RESUMEN
Glycogen synthase kinase-3 (GSK-3) is a critical molecule in Alzheimer's disease (AD) that modulates two histopathological hallmarks of AD: Amyloid beta (Aß) plaques and neurofibrillary tangles composed of aberrant hyper-phosphorylation of tau protein. This study was performed to investigate the protective effect of flavone apigenin through inhibition of GSK-3 and the involvement of this kinase in the inhibition of BACE1 expression and hyperphosphorylation of tau protein in an AD rat model. 15 nM of aggregated amyloid-beta 25-35 was microinjected into the left lateral ventricle of an AD rat. Apigenin (50 mg/kg) was administered orally 45 min before the Aß injection and continued daily for three weeks. Immunohistochemistry and western blot analysis showed that apigenin significantly reduced the hyperphosphorylation of tau levels in the hippocampus. Real-time PCR analysis revealed significant inhibition of the mRNA level of ß secretase (BACE1) and GSK-3ß, but Apigenin had no effect on the level of GSK-3α. The results demonstrate that apigenin has a protective effect against amyloid-beta 25-35 by decreasing the expression of GSK-3ß with the consequence of lowering the hyperphosphorylation of tau protein and suppressing BACE1 expression.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/prevención & control , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/toxicidad , Apigenina/farmacología , Glucógeno Sintasa Quinasa 3 beta/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/toxicidad , Secretasas de la Proteína Precursora del Amiloide/biosíntesis , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Ácido Aspártico Endopeptidasas/biosíntesis , Ácido Aspártico Endopeptidasas/genética , Inmunohistoquímica , Masculino , Fosforilación , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Wistar , Proteínas tau/metabolismoRESUMEN
Alzheimer's disease (AD)-associated neurodegeneration is triggered by different fragments of amyloid beta (Aß). Among them, Aß (25-35) fragment plays a critical role in the development of neurodegeneration-it reduces synaptic integrity by disruption of excitatory/inhibitory ratio across networks and alters the growth factors synthesis. Thus, in this study, we aimed to identify the involvement of neurotrophic factors-the insulin-like growth factor 1 (IGF-1) and nerve growth factor (NGF)-of AD-like neurodegeneration induced by Aß (25-35). Taking into account our previous findings on the neuroprotective effects of the mix of proteoglycans of embryonic genesis (PEG), it was suggested to test its regulatory effect on IGF-1 and NGF levels. To evaluate the progress of neurodegeneration, in vivo electrophysiological investigation of synaptic activity disruption of the entorhinal cortex-hippocampus circuit at AD was performed and the potential recovery effects of PEG with relative structural changes were provided. To reveal the direct effects of PEG on brain functional activity, the electrophysiological pattern of the single cells from nucleus supraopticus, sensomotor cortex and hippocampus after acute injection of PEG was examined. Our results demonstrated that after i.c.v. injection of Aß (25-35), the level of NGF decreased in cerebral cortex and hypothalamus, and, in contrast, increased in hippocampus, prompting its multidirectional role in case of brain damage. The concentration of IGF-1 significantly increased in all investigated brain structures. The administration of PEG balanced the growth factor levels accompanied by substantial restoration of neural tissue architecture and synaptic activity. Acute injection of PEG activated the hypothalamic nucleus supraopticus and hippocampal neurons. IGF-1 and NGF levels were found to be elevated in animals receiving PEG in an absence of amyloid exposure. We suggest that IGF-1 and NGF play a critical role in the development of AD. At the same time, it becomes clear that the neuroprotective effects of PEG are likely mediated via the regulation of neurotrophins.
Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides/metabolismo , Encéfalo , Electrocardiografía , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Fragmentos de Péptidos/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/fisiopatología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/fisiopatología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Alzheimer (AD) is a degenerative disease that can lead memory loss and behavioral dysfunction. Aß protein and phosphorylation of Tau protein are related to the onset of AD. However, at present, its treatment and drugs are limited. The purpose of our study is to evaluate whether phosphocreatine (PCr) could protect neuronal injury induced by Aß protein in vivo and in vitro through AKT/GSK-3ß/Tau/APP/CDK5 pathways. Differentiated PC-12 cells were cultured with Aß25-35 for 24 h, while the mice were injected with D-Galactose for eight weeks, both of them were pretreated with PCr for 2 h. The results showed PCr could obviously induce cells and hippocampus apoptosis using DAPI and TUNEL. PCr decreased the levels of intercellular reactive oxygen species (ROS) and malondialdehyde (MDA), and increased the activities of superoxide dismutase (SOD). Besides, the apoptosis pathway was detected using Western blot, showing that PCr could significantly reduce caspase-3, caspase-9, Bcl-2/Bax expression in vivo and in vitro. At the same time, PCr could decreased Ca2+ and apoptosis by Flow Cytometry in PC-12 cells. We observed that the morphological alteration of hippocampus injury was mitigated with the pretreatment of PCr. Furthermore, PCr pretreatment could decrease Aß25-35-induced PC-12 cells apoptosis with APP cDNA transfection, which up-regulated AKT/GSK-3ß/CDK5 pathways and induced Tau phosphorylation. In summary, PCr could reduce Aß25-35 toxicity to protect neuronal cells via AKT/GSK-3ß/CDK5 pathways.
