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Mitochondria as guardian of nuclear genome
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Abstracts / Mitochondrion 6 (2006) 263–288
or cultured kidney cells (MDCK and OK cells). Diclofenac was found to uncouple mitochondrial respiration, when complex I (glutamate/malate) and complex II (succinate) substrates were used. The rate of ATP biosynthesis in mitochondria energized with glutamate and/or malate or succinate was also inhibited by diclofenac. However, when complex I substrates were used, ATP biosynthesis was more compromised. A dose-dependent inhibition of MMP by diclofenac was also observed. Since the phosphorylation coupled to the oxidation of glutamate and/or malate was significantly more compromised than that of succinate, the effect of diclofenac on NADH dehydrogenase, glutamate dehydrogenase and malate dehydrogenase was examined but found to be unaffected by diclofenac. The transport system for the entry of glutamate/malate into mitochondria was next examined as a possible target as the malate–aspartate shuttle is the most important transport system for NAD(P)H in kidney mitochondria. Low micromolar concentration of diclofenac inhibited this shuttle, exhibiting mixed kinetics. This observation was supported by a decrease in intra-mitochondrial NAD(P)H generated from glutamate/malate in mitochondria pre-incubated with diclofenac, In conclusion, in addition to the known uncoupling effect of diclofenac, a decrease in intra-mitochondrial reducing equivalents resulting from an inhibition of the malate–aspartate shuttle could explain the compromise in ATP biosynthesis and decrease in MMP observed in isolated kidney mitochondria and in MDCK and OK cells. doi:10.1016/j.mito.2006.08.032
Therapeutic potential of dichloroacetate for pyruvate dehydrogenase complex deficiency K.M. Berendzen a, D.W. Theriaque b, J.J. Shuster b,c, P.W. Stacpoole a,b,d,* a Department of Medicine (Division of Endocrinology and Metabolism), University of Florida College of Medicine, Gainesville, FL, USA; b General Clinical Research Center, University of Florida College of Medicine, Gainesville, FL, USA; c Department of Statistics (Division of Biostatistics), University of Florida College of Medicine, Gainesville, FL, USA; d Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, FL, USA We reviewed the use of oral dichloroacetate (DCA) in the treatment of children with congenital lactic acidosis caused by mutations in the pyruvate dehydrogenase complex (PDC). DCA stimulates PDC by inhibiting the phosphorylation of the E1a subunit and, in some cases, by stabilizing the complex and decreasing its turnover. The case histories of 46 subjects were analyzed with regard to diagnosis, clinical presentation and response to DCA. Patients ranged in age from newborn to middle age, but 32 subjects were less than 10 years old at the time DCA
administration began. Residual mean ± SD PDC activity measured in patient cells was 29.7 ± 19.2% of normal. In 31 cases in which molecular genetic studies were performed, 22 (71%) had a mutation in the E1a subunit gene. Pretreatment lactate concentrations were elevated in blood (5.15 ± 3.85 mmol/l) and cerebrospinal fluid (CSF; 6.03 ± 4.14 mmol/l) and decreased with DCA treatment (p 6 0.005). Four children with a mutation in the E1a gene have received DCA for 5–9.5 years as part of a doubleblind, controlled trial and subsequent open label, long term investigation. These patients have demonstrated stabilization or improvement in their clinical courses without clinically significant toxicity. We conclude DCA may be particularly effective in children with PDC E1a deficiency by stimulating residual enzyme activity and, consequently, cellular energy metabolism. A controlled trial is needed to determine the definitive role of DCA in the management of this devastating disease. doi:10.1016/j.mito.2006.08.033
