RESUMO
Studies showed that JM-20, a benzodiazepine-dihydropyridine hybrid molecule, protects against rotenone and 6-hydroxydopamine neurotoxicity. However, its protective effects against cytotoxicity induced by endogenous neurotoxins involved in Parkinson's disease (PD) pathogenesis have never been investigated. In this study, we evaluated the ability of JM-20 to inhibit alpha-synuclein (aSyn) aggregation. We also evaluated the interactions of JM-20 with aSyn by molecular docking and molecular dynamics and assessed the protective effect of JM-20 against aminochrome cytotoxicity. We demonstrated that JM-20 induced the formation of heterogeneous amyloid fibrils, which were innocuous to primary cultures of mesencephalic cells. Moreover, JM-20 reduced the average size of aSyn positive inclusions in H4 cells transfected with SynT wild-type and synphilin-1-V5, but not in HEK cells transfected with synphilin-1-GFP. In silico studies showed the interaction between JM-20 and the aSyn-binding site. Additionally, we showed that JM-20 protects SH-SY5Y cells against aminochrome cytotoxicity. These results reinforce the potential of JM-20 as a neuroprotective compound for PD and suggest aSyn as a molecular target for JM-20.
Assuntos
Di-Hidropiridinas , Neuroblastoma , Doença de Parkinson , Humanos , alfa-Sinucleína , Benzodiazepinas , Simulação de Acoplamento Molecular , Doença de Parkinson/tratamento farmacológicoRESUMO
The number of people with dementia worldwide is estimated at 50 million by 2018 and continues to rise mainly due to increasing aging and population growth. Clinical impact of current interventions remains modest and all efforts aimed at the identification of new therapeutic approaches are therefore critical. Previously, we showed that JM-20, a dihydropyridine-benzodiazepine hybrid molecule, protected memory processes against scopolamine-induced cholinergic dysfunction. In order to gain further insight into the therapeutic potential of JM-20 on cognitive decline and Alzheimer's disease (AD) pathology, here we evaluated its neuroprotective effects after chronic aluminum chloride (AlCl3) administration to rats and assessed possible alterations in several types of episodic memory and associated pathological mechanisms. Oral administration of aluminum to rodents recapitulates several neuropathological alterations and cognitive impairment, being considered a convenient tool for testing the efficacy of new therapies for dementia. We used behavioral tasks to test spatial, emotional- associative and novel object recognition memory, as well as molecular, enzymatic and histological assays to evaluate selected biochemical parameters. Our study revealed that JM-20 prevented memory decline alongside the inhibition of AlCl3 -induced oxidative stress, increased AChE activity, TNF-α and pro-apoptotic proteins (like Bax, caspase-3, and 8) levels. JM-20 also protected against neuronal damage in the hippocampus and prefrontal cortex. Our findings expanded our understanding of the ability of JM-20 to preserve memory in rats under neurotoxic conditions and confirm its potential capacity to counteract cognitive impairment and etiological factors of AD by breaking the progression of key steps associated with neurodegeneration.
