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Michio Hirano - One of the best experts on this subject based on the ideXlab platform.
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MERRF and kearns sayre overlap Syndrome due to the mitochondrial dna m 3291t c mutation p07 209
Neurology, 2012Co-Authors: Valentina Emmanuele, David Silvers, Evangelia Sotiriou, Kurenai Tanji, Salvatore Dimauro, Michio HiranoAbstract:Objective: To report a patient with a complex phenotype of myoclonus epilepsy with ragged-red fibers (MERRF) Syndrome and Kearns–Sayre Syndrome (KSS), who harbored a m.3291T>C mutation in the tRNALeu(UUR) gene. This mutation has never been reported in association with a MERRF/KSS Syndrome. Background Individuals who harbor a pathogenic mtDNA mutation can manifest overlapping features of typical mitochondrial Syndromes. MERRF is a multisystem disorder characterized by myoclonic seizures, cerebellar ataxia, mitochondrial myopathy, and ragged-red fibers (RRF) on muscle biopsy, commonly caused by the m.8344A>G mutation in the tRNALys gene. KSS is a mitochondrial disorder characterized by onset before 20 years of age of progressive external ophthalmoplegia or pigmentary retinopathy, together with at least one of a triad of cerebellar ataxia, heart block, and elevated cerebrospinal fluid protein. Typically, KSS is due to single, large-scale deletions of mtDNA. Design/Methods: Clinical data were collected. Muscle histochemical and biochemical analysis was performed. Muscle DNA was screened for mtDNA large-scale rearrangements and point mutations. Mutation level was quantified by restriction fragment length polymorphism (RFLP) analysis. Results: A 48-year-old man presented with progressive myoclonus epilepsy, cerebellar ataxia, hearing loss, myopathic weakness, ophthalmoparesis, pigmentary retinopathy, and bifascicular heart block. Muscle biopsy demonstrated multiple RRF. Biochemical analysis of respiratory chain enzymes was normal. Genetic tests were negative for the common MERRF mtDNA mutations, and no deletions in mitochondrial DNA were detected. Direct sequencing mtDNA revealed a T-to-C transition at nucleotide position 3291 in the tRNALeu(UUR) gene. PCR RFLP showed 92% mutant genome in muscle. Conclusions: This is the first report associating a m.3291T>C mutation in the tRNALeu(UUR) gene with an adult-onset MERRF/KSS overlap. Our data reinforce the well-established concept that, in mtDNA-related disorders, the same genetic abnormality can be associated with a wide spectrum of phenotypes, and the observation that tRNALeu(UUR) is a hotspot for mtDNA mutations. Supported by: The National Institutes of Health (NIH HD32062) and by the Marriott Mitochondrial Disorders Clinical Research Fund (MMDCRF). M.H. was supported by NIH grants (R01HD57543, R01HD056103, and RCNS070232) and by the Muscular Dystrophy Association. Disclosure: Dr. Emmanuele has nothing to disclose. Dr. Silvers has nothing to disclose. Dr. Sotiriou has nothing to disclose. Dr. Tanji has nothing to disclose. Dr. DiMauro has nothing to disclose. Dr. Hirano has received personal compensation for activities with Athena Diagnostics as a speaker.Dr. Hirano has received personal compensation in an editorial capacity for Current Neurology and Neuroscience Reports.Dr. Hirano has received research support from Santhera Pharmaceutical and Edison Pharmaceuticals.
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MERRF and kearns sayre overlap Syndrome due to the mitochondrial dna m 3291t c mutation
Muscle & Nerve, 2011Co-Authors: Valentina Emmanuele, David Silvers, Evangelia Sotiriou, Kurenai Tanji, Salvatore Dimauro, Michio HiranoAbstract:A 48-year-old man presented with a complex phenotype of myoclonus epilepsy with ragged-red fibers (MERRF) Syndrome and Kearns–Sayre Syndrome (KSS), which included progressive myoclonus epilepsy, cerebellar ataxia, hearing loss, myopathic weakness, ophthalmoparesis, pigmentary retinopathy, bifascicular heart block, and ragged-red fibers. The m.3291T>C mutation in the tRNALeu(UUR) gene was found with 92% heteroplasmy in muscle. This mutation has been reported with MELAS, myopathy, and deafness with cognitive impairment. This is the first description with a MERRF/KSS Syndrome. Muscle Nerve 44: 448–451, 2011
Gonzalo Sanz Ricardo - One of the best experts on this subject based on the ideXlab platform.
