- Email: [email protected]
Motor neuron degeneration is linked with defects in mitochondrial dynamics in amyotrophic lateral sclerosis
648
Abstracts
Drosophila, recent studies have suggested that PINK1 plays a positive role in mitochondrial fission, thereby protecting against mitochondrial and cell death. However, preliminary analyses using live imaging of mitochondria in neurons do not show altered fusion and fission rates. Instead, our data indicates specific defects in the mitochondrial membrane potential and in the electron transport chain of pink1 deficient animals, providing an alternative explanation for the mitochondrial defects observed in the mutants and also providing an explanation for the reduced ATP levels in pink1 mutant flies. To further elucidate the function of Pink1 we performed a suppressor/ enhancer screen using EMS mutants. These EMS mutants have been previously created and selected for their defect in neuronal communication. This screen aims to identify multiple genes that are involved in the Pink1-Parkinson pathway and that might also affect other Parkinson related genes. For a fast and efficient screen we decided to test the flight capacity of pink1 mutant animals that harbor one mutant copy of the EMS induced mutants (dominant suppression or enhancement). We screened 200 mutants on the second chromosome that affect neuronal communication and identified 23 dominant suppressors and 13 dominant enhancers. Performing complementation tests among the suppressing and enhancing EMS mutants allowed us to identify 4 different complementation groups with more than one allele. Preliminary tests indicate that the dominant suppressors not only suppress the flight inability of pink1 but also the defects in mitochondrial membrane potential seen in pink1. Hence, we believe these suppressors will yield novel insight into the mitochondrial defects observed in pink1 mutants. doi:10.1016/j.mito.2011.03.041
29 Drosophila transgenic RNAi screen of genes implicated in human neurological disease Dominik M. Haddad a,b,⁎, Melissa Vos a,b, Vanessa A. Morais a,b, Maarten Leyssen a,b, Joke Allemeersch c, Bart De Strooper a,b, Patrik Verstreken a,b a VIB, Department of Molecular and Developmental Genetics, Herestraat 49 bus 602, 3000 Leuven, Belgium b K.U.Leuven, Center for Human Genetics, Program in Molecular and Developmental Genetics, Program in Cognitive and Molecular Neuroscience, Herestraat 49 bus 602, 3000 Leuven, Belgium c VIB, MicroArray Facility, Herestraat 49 bus 602, 3000 Leuven, Belgium Numerous genes have been implicated in neurological disease. However, whether these genes have a common outcome on cellular processes in the nervous system and affect synaptic function remains elusive. Large scale genetic and biochemical screens continue to isolate novel components that mediate neurotransmission, nevertheless, many additional proteins remain to be characterized. To identify novel genes that affect synaptic and neuronal functions we performed a nervous system-specific RNA interference screen in Drosophila targeting genes implicated in neurological disease. Using database analyses we have identified the Drosophila orthologues of the 162 evolutionary best conserved neurological disease genes. We tested these animals using behavioral assays, such as lethality, life span, stress sensitivity and flight ability. While nervous system specific RNA interference results in lethality of 6 of the 162 targeted genes, 44 genes shows consistent behavioral defects. To reveal functional mechanisms by which these 50 targeted genes affect the synapse, we tested their involvement in neuronal and synaptic development and function. For this, morphological analyses of mutant synapses and live imaging of synaptic vesicle trafficking were performed. The majority of the 50 targeted genes show clear deficits in synaptic development and function, and moreover, 18 of these targeted genes have not been implicated in these processes before. Interestingly the phenotypes we observe are remarkably reminiscent to
