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Mitochondrial DNA (mtDNA) 3243A > G tRNALeu((UUR)) heteroplasmic mutation (m.3243A > G) exhibits clinically heterogeneous phenotypes. While the high mtDNA heteroplasmy exceeding a critical threshold causes mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes (MELAS) syndrome, the low mtDNA heteroplasmy causes maternally inherited diabetes with or without deafness (MIDD) syndrome. How quantitative differences in mtDNA heteroplasmy produces distinct pathological states has remained elusive. Here we show that despite striking similarities in the energy metabolic gene expression signature, the mitochondrial bioenergetics, biogenesis and fuel catabolic functions are distinct in cells harboring low or high levels of the m.3243 A > G mutation compared to wild type cells. We further demonstrate that the low heteroplasmic mutant cells exhibit a coordinate induction of transcriptional regulators of the mitochondrial biogenesis, glucose and fatty acid metabolism pathways that lack in near homoplasmic mutant cells compared to wild type cells. Altogether, these results shed new biological insights on the potential mechanisms by which low mtDNA heteroplasmy may progressively cause diabetes mellitus.

Mitochondrial DNA (mtDNA) 3243A > G tRNALeu((UUR)) heteroplasmic mutation (m.3243A > G) exhibits clinically heterogeneous phenotypes. While the high mtDNA heteroplasmy exceeding a critical threshold causes mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes (MELAS) syndrome, the low mtDNA heteroplasmy causes maternally inherited diabetes with or without deafness (MIDD) syndrome. How quantitative differences in mtDNA heteroplasmy produces distinct pathological states has remained elusive. Here we show that despite striking similarities in the energy metabolic gene expression signature, the mitochondrial bioenergetics, biogenesis and fuel catabolic functions are distinct in cells harboring low or high levels of the m.3243 A > G mutation compared to wild type cells. We further demonstrate that the low heteroplasmic mutant cells exhibit a coordinate induction of transcriptional regulators of the mitochondrial biogenesis, glucose and fatty acid metabolism pathways that lack in near homoplasmic mutant cells compared to wild type cells. Altogether, these results shed new biological insights on the potential mechanisms by which low mtDNA heteroplasmy may progressively cause diabetes mellitus.

Mitochondrial DNA (mtDNA) 3243A > G tRNALeu((UUR)) heteroplasmic mutation (m.3243A > G) exhibits clinically heterogeneous phenotypes. While the high mtDNA heteroplasmy exceeding a critical threshold causes mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes (MELAS) syndrome, the low mtDNA heteroplasmy causes maternally inherited diabetes with or without deafness (MIDD) syndrome. How quantitative differences in mtDNA heteroplasmy produces distinct pathological states has remained elusive. Here we show that despite striking similarities in the energy metabolic gene expression signature, the mitochondrial bioenergetics, biogenesis and fuel catabolic functions are distinct in cells harboring low or high levels of the m.3243 A > G mutation compared to wild type cells. We further demonstrate that the low heteroplasmic mutant cells exhibit a coordinate induction of transcriptional regulators of the mitochondrial biogenesis, glucose and fatty acid metabolism pathways that lack in near homoplasmic mutant cells compared to wild type cells. Altogether, these results shed new biological insights on the potential mechanisms by which low mtDNA heteroplasmy may progressively cause diabetes mellitus.

Mitochondrial DNA (mtDNA) 3243A > G tRNALeu((UUR)) heteroplasmic mutation (m.3243A > G) exhibits clinically heterogeneous phenotypes. While the high mtDNA heteroplasmy exceeding a critical threshold causes mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes (MELAS) syndrome, the low mtDNA heteroplasmy causes maternally inherited diabetes with or without deafness (MIDD) syndrome. How quantitative differences in mtDNA heteroplasmy produces distinct pathological states has remained elusive. Here we show that despite striking similarities in the energy metabolic gene expression signature, the mitochondrial bioenergetics, biogenesis and fuel catabolic functions are distinct in cells harboring low or high levels of the m.3243 A > G mutation compared to wild type cells. We further demonstrate that the low heteroplasmic mutant cells exhibit a coordinate induction of transcriptional regulators of the mitochondrial biogenesis, glucose and fatty acid metabolism pathways that lack in near homoplasmic mutant cells compared to wild type cells. Altogether, these results shed new biological insights on the potential mechanisms by which low mtDNA heteroplasmy may progressively cause diabetes mellitus.

Mitochondrial DNA (mtDNA) 3243A > G tRNALeu((UUR)) heteroplasmic mutation (m.3243A > G) exhibits clinically heterogeneous phenotypes. While the high mtDNA heteroplasmy exceeding a critical threshold causes mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes (MELAS) syndrome, the low mtDNA heteroplasmy causes maternally inherited diabetes with or without deafness (MIDD) syndrome. How quantitative differences in mtDNA heteroplasmy produces distinct pathological states has remained elusive. Here we show that despite striking similarities in the energy metabolic gene expression signature, the mitochondrial bioenergetics, biogenesis and fuel catabolic functions are distinct in cells harboring low or high levels of the m.3243 A > G mutation compared to wild type cells. We further demonstrate that the low heteroplasmic mutant cells exhibit a coordinate induction of transcriptional regulators of the mitochondrial biogenesis, glucose and fatty acid metabolism pathways that lack in near homoplasmic mutant cells compared to wild type cells. Altogether, these results shed new biological insights on the potential mechanisms by which low mtDNA heteroplasmy may progressively cause diabetes mellitus.

Mitochondrial DNA (mtDNA) 3243A > G tRNALeu((UUR)) heteroplasmic mutation (m.3243A > G) exhibits clinically heterogeneous phenotypes. While the high mtDNA heteroplasmy exceeding a critical threshold causes mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes (MELAS) syndrome, the low mtDNA heteroplasmy causes maternally inherited diabetes with or without deafness (MIDD) syndrome. How quantitative differences in mtDNA heteroplasmy produces distinct pathological states has remained elusive. Here we show that despite striking similarities in the energy metabolic gene expression signature, the mitochondrial bioenergetics, biogenesis and fuel catabolic functions are distinct in cells harboring low or high levels of the m.3243 A > G mutation compared to wild type cells. We further demonstrate that the low heteroplasmic mutant cells exhibit a coordinate induction of transcriptional regulators of the mitochondrial biogenesis, glucose and fatty acid metabolism pathways that lack in near homoplasmic mutant cells compared to wild type cells. Altogether, these results shed new biological insights on the potential mechanisms by which low mtDNA heteroplasmy may progressively cause diabetes mellitus.

