RESUMEN
Mitochondrial disease is a devastating genetic disorder, with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and m.3243A>G being the most common phenotype and genotype, respectively. The treatment for MELAS patients is still less effective. Here, we performed transcriptomic and proteomic analysis in muscle tissue of MELAS patients, and discovered that the expression of molecules involved in serine catabolism were significantly upregulated, and serine hydroxymethyltransferase 2 (SHMT2) increased significantly in both the mRNA and protein levels. The SHMT2 protein level was also increased in myoblasts with m.3243A>G mutation, which was transdifferentiated from patients derived fibroblasts, accompanying with the decreased nicotinamide adenine dinucleotide (NAD+)/reduced NAD+ (NADH) ratio and cell viability. After treating with SHMT2 inhibitor (SHIN1), the NAD+/NADH ratio and cell viability in MELAS myoblasts increased significantly. Taken together, our study indicates that enhanced serine catabolism plays an important role in the pathogenesis of MELAS and that SHIN1 can be a potential small molecule for the treatment of this disease.
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Glicina Hidroximetiltransferasa , Síndrome MELAS , Serina , Humanos , Síndrome MELAS/metabolismo , Síndrome MELAS/genética , Síndrome MELAS/patología , Glicina Hidroximetiltransferasa/metabolismo , Glicina Hidroximetiltransferasa/genética , Serina/metabolismo , Mioblastos/metabolismo , NAD/metabolismo , Masculino , Proteómica/métodos , Femenino , Transcriptoma , MultiómicaRESUMEN
OBJECTIVES AND SCOPE: Primary mitochondrial diseases (PMDs) are rare genetic disorders resulting from mutations in genes crucial for effective oxidative phosphorylation (OXPHOS) that can affect mitochondrial function. In this review, we examine the bioenergetic alterations and oxidative stress observed in cellular models of primary mitochondrial diseases (PMDs), shedding light on the intricate complexity between mitochondrial dysfunction and cellular pathology. We explore the diverse cellular models utilized to study PMDs, including patient-derived fibroblasts, induced pluripotent stem cells (iPSCs) and cybrids. Moreover, we also emphasize the connection between oxidative stress and neuroinflammation. INSIGHTS: The central nervous system (CNS) is particularly vulnerable to mitochondrial dysfunction due to its dependence on aerobic metabolism and the correct functioning of OXPHOS. Similar to other neurodegenerative diseases affecting the CNS, individuals with PMDs exhibit several neuroinflammatory hallmarks alongside neurodegeneration, a pattern also extensively observed in mouse models of mitochondrial diseases. Based on histopathological analysis of postmortem human brain tissue and findings in mouse models of PMDs, we posit that neuroinflammation is not merely a consequence of neurodegeneration but a potential pathogenic mechanism for disease progression that deserves further investigation. This recognition may pave the way for novel therapeutic strategies for this group of devastating diseases that currently lack effective treatments. SUMMARY: In summary, this review provides a comprehensive overview of bioenergetic alterations and redox imbalance in cellular models of PMDs while underscoring the significance of neuroinflammation as a potential driver in disease progression.
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Metabolismo Energético , Enfermedades Mitocondriales , Enfermedades Neuroinflamatorias , Estrés Oxidativo , Humanos , Estrés Oxidativo/fisiología , Enfermedades Mitocondriales/fisiopatología , Enfermedades Mitocondriales/metabolismo , Enfermedades Neuroinflamatorias/fisiopatología , Enfermedades Neuroinflamatorias/metabolismo , Animales , Metabolismo Energético/fisiología , Fosforilación Oxidativa , Ratones , Mitocondrias/metabolismo , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Enfermedad de Leigh/metabolismo , Enfermedad de Leigh/genética , Enfermedad de Leigh/fisiopatología , Síndrome MELAS/metabolismo , Síndrome MELAS/fisiopatología , Síndrome MELAS/genética , Modelos Animales de EnfermedadRESUMEN
ABSTRACT: Pseudouridine is the most prevalent RNA modification, and its aberrant function is implicated in various human diseases. However, the specific impact of pseudouridylation on hematopoiesis remains poorly understood. Here, we investigated the role of transfer RNA (tRNA) pseudouridylation in erythropoiesis and its association with mitochondrial myopathy, lactic acidosis, and sideroblastic anemia syndrome (MLASA) pathogenesis. By using patient-specific induced pluripotent stem cells (iPSCs) carrying a genetic pseudouridine synthase 1 (PUS1) mutation and a corresponding mutant mouse model, we demonstrated impaired erythropoiesis in MLASA-iPSCs and anemia in the MLASA mouse model. Both MLASA-iPSCs and mouse erythroblasts exhibited compromised mitochondrial function and impaired protein synthesis. Mechanistically, we revealed that PUS1 deficiency resulted in reduced mitochondrial tRNA levels because of pseudouridylation loss, leading to aberrant mitochondrial translation. Screening of mitochondrial supplements aimed at enhancing respiration or heme synthesis showed limited effect in promoting erythroid differentiation. Interestingly, the mammalian target of rapamycin (mTOR) inhibitor rapamycin facilitated erythroid differentiation in MLASA-iPSCs by suppressing mTOR signaling and protein synthesis, and consistent results were observed in the MLASA mouse model. Importantly, rapamycin treatment partially ameliorated anemia phenotypes in a patient with MLASA. Our findings provide novel insights into the crucial role of mitochondrial tRNA pseudouridylation in governing erythropoiesis and present potential therapeutic strategies for patients with anemia facing challenges related to protein translation.
