RESUMEN
Amyloid beta-peptide (Abeta), a causative molecule in the pathogenesis of Alzheimer's disease and the main component of senile plaques, is known to be neurotoxic in vitro and in vivo. The mechanisms involved in this Ab-mediated neurotoxicity are not fully understood, although there is evidence to suggest the involvement of oxidative stress, alterations in calcium homeostasis, and/or of CDK activators. Many studies have suggested that Ab may exert its toxic effect via the activation of transcription factors. Therefore, we investigated Ab- responsive genes in human neuroblastoma CHP134 cells using 3.1K human DNA microarrays. Among the several genes overexpressed or repressed by Ab, RTP801, Hi95/sestrin 2, and stanniocalcin 2 were confirmed to be Ab-mediated overexpression in the cells by semiquantitative RT-PCR. Transient expression of the sense RTP801 gene in CHP134 cells increased sensitivity to Abeta cytotoxicity and the expression of the antisense RTP801 gene protected the cells from the Abeta toxicity. These results suggest that RTP801 might play important roles in Abeta toxicity and the pathogenesis of Alzheimer's disease.
Asunto(s)
Humanos , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/análisis , Secuencia de Bases , Línea Celular Tumoral , ADN Complementario/análisis , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Glicoproteínas/genética , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Alzheimer's disease is a neurodegenerative disorder characterized by the extracellular deposition of beta-amyloid peptide(Abeta) in the brain, presumed to play a pathogenic role. However, the precise molecular mechanisms of its neurotoxicity are not fully understood. METHODS: Abeta-mediated cytotoxicity in neuronal cell lines (PC12, SH-SY5Y, IMR32, and U87) was measured by an MTT assay. NF-kappaB activation by Abetawas examined by a luciferase assay and apoptosis induced by Abetawas measured by cytoplasmic DNA fragmentations. RESULTS: Abetacytotoxicity in the tested cell lines was more prominent in the absence of serum than in the presence of serum in culture media. PC12 cells showed the highest sensitivity to Abetacytotoxicity among the cell lines. The Abeta(25-35) cytotoxicity in PC12 cells was increased in a dose-dependent manner. For convincing oxidative stress involved in Abetacytotoxicity, antioxidants such as DTT, GSH, vitamin C, or NAC were pretreated. GSH protected PC12 cells from Abetacytotoxicity, but DTT or NAC did not. Abeta (25-35) treatment to PC12 cells increased the NF-kappaB activity in a dose-dependent manner. Cytoplasmic DNA fragmentations, one of the apoptotic indicators, were increased at lower concentrations of Abeta(25-35) from 0.01 to 0.1 microM, however, dose-dependent increments of DNA fragmentations were not observed at higher concentrations from 1 to 10 microM. CONCLUSIONS: From these results, Abeta-induced cytotoxicity in PC12 cells might be mediated by oxidative stress.