Amyloids and Are Generated Intracellularly in Cultured Human Neurons and Their Secretion Increases with Maturation

Abstract

Previous studies have demonstrated the presence of amyloid beta (Abeta) in neurons (NT2N) derived from a human embryonal carcinoma cell line (NT2) by steady state metabolic radiolabeling and immunoprecipitation. We show here that Abeta is present intracellularly since trypsin digestion of intact NT2N cells at 4 degrees C did not eliminate the Abeta recovered in cell lysates. To determine whether both Abeta40 and Abeta42 are produced intracellularly, quantitative sandwich enzyme-linked immunosorbent assay (ELISA) was performed using COOH-terminal end-specific anti-Abeta monoclonal antibodies. Sandwich ELISA detected intracellular Abeta40 and A++beta42 in NT2N cell lysates at a ratio of 3:1, whereas secreted Abeta40 and Abeta42 were recovered in medium conditioned by NT2N cells at a ratio of approximately 20:1. Metabolic steady state and pulse-chase labeling studies demonstrated a 2-h delay in the detection of cell-associated Abeta40/Abeta42 in the medium, suggesting that Abeta is generated at a slow rate intracellularly prior to its secretion. Finally, as NT2N cells mature over time in culture, the secretion of Abeta40 and Abeta42 increases more than 5-fold over 7 weeks. This increase in the secretion of Abeta40/Abeta42 in NT2N cells as a function of time may recapitulate a similar phenomenon in the aging brain.