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Mitochondrial DNA heteroplasmy shift in liver and retina using a virus-delivered mitochondrially targeted restriction endonuclease
286
Abstracts / Mitochondrion 6 (2006) 263–288
Methods: The medical records of several hundred patients evaluated by a single metabolic geneticist over a five-year period were reviewed for cases meeting established diagnostic criteria for CRPS. Quantitative pedigree analysis for maternal inheritance was determined by dividing the sum of neuroendocrine conditions in all first and second-degree relatives by the number of relatives, and comparing the maternal and paternal lineages. A ratio P3 is considered to be highly suggestive of maternal inheritance. Results: CRPS was present in 8 children in 7 families. Maternal inheritance of multiple functional (migraine, depression, irritable bowel, cyclic vomiting, etc.) and other neurological and endocrine conditions was present in 7/7 by quantitative pedigree analysis. Mitochondrial dysfunction was present in 7/7 by urine organic acid analysis (GC/MS) and 4/4 by enzymology (3 with low respiratory complex activity/ies). A heteroplasmic mtDNA change (all in the control region) was found in 3/5 cases. Triggers for the development of CRPS included illness and injury; some cases being idiopathic. Discussion: In some families, maternally inherited mtDNA sequence variants can predispose towards the development of CRPS, along with other functional conditions. The contribution of mtDNA sequence variation and/or mitochondrial dysfunction to the pathogenesis of CRPS in general is unknown. doi:10.1016/j.mito.2006.08.058
Mitochondrial involvement in selective vulnerability of motor neurons during glutamate stimulation Dinesh Chandra Joshi *, Indrani Sen, Nanda B. Joshi, Preeti G. Joshi National Institute of Mental Health and Neurosciences, Bangalore, India Glutamate is the major excitatory neurotransmitter in mammalian central nervous system and is involved in various brain functions such as synaptic plasticity, learning and memory etc. However, increase in extracellular glutamate is neurotoxic and has been implicated in the pathophysiology of various neurodegenerative disorders. Glutamate mediates fast excitatory neurotransmission via three types of ionotropic glutamate receptors NMDA, AMPA, and Kainate. Activation of glutamate receptor causes a massive increase in [Ca2+]i which may initiate the downstream events of cell death. During cytosolic Ca2+ overload mitochondria act as a buffering organelle and play an important role in Ca2+ homeostasis. Accumulating evidence suggests that mitochondrial Ca2+ overload plays a crucial role in glutamate-induced neurotoxicity. Present study was undertaken to understand the relationship between glutamate receptor stimulated changes in downstream events, such as rise in [Ca2+]i, mitochondrial Ca2+ overload, mitochondrial depolarization, generation
of reactive oxygen species (ROS), and the selective vulnerability of motor neurons. Glutamate induced rise in [Ca2+]i, [Ca2+]m, mitochondrial depolarization and ROS formation was significantly higher in motor neurons as compared to other spinal neurons. Among the agonists, AMPA was most potent and the order of potency was AMPA > NMDA > Kainate. Glutamate induced higher cytotoxicity to motor neurons as compared to other spinal neurons. AMPA receptor antagonist NBQX provided significant protection to both types of neurons than NMDA receptor antagonist APV. In glutamate-treated cultures motor neurons showed release of cytochrome c as evidenced by diffused cytoplasmic staining, while nontreated neurons showed punctate mitochondrial localization of cytochrome c, indicates activation of mitochondrial apoptotic pathway in motor neurons. Our results suggest that glutamate stimulation leads to enormous increase in [Ca2+]i preferentially through AMPA receptor, that impairs mitochondrial functions and activates apoptotic pathway, which may be crucial in selective vulnerability of motor neurons in spinal cord. doi:10.1016/j.mito.2006.08.059