Mitochondrial DNA (mtDNA) 3243A > G tRNALeu((UUR)) heteroplasmic mutation (m.3243A > G) exhibits clinically heterogeneous phenotypes. While the high mtDNA heteroplasmy exceeding a critical threshold causes mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes (MELAS) syndrome, the low mtDNA heteroplasmy causes maternally inherited diabetes with or without deafness (MIDD) syndrome. How quantitative differences in mtDNA heteroplasmy produces distinct pathological states has remained elusive. Here we show that despite striking similarities in the energy metabolic gene expression signature, the mitochondrial bioenergetics, biogenesis and fuel catabolic functions are distinct in cells harboring low or high levels of the m.3243 A > G mutation compared to wild type cells. We further demonstrate that the low heteroplasmic mutant cells exhibit a coordinate induction of transcriptional regulators of the mitochondrial biogenesis, glucose and fatty acid metabolism pathways that lack in near homoplasmic mutant cells compared to wild type cells. Altogether, these results shed new biological insights on the potential mechanisms by which low mtDNA heteroplasmy may progressively cause diabetes mellitus.

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Peripheral neuropathy in mitochondrial diseases (MDsBACKGROUND AND PURPOSE: Peripheral neuropathy in mitochondrial diseases (MDs) may vary from a subclinical finding in a multisystem syndrome to a severe, even isolated, manifestation in some patients. METHODS: To investigate the involvement of the peripheral nervous system in MDs extensive electrophysiological studies were performed in 109 patients with morphological, biochemical and genetic diagnosis of MD [12 A3243G progressive external ophthalmoplegia (PEO)/mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), 16 myoclonic epilepsy with ragged-red fibres (MERRF), four mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), 67 PEO with single or multiple deletions of mitochondrial DNA, 10 others]. RESULTS: A neuropathy was found in 49 patients (45%). The incidence was very high in MNGIE (100%), MELAS (92%) and MERRF (69%), whilst 28% of PEO patients had evidence of peripheral involvement. The most frequent abnormality was a sensory axonal neuropathy found in 32/49 patients (65%). A sensory-motor axonal neuropathy was instead detected in 16% of the patients and sensory-motor axonal demyelinating neuropathy in 16%. Finally one Leigh patient had a motor axonal neuropathy. It is interesting to note that the great majority had preserved tendon reflexes and no sensory disturbances. CONCLUSIONS: In conclusion, peripheral involvement in MD is frequent even if often mild or asymptomatic. The correct identification and characterization of peripheral neuropathy through electrophysiological studies represents another tile in the challenge of MD diagnosis.) may vary from a subclinical finding in a multisystem syndrome to a severe, even isolated, manifestation in some patients.

Mitochondria are a source of reactive oxygen species (ROS). Mitochondrial diseases are the result of inherited defects in mitochondrially expressed genes. One potential pathomechanism for mitochondrial disease is oxidative stress. Oxidative stress can occur as the result of increased ROS production or decreased ROS protection. The role of oxidative stress in the five most common inherited mitochondrial diseases, Friedreich ataxia, LHON, MELAS, MERRF, and Leigh syndrome (LS), is discussed. Published reports of oxidative stress involvement in the pathomechanisms of these five mitochondrial diseases are reviewed. The strongest evidence for an oxidative stress pathomechanism among the five diseases was for Friedreich ataxia. In addition, a meta-analysis was carried out to provide an unbiased evaluation of the role of oxidative stress in the five diseases, by searching for "oxidative stress" citation count frequency for each disease. Of the five most common mitochondrial diseases, the strongest support for oxidative stress is for Friedreich ataxia (6.42%), followed by LHON (2.45%), MELAS (2.18%), MERRF (1.71%), and LS (1.03%). The increased frequency of oxidative stress citations was significant relative to the mean of the total pool of five diseases (p<0.01) and the mean of the four non-Friedreich diseases (p<0.0001). Thus there is support for oxidative stress in all five most common mitochondrial diseases, but the strongest, significant support is for Friedreich ataxia.

