RESUMO
Parkinson disease (PD) is characterized by the loss of dopaminergic (DAergic) neurons linked to environmental toxicants that cause oxidative stress (OS). The aim of this investigation was to establish the molecular response of human mesenchymal stroma cells (MSCs) depleted of glutathione (GSH) by the specific inhibitor L-buthionine-sulfoximine (BSO) to 6-hydroxydopamine (6-OHDA) and/or N-acetylcysteine (NAC) co-treatment. We found that treatment with BSO (10 mM) plus 6-OHDA (200 µM) induced apoptosis in MSCs through an oxidative stress (OS) mechanism involving H2O2, reflected by the detection of dichlorofluorescein-positive (DCF+) cells and oxidation of DJ-1 Cys106-SH into DJ-1 Cys106-SO3; an almost complete reduction in glutathione peroxidase 1 (GPX1) expression; activation of the transcription factor c-JUN, the pro-apoptotic protein BAX and BH-3-only protein PUMA; loss of mitochondrial membrane potential (∆Ψm); activation of the protease caspase-3 (CASP3) and apoptosis-inducing factor (AIF); chromatin condensation; and DNA fragmentation. Strikingly, co-treatment of MSCs with NAC (5 mM) and BSO + 6-OHDA significantly reduced the expression of OS and cell death markers but were unable to restore the expression of GPX1 compared to the expression in untreated or treated cells with NAC only. These findings highlighted the importance of the maintenance of the GSH-dependent (e.g., GPX1, GSH synthesis) and -independent (e.g., ROS scavenger molecules and thiol reducing activity) antioxidant systems (e.g., NAC) in the protection of MSCs from detrimental stress stimuli, thereby increasing the survival of stromal cells.
Assuntos
Acetilcisteína/farmacologia , Apoptose/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Antioxidantes/metabolismo , Butionina Sulfoximina/metabolismo , Morte Celular/efeitos dos fármacos , Glutationa/metabolismo , Glutationa Peroxidase , Humanos , Peróxido de Hidrogênio/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Oxidopamina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Glutationa Peroxidase GPX1RESUMO
BACKGROUND AIMS: Stem cell transplantation is an excellent option for regenerative or replacement therapy. However, deleterious microenvironmental and endogenous factors (e.g., oxidative stress) compromise ongoing graft survival and longevity. Therefore, (transient or stable) genetically modified cells may be reasonably thought to resist oxidative stress-induced damage. Genetic engineering of mesenchymal stromal cells (MSCs) obtained from Wharton's jelly tissue may offer some therapeutic potential. PARKIN is a multifunctional ubiquitin ligase able to protect dopaminergic cells against stress-related signaling. We, therefore, evaluated the effect of the neurotoxicant 6-hydroxydopamine (6-OHDA) on regulated cell death signaling in MSCs and investigated whether overexpression of PARKIN in MSCs was capable of modulating the effect of 6-OHDA. METHODS: We transiently transfected Wharton's jelly-derived MSCs with an mCherry-PARKIN vector using the Lipofectamine LTX method. Naïve MSCs and MSCs overexpressing PARKIN were exposed to increasing concentrations of 6-OHDA. We used light and fluorescence microscopy, flow cytometry, immunocytochemistry staining, in-cell Western and Western blot analysis. RESULTS: After 12-24 h of 6-OHDA exposure, we detected dichlorofluorescein (DCF)-positive cells (80%) indicative of reactive oxygen species (H2O2) production, reduced cell viability (40-50%), decreased mitochondrial membrane potential (ΔΨm, ~35-45%), DNA fragmentation (18-30%), and G1-arrested cell cycle in the MSCs. 6-OHDA exposure increased the expression of the transcription factor c-JUN, increased the expression of the mitochondria maintenance Phosphatase and tensin homologue-induced putative kinase 1 (PINK1) protein and increased the expression of pro-apoptotic PUMA, caspase-3 and apoptosis-inducing factor (AIF). 6-OHDA exposure also significantly augmented the oxidation of the oxidative stress sensor, DJ-1. Overexpression of PARKIN in MSCs not only significantly reduced the expression of cell death and oxidative stress markers but also significantly reduced DCF-positive cells (~50% reduction). DISCUSSION: 6-OHDA induced apoptosis in MSCs via generation of H2O2, activation of c-JUN and PUMA, mitochondrial depolarization and nuclei fragmentation. Our findings suggest that PARKIN protects MSCs against 6-OHDA toxicity by partly interacting with H2O2, reducing the expression of c-JUN, PUMA, AIF and caspase-3, and maintaining the mitochondrial ΔΨm.
