Oxygen-glucose deprivation regulates BACE1 expression through induction of autophagy in Neuro-2a/APP695 cells

Rong-fu Chen, Ting Zhang, Yin-yi Sun, Ya-meng Sun, Wen-qi Chen, Nan Shi, Fang Shen, Yan Zhang, Kang-yong Liu,, Xiao-jiang Sun,

1 Department of Neurology, Sixth People’s Hospital Affi liated to Shanghai Jiao Tong University, Shanghai, China

2 Zhoupu Hospital, Pudong New District, Shanghai, China

Oxygen-glucose deprivation regulates BACE1 expression through induction of autophagy in Neuro-2a/APP695 cells

Rong-fu Chen1, Ting Zhang1, Yin-yi Sun1, Ya-meng Sun1, Wen-qi Chen1, Nan Shi2, Fang Shen1, Yan Zhang2, Kang-yong Liu1,2, Xiao-jiang Sun1,*

1 Department of Neurology, Sixth People’s Hospital Affi liated to Shanghai Jiao Tong University, Shanghai, China

2 Zhoupu Hospital, Pudong New District, Shanghai, China

Our previous fi ndings have demonstrated that autophagy regulation can alleviate the decline of learning and memory by eliminating deposition of extracellular beta-amyloid peptide (Aβ) in the brain after stroke, but the exact mechanism is unclear. It is presumed that the regulation of beta-site APP-cleaving enzyme 1 (BACE1), the rate-limiting enzyme in metabolism of Aβ, would be a key site. Neuro-2a/amyloid precursor protein 695 (APP695) cell models of cerebral ischemia were established by oxygen-glucose deprivation to investigate the eff ects of Rapamycin (an autophagy inducer) or 3-methyladenine (an autophagy inhibitor) on the expression of BACE1. Either oxygen-glucose deprivation or Rapamycin down-regulated the expression of BACE1 while 3-methyladenine up-regulated BACE1 expression. These results confi rm that oxygen-glucose deprivation down-regulates BACE1 expression in Neuro-2a/APP695 cells through the introduction of autophagy.

nerve regeneration; brain Injury; oxygen-glucose deprivation; cerebral ischemia; stroke; autophagy; beta-site APP-cleaving enzyme 1 (BACE1); beta-amyloid peptide; 3-methyladenine (3-MA); Rapamycin; neural regeneration

Funding: This work was supported by the National Natural Science Foundation of China, No. 31171014, 31371065; a grant from Shanghai Municipal Health Bureau, China, No. 20134125; a grant from Shanghai Pudong District Health Bureau of China, No. PDZz2013-10.

Chen RF, Zhang T, Sun YY, Sun YM, Chen WQ, Shi N, Shen F, Zhang Y, Liu KY, Sun XJ (2015) Oxygen-glucose deprivation regulates BACE1 expression through induction of autophagy in Neuro-2a/APP695 cells. Neural Regen Res 10(9):1433-1440.

Introduction

With increasing human life expectancy, the prevalence of ischemic stroke and associated disability has increased; ischemic stroke accounts for 80% of all occurrences of stroke and about two-thirds of stroke patients have cognitive impairment to diff erent degrees. Many studies have suggested that cognitive decline is closely related to the deposition of extracellular β-amyloid peptide (Aβ) in the brain (Henon et al., 2001; Pendlebury and Rothwell, 2009; Landau et al., 2012; Pimentel-Coelho and Rivest, 2012). Therefore, preventing Aβ production and/or promoting Aβ degradation can improve cognitive decline in patients or mice (Hachinski and Munoz, 1997; Zhang et al., 2007; McAllister et al., 2010; Chetelat et al., 2012; Kawahara et al., 2012). Aβ is a polypeptide of 37–43 amino acids and is derived from β-amyloid precursor protein (APP) by β- and α-secretase cleavage (Nunan and Small, 2000). APP is a type I transmembrane glycoprotein that can be cleaved by α-, β-, and γ-secretases, and Vassar (2005) identified the β-secretase as the β-site APP-cleaving enzyme 1 (BACE1), the key rate-limiting enzyme in Aβ metabolism (Vassar, 2001). BACE1 triggers Aβ generation and acts as a key regulator of Aβ metabolism. In addition, up-regulation of BACE1 expression has been shown to increase Aβ production (Sun et al., 2006), and regulation of BACE1 activity can aff ect Aβ generation (Gravenfors et al., 2012; Obregon et al., 2012; Zhu et al., 2012; Yun et al., 2013). Moreover, application of a BACE1 inhibitor is expected to decrease Aβ expression in patients with Alzheimer’s disease (Kacker et al., 2012). These data suggest that reduction of Aβ generation through down-regulation of BACE1 expression might be practicable and benefi cial.