Asunto(s)
Péptidos beta-Amiloides , Fármacos Neuroprotectores , Péptidos beta-Amiloides/toxicidad , Animales , Apoptosis , Muerte Celular , Glucógeno Sintasa Quinasa 3 beta/genética , Ratones , Fármacos Neuroprotectores/farmacología , Fosfocreatina/farmacología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
The search for new therapeutics for the treatment of Alzheimer's disease (AD) is still in progress. Aberrant pathways of synaptic transmission in basal forebrain cholinergic neural circuits are thought to be associated with the progression of AD. However, the effect of amyloid-beta (Aß) on short-term plasticity (STP) of cholinergic circuits in the nucleus basalis magnocellularis (NBM) is largely unknown. STP assessment in rat brain cholinergic circuitry may indicate a new target for AD cholinergic therapeutics. Thus, we aimed to study in vivo electrophysiological patterns of synaptic activity in NBM-hippocampus and NBM-basolateral amygdala circuits associated with AD-like neurodegeneration. The extracellular single-unit recordings of responses from the hippocampal and basolateral amygdala neurons to high-frequency stimulation (HFS) of the NBM were performed after intracerebroventricular injection of Aß 25-35. We found that after Aß 25-35 exposure the number of hippocampal neurons exhibiting inhibitory responses to HFS of NBM is decreased. The reverse tendency was seen in the basolateral amygdala inhibitory neural populations, whereas the number of amygdala neurons with excitatory responses decreased. The low intensity of inhibitory and excitatory responses during HFS and post-stimulus period is probably due to the anomalous basal synaptic transmission and excitability of hippocampal and amygdala neurons. These functional changes were accompanied by structural alteration of hippocampal, amygdala, and NBM neurons. We have thus demonstrated that Aß 25-35 induces STP disruption in NBM-hippocampus and NBM-basolateral amygdala circuits as manifested by unbalanced excitatory/inhibitory responses and their frequency. The results of this study may contribute to a better understanding of synaptic integrity. We believe that advancing our understanding of in vivo mechanisms of synaptic plasticity disruption in specific neural circuits could lead to effective drug searches for AD treatment.
RESUMEN
Justicidin A is a structurally defined arylnaphthalide lignan, which has been shown anti-cancer activity; however, the neuroprotective effect of justicidin A is still untested. In this study, we investigated the action of justicidin A on amyloid beta (Aß)25-35-induced neuronal cell death via inhibition of the hyperphosphorylation of tau and induction of autophagy in SH-SY5Y cells. Pretreatment with justicidin A significantly elevated cell viability in cells treated with Aß25-35. Western blot data demonstrated that justicidin A inhibited the Aß25-35-induced up-regulation the levels of hyperphosphorylation of tau in SH-SY5Y cells. In addition, treatment with justicidin A significantly induced autophagy as measured by the increasing LC3 II/I ratio, an important autophagy marker. These studies showed that justicidin A inhibited activity of glycogen synthase kinase-3beta (GSK-3ß), which is an important kinase in up-stream signaling pathways; inhibited hyperphosphorylation of tau in AD; and enhanced activity of AMP-activated protein kinase (AMPK), which is the key molecule for both hyperphosphorylation of tau and induction of autophagy. These data provide the first evidence that justicidin A protects SH-SY5Y cells from Aß25-35-induced neuronal cell death through inhibition of hyperphosphorylation of tau and induction of autophagy via regulation the activity of GSK-3ß and AMPK, and they also provide some insights into the relationship between tau protein hyperphosphorylation and autophagy. Thus, we conclude that justicidin A may have a potential role for neuroprotection and, therefore, may be used as a therapeutic agent for AD.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Autofagia/fisiología , Dioxolanos/farmacología , Lignanos/farmacología , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/toxicidad , Proteínas tau/metabolismo , Autofagia/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Humanos , Neuronas/efectos de los fármacos , Fosforilación/efectos de los fármacos , Fosforilación/fisiologíaRESUMEN
