RESUMEN
The development of the Artemis programme with the goal of returning to the moon is spurring technology advances that will eventually take humans to Mars and herald a new era of interplanetary space travel. However, long-term space travel poses unique challenges including exposure to ionising radiation from galactic cosmic rays and potential solar particle events, exposure to microgravity and specific nutritional challenges arising from earth independent exploration. Ionising radiation is one of the major obstacles facing future space travel as it can generate oxidative stress and directly damage cellular structures such as DNA, in turn causing genomic instability, telomere shortening, extracellular-matrix remodelling and persistent inflammation. In the gastrointestinal tract (GIT) this can lead to leaky gut syndrome, perforations and motility issues, which impact GIT functionality and affect nutritional status. While current countermeasures such as shielding from the spacecraft can attenuate harmful biological effects, they produce harmful secondary particles that contribute to radiation exposure. We hypothesised that induction of a torpor-like state would confer a radioprotective effect given the evidence that hibernation extends survival times in irradiated squirrels compared to active controls. To test this hypothesis, a torpor-like state was induced in zebrafish using melatonin treatment and reduced temperature, and radiation exposure was administered twice over the course of 10 days. The protective effects of induced-torpor were assessed via RNA sequencing and qPCR of mRNA extracted from the GIT. Pathway and network analysis were performed on the transcriptomic data to characterise the genomic signatures in radiation, torpor and torpor + radiation groups. Phenotypic analyses revealed that melatonin and reduced temperature successfully induced a torpor-like state in zebrafish as shown by decreased metabolism and activity levels. Genomic analyses indicated that low dose radiation caused DNA damage and oxidative stress triggering a stress response, including steroidal signalling and changes to metabolism, and cell cycle arrest. Torpor attenuated the stress response through an increase in pro-survival signals, reduced oxidative stress via the oxygen effect and detection and removal of misfolded proteins. This proof-of-concept model provides compelling initial evidence for utilizing an induced torpor-like state as a potential countermeasure for radiation exposure.
Asunto(s)
Exposición a la Radiación , Letargo/fisiología , Pez Cebra/fisiología , Animales , Ritmo Circadiano/genética , Ritmo Circadiano/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Degradación Asociada con el Retículo Endoplásmico/efectos de la radiación , Regulación de la Expresión Génica/efectos de la radiación , Redes Reguladoras de Genes/efectos de la radiación , Melatonina/farmacología , Modelos Animales , Fosforilación Oxidativa/efectos de la radiación , Reproducibilidad de los Resultados , Estrés Fisiológico/genética , Estrés Fisiológico/efectos de la radiación , Análisis de Supervivencia , Temperatura , Transcriptoma/genética , Transcriptoma/efectos de la radiación , Pez Cebra/genéticaRESUMEN
Despite the availability of more than 25 antiseizure drugs on the market, approximately 30% of patients with epilepsy still suffer from seizures. Thus, the epilepsy therapy market has a great need for a breakthrough drug that will aid pharmacoresistant patients. In our previous study, we discovered a vitamin K analogue, 2h, which displayed modest antiseizure activity in zebrafish and mouse seizure models. However, there are limitations to this compound due to its pharmacokinetic profile. In this study, we develop a new series of vitamin K analogues by modifying the structure of 2h. Among these, compound 3d shows full protection in a rodent pharmacoresistant seizure model with limited rotarod motor toxicity and favorable pharmacokinetic properties. Furthermore, the brain/plasma concentration ratio of 3d indicates its excellent permeability into the brain. The resulting data shows that 3d can be further developed as a potential antiseizure drug in the clinic.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Convulsiones/tratamiento farmacológico , Vitamina K/análogos & derivados , Administración Oral , Animales , Anticonvulsivantes/química , Anticonvulsivantes/farmacocinética , Anticonvulsivantes/farmacología , Encéfalo/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Semivida , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Masculino , Ratones , Convulsiones/patología , Relación Estructura-Actividad , Vitamina K/farmacocinética , Vitamina K/farmacología , Vitamina K/uso terapéutico , Pez CebraRESUMEN
Here, we present a new series of hydrazide-bearing class I selective HDAC inhibitors designed based on panobinostat. The cap, linker, and zinc-binding group were derivatized to improve HDAC affinity and antileukemia efficacy. Lead inhibitor 13a shows picomolar or low nanomolar IC50 values against HDAC1 and HDAC3 and exhibits differential toxicity profiles toward multiple cancer cells with different FLT3 and p53 statuses. 13a indirectly inhibits the FLT3 signaling pathway and down-regulates master antiapoptotic proteins, resulting in the activation of pro-caspase3 in wt-p53 FLT3-ITD MV4-11 cells. While in the wt-FLT3 and p53-null cells, 13a is incapable of causing apoptosis at a therapeutic concentration. The MDM2 antagonist and the proteasome inhibitor promote 13a-triggered apoptosis by preventing p53 degradation. Furthermore, we demonstrate that apoptosis rather than autophagy is the key contributing factor for 13a-triggered cell death. When compared to panobinostat, 13a is not mutagenic and displays superior in vivo bioavailability and a higher AUC0-inf value.
