RESUMO
Neuronal cell death occurs extensively during development and pathology, where it is especially important because of the limited capacity of adult neurons to proliferate or be replaced. The concept of cell death used to be simple as there were just two or three types, so we just had to work out which type was involved in our particular pathology and then block it. However, we now know that there are at least a dozen ways for neurons to die, that blocking a particular mechanism of cell death may not prevent the cell from dying, and that non-neuronal cells also contribute to neuronal death. We review here the mechanisms of neuronal death by intrinsic and extrinsic apoptosis, oncosis, necroptosis, parthanatos, ferroptosis, sarmoptosis, autophagic cell death, autosis, autolysis, paraptosis, pyroptosis, phagoptosis, and mitochondrial permeability transition. We next explore the mechanisms of neuronal death during development, and those induced by axotomy, aberrant cell-cycle reentry, glutamate (excitoxicity and oxytosis), loss of connected neurons, aggregated proteins and the unfolded protein response, oxidants, inflammation, and microglia. We then reassess which forms of cell death occur in stroke and Alzheimer's disease, two of the most important pathologies involving neuronal cell death. We also discuss why it has been so difficult to pinpoint the type of neuronal death involved, if and why the mechanism of neuronal death matters, the molecular overlap and interplay between death subroutines, and the therapeutic implications of these multiple overlapping forms of neuronal death.
Assuntos
Apoptose/fisiologia , Morte Celular/fisiologia , Microglia/metabolismo , Neurônios/metabolismo , Animais , Humanos , Fagocitose/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Background and Objectives: Recent findings suggest that neurodegeneration starts early in the course of multiple sclerosis (MS) and significantly contributes to the progression of patients' disability. Tau is a microtubule-binding protein that is known to play a role in the pathophysiology of many neurodegenerative disorders. Newly emerging data on tau protein-induced neurodegenerative processes and its possible involvement in MS suggest that it may be involved in the pathology of early-stage MS. Therefore, this study aimed to test this hypothesis in patients with newly diagnosed MS. Materials and Methods: Cerebrospinal fluid (CSF) was collected from 19 patients with newly diagnosed MS and 19 control subjects. All MS patients underwent neurological examination, lumbar punction, and brain magnetic resonance imaging (MRI). CSF concentrations of total and phosphorylated tau (phospho-tau-181) protein were measured using commercial enzyme-linked immunosorbent assay kits. Results: The total tau concentration was significantly higher in the CSF of MS patients compared to controls (141.67 pg/mL, IQR 77.79-189.17 and 68.77 pg/mL, IQR 31.24-109.17, p = 0.025). In MS patients, the total tau protein positively correlated with total CSF protein (r = 0.471, p = 0.048). Significantly higher total tau concentration was measured in MS patients with higher lesion load in brain MRI (≥9 versus <9 lesions; 168.33 pg/mL, IQR 111.67-222.32 and 73.33 pg/mL, IQR -32.13-139.29-, p = 0.021). The CSF concentration of phospho-tau-181 protein was below the detection limit in both MS and control subjects. Conclusions: The concentration of total tau protein level is elevated, whereas phospho-tau-181 is undetectable in the CSF of patients with early-stage MS.
