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

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