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

Research output: Contribution to journalArticle

158 Citations (Scopus)

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
StatePublished - Apr 12 1996

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Immunosorbents
Amyloid
Neurons
Assays
Enzymes
Conditioned Culture Medium
Trypsin
Labeling
Brain
Aging of materials
Monoclonal Antibodies
Cells
Enzyme-Linked Immunosorbent Assay
Embryonal Carcinoma Stem Cells
Immunoprecipitation
Digestion
Cell Line

ASJC Scopus subject areas

  • Biochemistry

Cite this

Amyloids β40 and β42 are generated intracellularly in cultured human neurons and their secretion increases with maturation. / Turner, R. Scott; Suzuki, Nobuhiro; Chyung, Abraham S C; Younkin, Steven G; Lee, Virginia M Y.

In: Journal of Biological Chemistry, Vol. 271, No. 15, 12.04.1996, p. 8966-8970.

Research output: Contribution to journalArticle

Turner, R. Scott ; Suzuki, Nobuhiro ; Chyung, Abraham S C ; Younkin, Steven G ; Lee, Virginia M Y. / Amyloids β40 and β42 are generated intracellularly in cultured human neurons and their secretion increases with maturation. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 15. pp. 8966-8970.
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