Amyloid Beta-Peptide Increases BACE1 Translation through the Phosphorylation of the Eukaryotic Initiation Factor-2α

Alzheimer's disease (AD) is tightly linked to oxidative stress since amyloid beta-peptide (Aβ) aggregates generate free radicals. Moreover, the aggregation of Aβ is increased by oxidative stress, and the neurotoxicity induced by the oligomers and fibrils is in part mediated by free radicals. Interestingly, it has been reported that oxidative stress can also induce BACE1 transcription and expression. BACE1 is the key enzyme in the cleavage of the amyloid precursor protein to produce Aβ, and the expression of this enzyme has been previously shown to be enhanced in the brains of Alzheimer's patients. Here, we have found that BACE1 expression is increased in the hippocampi from AD patients at both the early (Braak stage II) and late (Braak stage VI) stages of the disease as studied by immunohistochemistry and western blot. To address the role of Aβ and oxidative stress in the regulation of BACE1 expression, we have analyzed the effect of subtoxic concentrations of Aβ oligomers (0.25 μM) and H2O2 (10 mM) on a human neuroblastoma cell line. Firstly, our results show that Aβ oligomers and H2O2 induce an increase of BACE1 mRNA as we studied by qPCR. Regarding BACE1 translation, it is dependent on the phosphorylation of the eukaryotic initiation factor 2α (eIF2α), since BACE1 mRNA bears a 5′UTR that avoids its translation under basal conditions. BACE1 5′UTR contains four upstream initiating codons (uAUGs), and its translation is activated when eIF2α is phosphorylated. Consistently, we have obtained that Aβ oligomers and H2O2 increase the levels of BACE1 and p-eIF2α assayed by western blot and confocal microscopy. Our results suggest that Aβ oligomers increase BACE1 translation by phosphorylating eIF2α in a process that involves oxidative stress and conforms a pathophysiological loop, where the Aβ once aggregated favors its own production continuously by the increase in BACE1 expression as observed in AD patients.


Introduction
Alzheimer's disease (AD) is a neurodegenerative process that occurs in the elderly being the most prevalent type of human dementia. AD symptoms consist in dramatic memory deficits and an irreversible cognitive decline. They start with neuronal death in the hippocampus and the brain structure for learning and memory, and later, the neuronal loss progresses to other cortical areas. The histopathological characteristics of AD patients are extracellular senile plaques and intracellular neurofibrillary tangles. The senile plaques are mainly composed of amyloid β-protein (Aβ) and the neurofibrillary tangles of hyperphosphorylated tau protein [1,2].
Aβ is a peptide having from 36 to 43 amino acids [3]; however, Aβ 1-40 is the most abundant. Aβ is produced by the consecutive enzymatic action of the beta-and gammasecretase activities on an integral type I transmembrane glycoprotein termed amyloid precursor protein (APP) [4]. Aβ 1-42 production increases with aging [5], in patients of familiar AD bearing some mutations in the presenilins (PS1/PS2) [6], which are the catalytic core of the gammasecretase complex, or due to the nitrotyrosination of the gamma-secretase complex [7]. The pathophysiological relevance of this shift in the Aβ species production is due to the high aggregant properties of the Aβ 1-42 [8,9], which makes it more neurotoxic.
The oligomeric forms are the responsible for the most neurotoxic effects associated to the Aβ [10][11][12]. Aβ aggregates produce H 2 O 2 [13] inducing oxidative stress and starting an intracellular cascade of reactive oxygen species, which will compromise neuronal viability [14,15]. Moreover, oxidative stress has been reported to increase the transcription and expression of the enzyme Beta-site Amyloid precursor protein Cleaving Enzyme 1 (BACE1) [16,17]. BACE1 is the key enzyme that carries out the beta-secretase activity on APP to initiate the Aβ production pathway.
BACE1 expression is physiologically repressed at the transcriptional and translational level. There are different pathways, mostly related to different types of stress, that induce the nuclear translocation of transcription factors, such as cJun/cFos, to bind to BACE1 gene promoter [18,19]. However, the tightest control of BACE1 is at the translational level since BACE1 mRNA bears a particular 5′UTR that contains four upstream initiation codons (uAUGs) and possesses a rich GC content. These sequence motifs confer a particular secondary structure to that region of the mRNA that impairs ribosomes to reach the main AUG in order to start BACE1 translation [20][21][22][23]. In basal conditions, ribosomes remain in one of the uAUG, especially the second one. BACE1 mRNA translation is only activated when the eukaryotic initiation factor 2α (eIF2α) is phosphorylated at serine 51 [19,[24][25][26]. The physiological relevance of the eIF2α phosphorylation is to block the translation of the most of the proteins under stressful conditions and to induce only the translation of a group of special proteins that bear 3 or more uAUGs in the 5′UTR [27]. These stressful conditions are virus infection, reticular stress, nutrient deprivation, or oxidative stress. There are four eIF2α kinases: hemeregulated eukaryotic initiation (HRI), general control nonderepressible 2 kinase (GCN2), double-stranded RNAactivated protein kinase (PKR), and double-stranded RNAactivated protein kinase-like (PERK), which phosphorylate eIF2α at serine 51 blocking translation initiation [25][26][27][28][29][30].  (EC-IMIM-UPF). Hippocampal samples were obtained from 3 nondemented controls (1 man and 2 women; mean: 69 years old), 2 AD patients at Braak stage II (2 men; 83 and 76 years old), and 4 AD patients at Braak stage VI (2 men and 2 women; mean: 70 years old). Sections (5 μm) were treated with 4% H 2 O 2 and incubated o.n. at 4°C with 1 : 100 rabbit anti-BACE1 Antibody (Ab; Invitrogen). The secondary Ab was 1 : 500 donkey anti-rabbit peroxidase-conjugated Ab, which was incubated for 1 h at room temperature (RT). A Peroxidase Substrate Kit DAB (Vector) was used. Slides were stained with hematoxylin and fixed. The images were taken by a Leica DMR microscope.