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Eritropoyesis , Células Madre Pluripotentes Inducidas , Mitocondrias , ARN de Transferencia , Animales , Ratones , Humanos , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Mitocondrias/metabolismo , Mitocondrias/patología , Células Madre Pluripotentes Inducidas/metabolismo , Seudouridina/metabolismo , Anemia Sideroblástica/genética , Anemia Sideroblástica/metabolismo , Anemia Sideroblástica/patología , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismo , Hidroliasas/metabolismo , Hidroliasas/genética , Síndrome MELAS/genética , Síndrome MELAS/patología , Síndrome MELAS/metabolismo , Modelos Animales de EnfermedadRESUMEN
PURPOSE: MELAS syndrome is a genetic disorder caused by mitochondrial DNA mutations. We previously described that MELAS patients had increased CSF glutamate and decreased CSF glutamine levels and that oral glutamine supplementation restores these values. Proton magnetic resonance spectroscopy (1H-MRS) allows the in vivo evaluation of brain metabolism. We aimed to compare 1H-MRS of MELAS patients with controls, the 1H-MRS after glutamine supplementation in the MELAS group, and investigate the association between 1H-MRS and CSF lactate, glutamate, and glutamine levels. METHODS: We conducted an observational case-control study and an open-label, single-cohort study with single-voxel MRS (TE 144/35 ms). We assessed the brain metabolism changes in the prefrontal (PFC) and parieto-occipital) cortex (POC) after oral glutamine supplementation in MELAS patients. MR spectra were analyzed with jMRUI software. RESULTS: Nine patients with MELAS syndrome (35.8 ± 3.2 years) and nine sex- and age-matched controls were recruited. Lactate/creatine levels were increased in MELAS patients in both PFC and POC (0.40 ± 0.05 vs. 0, p < 0.001; 0.32 ± 0.03 vs. 0, p < 0.001, respectively). No differences were observed between groups in glutamate and glutamine (Glx/creatine), either in PFC (p = 0.930) or POC (p = 0.310). No differences were observed after glutamine supplementation. A positive correlation was found between CSF lactate and lactate/creatine only in POC (0.85, p = 0.003). CONCLUSION: No significant metabolite changes were observed in the brains of MELAS patients after glutamine supplementation. While we found a positive correlation between lactate levels in CSF and 1H-MRS in MELAS patients, we could not monitor treatment response over short periods with this tool. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04948138; initial release 24/06/2021; first patient enrolled on 1/07/2021. https://clinicaltrials.gov/ct2/show/NCT04948138.
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Glutamina , Síndrome MELAS , Humanos , Glutamina/metabolismo , Síndrome MELAS/diagnóstico por imagen , Síndrome MELAS/tratamiento farmacológico , Síndrome MELAS/metabolismo , Creatina/metabolismo , Estudios de Casos y Controles , Estudios de Cohortes , Espectroscopía de Resonancia Magnética/métodos , Ácido Glutámico/metabolismo , Espectroscopía de Protones por Resonancia Magnética/métodos , Lactatos , Suplementos DietéticosRESUMEN
Nuclease-mediated editing of heteroplasmic mitochondrial DNA (mtDNA) seeks to preferentially cleave and eliminate mutant mtDNA, leaving wild-type genomes to repopulate the cell and shift mtDNA heteroplasmy. Various technologies are available, but many suffer from limitations based on size and/or specificity. The use of ARCUS nucleases, derived from naturally occurring I-CreI, avoids these pitfalls due to their small size, single-component protein structure and high specificity resulting from a robust protein-engineering process. Here we describe the development of a mitochondrial-targeted ARCUS (mitoARCUS) nuclease designed to target one of the most common pathogenic mtDNA mutations, m.3243A>G. mitoARCUS robustly eliminated mutant mtDNA without cutting wild-type mtDNA, allowing for shifts in heteroplasmy and concomitant improvements in mitochondrial protein steady-state levels and respiration. In vivo efficacy was demonstrated using a m.3243A>G xenograft mouse model with mitoARCUS delivered systemically by adeno-associated virus. Together, these data support the development of mitoARCUS as an in vivo gene-editing therapeutic for m.3243A>G-associated diseases.