Assuntos
Esclerose Múltipla , Humanos , Esclerose Múltipla/líquido cefalorraquidiano , Proteínas tau/líquido cefalorraquidiano , Projetos Piloto , Biomarcadores/líquido cefalorraquidiano , EncéfaloRESUMO
Alzheimer's disease (AD) is the most common form of dementia worldwide, and it contributes up to 70% of cases. AD pathology involves abnormal amyloid beta (Aß) accumulation, and the link between the Aß1-42 structure and toxicity is of major interest. NMDA receptors (NMDAR) are thought to be essential in Aß-affected neurons, but the role of this receptor in glial impairment is still unclear. In addition, there is insufficient knowledge about the role of Aß species regarding mitochondrial redox states in neurons and glial cells, which may be critical in developing Aß-caused neurotoxicity. In this study, we investigated whether different Aß1-42 species-small oligomers, large oligomers, insoluble fibrils, and monomers-were capable of producing neurotoxic effects via microglial NMDAR activation and changes in mitochondrial redox states in primary rat brain cell cultures. Small Aß1-42 oligomers induced a concentration- and time-dependent increase in intracellular Ca2+ and necrotic microglial death. These changes were partially prevented by the NMDAR inhibitors MK801, memantine, and D-2-amino-5-phosphopentanoic acid (DAP5). Neither microglial intracellular Ca2+ nor viability was significantly affected by larger Aß1-42 species or monomers. In addition, the small Aß1-42 oligomers caused mitochondrial reactive oxygen species (mtROS)-mediated mitochondrial depolarization, glutamate release, and neuronal cell death. In microglia, the Aß1-42-induced mtROS overproduction was mediated by intracellular calcium ions and Aß-binding alcohol dehydrogenase (ABAD). The data suggest that the pharmacological targeting of microglial NMDAR and mtROS may be a promising strategy for AD therapy.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Ratos , Animais , Peptídeos beta-Amiloides/metabolismo , Microglia/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Doença de Alzheimer/metabolismo , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Recent studies have identified that under stimulation by bacterial lipopolysaccharide mammalian macrophages produce itaconic acid. Yet, it is unknown whether itaconate has any effect on viability of brain cells. Here we used extracellularly added itaconate to investigate its effects on viability of cerebellar granule cells (CGC) in cultures and respiratory functions of these cells and isolated brain mitochondria. We found that 3-5 mM itaconate had no effect on the viability of neurons, but 10 mM itaconate was toxic and induced neuronal apoptosis. Removal of itaconate after 24 h incubation resulted in further decrease in viability and number of neurons. Respiration of intact neurons was not affected by itaconate, but permeabilized cells as well as isolated brain mitochondria demonstrated decreased rates of respiration in the presence of itaconate. Using isolated adult rat brain mitochondria we found that itaconate decreased mitochondrial phosphorylating respiration, mitochondrial calcium retention capacity, production of reactive oxygen species with Complex I and Complex II substrates as well as inhibition of Complex I, Complex IV and ATP synthase. In conclusion, the results suggest that itaconic acid at millimolar concentrations affects mitochondrial functions and viability of neurons.
Assuntos
Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Succinatos/uso terapêutico , Animais , Humanos , Ratos , Espécies Reativas de Oxigênio , Succinatos/farmacologiaRESUMO
Microglial functioning depends on Ca2+ signaling. By using Ca2+ sensitive fluorescence dye, we studied how inhibition of mitochondrial respiration changed spontaneous Ca2+ signals in soma of microglial cells from 5-7-day-old rats grown under normoxic and mild-hypoxic conditions. In microglia under normoxic conditions, metformin or rotenone elevated the rate and the amplitude of Ca2+ signals 10-15 min after drug application. Addition of cyclosporin A, a blocker of mitochondrial permeability transition pore (mPTP), antioxidant trolox, or inositol 1,4,5-trisphosphate receptor (IP3R) blocker caffeine in the presence of rotenone reduced the elevated rate and the amplitude of the signals implying sensitivity to reactive oxygen species (ROS), and involvement of mitochondrial mPTP together with IP3R. Microglial cells exposed to mild hypoxic conditions for 24 h showed elevated rate and increased amplitude of Ca2+ signals. Application of metformin or rotenone but not phenformin before mild hypoxia reduced this elevated rate. Thus, metformin and rotenone had the opposing fast action in normoxia after 10-15 min and the slow action during 24 h mild-hypoxia implying activation of different signaling pathways. The slow action of metformin through inhibition of complex I could stabilize Ca2+ homeostasis after mild hypoxia and could be important for reduction of ischemia-induced microglial activation.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Metformina/farmacologia , Animais , Cafeína/farmacologia , Sinalização do Cálcio/fisiologia , Cromanos/farmacologia , Ciclosporina/farmacologia , Complexo I de Transporte de Elétrons/metabolismo , Feminino , Hipóxia/genética , Hipóxia/metabolismo , Masculino , Metformina/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Neurônios/metabolismo , Cultura Primária de Células , Ratos , Espécies Reativas de Oxigênio/metabolismo , Rotenona/farmacologiaRESUMO
Mitochondrial dysfunction during ischemic stroke ultimately manifests as ATP depletion. Mitochondrial ATP synthase upon loss of mitochondrial membrane potential during ischemia rapidly hydrolyses ATP and thus contributes to ATP depletion. Increasing evidence suggests that inhibition of ATP synthase limits ATP depletion and is protective against ischemic tissue damage. Bedaquiline (BDQ) is an anti-microbial agent, approved for clinical use, that inhibits ATP synthase of Mycobacteria; however recently it has been shown to act on mitochondrial ATP synthase, inhibiting both ATP synthesis and hydrolysis in low micromolar concentrations. In this study, we investigated whether preconditioning with BDQ can alleviate ischemia/reperfusion-induced brain injury in Wistar rats after middle cerebral artery occlusion-reperfusion and whether it affects mitochondrial functions. We found that BDQ was effective in limiting necrosis and neurological dysfunction during ischemia-reperfusion. BDQ also caused inhibition of ATPase activity, mild uncoupling of respiration, and stimulated mitochondrial respiration both in healthy and ischemic mitochondria. Mitochondrial calcium retention capacity was unaffected by BDQ preconditioning. We concluded that BDQ has neuroprotective properties associated with its action on mitochondrial respiration and ATPase activity.
