RESUMO
N-Formylation of the Met-tRNAMet by the nuclearly encoded mitochondrial methionyl-tRNA formyltransferase (MTFMT) has been found to be a key determinant of protein synthesis initiation in mitochondria. In humans, mutations in the MTFMT gene result in Leigh syndrome, a progressive and severe neurometabolic disorder. However, the absolute requirement of formylation of Met-tRNAMet for protein synthesis in mammalian mitochondria is still debated. Here, we generated a Mtfmt-KO mouse fibroblast cell line and demonstrated that N-formylation of the first methionine via fMet-tRNAMet by MTFMT is not an absolute requirement for initiation of protein synthesis. However, it differentially affected the efficiency of synthesis of mtDNA-coded polypeptides. Lack of methionine N-formylation did not compromise the stability of these individual subunits but had a marked effect on the assembly and stability of the OXPHOS complexes I and IV and on their supercomplexes. In summary, N-formylation is not essential for mitochondrial protein synthesis but is critical for efficient synthesis of several mitochondrially encoded peptides and for OXPHOS complex stability and assembly into supercomplexes.
Assuntos
Hidroximetil e Formil Transferases/genética , Metionina/genética , Mitocôndrias/genética , Biossíntese de Proteínas/genética , Animais , DNA Mitocondrial/genética , Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Mutação , Fosforilação Oxidativa , Aminoacil-RNA de Transferência/genéticaRESUMO
Acute pharmacological activation of adenosine monophosphate (AMP)-kinase using 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside (AICAR) has been shown to improve muscle mitochondrial function by increasing mitochondrial biogenesis. We asked whether prolonged AICAR treatment is beneficial in a mouse model of slowly progressing mitochondrial myopathy (Cox10-Mef2c-Cre), and whether the compensatory mechanism is indeed an increase in mitochondrial biogenesis. We treated the animals for 3 months and found that sustained AMP-dependent kinase activation improved cytochrome c oxidase activity, rescued the motor phenotype and delayed the onset of the myopathy. This improvement was observed whether treatment started before or after the onset of the disease. We found that AICAR increased skeletal muscle regeneration thereby decreasing the levels of deleted Cox10-floxed alleles. We conclude that although increase in mitochondrial biogenesis and other pathways may contribute, the main mechanism by which AICAR improves the myopathy phenotype is by promoting muscle regeneration.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Miopatias Mitocondriais/tratamento farmacológico , Miopatias Mitocondriais/metabolismo , Fibras Musculares Esqueléticas/fisiologia , Regeneração/efeitos dos fármacos , Ribonucleotídeos/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Aminoimidazol Carboxamida/farmacologia , Animais , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Mitocôndrias Musculares/genética , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/patologia , Miopatias Mitocondriais/genética , Miopatias Mitocondriais/patologiaRESUMO
The cre/loxP recombination system is a valuable tool used to generate tissue specific genomic rearrangements in mouse models. The deletion of a region of interest flanked by two loxP sites is accomplished by the recombinase (cre) enzyme, which binds to the inverted repeat segments of two loxP sites and recognition of a conserved TA sequence in the asymmetric central spacer region "ATAACTTCGTATA -NNNTANNN-TATACGAAGTTAT. In vivo, we found that a single T to C mutation at position 4 of the central spacer region in the distal (3') loxP site, completely inhibited the recombination reaction in two conditional mouse models. These mice were generated using a mitochondrial methionyl-tRNA formyltransferase (Mtfmt) gene targeted construct and cre transgene under the control of tissue-specific promoters: calcium/calmodulin-dependent kinase II alpha (Camk2a-cre) and myosin light polypeptide 1 (Myl1-cre). Surprisingly, transient transfection of a plasmid expressing cre in dermal fibroblasts derived from the same mutant floxed Mtfmt((loxP/loxP)) mice line, successfully deleted the region of interest. This study demonstrates the sequence specificity required in vivo, the possibility of bypassing this limitation by expressing high levels of cre recombinase ex vivo and raises concerns related to the quality control of large scale production of gene targeted constructs and mice. genesis 53:695-700, 2015. © 2015 Wiley Periodicals, Inc.
