RESUMO
Parkinson's disease (PD) is a neurodegenerative disease which results in damage in neuronal cells. Insulin-like growth factor (IGF)-1 was previously reported to play a role of neuroprotection in some diseases. Nitric oxide (NO) can also regulate neuronal cells. However, the mechanisms underlying IGF-1 and NO in PD still need to be elucidated. In present study, we explored the interaction between IGF-1 and inducible Nitric-Oxide Synthase (iNOS) in PD progression. We firstly constructed PD models by methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or MPP+ treatment. Then RT-qPCR revealed that IGF-1 expression was downregulated while iNOS expression was upregulated in MPTP model. Moreover, IGF-1 elevation or iNOS depletion enhanced cell viability and blocked cell apoptosis. Rescue assay disclosed iNOS overexpression reversed the effect on viability and apoptosis mediated by IGF-1 upregulation. Furthermore, IGF-1 was identified to positively regulate miR-302b-5p which could target iNOS. MiR-302b-5p could abolish the inhibitory function IGF-1 exerted on cell apoptosis and iNOS could counteract miR-302b-5p upregulation-triggered inhibition on cell apoptosis as well. Besides, we observed the deficiency of miR-302b-5p improved the lesioned neurobehavior of MPTP-treated mice. To sum up, present study proved that miR-302b-5p enhanced the neuroprotective effect of IGF-1 in MPTP-induced PD by regulating iNOS, recommending a novel therapeutic target for PD treatment. SIGNIFICANCE OF THE STUDY: In this study, we mainly explored that IGF-1 was decreased while iNOS was boosted in MPTP-induced PD mice model; IGF-1 suppressed while iNOS promoted MPP+ -induced toxicity and apoptosis in SH-SY5Y cells; miR-302b-5p ehanhced the neuroprotective effect of IGF-1 via targeting Inos; deficiency of miR-302b-5p improved the lesioned neurobehavior of MPTP-treated mice.
Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/análogos & derivados , Fator de Crescimento Insulin-Like I/metabolismo , MicroRNAs/metabolismo , Fármacos Neuroprotetores/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Doença de Parkinson Secundária/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/administração & dosagem , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Animais , Linhagem Celular Tumoral , Humanos , Fator de Crescimento Insulin-Like I/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Óxido Nítrico Sintase Tipo II/genética , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/genéticaRESUMO
Ever since its introduction 40 years ago l-3,4-dihydroxyphenylalanine (l-DOPA) therapy has retained its role as the leading standard medication for patients with Parkinson's disease. With time, however, the shortcomings of oral l-DOPA treatment have become apparent, particularly the motor fluctuations and troublesome dyskinetic side effects. These side effects, which are caused by the excessive swings in striatal dopamine caused by intermittent oral delivery, can be avoided by delivering l-DOPA in a more continuous manner. Local gene delivery of the l-DOPA synthesizing enzymes, tyrosine hydroxylase and guanosine-tri-phosphate-cyclohydrolase-1, offers a new approach to a more refined dopaminergic therapy where l-DOPA is delivered continuously at the site where it is needed i.e. the striatum. In this study we have explored the therapeutic efficacy of adeno-associated viral vector-mediated l-DOPA delivery to the putamen in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated rhesus monkeys, the standard non-human primate model of Parkinson's disease. Viral vector delivery of the two enzymes, tyrosine hydroxylase and guanosine-5'-tri-phosphate-cyclohydrolase-1, bilaterally into the dopamine-depleted putamen, induced a significant, dose-dependent improvement of motor behaviour up to a level identical to that obtained with the optimal dose of peripheral l-DOPA. Importantly, this improvement in motor function was obtained without any adverse dyskinetic effects. These results provide proof-of-principle for continuous vector-mediated l-DOPA synthesis as a novel therapeutic strategy for Parkinson's disease. The constant, local supply of l-DOPA obtained with this approach holds promise as an efficient one-time treatment that can provide long-lasting clinical improvement and at the same time prevent the appearance of motor fluctuations and dyskinetic side effects associated with standard oral dopaminergic medication.
