RESUMEN
The diagnosis of Mendelian disorders following uninformative exome and genome sequencing remains a challenging and often unmet need. Following uninformative exome and genome sequencing of a family quartet including two siblings with suspected mitochondrial disorder, RNA sequencing (RNAseq) was pursued in one sibling. Long-read amplicon sequencing was used to determine and quantify transcript structure. Immunoblotting studies and quantitative proteomics were performed to demonstrate functional impact. Differential expression analysis of RNAseq data identified significantly decreased expression of the mitochondrial OXPHOS Complex I subunit NDUFB10 associated with a cryptic exon in intron 1 of NDUFB10, that included an in-frame stop codon. The cryptic exon contained a rare intronic variant that was homozygous in both affected siblings. Immunoblot and quantitative proteomic analysis of fibroblasts revealed decreased abundance of Complex I subunits, providing evidence of isolated Complex I deficiency. Through multiomic analysis we present data implicating a deep intronic variant in NDUFB10 as the cause of mitochondrial disease in two individuals, providing further support of the gene-disease association. This study highlights the importance of transcriptomic and proteomic analyses as complementary diagnostic tools in patients undergoing genome-wide diagnostic evaluation.
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Enfermedades Mitocondriales , NADH Deshidrogenasa/genética , Proteómica , Complejo I de Transporte de Electrón/genética , Humanos , Intrones/genética , Enfermedades Mitocondriales/diagnóstico , Enfermedades Mitocondriales/genética , MutaciónRESUMEN
Reactive oxygen species (ROS) have already been demonstrated to impede the migratory ability in non-melanocytic cell lines by depleting mitochondrial ATP production. Therefore, understanding the mitochondrial metabolic response to migration in the presence of ROS should be a key to understanding repigmentation in vitiligo. This study aimed to investigate the energy mechanism associated with the ROS-mediated attenuation of melanocyte migration. After melanocytes were pretreated with H2 O2 , their ATP production, migratory ability, ultrastructural changes and Mitochondrial Permeability Potential were analysed. The results showed that, in parallel with the decreased ATP production, the migratory ability of melanocytes was significantly inhibited by oxidative stress. Supplementation with exogenous ATP reversed the suppressed ATP-dependent migration of melanocytes. Melanocytes were then stressed with H2 O2 and Agilent Whole Human Genome microarray analysis identified 763 up-regulated mRNAs and 1117 down-regulated mRNAs. Among them, 11 of the encoded proteins were involved in mitochondrial ATP production and their expression levels were verified. The decreased expression of NADH dehydrogenase 2(ND2) , cytochrome c oxidase 1(COX1) and cytochrome c oxidase 3(COX3) was shown to be involved in the depletion of mitochondrial ATP production, which was coupled with the impaired migratory potential. These results indicate that the migration of melanocytes relies heavily on an inexhaustible supply of ATP from mitochondria.
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Adenosina Trifosfato/biosíntesis , Movimiento Celular , Melanocitos/fisiología , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Adenosina Trifosfato/farmacología , Vías Biosintéticas/genética , Movimiento Celular/efectos de los fármacos , Ciclooxigenasa 1/genética , Ciclooxigenasa 1/metabolismo , Regulación hacia Abajo , Colorantes Fluorescentes/metabolismo , Perfilación de la Expresión Génica , Humanos , Peróxido de Hidrógeno/farmacología , Melanocitos/ultraestructura , NADH Deshidrogenasa/genética , NADH Deshidrogenasa/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxidantes/farmacología , Estrés Oxidativo/genética , Permeabilidad , ARN Mensajero/análisis , Regulación hacia Arriba , Vitíligo/fisiopatologíaRESUMEN
Signaling diversity and subsequent complexity in higher eukaryotes is partially explained by one gene encoding a polypeptide with multiple biochemical functions in different cellular contexts. For example, mouse double minute 2 (MDM2) is functionally characterized as both an oncogene and a tumor suppressor, yet this dual classification confounds the cell biology and clinical literatures. Identified via complementary biochemical, organellar, and cellular approaches, we report that MDM2 negatively regulates NADH:ubiquinone oxidoreductase 75 kDa Fe-S protein 1 (NDUFS1), leading to decreased mitochondrial respiration, marked oxidative stress, and commitment to the mitochondrial pathway of apoptosis. MDM2 directly binds and sequesters NDUFS1, preventing its mitochondrial localization and ultimately causing complex I and supercomplex destabilization and inefficiency of oxidative phosphorylation. The MDM2 amino-terminal region is sufficient to bind NDUFS1, alter supercomplex assembly, and induce apoptosis. Finally, this pathway is independent of p53, and several mitochondrial phenotypes are observed in Drosophila and murine models expressing transgenic Mdm2.
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Mitocondrias/metabolismo , NADH Deshidrogenasa/genética , Estrés Oxidativo/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteína p53 Supresora de Tumor/genética , Células A549 , Animales , Apoptosis/genética , Respiración de la Célula/genética , Citosol/metabolismo , Drosophila melanogaster/genética , Complejo I de Transporte de Electrón/genética , Humanos , Ratones , Ratones Transgénicos , Mitocondrias/genética , Transducción de Señal/genéticaAsunto(s)
Encéfalo/diagnóstico por imagen , Leucoencefalopatías/diagnóstico por imagen , Enfermedades Neurodegenerativas/diagnóstico por imagen , Complejo I de Transporte de Electrón , Femenino , Humanos , Lactante , Leucoencefalopatías/tratamiento farmacológico , Leucoencefalopatías/genética , Imagen por Resonancia Magnética , NADH Deshidrogenasa/genética , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/genética , Terapia OrtomolecularRESUMEN
Converting carcinomas in benign oncocytomas has been suggested as a potential anti-cancer strategy. One of the oncocytoma hallmarks is the lack of respiratory complex I (CI). Here we use genetic ablation of this enzyme to induce indolence in two cancer types, and show this is reversed by allowing the stabilization of Hypoxia Inducible Factor-1 alpha (HIF-1α). We further show that on the long run CI-deficient tumors re-adapt to their inability to respond to hypoxia, concordantly with the persistence of human oncocytomas. We demonstrate that CI-deficient tumors survive and carry out angiogenesis, despite their inability to stabilize HIF-1α. Such adaptive response is mediated by tumor associated macrophages, whose blockage improves the effect of CI ablation. Additionally, the simultaneous pharmacological inhibition of CI function through metformin and macrophage infiltration through PLX-3397 impairs tumor growth in vivo in a synergistic manner, setting the basis for an efficient combinatorial adjuvant therapy in clinical trials.
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Adenoma Oxifílico/tratamiento farmacológico , Adenoma Oxifílico/genética , Aminopiridinas/farmacología , Antineoplásicos/farmacología , Complejo I de Transporte de Electrón/antagonistas & inhibidores , Complejo I de Transporte de Electrón/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Metformina/farmacología , Pirroles/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Drosophila , Femenino , Técnicas de Inactivación de Genes , Células HCT116 , Humanos , Macrófagos/inmunología , Ratones , Ratones Noqueados , Ratones Desnudos , NADH Deshidrogenasa/genética , Neovascularización Patológica/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Araucaria angustifolia extract (AAE) is a polyphenol-rich extract that has gained interest as a natural anticancer agent. Recent work suggests that AAE induces oxidative damage and apoptosis through its action on decreasing complex I activity of the mitochondrial Electron Transport Chain (ETC). AIMS AND METHODS: In the present study, we aimed to further examine the specific targets by which AAE exerts proapoptotic effects in HEp-2 cancer cells. Specifically, the effect of AAE on the: 1) levels of pyruvate dehydrogenase was assessed by ELISA assay; 2) levels of mitochondrial ETC complexes, focusing on complex I at the gene transcript and protein level relevant to ROS generation was evaluated by multiplex ELISA followed by qRT-PCR and immunoblotting; 3) mitochondrial network distribution analysis was assessed by MitoTracker Red CMXRos; and 4) chemical variations on DNA was evaluated by dot-blotting in HEp-2 cells. RESULTS: Results demonstrated that AAE increased protein levels of PDH, switching energy metabolism to oxidative metabolism. Protein expression levels of complex I and III were found decreased in AAE-treated HEp-2 cells. Analyzing the subunits of complex I, changes in protein and gene transcript levels of NDUFS7 and NDUFV2 were found. Mitochondria staining after AAE incubation revealed changes in the mitochondrial network distribution. AAE was able to induce DNA hypomethylation and decreased DNA (cytosine-5)-methyltransferase 1 activity. CONCLUSION: Our data demonstrate for the first time that AAE alters expression of NDUFS7 and NDUFV2 mitochondrial subunits and induce epigenetic changes in HEp-2 cancer cells leading to a possible suppression of oncogenes.
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Antineoplásicos Fitogénicos/farmacología , Carcinoma de Células Escamosas/tratamiento farmacológico , Epigénesis Genética/efectos de los fármacos , Neoplasias Laríngeas/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Extractos Vegetales/farmacología , Polifenoles/farmacología , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Epigénesis Genética/genética , Humanos , Neoplasias Laríngeas/metabolismo , Neoplasias Laríngeas/patología , Mitocondrias/metabolismo , NADH Deshidrogenasa/antagonistas & inhibidores , NADH Deshidrogenasa/genética , NADH Deshidrogenasa/metabolismo , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Polifenoles/química , Polifenoles/aislamiento & purificación , Relación Estructura-Actividad , Tracheophyta/químicaRESUMEN
INTRODUCTION: Charles Bonnet syndrome is characterized by simple or complex visual hallucinations (VH) due to damage along the visual pathways. We report a functional MRI study of brain correlates of VH in the context of a severe optic atrophy in a patient with Leber's Hereditary Optic Neuropathy (LHON). CASE REPORT: A 62-year-old man was diagnosed with LHON (11778/ND4 mtDNA mutation) after subacute visual loss in left eye (right eye was amblyopic). One month later, he experienced VH of a few seconds consisting in "moving red and blue miniature cartoons". One year later VH content changed in colored mosaic (10-15 s duration), usually stress-related, and blue and white flashes (2-5 s), triggered by unexpected auditory stimuli. Audiometry revealed mild sensorineural hearing loss. Three block design functional MRI paradigms were administrated: 1) random "clap", 2) "checkerboard" and 3) non-random "beep". After random "claps" simple flashes were evoked with bilateral activation of primary and secondary visual cortex, cuneus, precuneus and insula. Neither hallucinations nor cortex activation were registered after "checkerboard" stimulation, due to the severe visual impairment. Primary and secondary auditory cortices were "beep"-activated, without eliciting VH by non-random "beep". CONCLUSIONS: The peculiarity of our case is that VH were triggered by random auditory stimuli, possibly due to a cross-modal plasticity between visual and auditory networks, likely influenced by the sensorineural deafness. Functional alterations of both networks in resting conditions have been demonstrated in LHON patients, even without an auditory deficit. Finally, the absence of VH triggered by expected stimuli is consistent with the "expectation suppression theory", based on increased neural activations after unexpected but not by predicted events.
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Corteza Cerebral/fisiopatología , Síndrome de Charles Bonnet/complicaciones , Síndrome de Charles Bonnet/fisiopatología , Atrofia Óptica Hereditaria de Leber/complicaciones , Estimulación Acústica , Adulto , ADN Mitocondrial/genética , Neuroimagen Funcional , Alucinaciones/complicaciones , Alucinaciones/fisiopatología , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , NADH Deshidrogenasa/genética , Atrofia Óptica Hereditaria de Leber/genética , Trastornos de la Visión/complicacionesRESUMEN
BACKGROUND: Leber's hereditary optic neuropathy (LHON) is a maternally inherited recessive disease rarely complicated with glaucoma. We conducted a clinical and genetic retrospective case series to describe three cases of juvenile open-angle glaucoma (JOAG) and an ND4 m11778G > A mitochondrial DNA (mtDNA) mutation, which is pathognomonic for LHON. CASE PRESENTATION: Patient 1 was a 16-year-old boy diagnosed with bilateral JOAG and high myopia. His intraocular pressure (IOP) was poorly controlled with the use of full topical anti-glaucoma medications. His best-corrected visual acuity (BCVA) decreased gradually over 5 years. Fundoscopic examination revealed bilateral enlarged disc cupping of the optic nerves with sectorial excavation and reduction of the neural rim in the left eye. His visual field (VF) was characterized by bilateral progressive central scotoma. Pattern visual evoked potentials (VEPs) and pattern electroretinograms (ERGs) showed extinguished responses in both eyes. Because of the non-specific visual field findings and the optic neuropathy disclosed by the pattern VEPs and pattern ERGs, we arranged a genetic test for the patient, which revealed an m11778G > A mtDNA mutation. Patient 2, the younger brother of Patient 1, was a 15-year-old boy who had been diagnosed with bilateral JOAG in 2010. The BCVA of both eyes remained at 1.0 during the follow-up period. Fundoscopic examination revealed bilateral mildly paled optic disc with enlarged cupping and reduction of the neural rim. The pattern ERG revealed a decreased N95 amplitude bilaterally. The genetic test revealed an m11778G > A mtDNA mutation. Patient 3 was a 35-year-old man with bilateral JOAG. His BCVA decreased gradually over 10 years. Fundoscopic examination revealed paled optic disc with enlarged disc cupping and reduction of the neural rim in both eyes. The pattern ERG revealed a decreased N95 amplitude bilaterally. The genetic test revealed an m11778G > A mtDNA mutation. CONCLUSIONS: This case series describes three patients with concomitant occurrence of JOAG and LHON. These two diseases may have a cumulative effect on oxidative stress and retinal ganglion cell death with the rapid deterioration of vision, which may occur during adolescence.