Autophagy, or cellular self-digestion, is a cellular catabolic pathway in which misfolded or aggregated proteins and damaged organelles are transported to lysosomes for degradation (Levine and Kroemer, 2008). Studies have shown that the β-site APP-cleaving enzyme can be degraded via the lysosomal pathway (Koh et al., 2005). Thus, reduction of Aβ generation might indirectly result from inhibition of BACE1 metabolism. Yu et al. (2005) discovered that autophagy regulation could modulate Aβ generation in the brain of mice, thus improving learning and memory. A previous study has shown that autophagy regulation aff ected Aβ generation (Zhang et al., 2011). The present study focused on the relationship between autophagy and BACE1.

In ischemic stroke, regional cerebral blood fl ow is inadequate or blocked. Neurons in the aff ected areas are short of oxygen and glucose, subsequently leading to neuronal loss in the central necrosis area and cellular stress injury in the penumbral region of brain infarction (Dirnagl et al., 1999). Therefore, a oxygen-glucose deprivation (OGD) model might mimic the pathological changes of cerebral ischemia and mouse neocortical cell cultures has become an in vitro model in its research (Goldberg and Choi, 1993). A cell model of cerebral ischemia was established as previously described (Wang et al., 2010). This was applied in the following experiments to evaluate BACE1 expression with autophagy regulators and to explore pathways involved in BACE1 metabolism. The results might provide some useful information for clinical treatment of post-stroke cognitive decline.

Materials and Methods

Creation of a cell model of cerebral ischemia in vitro

Neuro-2a/amyloid precursor protein 695 (Neuro-2a/ APP695) cell lines, stably over-expressing wild-type human APP695 protein, were provided by Professor Chun-jiu Zhong from the Department of Neurology of Zhongshan Hospital of Fudan University, China. Neuro-2a/APP695 cells over-expressed APP695 protein, so the intervention eff ect of β-site APP-cleaving enzyme by drugs or treatment could be induced easily. They were cultured in mixed media [44.5% Dulbecco’s modifi ed Eagle’s medium (DMEM), 44.5% Opti-MEM?, 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 U/mL streptomycin, 200 μg/mL G418; Thermo Fisher Scientifi c, Waltham, MA, USA) in an incubator with 100% saturated humidity and 5% CO2(Heraeus, Hanau, Germany) at 37°C. A cell model of OGD in vitro was established according to a previous report (Wang et al., 2010). Cells were passaged at around 80% confl uence, and cultured as usual. When cells were in the logarithmic growth phase, the cell culture medium was replaced with OGD culture medium. The study was approved by the Local Institutional Ethical Committee of the Sixth People’s Hospital of Shanghai, China.

Preparation of autophagy-inducing or -inhibiting drugs

Rapamycin (2.5 mg/mL), a liquid reagent itself, dissolves easily in cell culture medium (working concentration 200 ng/mL) while 3-methyladenine (3-MA), a poorly water-soluble drug, should be dissolved with sterilized phosphate buffered saline (PBS) solution beforehand. Before each trial, a stock solution of 100 mM 3-MA in PBS was heated in a water bath at 50°C, and then diluted into working concentration (5 mM) with culture medium, avoiding white fl ock formation.

