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Obtaining differentiated cells with high physiological functions by an efficient, but simple and rapid differentiation method is crucial for modeling neuronal diseases in vitro using human pluripotent stem cells (hPSCs). Currently, methods involving the transient expression of one or a couple of transcription factors have been established as techniques for inducing neuronal differentiation in a rapid, single step. It has also been reported that microRNAs can function as reprogramming effectors for directly reprogramming human dermal fibroblasts to neurons. In this study, we tested the effect of adding neuronal microRNAs, miRNA-9/9*, and miR-124 (miR-9/9*-124), for the neuronal induction method of hPSCs using Tet-On-driven expression of the Neurogenin2 gene (Ngn2), a proneural factor. While it has been established that Ngn2 can facilitate differentiation from pluripotent stem cells into neurons with high purity due to its neurogenic effect, a long or indefinite time is required for neuronal maturation with Ngn2 misexpression alone. With the present method, the cells maintained a high neuronal differentiation rate while exhibiting increased gene expression of neuronal maturation markers, spontaneous calcium oscillation, and high electrical activity with network bursts as assessed by a multipoint electrode system. Moreover, when applying this method to iPSCs from Alzheimer's disease (AD) patients with presenilin-1 (PS1) or presenilin-2 (PS2) mutations, cellular phenotypes such as increased amount of extracellular secretion of amyloid ß42, abnormal oxygen consumption, and increased reactive oxygen species in the cells were observed in a shorter culture period than those previously reported. Therefore, it is strongly anticipated that the induction method combining Ngn2 and miR-9/9*-124 will enable more rapid and simple screening for various types of neuronal disease phenotypes and promote drug discovery.
Assuntos
MicroRNAs/metabolismo , Doenças do Sistema Nervoso/genética , Neurogênese/fisiologia , Neurônios/metabolismo , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular , Humanos , Neurônios/citologia , Fenótipo , TransfecçãoRESUMO
BackgroundDiagnosing mitochondrial disease (MD) is a challenge. In addition to genetic analyses, clinical practice is to perform invasive procedures such as muscle biopsy for biochemical and histochemical analyses. Blood cell respirometry is rapid and noninvasive. Our aim was to explore its possible role in diagnosing MD.MethodsBlood samples were collected from 113 pediatric patients, for whom MD was a differential diagnosis. A respiratory analysis model based on ratios (independent of mitochondrial specific content) was derived from a group of healthy controls and tested on the patients. The diagnostic accuracy of platelet respirometry was evaluated against routine diagnostic investigation.ResultsMD prevalence in the cohort was 16%. A ratio based on the respiratory response to adenosine diphosphate in the presence of complex I substrates had 96% specificity for disease and a positive likelihood ratio of 5.3. None of the individual ratios had sensitivity above 50%, but a combined model had 72% sensitivity.ConclusionNormal findings of platelet respirometry are not able to rule out MD, but pathological results make the diagnosis more likely and could strengthen the clinical decision to perform further invasive analyses. Our results encourage further study into the role of blood respirometry as an adjunct diagnostic tool for MD.
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Plaquetas/metabolismo , Mitocôndrias/metabolismo , Doenças Mitocondriais/sangue , Doenças Mitocondriais/diagnóstico , Consumo de Oxigênio , Biópsia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Ácido Láctico/sangue , Masculino , Oxigênio/química , Prevalência , Sensibilidade e EspecificidadeRESUMO
Mitochondrial complex I (CI) deficiency is the most prevalent defect in the respiratory chain in paediatric mitochondrial disease. This heterogeneous group of diseases includes serious or fatal neurological presentations such as Leigh syndrome and there are very limited evidence-based treatment options available. Here we describe that cell membrane-permeable prodrugs of the complex II substrate succinate increase ATP-linked mitochondrial respiration in CI-deficient human blood cells, fibroblasts and heart fibres. Lactate accumulation in platelets due to rotenone-induced CI inhibition is reversed and rotenone-induced increase in lactate:pyruvate ratio in white blood cells is alleviated. Metabolomic analyses demonstrate delivery and metabolism of [(13)C]succinate. In Leigh syndrome patient fibroblasts, with a recessive NDUFS2 mutation, respiration and spare respiratory capacity are increased by prodrug administration. We conclude that prodrug-delivered succinate bypasses CI and supports electron transport, membrane potential and ATP production. This strategy offers a potential future therapy for metabolic decompensation due to mitochondrial CI dysfunction.