Assuntos
Antiparkinsonianos/administração & dosagem , GTP Cicloidrolase/administração & dosagem , Vetores Genéticos/uso terapêutico , Levodopa/biossíntese , Transtornos Parkinsonianos/terapia , Putamen/metabolismo , Tirosina 3-Mono-Oxigenase/administração & dosagem , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/efeitos adversos , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/análogos & derivados , Animais , Antiparkinsonianos/uso terapêutico , Dependovirus/genética , Avaliação Pré-Clínica de Medicamentos , Feminino , GTP Cicloidrolase/análise , GTP Cicloidrolase/genética , GTP Cicloidrolase/metabolismo , Genes Reporter , Genes Sintéticos , Vetores Genéticos/administração & dosagem , Humanos , Macaca mulatta , Masculino , Atividade Motora/efeitos dos fármacos , Transtornos Parkinsonianos/induzido quimicamente , Parte Compacta da Substância Negra/química , Parte Compacta da Substância Negra/patologia , Estudo de Prova de Conceito , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/análise , Proteínas Recombinantes/uso terapêutico , Tirosina 3-Mono-Oxigenase/análise , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Malignant gliomas, including glioblastomas, are extremely difficult to treat. The median survival for glioblastoma patients with optimal therapeutic intervention is 15 months. We developed a novel MAO-B-selectively activated prodrug, N,N-bis(2-chloroethyl)-2-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)propanamide (MP-MUS), for the treatment of gliomas based on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The design of neutral MP-MUS involved the use of a seeker molecule capable of binding to mitochondrial MAO-B, which is up-regulated ≥fourfold in glioma cells. Once the binding occurs, MP-MUS is converted into a positively charged moiety, P(+) -MUS, which accumulates inside mitochondria at a theoretical maximal value of 1000:1 gradient. The LD50 of MP-MUS against glioma cells is 75 µM, which is two- to threefold more potent than temozolomide, a primary drug for gliomas. Importantly, MP-MUS was found to be selectively toxic toward glioma cells. In the concentration range of 150-180 µM MP-MUS killed 90-95 % of glioma cells, but stimulated the growth of normal human astrocytes. Moreover, maturation of MP-MUS is highly dependent on MAO-B, and inhibition of MAO-B activity with selegiline protected human glioma cells from apoptosis.
Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/análogos & derivados , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Glioma/tratamento farmacológico , Pró-Fármacos/química , Pró-Fármacos/farmacologia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/administração & dosagem , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/metabolismo , Antineoplásicos/administração & dosagem , Antineoplásicos/metabolismo , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Glioma/metabolismo , Glioma/patologia , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Monoaminoxidase/metabolismo , Inibidores da Monoaminoxidase/farmacologia , Pró-Fármacos/administração & dosagem , Pró-Fármacos/metabolismo , Selegilina/farmacologiaRESUMO
Endoplasmic reticulum (ER) stress has been implicated in Parkinson disease. We previously reported that thioredoxin 1 (Trx-1) suppressed the ER stress caused by 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine; however, its molecular mechanism remains largely unknown. In the present study, we showed that 1-methyl-4-phenylpyridinium ion (MPP(+)) induced ER stress by activating glucose-regulated protein 78 (GRP78), inositol-requiring enzyme 1α (IRE1α), tumor necrosis factor receptor-associated factor 2 (TRAF2), c-Jun N-terminal kinase (JNK), caspase-12, and C/EBP homologous protein (CHOP) in PC12 cells. The downregulation of Trx-1 aggravated the ER stress and further increased the expression of the above molecules induced by MPP(+). In contrast, overexpression of Trx-1 attenuated the ER stress and repressed the expression of the above molecules induced by MPP(+). More importantly, the overexpression of Trx-1 in transgenic mice suppressed ER stress by inhibiting the activation of these molecules. We present, for the first time, the molecular mechanism of Trx-1 suppression of endoplasmic reticulum stress in Parkinson disease in vitro and in vivo. Based on our findings, we conclude that Trx-1 plays a neuroprotective role in Parkinson disease by suppressing ER stress by regulating the activation of GRP78, IRE1α, TRAF2, JNK, caspase-12, and CHOP.
Assuntos
Estresse do Retículo Endoplasmático/genética , Intoxicação por MPTP/genética , Tiorredoxinas/genética , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/análogos & derivados , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , 1-Metil-4-fenilpiridínio/farmacologia , Animais , Caspase 12/genética , Caspase 12/metabolismo , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endorribonucleases/genética , Endorribonucleases/metabolismo , Regulação da Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Intoxicação por MPTP/induzido quimicamente , Intoxicação por MPTP/metabolismo , Intoxicação por MPTP/fisiopatologia , Camundongos , Camundongos Transgênicos , Células PC12 , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Transdução de Sinais , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Tiorredoxinas/metabolismo , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismoRESUMO
AIM OF THE STUDY: Polygalae radix, the root of Polygala tenuifolia Willd, has commonly been used for the treatment of amnesia and anxiety in traditional Korean medicine. The aim of this study was to investigate its neuroprotective effects and possible mechanisms of action in models of Parkinson's disease. MATERIALS AND METHODS: This study utilized a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, a reactive oxygen species (ROS) assay, a nitric oxide (NO) production assay, and a caspase-3 activity test as measures of cell viability in PC12 cells damaged by 6-hydroxydopamine (6-OHDA). The protective effects of PRE against 1-methyl-4-phenylpyridium (MPP(+))-induced neurotoxicity were assessed in rat primary dopaminergic neurons and in a mouse PD model in which PRE was administered (100mg/kg/day, 3 days, p.o.) before acute 1-mehtyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity. Finally, TH immunohistochemistry tests were conducted in the substantia nigra pars compacta (SNpc) and striatum (ST). RESULTS AND CONCLUSIONS: PRE significantly inhibited 6-OHDA-induced cell damage at doses of 0.05-1µg/ml with a maximal effect at 0.1µg/ml. Caspase-3 activity and the production of ROS and NO were alleviated at 0.1µg/ml. Also at this dose, PRE protected mesencephalic dopaminergic neurons from MPP(+)-induced toxicity. In an in vivo mouse model of PD, PRE protected dopaminergic neurons and fibers from MPTP-induced toxicity in the SNpc and ST. These results demonstrate that PRE has protective effects on dopaminergic neurons via its anti-oxidant and anti-apoptotic activity.