[EN] The molecular mechanisms underlying human diseases, like MELAS and MERRF syndromes, are caused by mutations in mitochondrial (mt) tRNA genes, like mt-tRNALeu(UUR) and mt-tRNALys genes. These mutations are thought to exert their pathogenic role by hindering the modification of the uridine located at the anticodon wobble position (U34), leading to dysfunction of the oxidative phosphorylation system. Recently, it has being tested the hypothesis that regulation of the mt-tRNA modification enzymes may participate in the pathogenic mechanisms underlying these diseases. In fact, the wobble modification defects in MELAS are accompanied by an unexpected reduction in the steady-state levels of such enzymes. Since ROS is increased in MELAS cells and these species have been hypothesized to cause a microRNA-mediated response in mitochondrial disorders, it has being proved that the under-expression of the tRNA modifying enzymes in MELAS could be a consequence of a microRNA-directed regulation. An in silico analysis provided a group of microRNAs predicted to control the expression of mt-tRNA modifying proteins, eg. microRNA-9/9*.The overall goal of the submitted TFG project is to explore the role of other ROS-sensitive microRNAs in the expression of the mt-tRNA modifying proteins and to determine whether some of them construct specific signatures for each mitochondrial disease associated with mt-tRNA wobble modification defects. Thus, it is propose the following specific aims: -Perform a comparative analysis of the expression of predicted microRNAs in different cell lines carrying mutations in mt-tRNA genes and also compare the expression of the mttRNA modifying enzymes at transcriptional level by RT-qPCR. -Compare the expression of mt-tRNA modifying enzymes at translational level by immunoblotting assays. An overexpression of microRNAs miR-9/9*, miR-338 and miR-335 was observed, which was accompanied by the downregulation of their targets modifying enzymes. Furthermore, it c, [ES] Los mecanismos moleculares causantes de algunas enfermedades, como el síndrome MELAS o MERRF, se deben a mutaciones en genes mitocondriales (mt) de tRNA, como los genes mt-tRNALeu(UUR) y mt-tRNALys. Se piensa que estas mutaciones dificultan la modificación de la uridina situada en la tercera posición del anticodón o posición tambaleante (U34) de estos mt-tRNAs, lo que conduce a la disfunción del sistema de fosforilación oxidativa y a la manifestación de la enfermedad. Recientemente, se ha puesto a prueba la hipótesis de que la regulación de las enzimas modificadoras de mt-tRNAs puede participar en los mecanismos patogénicos que subyacen a estas enfermedades. De hecho, el defecto en la modificacion de la posición tambaleante en MELAS se ve acompañado de una reducción inesperada en los niveles basales de ciertas enzimas modificadoras de mt-tRNAs. Debido a la elevada presencia de especies reactivas del oxígeno (ROS) en células MELAS y su papel en la activación de microRNAs reguladores transcripcionales, se ha podido relacionar la baja expresión de dichas enzimas modificadoras de mt-tRNAs con la regulación directa mediante estos microRNAs sensibles a ROS. Un estudio in silico ha previsto de un grupo de microRNAs candidatos a ser reguladores negativos post-transcripcionales de estas enzimas, como por ejemplo el microRNA-9/9*. El principal objetivo de este TFG es estudiar la implicación de los microRNAs sensibles a ROS en la expresión de las enzimas modificadoras de mt-tRNAs e identificar si la sobreexpresión de estos microRNAS constituye una firma específica para estas enfermedades mitocondriales. Para ello realizarán: - Análisis comparativos mediante RT-qPCR de la expresión de los microRNAs candidatos en distintas líneas celulares portadoras de mutaciones en genes mt-tRNAs y de la expresión de sus enzimas diana a nivel transcripcional. - Ensayos de inmunotransferencia para comparar los niveles de expresión de las enzimas a nivel traduccional. Tras los estudios real

[EN] The molecular mechanisms underlying human diseases, like MELAS and MERRF syndromes, are caused by mutations in mitochondrial (mt) tRNA genes, like mt-tRNALeu(UUR) and mt-tRNALys genes. These mutations are thought to exert their pathogenic role by hindering the modification of the uridine located at the anticodon wobble position (U34), leading to dysfunction of the oxidative phosphorylation system. Recently, it has being tested the hypothesis that regulation of the mt-tRNA modification enzymes may participate in the pathogenic mechanisms underlying these diseases. In fact, the wobble modification defects in MELAS are accompanied by an unexpected reduction in the steady-state levels of such enzymes. Since ROS is increased in MELAS cells and these species have been hypothesized to cause a microRNA-mediated response in mitochondrial disorders, it has being proved that the under-expression of the tRNA modifying enzymes in MELAS could be a consequence of a microRNA-directed regulation. An in silico analysis provided a group of microRNAs predicted to control the expression of mt-tRNA modifying proteins, eg. microRNA-9/9*.The overall goal of the submitted TFG project is to explore the role of other ROS-sensitive microRNAs in the expression of the mt-tRNA modifying proteins and to determine whether some of them construct specific signatures for each mitochondrial disease associated with mt-tRNA wobble modification defects. Thus, it is propose the following specific aims: -Perform a comparative analysis of the expression of predicted microRNAs in different cell lines carrying mutations in mt-tRNA genes and also compare the expression of the mttRNA modifying enzymes at transcriptional level by RT-qPCR. -Compare the expression of mt-tRNA modifying enzymes at translational level by immunoblotting assays. An overexpression of microRNAs miR-9/9*, miR-338 and miR-335 was observed, which was accompanied by the downregulation of their targets modifying enzymes. Furthermore, it c, [ES] Los mecanismos moleculares causantes de algunas enfermedades, como el síndrome MELAS o MERRF, se deben a mutaciones en genes mitocondriales (mt) de tRNA, como los genes mt-tRNALeu(UUR) y mt-tRNALys. Se piensa que estas mutaciones dificultan la modificación de la uridina situada en la tercera posición del anticodón o posición tambaleante (U34) de estos mt-tRNAs, lo que conduce a la disfunción del sistema de fosforilación oxidativa y a la manifestación de la enfermedad. Recientemente, se ha puesto a prueba la hipótesis de que la regulación de las enzimas modificadoras de mt-tRNAs puede participar en los mecanismos patogénicos que subyacen a estas enfermedades. De hecho, el defecto en la modificacion de la posición tambaleante en MELAS se ve acompañado de una reducción inesperada en los niveles basales de ciertas enzimas modificadoras de mt-tRNAs. Debido a la elevada presencia de especies reactivas del oxígeno (ROS) en células MELAS y su papel en la activación de microRNAs reguladores transcripcionales, se ha podido relacionar la baja expresión de dichas enzimas modificadoras de mt-tRNAs con la regulación directa mediante estos microRNAs sensibles a ROS. Un estudio in silico ha previsto de un grupo de microRNAs candidatos a ser reguladores negativos post-transcripcionales de estas enzimas, como por ejemplo el microRNA-9/9*. El principal objetivo de este TFG es estudiar la implicación de los microRNAs sensibles a ROS en la expresión de las enzimas modificadoras de mt-tRNAs e identificar si la sobreexpresión de estos microRNAS constituye una firma específica para estas enfermedades mitocondriales. Para ello realizarán: - Análisis comparativos mediante RT-qPCR de la expresión de los microRNAs candidatos en distintas líneas celulares portadoras de mutaciones en genes mt-tRNAs y de la expresión de sus enzimas diana a nivel transcripcional. - Ensayos de inmunotransferencia para comparar los niveles de expresión de las enzimas a nivel traduccional. Tras los estudios real