Assuntos
Permeabilidade da Membrana Celular , Complexo I de Transporte de Elétrons/deficiência , Doenças Mitocondriais/metabolismo , Pró-Fármacos/farmacologia , Ácido Succínico/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Fibroblastos/patologia , Humanos , Lactatos/metabolismo , Doença de Leigh/patologia , Metabolômica , Modelos Biológicos , Pró-Fármacos/química , Ácido Succínico/químicaRESUMO
BACKGROUND: Patients with Huntington's disease display symptoms from both the central nervous system and peripheral tissues. Mitochondrial dysfunction has been implicated as part of the pathogenesis of the disease and has been reported in brain tissue and extracerebral tissues, such as muscle and blood cells, but the results are inconsistent. Therefore, the authors performed a refined evaluation of mitochondrial function in 2 types of peripheral blood cells from 14 patients with Huntington's disease and 21 control subjects. Several hypotheses were predefined, including impaired mitochondrial complex II function (primary), complex I function (secondary), and maximum oxidative phosphorylation capacity (secondary) in patient cells. METHODS: High-resolution respirometry was applied to viable platelets and mononuclear cells. Data were normalized to cell counts, citrate synthase activity, and mitochondrial DNA copy numbers. RESULTS: Normalized to citrate synthase activity, platelets from patients with Huntington's disease displayed respiratory dysfunction linked to complex I, complex II, and lower maximum oxidative phosphorylation capacity. No difference was seen in mononuclear cells or when platelet data were normalized to cell counts or mitochondrial DNA. The ratio of complex I respiration through maximum oxidative phosphorylation was significantly decreased in patients compared with controls. The corresponding ratio for complex II was unaffected. CONCLUSIONS: The data indicate decreased function of mitochondrial complex I in peripheral blood cells from patients with Huntington's disease, although this could not be uniformly confirmed. The results do not confirm a systemic complex II dysfunction and do not currently support the use of mitochondrial function in blood cells as a biomarker for the disease.
RESUMO
Mitochondrial dysfunction is implicated in amyotrophic lateral sclerosis, where the progressive degeneration of motor neurons results in muscle atrophy, paralysis and death. Abnormalities in both central nervous system and muscle mitochondria have previously been demonstrated in patient samples, indicating systemic disease. In this case-control study, venous blood samples were acquired from 24 amyotrophic lateral sclerosis patients and 21 age-matched controls. Platelets and peripheral blood mononuclear cells were isolated and mitochondrial oxygen consumption measured in intact and permeabilized cells with additions of mitochondrial substrates, inhibitors and titration of an uncoupler. Respiratory values were normalized to cell count and for two markers of cellular mitochondrial content, citrate synthase activity and mitochondrial DNA, respectively. Mitochondrial function was correlated with clinical staging of disease severity. Complex IV (cytochrome c-oxidase)-activity normalized to mitochondrial content was decreased in platelets from amyotrophic lateral sclerosis patients both when normalized to citrate synthase activity and mitochondrial DNA copy number. In mononuclear cells, complex IV-activity was decreased when normalized to citrate synthase activity. Mitochondrial content was increased in amyotrophic lateral sclerosis patient platelets. In mononuclear cells, complex I activity declined and mitochondrial content increased progressively with advancing disease stage. The findings are, however, based on small subsets of patients and need to be confirmed. We conclude that when normalized to mitochondria-specific content, complex IV-activity is reduced in blood cells from amyotrophic lateral sclerosis patients and that there is an apparent compensatory increase in cellular mitochondrial content. This supports systemic involvement in amyotrophic lateral sclerosis and suggests further study of mitochondrial function in blood cells as a future biomarker for the disease.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Células Sanguíneas/patologia , Células Sanguíneas/ultraestrutura , Mitocôndrias/patologia , Doenças Mitocondriais/etiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/complicações , Estudos de Casos e Controles , Citrato (si)-Sintase/metabolismo , DNA Mitocondrial/metabolismo , Progressão da Doença , Complexo IV da Cadeia de Transporte de Elétrons , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Complexos Multienzimáticos/metabolismo , Estatísticas não Paramétricas , Superóxido DismutaseRESUMO
Obesity is a major global public health problem, and understanding its pathogenesis is critical for identifying a cure. In this study, a gene knockout strategy was used in post-neonatal mice to delete synoviolin (Syvn)1/Hrd1/Der3, an ER-resident E3 ubiquitin ligase with known roles in homeostasis maintenance. Syvn1 deficiency resulted in weight loss and lower accumulation of white adipose tissue in otherwise wild-type animals as well as in genetically obese (ob/ob and db/db) and adipose tissue-specific knockout mice as compared to control animals. SYVN1 interacted with and ubiquitinated the thermogenic coactivator peroxisome proliferator-activated receptor coactivator (PGC)-1ß, and Syvn1 mutants showed upregulation of PGC-1ß target genes and increase in mitochondrion number, respiration, and basal energy expenditure in adipose tissue relative to control animals. Moreover, the selective SYVN1 inhibitor LS-102 abolished the negative regulation of PGC-1ß by SYVN1 and prevented weight gain in mice. Thus, SYVN1 is a novel post-translational regulator of PGC-1ß and a potential therapeutic target in obesity treatment.