those observed in other mutants known to affect mitochondria. Indeed, mutants that affect mitochondrial function also affect specific aspects of vesicle mobilization and neurotransmission, revealing the most ATPsensitive processes. Thus, we decided to analyze mitochondrial function upon neuronal knock-down of these 50 targeted genes. Interestingly, using quantitative measurements of mitochondrial membrane potential and biochemical assays to assess electron transport chain integrity, we were able to correlate specific aspects of synaptic dysfunction in several of these mutants to mitochondrial abnormalities. Defects in synaptic vesicle traffic during intense stimulation correlate very well with an inability of mitochondria to maintain a negative potential across their inner membrane. Comparison of the synaptic and mitochondrial phenotypes using hierarchical clustering identified several groups of related genes and suggests overlapping cellular effects of the diseases they are implicated in. While only few of the genes we analyzed here have been previously linked to mitochondria, our data strongly suggest that mitochondria take a central stage in the etiology of various human neurological diseases. doi:10.1016/j.mito.2011.03.042
30 The role of cardiolipin in molecular assemblies of tafazzin Ashim Malhotra a,⁎, Devrim Acehan c, Mindong Ren b, Michael Schlame a,b a Department of Anesthesiology, New York University School of Medicine, 550 First, Avenue, New York, NY 10016, United States b Department of Cell Biology, New York University School of Medicine, 550 First, Avenue, New York, NY 10016, United States c The Skirball Institute for Biomolecular Medicine, New York University School of Medicine, 550 First, Avenue, New York, NY 10016, United States Tafazzin is a mitochondrial transacylase that shuttles acyl groups between phospholipids and regulates the remodeling of cardiolipin, an inner mitochondrial membrane phospholipid. Apart from its biochemical significance, tafazzin mutations have been associated with Barth Syndrome, which is an X-linked, infantile cardiomyopathy characterized by deficiency of cardiolipin and mitochondrial abberations. We have found that though Drosophila tafazzin is about 55 kDa in weight, on Blue Native gels it appears to aggregate in huge (>400 kDa) molecular aggregates. This is also true of human tafazzin transcripts expressed in 293 cells in culture. Two dimensional Blue Native page followed by Western blotting for tafazzin of excised gel slices on SDS-PAGE confirms the presence of tafazzin in these molecular assemblies. Recombination purified tafazzin also exhibits aggregation in BN PAGE systems. Tomographic re-constructions of Electron micrographs of Wild-type and cardiolipin synthase lacking Drosophila mitochondria provide evidence of the need for cardiolipin for three dimensional packing of ATP synthase. Based on these data we hypothesize that cardiolipin may be involved in the packing of tafazzin in areas of negative mitochondrial curvatures. We provide the first such evidence. doi:10.1016/j.mito.2011.03.043
31 Motor neuron degeneration is linked with defects in mitochondrial dynamics in amyotrophic lateral sclerosis Wenjun Song⁎, Viviana de Assis, Sarah Lubitz, Ella Bossy-Wetzel Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, United States Mitochondria play an essential role in neuronal function. Their dynamic fission, fusion, and transport allow equal energy distribution, mixing of metabolites, effective Ca 2+ buffering, functional bioenergetics, and the ability to meet high energy demands in the nerve terminal. It is clear that mitochondrial dysfunction participates
Abstracts
in amyotrophic lateral sclerosis (ALS), which is a neurodegenerative disease characterized by motor neuron loss. Mitochondrial dysfunction is an early event and contributes to disease progression; however, the mechanism remains unclear. Although recent studies indicate impaired mitochondrial axonal transport and defects in mitochondrial fission and fusion machinery, the potential implications in the primary motor neuron have never been explored. To address this question, we co-cultured primary motor neurons with astrocytes and investigated the characteristics of the cultures using live cell imaging. We find that mitochondria become smaller in the mSOD1 G93A motor neurons co-cultured with the wide-type (wt) astrocytes, but mitochondrial fragmentation does not occur. In addition, both anterograde and retrograde axonal transports are defective in the mSOD1 G93A motor neurons. Moreover, the aggregation of smaller mitochondria in the nerve terminal correlates with decreased axonal sprouting. Finally, mitochondria in the mSOD1 G93A motor neurons are more sensitive to the glutamate-induced excitotoxicity, undergoing rapid mitochondrial fragmentation and cell death. These data suggest that motor neuron degeneration in ALS is linked with a unique set of defects in mitochondrial dynamics. Supported by: R01 NS047456, R01 EY016164, R01 NS055193. doi:10.1016/j.mito.2011.03.044
