Antagonistic Effects of β-Site Amyloid Precursor Protein-cleaving Enzymes 1 and 2 on β-Amyloid Peptide Production in Cells

Guriqbal Basi,Normand Frigon,Robin Barbour,Tam Doan,Grace Gordon,Lisa Mcconlogue,Sukanto Sinha,Michelle Zeller

doi:10.1074/jbc.m300169200

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

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

RESULTS

DISCUSSION