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Papel de las mutaciones del ADNmt en la producción de daño oxidativo mediado por ROS en un modelo de cíbridos transmitocondriales
Bellaterra : Universitat Autònoma de Barcelona, 2006Co-Authors: Gonzalo Sanz Ricardo, Universitat Autònoma De Barcelona. Departament De Bioquímica I De BioloAbstract:Consultable des del TDXTítol obtingut de la portada digitalitzadaEl genoma mitocondrial humano es una molécula circular de doble cadena de 16,5 kb. En su secuencia existe información para 13 polipéptidos de diferentes subunidades de los complejos de la cadena de transporte electrónico (CTE), para 22 ARNt y para 2 ARNr. Una mutación en cualquiera de estos genes puede provocar que la CTE no funcione correctamente, dando lugar a una disfunción del sistema de fosforilación oxidativa. Todo ello puede provocar por un lado un déficit de energía en las células o tejidos, o por otro lado un incremento de la producción de especies reactivas de oxígeno (ROS). Según la demanda energética de cada tejido este déficit de producción de energía será más o menos importante, pudiendo incluso provocar graves trastornos fisiopatológicos. El incremento de la producción de ROS por parte de la cadena de transporte electrónico puede ser eliminado con ayuda de las defensas antioxidantes celulares. Si la producción de ROS es más importante que la acción de estas defensas, ello puede llegar a provocar lesiones en diferentes componentes celulares tales como lípidos, proteínas o al propio ADNmt. Para profundizar en este campo, en este trabajo en primer lugar se han diagnosticado a cuatro pacientes con enfermedad mitocondrial, portadores de una mutación en su genoma mitocondrial. A partir de plaquetas de estos pacientes se han generado cíbridos transmitocondriales, que se han utilizado como modelo de estudio. Se han estudiado las siguientes mutaciones en genes mitocondriales: T14487C en la subunidad ND6 del complejo I, A3243G en el ARN de transferencia Leu (UUR), A8344G en el ARN de transferencia Lys y G6930A en la subunidad COXI del complejo IV. Analizando la producción de peróxido de hidrógeno como medida de la producción de ROS en estas cuatro líneas, hemos observado que las líneas portadoras de una mutación que afectase al funcionamiento del complejo I y III (descritos ampliamente en la literatura como principales productores de ROS en la mitocondria) es decir A3243G, A8344G y T144874C, sí provocan un incremento de la producción, mientras que la mutación que no afectaba a estos complejos (G6930A) no provocaba incremento. Posteriormente se ha estudiado si este incremento producía daño oxidativo a diferentes componentes celulares, tales como lípidos, proteínas y el propio ADNmt. Previamente, debido a que en la literatura no existía un consenso claro sobre el mejor método de análisis de la peroxidación lipídica, se realizó un pequeño estudio sobre cuál era el mejor inhibidor de la peroxidación lipídica a utilizar y en que concentración, obteniendo que el mejor a utilizar era el BHT a una concentración de 3mM. En cuanto los resultados de daño oxidativo se observó que en los lípidos solo se observaba daño oxidativo en la línea portadora de la mutación T144874C, mientras que las otras no lo presentaban. En la oxidación de proteínas no se observó daño en ninguna de las cuatro líneas portadoras de la mutación y en cuanto a la oxidación del ADNmt, se observó daño oxidativo en las líneas portadoras de las mutaciones A8344G y T14487C. Con estos resultados se observa que en algunas mutaciones en el genoma mitocondrial la producción de ROS generada es superior a la capacidad detoxificadora de la célula, provocando daño oxidativo, mientras que en otras la producción de ROS no supera la acción de las enzimas antioxidantes.Mitochondrial encephalomyopathies caused by mutations in mitochondrial DNA (mtDNA) are a heterogeneous group of disorders characterized by primary dysfunction of the oxidative phosphorylation system (OXPHOS) with a decrease in ATP production. Clinical and biochemical heterogeneity of mitochondrial disorders is due to the ubiquitous nature of mitochondria and the dual genetic (mitochondrial and nuclear DNA) control of OXPHOS. Some unique features of mitochondrial genetics, such as heteroplasmy and tissue segregation, contribute to this phenomenon. However, the precise mechanisms leading to this heterogeneity are still largely unclear. Mitochondria are the major source of reactive oxygen species (ROS), which are generated as toxic by-products of redox-coupled reactions in the electron transport chain (ETC). Inhibition of the ETC in vitro using some respiratory complex inhibitors results in a significant increase in the mitochondrial production of ROS. This increase suggests that when dysfunction of the respiratory chain complexes occurs, electrons can be transferred directly to the molecular oxygen. However, cells are well protected by antioxidant enzymes: the manganese superoxide dismutase (Mn-SOD) and copper-Zinc superoxide dismutase (CuZn-SOD) to eliminate superoxide anion (O2.