32 Mutant Huntington interaction with DRP1 impairs the mitochondrial fission and fusion balance and mediates neuronal injury Alejandra M. Petrilli a,⁎, Wenjun Song a, Blaise Bossy a, Geraldine Liot a, Sarah Lubitz a, Viviana de Assis a, John Johnson a, Patrick Poquiz b, Jonathan Tjong b, Mahmoud Pouladi c, Michael R. Hayden c, Eliezer Masliah d, Mark Ellisman b, Isabelle Rouiller e, Guy Perkins b, Ella Bossy-Wetzel a a Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, United States b NCMIR, University of California, San Diego, CA, United States c CMMT, University of British Columbia, BC, Canada d University of California, San Diego, La Jolla, CA, United States e McGill University, Montreal, QC, Canada Huntington's disease (HD) is a progressive and lethal hereditary neurodegenerative disorder caused by an expansion of CAG repeats in the huntingtin gene that codes for an abnormally elongated polyglutamine (polyQ) stretch in the huntingtin protein (Htt). It is becoming increasingly evident that mitochondrial dysfunction plays an early and central role in HD pathogenesis; however, the precise cause of this decline in mitochondrial function remains unclear. We therefore investigated whether mutant huntingtin (mtHtt) alters the mitochondrial fission/fusion balance and thus mediates cell death. We found that mtHtt per se triggers poly-Q length dependent mitochondrial fragmentation in primary neuronal cultures. Furthermore, EM tomography shows evidence of mitochondrial and cristae fragmentation in medium spiny neurons of mtHtt transgenic mice in vivo. This event is accompanied by an arrest in retrograde and anterograde transports of mitochondria, dendritic spine loss, and neuronal cell death. Interestingly, mtHtt abnormally interacts with the mitochondrial fission factor dynamin-related protein 1 (DRP1) in mtHtt transgenic mice and HD patient samples in vivo. In contrast, mtHtt interaction with the mitochondrial fusion factor Mitofusin 2 (MFN2) is the same as wild type Htt. In addition, recombinant mtHtt modifies the DRP1 ring structure in vitro and accelerates DRP1 GTPase activity. Finally, expression of MFN2 plus DRP1K38A restores mitochondrial trafficking and dendritic spine formation and increases survival in neurons expressing mtHtt. In summary, our findings might provide targets for new therapies to combat neurodegeneration in HD.
649
Supported by NIH grants: RO1 NS047456, RO1 EY016164, RO1 NS055193 (to EBW); P41RR04050, RO1 NS14718 (to MHE). doi:10.1016/j.mito.2011.03.045
33 Repair of H2O2-induced mtDNA damage requires the BER enzyme Apn1 Yaria Arroyo-Torres a,⁎, Karina Acevedo-Torres b, Sharon Fonseca-Williams a, Sylvette Ayala-Torres c, Carlos A. Torres-Ramos a a Department of Physiology and Biophysics, University of Puerto Rico Medical, Sciences Campus, San Juan, 00935-5067, Puerto Rico b Department of Biochemistry, San Juan Bautista School of Medicine, Caguas, 00726-4968, Puerto Rico c Department of Pharmacology and Toxicology, University of Puerto Rico Medical Sciences Campus, San Juan, 00936-5067, Puerto Rico Mitochondria are a prominent source of reactive oxygen species (ROS), which can damage macromolecules such as nucleic acids. Unrepaired DNA lesions can result in mutations. Mutations in the mitochondrial genome (mtDNA) have been associated to several neurodegenerative diseases, cancer, and aging. Therefore, understanding how cells repair mtDNA is of great relevance to human health. Base excision repair (BER) is the main repair mechanism responsible for the removal of oxidative damaged DNA in mitochondria. Recently, we showed that in the model organism Saccharomyces cerevisiae the BER abasic site endonuclease Apn1 is required for the repair of mtDNA damage induced by the alkylating agent methyl methanesulfonate (Environ Mol Mutagen 2009, 50:317). We have extended this study by determining the role of Apn1 in mtDNA repair of damage induced by oxidative damage. We applied a quantitative PCR (QPCR) assay that can detect a variety of DNA lesions