Assuntos
Cloreto de Alumínio/toxicidade , Benzodiazepinas/farmacologia , Transtornos da Memória/induzido quimicamente , Memória/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Niacina/análogos & derivados , Cloreto de Alumínio/antagonistas & inibidores , Animais , Hipocampo/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Teste do Labirinto Aquático de Morris/efeitos dos fármacos , Niacina/farmacologia , Teste de Campo Aberto/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacos , Ratos , Ratos Wistar , Teste de Desempenho do Rota-RodRESUMO
Cerebral ischemia constitutes the most frequent type of cerebrovascular disease. The reduction of blood supply to the brain initiates the ischemic cascade starting from ionic imbalance to subsequent glutamate excitotoxicity, neuroinflammation and oxidative stress, eventually causing neuronal death. Previously, the authors have demonstrated the in vitro cytoprotective and antioxidant effects of a new arylidene malonate derivative, KM-34, against oxidizing agents like hydrogen peroxide, glutamate or Fe3+/ascorbate. Here, we examined for the first time the neuroprotective effect of KM-34 on ischemia/reperfusion models. In vitro, treatment with 10 and 50 µM KM-34 reduced the cellular death (propidium iodide incorporation) induced by oxygen glucose deprivation (OGD) in rat organotypic hippocampal slices cultures. In vivo, stroke was induced in male Wistar rats through middle cerebral artery occlusion (MCAO), followed by 23 h of reperfusion. KM-34 was orally administered 105 min after MCAO onset. We noticed that 1 mg/kg KM-34 reduced infarct volume and neurological score, and increased the latency to fall in the Hanging Wire test compared to vehicle-treated ischemic animals. While ischemic and sham-operated groups showed similar horizontal locomotor activity, vertical counts decreased after MCAO, suggesting that vertical movements are more sensitive to the ischemic injury. Treatment with KM-34 also alleviated the mitochondrial impairment (ROS generation, swelling and membrane potential dissipation) induced by transient MCAO but not significant alterations were found in oxidative stress parameters. Overall, the study provides preclinical evidences confirming the neuroprotective effects of a novel synthetic molecule and paved the way for future investigations regarding its therapeutic potential against brain ischemia/reperfusion injury.
Assuntos
Encéfalo/efeitos dos fármacos , Catecóis/farmacologia , Infarto da Artéria Cerebral Média/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Locomoção/efeitos dos fármacos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Dilatação Mitocondrial/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ratos Wistar , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Técnicas de Cultura de TecidosRESUMO
We have previously shown that JM-20, a new chemical entity consisting of 1,5-benzodiazepine fused to a dihydropyridine moiety, protects against rotenone-induced neurotoxicity in an experimental model of Parkinson's disease (PD). The aim of this study was to investigate the effect of a novel hybrid molecule, named JM-20, in in vitro and in vivo models of PD induced by 6-hydroxydopamine (6-OHDA). PC-12 cells were exposed to 6-OHDA and treated with JM-20. Protection against mitochondrial damage induced by 6-OHDA was also investigated using isolated rat brain mitochondria. We found that JM-20 protected PC-12 cells against cytotoxicity induced by 6-OHDA and inhibited hydrogen peroxide generation, mitochondrial swelling and membrane potential dissipation. For in vivo experiments, adult male Wistar rats were lesioned in the substantia nigra pars compacta (SNpc) by 6-OHDA administration. JM-20 was orally administered (10, 20 or 40 mg/kg), intragastric via gavage, 24 h after surgery and daily for seven days. Treatment with JM-20 significantly reduced the percentage of motor asymmetry and increased vertical exploration. It improved the redox state of the SNpc and the striatal tissue of these animals. Also, JM-20 reduced glial fibrillary acidic protein overexpression and increased tyrosine hydroxylase-positive cell number, both in SNpc. Altogether, these results demonstrate that JM-20 is a potential neuroprotective agent against 6-OHDA-induced damage in both in vitro and in vivo models. The mechanism underlying JM-20 neuroprotection against 6-OHDA appears to be associated with the control of oxidative injury and mitochondrial impairment.
