RESUMO
Leigh syndrome (LS) is the most frequent infantile mitochondrial disorder (MD) and is characterized by neurodegeneration and astrogliosis in the basal ganglia or the brain stem. At present, there is no cure or treatment for this disease, partly due to scarcity of LS models. Current models generally fail to recapitulate important traits of the disease. Therefore, there is an urgent need to develop new human in vitro models. Establishment of induced pluripotent stem cells (iPSCs) followed by differentiation into neurons is a powerful tool to obtain an in vitro model for LS. Here, we describe the generation and characterization of iPSCs, neural stem cells (NSCs) and iPSC-derived neurons harboring the mtDNA mutation m.13513G>A in heteroplasmy. We have performed mitochondrial characterization, analysis of electrophysiological properties and calcium imaging of LS neurons. Here, we show a clearly compromised oxidative phosphorylation (OXPHOS) function in LS patient neurons. This is also the first report of electrophysiological studies performed on iPSC-derived neurons harboring an mtDNA mutation, which revealed that, in spite of having identical electrical properties, diseased neurons manifested mitochondrial dysfunction together with a diminished calcium buffering capacity. This could lead to an overload of cytoplasmic calcium concentration and the consequent cell death observed in patients. Importantly, our results highlight the importance of calcium homeostasis in LS pathology.
Assuntos
Cálcio/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Doença de Leigh/metabolismo , Consumo de Oxigênio/fisiologia , Western Blotting , Proliferação de Células/fisiologia , Células Cultivadas , Eletrofisiologia , Imunofluorescência , Humanos , Ácido Láctico/metabolismo , Doença de Leigh/patologia , Mitocôndrias/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Consumo de Oxigênio/genéticaRESUMO
Mitochondrial disorders (MDs) arise as a result of a respiratory chain dysfunction. While some MDs can affect a single organ, many involve several organs, the brain being the most affected, followed by heart and/or muscle. Many of these diseases are associated with heteroplasmic mutations in the mitochondrial DNA (mtDNA). The proportion of mutated mtDNA must exceed a critical threshold to produce disease. Therefore, understanding how embryonic development determines the heteroplasmy level in each tissue could explain the organ susceptibility and the clinical heterogeneity observed in these patients. In this report, the dynamics of heteroplasmy and the influence in cardiac commitment of the mutational load of the m.13513G>A mutation has been analyzed. This mutation has been reported as a frequent cause of Leigh syndrome (LS) and is commonly associated with cardiac problems. In this report, induced pluripotent stem cell (iPSc) technology has been used to delve into the molecular mechanisms underlying cardiac disease in LS. When mutation m.13513G>A is above a threshold, iPSc-derived cardiomyocytes (iPSc-CMs) could not be obtained due to an inefficient epithelial-mesenchymal transition. Surprisingly, these cells are redirected toward neuroectodermal lineages that would give rise to the brain. However, when mutation is below that threshold, dysfunctional CM are generated in a mutant-load dependent way. We suggest that distribution of the m.13513G>A mutation during cardiac differentiation is not at random. We propose a possible explanation of why neuropathology is a frequent feature of MD, but cardiac involvement is not always present.
Assuntos
DNA Mitocondrial/genética , Transporte de Elétrons/genética , Cardiopatias/genética , Doença de Leigh/genética , Doenças Mitocondriais/genética , Diferenciação Celular/genética , Complexo I de Transporte de Elétrons/genética , Desenvolvimento Embrionário/genética , Transição Epitelial-Mesenquimal/genética , Cardiopatias/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Doença de Leigh/patologia , Mitocôndrias/genética , Mitocôndrias/patologia , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Proteínas Mitocondriais/genética , Mutação , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Placa Neural/crescimento & desenvolvimento , Placa Neural/patologia , FenótipoRESUMO
We have generated a human iPSC line IISHDOi003-A from fibroblasts of a patient with a dominant optic atrophy 'plus' phenotype, harbouring a heterozygous mutation, c.1635C>A; p.Ser545Arg, in the OPA1 gene. Reprogramming factors Oct3/4, Sox2, Klf4, and c-Myc were delivered using Sendai virus.
Assuntos
GTP Fosfo-Hidrolases/genética , Atrofia Óptica Autossômica Dominante/genética , Linhagem Celular , GTP Fosfo-Hidrolases/farmacologia , Humanos , Fator 4 Semelhante a Kruppel , Masculino , Mutação , Atrofia Óptica Autossômica Dominante/metabolismo , Atrofia Óptica Autossômica Dominante/patologiaRESUMO
Human iPSC line Oex2054SV.4 was generated from fibroblasts of a patient with an optic atrophy 'plus' phenotype associated with a heterozygous mutation in the OPA1 gene. Reprogramming factors OCT3/4, SOX2, CMYC and KLF4 were delivered using a non-integrative methodology that involves the use of Sendai virus.
Assuntos
Fibroblastos/citologia , GTP Fosfo-Hidrolases/genética , Células-Tronco Pluripotentes Induzidas/citologia , Sequência de Bases , Diferenciação Celular , Células Cultivadas , Reprogramação Celular , Análise Mutacional de DNA , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariótipo , Fator 4 Semelhante a Kruppel , Masculino , Microscopia de Fluorescência , Mutação , Atrofia Óptica/genética , Atrofia Óptica/metabolismo , Atrofia Óptica/patologia , Fenótipo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Human iPSC line GFM1SV.25 was generated from fibroblasts of a child with a severe mitochondrial encephalopathy associated with mutations in the GFM1 gene, encoding the mitochondrial translation elongation factor G1. Reprogramming factors OCT3/4, SOX2, CMYC and KLF4 were delivered using a non integrative methodology that involves the use of Sendai virus.