such as modified bases, strand breaks, and abasic sites. In this assay, increased DNA lesions in a target mtDNA sequence results in decreased PCR product. Treatment of wild type (WT) yeast cells with increasing H2O2 doses (0.1, 0.2, 0.3, 0.4, and 0.5 mM) results in a dose-dependent decrease in the mtDNA amplicon (0.07, 19.2, 20.8, 20.5 and 53.1 percent inhibition, respectively). Deletion of the APN1 gene (apn1Δ cells) results in a greater inhibition of the mtDNA amplicon (4.7, 9.6, 61, 95, and 97 percent, inhibition, respectively). These results suggest that Apn1 plays a prominent role in the repair of mtDNA lesions induced by oxidative agents. We also performed experiments in cells grown in media containing the non-fermentable carbon source glycerol (as opposed to glucose) in order to induce growth conditions that require oxidative phosphorylation. Our results show that the effect of the H2O2 treatment in cells grown in glycerol is even greater than in cells grown in glucose. Specifically, the lesion number between the apn1Δ and WT yeast strains treated with 0.1, 0.2, and 0.3 mM H2O2 was a 2 to 31 fold higher in cells grown in conditions favoring oxidative phosphorylation. Taken together these results suggest that repair of mtDNA damage induced by mitochondrial generated ROS requires the BER enzyme Apn1. Sponsored by 2R25GM061838-09, SC3GM084759-02, G12RR03051, and U54NS43011. doi:10.1016/j.mito.2011.03.046
34 Fibroblast immuno-diagnosis of cytochrome oxidase (COX) deficiency in mitochondrial disease AiLian Du a,b, Thuy Le b, CongFeng Xu c, Robert Naviaux b, Richard Haas d,⁎ a Department of Neurology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, PR China b The Mitochondrial and Metabolic Disease Center, University of California San Diego, CA 92103–8467, USA
Abstracts
Drosophila, recent studies have suggested that PINK1 plays a positive role in mitochondrial fission, thereby protecting against mitochondrial and cell death. However, preliminary analyses using live imaging of mitochondria in neurons do not show altered fusion and fission rates. Instead, our data indicates specific defects in the mitochondrial membrane potential and in the electron transport chain of pink1 deficient animals, providing an alternative explanation for the mitochondrial defects observed in the mutants and also providing an explanation for the reduced ATP levels in pink1 mutant flies. To further elucidate the function of Pink1 we performed a suppressor/ enhancer screen using EMS mutants. These EMS mutants have been previously created and selected for their defect in neuronal communication. This screen aims to identify multiple genes that are involved in the Pink1-Parkinson pathway and that might also affect other Parkinson related genes. For a fast and efficient screen we decided to test the flight capacity of pink1 mutant animals that harbor one mutant copy of the EMS induced mutants (dominant suppression or enhancement). We screened 200 mutants on the second chromosome that affect neuronal communication and identified 23 dominant suppressors and 13 dominant enhancers. Performing complementation tests among the suppressing and enhancing EMS mutants allowed us to identify 4 different complementation groups with more than one allele. Preliminary tests indicate that the dominant suppressors not only suppress the flight inability of pink1 but also the defects in mitochondrial membrane potential seen in pink1. Hence, we believe these suppressors will yield novel insight into the mitochondrial defects observed in pink1 mutants. doi:10.1016/j.mito.2011.03.041
29 Drosophila transgenic RNAi screen of genes implicated in human neurological disease Dominik M. Haddad a,b,⁎, Melissa Vos a,b, Vanessa A. Morais a,b, Maarten Leyssen a,b, Joke Allemeersch c, Bart De Strooper a,b, Patrik Verstreken a,b a VIB, Department of Molecular and Developmental Genetics, Herestraat 49 bus 602, 3000 Leuven, Belgium b K.U.Leuven, Center for Human Genetics, Program in Molecular and Developmental Genetics, Program in Cognitive and Molecular Neuroscience, Herestraat 49 bus 602, 3000 Leuven, Belgium c VIB, MicroArray Facility, Herestraat 49 bus 602, 3000 Leuven, Belgium Numerous genes have been implicated in neurological disease. However, whether these genes have a common outcome on cellular processes in the nervous system and affect synaptic function remains