Mitochondrial DNA heteroplasmy shift in liver and retina using a virus-delivered mitochondrially targeted restriction endonuclease S.L. Williams a,*, S.R. Bacman a, M.P. Bayona-Bafaluy b, E.A. Shoubridge c, C.T. Moraes a a Department of Neurology, Miller School of Medicine, University of Miami, Miami, FL, USA; b Department of Biochemistry and Molecular and Cellular Biology, University of Zaragoza, Zaragoza, Spain; c Department of Human Genetics, Montreal Neurological Institute, McGill University, Montreal, Que., Canada Heteroplasmy shift to reduce mtDNA mutant load provides a therapeutic strategy for the treatment of heteroplasmic mtDNA disorders. Using the NZB/BALB-c heteroplasmic mouse which expresses two wild-type mtDNA haplotypes, we recently demonstrated that viral vectors can be used to deliver restriction enzymes to mitochondria and achieve a rapid and substantial heteroplasmy shift in vivo. We have now extended the tissue coverage of this model using systemic and local injections to induce heteroplasmy shift in clinically relevant tissues. In our model ApaL1 targeted to mitochondria is used to selectively cleave BALB-c mtDNA and induce heteroplasmy shift towards an abundance of NZB mtDNA. In young animals, systemic injection of adenovirus expressing our construct induced a shift from a pre-injection range of 16–64% NZB to 70–93% NZB mtDNA post-injection in liver (n = 6). Results for older animals were similar except that age-related genetic drift meant that the initial abundance of NZB mtDNA was significantly higher (72–95% NZB pre-injection to 89–97% NZB mtDNA post-injection,
Abstracts / Mitochondrion 6 (2006) 263–288
n = 8). Southern blotting demonstrated that there was no reduction in overall mtDNA levels in either age group, and staining for COX activity was normal. Injection of control vector (cytosolic eGFP) did not induce heteroplasmy shift. Comparable results were obtained in the eye. Intravitreal injection of adeno-associated virus carrying the ApaL1 construct induced a heteroplasmy shift from 4–7% NZB to 34–57% NZB mtDNA when comparing pooled retinal ganglion cells (n = 4) dissected from injected and uninjected eyes of a single animal. Immunostaining identified significant expression of our construct in retinal pigmented epithelium and optic nerve suggesting heteroplasmy shift may also occur in these tissues. It is hoped that these data may provide a foundation for further studies into the potential of gene therapy approaches to the treatment of mtDNA disorders. doi:10.1016/j.mito.2006.08.060
Progress towards transforming mammalian mitochondria with engineered mitochondrial genomes Young Yoon *, Michael Koob Institute of Human Genetics, University of Minnesota, Minneapolis, MN, USA Technical hurdles currently prevent us from engineering accurate mouse models of human mitochondria diseases, but we now report progress in our efforts to transform the mitochondrial network in mouse cells with engineered mtDNA. We have successfully generated strains of Saccharomyces cerevisiae that contain the entire mouse mitochondrial genome in their mitochondria and have found that these mouse mtDNAs are maintained stably in these cells. Because mtDNA is naturally transferred between mitochondria through the process of mitochondrial fusion, we are developing methods to use these transmitochondrial yeast mitochondria as vectors for transferring the engineered mtDNA back into the mitochondrial networks of mouse cells. In a separate project, we are also pursuing a method for transferring exogenous DNA molecules into mammalian mitochondria using bacterial conjugation. We tested this approach by transferring plasmid DNA containing a T7 promoter sequence into isolated mitochondria that we had engineered to contain T7 RNA polymerase. After conjugation between E. coli and mitochondria, we detected robust levels of RNA transcription from the DNA constructs that had been transferred into the isolated mitochondria. We are now testing this approach using enteroinvasive E. coli, which can invade and replicate in the cytoplasm of mammalian cells, with the goal of transferring DNA molecules into mitochondria in vivo. Finally, we are developing tools for selecting cells in which mitochondria have been transformed. We have tested some of the standard antibiotic resistance genes used as selectable marker proteins by targeting them into the mitochondrial matrix rather than the cytoplasm of mammalian cells and
287
found that they can confer drug-resistance to cells from within the mitochondrial network. Based on this data, we recoded the selectable marker genes to allow them to be translated in mammalian mitochondria but not in the cytoplasm and fused them in-frame with mitochondrial genes in our mtDNA clones. doi:10.1016/j.mito.2006.08.061