Asunto(s)
Antineoplásicos/metabolismo , Inhibidores de Histona Desacetilasas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Panobinostat/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Tirosina Quinasa 3 Similar a fms/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Diseño de Fármacos , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Masculino , Ratones , Panobinostat/química , Panobinostat/uso terapéutico , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidoresRESUMEN
Smoking is the largest preventable cause of death and disease in the United States. However, <5% of quit attempts are successful, underscoring the urgent need for novel therapeutics. Microglia are one untapped therapeutic target. While previous studies have shown that microglia mediate both inflammatory responses in the brain and brain plasticity, little is known regarding their role in nicotine dependence and withdrawal phenotypes. Here, we examined microglial changes in the striatum-a mesolimbic region implicated in the rewarding effects of drugs and the affective disruptions occurring during withdrawal. We show that both nicotine and withdrawal induce microglial morphological changes; however, proinflammatory effects and anxiogenic behaviors were observed only during nicotine withdrawal. Pharmacological microglial depletion during withdrawal prevented these effects. These results define differential effects of nicotine and withdrawal on inflammatory signaling in the brain, laying the groundwork for development of future smoking cessation therapeutics.
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Microglía/patología , Núcleo Accumbens/metabolismo , Síndrome de Abstinencia a Sustancias/patología , Animales , Ansiedad/etiología , Modelos Animales de Enfermedad , Locomoción , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/metabolismo , NADPH Oxidasa 2/metabolismo , Nicotina/administración & dosificación , Compuestos Orgánicos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Transducción de Señal/efectos de los fármacos , Síndrome de Abstinencia a Sustancias/complicaciones , Síndrome de Abstinencia a Sustancias/metabolismoRESUMEN
[This corrects the article DOI: 10.1371/journal.pgen.1007743.].
RESUMEN
Development and function of tissues and organs are powered by the activity of mitochondria. In humans, inherited genetic mutations that lead to progressive mitochondrial pathology often manifest during infancy and can lead to death, reflecting the indispensable nature of mitochondrial biogenesis and function. Here, we describe a zebrafish mutant for the gene mia40a (chchd4a), the life-essential homologue of the evolutionarily conserved Mia40 oxidoreductase which drives the biogenesis of cysteine-rich mitochondrial proteins. We report that mia40a mutant animals undergo progressive cellular respiration defects and develop enlarged mitochondria in skeletal muscles before their ultimate death at the larval stage. We generated a deep transcriptomic and proteomic resource that allowed us to identify abnormalities in the development and physiology of endodermal organs, in particular the liver and pancreas. We identify the acinar cells of the exocrine pancreas to be severely affected by mutations in the MIA pathway. Our data contribute to a better understanding of the molecular, cellular and organismal effects of mitochondrial deficiency, important for the accurate diagnosis and future treatment strategies of mitochondrial diseases.
