RESUMEN
The double-stranded RNA-dependent protein kinase (PKR) is switched on by a wide range of stimuli, including the amyloid peptide. Then, PKR transmits signals to the translational machinery, apoptosis and inflammatory signaling pathways by interacting with some adapters. In virus-infected cells, PKR engages the nucleus factor κB (NF-κB) pathway. In many models of Alzheimer's disease (AD) and patients with AD, PKR was activated. Furthermore, there is strong evidence implicating the inflammatory process in the AD brain. However, the PKR involvement in inflammatory responses in AD is not elucidated. Based on our previous in vitro results, the aim of this study was to evaluate the effects of a pharmacological inhibition of PKR in inflammation in APPswePS1dE9 transgenic mice. Our results showed that PKR inhibition prevented the NF-κB activation and production of tumor necrosis factor alpha (TNFα) and interleukin (IL)-1ß at 12 months of age without decrease of Aß42 levels and memory deficits. Surprisingly, PKR inhibition failed to prevent IL-1ß- mediated inflammation and induced a great increase in ß-amyloid peptide (Aß42) levels at 18 months of age. In this model, our findings highlight the lack of relationship between inflammation and Aß42 levels. Moreover, the age-dependent inflammatory response must be carefully taken into account in the establishment of an anti-inflammatory therapy in AD.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/metabolismo , Regulación hacia Arriba/genética , eIF-2 Quinasa/antagonistas & inhibidores , Envejecimiento/genética , Envejecimiento/patología , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/genética , Animales , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Encéfalo/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Transgénicos , Fragmentos de Péptidos/genética , eIF-2 Quinasa/metabolismoRESUMEN
For 10 years, research has focused on signaling pathways controlling translation to explain neuronal death in Alzheimer Disease (AD). Previous studies demonstrated in different cellular and animal models and AD patients that translation is down-regulated by the activation of double-stranded RNA-dependent protein kinase (PKR). Among downstream factors of PKR, the Fas-associated protein with a death domain (FADD) and subsequent activated caspase-8 are responsible for PKR-induced apoptosis in recombinant virus-infected cells. However, no studies have reported the role of PKR in death receptor signaling in AD. The aim of this project is to determine physical and functional interactions of PKR with FADD in amyloid-beta peptide (Abeta) neurotoxicity and in APP(SL)PS1 KI transgenic mice. In SH-SY5Y cells, results showed that Abeta42 induced a large increase in phosphorylated PKR and FADD levels and a physical interaction between PKR and FADD in the nucleus, also observed in the cortex of APP(SL)PS1 KI mice. However, PKR gene silencing or treatment with a specific PKR inhibitor significantly prevented the increase in pT(451)-PKR and pS(194)-FADD levels in SH-SY5Y nuclei and completely inhibited activities of caspase-3 and -8. The contribution of PKR in neurodegeneration through the death receptor signaling pathway may support the development of therapeutics targeting PKR to limit neuronal death in AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/farmacología , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Transducción de Señal/efectos de los fármacos , eIF-2 Quinasa/metabolismo , Enfermedad de Alzheimer/inducido químicamente , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/metabolismo , Animales , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 8/genética , Caspasa 8/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Corteza Cerebral/metabolismo , Inhibidores Enzimáticos/farmacología , Proteína de Dominio de Muerte Asociada a Fas/genética , Ratones , Ratones Transgénicos , Fosforilación/efectos de los fármacos , Fosforilación/genética , Transducción de Señal/genética , eIF-2 Quinasa/antagonistas & inhibidores , eIF-2 Quinasa/genéticaRESUMEN
Alzheimer's disease (AD), a neurodegenerative disorder, is the most common form of dementia in the elderly individuals. Among the pathogenic mechanisms in AD, chronic systemic inflammation is described and characterized by massive production of proinflammatory cytokines by peripheral blood mononuclear cells (PBMCs), which may contribute to an altered immune response and exacerbation of neurodegeneration. Studies have also reported increased double-stranded RNA-dependent protein kinase (PKR) activation in the PBMCs of patients with AD. Interestingly, PKR could be involved in NF-κB activation, leading to production of a wide range of cytokines. We proposed to decrease proinflammatory cytokines production and release by treating the PBMCs in 25 patients with AD with a specific inhibitor of PKR. Our results showed that PKR inhibition greatly decreased tumor necrosis factor , interleukin (IL)-1α, IL-1ß, and IL-6 production and release but did not affect the chemokine RANTES. Moreover, inhibition of the proinflammatory factors was correlated with prevention of caspase-3 activation. These results indicated that specific inhibition of PKR at the peripheral level might decrease the inflammatory response in AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Citocinas/antagonistas & inhibidores , Leucocitos Mononucleares/enzimología , Inhibidores de Proteínas Quinasas/farmacología , eIF-2 Quinasa/antagonistas & inhibidores , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/patología , Caspasa 3/metabolismo , Inhibidores de Caspasas , Línea Celular Tumoral , Supervivencia Celular/fisiología , Células Cultivadas , Quimiocina CCL5/biosíntesis , Quimiocina CCL5/metabolismo , Citocinas/biosíntesis , Citocinas/metabolismo , Femenino , Humanos , Inflamación/enzimología , Inflamación/patología , Inflamación/prevención & control , Leucocitos Mononucleares/efectos de los fármacos , Masculino , FN-kappa B/antagonistas & inhibidores , FN-kappa B/fisiología , eIF-2 Quinasa/metabolismoRESUMEN