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ADN Mitocondrial , Síndrome MELAS , Humanos , Animales , Ratones , ADN Mitocondrial/genética , Síndrome MELAS/genética , Síndrome MELAS/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , MutaciónRESUMEN
The 3243A > G in mtDNA is a representative mutation in mitochondrial diseases. Mitochondrial protein synthesis is impaired due to decoding disorder caused by severe reduction of 5-taurinomethyluridine (τm5U) modification of the mutant mt-tRNALeu(UUR) bearing 3243A > G mutation. The 3243A > G heteroplasmy in peripheral blood reportedly decreases exponentially with age. Here, we found three cases with mild respiratory symptoms despite bearing high rate of 3243A > G mutation (>90%) in blood mtDNA. These patients had the 3290T > C haplotypic mutation in addition to 3243A > G pathogenic mutation in mt-tRNALeu(UUR) gene. We generated cybrid cells of these cases to examine the effects of the 3290T > C mutation on mitochondrial function and found that 3290T > C mutation improved mitochondrial translation, formation of respiratory chain complex, and oxygen consumption rate of pathogenic cells associated with 3243A > G mutation. We measured τm5U frequency of mt-tRNALeu(UUR) with 3243A > G mutation in the cybrids by a primer extension method assisted with chemical derivatization of τm5U, showing that hypomodification of τm5U was significantly restored by the 3290T > C haplotypic mutation. We concluded that the 3290T > C is a haplotypic mutation that suppresses respiratory deficiency of mitochondrial disease by restoring hypomodified τm5U in mt-tRNALeu(UUR) with 3243A > G mutation, implying a potential therapeutic measure for mitochondrial disease associated with pathogenic mutations in mt-tRNAs.
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Síndrome MELAS , Enfermedades Mitocondriales , Humanos , Síndrome MELAS/genética , Síndrome MELAS/metabolismo , ARN de Transferencia de Leucina/metabolismo , Taurina , Haplotipos , Mutación , ADN Mitocondrial/genética , Enfermedades Mitocondriales/genéticaRESUMEN
Variants within the high copy number mitochondrial genome (mtDNA) can disrupt organelle function and lead to severe multisystem disease. The wide range of manifestations observed in patients with mitochondrial disease results from varying fractions of abnormal mtDNA molecules in different cells and tissues, a phenomenon termed heteroplasmy. However, the landscape of heteroplasmy across cell types within tissues and its influence on phenotype expression in affected patients remains largely unexplored. Here, we identify nonrandom distribution of a pathogenic mtDNA variant across a complex tissue using single-cell RNA-Seq, mitochondrial single-cell ATAC sequencing, and multimodal single-cell sequencing. We profiled the transcriptome, chromatin accessibility state, and heteroplasmy in cells from the eyes of a patient with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and from healthy control donors. Utilizing the retina as a model for complex multilineage tissues, we found that the proportion of the pathogenic m.3243A>G allele was neither evenly nor randomly distributed across diverse cell types. All neuroectoderm-derived neural cells exhibited a high percentage of the mutant variant. However, a subset of mesoderm-derived lineage, namely the vasculature of the choroid, was near homoplasmic for the WT allele. Gene expression and chromatin accessibility profiles of cell types with high and low proportions of m.3243A>G implicate mTOR signaling in the cellular response to heteroplasmy. We further found by multimodal single-cell sequencing of retinal pigment epithelial cells that a high proportion of the pathogenic mtDNA variant was associated with transcriptionally and morphologically abnormal cells. Together, these findings show the nonrandom nature of mitochondrial variant partitioning in human mitochondrial disease and underscore its implications for mitochondrial disease pathogenesis and treatment.
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Síndrome MELAS , Enfermedades Mitocondriales , Enfermedades de la Retina , Humanos , Heteroplasmia , Síndrome MELAS/genética , Síndrome MELAS/metabolismo , Síndrome MELAS/patología , Enfermedades Mitocondriales/genética , ADN Mitocondrial/genética , Retina/patología , CromatinaRESUMEN
Mutations in mitochondrial (mt-)tRNAs frequently cause mitochondrial dysfunction. Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), and myoclonus epilepsy associated with ragged red fibers (MERRF) are major clinical subgroups of mitochondrial diseases caused by pathogenic point mutations in tRNA genes encoded in mtDNA. We previously reported a severe reduction in the frequency of 5-taurinomethyluridine (τm5U) and its 2-thiouridine derivative (τm5s2U) in the anticodons of mutant mt-tRNAs isolated from the cells of patients with MELAS and MERRF, respectively. The hypomodified tRNAs fail to decode cognate codons efficiently, resulting in defective translation of respiratory chain proteins in mitochondria. To restore the mitochondrial activity of MELAS patient cells, we overexpressed MTO1, a τm5U-modifying enzyme, in patient-derived myoblasts. We used a newly developed primer extension method and showed that MTO1 overexpression almost completely restored the τm5U modification of the MELAS mutant mt-tRNALeu(UUR). An increase in mitochondrial protein synthesis and oxygen consumption rate suggested that the mitochondrial function of MELAS patient cells can be activated by restoring the τm5U of the mutant tRNA. In addition, we confirmed that MTO1 expression restored the τm5s2U of the mutant mt-tRNALys in MERRF patient cells. These findings pave the way for epitranscriptomic therapies for mitochondrial diseases.