[EN] The molecular mechanisms underlying human diseases, like MELAS and MERRF syndromes, are caused by mutations in mitochondrial (mt) tRNA genes, like mt-tRNALeu(UUR) and mt-tRNALys genes. These mutations are thought to exert their pathogenic role by hindering the modification of the uridine located at the anticodon wobble position (U34), leading to dysfunction of the oxidative phosphorylation system. Recently, it has being tested the hypothesis that regulation of the mt-tRNA modification enzymes may participate in the pathogenic mechanisms underlying these diseases. In fact, the wobble modification defects in MELAS are accompanied by an unexpected reduction in the steady-state levels of such enzymes. Since ROS is increased in MELAS cells and these species have been hypothesized to cause a microRNA-mediated response in mitochondrial disorders, it has being proved that the under-expression of the tRNA modifying enzymes in MELAS could be a consequence of a microRNA-directed regulation. An in silico analysis provided a group of microRNAs predicted to control the expression of mt-tRNA modifying proteins, eg. microRNA-9/9*.The overall goal of the submitted TFG project is to explore the role of other ROS-sensitive microRNAs in the expression of the mt-tRNA modifying proteins and to determine whether some of them construct specific signatures for each mitochondrial disease associated with mt-tRNA wobble modification defects. Thus, it is propose the following specific aims: -Perform a comparative analysis of the expression of predicted microRNAs in different cell lines carrying mutations in mt-tRNA genes and also compare the expression of the mttRNA modifying enzymes at transcriptional level by RT-qPCR. -Compare the expression of mt-tRNA modifying enzymes at translational level by immunoblotting assays. An overexpression of microRNAs miR-9/9*, miR-338 and miR-335 was observed, which was accompanied by the downregulation of their targets modifying enzymes. Furthermore, it c, [ES] Los mecanismos moleculares causantes de algunas enfermedades, como el síndrome MELAS o MERRF, se deben a mutaciones en genes mitocondriales (mt) de tRNA, como los genes mt-tRNALeu(UUR) y mt-tRNALys. Se piensa que estas mutaciones dificultan la modificación de la uridina situada en la tercera posición del anticodón o posición tambaleante (U34) de estos mt-tRNAs, lo que conduce a la disfunción del sistema de fosforilación oxidativa y a la manifestación de la enfermedad. Recientemente, se ha puesto a prueba la hipótesis de que la regulación de las enzimas modificadoras de mt-tRNAs puede participar en los mecanismos patogénicos que subyacen a estas enfermedades. De hecho, el defecto en la modificacion de la posición tambaleante en MELAS se ve acompañado de una reducción inesperada en los niveles basales de ciertas enzimas modificadoras de mt-tRNAs. Debido a la elevada presencia de especies reactivas del oxígeno (ROS) en células MELAS y su papel en la activación de microRNAs reguladores transcripcionales, se ha podido relacionar la baja expresión de dichas enzimas modificadoras de mt-tRNAs con la regulación directa mediante estos microRNAs sensibles a ROS. Un estudio in silico ha previsto de un grupo de microRNAs candidatos a ser reguladores negativos post-transcripcionales de estas enzimas, como por ejemplo el microRNA-9/9*. El principal objetivo de este TFG es estudiar la implicación de los microRNAs sensibles a ROS en la expresión de las enzimas modificadoras de mt-tRNAs e identificar si la sobreexpresión de estos microRNAS constituye una firma específica para estas enfermedades mitocondriales. Para ello realizarán: - Análisis comparativos mediante RT-qPCR de la expresión de los microRNAs candidatos en distintas líneas celulares portadoras de mutaciones en genes mt-tRNAs y de la expresión de sus enzimas diana a nivel transcripcional. - Ensayos de inmunotransferencia para comparar los niveles de expresión de las enzimas a nivel traduccional. Tras los estudios real

Las enfermedades mitocondriales son un grupo heterogéneo de trastornos en la fosforilación oxidativa, que se expresan según la naturaleza de la mutación, el fenotipo de las mitocondrias, el grado de complementación con el mtDNA normal y tejidos afectados. El diagnóstico se basa en la sospecha clínica y en la corroboración de fibras rojo rasgadas en la biopsia muscular con tricrómico de Gomori, entre otras técnicas. Presentamos un caso de una mujer de 41 años, con antecedentes de diabetes mellitus insulinodependiente, hipoacusia neurosensorial bilateral, intolerancia al ejercicio y debilidad muscular, que sufre un cuadro pseudovascular, con aumento del ácido láctico en sangre y LCR. TC y RM cerebral indican lesión isquémica temporoparietal derecha, y biopsia evidencia miopatía mitocondrial. Destacamos la importancia del diagnóstico diferencial de enfermedad cerebrovascular en adulto joven.

[Backgroun and Purpose]: MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is a mitochondrial disease most usually caused by point mutations in tRNA genes encoded by mitochondrial DNA (mtDNA). Approximately 80% of cases of MELAS syndrome are associated with a m.3243A > G mutation in the MT-TL1 gene, which encodes the mitochondrial tRNALeu (UUR). Currently, no effective treatments are available for this chronic progressive disorder. Treatment strategies in MELAS and other mitochondrial diseases consist of several drugs that diminish the deleterious effects of the abnormal respiratory chain function, reduce the presence of toxic agents or correct deficiencies in essential cofactors., [Experimental Approach]: We evaluated the effectiveness of some common pharmacological agents that have been utilized in the treatment of MELAS, in yeast, fibroblast and cybrid models of the disease. The yeast model harbouring the A14G mutation in the mitochondrial tRNALeu(UUR) gene, which is equivalent to the A3243G mutation in humans, was used in the initial screening. Next, the most effective drugs that were able to rescue the respiratory deficiency in MELAS yeast mutants were tested in fibroblasts and cybrid models of MELAS disease., [Key Results]: According to our results, supplementation with riboflavin or coenzyme Q10 effectively reversed the respiratory defect in MELAS yeast and improved the pathologic alterations in MELAS fibroblast and cybrid cell models., [Conclusions and Implications]: Our results indicate that cell models have great potential for screening and validating the effects of novel drug candidates for MELAS treatment and presumably also for other diseases with mitochondrial impairment.