-) and the glutahione peroxidase (GSH-Px) and catalase (CAT) to eliminate hydrogen peroxide. Oxidative stress results when the balance of prooxidants and antioxidants is altered in favour of the prooxidants. In turn, an excess of ROS may contribute to OXPHOS damage. Thus, to define the relationship between mtDNA mutations and production of ROS, several transmitochondrial cell lines (cybrids) carrying different mutations in their mtDNA were obtained from different mitochondrial patients. These included two common and well characterized mtDNA mutations in tRNA genes, the A3243G transition in the tRNALeu(UUR) derived from a patient with MELAS Syndrome (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes), and the A8344G mutation in the tRNALys, derived from a patient with the MERRF Syndrome (myoclonus epilepsy with ragged-red fibers). In addition, another two cybrids cell lines were studied, harbouring the G6930A mutation in the gene encoding the subunit I (COI) of the cytochrome c oxidase (COX). This mutation changes the amino acid glycine into a premature termination codon, resulting in the loss of the last 170 amino acids (33%) of the polypeptide, thus causing a complete disruption in the COX assembly. The last cybrid cell line studied carried the mutation T144874C in the subunit 6 of the complex I of the ETC. Hydrogen peroxide production was increased in cybrids harbouring tRNA and complex I mutations, but no changes were observed in cybrids harbouring the mutation in complex IV. No oxidative damage to lipids, proteins or mtDNA was detected in cybrids harbouring A3243G and G6930A mutations. In the cybrid cell line harbouring A8344G mutation, only oxidative damage to mtDNA was observed and in the cybrids harbouring the mutation in complex I, mtDNA and lipid oxidative damage were detected. These results suggest that some mutations in mtDNA may increase the production of hydrogen peroxide (i.e., those mutations which affect complex I or III of the ETC) meanwhile other mutations do not. Furthermore this increase can sometimes override the antioxidant defences of the cells and produce oxidative damage to key cellular components
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Papel de las mutaciones del ADNmt en la producción de daño oxidativo mediado por ROS en un modelo de cíbridos transmitocondriales
'Universitat Autonoma de Barcelona', 2005Co-Authors: Gonzalo Sanz RicardoAbstract:El genoma mitocondrial humano es una molécula circular de doble cadena de 16,5 kb. En su secuencia existe información para 13 polipéptidos de diferentes subunidades de los complejos de la cadena de transporte electrónico (CTE), para 22 ARNt y para 2 ARNr. Una mutación en cualquiera de estos genes puede provocar que la CTE no funcione correctamente, dando lugar a una disfunción del sistema de fosforilación oxidativa. Todo ello puede provocar por un lado un déficit de energía en las células o tejidos, o por otro lado un incremento de la producción de especies reactivas de oxígeno (ROS). Según la demanda energética de cada tejido este déficit de producción de energía será más o menos importante, pudiendo incluso provocar graves trastornos fisiopatológicos.El incremento de la producción de ROS por parte de la cadena de transporte electrónico puede ser eliminado con ayuda de las defensas antioxidantes celulares. Si la producción de ROS es más importante que la acción de estas defensas, ello puede llegar a provocar lesiones en diferentes componentes celulares tales como lípidos, proteínas o al propio ADNmt. Para profundizar en este campo, en este trabajo en primer lugar se han diagnosticado a cuatro pacientes con enfermedad mitocondrial, portadores de una mutación en su genoma mitocondrial. A partir de plaquetas de estos pacientes se han generado cíbridos transmitocondriales, que se han utilizado como modelo de estudio. Se han estudiado las siguientes mutaciones en genes mitocondriales: T14487C en la subunidad ND6 del complejo I, A3243G en el ARN de transferencia Leu (UUR), A8344G en el ARN de transferencia Lys y G6930A en la subunidad COXI del complejo IV. Analizando la producción de peróxido de hidrógeno como medida de la producción de ROS en estas cuatro líneas, hemos observado que las líneas portadoras de una mutación que afectase al funcionamiento del complejo I y III (descritos ampliamente en la literatura como principales productores de ROS en la mitocondria) es decir A3243G, A8344G y T144874C, sí provocan un incremento de la producción, mientras que la mutación que no afectaba