Mitochondria as guardian of nuclear genome Keshav K. Singh Department of Cancer Genetics, Cell and Virus Building, Room 247, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA A number of human diseases are attributed to pathogenic mutations of mitochondrial DNA (mtDNA). Using the simple unicellular eukaryote Saccharomyces cerevisiae and human culture as a models we have tested the hypothesis that mitochondria function as the guardian of the nuclear genome. Using these model systems we analyzed the consequences of disrupting mitochondrial function on nuclear genome stability. Our studies suggest that mitochondrial dysfunction leads to genomic instability in the nuclear genome. Using mammalian cell culture model and cybrid cell technology, we provide evidence that (i) inactivation of mitochondrial genes leads to characteristic chromosomal instability that is found in a variety of human tumors, (ii) mitochondrial gene knockout cells show transformed phenotype, and (iii) our study also revealed that tumorigenic phenotype can be reversed by exogenous transfer of wild type mitochondria in to mtDNA knock out cells. We complemented human cell culture studies with S. cerevisiae. Our data suggest that mitochondrial dysfunction leads to increased nuclear genomic instability after inhibition of oxidative phosphorylation (OXPHOS) by various chemical inhibitors. We also observed an increase in the frequency of mutations in the nuclear genome in mitochondrial mutant strains lacking the entire mitochondrial genome (rho0) or those with deleted mtDNA (rho ). Our study revealed that in rho0 cells the DNA polymerase f (encoded by REV3 and REV7 genes) and Rev1p proteins involved in error-prone translesion DNA synthesis (TLS) control mutagenesis in the nuclear
Abstracts / Mitochondrion 6 (2006) 263–288
genome. However, these proteins were not involved in nuclear DNA mutagenesis caused by chemical inhibition of OXPHOS. Surprisingly we found that both polymerase f and Rev1p localize in the mitochondria and these proteins participate in the mtDNA mutagenesis. This is the first report demonstrating that the DNA polymerase f and Rev1 proteins function in the mitochondria. Our studies suggest that REV proteins mediate inter-genomic cross talk between mitochondria and the nucleus and that mitochondria may function as the guardian of the nuclear genome. doi:10.1016/j.mito.2006.08.034
Functional analysis of mouse mTERF.D3, a novel mitochondrial transcription termination-like factor Corneliu C. Luca *, Carlos T. Moraes University of Miami, Cell Biology and Anatomy Department and Neurology Department, USA Mitochondrial transcription termination factor (mTERF) is involved in mitochondrial DNA transcription termination at the boundary of the 16S rRNA and tRNALeu genes. mTERF is responsible for production of a 50-fold ribosomal RNA excess relative to the downstream transcripts in a mechanism involving binding to both the transcription termination and initiation sites. In this study we have analyzed a novel mTERF-like protein with homologous mTERF-like leucine zipper domains. This protein, termed mTERF.D3, is conserved in higher eukaryotes and expressed at high levels in brain, heart, liver, lung, and spleen in the adult mouse. Immunohistochemistry experiments and subcellular fractionation analysis showed that mTERF.D3 is a mitochondrial matrix protein and is associated with the inner membrane. Biochemical characterization of mTERF.D3 suggests that this protein is present as a monomer and is able to bind polyanions. Together these findings suggest that mTERF.D3 may also play a role in mitochondrial DNA transcription regulation.To further investigate the function of this protein in vivo, we have created a mouse deficient in mTERF.D3 using a gene trapping strategy. We could not detect any mTERF.D3 mRNA or protein in the knockout mice. These mice have a normal life span and no apparent phenotype or OXPHOS defects. Further studies, including stress challenges, are under way to assess the mitochondrial function in mTERF.D3 / mice. doi:10.1016/j.mito.2006.08.035
Manipulating heteroplasmy by delivering restriction endonuclease to mitochondria in a ‘‘differential multiple cleavagesite’’ model Sandra R. Bacman a,*, Sion L. Williams a, Dayami a Hernandez , Brendan J. Battersby b, Eric A. Shoubridge b, Carlos T. Moraes a
277
a
Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, USA; b Department of Human Genetics, Montreal Neurological Institute, McGill University, Montreal, Que., Canada Manipulating mtDNA heteroplasmy by importing restriction endonucleases (RE) into mitochondria has great promise as a therapeutic approach when single mutated sites are recognized by specific endonucleases. However, this requirement limits this approach to very few pathogenic mutations. Therefore, we attempted to use a RE that recognizes multiple sites in the mtDNA to alter heteroplasmy. We took advantage of a heteroplasmic mouse model that carries two genotypes of mtDNA sequence variants (NZBxBALB/c). ScaI recognizes both mtDNA haplotypes (3 sites in BALB/c and 5 sites in NZB mtDNA). Cultured hepatocytes derived from these mice expressing a mefiprestone-inducible ScaI with a mitochondrial targeting sequence and an HA tag (mito-ScaI-HA) showed a significant shift in mtDNA heteroplasmy. This shift was in the predicted direction with a relative decrease in the 5-site-genotype (NZB). We also transduced cultured hepatocytes with adenovirus (Ad5) containing the mito-ScaI-HA construct and observed both a transient mtDNA depletion and a 20% of decrease of the NZB haplotype in 2 days, which persisted for 10 days. In vivo studies were also done in the mtDNA heteroplasmic mice (NZBxBALB/c) both in liver by injecting Ad5-mito-ScaI-HA intravenously, which efficiently transduces the liver, and in skeletal muscle by direct injections of the same recombinant virus in biceps femoris. Liver biopsies at different times and laser capture microscope (LCM) samples from muscle biopsies were analyzed for protein expression, respiratory chain activity, mtDNA heteroplasmy shift and mtDNA content. We were able to observe a directional shift of mtDNA heteroplasmy in both tissues, although a transient cytochrome oxidase deficiency accompanied the process. These results suggest that if carefully controlled, the expression of mito-RE in the context of multiple cleavage-sites has the potential to modulate mtDNA heteroplasmy in mitochondrial diseases.
doi:10.1016/j.mito.2006.08.036
Regenerate to survive: Cytochrome oxidase deficiency in hepatocytes Francisca Diaz *, Sofia Garcia, Dayami Hernandez, Carlos T. Moraes Department of Neurology, University of Miami, Miller School of Medicine, USA The biosynthesis of cytochrome c oxidase (COX) depends on 13 integral subunits and additional assembly factors. One of these factors, COX10, a protoheme:heme O farnesyl transferase, is involved in the synthesis of heme a, one of the prosthetic groups of COX. COX10 is required for COX activity and assembly, and it has been found
Abstracts / Mitochondrion 6 (2006) 263–288
or cultured kidney cells (MDCK and OK cells). Diclofenac was found to uncouple mitochondrial respiration, when complex I (glutamate/malate) and complex II (succinate) substrates were used. The rate of ATP biosynthesis in mitochondria energized with glutamate and/or malate or succinate was also inhibited by diclofenac. However, when complex I substrates were used, ATP biosynthesis was more compromised. A dose-dependent inhibition of MMP by diclofenac was also observed. Since the phosphorylation coupled to the oxidation of glutamate and/or malate was significantly more compromised than that of succinate, the effect of diclofenac on NADH dehydrogenase, glutamate dehydrogenase and malate dehydrogenase was examined but found to be unaffected by diclofenac. The transport system for the entry of glutamate/malate into mitochondria was next examined as a possible target as the malate–aspartate shuttle is the most important transport system for NAD(P)H in kidney mitochondria. Low micromolar concentration of diclofenac inhibited this shuttle, exhibiting mixed kinetics. This observation was supported by a decrease in intra-mitochondrial NAD(P)H generated from glutamate/malate in mitochondria pre-incubated with diclofenac, In conclusion, in addition to the known uncoupling effect of diclofenac, a decrease in intra-mitochondrial reducing equivalents resulting from an inhibition of the malate–aspartate shuttle could explain the compromise in ATP biosynthesis and decrease in MMP observed in isolated kidney mitochondria and in MDCK and OK cells. doi:10.1016/j.mito.2006.08.032