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Glaucoma de Ángulo Abierto/genética , NADH Deshidrogenasa/genética , Atrofia Óptica Hereditaria de Leber/genética , Adolescente , Adulto , ADN Mitocondrial/genética , Glaucoma de Ángulo Abierto/etiología , Humanos , Masculino , Atrofia Óptica Hereditaria de Leber/complicaciones , Estudios RetrospectivosRESUMEN
BACKGROUND: Nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit-2 237 leucine/methionine (ND2-237 Leu/Met) polymorphism has been shown to modify the association of coffee consumption with the risk of hypertension, dyslipidemia, and abnormal glucose tolerance, and low serum chloride levels have been shown to be associated with all-cause and cardiovascular disease mortality. Therefore, the purpose of the present study was to investigate whether ND2-237 Leu/Met polymorphism influences the association of coffee consumption with serum chloride levels in male Japanese health checkup examinees. METHODS: From among individuals visiting the hospital for a regular medical checkup, 402 men (mean age ± standard deviation, 53.9 ± 7.8 years) were selected for inclusion in the study. After ND2-237 Leu/Met genotyping, we conducted an exploratory cross-sectional study to examine the combined association of ND2-237 Leu/Met polymorphism and coffee consumption with serum electrolyte levels. RESULTS: After adjusting for age, body mass index, habitual smoking, alcohol consumption, green tea consumption, and antihypertensive medication, coffee consumption significantly increased serum chloride levels (p for trend = 0.001) in men with the ND2-237Leu genotype. After these adjustments, the odds ratios (ORs) for low levels of serum chloride, defined as <100 mEq/L, were found to be dependent on coffee consumption (p for trend = 0.001). In addition, the OR for low levels of serum chloride was significantly lower in men with the ND2-237Leu genotype who consumed ≥4 compared with <1 cup of coffee per day (OR = 0.096, 95% confidence interval = 0.010â»0.934; p = 0.044). However, neither serum chloride levels nor risk of low levels of serum chloride appeared to be dependent on coffee consumption. CONCLUSIONS: The results suggest that ND2-237 Leu/Met polymorphism modifies the association of coffee consumption with serum chloride levels in middle-aged Japanese men.
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Cloruros/sangre , Café , Conducta Alimentaria , NADH Deshidrogenasa/genética , Polimorfismo Genético , Factores de Edad , Estudios Transversales , Interacción Gen-Ambiente , Genotipo , Humanos , Japón , Masculino , Persona de Mediana Edad , Fenotipo , Factores SexualesRESUMEN
Oxidative damage is accrual of molecular deterioration from reactive oxygen species (ROS) while decrease in generation of ROS is related with free radical scavenging enzymes. Boerhaavia diffusa L. (Nyctaginaceae) derived novel molecule Boeravinone B (BOB) possesses a variety of pharmacological activities, yet their anti-aging potential has not been explored. The aim of the present study was to elucidate the mechanism of BOB mediated oxidative stress resistance and lifespan extension in Caenorhabditis elegans. The results showed that the BOB significantly extends the lifespan of C. elegans with its anti-oxidative potential via reducing accumulation of reactive oxygen species (ROS). BOB was found to recover the shortened lifespan of oxidative stress prone mutants mev-1 and gas-1 (14.75 and 16.11%, respectively). Additionally, this finding supported by the reduced ROS levels seen in BOB treated worms. Further, the effective concentration of BOB (25⯵M) significantly enhanced the expressions of target genes such as superoxide dismutase (SOD-3), glutathione-S-transferase (GST-4) and heat shock protein (HSP-16.2) fused to green fluorescent protein (GFP), and it does so by modulating the stress-related signaling pathways (SEK-1) and transcription factors (SKN-1/Nrf and DAF-16/Foxo). Moreover, BOB exposure (25⯵M) caused significant changes of age-dependent biomarkers such as pharyngeal pumping, body bend, locomotor activity and lipofuscin accumulation were also showed that BOB retards the aging. Overall, the findings highlight the antioxidant supplement triggering pharmaceutical potential of BOB which may serve as a new future perspective for healthy aging or delayed onset of oxidative related diseases.
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Caenorhabditis elegans/fisiología , Flavonoides/farmacología , Radicales Libres/metabolismo , Longevidad , Especies Reactivas de Oxígeno/metabolismo , Animales , Antioxidantes/farmacología , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Citocromos b/genética , Regulación de la Expresión Génica , Glutatión Transferasa/metabolismo , Proteínas de Choque Térmico/metabolismo , Calor , NADH Deshidrogenasa/genética , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal , Estrés Fisiológico/efectos de los fármacos , Superóxido Dismutasa/metabolismoRESUMEN
SIGNIFICANCE: The American Academy of Ophthalmology currently recommends against routine genetic testing for complex diseases such as age-related macular degeneration (AMD). The results of this study demonstrate that patients are very interested in predictive genetic testing for AMD, find the information useful, and make behavioral changes as a result of the information. PURPOSE: The goal of this project was to conduct a pilot AMD genomic medicine study. METHODS: Eligible patients were aged 50 to 65 years with no personal history of AMD. DNA samples were genotyped for five single-nucleotide polymorphisms (SNPs) in the CFH gene, one SNP in the ARMS-2 gene, one SNP in the C3 gene, and one SNP in the mitochondrial ND2 gene. A risk score was calculated utilizing a model based on odds ratios, lifetime risk of advanced AMD and known population prevalence of genotype, haplotype, and smoking risk. The study optometrist provided the patient's risk score and counseling for personal protective behaviors. Telephone interviews were conducted 1 to 3 months after the counseling visit. RESULTS: One hundred one subjects (85%) participated in the genetic testing; 78 (77.2%) were female. Follow-up interviews were conducted with 94 participants (93.1%). More than half (n = 48) of the participants said that they were motivated to participate in the study because they had a family member with AMD or another eye or genetic disorder. Despite low risk levels, many participants reported making changes as a result of the genetic testing. Twenty-seven people reported making specific changes, including wearing sunglasses and brimmed hat and taking vitamin supplements. Another 16 people said that they were already doing the recommended activities, including wearing glasses, quitting smoking, and/or taking vitamins. CONCLUSIONS: Interest in genetic testing for future risk of AMD was high in this population and resulted in support to continue current health behaviors or incentive to improve behaviors related to eye health.