Evaluation of proliferation rate of Neuro-2a/APP695 cells with OGD treatment

The cell proliferation rate was detected by a cell proliferation-cytotoxicity assay kit (CCK-8, CK04; Dojindo, Kumamoto, Japan). Grouping was designed as the following: control group, OGD group (OGD for 1 hour), OGD + Rapamycin group (intervention with 200 ng/mL Rapamycin, Sigma-Aldrich, St. Louis, MO, USA), and OGD + 3-MA group (intervention with 5 mM 3-MA, Sigma-Aldrich). Before OGD treatment, cells were pre-incubated with Rapamycin (200 ng/mL) or 3-MA (5 mM) for 1 hour, and then followed by OGD treatment along with a drug intervention (200 ng/mL Rapamycin or 5 mM 3-MA) for 1 hour respectively. At the same time, the cell culture medium was replaced with OGD culture medium (sugar-free DMEM, 1.0 mM NaCN (Sigma-Aldrich), penicillin, streptomycin, and 2% FBS). Cells in the logarithmic growth phase were collected and planted at a density of 1 × 104cells per well in a 96-well plate. After incubation for 24 hours, cells were treated with OGD or interventional drugs, and 10 μL CCK-8 solution (10 mg/mL) was added to the cell medium of all groups, and incubation was continued for 2 hours. Finally, the optical density value at 450 nm was measured with a multifunctional microplate reader (BioTek Instruments, Inc., VT, USA) using a reference wavelength of 630 nm to detect a yellow-colored product (formazan), which is soluble in the tissue culture medium. The amount of the formazan dye generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells. Relative cell viability was compared with the control group by percentage optical density.

Evaluation of apoptosis ratio of Neuro-2a/APP695 cells with OGD treatment

When a cell culture reached 80% confl uence, cells were trypsinized, resuspended, and then planted in 6-well plates at an appropriate density. After 24 hour incubation, cells were treated with OGD or an intervention drug (200 ng/mL Rapamycin or 5 mM 3-MA) for 1 hour. Next, cells were rinsed with ice-cold 1 × binding buff er once and digested for 20 minutes with ethylenediamine tetra acetic acid-free trypsin. Cell lysates were collected in fl ow cytometry tubes and centrifuged at 1,000 r/min at 4°C for 5 minutes. After centrifugation, 1 × binding buff er was added to each tube at 1 × 106cells /mL, and 100 μL of the cell re-suspension solution was transferred into a new fl ow cytometry tube. Then the cell suspensions were mixed with 5 μL of Annexin-V-fl uorescein isothiocyanate (20 μg/mL, V13241; Invitrogen, Carlsbad, CA, USA), shaken gently, incubated for 15 minutes at room temperature in the dark, and then re-suspended with 400 μL 1 × binding buff er, mixed with 1 μL propidium iodide (PI; 100 μg/mL, V13241; Invitrogen) for 5 minutes before detection by fl ow cytometry. A tube without propidium iodide was used as a negative control. After staining, apoptotic cells, dead cells and live cells exhibited diff erent fl uorescence. These populations can easily be distinguished using a fl ow cytometer (FACSAria, Becton Dickinson Company, Franklin Lakes, NJ, USA). The relative cell apoptosis ratio was evaluated by percentage of the control group. The total number of cells (all events) was 30,000.

Western blot assay

Target proteins in the cell lysates of all groups were detected by western blot assay. First, cells were collected after their