Assuntos
Peso Corporal/genética , Mitocôndrias/fisiologia , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Células 3T3-L1 , Animais , Células Cultivadas , Regulação para Baixo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Obesidade/genética , Obesidade/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genéticaRESUMO
BACKGROUND: The levels of nitric oxide (NO) and various cytokines are known to be increased during sepsis. These signaling molecules could potentially act as regulators and underlie the enhancement of mitochondrial function described in the later phase of sepsis. Therefore, we investigated the correlation between observed changes in platelet mitochondrial respiration and a set of pro- and anti-inflammatory cytokines as well as NO plasma levels in patients with sepsis. METHODS AND RESULTS: Platelet mitochondrial respiration and levels of TNFα, MCP-1 (monocyte chemotactic protein-1), INFγ (interferon-γ), IL-1ß, IL-4, IL-5, IL-6, IL-8, IL-10 and IL-17 and NO were analyzed in 38 patients with severe sepsis or septic shock at three time points during one week following admission to the ICU. Citrate synthase, mitochondrial DNA and cytochrome c were measured as markers of cellular mitochondrial content. All mitochondrial respiratory states increased over the week analyzed (p<0.001). IL-8 levels correlated with maximal mitochondrial respiration on day 6-7 (p = 0.02, r2 = 0.22) and was also higher in non-survivors compared to survivors on day 3-4 and day 6-7 (p = 0.03 respectively). Neither NO nor any of the other cytokines measured correlated with respiration or mortality. Cytochrome c levels were decreased at day 1-2 by 24 ± 5% (p = 0.03) and returned towards values of the controls at the last two time points. Citrate synthase activity and mitochondrial DNA levels were similar to controls and remained constant throughout the week. CONCLUSIONS: Out of ten analyzed cytokines and nitric oxide, IL-8 correlated with the observed increase in mitochondrial respiration. This suggests that cytokines as well as NO do not play a prominent role in the regulation of platelet mitochondrial respiration in sepsis. Further, the respiratory increase was not accompanied by an increase in markers of mitochondrial content, suggesting a possible role for post-translational enhancement of mitochondrial respiration rather than augmented mitochondrial mass.
Assuntos
Plaquetas/patologia , Citocinas/sangue , Mitocôndrias/patologia , Óxido Nítrico/sangue , Sepse/sangue , Respiração Celular , Humanos , Sepse/patologiaRESUMO
BACKGROUND: The levels of nitric oxide (NO) and various cytokines are known to be increased during sepsis. These signaling molecules could potentially act as regulators and underlie the enhancement of mitochondrial function described in the later phase of sepsis. Therefore, we investigated the correlation between observed changes in platelet mitochondrial respiration and a set of pro- and anti-inflammatory cytokines as well as NO plasma levels in patients with sepsis. METHODS AND RESULTS: Platelet mitochondrial respiration and levels of TNFα, MCP-1 (monocyte chemotactic protein-1), INFγ (interferon-γ), IL-1ß, IL-4, IL-5, IL-6, IL-8, IL-10 and IL-17 and NO were analyzed in 38 patients with severe sepsis or septic shock at three time points during one week following admission to the ICU. Citrate synthase, mitochondrial DNA and cytochrome c were measured as markers of cellular mitochondrial content. All mitochondrial respiratory states increased over the week analyzed (p<0.001). IL-8 levels correlated with maximal mitochondrial respiration on day 6-7 (pâ=â0.02, r2â=â0.22) and was also higher in non-survivors compared to survivors on day 3-4 and day 6-7 (pâ=â0.03 respectively). Neither NO nor any of the other cytokines measured correlated with respiration or mortality. Cytochrome c levels were decreased at day 1-2 by 24±5% (pâ=â0.03) and returned towards values of the controls at the last two time points. Citrate synthase activity and mitochondrial DNA levels were similar to controls and remained constant throughout the week. CONCLUSIONS: Out of ten analyzed cytokines and nitric oxide, IL-8 correlated with the observed increase in mitochondrial respiration. This suggests that cytokines as well as NO do not play a prominent role in the regulation of platelet mitochondrial respiration in sepsis. Further, the respiratory increase was not accompanied by an increase in markers of mitochondrial content, suggesting a possible role for post-translational enhancement of mitochondrial respiration rather than augmented mitochondrial mass.