RESUMEN
Copper is an essential cofactor of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain. Inherited loss-of-function mutations in several genes encoding proteins required for copper delivery to CcO result in diminished CcO activity and severe pathologic conditions in affected infants. Copper supplementation restores CcO function in patient cells with mutations in two of these genes, COA6 and SCO2, suggesting a potential therapeutic approach. However, direct copper supplementation has not been therapeutically effective in human patients, underscoring the need to identify highly efficient copper transporting pharmacological agents. By using a candidate-based approach, we identified an investigational anticancer drug, elesclomol (ES), that rescues respiratory defects of COA6-deficient yeast cells by increasing mitochondrial copper content and restoring CcO activity. ES also rescues respiratory defects in other yeast mutants of copper metabolism, suggesting a broader applicability. Low nanomolar concentrations of ES reinstate copper-containing subunits of CcO in a zebrafish model of copper deficiency and in a series of copper-deficient mammalian cells, including those derived from a patient with SCO2 mutations. These findings reveal that ES can restore intracellular copper homeostasis by mimicking the function of missing transporters and chaperones of copper, and may have potential in treating human disorders of copper metabolism.
Asunto(s)
Antineoplásicos/farmacología , Cobre/deficiencia , Drogas en Investigación/farmacología , Complejo IV de Transporte de Electrones/metabolismo , Hidrazinas/farmacología , Mitocondrias/efectos de los fármacos , Animales , Antineoplásicos/uso terapéutico , Transporte Biológico/genética , Proteínas Portadoras/genética , Línea Celular , Coenzimas/deficiencia , Cobre/uso terapéutico , Transportador de Cobre 1 , Suplementos Dietéticos , Modelos Animales de Enfermedad , Reposicionamiento de Medicamentos , Drogas en Investigación/uso terapéutico , Fibroblastos , Humanos , Hidrazinas/uso terapéutico , Proteínas de Transporte de Membrana/genética , Errores Innatos del Metabolismo/tratamiento farmacológico , Errores Innatos del Metabolismo/genética , Errores Innatos del Metabolismo/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Chaperonas Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Ratas , Saccharomyces cerevisiae , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
There are approximately 1500 proteins that are needed for mitochondrial structure and function, most of which are encoded in the nuclear genome (Calvo et al., 2006). Each mitochondrion has its own genome (mtDNA), which in humans encodes 13 polypeptides, 22 tRNAs and 2 rRNAs required for oxidative phosphorylation. The mitochondrial genome of humans and most vertebrates is approximately 16.5kbp, double-stranded, circular, with few non-coding bases. Thus, maintaining mtDNA stability, that is, the ability of the cell to maintain adequate levels of mtDNA template for oxidative phosphorylation is essential and can be impacted by the level of mtDNA mutation currently within the cell or mitochondrion, but also from errors made during normal mtDNA replication, defects in mitochondrial quality control mechanisms, and exacerbated by exposures to exogenous and/or endogenous genotoxic agents. In this review, we expand on the origins and consequences of mtDNA instability, the current state of research regarding the mechanisms by which mtDNA instability can be overcome by cellular and chemical interventions, and the future of research and treatments for mtDNA instability.