The recombination protein RAD52 cooperates with the base excision repair protein OGG1 in the repair of oxidative DNA damage Nadja C. de Souza-Pinto c,*, Kazunari Hashiguchi a, Jingping Hu b, Scott Maynard c, Meltem Muftuoglu c, Vilhelm A. Bohr c a Laboratory of Molecular Gerontology, NIA-IRP, NIH, Baltimore, MD 21224, USA; b Tohoku University, Sendai, Japan; c Laboratory of Experimental Gerontology, NIAIRP, NIH, Baltimore, USA Oxidative damage to the mitochondrial DNA has been causally linked to several pathological states and to aging. 8-hydroxyguanine (8-OHdG) is a mutagenic base lesion that is formed at high levels by reactive oxygen species, which are generated in very close proximity to the sites where mitochondrial DNA lie. 8-OHdG is repaired by the base excision repair (BER) pathway. BER has long been considered a ‘‘simple’’ pathway that can be accomplished by a core set of four enzymes. Recently, this concept has been challenged by the identification of auxiliary proteins that modulate BER efficiency. The oxoguanine DNA glycosylase (OGG1) initiates BER of oxidized purines. Very few protein interactions have been identified for OGG1, and so far only 3 seem to have functional consequences. We report here that OGG1 interacts with the recombination protein RAD52 in vitro and in vivo, detected by co-immunoprecipitation from cell extracts. This interaction has profound functional consequences as OGG1 inhibits RAD52 catalytic activities and RAD52 stimulates OGG1 8-oxoG incision activity. These effects are not associated with direct interactions with the DNA substrates, since OGG1 inhibitory effect is independent on its binding to the RAD52 substrate and RAD52 does not increase OGG1 loading to its substrate. We found that RAD52 decreased the amount of OGG1-substrate complexes trapped by sodium borohydride and decreased OGG1 affinity for an abasic site containing oligonucleotide. These observations suggest that RAD52 stimulates OGG1 by increasing its turnover rate. Our results imply that RAD52 cooperates with OGG1 to increase BER efficiency and suggest a coordinated action between BER and double strand brake repair. This coordination may be particularly relevant when 8oxoG lesions positioned close to double strand breaks may impose a hindrance to RAD52 catalytic activities. doi:10.1016/j.mito.2006.08.062
Abstracts / Mitochondrion 6 (2006) 263–288
Methods: The medical records of several hundred patients evaluated by a single metabolic geneticist over a five-year period were reviewed for cases meeting established diagnostic criteria for CRPS. Quantitative pedigree analysis for maternal inheritance was determined by dividing the sum of neuroendocrine conditions in all first and second-degree relatives by the number of relatives, and comparing the maternal and paternal lineages. A ratio P3 is considered to be highly suggestive of maternal inheritance. Results: CRPS was present in 8 children in 7 families. Maternal inheritance of multiple functional (migraine, depression, irritable bowel, cyclic vomiting, etc.) and other neurological and endocrine conditions was present in 7/7 by quantitative pedigree analysis. Mitochondrial dysfunction was present in 7/7 by urine organic acid analysis (GC/MS) and 4/4 by enzymology (3 with low respiratory complex activity/ies). A heteroplasmic mtDNA change (all in the control region) was found in 3/5 cases. Triggers for the development of CRPS included illness and injury; some cases being idiopathic. Discussion: In some families, maternally inherited mtDNA sequence variants can predispose towards the development of CRPS, along with other functional conditions. The contribution of mtDNA sequence variation and/or mitochondrial dysfunction to the pathogenesis of CRPS in general is unknown. doi:10.1016/j.mito.2006.08.058
Mitochondrial involvement in selective vulnerability of motor neurons during glutamate stimulation Dinesh Chandra Joshi *, Indrani Sen, Nanda B. Joshi, Preeti G. Joshi National Institute of Mental Health and Neurosciences, Bangalore, India Glutamate is the major excitatory neurotransmitter in mammalian central nervous system and is involved in various brain functions such as synaptic plasticity, learning and memory etc. However, increase in extracellular glutamate is neurotoxic and has been implicated in the pathophysiology of various neurodegenerative disorders. Glutamate mediates fast excitatory neurotransmission via three types of ionotropic glutamate receptors NMDA, AMPA, and Kainate. Activation of glutamate receptor causes a massive increase in [Ca2+]i which may initiate the downstream events of cell death. During cytosolic Ca2+ overload mitochondria act as a buffering organelle and play an important role in Ca2+ homeostasis. Accumulating evidence suggests that mitochondrial Ca2+ overload plays a crucial role in glutamate-induced neurotoxicity. Present study was undertaken to understand the relationship between glutamate receptor stimulated changes in downstream events, such as rise in [Ca2+]i, mitochondrial Ca2+ overload, mitochondrial depolarization, generation