Assuntos
Antioxidantes/farmacologia , Benzodiazepinas/farmacologia , Encéfalo/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Niacina/análogos & derivados , Oxidopamina/toxicidade , Transtornos Parkinsonianos/tratamento farmacológico , Animais , Masculino , Mitocôndrias/metabolismo , Niacina/farmacologia , Teste de Campo Aberto/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Células PC12/efeitos dos fármacos , Transtornos Parkinsonianos/induzido quimicamente , Ratos , Ratos WistarRESUMO
OBJECTIVE: JM-20, a novel hybrid synthetic molecule, has been reported to have antioxidant, mitoprotective, anti-excitotoxic, anti-apoptotic and anti-inflammatory properties. However, the neuroprotective effect of JM-20 against memory impairment in preclinical AD-like models has not been analyzed. The aim of this study was to evaluate the potential neuroprotection of JM-20 that preserves essential memory process from cholinergic dysfunction and other molecular damages. METHODS: The effects of JM-20 on scopolamine (1 mg/kg)-induced cognitive disorders were studied. Male Wistar rats (220-230 g) were treated with JM-20 and/or scopolamine, and behavioral tasks were performed. The AChE activity, superoxide dismutase activity, catalase activity, MDA and T-SH level on brain tissue were determined by spectrophotometric methods. Mitochondrial functionality parameters were measured after behavioral tests. Histological analyses on hippocampus and prefrontal cortex were processed with hematoxylin and eosin, and neuronal and axonal damage were determined. RESULTS: The behavioral, biochemical and histopathological studies revealed that oral pre-treatment with JM-20 (8 mg/kg) significantly attenuated the scopolamine-induced memory deficits, mitochondrial malfunction, oxidative stress, and prevented AChE hyperactivity probably due to specific inhibition of AChE enzyme. It was also observed marked histological protection on hippocampal and prefrontal-cortex regions. CONCLUSIONS: The multimodal action of this molecule could mediate the memory protection here observed and suggest that it may modulate different pathological aspects of memory deï¬cits associated with AD in humans.
Assuntos
Benzodiazepinas/farmacologia , Inibidores da Colinesterase/farmacologia , Disfunção Cognitiva/tratamento farmacológico , Memória/efeitos dos fármacos , Niacina/análogos & derivados , Nootrópicos/farmacologia , Acetilcolinesterase/metabolismo , Animais , Antioxidantes/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/fisiologia , Masculino , Memória/fisiologia , Transtornos da Memória/tratamento farmacológico , Transtornos da Memória/metabolismo , Transtornos da Memória/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Niacina/farmacologia , Distribuição Aleatória , Ratos Wistar , EscopolaminaRESUMO
The etiology of Parkinson's disease is not completely understood and is believed to be multifactorial. Neuronal disorders associated to oxidative stress and mitochondrial dysfunction are widely considered major consequences. The aim of this study was to investigate the effect of the synthetic arylidenmalonate derivative 5-(3,4-dihydroxybenzylidene)-2,2-dimethyl-1,3-dioxane-4,6-dione (KM-34), in oxidative stress and mitochondrial dysfunction induced by 6-hydroxydopamine (6-OHDA). Pretreatment (2 h) with KM-34 (1 and 10 µM) markedly attenuated 6-OHDA-induced PC12 cell death in a concentration-dependent manner. KM-34 also inhibited H2O2 generation, mitochondrial swelling, and membrane potential dissipation after 6-OHDA-induced mitochondrial damage. In vivo, KM-34 treatment (1 and 2 mg/Kg) reduced percentage of asymmetry (cylinder test) and increased the vertical exploration (open field) with respect to untreated injured animals; KM-34 also reduced glial fibrillary acidic protein overexpression and increased tyrosine hydroxylase-positive cell number, both in substantia nigra pars compacta. These results demonstrate that KM-34 present biological effects associated to mitoprotection and neuroprotection in vitro, moreover, glial response and neuroprotection in SNpc in vivo. We suggest that KM-34 could be a putative neuroprotective agent for inhibiting the progressive neurodegenerative disease associated to oxidative stress and mitochondrial dysfunction.
