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© The Rockefeller University Press, 0021-9525/1998//1031 $5.00
The Journal of Cell Biology, Volume 141, Number 4, , 1998 1031-1039

Articles

Detection of a Novel Intraneuronal Pool of Insoluble Amyloid β Protein that Accumulates with Time in Culture



Daniel M. Skovronsky, Robert W. Doms, and Virginia M.-Y. Lee

Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

The amyloid-β peptide (Aβ) is produced at several sites within cultured human NT2N neurons with Aβ1-42 specifically generated in the endoplasmic reticulum/intermediate compartment. Since Aβ is found as insoluble deposits in senile plaques of the AD brain, and the Aβ peptide can polymerize into insoluble fibrils in vitro, we examined the possibility that Aβ1-40, and particularly the more highly amyloidogenic Aβ1-42, accumulate in an insoluble pool within NT2N neurons. Remarkably, we found that formic acid extraction of the NT2N cells solubilized a pool of previously undetectable Aβ that accounted for over half of the total intracellular Aβ. Aβ1-42 was more abundant than Aβ1-40 in this pool, and most of the insoluble Aβ1-42 was generated in the endoplasmic reticulum/intermediate compartment pathway. High levels of insoluble Aβ were also detected in several nonneuronal cell lines engineered to overexpress the amyloid-β precursor protein. This insoluble intracellular pool of Aβ was exceptionally stable, and accumulated in NT2N neurons in a time-dependent manner, increasing 12-fold over a 7-wk period in culture. These novel findings suggest that Aβ amyloidogenesis may be initiated within living neurons rather than in the extracellular space. Thus, the data presented here require a reexamination of the prevailing view about the pathogenesis of Aβ deposition in the AD brain.

Abbreviations used in this paper: Aβ, amyloid-β peptide; AD, Alzheimer's disease; APP, amyloid-β precursor protein; BHK, baby hamster kidney; ER/IC, endoplasmic reticulum/intermediate compartment; FAD, Familial Alzheimer's disease; SFV, Semliki Forest Virus; SFV-APPwt, SFV expressing wild-type APP695; TGN, trans-Golgi network.

We gratefully thank Dr. N. Suzuki and Tekeda Pharmaceutical for providing the monoclonal antibodies for the Aβ sandwich-ELISA. We also thank Dr. J. Wang for her help with neuronal aging experiments, and Dr. J.Q. Trojanowski for critically reading the manuscript.

Address all correspondence to Dr. Virginia M.-Y. Lee, Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, Maloney 3, HUP, Philadelphia, PA 19104-4283. Tel.: 215-662-6427; Fax: 215-349-5909; E-mail: vmylee{at}mail.med.upenn.edu


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