Assuntos
Apoptose , Células-Tronco Mesenquimais/metabolismo , Doença de Parkinson/patologia , Doença de Parkinson/terapia , Ubiquitina-Proteína Ligases/metabolismo , Geleia de Wharton/citologia , Apoptose/efeitos dos fármacos , Caspase 3 , Sobrevivência Celular , Humanos , Peróxido de Hidrogênio/farmacologia , Potencial da Membrana Mitocondrial , Transplante de Células-Tronco Mesenquimais , Mitocôndrias/metabolismo , Estresse Oxidativo , Oxidopamina , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: The human mesenchymal stem cells derived from Wharton's jelly tissue (hWJ-MSCs) represent a tool for cell-based therapies and regenerative medicine. hWJ-MSCs form neurospheres (NSs) within 3-7 days. No data is available to establish the neuro-phenotypic markers and time of formation of nerve-like (NLCs) and glial cells from NSs derived from hWJ-MSCs. NEW METHOD: hWJ-MSCs were incubated with Fast-N-Spheres medium for 24 and 72h. The new formed NSs were in turn incubated with forskolin in neurogenic NeuroForsk medium for 1-7days. RESULTS: hWJ-MSCs cultured with Fast-N-Spheres medium trans-differentiated into NSs in just 24h compared to 72h for hWJ-MSCs cultured with classic growth factor medium. The NSs generated from the Fast-N-Spheres medium expressed reduced levels SOX2, OCT4 and NANOG, as markers of pluripotency compared to undifferentiated hWJ-MSCs. The formed NSs exposed to NeuroForsk medium differentiated into NLCs in 4days as evidenced by high levels of protein expression of the neuronal markers, and no expression of the glial marker GFAP. COMPARISON WITH EXISTING METHOD(S): Currently, the formation and harvest of NSs is expensive and time consuming. Published protocols require 3-7days to form NSs from whole human umbilical cord MSCs. We report for the first time, to our knowledge, the differentiation of NSs-derived from hWJ-MSCs into NLCs. CONCLUSIONS: The fastest method to obtain NSs and NLCs from hWJ-MSCs takes only five days using the two-step incubation media Fast-N-Spheres and NeuroForsk.
Assuntos
Técnicas de Cultura de Células/métodos , Transdiferenciação Celular/fisiologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Adipogenia/fisiologia , Western Blotting , Colforsina , Meios de Cultura , Imunofluorescência , Humanos , Cariotipagem , Neurogênese/fisiologia , Osteogênese/fisiologia , Fatores de Tempo , Geleia de Wharton/citologiaRESUMO
Parkinson's disease (PD) is a common progressive neurodegenerative disorder, for which at present no causal treatment is available. On the understanding that the causes of PD are mainly oxidative stress and mitochondrial dysfunction, antioxidants and other drugs are expected to be used. In the present study, we demonstrated for the first time that pure polyphenols such as gallic acid, ferulic acid, caffeic acid, coumaric acid, propyl gallate, epicatechin, epigallocatechin, and epigallocatechin gallate protect, rescue and, most importantly, restore the impaired movement activity (i.e., climbing capability) induced by paraquat in Drosophila melanogaster, a valid model of PD. We also showed for the first time that high concentrations of iron (e.g. 15 mM FeSO(4)) are able to diminish fly survival and movement to a similar extent as (20 mM) paraquat treatment. Moreover, paraquat and iron synergistically affect both survival and locomotor function. Remarkably, propyl gallate and epigallocatechin gallate protected and maintained movement abilities in flies co-treated with paraquat and iron. Our findings indicate that pure polyphenols might be potent neuroprotective agents for the treatment of PD against stressful stimuli.
Assuntos
Compostos Férricos/toxicidade , Flavonoides/farmacologia , Fármacos Neuroprotetores/farmacologia , Paraquat/toxicidade , Fenóis/farmacologia , Animais , Antioxidantes/farmacologia , Modelos Animais de Doenças , Drosophila melanogaster , Feminino , Atividade Motora/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Doença de Parkinson/tratamento farmacológico , Polifenóis , Taxa de SobrevidaRESUMO
Cannabinoids have been shown to function as protective agents via receptor-independent and/or receptor-dependent mechanisms against stressful conditions. However, the neuroprotective mechanism of cannabinoids is far from conclusive. Therefore, the genuine antioxidant impact of cannabinoids in vivo is still uncertain. In this study, we demonstrate for the first time that CP55,940, a nonselective CB(1)/CB(2) cannabinoid receptor agonist, significantly protects and rescues Drosophila melanogaster against paraquat (PQ) toxicity via a receptor-independent mechanism. Interestingly, CP55,940 restores the negative geotaxis activity (i.e., climbing capability) of the fly exposed to PQ. Moreover, Drosophila fed with (1-200 microM) SP600125, a specific inhibitor of the stress responsive Jun-N-terminal kinase (JNK) signaling, and 20 mM PQ increased survival percentage and movement function (i.e., climbing capability) when compared to flies only treated with PQ. Taken together our results suggest that exogenous antioxidant cannabinoids can protect against and rescue from locomotor dysfunction in wild type (Canton-S) Drosophila exposed to stress stimuli. Therefore, cannabinoids may offer promising avenues for the design of molecules to prevent, delay, or ameliorate the treatment of population at high risk of suffering Parkinson disease.
