory complex in the mitochondrial inner membrane with around 45 subunits in eukaryotes, of which 14 are of prokaryotic origin (core catalytic subunits). The emaining ‘‘accessory” subunits are also generally conserved. 60% of human patients with complex I defects carry no mutations in the structural genes, suggesting that mutations in yet-to-be discovered assembly factors of complex I as important causes of disease. We are using Chlamydomonas reinhardtii, a unicellular green alga as a model system to study complex I assembly. In Chlamydomonas, 33 out the 45 human complex I subunits, as well as the complex I assembly factor CIA30 are conserved, suggesting that the assembly pathway may also be generally conserved. In contrast to human and other model systems, Chlamydomonas mutants lacking complex I are viable. Chlamydomonas can be grown either autotrophically in the light, or heterotrophically on acetate in the dark and complex I-less mutants display a characteristic slow growth in the dark phenotype. To identify complex I assembly factors, we generated an insertional mutant library and specifically screened for the mutant phenotype. Two non-allelic nuclear mutants, amc1 and amc2 (assembly of mitochondrial complex I), obtained by insertional and chemical mutagenesis, respectively, lack a functional, fully assembled complex I, but accumulate a membrane-bound subcomplex with NADH dehydrogenase activity, indicating a defect in the assembly of the membrane domain of complex I. Preliminary results including sequencing and expression level analyses show that neither the nuclear genes encoding for the membrane domain subunits nor CIA30 are affected at the molecular level in the amc2 strain. Therefore, amc2 is likely mutated in a complex I assembly gene. To identify the AMC1 and AMC2 genes, we are currently performing a map-based cloning approach. doi:10.1016/j.mito.2008.12.028
33. Therapeutic potential of pyruvate for mitochondrial diseases Masashi Tanaka*, Yutaka Nishigaki, Noriyuki Fuku, Tohru Ibi, Ko Sahashi, Yasutoshi Koga Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, Japan Rationale 1. Pyruvate can eliminate hydrogen peroxide to form acetate, carbon dioxide, and water. Pyruvate prevents programmed cell death induced by hydrogen peroxide. The antioxidant activity of pyruvate may be also beneficial for patients with mitochondrial disorders. 2. Pyruvate has been used in culturing rho-0 cell. Pyruvate is preferentially taken up by the cell via the monocarboxylate transporter in exchange for lactate. Pyruvate oxidizes NADH to NAD+ via LDH reaction, which restores the ATP production by glycolysis. 3. Pyruvate activates PDHC through inhibition of pyruvate dehydrogenase kinases. Dichloroacetate (DCA) inhibits pyruvate dehydrogenase kinase and thereby activates PDHC. Although DCA has been used in treatment for lactic acidosis, it causes peripheral neuropathy. Because pyruvate is a physiological inhibitor of PDK, it should have the same lactate-lowering effect as DCA without any toxicity. Design and Results 1. We administered 5 g of sodium pyruvate to a patient with CPEO carrying deleted mtDNA. At 30 min after the administration, the L/P ratio decreased from 25.7 to 16.3. The decreased L/P ratio suggests that the intracellular redox state was improved and that ATP production by the glycolytic pathway was restored by the administration of pyruvate. 2. Adult-onset type II citrullinemia (CTLN2) is caused by a mutation in the gene encoding citrin, mitochondrial aspartate glutamate carrier. This defect impairs the import of NADH from the cytosol into mitochondria, resulting in fatty liver and liver cirrhosis. We demonstrated that perfusion of the liver of Citrin-/- mice with sodium pyruvate improved the formation of urea from ammonia.
69
Conclusion 1. Pyruvate administration would ameliorate the bioenergetic defects of paitients with mitochondrial disorders. 2. Pyruvate would prevent severe hepatic failure that necessitates liver transplantation in patients with citrin deficiency. 3. We have started an open clinical study on the effects of sodium pyruvate on mitochondrial diseases and type II citrullinemia. doi:10.1016/j.mito.2008.12.029
34. A transcriptional signature of primary mitochondrial disease is present across evolution Marni J. Falk*, Zhe Zhang, David L. Gasser, Marc Yudkoff The Children’s Hospital of Philadelphia and University of Pennsylvania School of Medicine, USA Mitochondrial disease diagnosis is complicated by the absence of a biomarker that sufficiently divulges all cases. Significant bioinformatic advances have made it feasible to consider a novel approach to address this diagnostic challenge. Specifically, applying metabolic pathway clustering to global genome transcriptional profiling presents a powerful opportunity to simultaneously survey very large datasets while reducing the inherent complexity of describing metabolic regulation. We have utilized this approach in two evolutionarily divergent animal models to identify cellular adaptations to single gene defects causing primary mitochondrial dysfunction. Results in C. elegans mutants for nuclear-encoded subunits of complexes I, II, and III are highly similar to those obtained in M. musculus mutants in the Coenzyme Q biosynthetic enzyme Pdss2. Specifically, primary mitochondrial mutants across evolution concordantly upregulate 15 basic cellular metabolic pathways involving carbohydrate, amino acid, and fatty acid metabolism, as well as cellular defenses. Unique transcriptional changes were also observed, as evidenced by only the mouse mutant having upregulation of the CoQ biosynthesis pathway (likely in compensation for their CoQ deficiency); biosynthetic pathways of cholesterol, bile acids, and steroids (as substrates immediately prior to the Pdss2 enzymatic block appear to get funneled toward alternative biochemical pathways); and regulation of autophagy (supported by histologic findings of mitophagy in these animals). Expression studies in CoQ mutant mice whose lethal kidney disease can be prevented with anti-oxidants are underway to determine if normalization occurs in previously upregulated metabolic pathways and to specify which metabolic alterations may be contributing to the disease phenotype. Validation of suggested metabolic alterations was performed through quantitative amino acid profiling in both species. Importantly, a ‘signature’ of nuclear mutations affecting respiratory chain function is emerging that appears to span evolution. This work suggests that identification of a similar systems biology-based ‘biomarker’ may be possible in human patients with primary mitochondrial disease. doi:10.1016/j.mito.2008.12.030
35. Blood-feeding reduces mitochondrial function in the flight muscle of Aedes aegypti mosquito A. Galina, C.P. Figueira, M.A. Vannier-Santos, P.L. Oliveira, M.F. Oliveira, R.L.S. Goncßalves* Federal University of Rio de Janeiro, Brazil Hematophagy poses a challenge to blood-feeding organisms since products of blood digestion, such as heme and iron, exert cellular deleterious effects [1] and, to overcome their toxicity, they evolved efficient adaptive mechanisms [2]. A hypothesis raised by our group [3] suggested that hematophagous organisms would shift their energy metabolism, from aerobic to fermentative, during blood digestion, as a preventive antioxidant defense, reducing both oxygen utilization and reactive oxygen species (ROS) generation in mitochondria. Here, we tested this hypothesis by investigating mitochondrial function obtained from flight muscles (FM)