Cell Line. Human neuroblastoma cells (SH-SY5Y cells)
were grown with Ham's F12 GlutaMax (F12 medium; Gibco) supplemented with 15% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Gibco). Cells were incubated at 37°C in a humidified atmosphere of 5% CO 2 .      Oxidative Medicine and Cellular Longevity phosphorylated proteins, the cells were lysed on ice with lysis solution as indicated in subsection 2.2. Extracts were homogenised using vortex for 30 min at 4°C; afterwards, samples were centrifuged at 10,000 for 5 min to obtain the supernatant. A Bio-Rad kit was used to calculate protein concentrations. Aliquots of 20 μL (for phosphorylated proteins) or 80 μg (for the other proteins) were loaded into a 10% SDS-PAGE gels. Afterwards, proteins were transferred onto 0.2 μm pore nitrocellulose membranes. Membranes were blocked for 1 h at RT with TTBS plus 5% bovine serum albumin (BSA) for phosphorylated proteins or 5% skimmed milk for the other proteins. Then, membranes were incubated o.n. at 4°C with 1 : 1,000 rabbit anti-BACE1 Ab (Invitrogen), 1 : 500 rabbit anti-p-eIF2α (Ser51) Ab (Invitrogen), 1 : 500 mouse anti-eIF2α Ab (Abcam), and 1 : 5,000 mouse antitubulin Ab (Sigma). 1 : 2,000 secondary Abs were horseradish peroxidase-conjugated donkey anti-mouse and anti-rabbit (GE Healthcare) for 1 h at RT. Bands were visualized with   Oxidative Medicine and Cellular Longevity Super Signal (Pierce) and analyzed with the Quantity One system in a BioRad Universal Hood II.

Translational Studies by Confocal Microscopy in Cells.
Cells were seeded on coverslips in 24-well plates at a density of 3 × 10 4 cells/well. After 12 h, the growth medium was removed, and cells were treated for 24 h with subtoxic concentrations of 0.

BACE1 Expression Is Increased in the Hippocampi from AD Patients.
There are previous works that reported increased expression of BACE1 in AD patients [31][32][33] relating this finding with the enhanced Aβ production that will induce the onset and progression of the disease. Consistently, Aβ 1-42 oligomers have been reported to be present in the hippocampi from AD patients since the early stages of the disease [34]. Here, we have studied the expression of BACE1 in the hippocampi from AD patients at two stages of the disease: Braak II (one of the initial stages showing the early symptoms of AD) and Braak VI (the final stage of the disease, when the Aβ is widespread in the brain and dementia is severe). We found in both stages an increased expression of BACE1 ( Figure 1(a)), linking the early enhancement of BACE1 expression with Aβ production. Furthermore, this increment in BACE1 expression was also demonstrated through western blot (Figure 1(b)) when the mature glycosylated form (70 kDa) was analysed. AD patient samples showed an increased expression of BACE1 compared with nondemented controls (p < 0:05). The maintained BACE1 expression even in the late stage of the disease suggest that oxidative stress, which has been reported to be significantly increased in AD [14], would be inducing BACE1 expression directly or by the indirect effect of Aβ 1-42 oligomers, which generate free radicals, as we address in the present work.