a estos complejos (G6930A) no provocaba incremento. Posteriormente se ha estudiado si este incremento producía daño oxidativo a diferentes componentes celulares, tales como lípidos, proteínas y el propio ADNmt. Previamente, debido a que en la literatura no existía un consenso claro sobre el mejor método de análisis de la peroxidación lipídica, se realizó un pequeño estudio sobre cuál era el mejor inhibidor de la peroxidación lipídica a utilizar y en que concentración, obteniendo que el mejor a utilizar era el BHT a una concentración de 3mM.En cuanto los resultados de daño oxidativo se observó que en los lípidos solo se observaba daño oxidativo en la línea portadora de la mutación T144874C, mientras que las otras no lo presentaban. En la oxidación de proteínas no se observó daño en ninguna de las cuatro líneas portadoras de la mutación y en cuanto a la oxidación del ADNmt, se observó daño oxidativo en las líneas portadoras de las mutaciones A8344G y T14487C. Con estos resultados se observa que en algunas mutaciones en el genoma mitocondrial la producción de ROS generada es superior a la capacidad detoxificadora de la célula, provocando daño oxidativo, mientras que en otras la producción de ROS no supera la acción de las enzimas antioxidantes.Mitochondrial encephalomyopathies caused by mutations in mitochondrial DNA (mtDNA) are a heterogeneous group of disorders characterized by primary dysfunction of the oxidative phosphorylation system (OXPHOS) with a decrease in ATP production. Clinical and biochemical heterogeneity of mitochondrial disorders is due to the ubiquitous nature of mitochondria and the dual genetic (mitochondrial and nuclear DNA) control of OXPHOS. Some unique features of mitochondrial genetics, such as heteroplasmy and tissue segregation, contribute to this phenomenon. However, the precise mechanisms leading to this heterogeneity are still largely unclear.Mitochondria are the major source of reactive oxygen species (ROS), which are generated as toxic by-products of redox-coupled reactions in the electron transport chain (ETC). Inhibition of the ETC in vitro using some respiratory complex inhibitors results in a significant increase in the mitochondrial production of ROS. This increase suggests that when dysfunction of the respiratory chain complexes occurs, electrons can be transferred directly to the molecular oxygen. However, cells are well protected by antioxidant enzymes: the manganese superoxide dismutase (Mn-SOD) and copper-Zinc superoxide dismutase (CuZn-SOD) to eliminate superoxide anion (O2.-) and the glutahione peroxidase (GSH-Px) and catalase (CAT) to eliminate hydrogen peroxide. Oxidative stress results when the balance of prooxidants and antioxidants is altered in favour of the prooxidants. In turn, an excess of ROS may contribute to OXPHOS damage. Thus, to define the relationship between mtDNA mutations and production of ROS, several transmitochondrial cell lines (cybrids) carrying different mutations in their mtDNA were obtained from different mitochondrial patients. These included two common and well characterized mtDNA mutations in tRNA genes, the A3243G transition in the tRNALeu(UUR) derived from a patient with MELAS Syndrome (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes), and the A8344G mutation in the tRNALys, derived from a patient with the MERRF Syndrome (myoclonus epilepsy with ragged-red fibers). In addition, another two cybrids cell lines were studied, harbouring the G6930A mutation in the gene encoding the subunit I (COI) of the cytochrome c oxidase (COX). This mutation changes the amino acid glycine into a premature termination codon, resulting in the loss of the last 170 amino acids (33%) of the polypeptide, thus causing a complete disruption in the COX assembly. The last cybrid cell line studied carried the mutation T144874C in the subunit 6 of the complex I of the ETC.Hydrogen peroxide production was increased in cybrids harbouring tRNA and complex I mutations, but no changes were observed in cybrids harbouring the mutation in complex IV. No oxidative damage to lipids, proteins or mtDNA was detected in cybrids harbouring A3243G and G6930A mutations. In the cybrid cell line harbouring A8344G mutation, only oxidative damage to mtDNA was observed and in the cybrids harbouring the mutation in complex I, mtDNA and lipid oxidative damage were detected.These results suggest that some mutations in mtDNA may increase the production of hydrogen peroxide (i.e., those mutations which affect complex I or III of the ETC) meanwhile other mutations do not. Furthermore this increase can sometimes override the antioxidant defences of the cells and produce oxidative damage to key cellular components
Valentina Emmanuele - One of the best experts on this subject based on the ideXlab platform.
