High glucose upregulates BACE1-mediated Aβ production through ROS-dependent HIF-1α and LXRα/ABCA1-regulated lipid raft reorganization in SK-N-MC cells

Abstract

There is an accumulation of evidence indicating that the risk of Alzheimer's disease is associated with diabetes mellitus, an indicator of high glucose concentrations in blood plasma. This study investigated the effect of high glucose on BACE1 expression and amyloidogenesis in vivo, and we present details of the mechanism associated with those effects. Our results, using ZLC and ZDF rat models, showed that ZDF rats have high levels of amyloid-beta (Aβ), phosphorylated tau, BACE1, and APP-C99. In vitro result with mouse hippocampal neuron and SK-N-MC, high glucose stimulated Aβ secretion and apoptosis in a dose-dependent manner. In addition, high glucose increased BACE1 and APP-C99 expressions, which were reversed by a reactive oxygen species (ROS) scavenger. Indeed, high glucose increased intracellular ROS levels and HIF-1α expression, associated with regulation of BACE1 and Liver X Receptor α (LXRα). In addition, high glucose induced ATP-binding cassette transporter A1 (ABCA1) down-regulation, was associated with LXR-induced lipid raft reorganization and BACE1 localization on the lipid raft. Furthermore, silencing of BACE1 expression was shown to regulate Aβ secretion and apoptosis of SK-N-MC. In conclusion, high glucose upregulates BACE1 expression and activity through HIF-1α and LXRα/ABCA1-regulated lipid raft reorganization, leading to Aβ production and apoptosis of SK-N-MC.

Figure 1. Effects of high glucose on Aβ secretion and neuronal apoptosis.

(a) Aβ, phosphorylated tau and β-actin protein expressions of the ZLC and ZDF brain tissues were detected by western blotting. (b) Slide samples for immunohistochemistry were immunostained with Aβ and p-tau (Ser³⁹⁶) specific antibodies and PI. Images shown in result are representative. All scale bars, 200 μm (magnification of low and high power field, ×100 and ×200). (c) Secreted Aβ from SK-N-MC in medium was detected by immunoprecipitation assay. Immunoprecipitated Aβ was analyzed by western blotting. (d) Cells were immunostained with p-tau (Ser³⁹⁶) and PI. Scale bars, 50 μm (magnification, ×800). Fluorescence intensity of p-tau was analyzed by using Image J software (developed by Wayne Rasband, National Institute of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/). The result images are representative of five independent experiments. Data are presented as a mean ± S.E. of six independent experiments. *p < 0.05 versus 5 mM of D-glucose treatment. (e) Cell viability was measured by trypan blue exclusion assay Data are presented as a mean ± S.E. of three independent experiments with duplex dishes. (f) Viable cells were detected by using annexin V/PI analysis. Data are presented as a mean ± S.E. of two independent duplex dishes. Each western blot result shown is representative images of three independent experiments. *p < 0.05 versus 5 mM of D-glucose treatment. All western blot data were cropped and acquired under same experimental conditions.

Figure 2. Effects of high glucose on BACE1 expression in diabetic rat models, mouse hippocampal neuron and SK-N-MC.

(a) BACE1 and APP-C99 expressions of the ZLC and ZDF brain tissues were detected by western blotting. Each data shown in the result is representative images of five independent experiments. (b) Tissue samples for immunohistochemistry were immunostained with BACE1 and APP-C99 specific antibodies. Images shown in result are representative. All scale bars, 200 μm (magnification of low and high power field, ×100 and ×200). (c) Mouse hippocampal neuron was incubated with 25 mM D-glucose for 24 h BACE1, APP- C99 and β-actin expressions of mouse hippocampal neuron were detected by western blotting. Each data shown in the result is representative image of five independent experiments. (d) Total mRNA extracted from SK-N-MC was reverse-transcribed, and subsequently bace1 and β-actin mRNA expressions were amplified by PCR. The mRNA expression of bace1 was analyzed by quantitative real-time PCR. The mRNA expression level was normalized by β-actin mRNA expression level. Data are presented as a mean ± S.E. of three independent duplex dishes. (e) bace1 and non-targeting (NT) siRNA was transfected to cell for 12 h prior to D-and L-glucose treatment for 24 h BACE1, APP-C99 and β-actin expressions were detected by western blotting. Each western blot image is representative of three independent experiments. Data are presented as a mean ± S.E. *p < 0.05 versus 5 mM of D-glucose treatment. All western blot data were cropped and acquired under same experimental conditions.

Figure 3. Role of ROS-induced HIF-1α in BACE1 expression by high glucose.

(a) DCF-DA–sensitive SK-N-MC cells were visualized by confocal microscopy. Scale bars, 50 μm (magnification, ×600). Intracellular ROS generation was quantified by using luminometer. Data are presented as a mean ± S.E. of two independent sixth dishes. (b) BACE1, APP-C99 and β-actin expressions were detected by western blotting. (c) Mouse hippocampal neuron was incubated with 25 mM D-glucose for 24 h HIF-1α and β-actin expressions of mouse hippocampal neuron were detected by western blotting. Each data shown in the result is representative image of five independent experiments. (d,e) NAC (5 mM) was pretreated to cells, and then HIF-1α and β-actin expressions of SK-N-MC were detected by western blotting. (f) Non-nuclear protein and nuclear expressions were normalized by β-tubulin and lamin A/C repectively. (g) Cells were immunostained with HIF-1α and PI. Scale bars, 50 μm (magnification, ×800). (h) DNA was immunoprecipitated with RNA polymerase, IgG and HIF-1α antibodies. The immunoprecipitation and input samples were amplified with primers of gapdh and bace1 gene promoters. (i) Quantatative data was analyzed by real time PCR of two independent experiments of triple dishes. (j) SK-N-MC cells were transfected by siRNAs for 24 h prior to D-glucose treatment. HIF-1α, APP-C99, BACE1 and β-actin were detected by western blotting. Each western blot image shown is representative of three independent experiments. Data are presented as a mean ± S.E. *p < 0.05 versus 5 mM of D-glucose treatment, ^#p < 0.05 versus 25 mM of D-glucose treatment. All western blot data were cropped and acquired under same experimental conditions.