Oxidative phosphorylation disorders are often associated with increased oxidative stress and antioxidant therapy is frequently given as treatment. However, the role of oxidative stress in oxidative phosphorylation disorders or patients is far from clear and consequently the preventive or therapeutic effect of antioxidants is highly anecdotic. Therefore, we performed a systematic study of a panel of oxidative stress parameters (reactive oxygen species levels, damage and defense) in fibroblasts of twelve well-characterized oxidative phosphorylation patients with a defect in the POLG1 gene, in the mitochondrial DNA-encoded tRNA-Leu gene (m.3243A > G or m.3302A > G) and in one of the mitochondrial DNA-encoded NADH dehydrogenase complex I (CI) subunits. All except two cell lines (one POLG1 and one tRNA-Leu) showed increased reactive oxygen species levels compared with controls, but only four (two CI and two tRNA-Leu) cell lines provided evidence for increased oxidative protein damage. The absence of a correlation between reactive oxygen species levels and oxidative protein damage implies differences in damage prevention or correction. This was investigated by gene expression studies, which showed adaptive and compensating changes involving antioxidants and the unfolded protein response, especially in the POLG1 group. This study indicated that patients display individual responses and that detailed analysis of fibroblasts enables the identification of patients that potentially benefit from antioxidant therapy. Furthermore, the fibroblast model can also be used to search for and test novel, more specific antioxidants or explore ways to stimulate compensatory mechanisms.

Med musiken till hands – Funktionsinriktad musikterapi med två ungdomar, en med Cerebral Pares och en med mitokondriell sjukdom. Funktionsinriktad musikterapi, FMT-metoden, är en neuromuskulär behandlingsmodell för barn och vuxna med olika funktionsnedsättningar. Jag har valt att i detta examensarbete för-klara vad metoden går ut på samt beskriva arbetet med två av mina elever, en med diagnosen Cerebral Pares och en med mitokondriell sjukdom. Jag har förklarat hur deras helhetsutveckling, främst arm-/handfunktionen, har påverkats. Båda ungdomarna har fått en utökad förmåga att greppa om olika föremål och sträcka ut för att nå ting som står långt ifrån dem.

Med musiken till hands – Funktionsinriktad musikterapi med två ungdomar, en med Cerebral Pares och en med mitokondriell sjukdom. Funktionsinriktad musikterapi, FMT-metoden, är en neuromuskulär behandlingsmodell för barn och vuxna med olika funktionsnedsättningar. Jag har valt att i detta examensarbete för-klara vad metoden går ut på samt beskriva arbetet med två av mina elever, en med diagnosen Cerebral Pares och en med mitokondriell sjukdom. Jag har förklarat hur deras helhetsutveckling, främst arm-/handfunktionen, har påverkats. Båda ungdomarna har fått en utökad förmåga att greppa om olika föremål och sträcka ut för att nå ting som står långt ifrån dem.

MELAS is commonly associated with peripheral hearing loss. Auditory agnosia is a rare cortical auditory impairment, usually due to bilateral temporal damage. We document, for the first time, auditory agnosia as the presenting hearing disorder in MELAS. A young woman with MELAS (A3243G mtDNA mutation) suffered from acute cortical hearing damage following a single stroke-like episode, in the absence of previous hearing deficits. Audiometric testing showed marked central hearing impairment and very mild sensorineural hearing loss. MRI documented bilateral, acute lesions to superior temporal regions. Neuropsychological tests demonstrated auditory agnosia without aphasia. Our data and a review of published reports show that cortical auditory disorders are relatively frequent in MELAS, probably due to the strikingly high incidence of bilateral and symmetric damage following stroke-like episodes. Acute auditory agnosia can be the presenting hearing deficit in MELAS and, conversely, MELAS should be suspected in young adults with sudden hearing loss.

The mitochondrial oxidative phosphorylation system (OXPHOS) generates energy but also deleterious reactive oxygen species (ROS). Changes in the cytoskeleton, composed mainly of microfilaments, microtubules and intermediate filaments, have been observed in OXPHOS deficiency. The 3243A>G point mutation in mitochondrial DNA (mtDNA) leads to mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), which is the most common mitochondrial disease. Interestingly, mitochondrial aberrations have been demonstrated in patients with a mutation in NOTCH3, the genetic cause of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Randomization of vimentin intermediate filament direction and length together with slower population growth was observed in myoblasts with 3243A>G, with no difference in the amount of apoptotic cell death. Upon complex IV inhibition (with or without the microtubule-depolymerizing compound nocodazole) or a lack of mtDNA (ρ0) in osteosarcoma cells the vimentin network collapsed perinuclearly, forming thick bundles, whereas complex I inhibition led to thinner vimentin network bundles. Furthermore, the amount of vimentin was increased in ρ0 cells. Mitochondria accumulated around the nucleus upon complex IV inhibition and in ρ0 cells. Analysis of the total proteome revealed that specific OXPHOS deficiencies led to changes in the expression of cytoskeletal proteins and proteins involved in apoptosis, OXPHOS, glycolysis and oxidative stress response. Muscle histochemical and genetic analysis showed ragged red fibres and cytochrome c oxidase-negative fibres to be associated with 5650G>A in a patient with R133C in NOTCH3 and 5650G>A in MTTA. Immunolabelling of cells with R133C and 5650G>A revealed a sparse tubulin network with asters and less abundant mitochondria by comparison with control cell lines. Comparison of nucleotide diversity between CADASIL pedigrees and controls showed

The mitochondrial DNA A3243G mutation causes neuromuscular disease. To investigate the muscle-specific pathophysiology of mitochondrial disease, rhabdomyosarcoma transmitochondrial hybrid cells (cybrids) were generated that retain the capacity to differentiate to myotubes. In some cases, striated muscle-like fibres were formed after innervation with rat embryonic spinal cord. Myotubes carrying A3243G mtDNA produced more reactive oxygen species than controls, and had altered glutathione homeostasis. Moreover, A3243G mutant myotubes showed evidence of abnormal mitochondrial distribution, which was associated with down-regulation of three genes involved in mitochondrial morphology, Mfn1, Mfn2 and DRP1. Electron microscopy revealed mitochondria with ultrastructural abnormalities and paracrystalline inclusions. All these features were ameliorated by anti-oxidant treatment, with the exception of the paracrystalline inclusions. These data suggest that rhabdomyosarcoma cybrids are a valid cellular model for studying muscle-specific features of mitochondrial disease and that excess reactive oxygen species production is a significant contributor to mitochondrial dysfunction, which is amenable to anti-oxidant therapy.

