Amyloid β-protein oligomers upregulate the β-secretase, BACE1, through a post-translational mechanism involving its altered subcellular distribution in neurons

Abstract

Background: β-Site amyloid precursor protein cleaving enzyme 1 (BACE1) is a membrane-bound aspartyl protease that initiates amyloid β-protein (Aβ) generation. Aberrant elevation of BACE1 levels in brains of Alzheimer's disease (AD) patients may involve Aβ. In the present study, we used a neuron culture model system to investigate the effects of Aβ on BACE1 expression as well as the underlying mechanisms.

Results: Rat primary cortical neurons were treated with relatively low concentrations (2.5 μM) of Aβ42 oligomers (Aβ-O) or fibrils (Aβ-F) for 2-3 days. Aβ-O induced a significant increase in protein levels of BACE1, while Aβ-F only had a marginal effect. Levels of amyloid precursor protein (APP) and the major α-secretase, ADAM10, remained unaltered upon treatment with both types of Aβ. Aβ-O treatment resulted in activation of eIF2α and caspase 3 in a time-dependent manner, with no changes in the endoplasmic reticulum (ER) stress marker, GRP78, indicating that a typical ER stress response is not induced under our experimental conditions. Furthermore, Aβ-O did not affect BACE1 mRNA expression but augmented the levels of exogenous BACE1 expressed via recombinant adenoviruses, indicating regulation of BACE1 protein expression, not at the transcriptional or translational but the post-translational level. Immunocytochemical analysis revealed that Aβ-O causes a significant increase in BACE1 immunoreactivity in neurites (both axons and dendrites), but not soma of neurons; this change appears relevant to the mechanism of Aβ-O-induced BACE1 elevation, which may involve impairment of BACE1 trafficking and degradation. In contrast, Aβ-O had no effect on APP immunoreactivity.

Conclusion: Our results collectively suggest that Aβ oligomers induce BACE1 elevation via a post-translational mechanism involving its altered subcellular distribution in neurons, which possibly triggers a vicious cycle of Aβ generation, thus contributing to the pathogenetic mechanism of AD.

Fig. 1

Aβ oligomers specifically enhance the BACE1 protein level in primary neurons. a Aβ42 oligomers (O) or fibril (F) preparations (2.3 or 4.5 μg) were separated with the Tris/Tricine gel, followed by Western blot analysis with anti-Aβ antibodies, 82E1 or 6E10. b Neurons grown on a 12-well plate were treated with 2.5 μM Aβ as above followed by cell survival assay using the Cell Counting Kit-8, as described in Methods. Relative levels of cell survival are presented as a graph. Data represent means ± SEM from four separate experiments. c Primary cortical neurons (9DIV) grown on a six-well plate were treated with 2.5 μM Aβ oligomers (O), fibrils (F) or vehicle (C) for 2 or 3 days, followed by Western blot analysis with antibodies against BACE1, APP, ADAM10 or β-actin. d Quantitative analysis of BACE1, APP and ADAM10 levels after normalization to β-actin. Data represent means ± SEM from three or four separate experiments. *p < 0.05, **p < 0.01, compared with control

Fig. 2

Aβ oligomers induce activation of caspase 3 and eIF2α, but do not alter GRP78 levels. a Primary neurons were treated with 2.5 μM Aβ oligomers (O), fibrils (F) or vehicle (C) for 2 or 3 days, followed by Western blot analysis of cell lysates. Membranes were probed with the indicated antibodies. b Quantitative analysis of p-eIF2α/total eIF2α ratios and cleaved caspase 3 levels. Data represent means ± SEM from three separate experiments. *p < 0.05 and **p < 0.01, compared with control. c Primary neurons were treated with 2.5 μM Aβ oligomers (O) for 3 days or 1 μM thapsigargin (Thap) for 1 day, followed by Western blot as above. Membranes were probed with the indicated antibodies. d Quantitation of GRP78 levels. Data represent means ± SEM from three independent experiments. **p < 0.01, compared with control

Fig. 3

Aβ oligomers augment BACE1 levels, not at the transcriptional or translational, but the post-translational level. a Total RNA was extracted from neurons treated with 2.5 μM Aβ oligomers (O), fibrils (F) or vehicle (C) for 1 or 2 days, followed by semi-quantitative RT-PCR. b Band intensities of BACE1 and vimentin in (A) were quantified, and BACE1 mRNA levels normalized to those of vimentin presented as a graph. Data represent means ± SEM from three separate experiments. c Primary cortical neurons (8DIV) were infected with recombinant BACE1 adenoviruses expressing rhodopsin-tagged BACE1. The next day, cells were exposed to Aβ oligomers for 1–3 days, followed by Western blot with anti-rhodopsin tag 1D4. Membranes were reprobed with the different antibodies specified. d Quantitative analysis of exogenous BACE1 levels. Data represent means ± SEM from three independent experiments. *p < 0.05, compared with control

Fig. 4

Immunocytochemical analysis of BACE1 and APP. a, b Primary cortical neurons grown on coverslips were treated with 2.5 μM Aβ oligomers (Aβ-O) for 3 days, followed by immunofluorescence staining with anti-BACE1 (a) or anti-APP (b) antibodies. Immunostaining of control and Aβ-O-treated cells was carried out under the same conditions, and their images were acquired at the same exposure time. Scale bar = 20 μm. Intensity of BACE1 immunoreactivity is relatively higher in neurites, but not soma of neurons treated with Aβ-O than control. c, d Fluorescence intensities of BACE1 (c) or APP (d) in soma and neurites were separately quantified as described in Methods, and the relative levels depicted on a graph. (n = 18 ~ 20, ***p < 0.001). e Fluorescence intensities of BACE1 in axons and dendrites were separately quantified as above in specimens doubly immunostained with BACE1 and MAP2, and the relative levels depicted on a graph (n = 24, *p < 0.05, **p < 0.01)

Fig. 5

A schema illustrating the mechanism by which Aβ oligomers (Aβ-O) induce BACE1 elevation in neurons. BACE1 levels in neurites, but not soma, are specifically increased in Aβ-O-treated neurons, compared with untreated control. Aβ-O possibly impairs the trafficking of BACE1 in neurites, leading to reduced transport to lysosomal compartments and augmentation in neurites. Aberrantly increased BACE1 consequently promotes Aβ production