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Síndrome MELAS , Síndrome MERRF , ARN de Transferencia , Humanos , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Síndrome MELAS/genética , Síndrome MELAS/metabolismo , Síndrome MELAS/terapia , Síndrome MERRF/genética , Síndrome MERRF/metabolismo , Síndrome MERRF/terapia , Mitocondrias/genética , Mitocondrias/metabolismo , Mutación , ARN de Transferencia/genética , ARN de Transferencia/metabolismoRESUMEN
BACKGROUND: The variant m.3571_3572insC/MT-ND1 thus far only reported in oncocytic tumors of different tissues. However, the role of m.3571_3572insC in inherited mitochondrial diseases has yet to be elucidated. METHODS: A patient diagnosed with MELAS syndrome was recruited, and detailed medical records were collected and reviewed. The muscle was biopsied for mitochondrial respiratory chain enzyme activity. Series of fibroblast clones bearing different m.3571_3572insC variant loads were generated from patient-derived fibroblasts and subjected to functional assays. RESULTS: Complex I deficiency was confirmed in the patient's muscle via mitochondrial respiratory chain enzyme activity assay. The m.3571_3572insC was filtered for the candidate variant of the patient according to the guidelines for mitochondrial mRNA variants interpretation. Three cell clones with different m.3571_3572insC variant loads were generated to evaluate mitochondrial function. Blue native PAGE analysis revealed that m.3571_3572insC caused a deficiency in the mitochondrial complex I. Oxygen consumption rate, ATP production, and lactate assays found an impairment of cellular bioenergetic capacity due to m.3571_3572insC. Mitochondrial membrane potential was decreased, and mitochondrial reactive oxygen species production was increased with the variant of m.3571_3572insC. According to the competitive cell growth assay, the mutant cells had impaired cell growth capacity compared to wild type. CONCLUSIONS: A novel variant m.3571_3572insC was identified in a patient diagnosed with MELAS syndrome, and the variant impaired mitochondrial respiration by decreasing the activity of complex I. In conclusion, the genetic spectrum of mitochondrial diseases was expanded by including m.3571_3572insC/MT-ND1.
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Síndrome MELAS , Enfermedades Mitocondriales , Humanos , ADN Mitocondrial/genética , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Síndrome MELAS/genética , Síndrome MELAS/metabolismo , Síndrome MELAS/patología , Enfermedades Mitocondriales/genética , Mutación , NADH Deshidrogenasa/genética , Mutación del Sistema de LecturaRESUMEN
BACKGROUND AND PURPOSE: Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome is a genetically heterogeneous disorder caused by mitochondrial DNA mutations. There are no disease-modifying therapies, and treatment remains mainly supportive. It has been shown previously that patients with MELAS syndrome have significantly increased cerebrospinal fluid (CSF) glutamate and significantly decreased CSF glutamine levels compared to controls. Glutamine has many metabolic fates in neurons and astrocytes, and the glutamate-glutamine cycle couples with many metabolic pathways depending on cellular requirements. The aim was to compare CSF glutamate and glutamine levels before and after dietary glutamine supplementation. It is postulated that high-dose oral glutamine supplementation could reduce the increase in glutamate levels. METHOD: This open-label, single-cohort study determined the safety and changes in glutamate and glutamine levels in CSF after 12 weeks of oral glutamine supplementation. RESULTS: Nine adult patients with MELAS syndrome (66.7% females, mean age 35.8 ± 3.2 years) were included. After glutamine supplementation, CSF glutamate levels were significantly reduced (9.77 ± 1.21 vs. 18.48 ± 1.34 µmol/l, p < 0.001) and CSF glutamine levels were significantly increased (433.66 ± 15.31 vs. 336.31 ± 12.92 µmol/l, p = 0.002). A side effect observed in four of nine patients was a mild sensation of satiety. One patient developed mild and transient elevation of transaminases, and another patient was admitted for an epileptic status without stroke-like episode. DISCUSSION: This study demonstrates that high-dose oral glutamine supplementation significantly reduces CSF glutamate and increases CSF glutamine levels in patients with MELAS syndrome. These findings may have potential therapeutic implications in these patients. TRIAL REGISTRATION INFORMATION: ClinicalTrials.gov Identifier: NCT04948138. Initial release 24 June 2021, first patient enrolled 1 July 2021. https://clinicaltrials.gov/ct2/show/NCT04948138.
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Acidosis Láctica , Síndrome MELAS , Accidente Cerebrovascular , Adulto , Femenino , Humanos , Masculino , Estudios de Cohortes , Suplementos Dietéticos , Ácido Glutámico/uso terapéutico , Glutamina/uso terapéutico , Síndrome MELAS/tratamiento farmacológico , Síndrome MELAS/genética , Síndrome MELAS/metabolismoRESUMEN
BACKGROUND: Mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage in the retinal pigment epithelium (RPE) have been implicated in the pathogenesis of age-related macular degeneration (AMD). However, a deeper understanding is required to determine the contribution of mitochondrial dysfunction and impaired mitochondrial autophagy (mitophagy) to RPE damage and AMD pathobiology. In this study, we model the impact of a prototypical systemic mitochondrial defect, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), in RPE health and homeostasis as an in vitro model for impaired mitochondrial bioenergetics. METHODS: We used induced pluripotent stem cells (iPSCs) derived from skin biopsies of MELAS patients (m.3243A > G tRNA leu mutation) with different levels of mtDNA heteroplasmy and differentiated them into RPE cells. Mitochondrial depletion of ARPE-19 cells (p0 cells) was also performed using 50 ng/mL ethidium bromide (EtBr) and 50 mg/ml uridine. Cell fusion of the human platelets with the p0 cells performed using polyethylene glycol (PEG)/suspension essential medium (SMEM) mixture to generate platelet/RPE "cybrids." Confocal microscopy, FLowSight Imaging cytometry, and Seahorse XF Mito Stress test were used to analyze mitochondrial function. Western Blotting was used to analyze expression of autophagy and mitophagy proteins. RESULTS: We found that MELAS iPSC-derived RPE cells exhibited key characteristics of native RPE. We observed heteroplasmy-dependent impairment of mitochondrial bioenergetics and reliance on glycolysis for generating energy in the MELAS iPSC-derived RPE. The degree of heteroplasmy was directly associated with increased activation of signal transducer and activator of transcription 3 (STAT3), reduced adenosine monophosphate-activated protein kinase α (AMPKα) activation, and decreased autophagic activity. In addition, impaired autophagy was associated with aberrant lysosomal function, and failure of mitochondrial recycling. The mitochondria-depleted p0 cells replicated the effects on autophagy impairment and aberrant STAT3/AMPKα signaling and showed reduced mitochondrial respiration, demonstrating phenotypic similarities between p0 and MELAS iPSC-derived RPE cells. CONCLUSIONS: Our studies demonstrate that the MELAS iPSC-derived disease models are powerful tools for dissecting the molecular mechanisms by which mitochondrial DNA alterations influence RPE function in aging and macular degeneration, and for testing novel therapeutics in patients harboring the MELAS genotype.
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Células Madre Pluripotentes Inducidas , Síndrome MELAS , Degeneración Macular , Autofagia/genética , ADN Mitocondrial/genética , Metabolismo Energético/genética , Células Epiteliales/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Síndrome MELAS/genética , Síndrome MELAS/metabolismo , Síndrome MELAS/patología , Degeneración Macular/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Pigmentos Retinianos/metabolismoRESUMEN
Maintaining mitochondrial function and dynamics is crucial for cellular health. In muscle, defects in mitochondria result in severe myopathies where accumulation of damaged mitochondria causes deterioration and dysfunction. Importantly, understanding the role of mitochondria in disease is a necessity to determine future therapeutics. One of the most common myopathies is mitochondrial encephalopathy lactic acidosis stroke-like episodes (MELAS), which has no current treatment. Recently, patients with MELAS treated with rapamycin exhibited improved clinical outcomes. However, the cellular mechanisms of rapamycin effects in patients with MELAS are currently unknown. In this study, we used cultured skin fibroblasts as a window into the mitochondrial dysfunction evident in MELAS cells, as well as to study the mechanisms of rapamycin action, compared with control, healthy individuals. We observed that mitochondria from patients were fragmented, had a threefold decline in the average speed of motility, a twofold reduced mitochondrial membrane potential, and a 1.5- to 2-fold decline in basal respiration. Despite the reduction in mitochondrial function, mitochondrial import protein Tim23 was elevated in patient cell lines. MELAS fibroblasts exhibited increased MnSOD levels and lysosomal function when compared with healthy controls. Treatment of MELAS fibroblasts with rapamycin for 24 h resulted in increased mitochondrial respiration compared with control cells, a higher lysosome content, and a greater localization of mitochondria to lysosomes. Our studies suggest that rapamycin has the potential to improve cellular health even in the presence of mtDNA defects, primarily via an increase in lysosomal content.
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Fibroblastos/efectos de los fármacos , Lisosomas/efectos de los fármacos , Síndrome MELAS/genética , Mitocondrias/efectos de los fármacos , Sirolimus/farmacología , Estudios de Casos y Controles , Preescolar , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Regulación de la Expresión Génica , Humanos , Lactante , Lisosomas/metabolismo , Síndrome MELAS/tratamiento farmacológico , Síndrome MELAS/metabolismo , Síndrome MELAS/patología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Mutación , Fosforilación Oxidativa/efectos de los fármacos , Cultivo Primario de Células , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo , Adulto JovenRESUMEN
Our purpose is to detect the metabolic alterations in acute stroke-like lesions (SLLs) and further investigate the correlations between metabolic concentrations and focal cerebral blood flow in patients with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) using proton MR spectroscopy (1H-MRS) and arterial spin labeling (ASL). A total of 23 patients with MELAS at acute stage of stroke-like episodes (SLEs) and 20 normal controls (NC) were recruited in this study, respectively. All subjects underwent conventional MRI and1H-MRS. In addition, ASL was performed in each patient. The measurements of creatine (Cr), choline (Cho), N-acetyl aspartate (NAA), lactate (Lac), glutamine/glutamate (Glx) levels and the ratios of Cho/Cr, NAA/Cr, Lac/Cr and Glx/Cr in acute SLLs for MELAS patients and left parietal and occipital lobes for NC were measured using LC-model software. Furthermore, in MELAS group, the associations between relative cerebral blood flow (rCBF) and metabolite concentrations in acute SLLs were also assessed. In MELAS group, acute SLLs were identified with metabolic abnormalities and increased rCBF. Specifically, compared with controls, MELAS patients exhibited significantly higher Lac, Cho levels and Lac/Cr, Cho/Cr ratios, and lower NAA, Glx levels and NAA/Cr and Glx/Cr ratios. Moreover, for MELAS patients, Lac concentration in acute SLLs was positively correlated with focal rCBF. This study exhibited the neural injury with increasing lactate and cerebral blood flow in the acute SLEs. It might shed light on the underlying biochemical mechanism of mitochondrial cytopathy and angiopathy in MELAS.