The 3243A>G mutation in mitochondrial DNA (mtDNA), the most common cause of the syndrome of mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes, is also associated with many other phenotypes such as hearing loss, diabetes mellitus, epilepsy, cognitive decline, myopathy and cardiomyopathy. The prevalence of the mutation has been shown to be 16.3/100 000 adults in Northern Finland. The present study was performed to estimate the frequency and progression of cardiac abnormalities and to examine causes of death in patients with 3243A>G. Left ventricular hypertrophy (LVH) was found in echocardiography in 56% of patients with 3243A>G and in 15% of age and sex-matched controls. The median thickness of the diastolic interventricular septum or posterior wall was 14 mm in the patients with LVH. The prevalence of LVH determined by echocardiography increased from 40% to 56% in 25 patients with 3243A>G during three years of follow-up, this trend being especially marked among the diabetic patients. The ultra-low-frequency (ULF) and very-low-frequency (VLF) components of the spectral analysis of heart rate variability (HRV) were lower among the patients with 3243A>G than in matched controls (p = 0.02 in ULF and p = 0.04 in VLF), and the short-term fractal scaling exponent in detrended fluctuation analysis of HRV was lower in the patients with 3243A>G (1.16 ± 0.18 vs. 1.28 ± 0.13) (p < 0.01). Survival analysis of a birth cohort from pedigrees with 3243A>G revealed excess mortality before the age of 50 years. Neurological and cardiovascular diseases accounted for 32% of all the underlying causes of death in families with 3243A>G. Death was sudden and unexpected in 31% of cases in which 3243A>G was considered to be involved in the cause of death. The results show that cardiac abnormalities are frequent and progressive in patients with the 3243A>G mtDNA mutation and that cardiac autonomic regulation is disturbed. Patients with the 3243A>G mutation and

Human mitochondria contain a circular genome called mitochondrial DNA (mtDNA). It encodes subunits of the respiratory chain enzymes involved in energy conservation in oxidative phosphorylation and the necessary RNA needed for their expression. Errors in these genes have been shown to cause diseases, called mitochondrial diseases, which mainly affect tissues with high energy-demand, such as brain, heart, and skeletal muscle, or to lead to the production of harmful by-products in the form of reactive oxygen species (ROS) during cellular respiration. ROS damage lipids, proteins, and DNA, especially mtDNA. Accumulation of mtDNA mutations has also been associated with aging. Mitochondrial complex I is located in the inner mitochondrial membrane and catalyzes NADH-ubiquinone oxidoreduction coupled to the translocation of four protons from the inside of the mitochondrion to the intermembranous space. Bacteria contain a homologous but simpler enzyme, NDH-1, with the same catalytic mechanism and which is therefore considered the catalytical core of mitochondrial complex I. Seven of the conserved membranous subunits in complex I are encoded in the mtDNA and are targets for mutations causing mitochondrial diseases, like MELAS syndrome or Leber hereditary optic neuropathy (LHON). We used Paracoccus denitrificans and Escherichia coli NDH-1 enzymes to reveal the role of selected conserved charged residues and MELAS or LHON amino acid substitutions in enzyme catalysis. The growth phenotypes and NDH-1-dependent activities in mutant bacterial membranes were characterized, in addition to the sensitivity to selected complex I inhibitors. In order to enable ROS production measurements in the bacterial model of human mitochondrial diseases, we evaluated the reliability of two superoxide detecting probes, lucigenin and coelenterazine. Elimination of the acidic residue in ND1 (position E228) previously found to cause MELAS, was found detrimental for NDH-1 assembly and activity. A

El Síndrome MELAS es una enfermedad mitocondrial cuyas características clínicas precisas y pruebas complementarias útiles para el diagnóstico aún son controvertidas. Puede simular una encefalitis herpética. Los niveles de ácido láctico en sangre y líquido cefalorraquídeo (elevados), las pruebas de neuroimagen (imágenes que simulan un infarto cerebral pero superan un territorio vascular), el electroencefalograma (enlentecimiento de fondo con posibles descargas periódicas lateralizadas), la biopsia muscular (fibras rojo rasgadas positivas para actividad COX) y los test de genética molecular (mutación puntual en el ADN mitocondrial) son útiles en el diagnóstico, pero ante la ausencia de hallazgos no se puede rechazar el diagnóstico de síndrome MELAS, tal y como sucede en el caso comunicado.

El Síndrome MELAS es una enfermedad mitocondrial cuyas características clínicas precisas y pruebas complementarias útiles para el diagnóstico aún son controvertidas. Puede simular una encefalitis herpética. Los niveles de ácido láctico en sangre y líquido cefalorraquídeo (elevados), las pruebas de neuroimagen (imágenes que simulan un infarto cerebral pero superan un territorio vascular), el electroencefalograma (enlentecimiento de fondo con posibles descargas periódicas lateralizadas), la biopsia muscular (fibras rojo rasgadas positivas para actividad COX) y los test de genética molecular (mutación puntual en el ADN mitocondrial) son útiles en el diagnóstico, pero ante la ausencia de hallazgos no se puede rechazar el diagnóstico de síndrome MELAS, tal y como sucede en el caso comunicado.

Two mutations (G8363A and A8296G) in the mtDNA (mitochondrial DNA) tRNA(Lys) gene have been associated with severe mitochondrial diseases in a number of reports. Their functional significance, however, remains unknown. We have already shown that homoplasmic cybrids harbouring the A8296G mutation display normal oxidative phosphorylation, although the possibility of a subtle change in mitochondrial respiratory capacity remains an open issue. We have now investigated the pathogenic mechanism of another mutation in the tRNA(Lys) gene (G8363A) by repopulating an mtDNA-less human osteosarcoma cell line with mitochondria harbouring either this genetic variant alone or an unusual combination of the two mutations (A8296G+G8363A). Cybrids homoplasmic for the single G8363A or the A8296G+G8363A mutations have defective respiratory-chain enzyme activities and low oxygen consumption, indicating a severe impairment of the oxidative phosphorylation system. Generation of G8363A cybrids within a wild-type or the A8296G mtDNA genetic backgrounds resulted in an important alteration in the conformation of the tRNA(Lys), not affecting tRNA steady-state levels. Moreover, mutant cybrids have an important decrease in the proportion of amino-acylated tRNA(Lys) and, consequently, mitochondrial protein synthesis is greatly decreased. Our results demonstrate that the pathogenicity of the G8363A mutation is due to a change in the conformation of the tRNA that severely impairs aminoacylation in the absence of changes in tRNA stability. The only effect detected in the A8296G mutation is a moderate decrease in the aminoacylation capacity, which does not affect mitochondrial protein biosynthesis.