Assuntos
Plaquetas/metabolismo , Citocinas/genética , Mitocôndrias/metabolismo , Óxido Nítrico/metabolismo , Choque Séptico/sangue , Idoso , Plaquetas/imunologia , Plaquetas/patologia , Estudos de Casos e Controles , Respiração Celular , Citrato (si)-Sintase/genética , Citrato (si)-Sintase/metabolismo , Citocromos c/genética , Citocromos c/metabolismo , Citocinas/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Feminino , Expressão Gênica , Humanos , Unidades de Terapia Intensiva , Masculino , Pessoa de Meia-Idade , Mitocôndrias/imunologia , Mitocôndrias/patologia , Fosforilação Oxidativa , Índice de Gravidade de Doença , Choque Séptico/imunologia , Choque Séptico/mortalidade , Choque Séptico/patologia , Análise de Sobrevida , Fatores de TempoRESUMO
BACKGROUND: Ischemic preconditioning has been proposed to involve changes in mitochondrial H(+) and K(+) fluxes, in particular through activation of uncoupling proteins and ATP-sensitive K(+) channels (MitoKATP). The objectives of the present study were to explore how increased H(+) and K(+) fluxes influence heart mitochondrial physiology with regard to production and scavenging of reactive oxygen species (ROS), volume changes and resistance to calcium-induced mitochondrial permeability transition (mPT). RESULTS: Isolated rat heart mitochondria were exposed to a wide concentration range of the protonophore CCCP or the potassium ionophore valinomycin to induce increased H(+) and K(+) conductance, respectively. Simultaneous monitoring of mitochondrial respiration and calcium retention capacity (CRC) demonstrated that the relative increase in respiration caused by valinomycin or CCCP correlated with a decrease in CRC, and that no level of respiratory uncoupling was associated with enhanced resistance to mPT. Mitochondria suspended in hyperosmolar buffer demonstrated a dose-dependent reduction in CRC with increasing osmolarity. However, mitochondria in hypoosmolar buffer to increase matrix volume did not display increased CRC. ROS generation was reduced by both K(+)- and H(+)-mediated respiratory uncoupling. The ability of heart mitochondria to detoxify H2O2 was substantially greater than the production rate. The H2O2 detoxification was dependent on respiratory substrates and was dramatically decreased following calcium-induced mPT, but was unaffected by uncoupling via increased K(+) and H(+) conductance. CONCLUSION: It is concluded that respiratory uncoupling is not directly beneficial to rat heart mitochondrial resistance to calcium overload irrespective of whether H(+) or K(+) conductance is increased. The negative effects of respiratory uncoupling thus probably outweigh the reduction in ROS generation and a potential positive effect by increased matrix volume, resulting in a net sensitization of heart mitochondria to mPT activation.
Assuntos
Mitocôndrias Cardíacas/metabolismo , Oxigênio/metabolismo , Canais de Potássio/metabolismo , Potássio/metabolismo , Prótons , Espécies Reativas de Oxigênio/metabolismo , Animais , Cálcio/metabolismo , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Cátions Monovalentes , Ciclosporina/farmacologia , Diazóxido/farmacologia , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Dilatação Mitocondrial/efeitos dos fármacos , Concentração Osmolar , Fosforilação Oxidativa/efeitos dos fármacos , Permeabilidade , Ionóforos de Próton/farmacologia , Ratos , Desacopladores/farmacologia , Valinomicina/farmacologiaRESUMO
INTRODUCTION: In sepsis, mitochondria have been associated with both initial dysfunction and subsequent upregulation (biogenesis). However, the evolvement of mitochondrial function in sepsis over time is largely unknown, and we therefore investigated mitochondrial respiration in peripheral blood immune cells (PBICs) in sepsis patients during the first week after admission to the intensive care unit (ICU). METHODS: PBICs from 20 patients with severe sepsis or septic shock were analyzed with high-resolution respirometry 3 times after admission to the ICU (within 48 hours, days 3 to 4 and days 6 to 7). Mitochondrial DNA (mtDNA), cytochrome c (Cyt c), and citrate synthase (CS) were measured as indicators of cellular mitochondrial content. RESULTS: In intact PBICs with endogenous substrates, a gradual increase in cellular respiration reached 173% of controls after 1 week (P = 0.001). In permeabilized cells, respiration using substrates of complex I, II, and IV were significantly increased days 1 to 2, reaching 137%, 130%, and 173% of controls, respectively. In parallel, higher levels of CS activity, mtDNA, and Cyt c content in PBICs (211%, 243%, and 331% of controls for the respective indicators were found at days 6 to 7; P < 0.0001). No differences in respiratory capacities were noted between survivors and nonsurvivors at any of the time points measured. CONCLUSIONS: PBICs from patients with sepsis displayed higher mitochondrial respiratory capacities compared with controls, due to an increased mitochondrial content, as indicated by increased mitochondrial DNA, protein content, and enzyme activity. The results argue against mitochondrial respiratory dysfunction in this type of cells in sepsis.
