Mild oxidative stress induces redistribution of BACE1 in non-apoptotic conditions and promotes the amyloidogenic processing of Alzheimer's disease amyloid precursor protein

Abstract

BACE1 is responsible for β-secretase cleavage of the amyloid precursor protein (APP), which represents the first step in the production of amyloid β (Aβ) peptides. Previous reports, by us and others, have indicated that the levels of BACE1 protein and activity are increased in the brain cortex of patients with Alzheimer's disease (AD). The association between oxidative stress (OS) and AD has prompted investigations that support the potentiation of BACE1 expression and enzymatic activity by OS. Here, we have established conditions to analyse the effects of mild, non-lethal OS on BACE1 in primary neuronal cultures, independently from apoptotic mechanisms that were shown to impair BACE1 turnover. Six-hour treatment of mouse primary cortical cells with 10-40 µM hydrogen peroxide did not significantly compromise cell viability but it did produce mild oxidative stress (mOS), as shown by the increased levels of reactive radical species and activation of p38 stress kinase. The endogenous levels of BACE1 mRNA and protein were not significantly altered in these conditions, whereas a toxic H2O2 concentration (100 µM) caused an increase in BACE1 protein levels. Notably, mOS conditions resulted in increased levels of the BACE1 C-terminal cleavage product of APP, β-CTF. Subcellular fractionation techniques showed that mOS caused a major rearrangement of BACE1 localization from light to denser fractions, resulting in an increased distribution of BACE1 in fractions containing APP and markers for trans-Golgi network and early endosomes. Collectively, these data demonstrate that mOS does not modify BACE1 expression but alters BACE1 subcellular compartmentalization to favour the amyloidogenic processing of APP, and thus offer new insight in the early molecular events of AD pathogenesis.

Figure 1. Establishment of mOS conditions for treatment of mouse primary cortical cultures. Cortical cells were treated for 6 h with various concentrations of H₂O₂.

A. MTT assay of primary cortical cells treated with up to 100 µM H₂O₂ (n = 3) showed that viability decreased significantly by treatment with 50 and 100 µM H₂O₂. B. MTT assay of cells treated with 10–40 µM H₂O₂ (n = 6) showed no significant loss in viability compared to untreated cells. C. DCF assay of lysates of cells treated with 10–40 µM H₂O₂ and untreated controls. DCF fluorescence values were normalised for protein concentration in the lysates. Experiments were performed three times. D. Immunoblot of phosphorylated p38 and total p38 using 20 µg of cell lysates. E. Quantitative analysis of phospho-p38 and total p38 band densities from three separate blots. Data were analysed with ANOVA, and the Tukey’s HSD (A and E) and Games-Howell (B and C) post-hoc tests. *p<0.05; **p<0.001, ***p<0.005. Error bars represent the standard deviation.

Figure 2. Analysis of BACE1 expression in primary neuronal cultures after mOS treatment.

A. Representative BACE1 immunoblot. Cell lysates (15 µg/lane) were subjected to SDS-PAGE on 8.5% Tris-Glycine gels, followed by immunoblotting with D10E5 rabbit monoclonal antibody that targets BACE1 C-terminal region. Blots were re-probed for actin. B. Densitometry analysis of blots from three individual experiments. BACE1 signal density was normalised to actin. No statistical difference was found between treated and control cells. C. Densitometry analysis of BACE1 blots. BACE1 signal was significantly increased in cells treated with 100 µM H₂O₂ compared to untreated cells or cells treated with 10 µM H₂O₂. Data were analysed by One-way ANOVA, and the Games-Howell post-hoc test. Error bars represent standard deviation. D. RT-qPCR analysis of BACE1 mRNA. 2 µg of RNA was used for first strand synthesis and the resulting cDNA (1 ng) subjected to quantitative real-time PCR. Levels of BACE1 mRNA transcripts were calculated using the comparative C_T method relative to either actin or β2-macroglobulin (B2M) housekeeping genes. qRT-PCR assays were carried out in triplicate. Data represent average data of two independent treatments carried out in triplicate. Error bars represent range of the fold-differences, determined by incorporating the standard deviation of the ΔΔC_T value into the fold-difference calculation.

Figure 3. Immunoblot of caspase-3 and GGA3 in lysates of primary cortical cultures treated with H₂O₂.

