β-Secretase Inhibitor Potency Is Decreased by Aberrant β-Cleavage Location of the “Swedish Mutant” Amyloid Precursor Protein

Hidekuni Yamakawa,Sosuke Yagishita,Eugene Futai,Shoichi Ishiura

doi:10.1074/jbc.m109.066753

Hidekuni Yamakawa, Sosuke Yagishita + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m109.066753

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Abstract

The amyloid-beta (Abeta) peptide, widely known as the causative molecule of Alzheimer disease (AD), is generated by the sequential cleavage of amyloid precursor protein (APP) by the aspartyl proteases BACE1/beta-secretase and presenilin/gamma-secretase. Inhibition of BACE1, therefore, is a promising strategy for preventing the progression of AD. However, beta-secretase inhibitors (BSIs) exhibit unexpectedly low potency in cells expressing "Swedish mutant" APP (APPswe) and in the transgenic mouse Tg2576, an AD model overexpressing APPswe. The Swedish mutation dramatically accelerates beta-cleavage of APP and hence the generation of Abeta; this acceleration has been assumed to underlie the poor inhibitory activity of BSI against APPswe processing. Here, we studied the mechanism by which the Swedish mutation causes this BSI potency decrease. Surprisingly, decreased BSI potency was not observed in an in vitro assay using purified BACE1 and substrates, indicating that the accelerated beta-cleavage resulting from the Swedish mutation is not its underlying cause. By focusing on differences between the cell-based and in vitro assays, we have demonstrated here that the potency decrease is caused by the aberrant subcellular localization of APPswe processing and not by accelerated beta-cleavage or the accumulation of the C-terminal fragment of beta-cleaved APP. Because most patients with sporadic AD express wild type APP, our findings suggest that the wild type mouse is superior to the Tg2576 mouse as a model for determining the effective dose of BSI for AD patients. This work provides novel insights into the potency decrease of BSI and valuable suggestions for its development as a disease-modifying agent.

Highlights

Several reports have shown that BACE1/␤-secretase inhibitors (BSIs) are less potent against A␤ generation in cells stably transfected with the APPswe variant than in those transfected with wild type amyloid precursor protein (APP) (APPwt) [22,23,24, 27], but the mechanism underlying this reduction in BSI inhibitory activity has not yet been elucidated
The reaction mixtures were appropriately diluted in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST) and 1% bovine serum albumin (BSA) and transferred to a detection plate coated with a monoclonal antibody specific for the N-terminal end generated by BACE1 cleavage (82E1; IBL Co., Ltd., Gunma, Japan)
We measured BACE1 activities at pH 4.5 and 6.2 and plotted the percentage of inhibition at each dose of Inhibitor IV (Table 1). Both the IC50 value and IC50 swe:wt remained unaffected by pH, indicating that the Swedish mutation-linked BSI potency decrease is not caused by a higher pH in the cellular compartment in which ␤-cleavage of APP occurs

Summary

To whom correspondence should be addressed

Tg2576 is a transgenic mouse expressing “Swedish mutant” APP (APPswe) [25] This two-amino acid mutation, which was discovered in Swedish familial AD patients [26], dramatically accelerates ␤-site processing of APP. This finding suggests that differences between the cell-based and in vitro enzymatic assays might underlie the apparent effect of the Swedish mutation on BSI potency Our analysis of these differences demonstrates that the potency decrease is caused by the aberrant subcellular localization of APPswe processing and not by accelerated ␤-cleavage or by the accumulation of the C-terminal fragment. Our findings suggest that the abnormal subcellular site of APPswe processing is responsible for the weakened inhibitory activity of BSIs against A␤ production in APPswe-expressing cells

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION