Beta-Amyloid peptide and a 3-kDa fragment are derived by distinct cellular mechanisms.

C Haass,A.Y Hung,M.G Schlossmacher,D.B Teplow,D.J Selkoe

doi:10.1016/s0021-9258(18)53650-4

Abstract

We have analyzed the cellular processing pathways which produce the 4-kDa amyloid beta-peptide (A beta) and a 3-kDa derivative (p3) of the beta-amyloid precursor protein (beta APP) found in conditioned media of tissue culture cells and in cerebrospinal fluid. Pulse-chase experiments reveal that both peptides are secreted in parallel with soluble beta APP (APPs); no precursor-product relation between A beta and p3 was found. The protease inhibitor leupeptin did not influence the production of either peptide. In contrast, the weak base ammonium chloride (NH4Cl) showed a dose-dependent inhibition of A beta production with less decrease in p3. A similar effect was observed using the monovalent ionophore monensin. Brefeldin A completely inhibited the generation of both peptides, indicating that proteases located in the endoplasmic reticulum or early Golgi are not sufficient for the production of the small peptides. Deletion of the beta APP cytoplasmic domain, which removes a consensus sequence that probably mediates reinternalization, caused an increase in secretion of both APPs and p3 and did not abolish A beta production. These observations suggest that completely mature beta APP within the late Golgi and/or at the cell surface is a prerequisite for A beta production but processing within the lysosome might not be directly required. p3 appears to derive from the 10-kDa C-terminal stub of beta APP following secretion of APPs.

Highlights

From the Department of Neurology and Program in Neuroscience, Harvard Medical School,and Center for Neurologic Diseases,Brigham and Women’s Hospital, Boston, Massachusetts 02115 plasmic reticulum (ER) andGolgi, the precursoris cleaved by an as yet unidentified protease designated bAPsePcretase to create the secreted form of pAPP (APP,) and a 10-kDa Cterminalfragmentthatremainsmembrane-bound (Weidemann et al, 1989; Oltersdorf et al, 1990; Esch et al, 1990; Wang et al, 1991)
Brefeldin A completely inhibited the generation of both peptides, indicating that proteases located in the endoplasmicreticulumor early Golgi are not sufficient for the production of the small peptides
To characterize the cellular pathways that produce the two peptides, we analyzed the formation of the peptides in pulse-chase experiments and after treatment of cultured cells with a variety of agentsthatinterfere withcellular processing pathways

Summary

Cellular Mechanisms*

Ported which contain an additionalexon encoding a protease inhibitory domain (@APP 563,751, an7d70) AB which is known to abolish H’, Na’, and K’ gradients and and p3 were generated at rates similar to thaot f APP, (Fig. inhibit late Golgi and lysosomal functions (Tartakoff, 1).The concomitant appearanceof AB and p3 suggests that 1983), had a strong dose-dependent inhibitory effect on the noprecursor-productrelationshipexistsbetweenthe two formation of A@(-96% at the maximum concentration), with peptides. This conclusion is supportedby the observation that less effect on p3 formation (-67% at the maximum conceneven after a 2-h label-freechase no decreaseof AB or increase tration; Fig. 20).

Momnsin IpMI

Our data do not exclude