Amyloids β40 and β42 are generated intracellularly in cultured human neurons and their secretion increases with maturation

R. Scott Turner, Nobuhiro Suzuki, Abraham S.C. Chyung, Steven G. Younkin, Virginia M.Y. Lee

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)8966-8970
Number of pages5
JournalJournal of Biological Chemistry
Volume271
Issue number15
DOIs
StatePublished - Apr 12 1996

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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