Therapeutic potential of dichloroacetate for pyruvate dehydrogenase complex deficiency K.M. Berendzen a, D.W. Theriaque b, J.J. Shuster b,c, P.W. Stacpoole a,b,d,* a Department of Medicine (Division of Endocrinology and Metabolism), University of Florida College of Medicine, Gainesville, FL, USA; b General Clinical Research Center, University of Florida College of Medicine, Gainesville, FL, USA; c Department of Statistics (Division of Biostatistics), University of Florida College of Medicine, Gainesville, FL, USA; d Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, FL, USA We reviewed the use of oral dichloroacetate (DCA) in the treatment of children with congenital lactic acidosis caused by mutations in the pyruvate dehydrogenase complex (PDC). DCA stimulates PDC by inhibiting the phosphorylation of the E1a subunit and, in some cases, by stabilizing the complex and decreasing its turnover. The case histories of 46 subjects were analyzed with regard to diagnosis, clinical presentation and response to DCA. Patients ranged in age from newborn to middle age, but 32 subjects were less than 10 years old at the time DCA
administration began. Residual mean ± SD PDC activity measured in patient cells was 29.7 ± 19.2% of normal. In 31 cases in which molecular genetic studies were performed, 22 (71%) had a mutation in the E1a subunit gene. Pretreatment lactate concentrations were elevated in blood (5.15 ± 3.85 mmol/l) and cerebrospinal fluid (CSF; 6.03 ± 4.14 mmol/l) and decreased with DCA treatment (p 6 0.005). Four children with a mutation in the E1a gene have received DCA for 5–9.5 years as part of a doubleblind, controlled trial and subsequent open label, long term investigation. These patients have demonstrated stabilization or improvement in their clinical courses without clinically significant toxicity. We conclude DCA may be particularly effective in children with PDC E1a deficiency by stimulating residual enzyme activity and, consequently, cellular energy metabolism. A controlled trial is needed to determine the definitive role of DCA in the management of this devastating disease. doi:10.1016/j.mito.2006.08.033
Mitochondria as guardian of nuclear genome Keshav K. Singh Department of Cancer Genetics, Cell and Virus Building, Room 247, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA A number of human diseases are attributed to pathogenic mutations of mitochondrial DNA (mtDNA). Using the simple unicellular eukaryote Saccharomyces cerevisiae and human culture as a models we have tested the hypothesis that mitochondria function as the guardian of the nuclear genome. Using these model systems we analyzed the consequences of disrupting mitochondrial function on nuclear genome stability. Our studies suggest that mitochondrial dysfunction leads to genomic instability in the nuclear genome. Using mammalian cell culture model and cybrid cell technology, we provide evidence that (i) inactivation of mitochondrial genes leads to characteristic chromosomal instability that is found in a variety of human tumors, (ii) mitochondrial gene knockout cells show transformed phenotype, and (iii) our study also revealed that tumorigenic phenotype can be reversed by exogenous transfer of wild type mitochondria in to mtDNA knock out cells. We complemented human cell culture studies with S. cerevisiae. Our data suggest that mitochondrial dysfunction leads to increased nuclear genomic instability after inhibition of oxidative phosphorylation (OXPHOS) by various chemical inhibitors. We also observed an increase in the frequency of mutations in the nuclear genome in mitochondrial mutant strains lacking the entire mitochondrial genome (rho0) or those with deleted mtDNA (rho ). Our study revealed that in rho0 cells the DNA polymerase f (encoded by REV3 and REV7 genes) and Rev1p proteins involved in error-prone translesion DNA synthesis (TLS) control mutagenesis in the nuclear
Abstracts / Mitochondrion 6 (2006) 263–288
genome. However, these proteins were not involved in nuclear DNA mutagenesis caused by chemical inhibition of OXPHOS. Surprisingly we found that both polymerase f and Rev1p localize in the mitochondria and these proteins participate in the mtDNA mutagenesis. This is the first report demonstrating that the DNA polymerase f and Rev1 proteins function in the mitochondria. Our studies suggest that REV proteins mediate inter-genomic cross talk between mitochondria and the nucleus and that mitochondria may function as the guardian of the nuclear genome. doi:10.1016/j.mito.2006.08.034
