TY - JOUR
T1 - Amyloids β40 and β42 are generated intracellularly in cultured human neurons and their secretion increases with maturation
AU - Turner, R. Scott
AU - Suzuki, Nobuhiro
AU - Chyung, Abraham S.C.
AU - Younkin, Steven G.
AU - Lee, Virginia M.Y.
PY - 1996/4/12
Y1 - 1996/4/12
N2 - 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 Aβ40 and Aβ42 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 Aβ40 and Aβ42 in NT2N cell lysates at a ratio of 3:1, whereas secreted Aβ40 and Aβ42 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 Aß40/Aß42 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 Aβ40 and Aβ42 increases more than 5-fold over 7 weeks. This increase in the secretion of Aβ40/Aβ42 in NT2N cells as a function of time may recapitulate a similar phenomenon in the aging brain.
AB - 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 Aβ40 and Aβ42 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 Aβ40 and Aβ42 in NT2N cell lysates at a ratio of 3:1, whereas secreted Aβ40 and Aβ42 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 Aß40/Aß42 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 Aβ40 and Aβ42 increases more than 5-fold over 7 weeks. This increase in the secretion of Aβ40/Aβ42 in NT2N cells as a function of time may recapitulate a similar phenomenon in the aging brain.
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U2 - 10.1074/jbc.271.15.8966
DO - 10.1074/jbc.271.15.8966
M3 - Article
C2 - 8621541
AN - SCOPUS:0029664624
SN - 0021-9258
VL - 271
SP - 8966
EP - 8970
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 15
ER -