MicroRNA-298 and microRNA-328 regulate expression of mouse beta-amyloid precursor protein-converting enzyme 1

Abstract

MicroRNAs (miRNAs) are key regulatory RNAs known to repress mRNA translation through recognition of specific binding sites located mainly in their 3'-untranslated region (UTR). Loss of specific miRNA control of gene expression is thus expected to underlie serious genetic diseases. Intriguingly, previous post-mortem analyses showed higher beta-amyloid precursor protein-converting enzyme (BACE) protein, but not mRNA, levels in the brain of patients that suffered from Alzheimer disease (AD). Here we also observed a loss of correlation between BACE1 mRNA and protein levels in the hippocampus of a mouse model of AD. Consistent with an impairment of miRNA-mediated regulation of BACE1 expression, these findings prompted us to investigate the regulatory role of the BACE1 3'-UTR element and the possible involvement of specific miRNAs in cultured neuronal (N2a) and fibroblastic (NIH 3T3) cells. Through various experimental approaches, we validated computational predictions and demonstrated that miR-298 and miR-328 recognize specific binding sites in the 3'-UTR of BACE1 mRNA and exert regulatory effects on BACE1 protein expression in cultured neuronal cells. Our results may provide the molecular basis underlying BACE1 deregulation in AD and offer new perspectives on the etiology of this neurological disorder.

Fig. 1

Deregulation of BACE1 mRNA and protein expression patterns in the hippocampus of APP_Swe/PS1 versus wild-type (WT) mice. A–B. BACE1 mRNA levels were determined by in situ hybridization on brain sections taken from WT (lane 1) or APP_Swe/PS1 (lane 3) at 4, 6 and 19 months of age (n = 4 for each time point). A, C & D. Protein levels were assessed on adjacent brain sections by means of immunohistochemistry using BACE1 PC529 antibody and DAB staining. Please note the different patterns of BACE1 mRNA and protein in aging APP mice; while the mRNA decreased, the protein levels increased in the hippocampus of the mouse model of AD at 19 months of age. Scale bars represent 500 μm.

Fig. 2

Mouse BACE1 3′UTR harbors a negative gene regulatory element. Cultured murine neuronal (N2a) and fibroblastic (NIH 3T3) cell lines were transfected with a reporter construct expressing Rluc coupled with the 3′UTR of either BACE1 or BACE2. The results of Rluc activity were normalized with Fluc reporter activity and expressed as a percentage of the results obtained with the empty reporter vector (set at 100%) (n = 6 experiments).

Fig. 3

Binding sites for miR-298 and miR-328 are present in BACE1 3′UTR. A. Computational prediction of binding sites (BS) for miR-298 and miR-298 in BACE1 3′UTR and their respective binding free energies (ΔG), as determined by the algorithm RNAhybrid. B & C. Electrophoretic mobility shift assays (EMSA) were performed to validate miRNA recognition of the putative BS for miR-298 (298 BS) (B) or miR-328 (328 BS) (C), using wild-type (WT) and mutated (mut) BS RNA sequences. Transfer RNA (tRNA) was used as control RNA. miRNA:298 BS and miRNA:328 BS duplex formation was monitored by nondenaturing PAGE and autoradiography. Conc., concentration; ORF, open reading frame.

Fig. 4

The miR-328 binding site element is functionally regulatable upon expression of pre-miR-328. A. N2a and NIH 3T3 cell lines were cotransfected with the pre-miR-328 expression construct and a reporter construct expressing Rluc coupled with a single copy of a target sequence perfectly complementary to miR-328 (n = 3 experiments). psiNEG, vector encoding an inactive Rluc short hairpin RNA; Targ, target. The results of Rluc activity were normalized with Fluc reporter activity and expressed as a percentage of the results obtained with a reporter vector lacking the regulatory sequence in the 3′UTR of its Rluc reporter gene (Rluc) (set at 100%). B. N2a and NIH 3T3 cell lines were cotransfected with a reporter construct expressing Rluc coupled with 1 (1 × BS) or 3 (3 × BS) copies of the miR-328 natural binding sites (BS) and the pre-miR-328 expression construct (n = 3 experiments).

Fig. 5

The miRNA binding sites for miR-298 and miR-328 mediate repression of gene expression. A & B. N2a and NIH 3T3 cell lines were cotransfected with synthetic duplexes of miR-298 (A) or miR-328 (B) and a reporter construct expressing Rluc coupled with 1 (1 × BS) or 3 (3 × BS) copies of their respective natural binding sites (BS). The results of Rluc activity were normalized with Fluc reporter activity and expressed as a percentage of the results obtained with a reporter vector lacking miR-298 or miR-328 BS in the 3′UTR of its Rluc reporter gene (no BS) (set at 100%) (n = 3 experiments).

Fig. 6

Binding sites for miR-298 and miR-328 are functional and act as negative regulators of gene expression. A. N2a and NIH 3T3 cell lines were cotransfected with miR-298 and/or miR-328 duplexes, or miR-196 control duplex, and a reporter construct expressing Rluc coupled with the 200-nt miRNA binding site (BS) module comprising both miRNA BS. The results of Rluc activity were normalized with Fluc reporter activity and expressed as a percentage of the results obtained with a duplex of miR-196, used as a control (set at 100%) (n = 3 experiments). B. N2a and NIH 3T3 cell lines were transfected with a reporter construct expressing Rluc coupled with the miRNA BS module wild-type (WT) or harboring mutations in the miRNA BS sequences recognized by the seed region of miR-298 (298mut) and/or miR-328 (328mut). The results of Rluc activity were normalized with Fluc reporter activity and expressed in fold increases as compared to the results obtained with the WT 3′UTR of BACE1 (set at 1) (n = 3 experiments). C. N2a and NIH 3T3 cell lines were cotransfected as described in A, but with a reporter construct expressing Rluc coupled with the 3′UTR of BACE1.

Fig. 7

miR-298 and miR-328 regulate endogenous BACE1 expression in N2a cells. A & C. N2a cells were transfected with duplexes of miR-298 and/or miR-328, or miR-196 (used as a negative control) (A), or 2′OMe oligoribonucleotide (2′OMe) antisenses (AS) directed against miR-298 and/or miR-328, miR-324 or Green Fluorescent Protein (GFP) (used as negative controls) (C). Protein extracts were analyzed by immunoblot analysis (IB) using the PC529 antibody recognizing BACE1 or AC-40 antibody recognizing actin, and the immunoreactive bands revealed by ECL (left panels). The intensity of the bands from 3 experiments was analyzed quantitatively by densitometry and the results obtained with the anti-BACE1 antibody were normalized with the anti-actin signal (right panels). Results are expressed as mean ± sem (ANOVA with Bonferroni’s post hoc test: * P < 0.05; ** P < 0.01; *** P < 0.001) B. Northern blot analysis of small RNAs (< 200 nt) isolated from N2a and NIH 3T3 cells. A ³²P-labeled probe complementary to miR-298 or miR-328 was hybridized following a Northern blot procedure improved for the detection of small RNAs (35) (upper panels). 5S RNA was probed as a control (lower panels).

Fig. 8

Decreased expression of miR-298 and miR-328 in the granular neurons of the hippocampus during aging of APP_Swe/PS1 mice. (A) In situ hybridization for miR-298 and miR-328 on brain sections taken from APP_Swe/PS1 mice at 3 and 13 months of age. The granular part of the hippocampus is delimited by a dashed line. (B) Quantification of the RNA hybridization signals. Results are expressed in relative optical density (O.D.) units as mean ± sem (Student’s t-test: * P < 0.05; ** P < 0.01).