BACKGROUND: Recently, an abnormal deposition of Amyloid-Beta (Aß) was considered the primary cause of the pathogenesis of Alzheimer's disease (AD). And how to inhibit the cytotoxicity is considered an important target for the treatment of AD. Triptolide (TP), a purified diterpenoid from the herb Tripterygium wilfordii Hook.f. (TWHF), has potential neuroprotective effects pertinent to disease of the nervous system. However, whether triptolide and its specific mechanisms have protective functions in differentiated PC12 cells treated with Aß25-35 remain unclear. AIMS: The purpose is to investigate the protective functions of triptolide in Aß25-35-stimulated differentiated PC12 cells. MAIN METHODS: In the study, we use 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) assay, flow cytometry assay, immunohistochemical staining and Western blot to observe the effects of triptolide on cytotoxicity induced by Aß25-35 and its mechanism of oxidative stress. KEY FINDINGS: The result of MTT and LDH assay indicates that triptolide protected PC12 cells against Aß25-35-induced cytotoxicity. The flow cytometry assay shows that triptolide attenuated Aß25-35-induced apoptosis in differentiated PC12 cells. Meanwhile, the results give a clear indication that triptolide could downregulate generation of reactive oxygen species (ROS), hydrogen peroxide (H2O2) and malondialdehyde (MDA) induced by Aß25-35. The apoptotic process triggered by triptolide involved the up-regulation of the activity of superoxide dismutase (SOD). SIGNIFICANCE: The results suggest that triptolide may serve as an important role in the inhibition of the cell apoptosis induced by Aß and the decreased oxidative stress is a key mechanism in the protective effect of triptolide in AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Diterpenos/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Fenantrenos/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Animales , Apoptosis/efectos de los fármacos , Diterpenos/química , Compuestos Epoxi/química , Compuestos Epoxi/farmacología , Peróxido de Hidrógeno/metabolismo , Malondialdehído/metabolismo , Neuronas/metabolismo , Fármacos Neuroprotectores/química , Células PC12 , Fenantrenos/química , Ratas , Especies Reactivas de Oxígeno/metabolismo , Tripterygium/químicaRESUMEN
Sulfuretin, one of the major flavonoid glycosides found in the stem bark of Albizzia julibrissin and heartwood of Rhus verniciflua, is a known anti-oxidant. We previously demonstrated that sulfuretin inhibits neuronal death via reactive oxygen species (ROS)-dependent mechanisms in human SH-SY5Y cells, although other relevant mechanisms of action of this compound remain largely uncharacterized. As part of our ongoing exploration of the pharmacological actions of sulfuretin, we studied the neuroprotective effects of sulfuretin against amyloid beta (Aß)-induced neurotoxicity in human SH-SY5Y and primary hippocampal neuron cells and investigated the possible mechanisms involved. Specifically, we found in the present study that sulfuretin significantly attenuates the decrease in cell viability, release of lactate dehydrogenase, and accumulation of ROS associated with Aß25-35-induced neurotoxicity in neuronal cells. Furthermore, sulfuretin stimulated the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a downstream target of phosphatidylinositol 3-kinases (PI3K)/Akt. We demonstrated that sulfuretin induces the expression of heme oxygenase-1 (HO-1), an anti-oxidant response gene. Notably, we found that the neuroprotective effects of sulfuretin were diminished by an Nrf2 small interfering RNA (siRNA), the HO-1 inhibitor zinc protoporphyrin IX (ZnPP), as well as the PI3K/Akt inhibitor LY294002. Taken together, these results indicated that sulfuretin protects neuronal cells from Aß25-35-induced neurotoxicity through activation of Nrf/HO-1 and PI3K/Akt signaling pathways. Our results also indicate that sulfuretin-induced induction of Nrf2-dependent HO-1 expression via the PI3K/Akt signaling pathway has preventive and/or therapeutic potential for the management of Alzheimer's disease.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Benzofuranos/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/toxicidad , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Flavonoides/farmacología , Técnicas de Silenciamiento del Gen , Hemo Oxigenasa (Desciclizante)/antagonistas & inhibidores , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo-Oxigenasa 1/antagonistas & inhibidores , Hemo-Oxigenasa 1/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Factor 2 Relacionado con NF-E2/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
The 25-35 fragment of the amyloid ß (Aß) peptide is a naturally occurring proteolytic by-product that retains the pathophysiology of its larger parent molecule, whose deposition has been shown to involve mitochondrial dysfunction. Hence, disruption of Aß(25-35) aggregates could afford an effective remedial strategy for Alzheimer's disease (AD). In the present study, the effect of a number of selected small-molecule natural products (polyphenols: resveratrol, quercetin, biochanin A, and indoles: indole-3-acetic acid, indole-3-carbinol (I3C)) on Aß(25-35) fibrillogenesis was explored under physiological conditions, and interaction of the resulting structures with rat brain mitochondria was investigated. Several techniques, including fluorescence, circular dichroism, and transmission electron microscopy were utilized to characterize the aggregation products, and possible mitochondrial membrane permeabilization was determined following release of marker enzymes. Results demonstrate