Assuntos
DNA Intergênico/metabolismo , Integrases/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Hidroximetil e Formil Transferases/genética , Camundongos Knockout , Camundongos Transgênicos , Mutação PuntualRESUMO
The ATPase Family AAA Domain Containing 3A (ATAD3A), is a mitochondrial inner membrane protein conserved in metazoans. ATAD3A has been associated with several mitochondrial functions, including nucleoid organization, cholesterol metabolism, and mitochondrial translation. To address its primary role, we generated a neuronal-specific conditional knockout (Atad3 nKO) mouse model, which developed a severe encephalopathy by 5 months of age. Pre-symptomatic mice showed aberrant mitochondrial cristae morphogenesis in the cortex as early as 2 months. Using a multi-omics approach in the CNS of 2-to-3-month-old mice, we found early alterations in the organelle membrane structure. We also show that human ATAD3A associates with different components of the inner membrane, including OXPHOS complex I, Letm1, and prohibitin complexes. Stochastic Optical Reconstruction Microscopy (STORM) shows that ATAD3A is regularly distributed along the inner mitochondrial membrane, suggesting a critical structural role in inner mitochondrial membrane and its organization, most likely in an ATPase-dependent manner.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Neurônios/metabolismo , Fosforilação Oxidativa , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Encefalopatias/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/genética , Deleção de Sequência , TranscriptomaRESUMO
Friedreich ataxia (FRDA) is caused by the reduced expression of the mitochondrial protein frataxin (FXN) due to an intronic GAA trinucleotide repeat expansion in the FXN gene. Although FRDA has no cure and few treatment options, there is research dedicated to finding an agent that can curb disease progression and address symptoms as neurobehavioral deficits, muscle endurance, and heart contractile dysfunctions. Because oxidative stress and mitochondrial dysfunctions are implicated in FRDA, we demonstrated the systemic delivery of catalysts activity of gold cluster superstructures (Au8-pXs) to improve cell response to mitochondrial reactive oxygen species and thereby alleviate FRDA-related pathology in mesenchymal stem cells from patients with FRDA. We also found that systemic injection of Au8-pXs ameliorated motor function and cardiac contractility of YG8sR mouse model that recapitulates the FRDA phenotype. These effects were associated to long-term improvement of mitochondrial functions and antioxidant cell responses. We related these events to an increased expression of frataxin, which was sustained by reduced autophagy. Overall, these results encourage further optimization of Au8-pXs in experimental clinical strategies for the treatment of FRDA.
Assuntos
Ataxia de Friedreich , Animais , Modelos Animais de Doenças , Ouro , Humanos , Camundongos , Espécies Reativas de Oxigênio , Expansão das Repetições de TrinucleotídeosRESUMO
Complex I (also known as NADH-ubiquinone oxidoreductase) deficiency is the most frequent mitochondrial disorder present in childhood. NADH-ubiquinone oxidoreductase iron-sulfur protein 3 (NDUFS3) is a catalytic subunit of the mitochondrial complex I; NDUFS3 is conserved from bacteria and essential for complex I function. Mutations affecting complex I, including in the Ndufs3 gene, cause fatal neurodegenerative diseases, such as Leigh syndrome. No treatment is available for these conditions. We developed and performed a detailed molecular characterization of a neuron-specific Ndufs3 conditional KO mouse model. We showed that deletion of Ndufs3 in forebrain neurons reduced complex I activity, altered brain energy metabolism, and increased locomotor activity with impaired motor coordination, balance, and stereotyped behavior. Metabolomics analyses showed an increase of glycolysis intermediates, suggesting an adaptive response to the complex I defect. Administration of metformin to these mice delayed the onset of the neurological symptoms but not of neuronal loss. This improvement was likely related to enhancement of glucose uptake and utilization, which are known effects of metformin in the brain. Despite reports that metformin inhibits complex I activity, our findings did not show worsening a complex I defect nor increases in lactic acid, suggesting that metformin should be further evaluated for use in patients with mitochondrial encephalopathies.