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Encéfalo/irrigación sanguínea , Ácido Láctico/metabolismo , Síndrome MELAS/diagnóstico por imagen , Metabolómica/métodos , Espectroscopía de Protones por Resonancia Magnética/métodos , Adolescente , Adulto , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Encéfalo/diagnóstico por imagen , Estudios de Casos y Controles , Colina/metabolismo , Creatina/metabolismo , Femenino , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Humanos , Síndrome MELAS/metabolismo , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Marcadores de Spin , Adulto JovenRESUMEN
BACKGROUND: Freezing human biopsies is common in clinical practice for storage. However, this technique disrupts mitochondrial membranes, hampering further analyses of respiratory function. To contribute to laboratorial diagnosis of mitochondrial diseases, this study sought to develop a respirometry approach using O2k (Oroboros Ins.) to measure the whole electron transport chain (ETC) activity in homogenates of frozen skeletal muscle biopsies. PATIENTS AND METHODS: We enrolled 16 patients submitted to muscle biopsy in the process of routine diagnostic investigation: four with mitochondrial disease and severe mitochondrial dysfunction; seven with exercise intolerance and multiple deletions of mitochondrial DNA, presenting mild to moderate mitochondrial dysfunction; five without mitochondrial disease, as controls. Whole homogenates of muscle fragments were prepared using grinder-type equipment. O2 consumption rates were normalized using citrate synthase activity. RESULTS: Transmission electron microscopy confirmed mitochondrial membrane discontinuation, indicating increased permeability of mitochondrial membranes in homogenates from frozen biopsies. O2 consumption rates in the presence of acetyl-CoA lead to maximum respiratory rates sensitive to rotenone, malonate and antimycin. This protocol of acetyl-CoA-driven respiration (ACoAR), applied in whole homogenates of frozen muscle, was sensitive enough to identify ETC abnormality, even in patients with mild to moderate mitochondrial dysfunction. We demonstrated adequate repeatability of ACoAR and found significant correlation between O2 consumption rates and enzyme activity assays of individual ETC complexes. CONCLUSIONS: We present preliminary data on a simple, low cost and reliable procedure to measure respiratory function in whole homogenates of frozen skeletal muscle biopsies, contributing to diagnosis of mitochondrial diseases in humans.
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Acetilcoenzima A/metabolismo , Mitocondrias Musculares/metabolismo , Enfermedades Mitocondriales/diagnóstico , Músculo Esquelético/metabolismo , Consumo de Oxígeno , Adolescente , Adulto , Biopsia , Respiración de la Célula , Niño , Técnicas de Laboratorio Clínico/métodos , Criopreservación , Transporte de Electrón , Femenino , Humanos , Síndrome MELAS/diagnóstico , Síndrome MELAS/metabolismo , Masculino , Potencial de la Membrana Mitocondrial , Enfermedades Mitocondriales/metabolismo , Membranas Mitocondriales/metabolismo , Músculo Esquelético/patología , Oftalmoplejía Externa Progresiva Crónica/diagnóstico , Oftalmoplejía Externa Progresiva Crónica/metabolismo , Fosforilación Oxidativa , Permeabilidad , Manejo de Especímenes , Adulto JovenRESUMEN
OBJECTIVE: The purpose of this study was to investigate correlations between brain proton magnetic resonance spectroscopy (1 H-MRS) findings with serum biomarkers and heteroplasmy of mitochondrial DNA (mtDNA) mutations. This study enrolled patients carrying mtDNA mutations associated with Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes (MELAS), and MELAS-Spectrum Syndrome (MSS). METHODS: Consecutive patients carrying mtDNA mutations associated with MELAS and MSS were recruited and their serum concentrations of lactate, alanine, and heteroplasmic mtDNA mutant load were evaluated. The brain protocol included single-voxel 1 H-MRS (1.5T) in the medial parieto-occipital cortex (MPOC), left cerebellar hemisphere, parieto-occipital white matter (POWM), and lateral ventricles. Relative metabolite concentrations of N-acetyl-aspartate (NAA), choline (Cho), and myo-inositol (mI) were estimated relative to creatine (Cr), using LCModel 6.3. RESULTS: Six patients with MELAS (age 28 ± 13 years, 3 [50%] female) and 17 with MSS (age 45 ± 11 years, 7 [41%] female) and 39 sex- and age-matched healthy controls (HC) were enrolled. These patients demonstrated a lower NAA/Cr ratio in MPOC compared to HC (p = 0.006), which inversely correlated with serum lactate (p = 0.021, rho = -0.68) and muscle mtDNA heteroplasmy (p < 0.001, rho = -0.80). Similarly, in the cerebellum patients had lower NAA/Cr (p < 0.001), Cho/Cr (p = 0.002), and NAA/mI (p = 0.001) ratios, which negatively correlated with mtDNA blood heteroplasmy (p = 0.001, rho = -0.81) and with alanine (p = 0.050, rho = -0.67). Ventricular lactate was present in 78.3% (18/23) of patients, correlating with serum lactate (p = 0.024, rho = 0.58). CONCLUSION: Correlations were found between the peripheral and biochemical markers of mitochondrial dysfunction and brain in vivo markers of neurodegeneration, supporting the use of both biomarkers as signatures of MELAS and MSS disease, to evaluate the efficacy of potential treatments.