respective treatment and fully lysed in radioimmunoprecipitation assay lysis buffer [1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1% NP-40, 50 mM Tris-HCl (pH 7.5), 250 mM sodium chloride] containing phenylmethanesulfonyl fluoride at 0°C for 20 minutes. Following centrifugation at 12,000 × g for 30 minutes at 4°C in a supercentrifuge (CP80 MX, Hitachi, Tokyo, Japan), supernatants were collected, and stored at ?80°C. After determining the protein concentration by a BCA protein assay kit (Thermos Fisher Scientific, Waltham, MA, USA), equal amounts of proteins were separated using SDS-polyacrylamide gel electrophoresis at 80 V constant voltage for 20 minutes, followed by 120 V constant voltage for 90 minutes. The proteins were then wet-transferred to polyvinylidene difluoride membranes (Merck Millipore: Calbiochem, Darmstadt, Germany) at 250 mA constant current for 90 minutes. Membranes were then blocked with 5% skim milk in Tris-buff ered saline and Tween 20 (TBS-T) at room temperature for 1 hour, followed by incubation at 4°C overnight with corresponding primary antibodies: (i) monoclonal anti-β-actin antibody produced in mouse (1:5,000; A5441; Sigma-Aldrich); (ii) rabbit anti-LC3-B monoclonal antibody, (1:1,000; L7543; Sigma-Aldrich). This antibody can detect protein of molecular weight major at 18 kDa and minor at the 16-kDa band. Immunoblotting of microtubule-associated protein 1-light chain 3 (LC3) usually gives two bands: LC3-I (18 kDa) and LC3-II (16 kDa). The amount of LC3-II or the LC3-II/LC3-I ratio correlates with the number of autophagosomes; (iii) rabbit anti-Beclin1 polyclonal antibody (1:1,000; sc-11427; Santa Cruz Biotechnology, Santa Cruz, CA, USA); and (iv) rabbit anti-BACE1 polyclonal antibody (1:500; 195111; Merck Millipore, Calbiochem, Darmstadt, Germany). Membranes were then rinsed three times for 10 minutes each using 1 × TBS-T solution, and then incubated with appropriate secondary antibodies (horse-radish peroxidase-labeled goat anti-rabbit or anti-mouse IgG, 1:10,000; Cell Signaling Technology, Danvers, MA, USA) at room temperature for 2 hours. Membranes were then rinsed three times for 10 minutes each using 1 × TBS-T solution. Finally, protein bands were detected using an electrochemiluminescence kit (Merck Millipore, Darmstadt, Germany) in the darkroom. Image J software (NIH, Bethesda, MA, USA) was used to compare the density of bands on a western blot image after fl atbed scanning. The relative optical density of the target protein of each group could be calculated compared to the optical density of β-actin protein. An ultrastructural observation of Neuro-2a/APP695 cells with OGD treatment was performed by transmission electron microscopy.

Cells in the logarithmic growth phase were seeded in plates. After 24 hour incubation, adherent cells were pretreated with intervention drugs (200 ng/mL Rapamycin or 5 mM 3-MA) for 1 hour, and followed by with OGD for 1 hour in the presence of Rapamycin (200 ng/mL) or 3-MA (5 mM) according to group. Cells were then digested with trypsin, collected in 1.5-mL Eppendorf tubes, centrifuged for 5 minutes at 2,000 r/min, and fi xed in 2.5% glutaraldehyde at 4°C overnight. Next day, the samples were rinsed three times with 0.1 M PBS for 15 minutes each, and then fixed with osmic acid for 2–3 hours. Afterwards, samples were rinsed three times with 0.1 M PBS for 15 minutes each. Finally, samples were dehydrated in 50–90% ethanol and 90% acetone and fi xed. The cells were embedded and the blocks were solidifi ed with 2.5% glutaraldehyde, sectioned with an ultramicrotome (70 nm thickness), and double stained with 3% uranyl acetate followed by lead citrate. Sections from diff erent groups were fi nally observed by transmission electron microscopy (TEM) (JEM-1230; Jeol, Tokyo, Japan) and photographed. Nuclear membrane, mitochondrial morphology, and autophagosome or autolysosome were observed in particular.

Statistical analysis

Data were expressed as the mean ± SD. SPSS 17.0 software (SPSS, Chicago, IL, USA) was used for statistical analysis. Diff erences among the groups were determined using oneway analysis of variance, and the least signifi cance diff erence post hoc tests were used for comparisons between groups. Results were statistically signifi cant at the α = 0.05 level.

Results

Signifi cant decrease of cell proliferation rates of Neuro-2a/ APP695 cells with OGD

Compared with the control group, cell proliferation rate of OGD group decreased signifi cantly by approximately 25% (P ＜ 0.01; Figure 1). Cells in OGD + Rapamycin group were pre-incubated with Rapamycin (200 ng/mL) for 1 hour, followed by OGD for 1 hour in the presence of Rapamycin (200 ng/mL). The same procedure was followed with cells of OGD + 3-MA group, with 3-MA (5 mM). Cell proliferation rates of both OGD + Rapamycin and OGD + 3-MA groups showed a decline compared with the control group, respectively (P ＜ 0.05; Figure 1), but no statistically signifi cant difference was found between the two groups (P ＞ 0.05).