Assuntos
Cicloexanóis/administração & dosagem , Herbicidas/toxicidade , Imunossupressores/administração & dosagem , Atividade Motora/efeitos dos fármacos , Paraquat/toxicidade , Animais , Antracenos/administração & dosagem , Antioxidantes/administração & dosagem , Comportamento Animal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Drosophila melanogaster , Interações Medicamentosas , Inibidores Enzimáticos/administração & dosagem , Comportamento Alimentar/efeitos dos fármacos , Feminino , Glucose/administração & dosagem , Masculino , Análise de Sobrevida , Fatores de Tempo , Tocoferóis/administração & dosagemAssuntos
Antígenos de Histocompatibilidade Classe I/genética , Sobrecarga de Ferro/genética , Proteínas de Membrana/genética , Mutação de Sentido Incorreto , Colômbia , Emigração e Imigração , Saúde da Família , Frequência do Gene , Genótipo , Proteína da Hemocromatose , Humanos , Sobrecarga de Ferro/etnologiaRESUMO
INTRODUCTION AND OBJECTIVES: Alzheimer s disease is a neurodegenerative disorder characterized neuropathologically by beta amyloid plaques, neurofibrillary tangles, gliosis and neuronal loss. Recently, we have elucidated a molecular cascade of cell death induced by A beta 25 35 involving the activation of nuclear factor kappa B (NF kB), p53, and c Jun transcription factors in vitro. At present, no comparative reports have been published to establish a similar cell death signalization pathway in in vitro and in in vivo. The aim of this investigation was to determine if AD brains might activate NF kB, p53, c Jun, Par 4 transcription factors and to establish whether there exist a relationship between neuronal DNA damage and transcription factors activation. PATIENTS AND METHODS: We investigated Ab plaques, neurofibrillary tangles and NF kB, p53, and c Jun transcription factor activation in five cerebral regions from 3 normal subjects and from six demented patient with sporadic AD and one patient with AD familiar according to CERAD criteria. Using TUNEL we determine neuronal damage. RESULTS: We demonstrated neuronal damage in 17 out of 50 regions evaluated as TUNEL positive, and their distribution was heterogeneous in all brain regions evaluated; and the activation of NF kB, p53, c Jun and Par 4 transcription factors from case # 24 and #22, corresponding to TUNEL positive. CONCLUSIONS: We found a correlation between severity of DNA damage and nuclear activation of the transcription factors. These findings suggest that the AD brain may induce cell death by a molecular signalization similar to a non neuronal model by Ab exposure. This in situ study might validate previous Ab induced cell death observations in vitro.
Assuntos
Doença de Alzheimer/metabolismo , Proteínas de Transporte/análise , Dano ao DNA , Peptídeos e Proteínas de Sinalização Intracelular , NF-kappa B/análise , Neurônios/química , Proteínas Proto-Oncogênicas c-jun/análise , Proteína Supressora de Tumor p53/análise , Adulto , Idoso , Peptídeos beta-Amiloides/análise , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Feminino , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Pessoa de Meia-Idade , Estatística como AssuntoRESUMO
OBJECTIVES: In this article, the authors analyzed critically the morphological and biochemical evidences of cell death by apoptosis from postmortem studies in Alzheimer s, Parkinson s, Huntington s and Wilson s diseases. DEVELOPMENT: During the last few years, apoptosis has been postulated as a type of neuronal death responsible for the neurodegenerative process in those heterogeneous, chronic and progressive neurological disorders, which are characterized by a selective and a symmetric loss of neurons in motor, sensory or cognitive systems. With regard to neuronal death mechanism and the contribution of the mutated or metabolic altered proteins such as betaA, P-tau, alpha-synuclein, Parkin, Huntingtin, ATP78B, proteins in the pathogenesis of those disorders are still unknown. CONCLUSIONS: We consider that the morphological (e.g. DNA fragmentation without showing classical apoptotic morphology) and biochemical evidences are still insufficient and contradictory to formally indict apoptosis as the mechanism of neuronal cell death in those neurological disorders. The establishment of the molecular mechanisms leading neurons to cell death (by apoptosis?) could provide significant information for the design of therapeutic strategies to retard or prevent the development of such neurodegenerative diseases in affected individuals.