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ADN Mitocondrial/genética , Síndrome MELAS/diagnóstico , Síndrome MELAS/genética , Síndrome MELAS/metabolismo , Espectroscopía de Protones por Resonancia Magnética , Adolescente , Adulto , Anciano , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Biomarcadores/metabolismo , Cerebelo/diagnóstico por imagen , Cerebelo/metabolismo , Corteza Cerebral/diagnóstico por imagen , Corteza Cerebral/metabolismo , Colina/metabolismo , Humanos , Inositol/metabolismo , Ventrículos Laterales/diagnóstico por imagen , Ventrículos Laterales/metabolismo , Síndrome MELAS/sangre , Masculino , Persona de Mediana Edad , Mutación , Sustancia Blanca/diagnóstico por imagen , Sustancia Blanca/metabolismo , Adulto JovenRESUMEN
INTRODUCTION: The m.3243A > G mitochondrial DNA mutation is one of the most common mitochondrial disease-causing mutations, with a carrier rate as high as 1:400. This point mutation affects the MT-TL1 gene, ultimately affecting the oxidative phosphorylation system and the cell's energy production. Strikingly, the m.3243A > G mutation is associated with different phenotypes, including mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), maternally inherited diabetes and deafness (MIDD) and myopathy. OBJECTIVES: We investigated urine metabolomes of MELAS, MIDD and myopathy patients in order to identify affected metabolic pathways and possible treatment options. METHODS: A multiplatform metabolomics approach was used to comprehensively analyze the metabolome and compare metabolic profiles of different phenotypes caused by the m.3243A > G mutation. Our analytical array consisted of NMR spectroscopy, LC-MS/MS and GC-TOF-MS. RESULTS: The investigation revealed phenotypic specific metabolic perturbations, as well as metabolic similarities between the different phenotypes. We show that glucose metabolism is highly disturbed in the MIDD phenotype, but not in MELAS or myopathy, remodeled fatty acid oxidation is characteristic of the MELAS patients, while one-carbon metabolism is strongly modified in both MELAS and MIDD, but not in the myopathy group. Lastly we identified increased creatine in the urine of the myopathy patients, but not in MELAS or MIDD. CONCLUSION: We conclude by giving novel insight on the phenotypes of the m.3243A > G mutation from a metabolomics point of view. Directives are also given for future investigations that could lead to better treatment options for patients suffering from this debilitating disease.
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Sordera/genética , Sordera/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Síndrome MELAS/genética , Síndrome MELAS/metabolismo , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Enfermedades Musculares/genética , Enfermedades Musculares/metabolismo , Mutación , Fenotipo , Cromatografía Liquida , Sordera/diagnóstico , Diabetes Mellitus Tipo 2/diagnóstico , Predisposición Genética a la Enfermedad , Humanos , Síndrome MELAS/diagnóstico , Espectroscopía de Resonancia Magnética , Metaboloma , Metabolómica/métodos , Enfermedades Mitocondriales/diagnóstico , Enfermedades Musculares/diagnóstico , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Increasing evidence from pathological and biochemical investigations suggests that mitochondrial metabolic impairment and oxidative stress play a crucial role in the pathogenesis of mitochondrial diseases, such as mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome, and various neurodegenerative disorders. Recent advances in molecular imaging technology with positron emission tomography (PET) and functional magnetic resonance imaging (MRI) have accomplished a direct and non-invasive evaluation of the pathophysiological changes in living patients. SCOPE OF REVIEW: In this review, we focus on the latest achievements of molecular imaging for mitochondrial metabolism and oxidative stress in mitochondrial diseases and neurodegenerative disorders. MAJOR CONCLUSIONS: Molecular imaging with PET and MRI exhibited mitochondrial metabolic changes, such as enhanced glucose utilization with lactic acid fermentation, suppressed fatty acid metabolism, decreased TCA-cycle metabolism, impaired respiratory chain activity, and increased oxidative stress, in patients with MELAS syndrome. In addition, PET imaging clearly demonstrated enhanced cerebral oxidative stress in patients with Parkinson's disease or amyotrophic lateral sclerosis. The magnitude of oxidative stress correlated well with clinical severity in patients, indicating that oxidative stress based on mitochondrial dysfunction is associated with the neurodegenerative changes in these diseases. GENERAL SIGNIFICANCE: Molecular imaging is a promising tool to improve our knowledge regarding the pathogenesis of diseases associated with mitochondrial dysfunction and oxidative stress, and this would facilitate the development of potential antioxidants and mitochondrial therapies.