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MERRF and kearns sayre overlap Syndrome due to the mitochondrial dna m 3291t c mutation p07 209
Neurology, 2012Co-Authors: Valentina Emmanuele, David Silvers, Evangelia Sotiriou, Kurenai Tanji, Salvatore Dimauro, Michio HiranoAbstract:Objective: To report a patient with a complex phenotype of myoclonus epilepsy with ragged-red fibers (MERRF) Syndrome and Kearns–Sayre Syndrome (KSS), who harbored a m.3291T>C mutation in the tRNALeu(UUR) gene. This mutation has never been reported in association with a MERRF/KSS Syndrome. Background Individuals who harbor a pathogenic mtDNA mutation can manifest overlapping features of typical mitochondrial Syndromes. MERRF is a multisystem disorder characterized by myoclonic seizures, cerebellar ataxia, mitochondrial myopathy, and ragged-red fibers (RRF) on muscle biopsy, commonly caused by the m.8344A>G mutation in the tRNALys gene. KSS is a mitochondrial disorder characterized by onset before 20 years of age of progressive external ophthalmoplegia or pigmentary retinopathy, together with at least one of a triad of cerebellar ataxia, heart block, and elevated cerebrospinal fluid protein. Typically, KSS is due to single, large-scale deletions of mtDNA. Design/Methods: Clinical data were collected. Muscle histochemical and biochemical analysis was performed. Muscle DNA was screened for mtDNA large-scale rearrangements and point mutations. Mutation level was quantified by restriction fragment length polymorphism (RFLP) analysis. Results: A 48-year-old man presented with progressive myoclonus epilepsy, cerebellar ataxia, hearing loss, myopathic weakness, ophthalmoparesis, pigmentary retinopathy, and bifascicular heart block. Muscle biopsy demonstrated multiple RRF. Biochemical analysis of respiratory chain enzymes was normal. Genetic tests were negative for the common MERRF mtDNA mutations, and no deletions in mitochondrial DNA were detected. Direct sequencing mtDNA revealed a T-to-C transition at nucleotide position 3291 in the tRNALeu(UUR) gene. PCR RFLP showed 92% mutant genome in muscle. Conclusions: This is the first report associating a m.3291T>C mutation in the tRNALeu(UUR) gene with an adult-onset MERRF/KSS overlap. Our data reinforce the well-established concept that, in mtDNA-related disorders, the same genetic abnormality can be associated with a wide spectrum of phenotypes, and the observation that tRNALeu(UUR) is a hotspot for mtDNA mutations. Supported by: The National Institutes of Health (NIH HD32062) and by the Marriott Mitochondrial Disorders Clinical Research Fund (MMDCRF). M.H. was supported by NIH grants (R01HD57543, R01HD056103, and RCNS070232) and by the Muscular Dystrophy Association. Disclosure: Dr. Emmanuele has nothing to disclose. Dr. Silvers has nothing to disclose. Dr. Sotiriou has nothing to disclose. Dr. Tanji has nothing to disclose. Dr. DiMauro has nothing to disclose. Dr. Hirano has received personal compensation for activities with Athena Diagnostics as a speaker.Dr. Hirano has received personal compensation in an editorial capacity for Current Neurology and Neuroscience Reports.Dr. Hirano has received research support from Santhera Pharmaceutical and Edison Pharmaceuticals.
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MERRF and kearns sayre overlap Syndrome due to the mitochondrial dna m 3291t c mutation
Muscle & Nerve, 2011Co-Authors: Valentina Emmanuele, David Silvers, Evangelia Sotiriou, Kurenai Tanji, Salvatore Dimauro, Michio HiranoAbstract:A 48-year-old man presented with a complex phenotype of myoclonus epilepsy with ragged-red fibers (MERRF) Syndrome and Kearns–Sayre Syndrome (KSS), which included progressive myoclonus epilepsy, cerebellar ataxia, hearing loss, myopathic weakness, ophthalmoparesis, pigmentary retinopathy, bifascicular heart block, and ragged-red fibers. The m.3291T>C mutation in the tRNALeu(UUR) gene was found with 92% heteroplasmy in muscle. This mutation has been reported with MELAS, myopathy, and deafness with cognitive impairment. This is the first description with a MERRF/KSS Syndrome. Muscle Nerve 44: 448–451, 2011
Universitat Autònoma De Barcelona. Departament De Bioquímica I De Biolo - One of the best experts on this subject based on the ideXlab platform.
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Papel de las mutaciones del ADNmt en la producción de daño oxidativo mediado por ROS en un modelo de cíbridos transmitocondriales
Bellaterra : Universitat Autònoma de Barcelona, 2006Co-Authors: Gonzalo Sanz Ricardo, Universitat Autònoma De Barcelona. Departament De Bioquímica I De BioloAbstract:Consultable des del TDXTítol obtingut de la portada digitalitzadaEl genoma mitocondrial humano es una molécula circular de doble cadena de 16,5 kb. En su secuencia existe información para 13 polipéptidos de diferentes subunidades de los complejos de la cadena de transporte electrónico (CTE), para 22 ARNt y para 2 ARNr. Una mutación en cualquiera de estos genes puede provocar que la CTE no funcione correctamente, dando lugar a una disfunción del sistema de fosforilación oxidativa. Todo ello puede provocar por un lado un déficit de energía en las células o tejidos, o por otro lado un incremento de la producción de especies reactivas de oxígeno (ROS). Según la demanda energética de cada tejido este déficit de producción de energía será más o menos importante, pudiendo incluso provocar graves trastornos fisiopatológicos. El incremento de la producción de ROS por parte de la cadena de transporte electrónico puede ser eliminado con ayuda de las defensas antioxidantes celulares. Si la producción de ROS es más importante que la acción de estas defensas, ello puede llegar a provocar lesiones en diferentes componentes celulares tales como lípidos, proteínas o al propio