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Daño del ADN , ADN Mitocondrial/genética , Contaminantes Ambientales/toxicidad , Inestabilidad Genómica , Mitocondrias/efectos de los fármacos , Animales , Ecotoxicología , Exposición a Riesgos Ambientales/efectos adversos , Interacción Gen-Ambiente , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Medición de RiesgoAsunto(s)
Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Enfermedades Mitocondriales/inducido químicamente , Enfermedades Mitocondriales/metabolismo , Animales , Arsénico/toxicidad , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Humanos , Mitocondrias/genética , Enfermedades Mitocondriales/genética , Especies Reactivas de Oxígeno/metabolismo , Inhibidores de la Transcriptasa Inversa/toxicidadRESUMEN
DNA polymerase gamma (POLG) is essential for replication and repair of mitochondrial DNA (mtDNA). Mutations in POLG cause mtDNA instability and a diverse range of poorly understood human diseases. Here, we created a unique Polg animal model, by modifying polg within the critical and highly conserved polymerase domain in zebrafish. polg(+/-) offspring were indistinguishable from WT siblings in multiple phenotypic and biochemical measures. However, polg(-/-) mutants developed severe mtDNA depletion by one week post-fertilization (wpf), developed slowly and had regenerative defects, yet surprisingly survived up to 4 wpf. An in vivo mtDNA polymerase activity assay utilizing ethidium bromide (EtBr) to deplete mtDNA, showed that polg(+/-) and WT zebrafish fully recover mtDNA content two weeks post-EtBr removal. EtBr further reduced already low levels of mtDNA in polg(-/-) animals, but mtDNA content did not recover following release from EtBr. Despite significantly decreased respiration that corresponded with tissue-specific levels of mtDNA, polg(-/-) animals had WT levels of ATP and no increase in lactate. This zebrafish model of mitochondrial disease now provides unique opportunities for studying mtDNA instability from multiple angles, as polg(-/-) mutants can survive to juvenile stage, rather than lose viability in embryogenesis as seen in Polg mutant mice.
Asunto(s)
ADN Mitocondrial/análisis , ADN Polimerasa Dirigida por ADN/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Adenosina Trifosfato/metabolismo , Aletas de Animales/fisiología , Animales , ADN Polimerasa gamma , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Ingeniería Genética , Glucólisis , Modelos Animales , Mutación , Consumo de Oxígeno , Regeneración , Análisis de Supervivencia , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/metabolismoRESUMEN
Organismal metabolic rate, a fundamental metric in biology, demonstrates an allometric scaling relationship with body size. Fractal-like vascular distribution networks of biological systems are proposed to underlie metabolic rate allometric scaling laws from individual organisms to cells, mitochondria, and enzymes. Tissue-specific metabolic scaling is notably absent from this paradigm. In the current study, metabolic scaling relationships of hearts and brains with body size were examined by improving on a high-throughput whole-organ oxygen consumption rate (OCR) analysis method in five biomedically and environmentally relevant teleost model species. Tissue-specific metabolic scaling was compared with organismal routine metabolism (RMO2), which was measured using whole organismal respirometry. Basal heart OCR and organismal RMO2 scaled identically with body mass in a species-specific fashion across all five species tested. However, organismal maximum metabolic rates (MMO2) and pharmacologically-induced maximum cardiac metabolic rates in zebrafish Danio rerio did not show a similar relationship with body mass. Brain metabolic rates did not scale with body size. The identical allometric scaling of heart and organismal metabolic rates with body size suggests that hearts, the power generator of an organism's vascular distribution network, might be crucial in determining teleost metabolic rate scaling under routine conditions. Furthermore, these findings indicate the possibility of measuring heart OCR utilizing the high-throughput approach presented here as a proxy for organismal metabolic rate-a useful metric in characterizing organismal fitness. In addition to heart and brain OCR, the current approach was also used to measure whole liver OCR, partition cardiac mitochondrial bioenergetic parameters using pharmacological agents, and estimate heart and brain glycolytic rates. This high-throughput whole-organ bioenergetic analysis method has important applications in toxicology, evolutionary physiology, and biomedical sciences, particularly in the context of investigating pathogenesis of mitochondrial diseases.
