RESUMO
Amyloid beta (Abeta) is a metabolic product of Abeta precursor protein (APP). Deposition of Abeta in the brain and neuronal degeneration are characteristic hallmarks of Alzheimer's disease (AD). Abeta induces neuronal degeneration, but the mechanism of neurotoxicity remains elusive. Increasing evidence implicates APP as a receptor-like protein for Abeta fibrils (fAbeta). In this study, we present further experimental support for the direct interaction of APP with fAbeta and for its involvement in Abeta neurotoxicity. Using recombinant purified holo-APP (h-APP), we have shown that it directly binds fAbeta. Employing deletion mutant forms of APP, we show that two different sequences are involved in the binding of APP to fAbeta. One sequence in the n-terminus of APP is required for binding of fAbeta to secreted APP (s-APP) but not to h-APP. In addition, the extracellular juxtamembrane Abeta-sequence mediates binding of fAbeta to h-APP but not to s-APP. Deletion of the extracellular juxtamembrane Abeta sequence abolishes abnormal h-APP accumulation and toxicity induced by fAbeta deposition, whereas deletions in the n-terminus of APP do not affect Abeta toxicity. These experiments show that interaction of toxic Abeta species with its membrane-anchored parental protein promotes toxicity in hippocampal neurons, adding further support to an Abeta-receptor-like function of APP directly implicated in neuronal degeneration in AD.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Precursor de Proteína beta-Amiloide/metabolismo , Hipocampo/citologia , Neurônios/efeitos dos fármacos , Precursor de Proteína beta-Amiloide/genética , Análise de Variância , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Cricetinae , Cricetulus , Embrião de Mamíferos , Proteínas de Fluorescência Verde/genética , Humanos , Imunoprecipitação/métodos , Mutação/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Ratos , TransfecçãoRESUMO
Amyloid beta (Abeta) is a metabolic product of amyloid-beta precursor protein (APP). Deposition of Abeta in the brain and neuronal degeneration are characteristic hallmarks of Alzheimer's disease (AD). Abeta induces neuronal degeneration, but the mechanism of neurotoxicity remains elusive. Here we show that overexpression of APP renders hippocampal neurons vulnerable to Abeta toxicity. Deletion of the extracellular Abeta sequence of APP prevents binding of APP to Abeta, and abolishes toxicity. Abeta toxicity is also abrogated by deletion of the cytoplasmic domain of APP, or by deletions comprising the Go protein-binding sequence of APP. Treatment with Pertussis toxin (PTX) abrogates APP-dependent toxicity of Abeta. Overexpression of PTX-insensitive Galpha-o subunit, but not Galpha-i subunit, of G protein restores Abeta toxicity in the presence of PTX, and this requires the integrity of APP-binding site for Go protein. Altogether, these experiments indicate that interaction of APP with toxic Abeta-species promotes toxicity in hippocampal neurons by a mechanism that involves APP-mediated Go protein activation, revealing an Abeta-receptor-like function of APP directly implicated in neuronal degeneration in AD.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/toxicidade , Animais , Sítios de Ligação/genética , Encéfalo/fisiopatologia , Células Cultivadas , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Humanos , Degeneração Neural/genética , Degeneração Neural/metabolismo , Degeneração Neural/fisiopatologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Toxina Pertussis/farmacologia , Ligação Proteica/genética , Estrutura Terciária de Proteína/fisiologia , RatosRESUMO
Deposition of fibrillar amyloid beta (fAbeta) plays a critical role in Alzheimer's disease (AD). We have shown recently that fAbeta-induced dystrophy requires the activation of focal adhesion proteins and the formation of aberrant focal adhesion structures, suggesting the activation of a mechanism of maladaptative plasticity in AD. Focal adhesions are actin-based structures that provide a structural link between the extracellular matrix and the cytoskeleton. To gain additional insight in the molecular mechanism of neuronal degeneration in AD, here we explored the involvement of LIM kinase 1 (LIMK1), actin-depolymerizing factor (ADF), and cofilin in Abeta-induced dystrophy. ADF/cofilin are actin-binding proteins that play a central role in actin filament dynamics, and LIMK1 is the kinase that phosphorylates and thereby inhibits ADF/cofilin. Our data indicate that treatment of hippocampal neurons with fAbeta increases the level of Ser3-phosphorylated ADF/cofilin and Thr508-phosphorylated LIMK1 (P-LIMK1), accompanied by a dramatic remodeling of actin filaments, neuritic dystrophy, and neuronal cell death. A synthetic peptide, S3 peptide, which acts as a specific competitor for ADF/cofilin phosphorylation by LIMK1, inhibited fAbeta-induced ADF/cofilin phosphorylation, preventing actin filament remodeling and neuronal degeneration, indicating the involvement of LIMK1 in Abeta-induced neuronal degeneration in vitro. Immunofluorescence analysis of AD brain showed a significant increase in the number of P-LIMK1-positive neurons in areas affected with AD pathology. P-LIMK1-positive neurons also showed early signs of AD pathology, such as intracellular Abeta and pretangle phosphorylated tau. Thus, LIMK1 activation may play a key role in AD pathology.