Assuntos
Unidades de Terapia Intensiva , Leucócitos Mononucleares/metabolismo , Mitocôndrias/metabolismo , Sepse/metabolismo , Adulto , Idoso , Respiração Celular/fisiologia , Feminino , Humanos , Imunidade Celular/fisiologia , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Mitocôndrias/imunologia , Sepse/imunologia , Adulto JovemRESUMO
The objective of the present study was to validate the presence and explore the characteristics of mitochondrial permeability transition (mPT) in isolated mitochondria from human heart tissue in order to investigate if previous findings in animal models of cardiac disorders are translatable to human disease. Mitochondria were rapidly isolated from fresh atrial tissue samples obtained from 14 patients undergoing Maze surgery due to atrial fibrillation. Human heart mitochondria exhibited typical mPT characteristics upon calcium overload such as swelling, evaluated by changes in light scattering, inhibition of respiration and loss of respiratory coupling. Swelling was a morphologically reversible event following transient calcium challenge. Calcium retention capacity (CRC), a quantitative measure of mPT sensitivity assayed by following extramitochondrial [Ca(2+)] and changes in respiration during a continuous calcium infusion, was significantly increased by cyclophilin D (CypD) inhibitors. The thiol-reactive oxidant phenylarsine oxide sensitized mitochondria to calcium-induced mPT. Release of the pro-apoptotic intermembrane protein cytochrome c was increased after, but not before, calcium discharge and respiratory inhibition in the CRC assay. From the present study, we conclude that adult viable heart mitochondria have a CypD- and oxidant-regulated mPT. The findings support that inhibition of mPT may be a relevant pharmacological target in human cardiac disease and may underlie the beneficial effect of cyclosporin A in reperfusion injury.
Assuntos
Coração/efeitos dos fármacos , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/fisiologia , Permeabilidade/efeitos dos fármacos , Idoso , Idoso de 80 Anos ou mais , Cálcio/metabolismo , Respiração Celular/efeitos dos fármacos , Respiração Celular/fisiologia , Peptidil-Prolil Isomerase F , Ciclofilinas/antagonistas & inibidores , Ciclofilinas/metabolismo , Ciclosporina/farmacologia , Citocromos c/metabolismo , Feminino , Coração/fisiologia , Humanos , Pessoa de Meia-Idade , Mitocôndrias Cardíacas/metabolismo , Dilatação Mitocondrial/efeitos dos fármacos , Dilatação Mitocondrial/fisiologia , Oxidantes/metabolismoRESUMO
Traumatic brain injury and ischemia can result in marked neuronal degeneration and residual impairment of cerebral function. However, no effective pharmacological treatment directed at tissues of the central nervous system (CNS) for acute intervention has been developed. The detailed pathophysiological cascade leading to -neurodegeneration in these conditions has not been elucidated, but cellular calcium overload and mitochondrial dysfunction have been implicated in a wide range of animal models involving degeneration of the CNS. In particular, activation of the calcium-induced mitochondrial permeability transition (mPT) is considered to be a major cause of cell death inferred by the broad and potent neuroprotective effects of -pharmacological inhibitors of mPT, especially modulators of cyclophilin activity and, more specifically, genetic inactivation of the mitochondrial cyclophilin, cyclophilin D. Reviewed are evidence and challenges that could bring on the dawning of mitochondrial medicine aimed at safeguarding energy supply following acute injury to the CNS.