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Encéfalo/metabolismo , Metabolismo Energético , Peces/metabolismo , Miocardio/metabolismo , Animales , Tamaño Corporal , Peces/clasificación , Fractales , Ensayos Analíticos de Alto Rendimiento , Especificidad de Órganos , Consumo de Oxígeno , Pez Cebra/metabolismoRESUMEN
Mitochondria are involved in key cellular functions including energy production, metabolic homeostasis, and apoptosis. Normal mitochondrial function is preserved by several interrelated mechanisms. One mechanism - intramitochondrial quality control (IMQC) - is represented by conserved proteases distributed across mitochondrial compartments. Many aspects and physiological roles of IMQC components remain unclear. Here, we show that the IMQC protease Oma1 is required for the stability of the respiratory supercomplexes and thus balanced and tunable bioenergetic function. Loss of Oma1 activity leads to a specific destabilization of respiratory supercomplexes and consequently to unbalanced respiration and progressive respiratory decline in yeast. Similarly, experiments in cultured Oma1-deficient mouse embryonic fibroblasts link together impeded supercomplex stability and inability to maintain proper respiration under conditions that require maximal bioenergetic output. Finally, transient knockdown of OMA1 in zebrafish leads to impeded bioenergetics and morphological defects of the heart and eyes. Together, our biochemical and genetic studies in yeast, zebrafish and mammalian cells identify a novel and conserved physiological role for Oma1 protease in fine-tuning of respiratory function. We suggest that this unexpected physiological role is important for cellular bioenergetic plasticity and may contribute to Oma1-associated disease phenotypes in humans.
Asunto(s)
Metaloproteasas/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Animales , Línea Celular , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Metabolismo Energético , Larva/metabolismo , Metaloproteasas/química , Metaloproteasas/genética , Ratones , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Morfolinos/farmacología , Fenotipo , Estabilidad Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The etiology of mitochondrial disease is poorly understood. Furthermore, treatment options are limited, and diagnostic methods often lack the sensitivity to detect disease in its early stages. Disrupted oxidative phosphorylation (OXPHOS) that inhibits ATP production is a common phenotype of mitochondrial disorders that can be induced in zebrafish by exposure to 2,4-dinitrophenol (DNP), a FDA-banned weight-loss agent and EPA-regulated environmental toxicant, traditionally used in research labs as an uncoupler of OXPHOS. Despite the DNP-induced OXPHOS inhibition we observed using in vivo respirometry, the development of the DNP-treated and control zebrafish were largely similar during the first half of embryogenesis. During this period, DNP-treated embryos induced gene expression of mitochondrial and nuclear genes that stimulated the production of new mitochondria and increased glycolysis to yield normal levels of ATP. DNP-treated embryos were incapable of sustaining this mitochondrial biogenic response past mid-embryogenesis, as shown by significantly lowered ATP production and ATP levels, decreased gene expression, and the onset of developmental defects. Examining neural tissues commonly affected by mitochondrial disease, we found that DNP exposure also inhibited motor neuron axon arbor outgrowth and the proper formation of the retina. We observed and quantified the molecular and physiological progression of mitochondrial dysfunction during development with this new model of OXPHOS dysfunction, which has great potential for use in diagnostics and therapies for mitochondrial disease.
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Desarrollo Embrionario/genética , Metabolismo Energético/genética , Mitocondrias/genética , Enfermedades Mitocondriales/genética , 2,4-Dinitrofenol/toxicidad , Adenosina Trifosfato/biosíntesis , Animales , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Mitocondrias/metabolismo , Mitocondrias/patología , Enfermedades Mitocondriales/inducido químicamente , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/patología , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Fosforilación Oxidativa/efectos de los fármacos , Retina/metabolismo , Retina/patología , Pez CebraRESUMEN
SIGNIFICANCE: Mitochondria are complex dynamic organelles pivotal for cellular physiology and human health. Failure to maintain mitochondrial health leads to numerous maladies that include late-onset neurodegenerative diseases and cardiovascular disorders. Furthermore, a decline in mitochondrial health is prevalent with aging. A set of evolutionary conserved mechanisms known as mitochondrial quality control (MQC) is involved in recognition and correction of the mitochondrial proteome. RECENT ADVANCES: Here, we review current knowledge and latest developments in MQC. We particularly focus on the proteolytic aspect of MQC and its impact on health and aging. CRITICAL ISSUES: While our knowledge about MQC is steadily growing, critical gaps remain in the mechanistic understanding of how MQC modules sense damage and preserve mitochondrial welfare, particularly in higher organisms. FUTURE DIRECTIONS: Delineating how coordinated action of the MQC modules orchestrates physiological responses on both organellar and cellular levels will further elucidate the current picture of MQC's role and function in health, cellular stress, and degenerative diseases.