Neurological features are common in mitochondrial diseases because tissues depending upon oxidative phosphorylation bear the brunt of the pathogenesis. The 3243A>G mutation in the MTTL1 gene in mitochondrial DNA is regarded as the most frequent mitchondrial point mutation and classically presents with mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS). Myopathy and peripheral neuropathy have been documented in patients with mitochondrial diseases, but not properly characterised in patients with the 3243A>G mutation. We have previously determined the prevalence of patients with this mutation in a defined population in northern Finland. The clinical spectrum and molecular aspects of myopathy and peripheral neuropathy are analysed here in a population-based cohort of patients with 3243A>G. Fifty patients were examined neurologically in order to define the frequency of myopathy and its histological, ultrastructural and clinical features. The frequency and phenotypic variability of peripheral neuropathy were determined in 32 patients and muscle computed tomography findings recorded in 24 patients. Finally, variations in mutation heteroplasmy were analysed in 10 patients using single muscle fibre PCR analysis. The frequency of peripheral neuropathy was 22% (95% confidence interval (CI), 9–40%) and that of clinical myopathy 50% (95% CI, 36–64%). Moderate limb weakness was the most common myopathic feature, but mild weakness and external ophthalmoplegia were also present. CT scans revealed myopathic changes in 54% of the patients (95% CI, 33–76%), most frequently in the pelvic muscles. The incidence of myopathy was highest in the fifth decade of life, and higher age and male gender increased the risk of neuropathy. Muscle histology was abnormal in 72% of the cases examined (95% CI, 55–86%). The presence of intramitochondrial crystals and COX-negative fibres and variations in the size and shape of mitochondria were more common in the musc

In most cases of the mitochondrial encephalomyopathies, the mutations of mtDNA usually appear in heteroplasmic states. The degree of mtDNA heteroplasmy has been suggested to play an important role in determining the clinical phenotypes and the organspecific defects. We devised a novel method for quantitative analysis of heteroplasmy using PCR-SSCP, which is useful to accurately quantitate heteroplasmy of very small amount of samples. Using this method, we analyzed the heteroplasmy of skeletal muscles or leucocytes from 12 cases of MERRF, 5 cases of MELAS, and 1 case of MERRF/MELAS overlap syndrome associated with 3243 tRNA^ mutation. We furthermore analyzed 5 autopsied cases (2 MERRF and 2 MELAS, 1 MERRF/MELAS overlap syndrome patients), and the cellular or organellar distributions of heteroplasmy of CNS in MERRF patients. Though there is a tendency that patients with high percentages of mutant mtDNAs in their skeletal muscles show severer clinical symptoms and earlier onset than those with low percentages, no obvious correlation was observed between the phenotype and genotype of leukocyte in MERRF and MELASpatients. And the percentages of mutant mtDNA were similarly high in both clinically affected and unaffected tissues in 2 MERRF, 2 MELAS, and 1 MERRF/MELAS overlap syndrome patients. Single muscle fiber analysis, however, revealed that the proportion of mutant mtDNA was significantly higher in ragged-red fibers than in non-ragged-red fibers.

Four members of a family were found to carry the A3243G mtDNA mutation. Clinical features varied from typical MELAS to myoclonic epilepsy to simple deafness without neurological signs. Several other members of the family had symptoms consistent with a mitochondrial disease. Muscle biopsy in 3 of the 4 patients showed the most prominent mitochondrial alterations with partial deficiency of cytochrome c oxidase in the case with the mildest phenotype. Mitochondrial DNA analysis detected a variable percentage of A3243G mutation, roughly correlating with the phenotype. The interesting feature of the family lies in the great intrafamilial variability of the severity of clinical expression, encompassing MELAS and MERRF features, associated with the A3243G mtDNA mutation. A search for the most common mtDNA mutations is recommended in all patients featuring incomplete MELAS or MERRF syndromes and in all familial cases presenting minimal clinical signs.

We performed morphological, biochemical, and genetic studies, including single-fiber PCR (sf PCR), on muscle biopsies obtained from a mother and daughter with MELAS syndrome due to the A3243G transition of mitochondrial DNA (mtDNA). The severity of muscle involvement appeared quite distinct, in spite of the fact that both patients segregated similar mutant mtDNA levels on total muscle DNA. The daughter did not show any clinical muscle involvement: muscle biopsy revealed many ragged red fibers (RRFs) mostly positive for cytochrome-c oxidase (COX) activity. In contrast, her mother had developed a generalized myopathy without progressive external ophthalmoplegia (PEO), morphologically characterized by many COX-negative RRFs. Single-muscle fiber PCR demonstrated in both patients significantly higher percentages of wild-type mtDNA in normal fibers (daughter: 23.25 ± 15.22; mother: 43.13 ± 26.11) than in COX-positive RRFs (daughter: 11.25 ± 5.22, P < 0.005; mother: 9.12 ± 5.9, P < 0.001) and in COX-negative RRFs (daughter: 8.9 ± 4.2, P < 0.001 mother: 4.8 ± 2.8, P < 0.001). Wild-type mtDNA levels resulted higher also in COX-positive vs. COX-negative RRFs (daughter: P < 0.05; mother: P < 0.001). Our data confirm a direct correlation between A3243G levels and impairment of COX function at the single-muscle fiber level. Moreover, the evidence of a clinical myopathy in the patient with higher amounts of COX-negative RRFs bolsters the concept that a differential distribution of mutant mtDNAs at the cellular level may have effects on the clinical involvement of individual tissues. However, the occurrence of a similar morphological and biochemical muscle phenotype also in PEO3243 patients suggests that other genetic factors involved in the interaction between mitochondrial and nuclear DNA, rather than the stochastic distribution of mtDNA genomes during embryogenesis, are primarily implicated in determining the various clinical expressions of the A3243G of mtDNA

Mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) is commonly associated with an A → G transition at position 3243 of the mitochondrial DNA. To determine the diversity of clinical syndromes associated with this mutation, 91 patients with mitochondrial encephalomyopathies that did not conform to the MELAS phenotype were screened. Twenty one patients with the 3243 mutation, most of whom had progressive external ophthalmoplegia (PEO) were found. Clinical features did not distinguish PEO patients with the 3243 mutation from those with large-scale deletions of mtDNA. However, most cases with single large-scale mtDNA deletions were sporadic, whereas most patients with the 3243 mutation had affected maternal relatives. Histochemical studies of muscle showed that cytochrome c oxidase (COX) deficiency was more severe in patients with PEO than in patients with typical MELAS, even though PEO patients had a lower percentage of mutant genomes in muscle. These data imply that the 3243 mutation is a major cause of familial PEO, and suggests that the threshold number of mtDNAs harboring the 3243 mutation necessary to affect a particular tissue vary in different patients. The proportion of mutant genomes in combination with other, still undefined, tissue-specific modulating factors seem to determine the overall clinical syndrome. © 1993.

Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) is a multisystemic mitochondrial disorder (Pavlakis et al. Advances in Contemporary Neurology. Philadelphia: Davis, 1988: 95-133) and most patients with the typical MELAS phenotype have a point mutation in mitochondrial DNA, an A to G transition at nucleotide 3243 (Goto et al. Nature 1990; 348: 651-653; Koboyashi et al. Biochem Biophys Res Commun 1990; 173: 816-822; Ciafaloni et al. Ann Neurol 1992; 31: 391-398). A 9-yr-old boy presenting with chronic asthma and depression was found to have abnormal mitochondria, partial defects of respiratory chain enzymes, and the MELAS point mutation. © 1993.

We studied 23 patients with clinically defined mitochondrial encephalomyopathy, lactic acidosis, and stroke‐like episodes (MELAS), 25 oligosymptomatic or asymptomatic maternal relatives, and 50 mitochondrial disease control subjects for the presence of a previously reported heteroplasmic point mutation at nt 3,243 in the transfer RNALeu(UUR) gene of mitochondrial DNA. We found a high concordance between clinical diagnosis of MELAS and transfer RNALeu(UUR) mutation, which was present in 21 of the 23 patients with MELAS, all 11 oligosymptomatic and 12 of 14 asymptomatic relatives, but in only five of 50 patients without MELAS. The proportion of mutant genomes in muscle ranged from 56 to 95% and was significantly higher in the patients with MELAS than in their oligosymptomatic or asymptomatic relatives. In subjects in whom both muscle and blood were studied, the percentage of mutations was significantly lower in blood and was not detected in three of 12 asymptomatic relatives. The activities of complexes I + III, II + III, and IV were decreased in muscle biopsies harboring the mutation, but there was no clear correlation between percentage of mutant mitochondrial DNAs and severity of the biochemical defect. Copyright © 1992 American Neurological Association

We documented the presence of a newly described point mutation in the tRNA Leu(UUR) gene of mitochondrial DNA in five postmortem tissues from a patient with MELAS syndrome. The mutation was heteroplasmic, but the percentage of mutant genomes was similar (79 to 88%) in both clinically affected and unaffected tissues. © 1991 American Academy of Neurology.

© 2018, The Author(s). Objective: To examine the efficacy and safety of the therapeutic regimen using oral and intravenous l-arginine for pediatric and adult patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Methods: In the presence and absence of an ictus of stroke-like episodes within 6 h prior to efficacy assessment, we correspondingly conducted the systematic administration of oral and intravenous l-arginine to 15 and 10 patients with MELAS in two, 2-year, prospective, multicenter clinical trials at 10 medical institutions in Japan. Subsequently, patients were followed up for 7 years. The primary endpoint in the clinical trial of oral l-arginine was the MELAS scale, while that for intravenous l-arginine was the improvement rates of headache and nausea/vomiting at 2 h after completion of the initial intravenous administration. The relationships between the ictuses of stroke-like episodes and plasma arginine concentrations were examined. Results: Oral l-arginine extended the interictal phase (p = 0.0625) and decreased the incidence and severity of ictuses. Intravenous l-arginine improved the rates of four major symptoms—headache, nausea/vomiting, impaired consciousness, and visual disturbance. The maximal plasma arginine concentration was 167 μmol/L when an ictus developed. Neither death nor bedriddenness occurred during the 2-year clinical trials, and the latter did not develop during the 7-year follow-up despite the progressively neurodegenerative and eventually life-threatening nature of MELAS. No treatment-related adverse events occurred, and the formulations of l-arginine were well tolerated. Conclusions: The systematic administration of oral and intravenous l-arginine may be therapeutically beneficial and clinically useful for patients with MELAS.

Official receipt for the transportation of tax wheat made out in the name of Melas, http://name.umdl.umich.edu/IC-APIS-X-1161%5D9924O.TIF, https://quod.lib.umich.edu/cgi/i/image/api/thumb/apis/1161/9924O.TIF/!250,250, http://quod.lib.umich.edu/a/apis?page=copyright, https://www.lib.umich.edu/about-us/policies/copyright-policy

Official receipt for the transportation of tax wheat made out in the name of Melas, http://name.umdl.umich.edu/IC-APIS-X-1161%5D9924O.TIF, https://quod.lib.umich.edu/cgi/i/image/api/thumb/apis/1161/9924O.TIF/!250,250, http://quod.lib.umich.edu/a/apis?page=copyright, https://www.lib.umich.edu/about-us/policies/copyright-policy

Official receipt for the transportation of tax wheat made out in the name of Melas, http://name.umdl.umich.edu/IC-APIS-X-1161%5D9924O.TIF, https://quod.lib.umich.edu/cgi/i/image/api/thumb/apis/1161/9924O.TIF/!250,250, http://quod.lib.umich.edu/a/apis?page=copyright, https://www.lib.umich.edu/about-us/policies/copyright-policy