elusive. Large scale genetic and biochemical screens continue to isolate novel components that mediate neurotransmission, nevertheless, many additional proteins remain to be characterized. To identify novel genes that affect synaptic and neuronal functions we performed a nervous system-specific RNA interference screen in Drosophila targeting genes implicated in neurological disease. Using database analyses we have identified the Drosophila orthologues of the 162 evolutionary best conserved neurological disease genes. We tested these animals using behavioral assays, such as lethality, life span, stress sensitivity and flight ability. While nervous system specific RNA interference results in lethality of 6 of the 162 targeted genes, 44 genes shows consistent behavioral defects. To reveal functional mechanisms by which these 50 targeted genes affect the synapse, we tested their involvement in neuronal and synaptic development and function. For this, morphological analyses of mutant synapses and live imaging of synaptic vesicle trafficking were performed. The majority of the 50 targeted genes show clear deficits in synaptic development and function, and moreover, 18 of these targeted genes have not been implicated in these processes before. Interestingly the phenotypes we observe are remarkably reminiscent to
those observed in other mutants known to affect mitochondria. Indeed, mutants that affect mitochondrial function also affect specific aspects of vesicle mobilization and neurotransmission, revealing the most ATPsensitive processes. Thus, we decided to analyze mitochondrial function upon neuronal knock-down of these 50 targeted genes. Interestingly, using quantitative measurements of mitochondrial membrane potential and biochemical assays to assess electron transport chain integrity, we were able to correlate specific aspects of synaptic dysfunction in several of these mutants to mitochondrial abnormalities. Defects in synaptic vesicle traffic during intense stimulation correlate very well with an inability of mitochondria to maintain a negative potential across their inner membrane. Comparison of the synaptic and mitochondrial phenotypes using hierarchical clustering identified several groups of related genes and suggests overlapping cellular effects of the diseases they are implicated in. While only few of the genes we analyzed here have been previously linked to mitochondria, our data strongly suggest that mitochondria take a central stage in the etiology of various human neurological diseases. doi:10.1016/j.mito.2011.03.042
30 The role of cardiolipin in molecular assemblies of tafazzin Ashim Malhotra a,⁎, Devrim Acehan c, Mindong Ren b, Michael Schlame a,b a Department of Anesthesiology, New York University School of Medicine, 550 First, Avenue, New York, NY 10016, United States b Department of Cell Biology, New York University School of Medicine, 550 First, Avenue, New York, NY 10016, United States c The Skirball Institute for Biomolecular Medicine, New York University School of Medicine, 550 First, Avenue, New York, NY 10016, United States Tafazzin is a mitochondrial transacylase that shuttles acyl groups between phospholipids and regulates the remodeling of cardiolipin, an inner mitochondrial membrane phospholipid. Apart from its biochemical significance, tafazzin mutations have been associated with Barth Syndrome, which is an X-linked, infantile cardiomyopathy characterized by deficiency of cardiolipin and mitochondrial abberations. We have found that though Drosophila tafazzin is about 55 kDa in weight, on Blue Native gels it appears to aggregate in huge (>400 kDa) molecular aggregates. This is also true of human tafazzin transcripts expressed in 293 cells in culture. Two dimensional Blue Native page followed by Western blotting for tafazzin of excised gel slices on SDS-PAGE confirms the presence of tafazzin in these molecular assemblies. Recombination purified tafazzin also exhibits aggregation in BN PAGE systems. Tomographic re-constructions of Electron micrographs of Wild-type and cardiolipin synthase lacking Drosophila mitochondria provide evidence of the need for cardiolipin for three dimensional packing of ATP synthase. Based on these data we hypothesize that cardiolipin may be involved in the packing of tafazzin in areas of negative mitochondrial curvatures. We provide the first such evidence. doi:10.1016/j.mito.2011.03.043
