RESUMEN
BACKGROUND: The FDA-approved small-molecule drug ibrutinib is an effective targeted therapy for patients with chronic lymphocytic leukemia (CLL). Ibrutinib inhibits Bruton's tyrosine kinase (BTK), a kinase involved in B cell receptor signaling. However, the potential regulation of neuroinflammatory responses in the brain by ibrutinib has not been comprehensively examined. METHODS: BV2 microglial cells were treated with ibrutinib (1 µM) or vehicle (1% DMSO), followed by lipopolysaccharide (LPS; 1 µg/ml) or PBS. RT-PCR, immunocytochemistry, and subcellular fractionation were performed to examine the effects of ibrutinib on neuroinflammatory responses. In addition, wild-type mice were sequentially injected with ibrutinib (10 mg/kg, i.p.) or vehicle (10% DMSO, i.p.), followed by LPS (10 mg/kg, i.p.) or PBS, and microglial and astrocyte activations were assessed using immunohistochemistry. RESULTS: Ibrutinib significantly reduced LPS-induced increases in proinflammatory cytokine levels in BV2 microglial and primary microglial cells but not in primary astrocytes. Ibrutinib regulated TLR4 signaling to alter LPS-induced proinflammatory cytokine levels. In addition, ibrutinib significantly decreased LPS-induced increases in p-AKT and p-STAT3 levels, suggesting that ibrutinib attenuates LPS-induced neuroinflammatory responses by inhibiting AKT/STAT3 signaling pathways. Interestingly, ibrutinib also reduced LPS-induced BV2 microglial cell migration by inhibiting AKT signaling. Moreover, ibrutinib-injected wild-type mice exhibited significantly reduced microglial/astrocyte activation and COX-2 and IL-1ß proinflammatory cytokine levels. CONCLUSIONS: Our data provide insights on the mechanisms of a potential therapeutic strategy for neuroinflammation-related diseases.
Asunto(s)
Antiinflamatorios/uso terapéutico , Citocinas/metabolismo , Inflamación/tratamiento farmacológico , Microglía/efectos de los fármacos , Pirazoles/uso terapéutico , Pirimidinas/uso terapéutico , Adenina/análogos & derivados , Animales , Animales Recién Nacidos , Línea Celular Transformada , Células Cultivadas , Medio de Cultivo Libre de Suero/farmacología , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Citocinas/genética , Modelos Animales de Enfermedad , Compuestos Heterocíclicos con 3 Anillos/farmacología , Inflamación/inducido químicamente , Lipopolisacáridos/efectos adversos , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/citología , Piperidinas , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/química , Pirimidinas/química , Ratas , Transducción de Señal/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacosRESUMEN
BACKGROUND: Neuroinflammation is associated with neurodegenerative diseases, including Alzheimer's disease (AD). Thus, modulating the neuroinflammatory response represents a potential therapeutic strategy for treating neurodegenerative diseases. Several recent studies have shown that dopamine (DA) and its receptors are expressed in immune cells and are involved in the neuroinflammatory response. Thus, we recently developed and synthesized a non-self-polymerizing analog of DA (CA140) and examined the effect of CA140 on neuroinflammation. METHODS: To determine the effects of CA140 on the neuroinflammatory response, BV2 microglial cells were pretreated with lipopolysaccharide (LPS, 1 µg/mL), followed by treatment with CA140 (10 µM) and analysis by reverse transcription-polymerase chain reaction (RT-PCR). To examine whether CA140 alters the neuroinflammatory response in vivo, wild-type mice were injected with both LPS (10 mg/kg, intraperitoneally (i.p.)) and CA140 (30 mg/kg, i.p.), and immunohistochemistry was performed. In addition, familial AD (5xFAD) mice were injected with CA140 or vehicle daily for 2 weeks and examined for microglial and astrocyte activation. RESULTS: Pre- or post-treatment with CA140 differentially regulated proinflammatory responses in LPS-stimulated microglia and astrocytes. Interestingly, CA140 regulated D1R levels to alter LPS-induced proinflammatory responses. CA140 significantly downregulated LPS-induced phosphorylation of ERK and STAT3 in BV2 microglia cells. In addition, CA140-injected wild-type mice exhibited significantly decreased LPS-induced microglial and astrocyte activation. Moreover, CA140-injected 5xFAD mice exhibited significantly reduced microglial and astrocyte activation. CONCLUSIONS: CA140 may be beneficial for preventing and treating neuroinflammatory-related diseases, including AD.