Assuntos
Diarilquinolinas/farmacologia , Inibidores Enzimáticos/farmacologia , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , Fármacos Neuroprotetores/farmacologia , Traumatismo por Reperfusão/metabolismo , Acidente Vascular Cerebral/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Respiração Celular/efeitos dos fármacos , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neuroproteção/efeitos dos fármacos , Ratos , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/patologia , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/patologiaRESUMO
Tau is a microtubule-associated protein, found at high levels in neurons, and its aggregation is associated with neurodegeneration. Recently, it was found that tau can be actively secreted from neurons, but the effects of extracellular tau on neuronal viability are unclear. In this study, we investigated whether extracellular tau2N4R can cause neurotoxicity in primary cultures of rat brain neurons and glial cells. Cell cultures were examined for neuronal loss, death, and phosphatidylserine exposure, as well as for microglial phagocytosis by fluorescence microscopy. Aggregation of tau2N4R was assessed by atomic force microscopy. We found that extracellular addition of tau induced a gradual loss of neurons over 1-2 days, without neuronal necrosis or apoptosis, but accompanied by proliferation of microglia in the neuronal-glial co-cultures. Tau addition caused exposure of the 'eat-me' signal phosphatidylserine on the surface of living neurons, and this was prevented by elimination of the microglia or by inhibition of neutral sphingomyelinase. Tau also increased the phagocytic activity of pure microglia, and this was blocked by inhibitors of neutral sphingomyelinase or protein kinase C. The neuronal loss induced by tau was prevented by inhibitors of neutral sphingomyelinase, protein kinase C or the phagocytic receptor MerTK, or by eliminating microglia from the cultures. The data suggest that extracellular tau induces primary phagocytosis of stressed neurons by activated microglia, and identifies multiple ways in which the neuronal loss induced by tau can be prevented.
Assuntos
Microglia/efeitos dos fármacos , Neurônios , Fagocitose/efeitos dos fármacos , Proteínas tau/farmacologia , Animais , Células Cultivadas , Técnicas de Cocultura , Microglia/metabolismo , Neurônios/patologia , Ratos , Proteínas tau/metabolismoRESUMO
Anthocyanins are considered as bioactive components of plant-based diets that provide protection against ischemic cardiovascular pathologies by mechanisms dependent on their antioxidant and reductive capacities. However, it is not clear whether similar anthocyanin-mediated mechanisms can provide protection against ischemia-induced brain mitochondrial injury and cell death. In this study, we compared effects of three cyanidin-3-glycosides - glucoside (Cy3G), galactoside (Cy3Gal) and rutinoside (Cy3R), with pelargonxidin-3-glucoside (Pg3G) and found that at 10-20 µM concentrations they have no direct effect on respiratory functions of mitochondria isolated from normal or ischemia-damaged rat brain slices. However, intravenous injection of Cy3Gal and Cy3G (0,025 mg/kg or 0,05 mg/kg what matches 10 µM or 20 µM respectively) but not Cy3R in rats protected against ischemia-induced caspase activation and necrotic cell death, and reduced infarct size in cerebral cortex and cerebellum. These effects correlated with cytochrome c reducing capacity of cyanidin-3-glycosides. In contrast, intravenous injection of 0,025 mg/kg Pg3G which has the lowest cytochrome c reducing capacity among investigated anthocyanins, had no effect on ischemia-induced caspase activation and necrosis but reduced brain infarct size whereas intravenous injection of 0,05 mg/kg of Pg3G slightly promoted necrosis in the brain. Our data suggest that reductive rather than antioxidant capacities of anthocyanins may be important components in providing protection against ischemic brain damage.
