Flavonoids and fibrate modulate apoE4-induced processing of amyloid precursor protein in neuroblastoma cells

Abstract

Introduction: Apolipoprotein (apo) E4, being a major genetic risk factor for Alzheimer's disease (AD), is actively involved in the proteolytic processing of amyloid precursor protein (APP) to amyloid β (Aβ) peptide, the principle constituent of amyloid plaques in Alzheimer Disease (AD) patients. ApoE4 is believed to affect APP processing through intracellular cholesterol homeostasis, whereas lowering the cholesterol level by pharmacological agents has been suggested to reduce Aβ production. This study has investigated the effects of hypolipidemic agents fenofibrate, and the flavonoids-naringenin and diosmetin-on apoE4-induced APP processing in rat neuroblastoma cells stably transfected with human wild-type APP 695 (B103-hAPP695wt).

Results: B103-hAPP695wt cells were pretreated with different doses of flavonoids and fenofibrate for 1 h prior to apoE4 exposure for 24 h. ApoE4-induced production of intra- and extracellular Aβ peptides has been reduced with fenofibrate, naringenin, and diosmetin treatments. Pretreatment with diosmetin has significantly reduced apoE4-induced full-length APP (fl- APP) expression, whereas naringenin and fenofibrate had no effect on it. In addition, the increase in the apoE4-induced secretion of sAPPtotal and sAPPα has been dose-dependently reduced with drug pretreatment. On the other hand, the decrease in the expression of both APP-carboxy terminal fragments (CTF)-α and -β (generated by the α- or β-secretase cleavage of APP) by apoE4 was dose-dependently increased in cells pretreated with fenofibrate and naringenin but not diosmetin.

Conclusion: Thus, we suggest that fenofibrate, naringenin, and diosmetin treatments can reduce apoE4- induced Aβ production by distinct mechanisms that may prove useful in developing drugs for AD patients.

Keywords: APP; amyloid β; apoE4; diosmetin; fibrates; flavonoids; naringenin; neuronal cells.

Figure 1

Comparative effect of different concentrations (Dose1, Dose2, and Dose3) of fenofibrate (25, 50, and 100 μM), naringenin (6.25, 12.5, and 25 μM), and diosmetin (25, 50, and 100 μM) (A) on the secretion of extracellular Aβ40 (B) and the production of intracellular Aβ 40 (C) and Aβ42 (D) as measured by ELISA, and cell viability (E) as measured by MTT assay, in apoE4- induced B103-hAPP695wt cells. Cells were treated with vehicle (0.1% DMSO), apoE4 (7.5 μg/mL), or drug control only (Dose3 (100 μM) fenofibrate, Dose3 (6.25 μM) naringenin, and Dose3 (100 μM) diosmetin) for 24 h. Pretreatment consisted of fenofibrate, naringenin, and diosmetin for 1 h prior to stimulation with apoE4 for 24 h. Quantitative evaluation for the secretion of extracellular Aβ and the production of intracellular Aβ was normalized against the respective cellular protein concentration of treated cells. For statistical evaluations, the two-way ANOVA analysis followed by Tukey’s range test was performed. Statistically significant differences (#p < 0.05) compared with vehicle; (*p < 0.05) compared with apoE4-treated cells only, not significantly different (ns) compared to vehicle. Data are expressed as percentage of protein expression compared with vehicle, and mean ± S.D. of at least three separate experiments.

Figure 2

Effect of fenofibrate (A), naringenin (B), and diosmetin (C) on fl-APP expression as measured by the Western blot analysis using mAB 3E9 and 6E10 Ab (D); and quantitative standardized data for comparing the fold changes in fl-APP mRNA expression as measured using RT-PCR (E). Cells were treated with vehicle (0.1% DMSO), apoE4 (7.5 μg/mL), or drug control (100 μM fenofibrate, 6.25 μM naringenin, and 100 μM diosmetin) for 24 h. Pretreatment consisted of fenofibrate, naringenin, and diosmetin at different doses (see Figure 1A) for 1 h prior to stimulation with apoE4 for 24 h. The top panels in the figure part labels A, B, and C are representative blots, and their corresponding bottom diagrams are the quantitative evaluation of blots for the expression of fl-APP normalized with their respective β-actin levels in cells. For statistical evaluations, the two-way ANOVA analysis followed by Tukey’s range test was performed. Statistically significant differences (#p < 0.05) compared with control; (*p < 0.05) compared with apoE4 treated cells only; not significantly different (ns) compared to control cells. Data are expressed as percentage of protein expression compared with control, and mean ± S.D. of at least three separate experiments.

Figure 3

Effect of fenofibrate (A), naringenin (B), and diosmetin (C) on the apoE4-induced secretion of sAPPtotal and sAPPα in the conditioned medium. Cells were treated with vehicle (0.1% DMSO), apoE4 (7.5 μg/mL), or drug control (100 μM fenofibrate, 6.25 μM naringenin, and 100 μM diosmetin) for 24 h. Pretreatment consisted of fenofibrate, naringenin, and diosmetin at different doses (see Figure 1A) for 1 h prior to stimulation with apoE4 for 24 h. The top panels in the figure part labels A, B, and C are representative blots, and their corresponding bottom diagrams are the quantitative evaluation of blots for the expression of sAPPtotal and sAPPα normalized with their respective β-actin levels in cells. For statistical evaluations, the two-way ANOVA analysis followed by Tukey’s range test was performed. Statistically significant differences (#p < 0.05) compared with control; (*p < 0.05) compared with apoE4 treated cells only; not significantly different (ns) compared to control cells. Data are expressed as a percentage of protein expression compared with control and mean ± S.D. of at least three separate experiments.

Figure 4

Effect of fenofibrate (A), naringenin (B), and diosmetin (C) on the apoE4-induced CTF–α and –β. Cells were treated with vehicle (0.1% DMSO), apoE4 (7.5 μg/mL), or drug control (100 μM fenofibrate, 6.25 μM naringenin, and 100 μM diosmetin) for 24 h. Pretreatment consisted of fenofibrate, naringenin, and diosmetin at different doses (see Figure 1A) for 1 h prior to stimulation with apoE4 for 24 h. The top panels in the figure part labels A, B, and C are representative blots, and their corresponding bottom diagrams are the quantitative evaluation of blots for the expression of CTFα and CTFβ normalized with their respective β-actin levels in cells. For statistical evaluations, the two-way ANOVA analysis followed by Tukey’s range test was performed. Statistically significant differences (#p < 0.05) compared with control; (*p < 0.05) compared with apoE4 treated cells only; significantly different (ns) compared to control cells. Data are expressed as percentage of protein expression compared with control, and mean ± S.D. of at least three separate experiments.