Assuntos
Antioxidantes/uso terapêutico , Encéfalo/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Doença de Parkinson/tratamento farmacológico , Fitoterapia , Polygala , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/análogos & derivados , Animais , Antioxidantes/farmacologia , Encéfalo/citologia , Encéfalo/metabolismo , Caspase 3/metabolismo , Modelos Animais de Doenças , Dopamina/metabolismo , Masculino , Medicina Tradicional Coreana , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Oxidopamina , Células PC12 , Doença de Parkinson/metabolismo , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Raízes de Plantas , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Previous reports have suggested that the herbal medicine Chunghyuldan (CHD, Qingxue-dan in Chinese and Daio-Orengedokuto in Japanese) has wide-ranging biological effects, including anti-hyperlipidaemic, anti-ischaemic, anti-inflammatory and antioxidant activities. Reactive oxygen species (ROS)-mediated mitochondrial dysfunction is thought to be one of the major pathological mechanisms responsible for Parkinson's disease (PD) and may underlie the selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) that is a hallmark of this disease. In this study, we examined the neuroprotective effects of CHD in PD models produced by treatment with neurotoxins that act via ROS-mediated mitochondrial dysfunction. In an in vitro PD model using 6-hydroxydopamine, CHD applied at concentrations of 10 and 100 µg/ml exhibited significant protective effects in PC12 cells by inhibiting intracellular ROS generation. CHD applied at 10 and 100 µg/ml also prevented 6-hydroxydopamine-induced mitochondrial depolarization and elevation of caspase-3 activity. At the same doses, CHD showed regulatory effects on the haem oxygenase-1 and gp91 phagocytic oxidase which have critical roles in generating ROS. In addition, CHD protected dopaminergic neurons in a primary mesencephalic culture against MPP+ neurotoxicity. In an in vivo PD model produced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment (20 mg/kg, 4 times, i.p.), co-administration of CHD (50 mg/kg, 5 days, p.o.) ameliorated PD-like behavioural symptoms (bradykinesia) and reduced dopaminergic neuronal damage in the SNpc and striatum as measured by immunocytochemistry. These results demonstrate the neuroprotective effects of CHD in PD models that are mediated through inhibition of ROS generation and associated mitochondrial dysfunction.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Doença de Parkinson/prevenção & controle , Espécies Reativas de Oxigênio/toxicidade , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/análogos & derivados , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Caspase 3/metabolismo , Morte Celular , Células Cultivadas , Modelos Animais de Doenças , Dopamina/metabolismo , Masculino , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/patologia , Neurotoxinas/metabolismo , Oxidopamina/farmacologia , Células PC12 , Doença de Parkinson/patologia , Ratos , Ratos Sprague-Dawley , Substância Negra/patologiaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: A traditional herb, Yi-Gan San, has been widely used for the management of neurodegenerative disorders in traditional East Asian Medicine. AIM OF THE STUDY: The present study investigated the neuroprotective effects of Yi-Gan San in 1-methyl-4-phenylpyridine/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced cytotoxicity in vitro and in vivo and sought to clarify its underlying mechanisms. MATERIALS AND METHODS: The effect of Yi-Gan San on 1-methyl-4-phenylpyridine was measured in terms of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays, caspase-3 activity, and western blot analysis of phosphorylated Akt, one of the survival-related signaling proteins in SH-SY5Y cells. The effects of Yi-Gan San were also confirmed in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonian mouse model using a rotarod test and tyrosine hydroxylase-immunohistochemistry. RESULTS: Pretreatment of Yi-Gan San with 1-methyl-4-phenylpyridine showed a significant protective effect on SH-SY5Y cells and significantly decreased the level of caspase-3 activity compared to the values for the 1-methyl-4-phenylpyridine-treated cells. This process increased the protein expressions of phosphorylated Akt, and an inhibitor of phosphatidylinositol 3-kinase (PI3K)/Akt, LY294002, significantly decreased this protective effect of Yi-Gan San. In the mouse Parkinson's disease model, treatment with Yi-Gan San also significantly improved motor functioning and prevented dopaminergic loss related to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine challenge. CONCLUSION: Using both in vitro and in vivo methods, this study revealed that Yi-Gan San has neuroprotective effects and rescues dopaminergic neurons from 1-methyl-4-phenylpyridine/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity, possibly via the PI3K/Akt pathway.