Eff ects of autophagy regulation and OGD on apoptosis ratios of Neuro-2a/APP695 cells

Cell apoptosis was measured by Annexin V-propidium iodide (PI) double staining (Figure 2). Cell apoptosis ratio of the control group (5.67 ± 0.91%) was signifi cantly lower than that of the other three groups (P ＜ 0.05). Moreover, there was no signifi cant diff erence in the apoptosis ratio between the other three groups (P ＞ 0.05). The highest apoptosis ratio occurred in the OGD group (11.20 ± 0.72%), less than 11.92%, which indicated that cells with OGD treatment in our experiments showed a predominating phenomenon of autophagy, not apoptosis.

Eff ects of autophagy regulation and OGD on LC3 expression in Neuro-2a/APP695 cells

As mentioned before, cells were pre-incubated with Rapa (200 ng/mL) or 3-MA (5 mM) for 1 hour, followed by OGD for 1 hour in the presence of Rapamycin (200 ng/mL) or 3-MA (5 mM) in its respective group. Cells were then collected and subjected to immunoblotting. Compared with the control group, LC3 expression increased in the cells of the

three other groups, There were signifi cant diff erences between the control and OGD and OGD + Rapamycin groups (each at P ＜ 0.01) but it was not signifi cant between the control and OGD + 3-MA groups (P ＞ 0.05; Figure 3). Rapa increased LC3 expression in cells between the OGD and OGD + Rapamycin groups, but no signifi cant statistical diff erence existed between them (P ＞ 0.05), however, 3-MA did significantly inhibit LC3 expression (OGD group versus OGD + 3-MA group, P ＜ 0.01). In addition, Rapamycin increased LC3 expression while 3-MA suppressed it (OGD + Rapamycin group versus OGD + 3-MA group, P ＜ 0.01).

Eff ects of autophagy regulators and oxygen-glucose deprivation on Beclin1 expression in Neuro-2a/APP695 cells

Compared with the control group, Beclin1 expression increased in cells of both OGD and OGD + Rapa groups (P ＜0.05), but decreased in OGD + 3-MA group (P ＜ 0.05; Figure 4). The level of Beclin1 was reduced remarkably in cells of OGD + 3-MA group compared with OGD or OGD + Rapamycin group (P ＜ 0.05, P ＜ 0.01, respectively).

Eff ects of autophagy regulation and OGD on BACE1 expression in Neuro-2a/APP695 cells

BACE1 expression decreased in cells of OGD and OGD + Rapamycin groups, and increased only in the OGD + 3-MA group. There was no statistical diff erence in BACE1 expression between the control and each of the other three groups (P ＞ 0.05; Figure 5). However, compared with the OGD group, BACE1 expression increased in cells of the OGD + 3-MA group (P ＜ 0.05; Figure 5). Moreover, BACE1 expression reduced in cells of OGD + Rapamycin group compared with OGD + 3-MA group (P ＜ 0.05; Figure 5).

Eff ects of autophagy regulation and OGD on the

morphology and ultrastructure of Neuro-2a/APP695 cells Under transmission electron microscope, cells in the control group displayed intact plasma and nuclear membranes, normal mitochondrial morphology, no autophagosome or autolysosome, and several small vacuoles (Figure 6). In the OGD group, cells were observed with approximately normal morphology, but autolysosomes were observed with a double-membrane structure (red arrows in Figure 6) and the phenomenon of nuclear margination in cells after OGD treatment. Cells of OGD + Rapamycin group presented with an intact morphology, no karyopyknosis, and no vacuolization in mitochondria. The formation of autophagosome was observed along with a double-membrane structure (not yet enclosed) in a cell (red arrows in Figure 6). In contrast, cells of OGD + 3-MA group displayed intact cell membranes, no obvious autophagosome or autolysosome, abundant vacuolization, and irregular mitochondrial morphology.