ADNmt. Para profundizar en este campo, en este trabajo en primer lugar se han diagnosticado a cuatro pacientes con enfermedad mitocondrial, portadores de una mutación en su genoma mitocondrial. A partir de plaquetas de estos pacientes se han generado cíbridos transmitocondriales, que se han utilizado como modelo de estudio. Se han estudiado las siguientes mutaciones en genes mitocondriales: T14487C en la subunidad ND6 del complejo I, A3243G en el ARN de transferencia Leu (UUR), A8344G en el ARN de transferencia Lys y G6930A en la subunidad COXI del complejo IV. Analizando la producción de peróxido de hidrógeno como medida de la producción de ROS en estas cuatro líneas, hemos observado que las líneas portadoras de una mutación que afectase al funcionamiento del complejo I y III (descritos ampliamente en la literatura como principales productores de ROS en la mitocondria) es decir A3243G, A8344G y T144874C, sí provocan un incremento de la producción, mientras que la mutación que no afectaba a estos complejos (G6930A) no provocaba incremento. Posteriormente se ha estudiado si este incremento producía daño oxidativo a diferentes componentes celulares, tales como lípidos, proteínas y el propio ADNmt. Previamente, debido a que en la literatura no existía un consenso claro sobre el mejor método de análisis de la peroxidación lipídica, se realizó un pequeño estudio sobre cuál era el mejor inhibidor de la peroxidación lipídica a utilizar y en que concentración, obteniendo que el mejor a utilizar era el BHT a una concentración de 3mM. En cuanto los resultados de daño oxidativo se observó que en los lípidos solo se observaba daño oxidativo en la línea portadora de la mutación T144874C, mientras que las otras no lo presentaban. En la oxidación de proteínas no se observó daño en ninguna de las cuatro líneas portadoras de la mutación y en cuanto a la oxidación del ADNmt, se observó daño oxidativo en las líneas portadoras de las mutaciones A8344G y T14487C. Con estos resultados se observa que en algunas mutaciones en el genoma mitocondrial la producción de ROS generada es superior a la capacidad detoxificadora de la célula, provocando daño oxidativo, mientras que en otras la producción de ROS no supera la acción de las enzimas antioxidantes.Mitochondrial encephalomyopathies caused by mutations in mitochondrial DNA (mtDNA) are a heterogeneous group of disorders characterized by primary dysfunction of the oxidative phosphorylation system (OXPHOS) with a decrease in ATP production. Clinical and biochemical heterogeneity of mitochondrial disorders is due to the ubiquitous nature of mitochondria and the dual genetic (mitochondrial and nuclear DNA) control of OXPHOS. Some unique features of mitochondrial genetics, such as heteroplasmy and tissue segregation, contribute to this phenomenon. However, the precise mechanisms leading to this heterogeneity are still largely unclear. Mitochondria are the major source of reactive oxygen species (ROS), which are generated as toxic by-products of redox-coupled reactions in the electron transport chain (ETC). Inhibition of the ETC in vitro using some respiratory complex inhibitors results in a significant increase in the mitochondrial production of ROS. This increase suggests that when dysfunction of the respiratory chain complexes occurs, electrons can be transferred directly to the molecular oxygen. However, cells are well protected by antioxidant enzymes: the manganese superoxide dismutase (Mn-SOD) and copper-Zinc superoxide dismutase (CuZn-SOD) to eliminate superoxide anion (O2.-) and the glutahione peroxidase (GSH-Px) and catalase (CAT) to eliminate hydrogen peroxide. Oxidative stress results when the balance of prooxidants and antioxidants is altered in favour of the prooxidants. In turn, an excess of ROS may contribute to OXPHOS damage. Thus, to define the relationship between mtDNA mutations and production of ROS, several transmitochondrial cell lines (cybrids) carrying different mutations in their mtDNA were obtained from different mitochondrial patients. These included two common and well characterized mtDNA mutations in tRNA genes, the A3243G transition in the tRNALeu(UUR) derived from a patient with MELAS Syndrome (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes), and the A8344G mutation in the tRNALys, derived from a patient with the MERRF Syndrome (myoclonus epilepsy with ragged-red fibers). In addition, another two cybrids cell lines were studied, harbouring the G6930A mutation in the gene encoding the subunit I (COI) of the cytochrome c oxidase (COX). This mutation changes the amino acid glycine into a premature termination codon, resulting in the loss of the last 170 amino acids (33%) of the polypeptide, thus causing a complete disruption in the COX assembly. The last cybrid cell line studied carried the mutation T144874C in the subunit 6 of the complex I of the ETC. Hydrogen peroxide production was increased in cybrids harbouring tRNA and complex I mutations, but no changes were observed in cybrids harbouring the mutation in complex IV. No oxidative damage to lipids, proteins or mtDNA was detected in cybrids harbouring A3243G and G6930A mutations. In the cybrid cell line harbouring A8344G mutation, only oxidative damage to mtDNA was observed and in the cybrids harbouring the mutation in complex I, mtDNA and lipid oxidative damage were detected. These results suggest that some mutations in mtDNA may increase the production of hydrogen peroxide (i.e., those mutations which affect complex I or III of the ETC) meanwhile other mutations do not. Furthermore this increase can sometimes override the antioxidant defences of the cells and produce oxidative damage to key cellular components
Yauhuei Wei - One of the best experts on this subject based on the ideXlab platform.
