Abstract

The deposition of extracellular beta-amyloid peptide (A beta) in the brain is a pathologic feature of Alzheimer's disease. The beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), an integral membrane aspartyl protease responsible for cleavage of amyloid precursor protein (APP) at the beta-site, promotes A beta production. A second integral membrane aspartyl protease related to BACE1, referred to as beta-site amyloid precursor protein cleaving enzyme 2 (BACE2) has also been demonstrated to cleave APP at the beta-cleavage site in transfected cells. The role of endogenous BACE2 in A beta production remains unresolved. We investigated the role of endogenous BACE2 in A beta production in cells by selective inactivation of its transcripts using RNA interference. We are able to reduce steady state levels for mRNA for each enzyme by >85%, and protein amounts by 88-94% in cells. Selective inactivation of BACE1 by RNA interference results in decreased beta-cleaved secreted APP and A beta peptide secretion from cells, as expected. Selective inactivation of BACE2 by RNAi results in increased beta-cleaved secreted APP and A beta peptide secretion from cells. Simultaneous targeting of both enzymes by RNA interference does not have any net effect on A beta released from cells. Our observations of changes in APP metabolism and A beta are consistent with a role of BACE2 in suppressing A beta production in cells that co-express both enzymes.

Highlights

  • The production and deposition of insoluble amyloid peptide (A␤)1 peptide in the brain results in the hallmark pathological feature of Alzheimer’s disease [1]

  • A discrepancy has been noted with regard to message and activity distribution of BACE1 [5, 6], we note that mRNA degradation is directly linked with protein expression, as well as with amyloid precursor protein (APP) metabolite production, in our experiments using RNA interference (RNAi) in HEK293 cells

  • We addressed the role of BACE1 and BACE2 in A␤ production in HEK293 cells by selective degradation of mRNA for each enzyme using RNAi

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Summary

The abbreviations used are

A␤, ␤-amyloid peptide; APP, amyloid precursor protein; APPsw, Swedish mutant allele of APP; BACE, ␤-site APP cleaving enzyme; CTFs, carboxyl-terminal fragments; GFP, green fluorescent protein; RNAi, RNA interference; sAPP, secreted APP; sAPP␤, ␤-cleaved sAPP; sAPP␣, ␣-cleaved sAPP; siRNA, small interfering RNA; wt, wild type; 293sw, HEK293 cells overexpressing APPsw; ELISA, enzyme-linked immunosorbent assay; RT, reverse transcription; sAPPtot, total sAPP. A second integral membrane aspartyl protease related to BACE1, referred to as BACE2 (ASP1, Memapsin 1) has been demonstrated to cleave APP at the ␤-cleavage site [7, 8, 12,13,14,15,16]. Both enzymes cleave APP at a second site within the A␤ region. Our studies suggest that BACE2 inhibition elevates secretion of A␤ in cells co-expressing BACE1 and BACE2 and are of significance for amyloid-based therapeutics targeting these enzymes

EXPERIMENTAL PROCEDURES
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