Discussion

Ischemic stroke is one of the leading causes of cognitive impairment; however, the mechanisms by which cognitive decline occurs after stroke are still not fully understood (Pluta et al., 2011). Cerebral ischemia has been reported to lead to intracellular deposition of Aβ in the brain (Bulbarelli et al., 2012), which eventually causes cognitive decline. However, related research on the mechanism of post-stroke cognitive decline is still in its infancy and needs urgent attention. Earlier experimental results showed that Aβ expression increased after cerebral ischemia (Zhang et al., 2011) and many studies have reported that BACE1 activity may aff ect Aβ generation (Sun et al., 2006; Cole and Vassar, 2007; Gravenfors et al., 2012; Obregon et al., 2012; Zhu et al., 2012; Yun et al., 2013). A prior study had reported that BACE1 was degraded through the lysosomal pathway (Koh et al., 2005). Therefore, BACE1 might be degraded through an autophagy-lysosomal pathway. There are three types of autophagy in mammalian cells: macroautophagy (usually termed autophagy), micro-autophagy, and chaperone-mediated autophagy (CMA). Macroautophagy (hereinafter referred to as autophagy) predominantly serves as a cell survival mechanism and occurs at low basal levels in all cells. CMA involves selective translocation of the cytosolic proteins. The mechanism of micro-autophagy is still unclear. Pharmacological agents (e.g., Rapamycin) or stress (e.g., starvation or various pathologies) can stimulate autophagy (Mizushima et al., 2008), but the target of Rapamycin in mammals (mTOR) negatively regulates autophagy. Under condition of nutrient deprivation or starvation, mTOR is inhibited, which activates the Atg13-ULK1/2-FIP200 complex to initiate autophagy. Rapamycin, selectively inhibits the mTOR complex 1 (mTORC1), inducing autophagy. The phosphoinositide-3-kinase (PI3K) pathway is the major downstream signaling pathway regulated by mTORC1, and up-regulation of PI3P (metabolite of the class III PI3K) can stimulate autophagy, while inhibition of PI3K by 3-MA can suppress autophagy (Levine and Kroemer, 2008; Ravikumar et al., 2010).

It was reported that moderate up-regulation of autophagy could decrease Aβ generation, thus alleviating cognitive impairment (Spilman et al., 2010; Majumder et al., 2011). Previous results showed that OGD also induced autophagy and the expression of LC3 and Beclin1 (an essential autophagy protein) increased (Tassa et al., 2003), which was consistent with previous reports (Klionsky et al., 2008; Mizushima et al., 2010). Beclin 1, a Bcl2 interacting protein, is the mammalian ortholog of yeast Atg6/Vps30. It is an essential autophagy protein that has been linked to multiple processes including tumor suppression, protection against some cardiac and neurological degenerative diseases. Oxygen-glucose deprivation and some drugs such as Rapamycin can introduce autophagy, and raise protein Beclin1expr ession. In the experiment, Beclin1 expression increased in cells of both OGD and OGD + Rapamycin groups. Meanwhile, LC3 is a soluble protein with a molecular mass of approximately 17 kDa that is distributed ubiquitously in mammalian tissues and cultured cells. During autophagy, autophagosomes engulf cytoplasmic components. Concomitantly, a cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to autophagosomal membranes. Autophagosomes fuse with lysosomes to form autolysosomes,

and components in the autophagosome are degraded by lysosomal hydrolases. At the same time, LC3-II in the autolysosomal lumen is degraded. So, lysosomal turnover of the autophagosomal marker LC3-II refl ects autophagic activity, and detecting LC3 by immunoblotting has become a reliable method for monitoring autophagy (Klionsky et al., 2008; Mizushima et al., 2010). In addition, autophagy inhibitor 3-MA could suppress the expression of autophagy-specifi c proteins markedly.

BACE1 might be degraded through the autophagy-lysosomal pathway, thereby impairing the fusion of autophagosome-lysosome or the lysosomal proteolytic function. This might lead to a reduction of BACE1 clearance and subsequently increase Aβ generation (Boland et al., 2008; Lai and McLaurin, 2012). Thus, BACE1 becomes one of the eff ective targets for reducing Aβ generation (Ohno et al., 2004, 2007; Laird et al., 2005; McConlogue et al., 2007). In our experiments, BACE1 decreased in cells of both OGD and OGD + Rapa groups, while it increased in OGD + 3-MA group, because autophagy was induced by OGD or Rapa but inhibited by 3-MA. Obregon et al. (2012) discovered that reduction of BACE1 activity could decrease Aβ production (Obregon et al., 2012), and in this present experiment we showed that up-regulation of autophagy could decrease BACE1 expression. Therefore, it will be necessary to check whether up-regulation of autophagy decreases Aβ expression by detection of the Aβ peptide in future experiments.