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Pruebas Genéticas , Degeneración Macular/genética , Degeneración Macular/psicología , Pacientes/psicología , Polimorfismo de Nucleótido Simple , Anciano , Complemento C3/genética , Factor H de Complemento/genética , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , NADH Deshidrogenasa/genética , Proyectos Piloto , Proteínas/genética , Medición de RiesgoRESUMEN
Mitochondrial complex I (CI) deficiency is the most frequent cause of oxidative phosphorylation (OXPHOS) disorders in humans. In order to benchmark the effects of CI deficiency on mitochondrial bioenergetics and dynamics, respiratory chain (RC) and endoplasmic reticulum (ER)-mitochondria communication, and superoxide production, fibroblasts from patients with mutations in the ND6, NDUFV1 or ACAD9 genes were analyzed. Fatty acid metabolism, basal and maximal respiration, mitochondrial membrane potential, and ATP levels were decreased. Changes in proteins involved in mitochondrial dynamics were detected in various combinations in each cell line, while variable changes in RC components were observed. ACAD9 deficient cells exhibited an increase in RC complex subunits and DDIT3, an ER stress marker. The level of proteins involved in ER-mitochondria communication was decreased in ND6 and ACAD9 deficient cells. |ΔΨ| and cell viability were further decreased in all cell lines. These findings suggest that disruption of mitochondrial bioenergetics and dynamics, ER-mitochondria crosstalk, and increased superoxide contribute to the pathophysiology in patients with ACAD9 deficiency. Furthermore, treatment of ACAD9 deficient cells with JP4-039, a novel mitochondria-targeted reactive oxygen species, electron and radical scavenger, decreased superoxide level and increased basal and maximal respiratory rate, identifying a potential therapeutic intervention opportunity in CI deficiency.
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Acil-CoA Deshidrogenasas/genética , Complejo I de Transporte de Electrón/deficiencia , Fibroblastos/enzimología , Enfermedades Mitocondriales/genética , NADH Deshidrogenasa/genética , Especies Reactivas de Oxígeno/metabolismo , Acil-CoA Deshidrogenasas/deficiencia , Adenosina Trifosfato/agonistas , Adenosina Trifosfato/biosíntesis , Transporte de Electrón/efectos de los fármacos , Transporte de Electrón/genética , Complejo I de Transporte de Electrón/genética , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Depuradores de Radicales Libres/farmacología , Expresión Génica , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Mitocondrias/patología , Enfermedades Mitocondriales/enzimología , Enfermedades Mitocondriales/patología , NADH Deshidrogenasa/deficiencia , Óxidos de Nitrógeno/farmacología , Fosforilación Oxidativa/efectos de los fármacos , Cultivo Primario de Células , Especies Reactivas de Oxígeno/antagonistas & inhibidoresRESUMEN
Evidence has demonstrated iron accumulation in specific brain regions of patients suffering from neurodegenerative disorders, and this metal has been recognized as a contributing factor for neurodegeneration. Using an experimental model of brain iron accumulation, we have shown that iron induces severe memory deficits that are accompanied by oxidative stress, increased apoptotic markers, and decreased synaptophysin in the hippocampus of rats. The present study aims to characterize iron loading effects as well as to determine the molecular targets of cannabidiol (CBD), the main non-psychomimetic compound of Cannabis sativa, on mitochondria. Rats received iron in the neonatal period and CBD for 14â¯days in adulthood. Iron induced mitochondrial DNA (mtDNA) deletions, decreased epigenetic modulation of mtDNA, mitochondrial ferritin levels, and succinate dehydrogenase activity. CBD rescued mitochondrial ferritin and epigenetic modulation of mtDNA, and restored succinate dehydrogenase activity in iron-treated rats. These findings provide new insights into molecular targets of iron neurotoxicity and give support for the use of CBD as a disease modifying agent in the treatment of neurodegenerative diseases.
