RESUMO
The mitochondrial transition pore (MTP) is implicated as a mediator of cell injury and death in many situations. The MTP opens in response to stimuli including reactive oxygen species and inhibition of the electron transport chain. Sporadic Parkinson's disease (PD) is characterized by oxidative stress and specifically involves a defect in complex I of the electron transport chain. To explore the possible involvement of the MTP in PD models, we tested the effects of the complex I inhibitor and apoptosis-inducing toxin N-methyl-4-phenylpyridinium (MPP+) on cyclosporin A (CsA)-sensitive mitochondrial swelling and release of cytochrome c. In the presence of Ca2+ and Pi, MPP+ induced a permeability transition in both liver and brain mitochondria. MPP+ also caused release of cytochrome c from liver mitochondria. Rotenone, a classic non-competitive complex I inhibitor, completely inhibited MPP(+)-induced swelling and release of cytochrome c. The MPP(+)-induced permeability transition was synergistic with nitric oxide and the adenine nucleotide translocator inhibitor atractyloside, and additive with phenyl arsine oxide cross-linking of dithiol residues. MPP(+)-induced pore opening and cytochrome c release were blocked by CsA, the Ca2+ uniporter inhibitor ruthenium red, the hydrophobic disulfide reagent N-ethylmaleimide, butacaine, and the free radical scavenging enzymes catalase and superoxide dismutase. MPP+ neurotoxicity may derive from not only its inhibition of complex I and consequent ATP depletion, but also from its ability to open the MTP and to release mitochondrial factors including Ca2+ and cytochrome c known to be involved in apoptosis.
Assuntos
1-Metil-4-fenilpiridínio/farmacologia , Grupo dos Citocromos c/metabolismo , Dopaminérgicos/farmacologia , Membranas Intracelulares/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , 1-Metil-4-fenilpiridínio/antagonistas & inibidores , 1-Metil-4-fenilpiridínio/metabolismo , Animais , Atractilosídeo/farmacologia , Encéfalo/efeitos dos fármacos , Ditiotreitol/farmacologia , Inibidores Enzimáticos/farmacologia , Glutationa/farmacologia , Masculino , Mitocôndrias/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Dilatação Mitocondrial , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , Oxirredução , Permeabilidade , Ratos , Ratos Sprague-DawleyRESUMO
The dopaminergic neurotoxin N-methyl,4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) causes a syndrome in primates and humans which mimics Parkinson's disease (PD) in clinical, pathological, and biochemical findings, including diminished activity of complex I in the mitochondrial electron transport chain. Reduced complex I activity is found in sporadic PD and can be transferred through mitochondrial DNA, suggesting a mitochondrial genetic etiology. We now show that MPTP treatment of mice and N-methylpyridinium (MPP+) exposure of human SH-SY5Y neuroblastoma cells increases oxygen free radical production and antioxidant enzyme activities. Cybrid cells created by transfer of PD mitochondria exhibit similar characteristics; however, PD cybrids' antioxidant enzyme activities are not further increased by MPP+ exposure, as are the activities in control cybrids. PD mitochondrial cybrids are subject to metabolic and oxidative stresses similar to MPTP parkinsonism and provide a model to determine mechanisms of oxidative damage and cell death in PD.