Hypoxia-inducible factor 1, a core transcription factor responsible for maintaining oxygen homeostasis, facilitates moderate responses of cells to hypoxia under physiological or pathological conditions. Hypoxia-induced autophagy is dependent on the HIF-1α/AMP-activated protein kinase (AMPK) signaling pathway (Mazure and Pouyssegur, 2010; Hu et al., 2012). However, during OGD, AMPK is activated, which can induce autophagy through the inhibition of mTOR. Moreover, Rapamycin (an inhibitor of FRAP, an inducer of autophagy) can suppress HIF-1 expression through the PI3K/PTEN/AKT/FRAP pathway and inhibit mTOR-dependent HIF-1α mRNA translation (Zhong et al., 2000; Semenza, 2010). A recent study reported that CMA or nutrient-defi cient conditions could lead to a reduced HIF-1α expression. Further studies have indicated that CMA decreased HIF-1α expression through the interaction between HIF-1α and HSC70 or LAMP2A, two major target proteins of CMA (Hubbi et al., 2013). It was thought that activation of autophagy could down-regulate HIF-1 expression. In addition, previous studies have shown that up-regulation of HIF-1α could increase BACE1 expression (Sun et al., 2006; Zhang et al., 2007). Our results show that BACE1 expression decreased in cells of OGD + Rapamycin group, but increased in OGD + 3-MA group, thus it was hypothesized that up-regulation of autophagy decreased BACE1 expression through the interaction between HIF-1 and CMA. Further research should be done to check these correlations and elucidate the mechanisms involved. Although CMA differs from autophagy, there might be a cross-talk between CMA and autophagy that has a synergistic eff ect to regulate BACE1 metabolism. This could be another area for further investigations.

The integrity of structure and function of lysosome is also crucial to autophagy. More interventions such as chloroquine or leupeptin should be made to check lysosomal function in these cells. BACE1 is a rate-limiting enzyme in the production of Aβ, thus the amount of Aβ should also be measured, the level of BACE1 does not fully represent Aβ production. Thus the quantity of Aβ metabolism should also be measured as well as its key rate-limiting enzyme BACE1.

The results of this study show that autophagy regulation could aff ect the expression of BACE1, and that regulation of BACE1 expression through OGD might be via an autophagy-dependent pathway in Neuro-2a/APP695 cells.

Acknowledgments: We would like to express our thanks to Jian-kang Huang at the Department of Neurology, Sixth People’s Hospital Affi liated to Shanghai Jiao Tong University, China, who provided assistance in experimental skills that were necessary for the entire research process.

Author contributions: RFC, KYL and XJS designed the study. RFC, TZ, YYS, YMS and WQC performed the experiments. KYL conducted statistical analysis. NS, FS, and YZ were responsible for cell culture and western blot analysis. XJS conceived the study, coordinated the entire procedure, and helped to write the paper. All authors approved the final version of the paper.

Confl icts of interest: None declared.

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Figure 1 Eff ects of autophagy regulation and oxygen-glucose deprivation (OGD) on proliferation rate in Neuro-2a/ amyloid precursor protein 695 cells (cell counting kit-8 assay).

Figure 2 Eff ects of autophagy regulation and oxygen-glucose deprivation (OGD) on apoptosis ratio in Neuro-2a/amyloid precursor protein 695 cells (fl ow cytometry).

Figure 4 Eff ects of autophagy regulation and oxygen-glucose deprivation (OGD) on Beclin1 expression in Neuro-2a/amyloid precursor protein 695 cells (western blot assay).

Figure 5 Eff ects of autophagy regulation and oxygen-glucose deprivation (OGD) on BACE1 expression in Neuro-2a/amyloid precursor protein 695 cells (western blot assay).

Figure 6 Eff ects of autophagy regulation and oxygen-glucose deprivation (OGD) on the morphology and ultrastructure of Neuro-2a/ amyloid precursor protein 695 cells (transmission electron microscopy).

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Copyedited by Dawes EA, Raye W, Wang J, Li CH, Song LP, Zhao M

*Correspondence to:

Xiao-jiang Sun, Ph.D.,

sunxj155@sohu.com.

orcid:

0000-0003-2061-7262 (Xiao-jiang Sun)

10.4103/1673-5374.165511

http://www.nrronline.org/

Accepted: 2015-08-05