title: Amyloids beta40 and beta42 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.
authors.affiliation: Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
authors.forename: R S
authors.initials: RS
authors.lastname: Turner
authors.forename: N
authors.initials: N
authors.lastname: Suzuki
authors.forename: A S
authors.initials: AS
authors.lastname: Chyung
authors.forename: S G
authors.initials: SG
authors.lastname: Younkin
authors.forename: V M
authors.initials: VM
authors.lastname: Lee
mesh_terms: D016229:Amyloid beta-Peptides / Q000378:metabolism*; D002478:Cells, Cultured; D016922:Cellular Senescence; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*
chemical_list: D016229:Amyloid beta-Peptides; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42)