Assuntos
Antocianinas/metabolismo , Encéfalo/metabolismo , Isquemia/metabolismo , Animais , Ratos , Ratos WistarRESUMO
Anthocyanins are water-soluble pigments providing certain color for various plant parts, especially in edible berries. Earlier these compounds were only known as natural food colorants, the stability of which depended on pH, light, storage temperature and chemical structure. However, due to the increase of the in vitro, in vivo experimental data, as well as of the epidemiological studies, today anthocyanins and their metabolites are also regarded as potential pharmaceutical compounds providing various beneficial health effects on either human or animal cardiovascular system, brain, liver, pancreas and kidney. Many of these effects are shown to be related to the free-radical scavenging and antioxidant properties of anthocyanins, or to their ability to modulate the intracellular antioxidant systems. However, it is generally overlooked that instead of acting exclusively as antioxidants certain anthocyanins affect the activity of mitochondria that are the main source of energy in cells. Therefore, the aim of the present review is to summarize the major knowledge about the chemistry and regulation of biosynthesis of anthocyanins in plants, to overview the facts on bioavailability, and to discuss the most recent experimental findings related to the beneficial health effects emphasizing mitochondria.
Assuntos
Antocianinas , Frutas , Animais , Antioxidantes/farmacologia , Humanos , Mitocôndrias , PigmentaçãoRESUMO
A growing number of studies suggest amyloid-ß and tau present in cerebrospinal fluid (CSF) and blood as putative biomarkers for Alzheimer's disease (AD). However, there is a question whether these compounds present in patients' bodily fluids can directly cause neurotoxic effects. We investigated effects of AD and other dementia (OD) patients' blood serum and CSF on viability of cells in primary cerebellar granule cell cultures. Overall, 59 individuals participated in the study from whom 55 samples of biological fluids were taken. Participants were classified into early (E-AD) and middle (M-AD) stages of AD, cognitively healthy control (HC) and OD groups. We found that concentrations of total and phosphorylated tau were higher in CSF from AD patients, while amyloid-ß42 and amyloid-ß40 in the serum was lower compared to HC. The most cytotoxic effects were induced by CSFs from M-AD patients which caused neuronal necrosis and suppressed microglial proliferation, whereas CSFs from the groups of other patients did not kill neurons. Serum and CSF from the E-AD group caused a reduction of neuronal numbers in cultures. There were no significant differences in levels of CSF biomarkers between the AD groups although both tau species in CSFs from M-AD patients were found to be significantly elevated compared to HC. Our data suggest that biological fluids from E-AD induce neuronal loss, whereas effects of CSF on the reduction in neuronal viability can serve as an indicator of M-AD and may be associated with extracellular tau.
Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Neurônios , Fragmentos de Peptídeos/líquido cefalorraquidiano , Proteínas tau/líquido cefalorraquidiano , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/sangue , Peptídeos beta-Amiloides/sangue , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/sangue , Proteínas tau/sangueRESUMO
Background and objective: Nitric oxide (NO) is known to exert cardioprotective effects against heart ischemic damage and may be involved in ischemic pre- and postconditioning. NO-triggered cardioprotective mechanisms are not well understood but may involve regulation of mitochondrial permeability transition pore (mPTP). In this study, we aimed to identify differentially phosphorylated mitochondrial proteins possibly involved in the NO/protein kinase G (PKG)/mPTP signaling pathway that can increase the resistance of cardiomyocytes to ischemic damage. Materials and methods: Isolated hearts from Wistar rats were perfused with NO donor NOC-18 prior to induction of stop-flow ischemia. To quantify and characterize the phosphoproteins, mitochondrial proteins were resolved and analyzed by two-dimensional gel electrophoresis followed by Pro-Q Diamond phosphoprotein gel staining, excision, trypsin digestions, and mass spectrometry. Quantitative proteomic analysis coupled with liquid chromatography-tandem mass spectrometry was also performed. Results: Mitochondrial protein phosphorylation patterns in NOC-18-pretreated ischemic hearts versus ischemic hearts were compared. Pretreatment of hearts with NOC-18 caused changes in mitochondrial phosphoproteome after ischemia which involved modifications of 10 mitochondrial membrane-bound and 10 matrix proteins. Among them, α-subunit of ATP synthase and adenine nucleotide (ADP/ATP) translocase 1, both of which are considered as potential structural components of mPTP, were identified. We also found that treatment of isolated non-ischemic mitochondria with recombinant PKG did not cause the same protein phosphorylation as pretreatment of hearts with NOC-18. Conclusions: Our study suggests that pretreatment of hearts with NOC-18 causes changes in mitochondrial phosphoproteome after ischemia which involves modifications of certain proteins thought to be involved in the regulation of mPTP opening and intracellular redox state. These proteins may be potential targets for pharmacological preconditioning of the heart.