Assuntos
Ciclofilinas/metabolismo , Mitocôndrias/efeitos dos fármacos , Fármacos Neuroprotetores , Animais , Arsenicais/farmacologia , Arsenicais/uso terapêutico , Lesões Encefálicas/tratamento farmacológico , Cálcio/metabolismo , Peptidil-Prolil Isomerase F , Ciclosporina/farmacologia , Ciclosporina/uso terapêutico , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Isquemia/tratamento farmacológicoRESUMO
Studying whole cell preparations with intact mitochondria and respiratory complexes has a clear benefit compared to isolated or disrupted mitochondria due to the dynamic interplay between mitochondria and other cellular compartments. Platelet mitochondria have a potential to serve as a source of human viable mitochondria when studying mitochondrial physiology and pathogenic mechanisms, as well as for the diagnostics of mitochondrial diseases. The objective of the present study was to perform a detailed evaluation of platelet mitochondrial respiration using high-resolution respirometry. Further, we aimed to explore the limits of sample size and the impact of storage as well as to establish a wide range of reference data from different pediatric and adult cohorts. Our results indicate that platelet mitochondria are well suited for ex-vivo analysis with the need for minute sample amounts and excellent reproducibility and stability.
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Plaquetas/metabolismo , Respiração Celular , Mitocôndrias/metabolismo , Adulto , Fatores Etários , Idoso , Criança , Pré-Escolar , Técnicas Citológicas/métodos , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Fatores Sexuais , Manejo de Espécimes/métodos , Adulto JovemRESUMO
The mitochondrial permeability transition (mPT) is considered to be a major cause of cell death under a variety of pathophysiological conditions of the central nervous system (CNS) and other organs. Pharmacological inhibition or genetic knockout of the matrix protein cyclophilin D (CypD) prevents mPT and cell degeneration in several models of brain injury. If these findings in animal models are translatable to human disease, pharmacological inhibition of mPT offers a promising therapeutic target. The objective of this study was to validate the presence of a CypD-sensitive mPT in adult human brain and liver mitochondria. In order to perform functional characterization of human mitochondria, fresh tissue samples were obtained during hemorrhage or tumor surgery and mitochondria were rapidly isolated. Mitochondrial calcium retention capacity, a quantitative assay for mPT, was significantly increased by the CypD inhibitor cyclosporin A in both human brain and liver mitochondria, whereas thiol-reactive compounds and oxidants sensitized mitochondria to calcium-induced mPT. Brain mitochondria underwent swelling upon calcium overload, which was reversible upon calcium removal. To further explore mPT of human mitochondria, liver mitochondria were demonstrated to exhibit several classical features of the mPT phenomenon, such as calcium-induced loss of membrane potential and respiratory coupling, as well as release of the pro-apoptotic protein cytochrome c. We concluded that adult viable human brain and liver mitochondria possess an active CypD-sensitive mPT. Our findings support the rationale of CypD and mPT inhibition as pharmacological targets in acute and chronic neurodegeneration.
Assuntos
Encéfalo/metabolismo , Encéfalo/ultraestrutura , Ciclofilinas/metabolismo , Membranas Intracelulares/metabolismo , Mitocôndrias Hepáticas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Respiração Celular/fisiologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Membranas Intracelulares/ultraestrutura , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Mitocôndrias Hepáticas/ultraestrutura , PermeabilidadeRESUMO
INTRODUCTION: Mitochondrial dysfunction has been suggested as a contributing factor to the pathogenesis of sepsis-induced multiple organ failure. Also, restoration of mitochondrial function, known as mitochondrial biogenesis, has been implicated as a key factor for the recovery of organ function in patients with sepsis. Here we investigated temporal changes in platelet mitochondrial respiratory function in patients with sepsis during the first week after disease onset. METHODS: Platelets were isolated from blood samples taken from 18 patients with severe sepsis or septic shock within 48 hours of their admission to the intensive care unit. Subsequent samples were taken on Day 3 to 4 and Day 6 to 7. Eighteen healthy blood donors served as controls. Platelet mitochondrial function was analyzed by high-resolution respirometry. Endogenous respiration of viable, intact platelets suspended in their own plasma or phosphate-buffered saline (PBS) glucose was determined. Further, in order to investigate the role of different dehydrogenases and respiratory complexes as well as to evaluate maximal respiratory activity of the mitochondria, platelets were permeabilized and stimulated with complex-specific substrates and inhibitors. RESULTS: Platelets suspended in their own septic plasma exhibited increased basal non-phosphorylating respiration (state 4) compared to controls and to platelets suspended in PBS glucose. In parallel, there was a substantial increase in respiratory capacity of the electron transfer system from Day 1 to 2 to Day 6 to 7 as well as compared to controls in both intact and permeabilized platelets oxidizing Complex I and/or II-linked substrates. No inhibition of respiratory complexes was detected in septic patients compared to controls. Non-survivors, at 90 days, had a more elevated respiratory capacity at Day 6 to 7 as compared to survivors. Cytochrome c increased over the time interval studied but no change in mitochondrial DNA was detected. CONCLUSIONS: The results indicate the presence of a soluble plasma factor in the initial stage of sepsis inducing uncoupling of platelet mitochondria without inhibition of the electron transfer system. The mitochondrial uncoupling was paralleled by a gradual and substantial increase in respiratory capacity. This may reflect a compensatory response to severe sepsis or septic shock, that was most pronounced in non-survivors, likely correlating to the severity of the septic insult.