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Cannabidiol/uso terapéutico , ADN Mitocondrial/metabolismo , Hipocampo/efectos de los fármacos , Compuestos de Hierro Carbonilo/toxicidad , Mitocondrias/efectos de los fármacos , Enfermedades Neurodegenerativas/inducido químicamente , Enfermedades Neurodegenerativas/tratamiento farmacológico , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animales , Animales Recién Nacidos , Creatina Quinasa/metabolismo , Metilación de ADN/efectos de los fármacos , ADN Mitocondrial/genética , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , NADH Deshidrogenasa/genética , NADH Deshidrogenasa/metabolismo , Enfermedades Neurodegenerativas/patología , Embarazo , Ratas , Ratas WistarRESUMEN
PURPOSE: Leber hereditary optic neuropathy (LHON) is a disorder characterized by severe and rapidly progressive visual loss when caused by a mutation in the mitochondrial gene encoding NADH:ubiquinone oxidoreductase subunit 4 (ND4). We have initiated a gene therapy trial to determine the safety and tolerability of escalated doses of an adeno-associated virus vector (AAV) expressing a normal ND4 complementary DNA in patients with a G to A mutation at nucleotide 11778 of the mitochondrial genome. DESIGN: In this prospective open-label trial (NCT02161380), the study drug (self-complementary AAV [scAAV]2(Y444,500,730F)-P1ND4v2) was intravitreally injected unilaterally into the eyes of 5 blind participants with G11778A LHON. Four participants with visual loss for more than 12 months were treated. The fifth participant had visual loss for less than 12 months. The first 3 participants were treated at the low dose of vector (5 × 10(9) vg), and the fourth participant was treated at the medium dose (2.46 × 10(10) vg). The fifth participant with visual loss for less than 12 months received the low dose. Treated participants were followed for 90 to 180 days and underwent ocular and systemic safety assessments along with visual structure and function examinations. PARTICIPANTS: Five legally blind patients with G11778A LHON. MAIN OUTCOME MEASURES: Loss of visual acuity. RESULTS: Visual acuity as measured by the Early Treatment Diabetic Retinopathy Study (ETDRS) eye chart remained unchanged from baseline to 3 months in the first 3 participants. For 2 participants with 90-day follow-up, acuity increased from hand movements to 7 letters in 1 and by 15 letters in 1, representing an improvement equivalent to 3 lines. No one lost vision, and no serious adverse events were observed. Minor adverse events included a transient increase of intraocular pressure (IOP), exposure keratitis, subconjunctival hemorrhage, a sore throat, and a transient increase in neutralizing antibodies (NAbs) against AAV2 in 1 participant. All blood samples were negative for vector DNA. CONCLUSIONS: No serious safety problems were observed in the first 5 participants enrolled in this phase I trial of virus-based gene transfer in this mitochondrial disorder. Additional study follow-up of these and additional participants planned for the next 4 years is needed to confirm these preliminary observations.
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ADN Mitocondrial/genética , Dependovirus/genética , Terapia Genética , Vectores Genéticos , NADH Deshidrogenasa/genética , Atrofia Óptica Hereditaria de Leber/terapia , Adulto , Electrorretinografía , Femenino , Humanos , Inyecciones Intravítreas , Masculino , Persona de Mediana Edad , Atrofia Óptica Hereditaria de Leber/genética , Atrofia Óptica Hereditaria de Leber/fisiopatología , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple , Estudios Prospectivos , Tomografía de Coherencia Óptica , Agudeza Visual/fisiología , Pruebas del Campo Visual , Campos VisualesRESUMEN
Mitochondria play an important role in maintaining metabolic and energy homeostasis in the cell. In plants, impairment in mitochondrial functions usually has detrimental effects on growth and development. To study genes that are important for plant growth, we have isolated a collection of slow growth (slo) mutants in Arabidopsis (Arabidopsis thaliana). One of the slo mutants, slo3, has a significant reduction in mitochondrial complex I activity. The slo3 mutant has a four-nucleotide deletion in At3g61360 that encodes a pentatricopeptide repeat (PPR) protein. The SLO3 protein contains nine classic PPR domains belonging to the P subfamily. The small deletion in the slo3 mutant changes the reading frame and creates a premature stop codon in the first PPR domain. We demonstrated that the SLO3-GFP is localized to the mitochondrion. Further analysis of mitochondrial RNA metabolism revealed that the slo3 mutant was defective in splicing of NADH dehydrogenase subunit7 (nad7) intron 2. This specific splicing defect led to a dramatic reduction in complex I activity in the mutant as revealed by blue native gel analysis. Complementation of slo3 by 35S:SLO3 or 35S:SLO3-GFP restored the splicing of nad7 intron 2, the complex I activity, and the growth defects of the mutant. Together, these results indicate that the SLO3 PPR protein is a splicing factor of nad7 intron 2 in Arabidopsis mitochondria.