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Generation of two isogenic human induced pluripotent stem cell lines from a 15 year-old female patient with MERRF Syndrome and A8344G mutation of mitochondrial DNA
Elsevier, 2018Co-Authors: Shih-jie Chou, Yauhuei Wei, Yu-hsuan Yang, Aliaksandr A. Yarmishyn, Chien-tsun Chen, Hsin-chen Lee, Shih-hwa ChiouAbstract:MERRF Syndrome is predominantly caused by A8344G mutation in the mitochondrial DNA (mtDNA), affecting MT-TK gene, which impairs the mitochondrial electron transport chain function. Here, we report the generation of two isogenic induced pluripotent stem cell (iPSC) lines, TVGH-iPSC-MRF-Mlow and TVGH-iPSC-MRF-Mhigh, from the skin fibroblasts of a female MERRF patient harboring mtDNA A8344G mutation by using retrovirus transduction system. Both cell lines share the same genetic background except containing different proportions of mtDNA with the A8344G mutation. Both cell lines exhibited the pluripotency and capacity to differentiate into three germ layers
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ATOH1/RFX1/RFX3 transcription factors facilitate the differentiation and characterisation of inner ear hair cell-like cells from patient-specific induced pluripotent stem cells harbouring A8344G mutation of mitochondrial DNA
Nature Publishing Group, 2018Co-Authors: Yen-chun Chen, Yauhuei Wei, Chia-ling Tsai, Wei-ting Hsu, Hung-ching Lin, Yi-chao HsuAbstract:Abstract Degeneration or loss of inner ear hair cells (HCs) is irreversible and results in sensorineural hearing loss (SHL). Human-induced pluripotent stem cells (hiPSCs) have been employed in disease modelling and cell therapy. Here, we propose a transcription factor (TF)-driven approach using ATOH1 and regulatory factor of x-box (RFX) genes to generate HC-like cells from hiPSCs. Our results suggest that ATOH1/RFX1/RFX3 could significantly increase the differentiation capacity of iPSCs into MYO7A mCherry-positive cells, upregulate the mRNA expression levels of HC-related genes and promote the differentiation of HCs with more mature stereociliary bundles. To model the molecular and stereociliary structural changes involved in HC dysfunction in SHL, we further used ATOH1/RFX1/RFX3 to differentiate HC-like cells from the iPSCs from patients with myoclonus epilepsy associated with ragged-red fibres (MERRF) Syndrome, which is caused by A8344G mutation of mitochondrial DNA (mtDNA), and characterised by myoclonus epilepsy, ataxia and SHL. Compared with isogenic iPSCs, MERRF-iPSCs possessed ~42–44% mtDNA with A8344G mutation and exhibited significantly elevated reactive oxygen species (ROS) production and CAT gene expression. Furthermore, MERRF-iPSC-differentiated HC-like cells exhibited significantly elevated ROS levels and MnSOD and CAT gene expression. These MERRF-HCs that had more single cilia with a shorter length could be observed only by using a non-TF method, but those with fewer stereociliary bundle-like protrusions than isogenic iPSCs-differentiated-HC-like cells could be further observed using ATOH1/RFX1/RFX3 TFs. We further analysed and compared the whole transcriptome of M1ctrl-HCs and M1-HCs after treatment with ATOH1 or ATOH1/RFX1/RFX3. We revealed that the HC-related gene transcripts in M1ctrl-iPSCs had a significantly higher tendency to be activated by ATOH1/RFX1/RFX3 than M1-iPSCs. The ATOH1/RFX1/RFX3 TF-driven approach for the differentiation of HC-like cells from iPSCs is an efficient and promising strategy for the disease modelling of SHL and can be employed in future therapeutic strategies to treat SHL patients
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disruption of the human coq5 containing protein complex is associated with diminished coenzyme q10 levels under two different conditions of mitochondrial energy deficiency
Biochimica et Biophysica Acta, 2016Co-Authors: Hsiuchuan Yen, Yichun Liu, Chiachi Kan, Hsingju Wei, Szuhsien Lee, Yauhuei Wei, Yuhsiu Feng, Chihwei Chen, Chinchang HuangAbstract:Abstract Background The Coq protein complex assembled from several Coq proteins is critical for coenzyme Q6 (CoQ6) biosynthesis in yeast. Secondary CoQ10 deficiency is associated with mitochondrial DNA (mtDNA) mutations in patients. We previously demonstrated that carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) suppressed CoQ10 levels and COQ5 protein maturation in human 143B cells. Methods This study explored the putative COQ protein complex in human cells through two-dimensional blue native-polyacrylamide gel electrophoresis and Western blotting to investigate its status in 143B cells after FCCP treatment and in cybrids harboring the mtDNA mutation that caused myoclonic epilepsy with ragged-red fibers (MERRF) Syndrome. Ubiquinol-10 and ubiquinone-10 levels were detected by high-performance liquid chromatography. Mitochondrial energy status, mRNA levels of various PDSS and COQ genes, and protein levels of COQ5 and COQ9 in cybrids were examined. Results A high-molecular-weight protein complex containing COQ5, but not COQ9, in the mitochondria was identified and its level was suppressed by FCCP and in cybrids with MERRF mutation. That was associated with decreased mitochondrial membrane potential and mitochondrial ATP production. Total CoQ10 levels were decreased under both conditions, but the ubiquinol-10:ubiquinone-10 ratio was increased in mutant cybrids. The expression of COQ5 was increased but COQ5 protein maturation was suppressed in the mutant cybrids. Conclusions A novel COQ5-containing protein complex was discovered in human cells. Its destabilization was associated with reduced CoQ10 levels and mitochondrial energy deficiency in human cells treated with FCCP or exhibiting MERRF mutation. General significance The findings elucidate a possible mechanism for mitochondrial dysfunction-induced CoQ10 deficiency in human cells.