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Plaquetas/fisiologia , Respiração Celular/fisiologia , Mitocôndrias/patologia , Sepse/metabolismo , Sepse/patologia , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sepse/mortalidade , Taxa de Sobrevida/tendências , Fatores de Tempo , Resultado do TratamentoAssuntos
Minociclina/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Animais , Cálcio/metabolismo , Humanos , Transplante de Fígado/efeitos adversos , Mitocôndrias Hepáticas/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Permeabilidade , Ratos , Traumatismo por Reperfusão/prevenção & controle , TemperaturaRESUMO
Modulation of K(+) conductance of the inner mitochondrial membrane has been proposed to mediate preconditioning in ischemia-reperfusion injury. The mechanism is not entirely understood, but it has been linked to a decreased activation of mitochondrial permeability transition (mPT). In the present study K(+) channel activity was mimicked by picomolar concentrations of valinomycin. Isolated brain mitochondria were exposed to continuous infusions of calcium. Monitoring of extramitochondrial Ca(2+) and mitochondrial respiration provided a quantitative assay for mPT sensitivity by determining calcium retention capacity (CRC). Valinomycin and cyclophilin D inhibition separately and additively increased CRC. Comparable degrees of respiratory uncoupling induced by increased K(+) or H(+) conductance had opposite effects on mPT sensitivity. Protonophores dose-dependently decreased CRC, demonstrating that so-called mild uncoupling was not beneficial per se. The putative mitoK(ATP) channel opener diazoxide did not mimic the effect of valinomycin. An alkaline matrix pH was required for mitochondria to retain calcium, but increased K(+) conductance did not result in augmented DeltapH. The beneficial effect of valinomycin on CRC was not mediated by H(2)O(2)-induced protein kinase Cepsilon activation. Rather, increased K(+) conductance reduced H(2)O(2) generation during calcium infusion. Lowering the osmolarity of the buffer induced an increase in mitochondrial volume and improved CRC similar to valinomycin without inducing uncoupling or otherwise affecting respiration. We propose that increased potassium conductance in brain mitochondria may cause a direct physiological effect on matrix volume inducing resistance to pathological calcium challenges.
Assuntos
Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Tamanho Mitocondrial , Potássio/metabolismo , Álcalis/metabolismo , Animais , Cálcio/metabolismo , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Respiração Celular/efeitos dos fármacos , Diazóxido/farmacologia , Ativação Enzimática/efeitos dos fármacos , Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , Masculino , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Tamanho Mitocondrial/efeitos dos fármacos , Mimetismo Molecular/efeitos dos fármacos , Nigericina/farmacologia , Canais de Potássio/metabolismo , Proteína Quinase C/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Valinomicina/farmacologiaRESUMO
Inhibition of mitochondrial permeability transition (mPT) has emerged as a promising approach for neuroprotection and development of well-tolerated mPT inhibitors with favorable blood-brain barrier penetration is highly warranted. In a recent study, 28 clinically available drugs with a common heterocyclic structure were identified as mPT inhibitors e.g. trifluoperazine, promethazine and nortriptyline. In addition, neuroprotection by structurally unrelated drugs e.g. neurosteroids, 4-hydroxy-tamoxifen and trimetazidine has been attributed to direct inhibition of mPT. The regulation of mPT is complex and highly dependent on the prevailing experimental conditions. Several features of mPT, such as swelling, depolarization or NADH oxidation, can also occur independently of the mPT phenomenon. Here, in isolated rodent brain-derived and human liver mitochondria, we re-evaluate drugs promoted as potent mPT inhibitors. We address the definition of an mPT inhibitor and present strategies to reliably detect mPT inhibition in vitro. Surprisingly, none of the 12 compounds tested displayed convincing mPT inhibition or effects comparable to cyclophilin D inhibition by the non-immunosuppressive cyclophilin inhibitor D-MeAla(3)-EtVal(4)-Cyclosporin (Debio 025). Propofol and 2-aminoethoxydiphenyl borate (2-APB) inhibited swelling in de-energized mitochondria but did not increase calcium retention capacity (CRC). Progesterone, trifluoperazine, allopregnanolone and 4-hydroxy-tamoxifen dose-dependently reduced CRC and respiratory control and were thus toxic rather than beneficial to mitochondrial function. Interestingly, topiramate increased CRC at high concentrations likely by a mechanism separate from direct mPT inhibition. We conclude that a clinically relevant mPT inhibitor should have a mitochondrial target and increase mitochondrial calcium retention at concentrations which can be translated to human use.