Assuntos
Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Isquemia Miocárdica/metabolismo , Doadores de Óxido Nítrico/farmacologia , Compostos Nitrosos/farmacologia , Fosfoproteínas/metabolismo , Proteoma/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Coração/efeitos dos fármacos , Masculino , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial , Miocárdio/metabolismo , Fosforilação , Proteoma/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacosRESUMO
Constantly growing experimental data from in vitro, in vivo and epidemiological studies show the great potential of anthocyanin-containing fruit and berry extracts or pure individual anthocyanins as cardioprotective food components or pharmacological compounds. In general it is regarded that the cardioprotective activity of anthocyanins is related to their antioxidant properties. However there are recent reports that certain anthocyanins may protect the heart against ischemia/reperfusion-induced injury by activating signal transduction pathways and sustaining mitochondrial functions instead of acting solely as antioxidants. In this review, we summarize the proposed mechanisms of direct or indirect actions of anthocyanins within cardiac cells with the special emphasis on recently discovered their pharmacological effects on mitochondria in cardioprotection: reduction of cytosolic cytochrome c preventing apoptosis and sustainment of electron transfer between NADH dehydrogenase and cytochrome c supporting oxidative phosphorylation in ischemia-damaged mitochondria.
Assuntos
Antocianinas/farmacologia , Antocianinas/uso terapêutico , Cardiotônicos/farmacologia , Cardiotônicos/uso terapêutico , Mitocôndrias Cardíacas/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Humanos , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêuticoRESUMO
Although it is well documented that soluble beta amyloid (Aß) oligomers are critical factors in the pathogenesis of Alzheimer's disease (AD) by causing synaptic dysfunction and neuronal death, the primary mechanisms by which Aß oligomers trigger neurodegeneration are not entirely understood. We sought to investigate whether toxic small Aß(1-42) oligomers induce changes in plasma membrane potential of cultured neurons and glial cells in rat cerebellar granule cell cultures leading to neuronal death and whether these effects are sensitive to the N-methyl-D-aspartate receptor (NMDA-R) antagonist MK801. We found that small Aß(1-42) oligomers induced rapid, protracted membrane depolarization of both neurons and microglia, whereas there was no change in membrane potential of astrocytes. MK801 did not modulate Aß-induced neuronal depolarization. In contrast, Aß1(-42) oligomer-induced decrease in plasma membrane potential of microglia was prevented by MK801. Small Aß(1-42) oligomers significantly elevated extracellular glutamate and caused neuronal necrosis, and both were prevented by MK801. Also, small Aß(1-42) oligomers decreased resistance of isolated brain mitochondria to calcium-induced opening of mitochondrial permeability transition pore. In conclusion, the results suggest that the primary effect of toxic small Aß oligomers on neurons is rapid, NMDA-R-independent plasma membrane depolarization, which leads to neuronal death. Aß oligomers-induced depolarization of microglial cells is NMDA-R dependent.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Microglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Animais , Morte Celular/efeitos dos fármacos , Cerebelo/efeitos dos fármacos , Maleato de Dizocilpina/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Microglia/metabolismo , Neurônios/metabolismo , RatosRESUMO
BACKGROUND: Ischemic brain injury due to stroke and/or cardiac arrest is a major health issue in modern society requiring urgent development of new effective therapies. The aim of this study was to evaluate mitochondrial, microcirculatory, and histological changes in a swine model of global cerebral ischemia. RESULTS: In our model, significant microcirculatory changes, but only negligible histological cell alterations, were observed 3 h after bilateral carotid occlusion, and were more pronounced if the vascular occlusion was combined with systemic hypotension. Analysis of mitochondrial function showed that LEAK respiration (measured in the presence of pyruvate + malate but without ADP) was not affected in any model of global cerebral ischemia in pigs. The OXPHOS capacity with pyruvate + malate as substrates decreased compared with the control levels after bilateral carotid artery occlusion, and bilateral carotid artery occlusion + hypotension by 20% and 79%, respectively, resulting in decreases in the respiratory control index of 14% and 73%, respectively. OXPHOS capacity with succinate as a substrate remained constant after unilateral carotid artery occlusion or bilateral carotid artery occlusion, but decreased by 53% after bilateral carotid artery occlusion and hypotension compared with controls (p < 0.05, n = 3-6). Addition of exogenous cytochrome c to mitochondria isolated from ischemia brains had no effect on respiration in all models used in this study. CONCLUSIONS: We found a decrease in microcirculation and mitochondrial oxidative phosphorylation activity, but insignificant neuronal death, after 3 h ischemia in all our pig models of global cerebral ischemia. Dysfunction of the mitochondrial oxidative phosphorylation system, particularly damage to complex I of the respiratory chain, may be the primary target of the ischemic insult, and occurs before signs of neuronal death can be detected.
Assuntos
Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Encéfalo/patologia , Encéfalo/fisiopatologia , Microcirculação , Mitocôndrias/patologia , Mitocôndrias/fisiologia , Animais , Velocidade do Fluxo Sanguíneo , Circulação Cerebrovascular , Feminino , Consumo de Oxigênio , SuínosRESUMO
In this work, the effects of standardized dry extract of Ginkgo biloba leaves, EGb761, on the respiration of rat heart and liver mitochondria were investigated. We revealed uncoupling of oxidative phosphorylation in rat heart mitochondria by EGb761 which was not observed in liver mitochondria respiring on pyruvate + malate; oxidation of succinate in heart mitochondria was inhibited by EGb761, concentration-dependently, almost completely at 1.00 mg/mL. Uncoupling effect of EGb761 was found to be due to increase in H(+) and K(+) permeability of inner membrane of mitochondria which is most likely to be mediated by the ATP/ADP-translocator and uncoupling proteins. EGb761 depressed State 3 respiration with pyruvate + malate (similarly in heart and liver mitochondria) and succinate (stronger than with pyruvate + malate) but not respiratory chain Complex IV; inhibition of respiration was not restored by uncoupler indicating the inhibitory action of EGb761 on the respiratory complexes preceding to Complex IV and/or on the substrate transport. Moreover, EGb761 rapidly reduced pure cytochrome c. This property of EGb761 together with the observed uncoupling of oxidative phosphorylation and reduction of H2O2 accumulation may be beneficial for the cell in the prevention of apoptosis and protection of cellular functions in pathological situations.
Assuntos
Respiração Celular/efeitos dos fármacos , Mitocôndrias/metabolismo , Extratos Vegetais/química , Folhas de Planta/química , Animais , Ginkgo biloba , Masculino , Folhas de Planta/metabolismo , Ratos , Ratos WistarRESUMO
Beta amyloid (Aß) oligomers are thought to contribute to the pathogenesis of Alzheimer's disease. However, clinical trials using Aß immunization were unsuccessful due to strong brain inflammation, the mechanisms of which are poorly understood. In this study we tested whether monoclonal antibodies to oligomeric Aß would prevent the neurotoxicity of Aß oligomers in primary neuronal-glial cultures. However, surprisingly,the antibodies dramatically increased the neurotoxicity of Aß. Antibodies bound to monomeric Aß fragments were non-toxic to cultured neurons, while antibodies to other oligomeric proteins: hamster polyomavirus major capsid protein, human metapneumovirus nucleocapsid protein, and measles virus nucleocapsid protein, strongly potentiated the neurotoxicity of their antigens. The neurotoxicity of antibody-antibody oligomeric antigen complexes was abolished by removal of the Fc region from the antibodies or by removal of microglia from cultures, and was accompanied by inflammatory activation and proliferation of the microglia in culture. In conclusion, we find that immune complexes formed by Aß oligomers or other oligomeric/multimeric antigens and their specific antibodies can cause death and loss of neurons in primary neuronal-glial cultures via Fc-dependent microglial activation. The results suggest that therapies resulting in antibodies to oligomeric Aß or oligomeric brain virus proteins should be used with caution or with suppression of microglial activation.