Assuntos
Antioxidantes/metabolismo , Encéfalo/enzimologia , Neurônios/metabolismo , Doença de Parkinson Secundária/metabolismo , Espécies Reativas de Oxigênio/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Animais , Encéfalo/metabolismo , Catalase/metabolismo , Modelos Animais de Doenças , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Humanos , Células Híbridas , Radical Hidroxila/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Neuroblastoma , Neurônios/enzimologia , Estresse Oxidativo , Doença de Parkinson Secundária/enzimologia , Superóxido Dismutase/metabolismo , Células Tumorais CultivadasRESUMO
The mitochondrial electron transport chain enzyme cytochrome c oxidase (COX) is defective in patients with sporadic Alzheimer's disease (AD). This defect arises from the mutation of mitochondrial DNA (mtDNA). To develop a tissue culture system that would express this genetically derived bioenergetic lesion and permit characterization of its functional consequences, we depleted Ntera2/D1 (NT2) teratocarcinoma cells of endogenous mtDNA and repopulated them with platelet mtDNA from AD patients. Cytochrome c oxidase activity was depressed in the resulting AD cytoplasmic hybrids (cybrids) compared with cybrids prepared with mtDNA from non-AD controls. Reactive oxygen species (ROS) production and free radical scavenging enzyme activities were significantly elevated in AD cybrids. A COX defect in NT2 AD cybrid lines indicates that AD patients possess mtDNA COX gene mutations that are sufficient for determining this biochemical lesion. Expression of unique functional characteristics (increased ROS production and free radical scavenging enzyme activities) relevant to neurodegeneration demonstrates the utility of these cells in defining AD pathophysiology at a cellular level. This in vitro tissue culture model of AD may prove useful in drug screening.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Encéfalo/patologia , Técnicas Genéticas , Citoplasma/fisiologia , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Técnicas de Transferência de Genes , Humanos , Hibridização Genética , Mutação , Espécies Reativas de Oxigênio/metabolismo , Valores de Referência , Teratocarcinoma/genética , Teratocarcinoma/metabolismo , Teratocarcinoma/patologia , Células Tumorais Cultivadas/metabolismoRESUMO
There is mounting evidence for mitochondrial involvement in neurodegenerative diseases including Alzheimer's and Parkinson's disease and amyotrophic lateral sclerosis. Mitochondrial DNA mutations, whether inherited or acquired, lead to impaired electron transport chain (ETC) functioning. Impaired electron transport, in turn, leads to decreased ATP production, formation of damaging free-radicals, and altered calcium handling. These toxic consequences of ETC dysfunction lead to further mitochondrial damage including oxidation of mitochondrial DNA, proteins, and lipids, and opening of the mitochondrial permeability transition pore, an event linked to cell death in numerous model systems. Although protective nuclear responses such as antioxidant enzymes and bcl-2 may be induced to combat these pathological changes, such a vicious cycle of increasing oxidative damage may insidiously damage neurons over a period of years, eventually leading to neuronal cell death. This hypothesis, a synthesis of the mitochondrial mutations and oxidative stress hypotheses of neurodegeneration, is readily tested experimentally, and clearly points out many potential therapeutic targets for preventing or ameliorating these diseases.
Assuntos
Núcleo Celular/fisiologia , Mitocôndrias/fisiologia , Mutação/fisiologia , Doenças Neurodegenerativas/fisiopatologia , Estresse Oxidativo/fisiologia , Animais , Morte Celular/fisiologia , Humanos , Mitocôndrias/metabolismoRESUMO
Sporadic Parkinson's disease is associated with a defect in the activity of complex I of the mitochondrial electron transport chain. This electron transport chain defect is transmitted through mitochondrial DNA, and when expressed in host cells leads to increased oxygen free radical production, increased antioxidant enzyme activities, and increased susceptibility to programmed cell death. Pramipexole, a chemically novel dopamine agonist used for the treatment of Parkinson's disease symptoms, possesses antioxidant activity and is neuroprotective toward substantia nigral dopamine neurons in hypoxic-ischemic and methamphetamine models. We found that pramipexole reduced the levels of oxygen radicals produced by methylpyridinium ion (MPP+) both when incubated with SH-SY5Y cells and when perfused into rat striatum. Pramipexole also exhibited a concentration-dependent inhibition of opening of the mitochondrial transition pore induced by calcium and phosphate or MPP+. These results suggest that pramipexole may be neuroprotective in Parkinson's disease by attenuating intracellular processes such as oxygen radical generation and the mitochondrial transition pore opening, which are associated with programmed cell death.