31 Motor neuron degeneration is linked with defects in mitochondrial dynamics in amyotrophic lateral sclerosis Wenjun Song⁎, Viviana de Assis, Sarah Lubitz, Ella Bossy-Wetzel Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, United States Mitochondria play an essential role in neuronal function. Their dynamic fission, fusion, and transport allow equal energy distribution, mixing of metabolites, effective Ca 2+ buffering, functional bioenergetics, and the ability to meet high energy demands in the nerve terminal. It is clear that mitochondrial dysfunction participates
Abstracts
in amyotrophic lateral sclerosis (ALS), which is a neurodegenerative disease characterized by motor neuron loss. Mitochondrial dysfunction is an early event and contributes to disease progression; however, the mechanism remains unclear. Although recent studies indicate impaired mitochondrial axonal transport and defects in mitochondrial fission and fusion machinery, the potential implications in the primary motor neuron have never been explored. To address this question, we co-cultured primary motor neurons with astrocytes and investigated the characteristics of the cultures using live cell imaging. We find that mitochondria become smaller in the mSOD1 G93A motor neurons co-cultured with the wide-type (wt) astrocytes, but mitochondrial fragmentation does not occur. In addition, both anterograde and retrograde axonal transports are defective in the mSOD1 G93A motor neurons. Moreover, the aggregation of smaller mitochondria in the nerve terminal correlates with decreased axonal sprouting. Finally, mitochondria in the mSOD1 G93A motor neurons are more sensitive to the glutamate-induced excitotoxicity, undergoing rapid mitochondrial fragmentation and cell death. These data suggest that motor neuron degeneration in ALS is linked with a unique set of defects in mitochondrial dynamics. Supported by: R01 NS047456, R01 EY016164, R01 NS055193. doi:10.1016/j.mito.2011.03.044
32 Mutant Huntington interaction with DRP1 impairs the mitochondrial fission and fusion balance and mediates neuronal injury Alejandra M. Petrilli a,⁎, Wenjun Song a, Blaise Bossy a, Geraldine Liot a, Sarah Lubitz a, Viviana de Assis a, John Johnson a, Patrick Poquiz b, Jonathan Tjong b, Mahmoud Pouladi c, Michael R. Hayden c, Eliezer Masliah d, Mark Ellisman b, Isabelle Rouiller e, Guy Perkins b, Ella Bossy-Wetzel a a Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, United States b NCMIR, University of California, San Diego, CA, United States c CMMT, University of British Columbia, BC, Canada d University of California, San Diego, La Jolla, CA, United States e McGill University, Montreal, QC, Canada Huntington's disease (HD) is a progressive and lethal hereditary neurodegenerative disorder caused by an expansion of CAG repeats in the huntingtin gene that codes for an abnormally elongated polyglutamine (polyQ) stretch in the huntingtin protein (Htt). It is becoming increasingly evident that mitochondrial dysfunction plays an early and central role in HD pathogenesis; however, the precise cause of this decline in mitochondrial function remains unclear. We therefore investigated whether mutant huntingtin (mtHtt) alters the mitochondrial fission/fusion balance and thus mediates cell death. We found that mtHtt per se triggers poly-Q length dependent mitochondrial fragmentation in primary neuronal cultures. Furthermore, EM tomography shows evidence of mitochondrial and cristae fragmentation in medium spiny neurons of mtHtt transgenic mice in vivo. This event is accompanied by an arrest in retrograde and anterograde transports of mitochondria, dendritic spine loss, and neuronal cell death. Interestingly, mtHtt abnormally interacts with the mitochondrial fission factor dynamin-related protein 1 (DRP1) in mtHtt transgenic mice and HD patient samples in vivo. In contrast, mtHtt interaction with the mitochondrial fusion factor Mitofusin 2 (MFN2) is the same as wild type Htt. In addition, recombinant mtHtt modifies the DRP1 ring structure in vitro and accelerates DRP1 GTPase activity. Finally, expression of MFN2 plus DRP1K38A restores mitochondrial trafficking and dendritic spine formation and increases survival in neurons expressing mtHtt. In summary, our findings might provide targets for new therapies to combat neurodegeneration in HD.