Assuntos
Apoptose/fisiologia , Doenças Neurodegenerativas/patologia , Neurônios/patologia , Doença de Alzheimer/patologia , Degeneração Hepatolenticular/patologia , Humanos , Doença de Huntington/patologia , Doença de Parkinson/patologiaRESUMO
Although there have been experimental approaches to understanding the etiology of Parkinson's disease, the cause of cell degeneration in this neurological disorder remains a mystery. Herein, a hypothetical model is proposed to explain the mechanism leading neurons to die. The model is based on recent experimental evidence and it attempts to dissect the actions of dopamine and metal ions as potential triggers for the activation of an ordered cascade of events of the cell death machinery.
Assuntos
Monoaminas Biogênicas/toxicidade , Modelos Neurológicos , Neurônios/patologia , Doença de Parkinson/patologia , Doença de Parkinson/fisiopatologia , Animais , Monoaminas Biogênicas/fisiologia , Morte Celular , Dopamina/fisiologia , Humanos , MetaisRESUMO
Recent studies have shown that the missense mutation in presenilin-1 [E280A] increases deposition of amyloid-beta (Abeta) and neurofibrillary tangle (NFT) formation. Indeed, we only observed 10 out of 48 FAD brain sections displaying TUNEL (+) labeling, and none with the classical apoptotic morphology. These results may indicate that DNA fragmentation is not a generalized phenomenon in early-onset FAD PS1 [E280A] patients or that neuronal cells are dying by a different mechanism of cell death. Taking together these findings suggest that Abeta and NFTs are not per se a causative factor to damage neuronal cells but their damage could be more related with individual neuronal vulnerability and brain aging
RESUMO
Dopamine (DA) in combination with iron (Fe(2+)) has been demonstrated to induce apoptosis in neuronal-like PC12 cells by an oxidative stress mechanism. To get a better insight of cell death and protective mechanisms in DA/Fe(2+)-induced toxicity, we investigated the effects of DA/Fe(2+) and the antioxidant action of 17 beta-estradiol (E2) in peripheral blood lymphocytes (PBL). We found that DA/Fe(2+)-induces apoptosis in PBL via a hydrogen peroxide (H(2)O(2))-mediated oxidative mechanism, which in turn triggers a cascade of molecular events requiring RNA and de novo protein synthesis. We have also demonstrated that E2 prevents significantly DA/Fe(2+)-induced apoptosis in PBL by directly inhibiting the intracellular accumulation of peroxides generated by DA/Fe(2+)-reaction. This protective activity is independent of the presence or activation of the estrogen receptors (ERs). These data further support and validate our previous hypothesis that DA/Fe(2+)/H(2)O(2) could be a general mediator of oxidative stress through a common cell death mechanism in both neuronal and nonneuronal cells. These findings may be particularly relevant to the potential approaches to rescue and prolong the survival of neurons by estrogens in patients with Parkinson's disease (PD).
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/genética , Dopamina/farmacologia , Estradiol/genética , Estradiol/farmacologia , Genômica , Ferro/farmacologia , Linfócitos/efeitos dos fármacos , Doença de Parkinson/genética , Adulto , Cicloeximida/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Fragmentação do DNA/genética , Dactinomicina/farmacologia , Eletroforese em Gel de Ágar , Humanos , Peróxido de Hidrogênio/toxicidade , Linfócitos/sangue , Masculino , Doença de Parkinson/sangue , Valor Preditivo dos Testes , Inibidores da Síntese de Proteínas/farmacologia , Receptores de Estrogênio/efeitos dos fármacosRESUMO
1. The basic etiology of Alzheimer's disease remains unknown, although four genes have so far been involved: beta-amyloid precursor protein, presenilin-1, presenilin-2 and apolipoprotein E genes. 2. The largest familial Alzheimer's disease (FAD) kindred so far reported belong to a point mutation in codon 280 that results in a glutamic acid-to-alanine substitution in presenilin-1 characterized in Antioquia, Colombia. 3. A hypothetical unified molecular mechanism model of cell death in FAD mediated by presenilin-1, beta-amyloid, and oxidative stress is proposed as an attempt to explain the mechanisms of neuronal loss in this neurodegenerative disorder.