Assuntos
Encefalopatias/metabolismo , Encéfalo/metabolismo , Cálcio/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Isomerases de Aminoácido/efeitos adversos , Isomerases de Aminoácido/farmacologia , Animais , Compostos de Boro/efeitos adversos , Compostos de Boro/farmacologia , Encéfalo/efeitos dos fármacos , Encefalopatias/tratamento farmacológico , Peptidil-Prolil Isomerase F , Ciclofilinas/efeitos adversos , Ciclofilinas/farmacologia , Relação Dose-Resposta a Droga , Humanos , Masculino , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Pregnanolona/efeitos adversos , Pregnanolona/farmacologia , Progesterona/efeitos adversos , Progesterona/farmacologia , Propofol/efeitos adversos , Propofol/farmacologia , Ratos , Ratos Wistar , Tamoxifeno/efeitos adversos , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Trifluoperazina/efeitos adversos , Trifluoperazina/farmacologia , Trimetazidina/efeitos adversos , Trimetazidina/farmacologiaRESUMO
Cerebral injury is a critical aspect of the management of patients in intensive care. Pathological conditions induced by cerebral ischemia, hypoxia, head trauma, and seizure activity can result in marked residual impairment of cerebral function. We have investigated the potential mechanisms leading to neuronal cell death in pathological conditions, with the aim of discovering therapeutic targets and methods to minimize neuronal damage resulting from insults directed at the central nervous system (CNS). Over the years, deeper understanding of the mechanisms of neuronal cell death has indeed evolved, enabling clinical critical care management to salvage neurons that are at the brink of degeneration and to support recovery of brain function. However, no substantial breakthrough has been achieved in the quest to develop effective pharmacological neuroprotective therapy directed at tissues of the CNS. The current situation is unacceptable, and preservation of function and protection of the brain from terminal impairment will be a vital medical issue in the twenty-first century. To achieve this goal, it is critical to clarify the key mechanisms leading to neuronal cell death. Here, we discuss the importance of the calcineurin/immunophilin signal transduction pathway and mitochondrial involvement in the detrimental chain of events leading to neuronal degeneration.
Assuntos
Lesões Encefálicas/metabolismo , Calcineurina/metabolismo , Doenças Mitocondriais/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Membranas Mitocondriais/metabolismo , Degeneração Neural/metabolismo , Inibidores de Calcineurina , Cálcio/metabolismo , Morte Celular/fisiologia , Peptidil-Prolil Isomerase F , Ciclofilinas/antagonistas & inibidores , Ciclofilinas/metabolismo , Humanos , Poro de Transição de Permeabilidade Mitocondrial , Transdução de Sinais , Canais de Ânion Dependentes de Voltagem/metabolismoRESUMO
Mitochondrial uptake of calcium in excitotoxicity is associated with subsequent increase in reactive oxygen species (ROS) generation and delayed cellular calcium deregulation in ischemic and neurodegenerative insults. The mechanisms linking mitochondrial calcium uptake and ROS production remain unknown but activation of the mitochondrial permeability transition (mPT) may be one such mechanism. In the present study, calcium increased ROS generation in isolated rodent brain and human liver mitochondria undergoing mPT despite an associated loss of membrane potential, NADH and respiration. Unspecific permeabilization of the inner mitochondrial membrane by alamethicin likewise increased ROS independently of calcium, and the ROS increase was further potentiated if NAD(H) was added to the system. Importantly, calcium per se did not induce a ROS increase unless mPT was triggered. Twenty-one cyclosporin A analogs were evaluated for inhibition of calcium-induced ROS and their efficacy clearly paralleled their potency of inhibiting mPT-mediated mitochondrial swelling. We conclude that while intact respiring mitochondria possess powerful antioxidant capability, mPT induces a dysregulated oxidative state with loss of GSH- and NADPH-dependent ROS detoxification. We propose that mPT is a significant cause of pathological ROS generation in excitotoxic cell death.