Assuntos
Complexo I de Transporte de Elétrons/deficiência , Metformina/farmacologia , Doenças Mitocondriais/complicações , Encefalomiopatias Mitocondriais/tratamento farmacológico , NADH Desidrogenase/fisiologia , Neurônios/efeitos dos fármacos , Animais , Metabolismo Energético , Feminino , Glicólise , Hipoglicemiantes/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Encefalomiopatias Mitocondriais/etiologia , Encefalomiopatias Mitocondriais/metabolismo , Encefalomiopatias Mitocondriais/patologia , Neurônios/metabolismo , Neurônios/patologiaRESUMO
Myopathies are common manifestations of mitochondrial diseases. To investigate whether gene replacement can be used as an effective strategy to treat or cure mitochondrial myopathies, we have generated a complex I conditional knockout mouse model lacking NDUFS3 subunit in skeletal muscle. NDUFS3 protein levels were undetectable in muscle of 15-day-old smKO mice, and myopathy symptoms could be detected by 2 months of age, worsening over time. rAAV9-Ndufs3 delivered systemically into 15- to 18-day-old mice effectively restored NDUFS3 levels in skeletal muscle, precluding the development of the myopathy. To test the ability of rAAV9-mediated gene replacement to revert muscle function after disease onset, we also treated post-symptomatic, 2-month-old mice. The injected mice showed a remarkable improvement of the mitochondrial myopathy and biochemical parameters, which remained for the duration of the study. Our results showed that muscle pathology could be reversed after restoring complex I, which was absent for more than 2 months. These findings have far-reaching implications for the ability of muscle to tolerate a mitochondrial defect and for the treatment of mitochondrial myopathies.
Assuntos
Complexo I de Transporte de Elétrons/genética , Terapia Genética , Miopatias Mitocondriais , Animais , Complexo I de Transporte de Elétrons/deficiência , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias , Miopatias Mitocondriais/genética , Miopatias Mitocondriais/metabolismo , Músculo Esquelético/metabolismo , NADH Desidrogenase/genéticaRESUMO
Photobiomodulation (PBM) therapy is based on the use of specific light parameters to promote tissue repair. Although demonstrated in different cell models and tissues, the mechanism by which photobiomodulation operates is not well understood. Previous studies suggested that the cell proliferation enhancement triggered by red and near-infrared PBM involves the activation of the mitochondrial respiratory chain enzyme cytochrome c oxidase (CCO). It was suggested that light in this range would displace inhibitory nitric oxide bound to CCO. To test this mechanism, we took advantage of cell lines lacking CCO, including a mouse line knockout for Cox10 (a gene required for the synthesis of heme a, the prosthetic group of CCO) and a human cell line with an mtDNA mutation in the tRNA Lysine gene, leading to mitochondrial protein synthesis impairment and the lack of three critical CCO subunits. In both models we showed the complete absence of assembled CCO. PBM (660â¯nm) was applied to these proliferating cells using various parameters. In most of the conditions tested, increased cell proliferation was associated with PBM in both control and CCO negative cells, demonstrating that CCO is not required for PBM enhancement of cellular proliferation. Additional experiments showed that PBM increased both ATP levels and citrate synthase activity and levels. These results showed that although metabolic changes are associated with PBM, CCO is not required for its cell proliferation enhancing effect.
Assuntos
Proliferação de Células/efeitos da radiação , Terapia com Luz de Baixa Intensidade , Linhagem Celular , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiaçãoRESUMO
Pathogenic mitochondrial DNA (mtDNA) mutations often co-exist with wild-type molecules (mtDNA heteroplasmy). Phenotypes manifest when the percentage of mutant mtDNA is high (70-90%). Previously, our laboratory showed that mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs) can eliminate mutant mtDNA from heteroplasmic cells. However, mitoTALENs are dimeric and relatively large, making it difficult to package their coding genes into viral vectors, limiting their clinical application. The smaller monomeric GIY-YIG homing nuclease from T4 phage (I-TevI) provides a potential alternative. We tested whether molecular hybrids (mitoTev-TALEs) could specifically bind and cleave mtDNA of patient-derived cybrids harboring different levels of the m.8344A>G mtDNA point mutation, associated with myoclonic epilepsy with ragged-red fibers (MERRF). We tested two mitoTev-TALE designs, one of which robustly shifted the mtDNA ratio toward the wild type. When this mitoTev-TALE was tested in a clone with high levels of the MERRF mutation (91% mutant), the shift in heteroplasmy resulted in an improvement of oxidative phosphorylation function. mitoTev-TALE provides an effective architecture for mtDNA editing that could facilitate therapeutic delivery of mtDNA editing enzymes to affected tissues.