Assuntos
1-Metil-4-fenilpiridínio/farmacologia , Antiparkinsonianos/farmacologia , Dopaminérgicos/farmacologia , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tiazóis/farmacologia , Animais , Benzotiazóis , Transporte Biológico/efeitos dos fármacos , Cálcio/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Fígado/química , Masculino , Microdiálise , Mitocôndrias/química , Mitocôndrias/efeitos dos fármacos , Neuroblastoma , Fármacos Neuroprotetores/farmacologia , Neurotoxinas/farmacologia , Doença de Parkinson Secundária/tratamento farmacológico , Doença de Parkinson Secundária/metabolismo , Fósforo/farmacologia , Porinas/metabolismo , Pramipexol , Ratos , Ratos Sprague-Dawley , Células Tumorais CultivadasRESUMO
Alzheimer's disease (AD) brains exhibit oxidative stress and a biochemical defect of complex IV (cytochrome oxidase, COX) of the mitochondrial electron transport chain (ETC). This defect can be transferred through mitochondrial DNA (mtDNA) into clonal SY5Y cells depleted of their mtDNA. The resulting cytoplasmic hybrids or "cybrids" retain the complex IV defect and exhibit oxidative stress. We measured the mitochondrial membrane potential (delta psi m) in AD and control cybrids via H3-tetraphenylphosphonium ion (H3-TPP+) accumulation. AD cybrids exhibited a significant (about 30%) decrease in H3-TPP+ accumulation relative to controls. Acute treatment of normal SY5Ys with azide, a COX inhibitor, moderately decreased H3-TPP+ retention and strongly inhibited COX activity in a dose-dependent manner. As the mitochondrial transition pore (MTP) can be activated by reactive oxygen species and ETC inhibitors, and its opening causes delta psi m dissipation, we tested the effects of the MTP inhibitor cyclosporin A (CsA) on TPP+ accumulation. 5mM CsA increased basal H3-TPP+ accumulation in SY5Y cells about 10-fold, corresponding to about a 2-fold increase in delta psi m. In the AD cybrids, CsA increased the apparent delta psi m to the same final levels as it did in controls. These results indicate that low-conductance MTP activity contributes significantly to resting delta psi m in SY5Y cells. We propose the novel hypothesis that the COX defect and resulting oxidative stress in AD may pathologically activate the MTP, resulting in lower delta psi m and the release of mitochondrial factors involved in apoptosis.
Assuntos
Doença de Alzheimer/fisiopatologia , Ciclosporina/farmacologia , Membranas Intracelulares/fisiologia , Mitocôndrias/fisiologia , Doença de Alzheimer/genética , Atractilosídeo/farmacologia , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Membranas Intracelulares/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Monoaminoxidase/metabolismo , Oniocompostos/metabolismo , Compostos Organofosforados/metabolismo , Azida Sódica/farmacologia , Células Tumorais CultivadasRESUMO
Complex I activity is reduced in cytoplasmic hybrid (cybrid) cell lines that contain mitochondrial DNA (mtDNA) from sporadic Parkinson's disease (PD) patients. This implies that mtDNA aberration occurs in sporadic PD. To assess the integrity of mtDNA in autosomal dominant PD arising from mutation of the alpha-synuclein gene, we transferred mitochondrial genes from PD-affected members of the Italian-American Contursi kindred to cells previously depleted of their endogenous mtDNA. Unlike cybrid cell lines expressing mtDNA from persons with sporadic or maternally inherited PD, the resultant Contursi cybrid lines did not manifest complex I deficiency, indicating that in Contursi PD mtDNA integrity is relatively preserved. Compared to control cybrids, however, Contursi cybrid lines did show some evidence of oxidative stress. For reasons that are unclear, at least a limited amount of mtDNA damage may nevertheless develop in PD patients with alpha-synuclein mutation.
Assuntos
DNA Mitocondrial/genética , NADH NADPH Oxirredutases/genética , Doença de Parkinson/genética , Adulto , Idoso , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Catalase/metabolismo , DNA Mitocondrial/sangue , Transporte de Elétrons , Complexo I de Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Genes Dominantes , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Humanos , Células Híbridas/metabolismo , Masculino , Pessoa de Meia-Idade , NADH NADPH Oxirredutases/metabolismo , Neuroblastoma , Valores de Referência , Células Tumorais CultivadasRESUMO
Oxidative stress induced by acute complex I inhibition with 1-methyl-4-phenylpyridinium ion activated biphasically the stress-activated c-Jun N-terminal kinase (JNK) and the early transcription factor nuclear factor-kappaB (NF-kappaB) in SH-SY5Y neuroblastoma cells. Early JNK activation was dependent on mitochondrial adenine nucleotide translocator (ANT) activity, whereas late-phase JNK activation and the cleavage of signaling proteins Raf-1 and mitogen-activated protein kinase (MAPK) kinase (MEK) kinase (MEKK)-1 appeared to be ANT-independent. Early NF-kappaB activation depended on MEK, later activation required an intact electron transport chain (ETC), and Parkinson's disease (PD) cybrid (mitochondrial transgenic cytoplasmic hybrid) cells had increased basal NF-kappaB activation. Mitochondria appear capable of signaling ETC impairment through MAPK modules and inducing protective NF-kappaB responses, which are increased by PD mitochondrial genes amplified in cybrid cells. Irreversible commitment to apoptosis in this cell model may derive from loss of Raf-1 and cleavage/activation of MEKK-1, processes reported in other models to be caspase-mediated. Therapeutic strategies that reduce mitochondrial activation of proapoptotic MAPK modules, i.e., JNK, and enhance survival pathways, i.e., NF-kappaB, may offer neuroprotection in this debilitating disease.