Assuntos
Peptídeos beta-Amiloides/imunologia , Peptídeos beta-Amiloides/toxicidade , Microglia/efeitos dos fármacos , Síndromes Neurotóxicas/patologia , Animais , Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo/imunologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Ativação de Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microglia/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/toxicidade , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: The central molecule in the pathogenesis of Alzheimer's disease (AD) is believed to be a small-sized polypeptide - beta amyloid (Aß) which has an ability to assemble spontaneously into oligomers. Various studies concerning therapeutic and prophylactic approaches for AD are based on the immunotherapy using antibodies against Aß. It has been suggested that either active immunization with Aß or passive immunization with anti-Aß antibodies might help to prevent or reduce the symptoms of the disease. However, knowledge on the mechanisms of Aß-induced immune response is rather limited. Previous research on Aß1-42 oligomers in rat brain cultures showed that the neurotoxicity of these oligomers considerably depends on their size. In the current study, we evaluated the dependence of immunogenicity of Aß1-42 oligomers on the size of oligomeric particles and identified the immunodominant epitopes of the oligomers. RESULTS: Mice were immunized with various Aß1-42 oligomers. The analysis of serum antibodies revealed that small Aß1-42 oligomers (1-2 nm in size) are highly immunogenic. They induced predominantly IgG2b and IgG2a responses. In contrast, larger Aß1-42 oligomers and monomers induced weaker IgG response in immunized mice. The monoclonal antibody against 1-2 nm Aß1-42 oligomers was generated and used for antigenic characterization of Aß1-42 oligomers. Epitope mapping of both monoclonal and polyclonal antibodies demonstrated that the main immunodominant region of the 1-2 nm Aß1-42 oligomers is located at the amino-terminus (N-terminus) of the peptide, between amino acids 1 and 19. CONCLUSIONS: Small Aß1-42 oligomers of size 1-2 nm induce the strongest immune response in mice. The N-terminus of Aß1-42 oligomers represents an immunodominant region which indicates its surface localization and accessibility to the B cells. The results of the current study may be important for further development of Aß-based vaccination and immunotherapy strategies.
Assuntos
Doença de Alzheimer/imunologia , Peptídeos beta-Amiloides/administração & dosagem , Imunidade Inata/efeitos dos fármacos , Imunoterapia , Fragmentos de Peptídeos/administração & dosagem , Doença de Alzheimer/tratamento farmacológico , Vacinas contra Alzheimer/imunologia , Peptídeos beta-Amiloides/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Linfócitos B/imunologia , Modelos Animais de Doenças , Mapeamento de Epitopos , Camundongos , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , RatosRESUMO
BACKGROUND/AIMS: Excessive systemic inflammatory response syndrome during severe acute pancreatitis (AP) leads to multiple organ dysfunction syndrome, which is the main cause of death and may be associated with primary mitochondrial disturbances. The aim of our study was to evaluate the role of mitochondria during experimental AP in pancreas and vital organs like kidney, lungs and liver within the first 48 h. METHODS: AP was induced in 39 male Wistar rats by intraductal application of sodium taurocholate (5%, 1.75 ml/kg). Animals were divided into groups reflecting the time from induction of the AP till collection of tissues (control and 1, 3, 6, 12, 24, 48 h). Mitochondria were isolated by differential centrifugation and mitochondrial respiration rates were measured oxygraphically. RESULTS: (1) Mitochondria in pancreas are affected within the first 6 h after onset of AP, (2) kidney mitochondria are affected 24 h after onset of AP, (3) lungs mitochondria are affected within 48 h after onset of AP whereas (4) liver mitochondria remain well preserved within the first 48 h. Severe AP-induced decrease in the oxidative phosphorylation of pancreas, kidney and lungs mitochondria was more pronounced with Complex I-linked (glutamate/malate) than with Complex II-linked (succinate) substrates and was associated with inhibition of Complex I. CONCLUSION: Our data show that the disturbances of mitochondrial energy metabolism in pancreas, kidney and lungs may play an important role in the development and progression of AP as a systemic disease.