Asunto(s)
Enfermedad de Alzheimer/complicaciones , Antiinflamatorios/uso terapéutico , Dopamina/análogos & derivados , Encefalitis/tratamiento farmacológico , Encefalitis/etiología , Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/genética , Animales , Animales Recién Nacidos , Encéfalo/metabolismo , Encéfalo/patología , Células Cultivadas , Modelos Animales de Enfermedad , Dopamina/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/efectos de los fármacos , Mutación/genética , Proteínas del Tejido Nervioso/metabolismo , Polisacáridos/farmacología , Presenilina-1/genética , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacosRESUMEN
Mucin 1 (Muc1) is a glycoprotein expressed on most epithelial cell surfaces, which has been confirmed as a useful biomarker for the diagnosis of early cancers. In this study, we demonstrate that a quantum dot (QD)-aptamer beacon acts by folding-induced dissociation of a DNA intercalating dye, BOBO-3, in the presence of the target molecules, Muc1. Release of intercalated BOBO-3s from the QD-conjugated aptamers results in a decrease in QD fluorescence resonance energy transfer (FRET)-mediated BOBO-3 emission, allowing for label-free Muc1 detection and quantitation. We attain highly specific and wide-range detection (from 50nM to 20µM) of Muc1, suggesting that our QD-aptamer beacon can be a potential alternative to immuno-based assays for Muc1 detection. The detection methodology is expected to be improved for the early diagnosis of different types of epithelial cancers of large populations.
Asunto(s)
Biomarcadores de Tumor/análisis , Sondas Moleculares/análisis , Mucina-1/análisis , Neoplasias Glandulares y Epiteliales/química , Aptámeros de Nucleótidos/análisis , Aptámeros de Nucleótidos/química , Carbocianinas/análisis , Carbocianinas/química , Transferencia Resonante de Energía de Fluorescencia , Humanos , Sondas Moleculares/química , Neoplasias Glandulares y Epiteliales/diagnóstico , Puntos CuánticosRESUMEN
We previously demonstrated that ibrutinib modulates LPS-induced neuroinflammation in vitro and in vivo, but its effects on the pathology of Alzheimer's disease (AD) and cognitive function have not been investigated. Here, we investigated the effects of ibrutinib in two mouse models of AD. In 5xFAD mice, ibrutinib injection significantly reduced Aß plaque levels by promoting the non-amyloidogenic pathway of APP cleavage, decreased Aß-induced neuroinflammatory responses, and significantly downregulated phosphorylation of tau by reducing levels of phosphorylated cyclin-dependent kinase-5 (p-CDK5). Importantly, tau-mediated neuroinflammation and tau phosphorylation were also alleviated by ibrutinib injection in PS19 mice. In 5xFAD mice, ibrutinib improved long-term memory and dendritic spine number, whereas in PS19 mice, ibrutinib did not alter short- and long-term memory but promoted dendritic spinogenesis. Interestingly, the induction of dendritic spinogenesis by ibrutinib was dependent on the phosphorylation of phosphoinositide 3-kinase (PI3K). Overall, our results suggest that ibrutinib modulates AD-associated pathology and cognitive function and may be a potential therapy for AD.
Asunto(s)
Adenina/análogos & derivados , Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/metabolismo , Encéfalo/patología , Cognición , Inflamación/patología , Piperidinas/farmacología , Proteínas tau/metabolismo , Adenina/farmacología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/fisiopatología , Cognición/efectos de los fármacos , Quinasa 5 Dependiente de la Ciclina/metabolismo , Citocinas/metabolismo , Espinas Dendríticas/efectos de los fármacos , Espinas Dendríticas/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Gliosis/complicaciones , Mediadores de Inflamación/metabolismo , Memoria a Largo Plazo/efectos de los fármacos , Ratones Transgénicos , Neurogénesis/efectos de los fármacos , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuroglía/patología , Fosforilación/efectos de los fármacos , Placa Amiloide/patologíaRESUMEN
Recent studies have shown that Liuwei Dihuang pills (LWPs) can positively affect learning, memory and neurogenesis. However, the underlying molecular mechanisms are not understood. In the present study, we developed ALWPs, a mixture of Antler and LWPs, and investigated whether ALWPs can affect neuroinflammatory responses. We found that ALWPs (500 mg/ml) inhibited lipopolysaccharide (LPS)-induced proinflammatory cytokine IL-1ß mRNA levels in BV2 microglial cells but not primary astrocytes. ALWPs significantly reduced LPS-induced cell-surface levels of TLR4 to alter neuroinflammation. An examination of the molecular mechanisms by which ALWPs regulate the LPS-induced proinflammatory response revealed that ALWPs significantly downregulated LPS-induced levels of FAK phosphorylation, suggesting that ALWPs modulate FAK signaling to alter LPS-induced IL-1ß levels. In addition, treatment with ALWPs followed by LPS resulted in decreased levels of the transcription factor NF-κB in the nucleus compared with LPS alone. Moreover, ALWPs significantly suppressed LPS-induced BV2 microglial cell migration. To examine whether ALWPs modulate learning and memory in vivo, wild-type C57BL/6J mice were orally administered ALWPs (200 mg/kg) or PBS daily for 3 days, intraperitoneally injected (i.p.) with LPS (250 µg/kg) or PBS, and assessed in Y maze and NOR tests. We observed that oral administration of ALWPs to LPS-injected wild-type C57BL/6J mice significantly rescued short- and long-term memory. More importantly, oral administration of ALWPs to LPS-injected wild-type C57BL/6J mice significantly reduced microglial activation in the hippocampus and cortex. Taken together, our results suggest that ALWPs can suppress neuroinflammation-associated cognitive deficits and that ALWPs have potential as a drug for neuroinflammation/neurodegeneration-related diseases, including Alzheimer's disease (AD).