Assuntos
Caspase 3/metabolismo , Medicamentos de Ervas Chinesas/uso terapêutico , Magnoliopsida , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Doença de Parkinson/tratamento farmacológico , Fitoterapia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/análogos & derivados , 1-Metil-4-fenilpiridínio , Animais , Linhagem Celular Tumoral , Cromonas/farmacologia , Citotoxinas , Modelos Animais de Doenças , Dopamina/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Morfolinas/farmacologia , Atividade Motora/efeitos dos fármacos , Neuroblastoma/metabolismo , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Doença de Parkinson/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Monocrotaline (MCT) causes pulmonary hypertension in the rat by a mechanism characterized by megalocytosis (enlarged cells with enlarged endoplasmic reticulum and Golgi and a cell cycle arrest) of pulmonary arterial endothelial (PAEC), arterial smooth muscle, and type II alveolar epithelial cells. In cell culture, although megalocytosis is associated with a block in entry into mitosis in both lung endothelial and epithelial cells, DNA synthesis is stimulated in endothelial but inhibited in epithelial cells. The molecular mechanism(s) for this dichotomy are unclear. While MCTP-treated PAEC and lung epithelial (A549) cells both showed an increase in the "promitogenic" transcription factor STAT3 levels and in the IL-6-induced nuclear pool of PY-STAT3, this was transcriptionally inactive in A549 but not in PAEC cells. This lack of transcriptional activity of STAT3 in A549 cells correlated with the cytoplasmic sequestration of the STAT3 coactivators CBP/p300 and SRC1/NcoA in A549 cells but not in PAEC. Both cell types displayed a Golgi trafficking block, loss of caveolin-1 rafts, and increased nuclear Ire1alpha, but an incomplete unfolded protein response (UPR) with little change in levels of UPR-induced chaperones including GRP78/BiP. There were discordant alterations in cell cycle regulatory proteins in the two cell types such as increase in levels of both cyclin D1 and p21 simultaneously, but with a decrease in cdc2/cdk1, a kinase required for entry into mitosis. While both cell types showed increased cytoplasmic geminin, the DNA synthesis-initiating protein Cdt1 was predominantly nuclear in PAEC but remained cytoplasmic in A549 cells, consistent with the stimulation of DNA synthesis in the former but an inhibition in the latter cell type. Thus differences in cell type-specific alterations in subcellular trafficking of critical regulatory molecules (such as CBP/p300, SRC1/NcoA, Cdt1) likely account for the dichotomy of the effects of MCTP on DNA synthesis in endothelial and epithelial cells.
Assuntos
Monocrotalina/farmacologia , Artéria Pulmonar/fisiologia , Mucosa Respiratória/citologia , Mucosa Respiratória/fisiologia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/análogos & derivados , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Separação Celular , Chaperona BiP do Retículo Endoplasmático , Humanos , Interleucina-6/farmacologia , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/fisiologia , Artéria Pulmonar/efeitos dos fármacos , Mucosa Respiratória/efeitos dos fármacos , Fator de Transcrição STAT3/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
The autoinhibitory control of electrically evoked release of [3H]-dopamine and the properties of that induced by nicotinic receptor (nAChR) stimulation were studied in slices of the human neocortex. In both models [3H]-dopamine release was action potential-induced and exocytotic. The selective dopamine D2 receptor agonist (-)-quinpirole reduced electrically evoked release of [3H]-dopamine, yielding IC50 and I(max) values of 23 nM and 76%, respectively. Also, the effects of several other subtype-selective dopamine receptor ligands confirmed that the terminal dopamine autoreceptor belongs to the D2 subtype. The autoinhibitory feedback control was slightly operative under stimulation conditions of 90 pulses and 3 Hz, with a biophase concentration of endogenous dopamine of 3.6 nM, and was enhanced under blockade of dopamine reuptake. [3H]-dopamine release evoked in an identical manner in mouse neocortical slices was not inhibited by (-)-quinpirole, suggesting the absence of dopamine autoreceptors in this tissue and underlining an important species difference. Also, nAChR stimulation-induced release of [3H]-dopamine revealed a species difference: [3H]-dopamine release was evoked in human, but not in rat neocortical slices. The nAChRs inducing [3H]-dopamine release most probably belong to the alpha3/beta2subtype, according to the potencies and efficacies of subtype-selective nAChR ligands. Part of these receptors may be located on glutamatergic neurons.