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Acidosis Láctica/diagnóstico por imagen , Encefalopatías/diagnóstico por imagen , Síndrome MELAS/diagnóstico por imagen , Mitocondrias/metabolismo , Miopatías Mitocondriales/diagnóstico por imagen , Enfermedades Neurodegenerativas/diagnóstico por imagen , Acidosis Láctica/metabolismo , Acidosis Láctica/patología , Encefalopatías/metabolismo , Encefalopatías/patología , Transporte de Electrón/genética , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Humanos , Ácido Láctico/metabolismo , Síndrome MELAS/metabolismo , Síndrome MELAS/patología , Imagen por Resonancia Magnética , Mitocondrias/genética , Mitocondrias/patología , Miopatías Mitocondriales/metabolismo , Miopatías Mitocondriales/patología , Imagen Molecular , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Estrés Oxidativo , Tomografía de Emisión de Positrones , Índice de Severidad de la EnfermedadRESUMEN
Humanin is a member of a new family of peptides that are encoded by short open reading frames within the mitochondrial genome. It is conserved in animals and is both neuroprotective and cytoprotective. Here we report that in C. elegans the overexpression of humanin is sufficient to increase lifespan, dependent on daf-16/Foxo. Humanin transgenic mice have many phenotypes that overlap with the worm phenotypes and, similar to exogenous humanin treatment, have increased protection against toxic insults. Treating middle-aged mice twice weekly with the potent humanin analogue HNG, humanin improves metabolic healthspan parameters and reduces inflammatory markers. In multiple species, humanin levels generally decline with age, but here we show that levels are surprisingly stable in the naked mole-rat, a model of negligible senescence. Furthermore, in children of centenarians, who are more likely to become centenarians themselves, circulating humanin levels are much greater than age-matched control subjects. Further linking humanin to healthspan, we observe that humanin levels are decreased in human diseases such as Alzheimer's disease and MELAS (Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like episodes). Together, these studies are the first to demonstrate that humanin is linked to improved healthspan and increased lifespan.
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Enfermedad de Alzheimer/sangre , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Longevidad/fisiología , Síndrome MELAS/sangre , Mitocondrias/metabolismo , Adulto , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Estudios de Casos y Controles , Niño , Estudios de Cohortes , ADN Mitocondrial/genética , Femenino , Factores de Transcripción Forkhead/metabolismo , Dosificación de Gen , Humanos , Recién Nacido , Péptidos y Proteínas de Señalización Intracelular/sangre , Péptidos y Proteínas de Señalización Intracelular/genética , Síndrome MELAS/metabolismo , Macaca mulatta , Ratones , Persona de Mediana Edad , Modelos Animales , Ratas Topo , Embarazo , Adulto JovenRESUMEN
Mitochondria are highly dynamic organelles that constantly fuse, divide, and move, and their function is regulated and maintained by their morphologic changes. Mitochondrial disease (MD) comprises a group of disorders involving mitochondrial dysfunction. However, it is not clear whether changes in mitochondrial morphology are related to MD. In this study, we examined mitochondrial morphology in fibroblasts from patients with MD (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and Leigh syndrome). We observed that MD fibroblasts exhibited significant mitochondrial fragmentation by upregulation of Drp1, which is responsible for mitochondrial fission. Interestingly, the inhibition of mitochondrial fragmentation by Drp1 knockdown enhanced cellular toxicity and led to cell death in MD fibroblasts. These results suggest that mitochondrial fission plays a critical role in the attenuation of mitochondrial damage in MD fibroblasts.
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Dinaminas/metabolismo , Fibroblastos/metabolismo , Enfermedad de Leigh/metabolismo , Síndrome MELAS/metabolismo , Mitocondrias/metabolismo , Piel/metabolismo , Muerte Celular , Células Cultivadas , Fibroblastos/patología , Humanos , Enfermedad de Leigh/patología , Síndrome MELAS/patología , Mitocondrias/patología , Piel/patologíaRESUMEN
Mitochondrial myopathy, Encephalopathy, Lactic Acidosis, and Stroke-like episodes (MELAS) is a common subtype of mitochondrial disease with high disability and mortality rate. The molecular mechanisms of MELAS are largely unknown and whether autophagy is activated in this disease remains controversial. In this work, we reported whole transcriptome profiling of skeletal muscle of MELAS patients and age-matched controls. Analyses revealed that MELAS patients had 224 differentially expressed genes (174 down-regulated, 50 up-regulated) compared to age-matched controls. Most of these genes relevant to MELAS are involved in signal transduction, metabolic process and immune system process. However, the RNA-seq data indicated that autophagy was not altered in MELAS. Functional assays showed that increased reactive oxygen species (ROS), decreased ATP production and decreased lysosome content in fibroblasts derived from MELAS patients, suggesting that mitochondrial dysfunction and degradation deficiency in MELAS. Furthermore, Western-blot analyses using skeletal muscle and fibroblasts derived from MELAS patients showed that autophagy was impaired in MEALS since two important autophagic genes: Beclin-1 and LC3-II, were significantly down-regulated. In conclusion, our study identified molecules and pathways involved in the mechanisms of MELAS, and the impairment of autophagy in this disease, which may serve as the potential therapeutic target for MELAS.