The control of translation is disturbed in Alzheimer's disease (AD). This study analysed the crosslink between the up regulation of double-stranded RNA-dependent-protein kinase (PKR) and the down regulation of mammalian target of rapamycin (mTOR) signalling pathways via p53, the protein Regulated in the Development and DNA damage response 1 (Redd1) and the tuberous sclerosis complex (TSC2) factors in two beta-amyloid peptide (Abeta) neurotoxicity models. In SH-SY5Y cells, Abeta42 induced an increase of P(T451)-PKR and of the ratio p66/(p66+p53) in nuclei and a physical interaction between these proteins. Redd1 gene levels increased and P(T1462)-TSC2 decreased. These disturbances were earlier in rat primary neurons with nuclear co-localization of Redd1 and PKR. The PKR gene silencing in SH-SY5Y cells prevented these alterations. p53, Redd1 and TSC2 could represent the molecular links between PKR and mTOR in Abeta neurotoxicity. PKR could be a critical target in a therapeutic program of AD.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/toxicidad , Proteínas Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Supresoras de Tumor/metabolismo , eIF-2 Quinasa/metabolismo , Análisis de Varianza , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Corteza Cerebral/citología , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Embrión de Mamíferos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Inmunoprecipitación/métodos , Neuroblastoma/patología , Neuronas/citología , Fosforilación/efectos de los fármacos , Proteínas Quinasas/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Wistar , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Serina-Treonina Quinasas TOR , Factores de Transcripción , Transfección/métodos , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , eIF-2 Quinasa/genéticaRESUMEN
Previous studies demonstrated that the PKR (double-stranded RNA-activated protein kinase) pathway was activated while the mTOR (mammalian target of rapamycin) pathway was inhibited in Alzheimer's disease (AD). Here, we analysed upstream and downstream factors of mTOR in brain of APP(SL)/PS1 KI mice displaying a massive neuronal loss in hippocampus. While mTOR levels were not modified, we found a great activation of Akt with a robust accumulation of P-Akt((T308)) in non-apoptotic neurons at 6 months of age. At the opposite, a significant decrease of the p70/85S6K activation was observed in brain of PS1 KI and APP(SL)/PS1 KI mice with a very weak or no nucleocytoplasmic P-p70/85S6K((T389)) staining in apoptotic neurons of APP(SL)/PS1 KI mice. Furthermore, the activation of Erk1/2, 4E-BP1 and p70S6K((T421/S424)) (substrate of Erk1/2), except eIF4E, was not modified. These findings demonstrate a clear dissociation between Akt and ribosomal S6K signaling markers in these mice which could be involved in the AD pathological process.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Encéfalo/metabolismo , Proteína Oncogénica v-akt/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal/fisiología , Factores de Edad , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/genética , Animales , Modelos Animales de Enfermedad , Regulación Enzimológica de la Expresión Génica/fisiología , Etiquetado Corte-Fin in Situ , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Presenilina-1/genética , Transducción de Señal/genéticaRESUMEN
Fluoro-Jade B is known as a high affinity fluorescent marker for the localization of neuronal degeneration during acute neuronal distress. However, one study suggested that fluoro-Jade B stains reactive astroglia in the primate cerebral cortex. In this study, we analyzed the staining of fluoro-Jade B alone or combined with specific markers for detection of glial fibrillary acidic protein (GFAP) or activated CD68 microglia in the double APP(SL)/PS1 KI transgenic mice of Alzheimer's disease (AD), which display a massive neuronal loss in the CA1 region of the hippocampus. Our results showed that fluoro-Jade B did not stain normal and degenerating neurons in this double mouse transgenic model. Fluoro-Jade B was co-localized with Abeta in the core of amyloid deposits and in glia-like cells expressing Abeta. Furthermore, fluoro-Jade B was co-localized with CD68/macrosialin, a specific marker of activated microglia, and with GFAP for astrocytes in APP(SL)/PS1 KI transgenic mice of AD. Taken together, these findings showed that fluoro-Jade B can be used to label activated microglia and astrocytes which are abundant in the brain of these AD transgenic mice. It could stain degenerating neurons as a result of acute insult while it could label activated microglia and astrocytes during a chronic neuronal degenerative process such as AD for example.