Assuntos
DNA Mitocondrial/metabolismo , Endonucleases/metabolismo , Terapia de Alvo Molecular/métodos , Proteínas Recombinantes/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Células Cultivadas , Reparo do DNA , Endonucleases/genética , Humanos , Hidrólise , Síndrome MERRF/tratamento farmacológico , Ligação Proteica , Proteínas Recombinantes/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Proteínas Virais/genéticaRESUMO
PURPOSE: To phenotypically and genotypically characterize a large Puerto Rican kindred with X-linked retinitis pigmentosa associated with a novel RP GTPase regulator (RPGR) genotype. METHODS: A total of 100 family members of a single kindred with X-linked RP were evaluated with ophthalmic examinations and blood DNA analysis. Visual fields, OCT, and full-field ERG were obtained on all affected males and carriers. RESULTS: Of the 100 family members examined, 13 were affected males and 18 were carriers. A deletion of 2 base pair of the RPGR gene in the ORF15 region at position c.2267-2268 (Lys756del2aaAG hemi) was identified with the affected and carriers. Best eye visual acuity was correlated with age (Spearman coefficient = 0.95) with hand-motion acuity by age 35 and light perception to no light perception by age 50-60. Visual fields were minimally plottable by age 40, and ERG responses reached non-detectable levels by late teens. Carriers had no or mild visual symptoms. All carriers had visual acuity of at least 20/50 or better in one eye, and the amount of retinal degeneration was variable with ERG responses ranging from severely impaired to normal. CONCLUSIONS: Profound visual loss occurred by the second decade of life with progression to near no light perception by age 60 in this kindred of X-linked RP associated with the RPGR genotype. Female carriers maintained visual acuity with age and were identifiable by clinical and ERG examination. The information from this study is important to determine the optimal age for intervention, as new RP treatments are being developed and tested.
Assuntos
Proteínas do Olho/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Retinose Pigmentar/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Eletrorretinografia , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Doenças Genéticas Ligadas ao Cromossomo X/fisiopatologia , Genótipo , Heterozigoto , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Fases de Leitura Aberta/genética , Fenótipo , Retinose Pigmentar/diagnóstico , Retinose Pigmentar/fisiopatologia , Tomografia de Coerência Óptica , Transtornos da Visão/diagnóstico , Transtornos da Visão/genética , Transtornos da Visão/fisiopatologia , Acuidade Visual/fisiologia , Campos Visuais/fisiologiaRESUMO
PURPOSE: To evaluate the efficiency and safety of AAV-mediated gene delivery of a normal human ND4 complex I subunit in the mouse visual system. METHODS: A nuclear encoded human ND4 subunit fused to the ATPc mitochondrial targeting sequence and FLAG epitope were packaged in AAV2 capsids that were injected into the right eyes of mice. AAV-GFP was injected into the left eyes. One month later, pattern electroretinography (PERG), rate of ATP synthesis, gene expression, and incorporation of the human ND4 subunit into the murine complex I were evaluated. Quantitative analysis of ND4FLAG-injected eyes was assessed compared with green fluorescent protein (GFP)-injected eyes. RESULTS: Rates of ATP synthesis and PERG amplitudes were similar in ND4FLAG- and GFP-inoculated eyes. PERG latency was shorter in eyes that received ND4FLAG. Immunoprecipitated murine complex I gave the expected 52-kDa band of processed human ND4FLAG. Confocal microscopy revealed perinuclear expression of FLAG colocalized with mitochondria-specific fluorescent dye. Transmission electron microscopy revealed FLAG immunogold within mitochondria. Compared with Thy1.2-positive retinal ganglion cells (RGCs), quantification was 38% for FLAG-positive RGCs and 65% for GFP-positive RGCs. Thy1.2 positive-RGC counts in AAV-ND4FLAG were similar to counts in control eyes injected with AAV-GFP. CONCLUSIONS: Human ND4 was properly processed and imported into the mitochondria of RGCs and axons of mouse optic nerve after intravitreal injection. Although it had approximately two-thirds the efficiency of GFP, the expression of normal human ND4 in murine mitochondria did not induce the loss of RGCs, ATP synthesis, or PERG amplitude, suggesting that allotopic ND4 may be safe for the treatment of patients with Leber hereditary optic neuropathy.