Amyloid Beta-Mediated Epigenetic Alteration of Insulin-Like Growth Factor Binding Protein 3 Controls Cell Survival in Alzheimer's Disease

<div><p>Swedish double mutation (KM670/671NL) of amyloid precursor protein (APP) is reported to increase toxic amyloid β (Aβ) production via aberrant cleavage at the β-secretase site and thereby cause early-onset Alzheimer's disease (AD). However, the underlying molecular mechanisms leading to AD pathogenesis remains largely unknown. Previously, our transcriptome sequence analyses revealed global expressional modifications of over 600 genes in APP-Swedish mutant-expressing H4 (H4-sw) cells compared to wild type H4 cells. Insulin-like growth factor binding protein 3 (<i>IGFBP3</i>) is one gene that showed significantly decreased mRNA expression in H4-sw cells. In this study, we investigated the functional role of <i>IGFBP3</i> in AD pathogenesis and elucidated the mechanisms regulating its expression. We observed decreased <i>IGFBP3</i> expression in the H4-sw cell line as well as the hippocampus of AD model transgenic mice. Treatment with exogenous IGFBP3 protein inhibited Aβ_{1<b>–</b>42}- induced cell death and caspase-3 activity, whereas siRNA-mediated suppression of IGFBP3 expression induced cell death and caspase-3 cleavage. In primary hippocampal neurons, administration of IGFBP3 protein blocked apoptotic cell death due to Aβ_{1<b>–</b>42} toxicity. These data implicate a protective role for IGFBP3 against Aβ_{1<b>–</b>42}-mediated apoptosis. Next, we investigated the regulatory mechanisms of IGFBP3 expression in AD pathogenesis. We observed abnormal <i>IGFBP3</i> hypermethylation within the promoter CpG island in H4-sw cells. Treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine restored <i>IGFBP3</i> expression at both the mRNA and protein levels. Chronic exposure to Aβ_{1<b>–</b>42} induced <i>IGFBP3</i> hypermethylation at CpGs, particularly at loci −164 and −173, and subsequently suppressed <i>IGFBP3</i> expression. Therefore, we demonstrate that expression of anti-apoptotic <i>IGFBP3</i> is regulated by epigenetic DNA methylation, suggesting a mechanism that contributes to AD pathogenesis.</p></div

IGFBP3 protects cells from Aβ_1–42 induced apoptosis H4 and H4-sw cells were treated for 24 h in serum-free media (SF) with 5 µM soluble Aβ₁_–42 in the presence or absence of exogenous recombinant human IGFBP3 (BP3) protein.

<p><i>IGFBP3</i> knockdown H4 cells generated by siRNA transfection or non-targeted siRNA-transfected H4 cells were treated with or without 5 µM soluble Aβ_{1<b>–</b>42} in SF media for 24 h. Apoptosis was evaluated after propidium iodide staining using FACS analyses. Compiled FACS results from three independent experiments are graphically illustrated in (A) and (B). Caspase 3 activation was examined in H4 cells by detecting cleaved forms of caspase 3 using western blot analyses. Representative results are illustrated and graphical values from densitometric analyses after normalization to β-actin are reported as relative values to that of untreated control (C). Rat hippocampal neuronal primary cells in supplements-free media were treated for 24 h with 500 nM oligomeric Aβ_{1<b>–</b>42} in the presence or absence of exogenous recombinant human IGFBP3 (BP3) protein. Caspase 3 activation was determined by detecting cleaved forms of caspase 3 using western blot analyses (D). Data are the mean ± SD of three independent experiments. Statistical analyses were performed using two-way analysis of variance (ANOVA) and Bonferroni post-tests. * indicates p<0.05. H4-sw, APP-Swedish mutant H4 cells; BP3, IGFBP3; siNC, non-targeting siRNA; siBP3, IGFBP3 siRNA.</p

<i>IGFBP3</i> expression changes following demethylation in APP-Swedish mutant cells H4-sw cells were treated for 3 days with 10 µM 5-aza-2′-deoxycytidine.

<p>DNA methylation status at specific CpG sites were analyzed using bisulfite pyrosequence analyses. The average percent methylation of triplicate pyrosequencing analyses from each of the five CpG sites are presented graphically (A). After treatment with 5-aza-dC, <i>IGFBP3</i> mRNA expression was measured using qPCR (B). IGFBP3 protein expression was detected using western blot analyses. Representative results are illustrated and values from densitometric analyses after normalization to β-actin are reported relative to that of untreated controls (C). Data are shown as the mean ± SD (n = 3). Statistical analyses were performed using <i>t</i>-tests (* indicates p<0.05). 5aza, 5-aza-2′-deoxycytidine.</p

CpG island methylation is altered in the <i>IGFBP3</i> promoter region in H4 cells treated with Aβ_1–42 H4 cells were treated for 5 days with different concentrations of Aβ₁_–42.

<p>DNA methylation at the -164 and -173 CpG sites was analyzed using bisulfite sequencing analyses (A). Each circle represents CpG dinucleotides. The methylation status of each CpG site is illustrated by black (methylated) and white (unmethylated) circles. The total percentage of methylation at specific CpG sites is indicated as a pie graph. The black segment of the pie graph indicates methylated CpG percentage whereas the white segment represents the unmethylated CpG percentage (A). <i>IGFBP3</i> expression after treatment with Aβ_{1<b>–</b>42} was determined using qPCR (B) and western blot analyses (C). Graphs depict compiled data from three independent experiments and values are relative to those of untreated controls. Data are shown as the mean ± SD (n = 3). Statistical analyses were performed using one-way ANOVA and Bonferroni post-tests (* indicates p<0.05).</p

IGFBP3 expression is down-regulated in an APP-mutant cell line and transgenic mice <i>IGFBP3</i> mRNA expression in normal and APP-Swedish mutant H4 cells (A), and the brain of wild type and PSEN1-APP transgenic mice (B6C3-Tg(APP695)85Dbo Tg(PSEN1)85Dbo) (C) was measured by transcriptome sequencing analyses and qPCR.

<p>Expression of IGFBP3 protein in normal and APP-Swedish mutant H4 cells was detected using western blot analyses (B). IGFBP3 protein in APP-Swedish mutant H4 cells is expressed relative to IGFBP3 protein levels in normal H4 cells. Data are represented as the mean ± standard deviation (SD) of triplicate experiments. Statistical analyses were performed using <i>t</i>-tests (* indicates p<0.05). H4-sw, APP-Swedish mutant H4 cells; Hippo, hippocampus; FC, frontal cortex; CB, cerebellum.</p

Hypermethylation of CpG islands within the <i>IGFBP3</i> promoter in APP-Swedish mutant cells Schematic diagram of the genomic region (+60 to −290) of IGFBP3 that was analyzed for methylation status.

<p>The CpG island is represented as a box (−240 to +10). Thin horizontal lines indicate each CpG site. The bent arrow indicates the transcription start site (+1) and the thick vertical solid line indicates the target CpG sites for pyrosequencing analyses (A). Methylation status analyses were conducted using bisulfite sequencing analyses, the 454 GS-FLX system, and bisulfite pyrosequencing. Individual bars represent the percentage of methylation at the corresponding CpG site within the <i>IGFBP3</i> promoter (A). Representative pyrograms are shown for each sample with the percentage methylation at each of the five CpG sites tested (B). Average percent methylation of triplicate pyrosequencing analyses at each of the five CpG sites are presented graphically (B). Data are shown as the mean ± SD of triplicate experiments. Statistical analyses were performed using <i>t</i>-tests (* indicates p<0.05). H4-sw, APP-Swedish mutant H4 cells.</p