Enhanced Release of Amyloid -Protein from Codon 670/671 Swedish Mutant -Amyloid Precursor Protein Occurs in Both Secretory and Endocytic Pathways

Ruth G Perez,Sharon L Squazzo,Edward H Koo

doi:10.1074/jbc.271.15.9100

Ruth G Perez, Sharon L Squazzo + Show 1 more

Open Access

https://doi.org/10.1074/jbc.271.15.9100

Copy DOI

Abstract

The mutation at codons 670/671 of beta-amyloid precursor protein (betaPP) dramatically elevates amyloid beta-protein (Abeta) production. Since increased Abeta may be responsible for the disease phenotype identified from a Swedish kindred with familial Alzheimer's disease, evaluation of the cellular mechanism(s) responsible for the enhanced Abeta release may suggest potential therapies for Alzheimer's disease. In this study, we analyzed Chinese hamster ovary cells stably transfected with either wild type betaPP (betaPP-wt) or "Swedish" mutant betaPP (betaPP-sw) for potential differences in betaPP processing. We confirmed that increased amounts of Abeta and a beta-secretase-cleaved COOH-terminally truncated soluble betaPP (betaPPs) were secreted from betaPP-sw cells. As shown previously for betaPP-wt cells, Abeta was released more slowly than the secretion of betaPPs from surface-labeled betaPP-sw cells, indicating that endocytosis of cell surface betaPP is one source of Abeta production. In contrast, by [35S]methionine metabolic labeling, the rates of Abeta and betaPPs release were virtually identical for both cell lines. In addition, the identification of intracellular betaPPs and Abeta shortly after pulse labeling suggests that Abeta is produced in the secretory pathway. Interestingly, more Abeta was present in medium from betaPP-sw cells than betaPP-wt cells after either cell surface iodination or [35S]methionine labeling, indicating that betaPP-sw cells have enhanced Abeta release in both the endocytic and secretory pathways. Furthermore, a variety of drug treatments known to affect protein processing similarly reduced Abeta release from both betaPP-wt and betaPP-sw cells. Taken together, the data suggest that the processing pathway for betaPP is similar for both betaPP-wt and betaPP-sw cells and that increased Abeta production by betaPP-sw cells arises from enhanced cleavage of mutant betaPP by beta-secretase, the as-yet unidentified enzyme(s) that cleaves at the NH2 terminus of Abeta.

Highlights

In Alzheimer’s disease a characteristic pathological finding in the brains of affected individuals is the deposition of amyloid ␤-protein (A␤)1 in senile plaques [1]
All cells reported to date which express the ␤PP mutation produce dramatically more A␤ peptide than do cells expressing wild type ␤PP [12, 22, 23, 31,32,33]
Since excess A␤ production may be causally related to the Alzheimer’s phenotype in individuals affected with the “Swedish” mutation [21], it is important to evaluate the mechanism by which A␤ is produced from ␤PP with this alteration

Summary

The abbreviations used are

A␤, amyloid ␤-protein; ␤PP, ␤-amyloid precursor protein; ␤PPs, soluble ␤PP; ␤PP-sw, “Swedish” mutant ␤PP; ␤PP-wt, wild type ␤PP; CHO, Chinese hamster ovary; DMEM, Dulbecbrane protein ␤-amyloid precursor protein (␤PP). Cleavage of ␤PP at this position creates a soluble ϳ100 –120-kDa NH2-terminal fragment (␤PPs) [5] and a COOH-terminal membrane-retained fragment of ϳ10 kDa [6] Generation of these fragments by ␣-secretase precludes formation of an intact A␤ sequence from full-length ␤PP. Cleavage of ␤PP at the NH2 terminus of the A␤ sequence by an enzyme designated ␤-secretase creates a shortened form of ␤PPs and the ϳ12-kDa COOH-terminal fragment [11, 12]. A detailed analysis of cellular processing of ␤PP with this mutation has not been co’s modified Eagle’s medium; MMAb, monoclonal antibodies 5A3 and 1G7 used together; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; DPBS, Dulbecco’s phosphate-buffered saline

EXPERIMENTAL PROCEDURES

RESULTS

Findings

DISCUSSION