Assuntos
Mitocôndrias Hepáticas/metabolismo , Mitocôndrias/metabolismo , Doenças Mitocondriais/etiologia , Pâncreas/fisiopatologia , Pancreatite/fisiopatologia , Animais , Modelos Animais de Doenças , Metabolismo Energético , Rim/fisiopatologia , Pulmão/fisiopatologia , Masculino , Insuficiência de Múltiplos Órgãos/etiologia , Pancreatite/induzido quimicamente , Ratos , Ratos Wistar , Ácido TaurocólicoRESUMO
BACKGROUND: The aim of this study is to evaluate the role of NADPH oxidase in primary intestinal epithelial cells during the active phase of UC. METHODS: The primary human colonic epithelial cells were isolated from 19 patients with mild to moderate inflammatory activity of UC and 14 controls using chelation method. The cells were cultivated under the effect of mediators. Viability of cells was assessed by fluorescent microscopy. Production of reactive oxygen species (ROS) by the cells was measured fluorimetrically using Amplex Red. Production of TNF-α cytokine by the colonic epithelial cells was analysed by ELISA. RESULTS: The results of our study showed that unstimulated cells of UC patients had a decreased viability, increased ROS production, but similar TNF-α level when compared to the controls. Stimulation with LPS increased hydrogen peroxide and TNF-α level in the UC group. Treatment of colonic epithelial cells with NADPH oxidase inhibitor increased cell viability decreased the levels of ROS and TNF-α in the LPS-treated cells isolated from UC patients. CONCLUSIONS: Our study showed that bacterial endotoxins induced NADPH oxidase activation in the colonic epithelial cells. Moreover, we revealed that treatment with NADPH oxidase inhibitors had a protective effect against pro-inflammatory action of LPS in human colonic epithelium cells during inflammation.
Assuntos
Colite Ulcerativa/metabolismo , Colo/metabolismo , Células Epiteliais/metabolismo , Peróxido de Hidrogênio/metabolismo , Mucosa Intestinal/metabolismo , NADPH Oxidases/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Estudos de Casos e Controles , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Colite Ulcerativa/imunologia , Colo/efeitos dos fármacos , Colo/imunologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Feminino , Humanos , Peróxido de Hidrogênio/imunologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Lipopolissacarídeos/farmacologia , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , NADPH Oxidases/antagonistas & inibidores , Oniocompostos/farmacologia , Espécies Reativas de Oxigênio/imunologia , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/imunologia , Adulto JovemRESUMO
BACKGROUND AND OBJECTIVE: It is well known that cardiomyocyte apoptosis contributes to ischemic heart damage. There is also increasing evidence that the polyphenolic compounds of natural origin, such as anthocyanins, may attenuate ischemia/reperfusion injury though the mechanisms of such protection are not clear. Following our previous studies showing the effect of certain anthocyanins on cytochrome c redox state, mitochondrial functions, and ischemia-induced caspase activation in the heart, here we investigated whether these anthocyanins can rescue cardiac cells from death by the mechanism involving the reduction of cytosolic cytochrome c. MATERIAL AND METHODS: Before global ischemia and reperfusion, isolated rat hearts were preloaded with cyanidin-3-O-glucoside (Cy3G) that has high cytochrome c-reducing capacity or pelargonidin-3-O-glucoside (Pg3G) that possesses low reducing activity. Cell death was evaluated assessing apoptosis by the TUNEL method or necrosis measuring the release of lactate dehydrogenase into perfusate. RESULTS: The perfusion of hearts with 20-µM Cy3G prevented ischemia/reperfusion-induced apoptosis of cardiomyocytes: the number of TUNEL-positive myocytes was decreased by 73% if compared with the untreated ischemic group. The same effect was observed measuring the activity of lactate dehydrogenase as the measure of necrosis: perfusion with 20-µM Cy3G reduced the level of LDH release into the perfusate to the control level. The perfusion of hearts with 20-µM Pg3G did not prevent ischemia/reperfusion-induced apoptosis as well as necrosis. CONCLUSIONS: Cy3G protected the rat heart from ischemia/reperfusion-induced apoptosis and necrosis; meanwhile, Pg3G did not exert any protective effect. The protective effect of Cy3G may be related due to its high capacity to reduce cytosolic cytochrome c.