of reactive oxygen species (ROS), and the selective vulnerability of motor neurons. Glutamate induced rise in [Ca2+]i, [Ca2+]m, mitochondrial depolarization and ROS formation was significantly higher in motor neurons as compared to other spinal neurons. Among the agonists, AMPA was most potent and the order of potency was AMPA > NMDA > Kainate. Glutamate induced higher cytotoxicity to motor neurons as compared to other spinal neurons. AMPA receptor antagonist NBQX provided significant protection to both types of neurons than NMDA receptor antagonist APV. In glutamate-treated cultures motor neurons showed release of cytochrome c as evidenced by diffused cytoplasmic staining, while nontreated neurons showed punctate mitochondrial localization of cytochrome c, indicates activation of mitochondrial apoptotic pathway in motor neurons. Our results suggest that glutamate stimulation leads to enormous increase in [Ca2+]i preferentially through AMPA receptor, that impairs mitochondrial functions and activates apoptotic pathway, which may be crucial in selective vulnerability of motor neurons in spinal cord. doi:10.1016/j.mito.2006.08.059
Mitochondrial DNA heteroplasmy shift in liver and retina using a virus-delivered mitochondrially targeted restriction endonuclease S.L. Williams a,*, S.R. Bacman a, M.P. Bayona-Bafaluy b, E.A. Shoubridge c, C.T. Moraes a a Department of Neurology, Miller School of Medicine, University of Miami, Miami, FL, USA; b Department of Biochemistry and Molecular and Cellular Biology, University of Zaragoza, Zaragoza, Spain; c Department of Human Genetics, Montreal Neurological Institute, McGill University, Montreal, Que., Canada Heteroplasmy shift to reduce mtDNA mutant load provides a therapeutic strategy for the treatment of heteroplasmic mtDNA disorders. Using the NZB/BALB-c heteroplasmic mouse which expresses two wild-type mtDNA haplotypes, we recently demonstrated that viral vectors can be used to deliver restriction enzymes to mitochondria and achieve a rapid and substantial heteroplasmy shift in vivo. We have now extended the tissue coverage of this model using systemic and local injections to induce heteroplasmy shift in clinically relevant tissues. In our model ApaL1 targeted to mitochondria is used to selectively cleave BALB-c mtDNA and induce heteroplasmy shift towards an abundance of NZB mtDNA. In young animals, systemic injection of adenovirus expressing our construct induced a shift from a pre-injection range of 16–64% NZB to 70–93% NZB mtDNA post-injection in liver (n = 6). Results for older animals were similar except that age-related genetic drift meant that the initial abundance of NZB mtDNA was significantly higher (72–95% NZB pre-injection to 89–97% NZB mtDNA post-injection,
Abstracts / Mitochondrion 6 (2006) 263–288
n = 8). Southern blotting demonstrated that there was no reduction in overall mtDNA levels in either age group, and staining for COX activity was normal. Injection of control vector (cytosolic eGFP) did not induce heteroplasmy shift. Comparable results were obtained in the eye. Intravitreal injection of adeno-associated virus carrying the ApaL1 construct induced a heteroplasmy shift from 4–7% NZB to 34–57% NZB mtDNA when comparing pooled retinal ganglion cells (n = 4) dissected from injected and uninjected eyes of a single animal. Immunostaining identified significant expression of our construct in retinal pigmented epithelium and optic nerve suggesting heteroplasmy shift may also occur in these tissues. It is hoped that these data may provide a foundation for further studies into the potential of gene therapy approaches to the treatment of mtDNA disorders. doi:10.1016/j.mito.2006.08.060