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ampk mediated increase of glycolysis as an adaptive response to oxidative stress in human cells implication of the cell survival in mitochondrial diseases
Biochimica et Biophysica Acta, 2012Co-Authors: Yauhuei WeiAbstract:Abstract We report that the energy metabolism shifts to anaerobic glycolysis as an adaptive response to oxidative stress in the primary cultures of skin fibroblasts from patients with MERRF Syndrome. In order to unravel the molecular mechanism involved in the alteration of energy metabolism under oxidative stress, we treated normal human skin fibroblasts (CCD-966SK cells) with sub-lethal doses of H2O2. The results showed that several glycolytic enzymes including hexokinase type II (HK II), lactate dehydrogenase (LDH) and glucose transporter 1 (GLUT1) were up-regulated in H2O2-treated normal skin fibroblasts. In addition, the glycolytic flux of skin fibroblasts was increased by H2O2 in a dose-dependent manner through the activation of AMP-activated protein kinase (AMPK) and phosphorylation of its downstream target, phosphofructokinase 2 (PFK2). Moreover, we found that the AMPK-mediated increase of glycolytic flux by H2O2 was accompanied by an increase of intracellular NADPH content. By treatment of the cells with glycolysis inhibitors, an AMPK inhibitor or genetic knockdown of AMPK, respectively, the H2O2-induced increase of NADPH was abrogated leading to the overproduction of intracellular ROS and cell death. Significantly, we showed that phosphorylation levels of AMPK and glycolysis were up-regulated to confer an advantage of survival for MERRF skin fibroblasts. Taken together, our findings suggest that the increased production of NADPH by AMPK-mediated increase of the glycolytic flux contributes to the adaptation of MERRF skin fibroblasts and H2O2-treated normal skin fibroblasts to oxidative stress.
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Functional Recovery of Human Cells Harbouring the Mitochondrial DNA Mutation MERRF A8344G via Peptide-Mediated Mitochondrial Delivery
Karger Publishers, 2012Co-Authors: Jui-chih Chang, Yauhuei Wei, Ko-hung Liu, Shou-jen Kou, Chieh-sen Chuang, Mingli Hsieh, Chin-san LiuAbstract:We explored the feasibility of mitochondrial therapy using the cell-penetrating peptide Pep-1 to transfer mitochondrial DNA (mtDNA) between cells and rescue a cybrid cell model of the mitochondrial disease myoclonic epilepsy with ragged-red fibres (MERRF) Syndrome. Pep-1-conjugated wild-type mitochondria isolated from parent cybrid cells incorporating a mitochondria-specific tag were used as donors for mitochondrial delivery into MERRF cybrid cells (MitoB2) and mtDNA-depleted Rho-zero cells (Mitoρ°). Forty-eight hours later, translocation of Pep-1-labelled mitochondria into the mitochondrial regions of MitoB2 and Mitoρ° host cells was observed (delivery efficiencies of 77.48 and 82.96%, respectively). These internalized mitochondria were maintained for at least 15 days in both cell types and were accompanied by mitochondrial function recovery and cell survival by preventing mitochondria-dependent cell death. Mitochondrial homeostasis analyses showed that peptide-mediated mitochondrial delivery (PMD) also increased mitochondrial biogenesis in both cell types, but through distinct regulatory pathways involving mitochondrial dynamics. Dramatic decreases in mitofusin-2 (MFN2) and dynamin-related protein 1/fission 1 were observed in MitoB2 cells, while Mitoρ° cells showed a significant increase in optic atrophy 1 and MFN2. These findings suggest that PMD can be used as a potential therapeutic intervention for mitochondrial disorders