Figure 4. Role of high glucose-induced ROS in LXR reduction.

(a) LXRα, LXRβ and β-actin were detected by western blotting. (b) Cells were immunostained with LXRα and PI. Scale bars, 50 μm (magnification, ×800). (c) LXRα, lamin a/c and β-tubulin in the non-nuclear and nuclear fractions were detected by western blotting. Non-nuclear protein and nuclear expressions were normalized by β-tubulin and lamin A/C repectively. (d) LXRα and β-actin protein expressions of the ZLC and ZDF brain tissues were detected by western blotting. (e) Immunohistochemistry with ZLC and ZDF tissues were performed with LXRα-specific antibody and PI. All scale bars, 200 μm (magnification of low and high power field, ×100 and ×200). (f) Mouse hippocampal neuron was incubated with 25 mM D-glucose for 24 h LXRα and β-actin expressions of mouse hippocampal neuron were detected by western blotting. Each data shown in the result is representative image of five independent experiments. (g) SK-N-MC was incubated with NAC (5 mM) for 30 min before D-glucose treatment. LXRα and β-actin expressions were analyzed by western blotting. (h) Phosphorylated JNK, JNK and β-actin expression were detected by western blotting. (i) SK-N-MC was incubated with SP600125 (1 μM) for 30 min before D-glucose treatment. LXRα expression was detected by western blotting. Each western blot image is representative of three independent experiments. Data are presented as a mean ± S.E. *p < 0.05 versus 5 mM of D-glucose treatment, ^#p < 0.05 versus 25 mM of D-glucose treatment. All western blot data were cropped and acquired under same experimental conditions.

Figure 5. Role of high glucose-reduced LXRα in ABCA1 expression and cholesterol accumulation.

(a) Total cholesterol level is measured by using cholesterol detection kit described in Materials & Methods. Data are presented as a mean ± S.E. of three independent experiments of duplex dishes. (b) Intracellular cholesterol was stained with fillipinIII, and visulalized by confocal microscopy. Scale bars, 50 μm (magnification, ×800). (c) TO901317 (1 μM) pretreated to SK-N-MC prior to D-glucose treatment. Total mRNA expressions of abca11 and β-actin were analyzed by quantitative real-time PCR. The mRNA expression level was normalized by β-actin mRNA expression level. Data are presented as a mean ± S.E. of three independent duplex dishes. (d) ABCA1 and β-actin expressions were detected by western blotting. (e) Cells were immunostained with ABCA1 and PI. Scale bars, 50 μm (magnification, ×800). (f) DNA was immunoprecipitated with RNA polymerase, IgG and LXRα antibodies. The immunoprecipitation and input samples were amplified with primers of gapdh and abca1 gene promoters. (g) CHIP data was quantified by real time PCR of two independent experiments of triple dishes. Each western blot image is representative of three independent experiments. Data are presented as a mean ± S.E. *p < 0.05 versus 5 mM of D-glucose treatment, ^#p < 0.05 versus 25 mM of D-glucose treatment. All western blot data were cropped and acquired under same experimental conditions.

Figure 6. Role of high glucose-induced BACE1 localization on the lipid raft in amyloidogenesis and apoptosis of SK-N-MC.

(a) SK-N-MC was incubated with TO901317 (1 μM) for 30 min prior to D-glucose treatment. Sucrose gradient-fractionized samples were blotted with BACE1, caveolin-1, flotillin-2 and β-actin-specific antibodies. (b) The same volumes of lipid raft fraction (#4–6) were loaded to SDS-PAGE gel, blotted with BACE1 and caveolin-1-specific antibodies. (c) Cells were stained with APP-C99, BACE1 and CTB, visualized by confocal microscopy. Scale bars, 50 μm (magnification, ×800). (d,e) Cells were incubated with MβCD and transfected with bace1 and NT siRNAs prior to D-glucose treatment. Secreted Aβ in medium was analyzed by immunoprecipitation assay. (f) The bace1 and NT siRNAs-transfected SK-N-MC samples were blotted with BACE1, cleaved caspase-9, cleaved caspase-3 and β-actin-specific antibodies. (g) Cell viability was measured by trypan blue exclusion assay. Data are presented as a mean ± S.E. of three independent experiments with duplex dishes. (h) Viable cells were detected by using annexin V/PI analysis. Data are presented as a mean ± S.E. of two independent duplex dishes. Each western blot image is representative of three independent experiments. *p < 0.05 versus 5 mM of D-glucose treatment, ^#p < 0.05 versus 25 mM of D-glucose treatment. All western blot data were cropped and acquired under same experimental conditions.

Figure 7. The schematic model for mechanism involved in high glucose-induced BACE1 expression and amyloidogenesis of SK-N-MC.

High glucose stimulates ROS production, leads to increase of HIF-1α for nuclear translocation and phosphorylation of JNK. Translocated HIF-1α is bound to HRE promoter region of bace1 gene, subsequently increases BACE1 expression. Phosphorylated JNK stimulates decrease in LXRα expression, reduced binding LXRα to the LXE promoter region of abca1 gene, leads to reduction of ABCA1 expression and intracellular cholesterol accumulation which may be involved in high glucose-induced lipid raft reorganization. In addition, high glucose-lipid raft modification induces BACE1 expression on the lipid raft, stimulates to Aβ secretion in SK-N-MC.