Progress towards transforming mammalian mitochondria with engineered mitochondrial genomes Young Yoon *, Michael Koob Institute of Human Genetics, University of Minnesota, Minneapolis, MN, USA Technical hurdles currently prevent us from engineering accurate mouse models of human mitochondria diseases, but we now report progress in our efforts to transform the mitochondrial network in mouse cells with engineered mtDNA. We have successfully generated strains of Saccharomyces cerevisiae that contain the entire mouse mitochondrial genome in their mitochondria and have found that these mouse mtDNAs are maintained stably in these cells. Because mtDNA is naturally transferred between mitochondria through the process of mitochondrial fusion, we are developing methods to use these transmitochondrial yeast mitochondria as vectors for transferring the engineered mtDNA back into the mitochondrial networks of mouse cells. In a separate project, we are also pursuing a method for transferring exogenous DNA molecules into mammalian mitochondria using bacterial conjugation. We tested this approach by transferring plasmid DNA containing a T7 promoter sequence into isolated mitochondria that we had engineered to contain T7 RNA polymerase. After conjugation between E. coli and mitochondria, we detected robust levels of RNA transcription from the DNA constructs that had been transferred into the isolated mitochondria. We are now testing this approach using enteroinvasive E. coli, which can invade and replicate in the cytoplasm of mammalian cells, with the goal of transferring DNA molecules into mitochondria in vivo. Finally, we are developing tools for selecting cells in which mitochondria have been transformed. We have tested some of the standard antibiotic resistance genes used as selectable marker proteins by targeting them into the mitochondrial matrix rather than the cytoplasm of mammalian cells and
287
found that they can confer drug-resistance to cells from within the mitochondrial network. Based on this data, we recoded the selectable marker genes to allow them to be translated in mammalian mitochondria but not in the cytoplasm and fused them in-frame with mitochondrial genes in our mtDNA clones. doi:10.1016/j.mito.2006.08.061
The recombination protein RAD52 cooperates with the base excision repair protein OGG1 in the repair of oxidative DNA damage Nadja C. de Souza-Pinto c,*, Kazunari Hashiguchi a, Jingping Hu b, Scott Maynard c, Meltem Muftuoglu c, Vilhelm A. Bohr c a Laboratory of Molecular Gerontology, NIA-IRP, NIH, Baltimore, MD 21224, USA; b Tohoku University, Sendai, Japan; c Laboratory of Experimental Gerontology, NIAIRP, NIH, Baltimore, USA Oxidative damage to the mitochondrial DNA has been causally linked to several pathological states and to aging. 8-hydroxyguanine (8-OHdG) is a mutagenic base lesion that is formed at high levels by reactive oxygen species, which are generated in very close proximity to the sites where mitochondrial DNA lie. 8-OHdG is repaired by the base excision repair (BER) pathway. BER has long been considered a ‘‘simple’’ pathway that can be accomplished by a core set of four enzymes. Recently, this concept has been challenged by the identification of auxiliary proteins that modulate BER efficiency. The oxoguanine DNA glycosylase (OGG1) initiates BER of oxidized purines. Very few protein interactions have been identified for OGG1, and so far only 3 seem to have functional consequences. We report here that OGG1 interacts with the recombination protein RAD52 in vitro and in vivo, detected by co-immunoprecipitation from cell extracts. This interaction has profound functional consequences as OGG1 inhibits RAD52 catalytic activities and RAD52 stimulates OGG1 8-oxoG incision activity. These effects are not associated with direct interactions with the DNA substrates, since OGG1 inhibitory effect is independent on its binding to the RAD52 substrate and RAD52 does not increase OGG1 loading to its substrate. We found that RAD52 decreased the amount of OGG1-substrate complexes trapped by sodium borohydride and decreased OGG1 affinity for an abasic site containing oligonucleotide. These observations suggest that RAD52 stimulates OGG1 by increasing its turnover rate. Our results imply that RAD52 cooperates with OGG1 to increase BER efficiency and suggest a coordinated action between BER and double strand brake repair. This coordination may be particularly relevant when 8oxoG lesions positioned close to double strand breaks may impose a hindrance to RAD52 catalytic activities. doi:10.1016/j.mito.2006.08.062