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Intrones/genética , Mitocondrias/enzimología , Proteínas Mitocondriales/metabolismo , NADH Deshidrogenasa/genética , Empalme del ARN/genética , Secuencias Repetitivas de Aminoácido , Secuencia de Aminoácidos , Arabidopsis/citología , Arabidopsis/enzimología , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Secuencia de Bases , Proliferación Celular , ADN Complementario/genética , Complejo I de Transporte de Electrón/metabolismo , Genes de Plantas , Prueba de Complementación Genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Datos de Secuencia Molecular , Mutación/genética , NADH Deshidrogenasa/metabolismo , Fenotipo , Raíces de Plantas , Transporte de Proteínas , Edición de ARN , Factores de Empalme de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Plantones/metabolismoRESUMEN
Mitochondrial disorders are devastating genetic diseases for which efficacious therapies are still an unmet need. Recent studies report that increased availability of intracellular NAD obtained by inhibition of the NAD-consuming enzyme poly(ADP-ribose) polymerase (PARP)-1 or supplementation with the NAD-precursor nicotinamide riboside (NR) ameliorates energetic derangement and symptoms in mouse models of mitochondrial disorders. Whether these pharmacological approaches also improve bioenergetics of human cells harboring mitochondrial defects is unknown. It is also unclear whether the same signaling cascade is prompted by PARP-1 inhibitors and NR supplementation to improve mitochondrial homeostasis. Here, we show that human fibroblasts mutant for the NADH dehydrogenase (ubiquinone) Fe-S protein 1 (NDUFS1) subunit of respiratory complex I have similar ATP, NAD, and mitochondrial content compared with control cells, but show reduced mitochondrial membrane potential. Interestingly, mutant cells also show increased transcript levels of mitochondrial DNA but not nuclear DNA respiratory complex subunits, suggesting activation of a compensatory response. At variance with prior work in mice, however, NR supplementation, but not PARP-1 inhibition, increased intracellular NAD content in NDUFS1 mutant human fibroblasts. Conversely, PARP-1 inhibitors, but not NR supplementation, increased transcription of mitochondrial transcription factor A and mitochondrial DNA-encoded respiratory complexes constitutively induced in mutant cells. Still, both NR and PARP-1 inhibitors restored mitochondrial membrane potential and increased organelle content as well as oxidative activity of NDUFS1-deficient fibroblasts. Overall, data provide the first evidence that in human cells harboring a mitochondrial respiratory defect exposure to NR or PARP-1, inhibitors activate different signaling pathways that are not invariantly prompted by NAD increases, but equally able to improve energetic derangement.
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Fibroblastos/efectos de los fármacos , Mitocondrias/efectos de los fármacos , NADH Deshidrogenasa/genética , NAD/metabolismo , Niacinamida/análogos & derivados , Metabolismo Energético , Fibroblastos/metabolismo , Homeostasis , Humanos , Lactante , Leucoencefalopatías/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Mutación , Niacinamida/farmacología , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Compuestos de Piridinio , Transducción de SeñalRESUMEN
Obesity and metabolic syndrome are growing public health problems. We investigated the effects of decaffeinated green tea extract (GTE) and voluntary running exercise (Ex) alone or in combination against obesity and metabolic syndrome in high fat (HF) fed C57BL/6J mice. After 16 wk, GTE + Ex treatment reduced final body mass (27.1% decrease) and total visceral fat mass (36.6% decrease) compared to HF-fed mice. GTE + Ex reduced fasting blood glucose (17% decrease), plasma insulin (65% decrease), and insulin resistance (65% decrease) compared to HF-fed mice. GTE or Ex alone had less significant effects. In the skeletal muscle, the combination of Ex and GTE increased the expression of peroxisome proliferator-activated receptor-γ coactivator-1α (Ppargc1a), mitochondrial NADH dehydrogenase 5 (mt-Nd5), mitochondrial cytochrome b (mt-Cytb), and mitochondrial cytochrome c oxidase III (mt-Co3). An increase in hepatic expression of peroxisome proliferator-activated receptor-α (Ppara) and liver carnitine palmitoyl transferase-1α (Cpt1a) and a decrease in hepatic expression of stearoyl-CoA desaturase 1 (Scd1) mRNA was observed in GTE + Ex mice. GTE + Ex was more effective than either treatment alone in reducing diet-induced obesity. These effects are due in part to modulation of genes related to energy metabolism and de novo lipogenesis.
