Additional file 4: Figure S4. of Statins induce insulin-degrading enzyme secretion from astrocytes via an autophagy-based unconventional secretory pathway

Cell death was not induced in this study. (A) MTS assay was used for checking cell viability under simvastatin, 3MA and/or bafilomycin treated condition. N = 5 experiments. (PDF 57 kb

Additional file 1: Figure S1. of Statins induce insulin-degrading enzyme secretion from astrocytes via an autophagy-based unconventional secretory pathway

Statins regulate cholesterol levels in astrocytes. (A) Cellular cholesterol levels were measured by filipin staining. MβCD is a positive control. (B) Quantitative analysis of Figure S1A using the Image J program (N = 3 experiments). ** p < 0.01, *** p < 0.001 vs. vehicle-treated cells. (PDF 217 kb

Additional file 2: Figure S2. of Statins induce insulin-degrading enzyme secretion from astrocytes via an autophagy-based unconventional secretory pathway

Fluvastatin induces IDE secretion from astrocytes. (A) Increased IDE levels secreted from the primary astrocytes by fluvastatin in a concentration-dependent manner. Blots are representative of at least 3 independent experiments (N = 3 experiments). (B) Quantitative analysis of Figure S2A. ** p < 0.01 vs. vehicle-treated cells. (PDF 71 kb

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

PS dKO MEFs are vulnerable to serum deprivation-induced cell death.

<p>(A) Western blot analysis of nicastrin and PS1 showing that PS dKO MEFs prevent nicastrin maturation. hPS1 MEFs were rescued PS1 and nicastrin maturation. Calnexin served as a loading control for membrane fractions. Filled and blank arrows indicate mature and immature nicastrin, respectively. Filled and blank arrowheads indicate full-length PS1 and PS1-CTF, respectively. (B) LDH release assay showing that PS dKO MEFs are injured by serum deprivation-induced stress. All MEFs were incubated DMEM media without serum for 36 hr. The graph represents (%) of positive control. Data are means±SEM values of three independent experiments. * represents significant differences from PS WT MEFs. ***P<0.001. # represents significant differences from PS dKO MEFs. ###P<0.001.</p

Accumulation of phospho-β-catenin induces cytotoxicity in H4 neuroglioma cells.

<p>(A) H4 cells were transiently transfected with EYFP vector, WT, and mutant β-catenin constructs in DMEM for 16 hr. K19/49R β-catenin-transfected cells showed higher immunoreactivity against anti-phospho-β-catenin (S33, 37, T41) without affecting endogenous β-catenin and β-actin levels. Filled and blank arrows indicate exogenous β-catenin-EYFP fusion protein and endogenous β-catenin, respectively. Arrowhead indicates nonspecific bands. (B) LDH release assay showed that K19/49R β-catenin induced cytotoxicity in the transfectants. The graph represents (%) of positive control. Data are means±SEM values of three independent experiments. * represents significant differences from EYFP vector-transfected H4 cells. *P<0.05, **P<0.01, ***P<0.001. # represents significant differences from WT β-catenin-EYFP-transfected H4 cells. ###P<0.001. (C) DCFDA staining showing ROS generation of mock and β-catenin-transfected H4 cells. Although fewer cells remained among K19/49R β-catenin tranfectants, the DCFDA signal was much higher than in other transfectants. The white bar represents 200 µm.</p

Phospho-β-catenin was accumulated in PS dKO MEFs under serum deprivation conditions.

<p>(A) β-catenin was accumulated in PS dKO MEFs, not in PS WT under normal and serum deprivation conditions. β-actin served as a loading control. (B) PS WT and PS dKO MEFs were incubated in growth media or DMEM for 30 hr, followed by immunofluorescent staining. PS dKO MEFs in DMEM labeled more brightly against anti-phospho-β-catenin (S33, 37, T41) antibody. The white bar represents 200 µm. (C) PS dKO MEFs were incubated w/wo 10 mM LiCl or 200 µM trolox in DMEM for 30 hr and labeled with phospho-β-catenin (S33, 37, T41)-specific antibody (Red). Many cells contained phosphorylated forms of β-catenin in DMEM- and trolox-treated PS dKO MEFs by fluorescence microscopy. But, LiCl-treated PS dKO MEFs stained weakly. DAPI staining was used to visualize cell nuclei (Blue). The white bar represents 200 µm. (D) Western blot analysis showed that LiCl effectively decreased phosphorylation of β-catenin at the S33, 37, and T41 sites without altering total β-catenin or β-actin levels. However, trolox-treated cells showed similar levels of phospho-β-catenin compared with DMEM alone.</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

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