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Envejecimiento/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/biosíntesis , Salud , HumanosRESUMEN
Mitochondrial respiratory chain biogenesis is orchestrated by hundreds of assembly factors, many of which are yet to be discovered. Using an integrative approach based on clues from evolutionary history, protein localization and human genetics, we have identified a conserved mitochondrial protein, C1orf31/COA6, and shown its requirement for respiratory complex IV biogenesis in yeast, zebrafish and human cells. A recent next-generation sequencing study reported potential pathogenic mutations within the evolutionarily conserved Cx9CxnCx10C motif of COA6, implicating it in mitochondrial disease biology. Using yeast coa6Δ cells, we show that conserved residues in the motif, including the residue mutated in a patient with mitochondrial disease, are essential for COA6 function, thus confirming the pathogenicity of the patient mutation. Furthermore, we show that zebrafish embryos with zfcoa6 knockdown display reduced heart rate and cardiac developmental defects, recapitulating the observed pathology in the human mitochondrial disease patient who died of neonatal hypertrophic cardiomyopathy. The specific requirement of Coa6 for respiratory complex IV biogenesis, its intramitochondrial localization and the presence of the Cx9CxnCx10C motif suggested a role in mitochondrial copper metabolism. In support of this, we show that exogenous copper supplementation completely rescues respiratory and complex IV assembly defects in yeast coa6Δ cells. Taken together, our results establish an evolutionarily conserved role of Coa6 in complex IV assembly and support a causal role of the COA6 mutation in the human mitochondrial disease patient.
Asunto(s)
Cobre/farmacología , Complejo IV de Transporte de Electrones/metabolismo , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Animales , Humanos , Mutación , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Piel/citología , Pez CebraRESUMEN
Opa1 catalyzes fusion of inner mitochondrial membranes and formation of the cristae. OPA1 mutations in humans lead to autosomal dominant optic atrophy. OPA1 knockout mice lose viability around embryonic day 9 from unknown reasons, indicating that OPA1 is essential for embryonic development. Zebrafish are an attractive model for studying vertebrate development and have been used for many years to describe developmental events that are difficult or impractical to view in mammalian models. In this study, Opa1 was successfully depleted in zebrafish embryos using antisense morpholinos, which resulted in disrupted mitochondrial morphology. Phenotypically, these embryos exhibited abnormal blood circulation and heart defects, as well as small eyes and small pectoral fin buds. Additionally, startle response was reduced and locomotor activity was impaired. Furthermore, Opa1 depletion caused bioenergetic defects, without impairing mitochondrial efficiency. In response to mitochondrial dysfunction, a transient upregulation of the master regulator of mitochondrial biogenesis, pgc1a, was observed. These results not only reveal a new Opa1-associated phenotype in a vertebrate model system, but also further elucidates the absolute requirement of Opa1 for successful vertebrate development.
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Mitocondrias/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Western Blotting , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Morfolinos/farmacología , Pez Cebra/genética , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genéticaRESUMEN
Many debilitating conditions are linked to bioenergetic defects. Developing screens to probe the genetic and/or chemical basis for such links has proved intractable. Furthermore, there is a need for a physiologically relevant assay of bioenergetics in whole organisms, especially for early stages in life where perturbations could increase disease susceptibility with aging. Thus, we asked whether we could screen bioenergetics and mitochondrial function in the developing zebrafish embryo. We present a multiplexed method to assay bioenergetics in zebrafish embryos from the blastula period (3 hours post-fertilization, hpf) through to hatching (48 hpf). In proof of principle experiments, we measured respiration and acid extrusion of developing zebrafish embryos. We quantified respiratory coupling to various bioenergetic functions by using specific pharmacological inhibitors of bioenergetic pathways. We demonstrate that changes in the coupling to ATP turnover and proton leak are correlated with developmental stage. The multiwell format of this assay enables the user to screen for the effects of drugs and environmental agents on bioenergetics in the zebrafish embryo with high sensitivity and reproducibility.
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Embrión no Mamífero/metabolismo , Metabolismo Energético , Análisis por Micromatrices/métodos , Pez Cebra/embriología , Adenosina Trifosfato/metabolismo , Animales , Respiración de la Célula/efectos de los fármacos , Embrión no Mamífero/citología , Embrión no Mamífero/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Concentración de Iones de Hidrógeno , Modelos Lineales , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Protones , Reproducibilidad de los Resultados , Pez Cebra/metabolismoRESUMEN
CONTEXT: Primary mitochondrial dysfunction is one of the most common causes of inherited disorders predominantly involving the neuromuscular system. Advances in the molecular study of mitochondrial DNA have changed our vision and our approach to primary mitochondrial disorders. Many of the mitochondrial disorders are caused by mutations in nuclear genes and are inherited in an autosomal recessive pattern. Among the autosomal inherited mitochondrial disorders, those related to DNA polymerase γ dysfunction are the most common and the best studied. Understanding the molecular mechanisms and being familiar with the recent advances in laboratory diagnosis of this group of mitochondrial disorders are essential for pathologists to interpret abnormal histopathology and laboratory results and to suggest further studies for a definitive diagnosis. OBJECTIVES: To help pathologists better understand the common clinical syndromes originating from mutations in DNA polymerase γ and its associated proteins and use the stepwise approach of clinical, laboratory, and pathologic diagnosis of these syndromes. DATA SOURCES: Review of pertinent published literature and relevant Internet databases. CONCLUSIONS: Mitochondrial disorders are now better recognized with the development of molecular tests for clinical diagnosis. A cooperative effort among primary physicians, diagnostic pathologists, geneticists, and molecular biologists with expertise in mitochondrial disorders is required to reach a definitive diagnosis.
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ADN Mitocondrial/genética , ADN Polimerasa Dirigida por ADN/genética , Mitocondrias/genética , Enfermedades Mitocondriales/diagnóstico , ADN Polimerasa gamma , ADN Mitocondrial/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Mitocondrias/metabolismo , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Técnicas de Diagnóstico MolecularRESUMEN
DNA polymerase gamma (pol gamma) is the only DNA polymerase within the mitochondrion and is thus essential for replication and repair of mtDNA. POLG, the gene encoding the catalytic subunit of pol gamma, is a major locus for a wide spectrum of mitochondrial diseases with more than 100 known disease mutations. Thus, we need to understand how and why pol gamma defects lead to disease. By using an extensive array of methods, we are developing a clearer understanding of how defects in pol gamma contribute to disease. Furthermore, crucial knowledge concerning the role of pol gamma in mtDNA replication and repair can be acquired. Here we present the protocols to characterize mutant DNA pol gamma proteins, namely, assays for processive DNA synthesis, exonuclease activity, DNA binding, subunit interaction, and protein stability.
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ADN Mitocondrial/metabolismo , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/aislamiento & purificación , Enfermedades Mitocondriales/genética , Proteínas Mutantes/aislamiento & purificación , Mutación/genética , Dicroismo Circular , ADN Polimerasa gamma , ADN Mitocondrial/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Estabilidad de Enzimas , Exonucleasas/metabolismo , Humanos , Inmunoprecipitación , Enfermedades Mitocondriales/metabolismo , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Subunidades de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Mutations in POLG are a major contributor to pediatric and adult mitochondrial diseases. However, the consequences of many POLG mutations are not well understood. We investigated the molecular cause of Alpers syndome in a patient harboring the POLG mutations A467T in trans with c.2157+5_+6 gc-->ag in intron 12. Analysis of transcripts arising from the c.2157+5_+6 gc-->ag allele revealed alternative splicing with an insertion of 30 intronic nucleotides leading to a premature termination codon. These transcripts were subsequently removed through nonsense-mediated decay, leading to haplotype insufficiency due to expression of the A467T allele and decreased expression of the c.2157+5_+6 gc-->ag allele, which is likely responsible for the Alpers syndrome phenotype.