Asunto(s)
Enfermedad de Alzheimer/patología , Astrocitos/patología , Microglía/patología , Factores de Edad , Secretasas de la Proteína Precursora del Amiloide/genética , Péptidos beta-Amiloides/metabolismo , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Astrocitos/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Fluoresceínas , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Inmunohistoquímica/métodos , Ratones , Ratones Transgénicos , Microglía/metabolismo , Compuestos Orgánicos , Fosfopiruvato Hidratasa/metabolismo , Presenilina-1/genéticaRESUMEN
The neuronal dopamine transporter (DAT) is a presynaptic plasma membrane protein mediating the re-uptake of dopamine released from synaptic cleft into the nerve terminals. While the regulation of its activity by protein kinase C signalling is well-characterized, there is controversial debate about its regulation by protein kinase A (PKA) signalling. In rat striatal synaptosomes, we showed that a cell-permeable cyclic adenosine 3',5'-monophosphate analogue up-regulated the DAT capacity without modification of its efficiency. This acute effect was PKA-, calcium calmodulin dependent kinase II- and phosphatase-dependent. Together, these results suggest that the activity of DAT may depend on a state of the transporter with both specific phosphorylated and dephosphorylated sites.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Neostriado/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Regulación hacia Arriba/efectos de los fármacos , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Dopamina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Técnicas In Vitro , Masculino , Neostriado/efectos de los fármacos , Neostriado/enzimología , Ratas , Transducción de Señal/efectos de los fármacos , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismoRESUMEN
The vinyl ether bond of plasmalogens could be among the first target of free radicals attack. Consequently, because of their location in the membranes of cells, plasmalogens represent a first shield against oxidative damages by protecting other macromolecules and are often considered as antioxidant molecules. However, under oxidative conditions their disruption leads to the release of fatty aldehydes. In this paper, we showed using gas chromatography-mass spectrometry (GC-MS) analyses that fatty aldehydes released from plasmalogens after oxidation (UV irradiation and Fe2+/ascorbate) of cerebral cortex homogenates can generate covalent modifications of endogenous macromolecules such as phosphatidylethanolamine (PE), like the very reactive and toxic malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE). These newly formed Schiff base adducts could be responsible for deleterious effects on cells thus making the protective role of plasmalogens potentially questionable.
Asunto(s)
Aldehídos/metabolismo , Encéfalo/metabolismo , Ácidos Grasos/metabolismo , Fosfatidiletanolaminas/metabolismo , Plasmalógenos/metabolismo , Animales , Ácido Ascórbico/metabolismo , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Ácidos Grasos/análisis , Cromatografía de Gases y Espectrometría de Masas , Radical Hidroxilo/metabolismo , Hierro/metabolismo , Peroxidación de Lípido , Modelos Químicos , Estrés Oxidativo/fisiología , Plasmalógenos/química , Ratas , Oxígeno Singlete/metabolismo , Factores de Tiempo , Rayos UltravioletaRESUMEN
Several experimental studies have shown that acidosis impairs neurotransmitter uptake processes. The purpose of this study was to determine the mechanism underlying acidosis-induced alterations of the high-affinity dopamine (DA) uptake in rat striatal synaptosomes and slices. Acidosis (pH 5.5) performed either by lactic acid or phosphoric acid induced a decrease in the high-affinity DA uptake in the two striatal models, slices being lesser affected than synaptosomes. Addition of the acid prior to uptake measurement led to a strong reduction of the DA uptake velocity. This early inhibitory effect was completely reversed when acid was removed from the medium by washings. Conversely, when slices and synaptosomes were pre-incubated for different times with each acid, DA uptake remained inhibited in spite of washings. This later inhibition was accompanied by the production of thiobarbituric acid reactive substances, a marker of lipid peroxidation, and was partially prevented by the antioxidant Trolox. Taken together, these results suggest that acidosis, in a degree encountered during ischemia, alters the high-affinity DA uptake by at least two ways: an early and direct effect of H(+) ions on the DA transporters, and subsequently an inhibition partially mediated by free radical damage.