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Dieta Alta en Grasa , Síndrome Metabólico/prevención & control , Condicionamiento Físico Animal , Té/química , Animales , Carnitina O-Palmitoiltransferasa/genética , Carnitina O-Palmitoiltransferasa/metabolismo , Metabolismo Energético , Resistencia a la Insulina , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , NADH Deshidrogenasa/genética , NADH Deshidrogenasa/metabolismo , Obesidad/prevención & control , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estearoil-CoA Desaturasa/genética , Estearoil-CoA Desaturasa/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: NADH dehydrogenase subunit-2 237 leucine/methionine (ND2-237 Leu/Met) polymorphism is associated with longevity in Japanese. A previous study has shown that ND2-237 Leu/Met polymorphism modulates the effects of green tea consumption on risk of hypertension. For men with ND2-237Leu, habitual green tea consumption may reduce the risk of hypertension. Moreover, there is a combined effect of ND2-237 Leu/Met polymorphism and alcohol consumption on risk of mildly decreased estimated glomerular filtration rate (eGFR) (<90 ml/min/1.73 m2). Several beneficial effects of green tea on the kidney have been reported. The objective of this study was to investigate whether ND2-237 Leu/Met polymorphism modifies the effects of green tea consumption on risk of mildly decreased eGFR in male Japanese health check-up examinees. RESULTS: For ND2-237Leu genotypic men, after adjustment for confounding factors, green tea consumption may increase the risk of mildly decreased eGFR (P for trend = 0.016). The adjusted odds ratio (OR) for mildly decreased eGFR was significantly higher in subjects with ND2-237Leu who consume ≥6 cups of green tea per day than those who consume ≤1 cup of green tea per day (adjusted OR = 5.647, 95% confidence interval: 1.528-20.88, P = 0.009). On the other hand, for ND2-237Met genotypic men, green tea consumption does not appear to determine the risk of mildly decreased eGFR. CONCLUSION: The present results suggest that ND2-237 Leu/Met polymorphism unexpectedly modifies the effects of green tea consumption on eGFR and the risk of mildly decreased eGFR in male Japanese subjects.
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Pueblo Asiatico/genética , Pruebas de Función Renal , NADH Deshidrogenasa/genética , Examen Físico , Polimorfismo de Nucleótido Simple/genética , Té , Intervalos de Confianza , Estudios Transversales , Genotipo , Tasa de Filtración Glomerular , Humanos , Japón , Masculino , Persona de Mediana Edad , Oportunidad RelativaRESUMEN
NDUFS3 is an integral subunit of the Q module of the mitochondrial respiratory Complex-I. The combined mutation (T145I + R199W) in the subunit is reported to cause optic atrophy and Leigh syndrome accompanied by severe Complex-I deficiency. In the present study, we have cloned and overexpressed the human NDUFS3 subunit and its double mutant in a soluble form in Escherichia coli. The wild-type (w-t) and mutant proteins were purified to homogeneity through a serial two-step chromatographic purification procedure of anion exchange followed by size exclusion chromatography. The integrity and purity of the purified proteins was confirmed by Western blot analysis and MALDI-TOF/TOF. The conformational transitions of the purified subunits were studied through steady state as well as time resolved fluorescence and CD spectroscopy under various denaturing conditions. The mutant protein showed altered polarity around tryptophan residues, changed quenching parameters and also noticeably altered secondary and tertiary structure compared to the w-t protein. Mutant also exhibited a higher tendency than the w-t protein for aggregation which was examined using fluorescent (Thioflavin-T) and spectroscopic (Congo red) dye binding techniques. The pH stability of the w-t and mutant proteins varied at extreme acidic pH and the molten globule like structure of w-t at pH1 was absent in case of the mutant protein. Both the w-t and mutant proteins showed multi-step thermal and Gdn-HCl induced unfolding. Thus, the results provide insight into the alterations of NDUFS3 protein structure caused by the mutations, affecting the overall integrity of the protein and finally leading to disruption of Complex-I assembly.
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NADH Deshidrogenasa/química , NADH Deshidrogenasa/genética , Estructura Cuaternaria de Proteína , Dicroismo Circular , Clonación Molecular , Complejo I de Transporte de Electrón/genética , Humanos , Concentración de Iones de Hidrógeno , Enfermedad de Leigh/genética , Mitocondrias/genética , Mutación , Conformación Proteica/efectos de los fármacos , Desnaturalización Proteica , Estabilidad Proteica , Espectrometría de Fluorescencia , Triptófano/químicaRESUMEN
Interferon beta (IFNß) reduces disease burden in relapsing-remitting multiple sclerosis (MS) patients. In this study, IFNß-1b-treated MS patient gene expression profiles and biological knowledgebases were integrated to study IFNß's pleiotropic mechanisms of action. Genes involved in immune regulation, mitochondrial fatty acid metabolism and antioxidant activity were discovered. Plausible mediators of neuronal preservation included NRF2, downregulation of OLA1, an antioxidant suppressor, and the antioxidant gene ND6, implicated in optic neuropathy and MS-like lesions. Network analysis highlighted IKBKE, which likely has a role in both viral response and energy metabolism. A comparative analysis of therapy-naive MS- and IFNß-associated gene expression suggests an IFNß insufficiency in MS. We observed more gene expression changes in long-term treatment than during acute dosing. These distinct short- and long-term effects were driven by different transcription factors. Multi-gene biomarker signatures of IFNß treatment effects were developed and subsequently confirmed in independent IFNß-1b-treated MS studies, but not in glatiramer acetate-treated patients.