Assuntos
Autorreceptores/fisiologia , Dopamina/metabolismo , Neocórtex/metabolismo , Receptores Nicotínicos/fisiologia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/análogos & derivados , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Adolescente , Adulto , Idoso , Alcaloides/farmacologia , Análise de Variância , Animais , Azocinas/farmacologia , Cálcio/farmacologia , Criança , Pré-Escolar , Domperidona/farmacologia , Antagonistas de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Estimulação Elétrica/métodos , Retroalimentação/efeitos dos fármacos , Feminino , Fluvoxamina/farmacologia , Humanos , Técnicas In Vitro , Isoxazóis/farmacologia , Masculino , Maprotilina/análogos & derivados , Maprotilina/farmacologia , Camundongos , Pessoa de Meia-Idade , Neocórtex/efeitos dos fármacos , Neocórtex/efeitos da radiação , Nicotina/farmacologia , Antagonistas Nicotínicos/farmacologia , Potássio/farmacologia , Pirrolidinas/farmacologia , Quinolizinas/farmacologia , Ratos , Sulpirida/farmacologia , Fatores de Tempo , Trítio/metabolismoRESUMO
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is known to induce parkinsonism in humans when it is oxidized to the 1-methyl-4-phenylpyridinium salt (MPP+). We previously reported the syntheses of 1-amino-4-phenyl-1,2,3,6-tetrahydropyridine (APTP) and 1-amino-4-phenyl-pyridinium salt (APP+), the 1-amino analogues of the dopaminergic neurotoxins, MPTP and MPP+, respectively, and demonstrated that both APTP and APP+ are cytotoxic to PC12 cells. In this study, we found that both APTP and APP+ induce apoptotic cell death in PC12 cells. Apoptosis was determined by the Comet assay and flow cytometric analysis. Prior to using the Comet assay for detection of apoptotic PC12 cells, Comet images of apoptotic and necrotic cells were first distinguished by using several standards. Comet images were classified into four groups (A to D) according to their shapes. Class D consisted of the apoptotic cells and was easily distinguished. We also demonstrated that apoptotic and necrotic PC12 cells can be easily differentiated and quantified using the convenient Comet assay.
Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/análogos & derivados , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , 1-Metil-4-fenilpiridínio/farmacologia , Apoptose/efeitos dos fármacos , Dano ao DNA , Células PC12/efeitos dos fármacos , 1-Metil-4-fenilpiridínio/análogos & derivados , Animais , Ensaio Cometa , Fragmentação do DNA , DNA de Neoplasias/análise , DNA de Neoplasias/efeitos dos fármacos , Citometria de Fluxo , Células PC12/patologia , Compostos de Piridínio , RatosAssuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Biologia Molecular/métodos , Proteínas Quinases/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/química , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/metabolismo , Células 3T3 , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Animais , Humanos , Camundongos , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Fosforilação , Proteínas Quinases/genética , Alinhamento de Sequência , Transdução de Sinais/fisiologia , Especificidade por SubstratoAssuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/análogos & derivados , Hipóxia-Isquemia Encefálica/cirurgia , Chaperonas Moleculares/metabolismo , Regeneração Nervosa/fisiologia , Transtornos Parkinsonianos/fisiopatologia , Transplante de Células-Tronco , Substância Negra/fisiopatologia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/administração & dosagem , Animais , Materiais Biocompatíveis , Sobrevivência Celular , Dextroanfetamina/farmacologia , Feminino , Hipóxia-Isquemia Encefálica/complicações , Hipóxia-Isquemia Encefálica/patologia , Intoxicação por MPTP/patologia , Intoxicação por MPTP/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Neural/etiologia , Degeneração Neural/patologia , Degeneração Neural/cirurgia , Vias Neurais/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/patologia , Transtornos Parkinsonianos/cirurgia , Ácido Poliglicólico , Próteses e Implantes , Recuperação de Função Fisiológica , Transdução de Sinais , Células-Tronco/efeitos dos fármacos , Células-Tronco/patologia , Células-Tronco/fisiologia , Substância Negra/efeitos dos fármacos , Substância Negra/patologia , Substância Negra/cirurgia , Têxteis , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
We investigated the hypothesis that neural stem cells (NSCs) possess an intrinsic capacity to "rescue" dysfunctional neurons in the brains of aged mice. The study focused on a neuronal cell type with stereotypical projections that is commonly compromised in the aged brain-the dopaminergic (DA) neuron. Unilateral implantation of murine NSCs into the midbrains of aged mice, in which the presence of stably impaired but nonapoptotic DA neurons was increased by treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), was associated with bilateral reconstitution of the mesostriatal system. Functional assays paralleled the spatiotemporal recovery of tyrosine hydroxylase (TH) and dopamine transporter (DAT) activity, which, in turn, mirrored the spatiotemporal distribution of donor-derived cells. Although spontaneous conversion of donor NSCs to TH(+) cells contributed to nigral reconstitution in DA-depleted areas, the majority of DA neurons in the mesostriatal system were "rescued" host cells. Undifferentiated donor progenitors spontaneously expressing neuroprotective substances provided a plausible molecular basis for this finding. These observations suggest that host structures may benefit not only from NSC-derived replacement of lost neurons but also from the "chaperone" effect of some NSC-derived progeny.
Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/análogos & derivados , Regeneração Nervosa/fisiologia , Neurônios/patologia , Transtornos Parkinsonianos/patologia , Transtornos Parkinsonianos/fisiopatologia , Transplante de Células-Tronco , Substância Negra/fisiopatologia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/administração & dosagem , Envelhecimento , Animais , Contagem de Células , Sobrevivência Celular , Dextroanfetamina/farmacologia , Feminino , Intoxicação por MPTP/patologia , Intoxicação por MPTP/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/cirurgia , Recuperação de Função Fisiológica , Valores de Referência , Transdução de Sinais , Células-Tronco/efeitos dos fármacos , Células-Tronco/patologia , Células-Tronco/fisiologia , Substância Negra/efeitos dos fármacos , Substância Negra/patologia , Substância Negra/cirurgia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
A 1-amino analog of MPTP, 1(N)-amino-4-phenyl-1,2,3,6-tetrahydropyridine, was synthesized and the oxidation was examined using human synaptosomal mitochondria as sources of type A and B monoamine oxidase. An oxidation product, 1-amino-4-phenylpyridinium ion, was quantified by high-performance liquid chromatography-fluorometric detection. The amino analog was a substrate of both type A and B monoamine oxidase and the oxidation depended linearly on the enzyme amount and the reaction time with an optimal pH around 7.5. After the systemic injection of the amino analog in C57/black mice for one week, 1-amino-4-phenylpyridinium ion was detected in the brain. 1(N)-Amino-4-phenyl-1,2,3,6-tetrahydropyridine was proved to be cytotoxic to pheochromocytoma PC12 cells, and it may be a new neurotoxin bioactivated through the oxidation by type A and B monoamine oxidase.
Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/análogos & derivados , Monoaminoxidase/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/metabolismo , Animais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Inibidores da Monoaminoxidase/farmacologia , Oxirredução , Células PC12 , Ratos , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/enzimologia , Sinaptossomos/metabolismoRESUMO
Oxidative stress is thought to play a key role in the apoptotic death of several cellular systems, including neurons. Oxidative stress is proposed also as a mechanism of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- and manganese (Mn)-induced neuronal death. We have recently shown that Mn and the MPTP analogue 1-methyl-4-(2'-ethylphenyl)-1,2,3,6-tetrahydropyridine (2'Et-MPTP), which is metabolized by MAO-A to 1-methyl-4-(2'-ethylphenyl)-pyridinium ion, induce apoptosis in PC12 cells. In the present study, we evaluated the effects of deprenyl and the antioxidant drugs N-acetylcysteine (NAC) and ascorbic acid (AA) on Mn- and 2'Et-MPTP-induced apoptosis in PC12 cells. Apoptosis was tested by terminal deoxynucleotidyl transferase-mediated 2'-deoxy-uridine-5'-triphosphate nick end labelling (TUNEL) technique, flow cytometry and fluorescence microscopy. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Mn-induced apoptosis and decrease in cell viability was inhibited by the antioxidants NAC and AA. Deprenyl failed to inhibit the above Mn effects. Neither NAC, AA nor deprenyl were able to inhibit both 2'Et-MPTP-induced apoptosis and decrease in cell viability. These results confirm that apoptosis may be an important mechanism of cell death in MPTP- and Mn-induced parkinsonism. However, an oxidative stress mechanism may be recognized, at least in vitro, only in the Mn-induced apoptosis.
Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/análogos & derivados , Apoptose/fisiologia , Manganês/farmacologia , Estresse Oxidativo , Células PC12/efeitos dos fármacos , Células PC12/fisiologia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Acetilcisteína/farmacologia , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Ácido Ascórbico/farmacologia , Sobrevivência Celular/efeitos dos fármacos , RatosRESUMO
The possibility of imaging monoamine oxidase (MAO) containing neurons through the MAO-mediated conversion of the nonfluorescent tetrahydropyridine compound trans-1-methyl-4-[4-dimethylaminophenylethenyl]-1,2,3,6-tetrahydro pyridine (t-THP) to the corresponding fluorescent trans-1-methyl-4-[4-dimethylaminophenylethenyl]pyridinium species (t-P+) was examined with the aid of human neuroblastoma cells (SH-SY5Y). Fluorescence microscopy and fluorescence measurements established the intracellular formation of a fluorescent species with maximal excitation/emission wavelengths of 485/620 and 530/620 nm corresponding to the fluorescence characteristics of synthetic t-P+. An independent assay confirmed the presence of both MAO-A and MAO-B in these cells. As expected, the development of the fluorescence was inhibited by both clorgyline (an MAO-A inhibitor) and deprenyl (an MAO-B inhibitor). Cytotoxic effects, as determined by trypan blue dye exclusion for viability and by the MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] assay for mitochondrial integrity, were not observed in cells incubated with concentrations of t-THP as high as 10(-3) M for 4 hr. The results from these studies with a neuronal cell line of human origin suggest: (1) that SH-SY5Y cells metabolize and, therefore, can be used for study of tetrahydropyridine compounds in vitro, and (2) that t-THP may be a useful agent to monitor neurodegenerative processes in MAO-rich neurons, including the dopaminergic nigrostriatal neurons that are damaged by the parkinsonian-inducing tetrahydropyrridine MPTP. The potential advantage of using t-THP over related imaging techniques is the possibility of assessing neuronal function by an in vivo processing of the reporter molecule rather than by postmortem immunofluorescent or formaldehyde-based procedures.
Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/análogos & derivados , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacocinética , Corantes Fluorescentes/farmacocinética , Neuroblastoma/metabolismo , Biotransformação , Corantes Fluorescentes/toxicidade , Humanos , Isoenzimas/metabolismo , Intoxicação por MPTP , Microscopia de Fluorescência , Monoaminoxidase/metabolismo , Neuroblastoma/enzimologia , Pirimidinas/farmacocinética , Pirimidinas/toxicidade , Espectrometria de Fluorescência , Células Tumorais CultivadasRESUMO
A novel dopamine (DA) autoreceptor agonist, 1,2,3,6-tetrahydro-4-phenyl-1- [(3-phenyl-3-cyclohexen-1-yl)methyl]pyridine (14), was identified. The structure-activity relationships surrounding this compound were studied by synthesis of analogues and evaluation of their dopaminergic activity. The cyclohexene substitution pattern was varied along with the length of the chain connecting the 1,2,3,6-tetrahydro-4-phenylpyridine to the cyclohexene. Compound 14, having the 1,3-substitution pattern and a single methylene chain, was the most potent. The 1,2,3,6-tetrahydro-4-phenylpyridine could be replaced by other aryl-cyclic amines with a slight loss in activity. The phenyl group on the cyclohexene ring could be para substituted; electron-donating groups were better tolerated than electron-withdrawing groups. Finally, the enantiomers of 14 were resolved via the 1,1'-binaphthyl-2,2'-diyl hydrogen phosphate salts. Although both isomers were partial DA agonists, the (+)-enantiomer had higher intrinsic activity than the (-)-enantiomer. Syntheses were developed that allowed rapid preparation of analogues. An X-ray crystal structure determination of an intermediate identified the (+)-isomer of 14 as having R configuration. This compound, designated CI-1007 (PD 143188), was found to have antipsychotic-like activity in behavioral tests; in particular, it was orally active in the conditioned avoidance test in squirrel monkeys with an ED50 of 0.6 mg/kg. The overall profile suggests that (R)-(+)-14 may be a clinically useful antipsychotic agent.
Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/análogos & derivados , Antipsicóticos/química , Agonistas de Dopamina/química , Piridinas/química , Potenciais de Ação/efeitos dos fármacos , Animais , Antipsicóticos/farmacologia , Aprendizagem da Esquiva/efeitos dos fármacos , Encéfalo/metabolismo , Células CHO , Cricetinae , Cristalografia por Raios X , AMP Cíclico/metabolismo , Cicloexanos/química , Cicloexenos , Dopamina/biossíntese , Agonistas de Dopamina/farmacologia , Camundongos , Modelos Moleculares , Estrutura Molecular , Atividade Motora , Piridinas/metabolismo , Piridinas/farmacologia , Ratos , Receptores Dopaminérgicos/metabolismo , Saimiri , Estereoisomerismo , Relação Estrutura-Atividade , Substância Negra/fisiologiaRESUMO
In 6-month-old male Wistar rats, levels of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), ascorbic acid (AA), dehydroascorbic acid (DHAA), uric acid, glutathione (GSH) and 1-methyl-4-phenylpyridinium ion (MPP+) were determined by HPLC in the crude striatal synaptosomal fraction after single injections of MPTP 35 mg/kg i.p. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced a 32.5% death rate within 15 min to 10 h. Groups of surviving rats were sacrificed 1, 3, 8 and 24 h after MPTP. MPTP significantly increased levels of DHAA and uric acid and decreased levels of DOPAC and GSH. Individual synaptosomal levels of MPP+ were correlated inversely with DOPAC (r = -0.601, P < 0.002) and GSH levels (r = -0.496, P < 0.02) and directly with levels of uric acid (r = +0.627, P < 0.001); these latter, in turn, were correlated with DHAA (r = +0.418, P < 0.05) and GSH levels (r = -0.357, P = 0.07). In conclusion, the response of the endogenous antioxidant system (increase in AA oxidation, decrease in GSH levels) correlates well with the MPTP-induced increase in uric acid levels and provides further evidence for a mechanism of MPTP neurotoxicity involving oxidative stress produced by xanthine oxidase.
Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/análogos & derivados , Ácido Ascórbico/metabolismo , Corpo Estriado/metabolismo , Dopaminérgicos/farmacologia , Glutationa/metabolismo , Compostos de Piridínio/metabolismo , Sinaptossomos/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Corpo Estriado/efeitos dos fármacos , Dopamina/metabolismo , Dopaminérgicos/metabolismo , Dopaminérgicos/toxicidade , Intoxicação por MPTP , Masculino , Terminações Nervosas/efeitos dos fármacos , Terminações Nervosas/metabolismo , Oxirredução , Ratos , Ratos Wistar , Sinaptossomos/efeitos dos fármacos , Ácido Úrico/metabolismoRESUMO
A pharmacological study using monkeys, in which parkinsonism was induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), was undertaken to elucidate the mechanism underlying urinary bladder dysfunctions in Parkinson's disease. Under ketamine anesthesia, cystometrograms showed that, in MPTP-treated monkeys, a contraction of the urinary bladder was induced with smaller bladder volume than that in normal monkeys. In MPTP-treated monkeys, subcutaneously injected SKF 38393, a dopamine D1 receptor agonist, significantly increased the bladder volume and pressure thresholds for inducing the micturition reflex without affecting those in normal monkeys. In contrast, subcutaneous injections of quinpirole, a dopamine D2 receptor agonist, and apomorphine, a dopamine D1 and D2 receptor agonist, slightly, but significantly reduced the volume threshold of the bladder for the micturition reflex in both normal and MPTP-treated groups. These results indicate that, in parkinsonism, the degeneration of dopaminergic neurons in the substantia nigra leads to the detrusor hyperreflexia, probably due to a failure of activation of dopamine D1 receptors.
Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/análogos & derivados , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/farmacologia , Dopaminérgicos , Doença de Parkinson Secundária/fisiopatologia , Reflexo/efeitos dos fármacos , Bexiga Urinária/inervação , Animais , Apomorfina/farmacologia , Dopaminérgicos/farmacologia , Ergolinas/farmacologia , Macaca fascicularis , Masculino , Doença de Parkinson Secundária/induzido quimicamente , Quimpirol , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/patologia , Micção/efeitos dos fármacosRESUMO
The study of oxygen radical generation and effects during 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) metabolism was undertaken in an in vitro test system. Three neurochemically discrete neuronal cell lines, B50 (cholinergic) and B65 rat cell lines and SKNSH human neuroblastoma (both catecholaminergic), were exposed to MPTP (0-200 microM). Parallel experiments were performed using reagent H2O2, an intermediate which may be generated during MPTP metabolism, to determine whether MPTP and H2O2 had any selectivity of toxicity and whether the mechanisms of cell death were similar. MPTP toxicity was shown to be reduced by monoamine oxidase B inhibitors, pargyline (P < 0.01) and selegiline (P < 0.05), indicating that toxicity was due to metabolism of MPTP rather than the parent compound. Cytotoxicity was also decreased in the presence of antioxidants, most notably in the presence of superoxide dismutase and catalase together (P < 0.01), suggesting that reactive oxygen species (ROS) play a role in MPTP-induced cell death. Attempts to determine the intracellular target for oxidative attack did not identify significant levels of lipid peroxidation products, but did demonstrate nucleoid expansion, possibly the result of double stranded DNA breaks induced by ROS.