the capacity of Aß(25-35) fibrils to damage mitochondria and suggest how small molecules may afford protection. While I3C appeared more effective in inhibiting the fibrillation process, all natural products behaved similarly in destabilizing preformed aggregates. It is concluded that elucidation of such protection may provide important insights into the development of preventive and therapeutic agents for AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Productos Biológicos/farmacología , Mitocondrias/metabolismo , Fragmentos de Péptidos/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Amiloide/metabolismo , Animales , Productos Biológicos/química , Dicroismo Circular , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Mitocondrias/ultraestructura , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Secundaria de Proteína , Ratas , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Molecular chaperones, or heat shock proteins (HSP), have been implicated in numerous neurodegenerative disorders characterized by the accumulation of protein aggregates, such as Alzheimer disease. The agglomeration of insoluble structures of Aß is thought to be responsible for neuronal death, which in turn leads to the loss of cognitive functions. Recent findings have shown that the induction of HSP decreases the level of abnormal protein aggregates, as well as demonstrating that 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), an analogue of geldanamycin (GA), increases Aß clearance through the induction of molecular chaperones in cell culture. In light of this discovery that HSP overexpression can be neuroprotective, the search for a way to pharmacologically induce the overexpression of HSP and other associated chaperones may lead to a promising approach for the treatment of neurodegenerative diseases. The aim of our study was to evaluate both the effect of 17-AAG on the cognitive process and the HSP response in rats injected with Aß25-35 into the CA1 of the hippocampus. The results show that the injection of Aß caused a significant increase in the expression of the HSP involved in the regulation of cellular proteostasis. While the HSP did not reverse excitotoxic damage, given that experimental subjects showed learning and memory deficits, the administration of 17-AAG prior to the injection of Aß25-35 did show an improvement in the behavioral assessment that correlated with the upregulation of HSP70 in subjects injured with Aß. Overall, our data shows that the pharmacological induction of HSP using 17-AAG may be an alternative treatment of neurodegenerative diseases.
Asunto(s)
Péptidos beta-Amiloides , Benzoquinonas/uso terapéutico , Trastornos del Conocimiento/inducido químicamente , Trastornos del Conocimiento/tratamiento farmacológico , Cognición/efectos de los fármacos , Proteínas de Choque Térmico/biosíntesis , Lactamas Macrocíclicas/uso terapéutico , Nootrópicos/uso terapéutico , Fragmentos de Péptidos , Péptidos beta-Amiloides/antagonistas & inhibidores , Animales , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/patología , Trastornos del Conocimiento/psicología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Fragmentos de Péptidos/antagonistas & inhibidores , Ratas , Ratas WistarRESUMEN
In Alzheimer's disease (AD), amyloid-ß (Aß) deposits in the cerebrovasculature can result in neurovascular dysfunction and/or cerebral amyloid angiopathy. The accumulation of Aß in blood vessels can cause endothelial cell damage, resulting in impaired Aß clearance by the blood-brain barrier. Additionally, impaired endothelial cell function can result in decreased angiogenesis in the brains of AD patients, affecting cognitive function. VEGF is a crucial mediator of angiogenesis and is deficient in AD brains thus promoting angiogenesis could be an important component of successful AD treatment. The C-terminal portion of the extracellular matrix proteoglycan perlecan, Domain V (DV), promotes brain-derived endothelial cell proliferation and is proangiogenic in that it increases VEGFR2 expression and production of VEGF. In this study, we show that Aß25-35 reduces proliferation of a mouse brain microvascular endothelial cell line (MBEC) in vitro and that DV and mouse LG3 (C-terminal fragment of DV) block these effects of Aß25-35. Additionally, we show that DV restores the ability of MBECs to form tube-like structures on Matrigel in the presence of Aß25-35 and that this is α5ß1 dependent. Interestingly, the reduction in tube-like structure formation by Aß25-35 was not due to endothelial cell death, suggesting that Aß25-35 induces the downregulation of a cell surface molecule required for adhesion events critical to the angiogenic process. We propose a model suggesting that DV works through both the α5ß1 integrin receptor and VEGFR2 to increase VEGF production, causing competition with Aß25-35 for VEGFR2 binding, thus ultimately increasing VEGF expression and restoring angiogenesis. This supports DV as a potential anti-amyloid therapy.