Aβ 1-42 Oligomers and H 2 O 2 Decrease Cell Viability at
High Concentrations. Human neuroblastoma cells were treated with increased concentration of oligomeric Aβ 1-42 for 24 h (Figure 2(a)) in order to study their neurotoxic abilities and to find the range of concentrations that do not produce a significant cytotoxicity to carry out the experiments on BACE1 regulation. Aβ 1-42 oligomers were significantly neurotoxic at 5 μM (p < 0:005), 10 μM (p < 0:001), and 15 μM (p < 0:001). The production of H 2 O 2 by the Aβ 1-42 oligomers is a continuous process, while the Aβ 1-42 is present, along 24 h in our experiments. Therefore, we have also studied the effect of the treatment with H 2 O 2 on cell viability (Figure 2(d)). We have obtained that H 2 O 2 is cytotoxic at 100 μM (p < 0:001).
The cell cytotoxicity was also studied by images obtained after the reduction of the MTT to blue formazan by bright field microscopy (Figures 2(b) and 2(e)) and with an Ab against active caspase-3 (Figures 2(c) and 2(f)). In both studies, 15 μM Aβ 1-42 oligomers and 100 μM H 2 O 2 showed a low reduction of MTT and high caspase activation as expected.
In order to mimic the pathophysiological effects of a continuous and insidious damage on neurons along the life, we decided to carry out the experiments on BACE1 regulation with the subtoxic concentration of 0.25 μM Aβ 1-42 oligomers and 10 μM H 2 O 2 . These concentrations were producing neither cytotoxicity when assayed by MTT reduction (Figures 2(a), 2(d), 2(b), and 2(e)) nor significant caspase activation (Figures 2(c) and 2(f)).

Discussion
There are many evidences that relate oxidative stress with the ethiopathogenesis of AD, a devastating neurodegenerative disease whose major risk factor is aging. This tight relationship starts with the aggregation of Aβ, which  Oxidative Medicine and Cellular Longevity increases by oxidative stress [35]. Once the fibrils are formed, they also produce H 2 O 2 [13] and hydroxyl radical [36,37]. In fact, the neurotoxicity of Aβ aggregates has been reported to be mediated by oxidative stress [14,15,38,39]. Moreover, oxidative stress has been demonstrated to increase BACE1 transcription and translation [16,17]. We have demonstrated in this work that BACE1 expression in increased even in the early stages of the disease. Since oxidative stress is a concomitant process with aging and Aβ aggregation is considered the key factor of AD, according to the amyloid cascade hypothesis, we have studied the effect of Aβ oligomers and oxidative stress in BACE1 expression and the mechanisms that control its pathophysiological expression. BACE1 is an enzyme that accomplishes some physiological functions as dendritic spine growth in the hippocampus where contributes to memory formation [26]. In fact, BACE1 knockout mice show synaptic plasticity deficits and cognitive impairment [40,41]. However, the dysregulation of BACE1 has harmful effects. In our study, we have found that long treatments with low nanomolar concentration of Aβ 1-42 oligomers, mimicking the environment of neurons in AD patients, increase BACE1 transcription and expression in vitro. The relevance of this finding is supported by the increased expression of BACE1 found in AD hippocampi. This would suggest the existence of a loop of amyloid production that will activate BACE1 to release more amyloid contributing to accelerate the dysregulation of BACE1 and the characteristic amyloidosis of AD. This effect should be based in the capability of Aβ 1-42 oligomers to produce oxidative stress since similar results has been obtained when we used hydrogen peroxide.
BACE1 expression is controlled physiologically by the phosphorylation of eIF2α carried out by the enzyme HRI  9 Oxidative Medicine and Cellular Longevity [26]. HRI is activated throughout the nitric oxide released after NMDA receptors activation in glutamatergic neurons. However, aging produces an increase in oxidative stress in brain [42,43] and would result in the loss of BACE1 physiological control. It is due to the fact that HRI, PKR, and PERK, three of the four kinases that phosphorylates eIF2α, can be activated by oxidative stress [44][45][46][47][48]. In our study, we have found that both oxidative stress and Aβ 1-42 oligomers yield to the phosphorylation of the eIF2α in vitro. The dysregulation of the different eIF2α kinases by oxidative stress would explain these results.
Summarizing, our results suggest that oxidative stress induced by Aβ oligomers increase BACE1 transcription and translation. Our data support that the mechanism involved in the increased BACE1 expression is the dysregulation of the phosphorylation of the eIF2α that would generate the amyloid burden in AD.

Data Availability
All the data used to support the findings of this study are available from the corresponding author upon request.