The A673T mutation in the amyloid precursor protein reduces the production of β-amyloid protein from its β-carboxyl terminal fragment in cells

Introduction: The A6731 mutation in the amylo d precursor protein (APP) protects against Alzheimer's disease by reducing beta-amyloid protein (AN production. This mutation reduced the release of the soluble APP fragment (sAPP beta), which is processed by beta-secretase, suggesting a concomitant decrease in the beta-carboxyl fragment of APP (C99), which is a direct substrate of gamma-secretase for q, production. However, it remains controversial whether the level of C99 is significantly reduced in cells expressing APP that carry A6731 as the cause of reduced /V production. Here, we investigated the effect of the A673T mutation in C99 on gamma-cleavage in cells. Results: We found that the level of e99 in cells expressing APP A673T was indistinctive of that observed in cells expressing wild type APP, although the release of sAPP beta was significantly reduced in the APP A673T cells. In addition, our reconstituted --secretase assay demonstrated no significant difference in,-cleavage on an APP fragment carrying the A673T mutation compared with the wild type fragment. Importantly, cells expressing C99 containing the A673T mutation (C99 A2T; in accordance with the PO numbering) produced roughly half the level of A beta compared with the wild type e99, suggesting that the e99 A2T is an insufficient substrate of y-secretase in cells. A cell free y-secretase assay revealed that A beta, production from the microsomal fraction of cells expressing C99 A2T was diminished. A sucrose gradient centrifugation analysis indicated that the levels of the C99 A2T that was codistributed with y-secretase components in the raft fractions were reduced significantly. Conclusions: Our data indicate that the A673T mutation in APP alters the release of sAPPri, but not the C99 level, and that the C99 A2T is an inefficient substrate for gamma-secretase in cell based assay.