Apolipoprotein E lipoprotein particles inhibit amyloid-β uptake through cell surface heparan sulphate proteoglycan

Abstract

Background: The accumulation, aggregation and deposition of amyloid-β (Aβ) peptides in the brain are central to the pathogenesis of Alzheimer's disease (AD). Alzheimer's disease risk increases significantly in individuals carrying one or two copies of APOE ε4 allele compared to individuals with an ε3/ε3 genotype. Growing evidence has demonstrated that apolipoprotein E (apoE) strongly influences AD pathogenesis by controlling Aβ aggregation and metabolism. Heparan sulphate proteoglycans (HSPGs) are abundant cell surface molecules that bind to both apoE and Aβ. HSPGs have been associated with Aβ aggregation and deposition. Although several lines of research have shown that apoE influences Aβ clearance in the brain, it is not clear how apoE influences HSPG-mediated cellular uptake of Aβ.

Results: In this study, we show that apoE lipoprotein particles from conditioned media of immortalized astrocytes isolated from human APOE-targeted replacement (TR) mice significantly suppress cellular Aβ42 and Aβ40 uptake through cell surface HSPG. ApoE3 and apoE4 particles have similar binding affinity to heparin, while apoE4 particles are likely hypolipidated compared to apoE particles. We also found that the apoE particles antagonize Aβ binding to cell surface, and inhibited Aβ uptake in a concentration-dependent manner in Chinese hamster ovary (CHO) cells. While the effect was not apoE isoform-dependent, the suppressive effect of apoE particles on Aβ uptake was not observed in HSPG-deficient CHO cells. We further demonstrated that apoE particles reduced the internalization of Aβ in mouse primary neurons, an effect that is eliminated by the presence of heparin.

Conclusions: Taken together, our findings indicate that apoE particles irrespective of isoform inhibit HSPG-dependent cellular Aβ uptake. Modulating the ability of apoE particles to affect Aβ cellular uptake may hold promises for developing new strategies for AD therapy.

Keywords: Alzheimer’s disease; Aβ; Cellular uptake; HSPG; apoE.

Fig. 1

Characterization of astrocyte-secreted apoE particles. Immunoaffinity purified apoE3 (500 ng) and apoE4 particles (500 ng) were analyzed by non-denaturing gradient gel electrophoresis (4–20 %) (a) and SDS-PAGE (b) followed by Western blot for apoE. Numbers on the left are molecular size markers expressed in kDa. c Total cholesterol concentrations of apoE3 and apoE4 particles were measured using the Amplex Red cholesterol assay kit and normalized against apoE concentrations. Data represent mean ± S.D. (n = 3). ***, p < 0.001

Fig. 2

ApoE particles bind to heparin and cell surface HSPG. ApoE3 particles (3.4 μg) and apoE4 particles (3.4 μg) were applied to HiTrap Heparin HP columns attached to a FPLC and eluted with a linear salt gradient (0–1.0 M NaCl). Concentrations of apoE in each fraction were determined by ELISA (a). An average of n = 3 experiments are plotted in the graph. CHO-K1 or CHO-M1 cells were incubated with apoE3 (200 nM) (b) or apoE4 (200 nM) (c) for 18 h at 37 °C. Internalization of apoE was analyzed by ELISA. Data represent mean ± S.D. (n = 3). **, p < 0.01; ***, p < 0.001

Fig. 3

ApoE particles inhibit Aβ cellular uptake through HSPG in CHO cells. CHO-K1 (wild-type) cells or CHO-M1 (HS-deficient) cells were incubated with 50 nM Aβ40 (a) or 50 nM Aβ42 (b) in the presence or absence of apoE3 particle (200 nM) or apoE4 particle (200 nM) for 18 h at 37 °C. The amount of internalized Aβ was quantified by ELISA. Data represent mean ± S.D. (n = 3). **, p < 0.01

Fig. 4

ApoE particles inhibit HSPG-mediated cellular uptake of Aβ in a concentration-dependent manner. CHO-K1 cells or CHO-M1 cells were incubated with Aβ42 (50 nM), together with various concentrations of apoE3 particles (0.2 nM to 200 nM) or apoE4 particles (0.2 nM to 200 nM) for 18 h at 37 °C. The amount of internalized Aβ was quantified by ELISA. Data represent mean ± S.D. (n = 3)

Fig. 5

ApoE particles suppress Aβ binding to HSPG on the cell surface. CHO-K1 or CHO-M1 cells were incubated with Aβ42 (50 nM) with or without apoE3 particles (200 nM) or apoE4 particles (200 nM) for 3 h at 4 °C. Cell-bound Aβ42 was quantified by ELISA. Data represent mean ± S.D. (n = 3). *, p < 0.05

Fig. 6

ApoE particles decrease HSPG-mediated cellular uptake of Aβ in primary neurons. a Mouse primary cortical neurons were incubated with FAM-Aβ42 (500 nM) with or without apoE3 particles (200 nM) in the presence or absence of heparin (15 U/ml) for 6 h at 37 °C and analyzed by confocal microscopy. Left, middle, and right columns indicate Lysotracker, FAM-Aβ42, and merged images, respectively. b Internalization of Aβ was quantified by FACS after incubation with FAM–Aβ42 (500 nM) for 6 h. Data represent mean ± S.D. (n = 3). N.S., not significant; **, p < 0.01; ***, p < 0.001