RESUMO
Oligomer formation is considered as a critical process for the neurotoxic effects of Alzheimer's amyloid ß (Aß) peptide. Previously we have demonstrated that lysophosphatidylcholine (LPC) increases the oligomer formation of Aß1-42, the major Aß peptide found Alzheimer's disease (AD) lesions. In this study, we have investigated whether LPC affects the neurotoxic effects of Aß1-42 in a neuronal cell line (A1) culture. Dimethyl thiazolyl diphenyl tetrazolium (MTT) assay revealed that up to 10µM concentration, LPC did not affect A1 cell viability. Aß1-42 decreased the cell viability, and such effect was dose dependently enhanced by LPC. However, neither LPC nor Aß1-42, alone or in combination increased lactate dehydrogenase (LDH) release from A1 cells after 24-h treatment. Terminal deoxynucleotidyl transferase dUTP-biotin nick-end-labeling (TUNEL) assay showed that LPC increased Aß1-42-induced apoptotic cell number. To determine the underlying mechanisms, the proteins implicated in apoptosis pathways including Bcl-2- and caspase-family were analyzed by Western blotting. The results demonstrated that Aß1-42 decreased Bcl-2 in A1 cells at 24h, whereas LPC had no effect at any time point. Both LPC and Aß1-42 increased Bax level at 24h, and their combined stimulation showed a synergistic effect. Similar synergistic effect of LPC and Aß1-42 on caspase9 activation was observed. Dot blot immunoassay and Western blotting showed that LPC augmented Aß1-42 oligomer formation in cell culture medium. Removing LPC-induced early-formed Aß1-42 oligomer from the culture medium by immunoprecipitation decreased active caspase9 level and neurotoxicity, as revealed by Western blotting and MTT assay. Furthermore, dihydroethidium (DHE) assay showed that Aß1-42 increased reactive oxygen species level in A1 cells, such effect was further enhanced by LPC. Thus, our results demonstrated that LPC increased the oligomer formation process of Aß1-42 peptide in culture condition, and consequently increased apoptotic neuronal death. Such process might be important for the pathogenesis of AD, and inhibition of LPC generation could be a therapeutic target for the disease.