20 µg of lysate was loaded per lane. A. Representative blots of caspase-3 and GGA3 from three experiments. Densitometry analyses of full-length proCaspase-3 35 kDa signal. C. Densitometry analysis of 75 kDa GGA3 signal. For both caspase-3 and GGA3, data were normalised to actin. One-way ANOVA indicated no significant change in levels of either protein in response to H₂O₂ treatments.

Figure 4. Quantitative analysis of APP C-terminal fragments in mouse primary cortical lysates after H₂O₂ treatment.

A, 20 µg of lysate samples were electrophoresed on 10–12% Tris-Tricine gels and transferred to nitrocellulose. Blots were probed with anti-APP C-terminal antibody 369. Brain homogenates from an APP-null mouse expressing the last 100 C-terminal amino acids of human APP (C100/APP−/−) and from a DBA/B6 mouse (background strain to the transgenic) were co-electrophoresed as references for β-CTF and APP, respectively. The blots shown are representative of three separate experiments. The * denotes non-specific or uncharacterized bands. B. Densitometry analysis of APP signal relative to actin suggests a decreasing trend but this is not statistically significant. C. Densitometry analysis of α-CTF signal relative to actin also suggests a decreasing trend that is not statistically significant. D. Densitometry analysis of β-CTF signal relative to actin indicates a statistically significant increase after treatment with 20 µM H₂O₂ (p = 0.045). Experiments were performed three times with duplicates. Statistical significance was assessed using One-way ANOVA and the Tukey’s HSD post-hoc test used for pair-wise comparison.

Figure 5. Immunofluorescence analysis of BACE1and colocalisation with early endosomal marker in mouse primary cortical neurons treated or untreated with H₂O₂.

Immunolabelling of untreated cells (a,c,e) and H₂O₂-treated cells (b,d,f). Cells were stained for BACE1 (a,b) with mouse monoclonal antibody 61-3E7 and for early endosomal marker, EEA1 (c,d). Partial colocalisation of BACE1 and EEA1 was observed in both untreated cells (e), and treated cells (f). Bar = 20 µm.

Figure 6. Immunofluorescence analysis of BACE1 and APP colocalisation. in mouse primary cortical neurons treated or untreated with H₂O₂.

Immunolabelling of untreated cells (a,c,e) and H₂O₂-treated cells (B). BACE1 was labelled with mouse antibody 61-3E7 (a,b) and APP was revealed with rabbit antibody 369 (c,d). Since Ab 369 targets the cytosolic domain of APP, it can stain both APP full-length and C-terminal fragments. Colocalisation of BACE1 and APP was observed in untreated cells (e) and this colocalisation may be increased in the treated cells (f). Bar = 20 µm.

Figure 7. Subcellular fractionation of mouse primary cortical cells on a discontinuous sucrose density gradient.

Cells were incubated for 6 h in the presence or absence of 40 µM H₂O₂ and lysed under isotonic conditions. Post-nuclear supernatants were centrifuged at 100,000×g on a discontinuous sucrose gradient. Fractions were analysed by immunoblotting with antibodies for BACE1 (D10E5), APP (22C11) and for organelle markers (Transferrin receptor, TfR for plasma membrane and endocytic vesicles; TGN38, for trans-Golgi; EEA1 for early endosomes). Signal density was determined for all fractions, and specific protein levels in each fraction were expressed as a percentage of the total signals from all thirteen fractions. Representative blots from two experiments with untreated cells (A) and H₂O₂-treated cells (B). Graphs (C and D) represent signal intensity % of each protein plotted against fraction number. Noticeable changes in BACE1 distribution were observed after H₂O₂-treatment, with BACE1 protein redistributing from light to denser fractions, enriched in EEA1 and TGN38 markers.

Figure 8. Schematic proposing a biphasic effect of oxidative stress on BACE1.

A. Effect of mOS on BACE1 expression. mOS does not alter BACE1 levels but induces its subcellular redistribution to enhance colocalisation. with APP and favour β-CTF production. It may be speculated that this also leads to Aβ production. mOS also induces p38 stress kinase phosphorylation, which can trigger cell survival signaling. B. Effect of severe OS. This increases BACE mRNA and protein expression, thereby increasing APP amyloidogenic processing. MAP kinases also become activated, as well as apoptotic mechanisms. These events cause further cellular accumulation of BACE1, particularly in cellular compartments where APP is present, hence resulting in higher Aβ production and leading to formation of toxic species that induce further OS, by way of a feed-back mechanism.