Abstract
Several lines of evidence implicate lipid raft microdomains in Alzheimer disease-associated β-amyloid peptide (Aβ) production. Notably, targeting β-secretase (β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1)) exclusively to lipid rafts by the addition of a glycosylphosphatidylinositol (GPI) anchor to its ectodomain has been reported to elevate Aβ secretion. Paradoxically, Aβ secretion is not reduced by the expression of non-raft resident S-palmitoylation-deficient BACE1 (BACE1-4C/A (C474A/C478A/C482A/C485A)). We addressed this apparent discrepancy in raft microdomain-associated BACE1 processing of APP in this study. As previously reported, we found that expression of BACE1-GPI elevated Aβ secretion as compared with wild-type BACE1 (WTBACE1) or BACE1-4C/A. However, this increase occurred without any difference in the levels of APP ectodomain released following BACE1 cleavage (soluble APPβ), arguing against an overall increase in BACE1 processing of APP per se. Further analysis revealed that WTBACE1 cleaves APP at β- and β'-sites, generating +1 and +11 β-C-terminal fragments and secreting intact as well as N-terminally truncated Aβ. In contrast, three different BACE1-GPI chimeras preferentially cleaved APP at the β-site, mainly generating +1 β-C-terminal fragment and secreting intact Aβ. As a consequence, cells expressing BACE1-GPI secreted relatively higher levels of intact Aβ without an increase in BACE1 processing of APP. Markedly reduced cleavage at β'-site exhibited by BACE1-GPI was cell type-independent and insensitive to subcellular localization of APP or the pathogenic KM/NL mutant. We conclude that the apparent elevation in Aβ secretion by BACE1-GPI is mainly attributed to preferential cleavage at the β-site and failure to detect +11 Aβ species secreted by cells expressing WTBACE1.
Highlights
In cultured cells, BACE1 localizes to late Golgi/TGN and endosomes at steady state, and a small percentage of BACE1 cycles between the cell surface and endosomes [11, 12]
BACE1-GPI Expression Elevates A Production without Altering sAPP Levels—To gain insights into the reported elevation of A production by BACE1 targeted to lipid rafts via a GPI anchor [19], we generated a BACE1-GPI construct by replacing the transmembrane domain and cytoplasmic tail of mouse BACE1 with the GPI anchor region from human placental alkaline phosphatase (PLAP) (Fig. 1A)
Because targeting BACE1 to lipid rafts through the attachment of a GPI anchor elevated A secretion, we expected that interfering with raft association of BACE1 by mutation of the palmitoylated Cys residues will lower A secretion
Summary
A, -amyloid peptide(s); APP, amyloid precursor protein; BACE1, -site APP-cleaving enzyme 1 (-secretase); CTF, C-terminal fragment; GPI, glycosylphosphatidylinositol; PLAP, placental alkaline phosphatase; TGN, trans-Golgi network; FL, full length; HEP, hephaestin; 4C/A, C474A/C478A/C482A/C485A; sAPP, soluble APP; ER, endoplasmic reticulum; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; NHS, N-hydroxysulfosuccinimide; Nav2, voltage-gated sodium channel 2 subunit; APPSwe, Swedish variant of APP. Displacing BACE1 from the lipid rafts by the Ala mutation of these 4 Cys residues had no influence on APP processing and A production in cultured cell lines [20]. This later finding is in contrast to the data from the analysis of BACE1GPI chimera, which was thought to increase A production due to its ability to exclusively associate with lipid rafts [19]. Our data reveal that the apparent increase in A production by BACE1-GPI previously attributed to raft-associated APP processing is a result of compromised cleavage at Ј-site and the inability to experimentally detect ϩ11 A species
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