Assuntos
MAP Quinase Quinase Quinase 1 , Mitocôndrias/enzimologia , NF-kappa B/metabolismo , Neurônios/enzimologia , Doença de Parkinson/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , 1-Metil-4-fenilpiridínio/farmacologia , Nucleotídeos de Adenina/metabolismo , Benzotiazóis , Transporte de Elétrons , Ativação Enzimática/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Herbicidas/farmacologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/antagonistas & inibidores , Neuroblastoma , Neurônios/química , Neurônios/citologia , Estresse Oxidativo/fisiologia , Peptídeos/farmacologia , Pramipexol , Proteínas Serina-Treonina Quinases/análise , Proteínas Proto-Oncogênicas c-raf/análise , Proteínas Proto-Oncogênicas c-raf/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Superóxido Dismutase/metabolismo , Tiazóis/farmacologia , Células Tumorais CultivadasRESUMO
Electron transport chain (ETC) dysfunction may arise from mitochondrial genetic, nuclear genetic, or toxic etiologies. Cytoplasmic hybrid (cybrid) systems can help distinguish between these possibilities by facilitating expression of suspect mitochondrial DNA (mtDNA) within a nuclear and environmentally controlled context. Perpetuation of ETC dysfunction in cybrids is consistent with an mtDNA pathogenesis while defect correction is not. We previously used cybrids to screen sporadic Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis patients for mtDNA mutation with positive results. To further address the fidelity of these experiments, we created and characterized cybrids expressing mtDNA from persons with Huntington's disease (HD), an autosomal dominant, nuclear DNA-determined disorder in which mitochondrial ETC functioning is abnormal. On ETC, oxidative stress, and calcium homeostasis assays HD cybrid lines were indistinguishable from control cybrid lines. These data support the use of the cybrid technique for mtDNA mutation screening in candidate diseases.
Assuntos
DNA Mitocondrial/genética , Doença de Huntington/genética , Células Híbridas/ultraestrutura , Mitocôndrias/metabolismo , Mutação , Adulto , Cálcio/metabolismo , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Citoplasma/ultraestrutura , DNA Mitocondrial/análise , Transporte de Elétrons , Expressão Gênica , Humanos , Células Híbridas/metabolismo , Pessoa de Meia-Idade , Quinona Redutases , Desacopladores/farmacologiaRESUMO
Progressive supranuclear palsy (PSP) is a neurodegenerative movement disorder of unknown etiology. We hypothesized that mitochondrial DNA (mtDNA) aberration could occur in this disease and contribute to its pathogenesis. To address this we created transmitochondrial cytoplasmic hybrid (cybrid) cell lines expressing mitochondrial genes from persons with PSP. The presence of cybrid mtDNA aberration was screened for by biochemical assay of mitochondrial gene products. Relative to a control cybrid set, complex I activity was reduced in PSP cybrid lines (p<0.005). Antioxidant enzyme activities were elevated in PSP cybrid lines. These data suggest that mtDNA aberration occurs in PSP, causes electron transport chain pathology, and can produce oxidative stress. Further study of mitochondrial dysfunction in PSP may yield insights into why neurodegeneration occurs in this disease.
Assuntos
DNA Mitocondrial/genética , Células Híbridas/metabolismo , Mitocôndrias/enzimologia , Mitocôndrias/genética , Paralisia Supranuclear Progressiva/diagnóstico , Paralisia Supranuclear Progressiva/genética , Idoso , Antioxidantes/metabolismo , Plaquetas/citologia , Catalase/metabolismo , Fusão Celular , Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Humanos , Células Híbridas/citologia , Masculino , NADH NADPH Oxirredutases/metabolismo , Estresse Oxidativo/genética , Superóxido Dismutase/metabolismo , Paralisia Supranuclear Progressiva/etiologia , Células Tumorais CultivadasRESUMO
Alzheimer's disease (AD) is characterized by the deposition in brain of beta-amyloid (Abeta) peptides, elevated brain caspase-3, and systemic deficiency of cytochrome c oxidase. Although increased Abeta deposition can result from mutations in amyloid precursor protein or presenilin genes, the cause of increased Abeta deposition in sporadic AD is unknown. Cytoplasmic hybrid ("cybrid") cells made from mitochondrial DNA of nonfamilial AD subjects show antioxidant-reversible lowering of mitochondrial membrane potential (delta(gYm), secrete twice as much Abeta(1-40) and Abeta(1-42), have increased intracellular Abeta(1-40) (1.7-fold), and develop Congo red-positive Abeta deposits. Also elevated are cytoplasmic cytochrome c (threefold) and caspase-3 activity (twofold). Increased AD cybrid Abeta(1-40) secretion was normalized by inhibition of caspase-3 or secretase and reduced by treatment with the antioxidant S(-)pramipexole. Expression of AD mitochondrial genes in cybrid cells depresses cytochrome c oxidase activity and increases oxidative stress, which, in turn, lowers delta(psi)m. Under stress, cells with AD mitochondrial genes are more likely to activate cell death pathways, which drive caspase 3-mediated Abeta peptide secretion and may account for increased Abeta deposition in the AD brain. Therapeutic strategies for reducing neurodegeneration in sporadic AD can address restoration of delta(psi)m and reduction of elevated Abeta secretion.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/metabolismo , Morte Celular/fisiologia , Células Híbridas/metabolismo , DNA Mitocondrial/metabolismo , Humanos , Imuno-Histoquímica , Neuroblastoma , Células Tumorais CultivadasRESUMO
Recent data suggesting complex I dysfunction in Parkinson's disease (PD) arises from mitochondrial DNA (mtDNA) mutation does not conclusively answer whether the responsible genetic lesion is inherited (primary) or somatic (secondary). To address this question, we identified a family in which multiple members over three generations are affected with PD through exclusively maternal lines. Cytoplasmic hybrids (cybrids) were created for 15 family members over two generations by transferring each individual's mtDNA to mtDNA-depleted human neuroblastoma cells. Eight of the 15 cybrid lines contained mtDNA obtained from maternally descended family members and seven contained mtDNA from paternally descended family members. After 6 weeks of culture, cybrid cell lines were assayed for complex I activity and oxidative stress, and mitochondrial morphology was analyzed by electron microscopy. Compared with the cybrid lines containing mtDNA from paternal descendants, cybrid lines containing mtDNA from maternal descendants had lower complex I activity, increased reactive oxygen species production, increased radical scavenging enzyme activities, and more abnormal mitochondrial morphologic features. These findings were present in cybrid lines containing mtDNA from maternal descendants with PD as well as in currently asymptomatic young maternal descendants, and support a precedent for inherited mtDNA mutation in some persons with PD.
Assuntos
NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Adulto , DNA Mitocondrial/genética , Feminino , Sequestradores de Radicais Livres/metabolismo , Humanos , Células Híbridas , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Mitocôndrias/ultraestrutura , Mutação/genética , Estresse Oxidativo/fisiologia , Doença de Parkinson/patologia , Linhagem , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais CultivadasRESUMO
Mitochondria are abnormal in persons with amyotrophic lateral sclerosis (ALS) for unknown reasons. We explored whether aberration of mitochondrial DNA (mtDNA) could play a role in this by transferring mitochondrial DNA (mtDNA) from ALS subjects to mtDNA-depleted human neuroblastoma cells. Resulting ALS cytoplasmic hybrids (cybrids) exhibited abnormal electron transport chain functioning, increases in free radical scavenging enzyme activities, perturbed calcium homeostasis, and altered mitochondrial ultrastructure. Recapitulation of defects previously observed in ALS subjects and ALS transgenic mice by expression of ALS mtDNA support a pathophysiologic role for mtDNA mutation in some persons with this disease.