MicroRNA-339-5p Down-regulates Protein Expression of β-Site Amyloid Precursor Protein-Cleaving Enzyme 1 (BACE1) in Human Primary Brain Cultures and Is Reduced in Brain Tissue Specimens of Alzheimer Disease Subjects

Justin M Long,Balmiki Ray,Debomoy K Lahiri

doi:10.1074/jbc.m113.518241

Abstract

Alzheimer disease (AD) results, in part, from the excess accumulation of the amyloid-β (Aβ) peptide as neuritic plaques in the brain. The short Aβ peptide is derived from the large transmembrane Aβ precursor protein (APP). The rate-limiting step in the production of Aβ from APP is mediated by the β-site APP-cleaving enzyme 1 (BACE1). Dysregulation of BACE1 levels leading to excess Aβ deposition is implicated in sporadic AD. Thus, elucidating the full complement of regulatory pathways that control BACE1 expression is key to identifying novel drug targets central to the Aβ-generating process. MicroRNAs (miRNAs) are expected to participate in this molecular network. Here, we identified a known miRNA, miR-339-5p, as a key contributor to this regulatory network. Two distinct miR-339-5p target sites were predicted in the BACE1 3'-UTR by in silico analyses. Co-transfection of miR-339-5p with a BACE1 3'-UTR reporter construct resulted in significant reduction in reporter expression. Mutation of both target sites eliminated this effect. Delivery of the miR-339-5p mimic also significantly inhibited expression of BACE1 protein in human glioblastoma cells and human primary brain cultures. Delivery of target protectors designed against the miR-339-5p BACE1 3'-UTR target sites in primary human brain cultures significantly elevated BACE1 expression. Finally, miR-339-5p levels were found to be significantly reduced in brain specimens isolated from AD patients as compared with age-matched controls. Therefore, miR-339-5p regulates BACE1 expression in human brain cells and is most likely dysregulated in at least a subset of AD patients making this miRNA a novel drug target.

Highlights

by the ␤-site A␤ precursor protein (APP)-cleaving enzyme 1 (BACE1) is the rate-limiting enzyme in the synthesis of A␤ from amyloid precursor protein
We examined the temporal profile of BACE1 expression in primary human fetal brain (HFB) cultures
In this mixed cell type culture system, we consistently observe multiple isoforms of BACE1 that are closely spaced on Western blot and difficult to discern as individual bands

Summary

Background

BACE1 is the rate-limiting enzyme in the synthesis of A␤ from amyloid precursor protein. Multiple studies have identified the second uORF in the BACE1 5Ј-UTR as a potent inhibitor of BACE1 translation (30 –33) Another post-transcriptional mechanism employed by human cells to control BACE1 levels is the expression of a BACE1 antisense noncoding RNA [34]. Many additional miRNA target sites are predicted in the BACE1 3Ј-UTR These miRNAs may mediate potent inhibitory effects and participate in the network of molecular regulators that control APP expression. We demonstrate that hsa-miR-339-5p, or miR339-5p, inhibits expression of BACE1 in a human glioblastoma cell line and in human primary brain cultures via two specific target sites in the BACE1 3Ј-UTR and is a participant in the endogenous molecular network that controls physiological BACE1 expression. We further show that miR-339-5p is dysregulated in a subset of AD patients

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION