RESUMO
Aggregation of beta-amyloid protein (Abeta) to form oligomers is considered to be a key step in generating neurotoxicity in the Alzheimer's disease brain. Agents that bind to Abeta and inhibit oligomerization have been proposed as Alzheimer's disease therapeutics. In this study, we investigated the binding of fluorescein-labeled Abeta(1-42) (FluoAbeta(1-42)) to SH-SY5Y neuroblastoma cells and examined the effect of the 39-kDa receptor-associated protein (RAP), on the Abeta cell interaction. FluoAbeta(1-42) bound to the cells in a punctate pattern. Surprisingly, when RAP was added to the incubations, FluoAbeta(1-42) and RAP were found to be co-localized on the cell surface, suggesting that RAP and Abeta may bind to each other. Experiments using the purified proteins confirmed that a RAP-Abeta complex was stable and resistant to sodium dodecyl sulfate. RAP also inhibited Abeta oligomerization. We next examined whether RAP could inhibit the neurotoxic effects of Abeta. Addition of Abeta(1-42) to SH-SY5Y cells caused an increase in intracellular Ca2+ that was inhibited by treatment of the Abeta peptide with RAP. RAP also blocked an Abeta-induced inhibition of long-term memory consolidation in 1-day-old chicks. This study demonstrates that RAP binds to Abeta and is an inhibitor of the neurotoxic effects of Abeta.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Proteína Associada a Proteínas Relacionadas a Receptor de LDL/uso terapêutico , Síndromes Neurotóxicas/tratamento farmacológico , Síndromes Neurotóxicas/metabolismo , Peptídeos beta-Amiloides/farmacologia , Análise de Variância , Animais , Animais Recém-Nascidos , Aprendizagem da Esquiva/efeitos dos fármacos , Comportamento Animal , Linhagem Celular Tumoral , Galinhas , Aprendizagem por Discriminação/efeitos dos fármacos , Modelos Animais de Doenças , Citometria de Fluxo/métodos , Fluoresceína/metabolismo , Humanos , Imunoprecipitação/métodos , Proteína Associada a Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteína Associada a Proteínas Relacionadas a Receptor de LDL/farmacologia , Memória/efeitos dos fármacos , Microscopia de Força Atômica/métodos , Microscopia Confocal/métodos , Peso Molecular , Neuroblastoma , Síndromes Neurotóxicas/fisiopatologia , Proteínas Nucleares/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Transporte Proteico/efeitos dos fármacosRESUMO
Neuritic abnormalities are a major hallmark of Alzheimer's disease (AD) pathology. Accumulation of beta-amyloid protein (Abeta) in the brain causes changes in neuritic processes in individuals with this disease. In this study, we show that Abeta decreases neurite outgrowth from SH-SY5Y human neuroblastoma cells. To explore molecular pathways by which Abeta alters neurite outgrowth, we examined the activation and localization of RhoA and Rac1 which regulate the level and phosphorylation of the collapsin response mediator protein-2 (CRMP-2). Abeta increased the levels of the GTP-bound (active) form of RhoA in SH-SY5Y cells. This increase in GTP-RhoA correlated with an increase in an alternatively spliced form of CRMP-2 (CRMP-2A) and its threonine phosphorylated form. Both a constitutively active form of Rac1 (CA-Rac1) and the Rho kinase inhibitor, Y27632, decreased levels of the CRMP-2A variant and decreased threonine phosphorylation caused by Abeta stimulation. The amount of tubulin bound to CRMP-2 was decreased in the presence of Abeta but Y27632 increased the levels of tubulin bound to CRMP-2. Increased levels of both RhoA and CRMP-2 were found in neurons surrounding amyloid plaques in the cerebral cortex of the APP(Swe) Tg2576 mice. We found that there was an increase in threonine phosphorylation of CRMP-2 in Tg2576 mice and the increase correlated with a decrease in the ability of CRMP-2 to bind tubulin. The results suggest that Abeta-induced neurite outgrowth inhibition may be initiated through a mechanism in which Abeta causes an increase in Rho GTPase activity which, in turn, phosphorylates CRMP-2 to interfere with tubulin assembly in neurites.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuritos/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/fisiologia , Doença de Alzheimer/patologia , Animais , Encéfalo/metabolismo , Tamanho Celular/efeitos dos fármacos , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Neuritos/metabolismo , Neuritos/ultraestrutura , Fosforilação , Transdução de Sinais , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Células Tumorais Cultivadas , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Accumulation of the amyloid protein (Abeta) in the brain is an important step in the pathogenesis of Alzheimer's disease. However, the mechanism by which Abeta exerts its neurotoxic effect is largely unknown. It has been suggested that the peptide can bind to the alpha7 nicotinic acetylcholine receptor (alpha7nAChR). In this study, we examined the binding of Abeta1-42 to endogenous and recombinantly expressed alpha7nAChRs. Abeta1-42 did neither inhibit the specific binding of alpha7nAChR ligands to rat brain homogenate or slice preparations, nor did it influence the activity of alpha7nAChRs expressed in Xenopus oocytes. Similarly, Abeta1-42 did not compete for alpha-bungarotoxin-binding sites on SH-SY5Y cells stably expressing alpha7nAChRs. The effect of the Abeta1-42 on tau phosphorylation was also examined. Although Abeta1-42 altered tau phosphorylation in alpha7nAChR-transfected SH-SY5Y cells, the effect of the peptide was unrelated to alpha7nAChR expression or activity. Binding studies using surface plasmon resonance indicated that the majority of the Abeta bound to membrane lipid, rather than to a protein component. Fluorescence anisotropy experiments indicated that Abeta may disrupt membrane lipid structure or fluidity. We conclude that the effects of Abeta are unlikely to be mediated by direct binding to the alpha7nAChR. Instead, we speculate that Abeta may exert its effects by altering the packing of lipids within the plasma membrane, which could, in turn, influence the function of a variety of receptors and channels on the cell surface.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Lipídeos de Membrana/metabolismo , Receptores Nicotínicos/metabolismo , Aconitina/análogos & derivados , Aconitina/metabolismo , Animais , Bungarotoxinas/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Feminino , Polarização de Fluorescência , Hipocampo/metabolismo , Humanos , Fluidez de Membrana/efeitos dos fármacos , Neuroblastoma/metabolismo , Oócitos/metabolismo , Ratos , Ratos Sprague-Dawley , Ressonância de Plasmônio de Superfície , Transfecção , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa7 , Proteínas tau/metabolismoRESUMO
The beta-amyloid protein precursor (APP) has been extensively studied for its role in amyloid production and the pathogenesis of Alzheimer's disease (AD). However, little is known about the normal function of APP and its biological interactions. In this Mini-Review, the role of the cytoplasmic domain of APP in APP trafficking and proteolysis is described. These studies suggest that proteins that bind to the cytoplasmic domain may be important targets for drug development in AD.