Assuntos
Antioxidantes/uso terapêutico , Catecóis/uso terapêutico , Mitocôndrias/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Oxidopamina/toxicidade , Transtornos Parkinsonianos/prevenção & controle , Animais , Antioxidantes/farmacologia , Catecóis/farmacologia , Relação Dose-Resposta a Droga , Masculino , Mitocôndrias/metabolismo , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Células PC12 , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/metabolismo , Ratos , Ratos WistarRESUMO
Oxidative stress and mitochondrial dysfunction are two pathophysiological factors often associated with the neurodegenerative process involved in Parkinson's disease (PD). The aim of this study was to investigate the effects of a novel hybrid molecule, named JM-20, in different in vitro and in vivo models of PD induced by rotenone. To perform in vitro studies, SHSY-5Y cells were exposed to rotenone and/or treated with JM-20. To perform in vivo studies male Wistar rats were intoxicated with rotenone (2.5 mg/kg) via intraperitoneal injection and/or treated with JM-20 (40 mg/kg) administered via oral (for 25 days, both treatment). Rats were evaluated for global motor activity by measurement of locomotor activity. In addition, the effects on mortality, general behavior and redox parameters were also investigated. JM-20 protected SHSY-5Y cells against rotenone-induced cytotoxicity, evidenced by a significant diminution of cell death. In in vivo studies, JM-20 prevented rotenone-induced vertical exploration and locomotion frequency reductions, moreover prevented body weight loss and mortality induced by rotenone. It also improved the redox state of rotenone-exposured animals by increasing superoxide dismutase and catalase activities, total tissue-SH levels and decreasing malondialdehyde concentrations. Finally, JM-20 inhibited spontaneous mitochondrial swelling and membrane potential dissipation in isolated rats brain mitochondria. These results demonstrate that JM-20 is a potential neuroprotective agent against rotenone-induced damage in both in vitro and in vivo models, resulting in reduced neuronal oxidative injury and protection of mitochondria from impairment.
Assuntos
Benzodiazepinas/farmacologia , Fármacos Neuroprotetores/farmacologia , Síndromes Neurotóxicas/prevenção & controle , Niacina/análogos & derivados , Rotenona/toxicidade , Animais , Peso Corporal/efeitos dos fármacos , Encéfalo/metabolismo , Catalase/metabolismo , Morte Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Masculino , Mitocôndrias/metabolismo , Atividade Motora/efeitos dos fármacos , Niacina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Ratos , Superóxido Dismutase/metabolismoRESUMO
Cerebral ischemia is the third most common cause of death and a major cause of disability worldwide. Beyond a shortage of essential metabolites, ischemia triggers many interconnected pathophysiological events, including excitotoxicity, oxidative stress, inflammation and apoptosis. Here, we investigated the neuroprotective mechanisms of JM-20, a novel synthetic molecule, focusing on the phosphoinositide-3-kinase (PI3K)/Akt survival pathway and glial cell response as potential targets of JM-20. For this purpose, we used organotypic hippocampal slice cultures exposed to oxygen-glucose deprivation (OGD) to achieve ischemic/reperfusion damage in vitro. Treatment with JM-20 at 0.1 and 10 µM reduced PI incorporation (indicative of cell death) after OGD. OGD decreased the phosphorylation of Akt (pro-survival) and GSK 3ß (pro-apoptotic), resulting in respective inhibition and activation of these proteins. Treatment with JM20 prevented the reduced phosphorylation of these proteins after OGD, representing a shift from pro-apoptotic to pro-survival signaling. The OGD-induced activation of caspase-3 was also attenuated by JM-20 treatment at 10 µM. Moreover, in cultures treated with JM-20 and exposed to OGD conditioning, we observed a decrease in activated microglia, as well as a decrease in interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α release into the culture medium, while the level of the anti-inflammatory IL-10 increased. GFAP immunostaining and IB4 labeling showed that JM-20 treatment significantly augmented GFAP immunoreactivity after OGD, when compared with cultures exposed to OGD only, suggesting the activation of astroglial cells. Our results confirm that JM-20 has a strong neuroprotective effect against ischemic injury and suggest that the mechanisms involved in this effect may include the modulation of reactive astrogliosis, as well as neuroinflammation and the anti-apoptotic cell signaling pathway.