RESUMEN
Oleamide was first known as a sleep-inducing fatty acid amide, and later shown to have wide range of neuropharmacological effects upon different neurochemical systems. However, the effects of oleamide on brain damage have scarcely been studied, and the molecular mechanisms and sites of its action remain elusive. Kainic acid (KA) has been used to produce an epileptic animal model that mimics human temporal lobe epilepsy and to induce calpain-activated excitotoxicity, which occurs in numerous neurodegenerative disorders. In this study, we examined whether oleamide protects against the KA-induced excitotoxic brain damage accompanied by behavioral seizure activity and neuronal cell death. Moreover, whether these effects of oleamide were mediated by calpain activity-related cellular mechanisms was investigated. KA-induced epileptic rats were produced by an intrastriatal injection of KA (5 nmole). Oral administration of oleamide (0.5, 2, and 10 mg/kg) 30 min prior to the KA injection showed dose-dependent inhibition of the KA-induced behavioral seizure activities that were monitored starting from 60 to 180 min post-surgery. Further repetitive oral administration of oleamide (once per day) for the next 4 consecutive days post-KA injection produced significant neuroprotection against the disrupted neuronal integrity that resulted from KA-induced excitotoxic damage that was also demonstrated by staining of striatal tissue sections with cresyl violet, hematoxylin/eosin, and fluoro-Jade B. In addition, oleamide blocked the KA-induced cleavage of cyclin-dependent kinase-5 coactivator (Cdk5-p35) and collapsin response mediator protein-2, which are believed to be mediated by calpain activation in striatal tissues dissected from KA-induced epileptic rats. Oleamide also reversed the KA-induced reduction in expression of an endogenous calpain inhibitory protein, calpastatin, and a marker of synaptic activity, synapsin-II. The hypothesis that oleamide could induce direct calpain inhibition was further investigated using in vitro calpain assays in both brain tissue and a cell-free and calpain-overexpressed neuronal cell system. These findings together suggest that oleamide has protective effects against excitotoxicity-induced neuronal death and behavioral seizure, partly via its direct calpain inhibitory activity.
RESUMEN
Collapsin response mediator protein (CRMP)-2 and the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway are associated with common physiological functions such as neuronal polarity, axonal outgrowth and synaptic strength, as well as various brain disorders including epilepsy. But, their regulatory and functional links are unclear. Alterations in CRMP-2 expression that lead to its functional changes are implicated in brain disorders such as epilepsy. Here, we investigate whether changes in CRMP-2 expression, possibly regulated by mTOR-related signaling, correlates with neuronal growth and viability. Inhibition of mTOR and/or phosphoinositol-3-kinase (PI3K) led to deceased p-S6K, and p-S6 signals also reduced CRMP-2 expression. These changes corresponded to inhibition of neuronal viability and proliferation in cultured hippocampal HT-22 cells under both basal serum-free and serum- or insulin-induced mTOR pathway-activated conditions. CRMP-2 expression tended to be increased by mTOR activation, indicated by an increase in p-S6/S6 level, in pentylentetrazole (PTZ)-induced epileptic rat hippocampal tissues was also significantly reduced by mTOR inhibition. Knockdown of CRMP-2 by si-RNA reduced the neuronal viability without changes in mTOR signaling, and overexpression of CRMP-2 recovered the glutamate-induced neurotoxicity and decrease of mTOR signaling in HT-22 cells. In conclusion, CRMP-2 protein expression controlled by the PI3K-mTOR-S6K signaling axis exerts its important functional roles in neuronal growth and survival.