649
Supported by NIH grants: RO1 NS047456, RO1 EY016164, RO1 NS055193 (to EBW); P41RR04050, RO1 NS14718 (to MHE). doi:10.1016/j.mito.2011.03.045
33 Repair of H2O2-induced mtDNA damage requires the BER enzyme Apn1 Yaria Arroyo-Torres a,⁎, Karina Acevedo-Torres b, Sharon Fonseca-Williams a, Sylvette Ayala-Torres c, Carlos A. Torres-Ramos a a Department of Physiology and Biophysics, University of Puerto Rico Medical, Sciences Campus, San Juan, 00935-5067, Puerto Rico b Department of Biochemistry, San Juan Bautista School of Medicine, Caguas, 00726-4968, Puerto Rico c Department of Pharmacology and Toxicology, University of Puerto Rico Medical Sciences Campus, San Juan, 00936-5067, Puerto Rico Mitochondria are a prominent source of reactive oxygen species (ROS), which can damage macromolecules such as nucleic acids. Unrepaired DNA lesions can result in mutations. Mutations in the mitochondrial genome (mtDNA) have been associated to several neurodegenerative diseases, cancer, and aging. Therefore, understanding how cells repair mtDNA is of great relevance to human health. Base excision repair (BER) is the main repair mechanism responsible for the removal of oxidative damaged DNA in mitochondria. Recently, we showed that in the model organism Saccharomyces cerevisiae the BER abasic site endonuclease Apn1 is required for the repair of mtDNA damage induced by the alkylating agent methyl methanesulfonate (Environ Mol Mutagen 2009, 50:317). We have extended this study by determining the role of Apn1 in mtDNA repair of damage induced by oxidative damage. We applied a quantitative PCR (QPCR) assay that can detect a variety of DNA lesions such as modified bases, strand breaks, and abasic sites. In this assay, increased DNA lesions in a target mtDNA sequence results in decreased PCR product. Treatment of wild type (WT) yeast cells with increasing H2O2 doses (0.1, 0.2, 0.3, 0.4, and 0.5 mM) results in a dose-dependent decrease in the mtDNA amplicon (0.07, 19.2, 20.8, 20.5 and 53.1 percent inhibition, respectively). Deletion of the APN1 gene (apn1Δ cells) results in a greater inhibition of the mtDNA amplicon (4.7, 9.6, 61, 95, and 97 percent, inhibition, respectively). These results suggest that Apn1 plays a prominent role in the repair of mtDNA lesions induced by oxidative agents. We also performed experiments in cells grown in media containing the non-fermentable carbon source glycerol (as opposed to glucose) in order to induce growth conditions that require oxidative phosphorylation. Our results show that the effect of the H2O2 treatment in cells grown in glycerol is even greater than in cells grown in glucose. Specifically, the lesion number between the apn1Δ and WT yeast strains treated with 0.1, 0.2, and 0.3 mM H2O2 was a 2 to 31 fold higher in cells grown in conditions favoring oxidative phosphorylation. Taken together these results suggest that repair of mtDNA damage induced by mitochondrial generated ROS requires the BER enzyme Apn1. Sponsored by 2R25GM061838-09, SC3GM084759-02, G12RR03051, and U54NS43011. doi:10.1016/j.mito.2011.03.046
34 Fibroblast immuno-diagnosis of cytochrome oxidase (COX) deficiency in mitochondrial disease AiLian Du a,b, Thuy Le b, CongFeng Xu c, Robert Naviaux b, Richard Haas d,⁎ a Department of Neurology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, PR China b The Mitochondrial and Metabolic Disease Center, University of California San Diego, CA 92103–8467, USA