Functional analysis of mouse mTERF.D3, a novel mitochondrial transcription termination-like factor Corneliu C. Luca *, Carlos T. Moraes University of Miami, Cell Biology and Anatomy Department and Neurology Department, USA Mitochondrial transcription termination factor (mTERF) is involved in mitochondrial DNA transcription termination at the boundary of the 16S rRNA and tRNALeu genes. mTERF is responsible for production of a 50-fold ribosomal RNA excess relative to the downstream transcripts in a mechanism involving binding to both the transcription termination and initiation sites. In this study we have analyzed a novel mTERF-like protein with homologous mTERF-like leucine zipper domains. This protein, termed mTERF.D3, is conserved in higher eukaryotes and expressed at high levels in brain, heart, liver, lung, and spleen in the adult mouse. Immunohistochemistry experiments and subcellular fractionation analysis showed that mTERF.D3 is a mitochondrial matrix protein and is associated with the inner membrane. Biochemical characterization of mTERF.D3 suggests that this protein is present as a monomer and is able to bind polyanions. Together these findings suggest that mTERF.D3 may also play a role in mitochondrial DNA transcription regulation.To further investigate the function of this protein in vivo, we have created a mouse deficient in mTERF.D3 using a gene trapping strategy. We could not detect any mTERF.D3 mRNA or protein in the knockout mice. These mice have a normal life span and no apparent phenotype or OXPHOS defects. Further studies, including stress challenges, are under way to assess the mitochondrial function in mTERF.D3 / mice. doi:10.1016/j.mito.2006.08.035
Manipulating heteroplasmy by delivering restriction endonuclease to mitochondria in a ‘‘differential multiple cleavagesite’’ model Sandra R. Bacman a,*, Sion L. Williams a, Dayami a Hernandez , Brendan J. Battersby b, Eric A. Shoubridge b, Carlos T. Moraes a
277
a
Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, USA; b Department of Human Genetics, Montreal Neurological Institute, McGill University, Montreal, Que., Canada Manipulating mtDNA heteroplasmy by importing restriction endonucleases (RE) into mitochondria has great promise as a therapeutic approach when single mutated sites are recognized by specific endonucleases. However, this requirement limits this approach to very few pathogenic mutations. Therefore, we attempted to use a RE that recognizes multiple sites in the mtDNA to alter heteroplasmy. We took advantage of a heteroplasmic mouse model that carries two genotypes of mtDNA sequence variants (NZBxBALB/c). ScaI recognizes both mtDNA haplotypes (3 sites in BALB/c and 5 sites in NZB mtDNA). Cultured hepatocytes derived from these mice expressing a mefiprestone-inducible ScaI with a mitochondrial targeting sequence and an HA tag (mito-ScaI-HA) showed a significant shift in mtDNA heteroplasmy. This shift was in the predicted direction with a relative decrease in the 5-site-genotype (NZB). We also transduced cultured hepatocytes with adenovirus (Ad5) containing the mito-ScaI-HA construct and observed both a transient mtDNA depletion and a 20% of decrease of the NZB haplotype in 2 days, which persisted for 10 days. In vivo studies were also done in the mtDNA heteroplasmic mice (NZBxBALB/c) both in liver by injecting Ad5-mito-ScaI-HA intravenously, which efficiently transduces the liver, and in skeletal muscle by direct injections of the same recombinant virus in biceps femoris. Liver biopsies at different times and laser capture microscope (LCM) samples from muscle biopsies were analyzed for protein expression, respiratory chain activity, mtDNA heteroplasmy shift and mtDNA content. We were able to observe a directional shift of mtDNA heteroplasmy in both tissues, although a transient cytochrome oxidase deficiency accompanied the process. These results suggest that if carefully controlled, the expression of mito-RE in the context of multiple cleavage-sites has the potential to modulate mtDNA heteroplasmy in mitochondrial diseases.
doi:10.1016/j.mito.2006.08.036
Regenerate to survive: Cytochrome oxidase deficiency in hepatocytes Francisca Diaz *, Sofia Garcia, Dayami Hernandez, Carlos T. Moraes Department of Neurology, University of Miami, Miller School of Medicine, USA The biosynthesis of cytochrome c oxidase (COX) depends on 13 integral subunits and additional assembly factors. One of these factors, COX10, a protoheme:heme O farnesyl transferase, is involved in the synthesis of heme a, one of the prosthetic groups of COX. COX10 is required for COX activity and assembly, and it has been found