Scavenging ROS Decreases Amyloid-beta Levels via Activation of PI3K/Akt/GLUT1 pathway in N2a/APP695swe Cells

Dysregulated glucose metabolism in the brain is considered to be the underlying cause of Alzheimer's disease (AD). Abnormal glucose metabolism in AD is associated with decreased glucose transporter 1 (GLUT1) and GLUT3 in the brain, but the underlying mechanisms remains unclear. Here, we reported that GLUT1 expression was decreased in N2a/APP695swe cells and GLUT3 expression was not signicantly changed. Flow Cytometry analysis showed a signicant increase of intracellular ROS content in N2a/APP695swe cells and GLUT1 expression was upregulated after treatment with the ROS scavenger N-acetyl-L-Cysteine (NAC). Cellular glucose uptake and ATP levels were reduced following decreased GLUT1 expression and increased after upregulating GLUT1. Western blot analyses showed that phosphorylation of PI3K/Akt pathway decreased in N2a/APP695swe cells. Aβ levels decreased after upregulation of GLUT1 expression and increased after downregulation of GLUT1. After NAC treatment, PI3K/Akt pathway phosphorylation levels and GLUT1 expression were upregulated, glucose uptake and ATP contents were increased, and Aβ levels were decreased. After adding PI3K/Akt pathway inhibitor LY29004, GLUT1 expression was reduced and Aβ levels were increased. Besides, the increased glucose uptake and ATP contents by the Akt activator SC79 were hindered with the GLUT1 inhibitor WZB117. Aβ levels decreased after SC79 treatment and increased after WZB117 treatment. Overall, our data suggest that ROS reduced GLUT1 expression by inhibiting PI3K/Akt pathway activity resulting in impaired glucose metabolism and scavenging ROS prevents Aβ via activation of PI3K/Akt/GLUT1 pathway in N2a/APP695swe cells.


Introduction
Alzheimer's disease (AD) is a progressive degenerative disease of the central nervous system and the main cause of dementia in the elderly [1] . The pathological characteristics of AD mainly include β-amyloid (Aβ) deposition, neuro brillary tangles (NFTs), of which the neurotoxicity of Aβ is currently recognized as the central part of AD [2] . Recent studies have found that the development of AD is associated with metabolic abnormalities. Particularly, glucose metabolism disorders in the brain are considered to be responsible for the development of AD [3] . Impaired brain glucose uptake and utilization are closely associated with AD pathological deterioration and cognitive impairment [4,5] .
Several studies have shown that the abnormal glucose metabolism in AD is mainly associated with a decrease in glucose transporter 1 (GLUT1) and GLUT3 in the brain, the main glucoses transporter responsible for glucose uptake in the mammalian brain [6] . The reduction of GLUT1 and GLUT3 might result in insu cient glucose uptake, compromised glucose metabolism, and ultimately lead to neuronal degeneration [3] [7,6] . The Phosphoinositide 3-Kinase-Akt (PI3K/Akt) pathway has been suggested to play essential roles in the regulation of GLUT1 and GLUT3 [8] . Consistently, previous studies have found that abnormal PI3K/Akt signaling is involved in the development of AD [9,10] . However, little is currently known about the mechanisms underlying the abnormal PI3K/Akt signaling and reduced expression of GLUT1 and GLUT3 in AD.
Reactive oxygen species (ROS) is an important factor in the early pathological development of AD [11] .
ROS triggers the formation and accumulation of Aβ, meanwhile, Aβ also promotes ROS production and enhances oxidative stress [12] . In addition, increased level of ROS is associated with decreased expression or activity of key proteins involved in the glucose metabolism pathway, which might result in a low energy state in neurons [13,14] . It has been reported that ROS affects brain energy metabolism by indirectly regulating neuronal cell permeability to glucose [1] . This study investigated the potential roles of ROS in the regulation of GLUT1 and GLUT3 and the underlying mechanisms in N2a/APP695swe cells.

Cell culture and treatment
The wild-type mouse neuroblastoma cells (N2a/WT) were purchased from Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). N2a/APP695swe cells were purchased from Obio Technology (Shanghai, China). All cells were cultured in Dulbecco's modi ed Eagle medium (DMEM; Gibco, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Gibco), and N2a/APP695swe cells medium were supplemented with an additional 0.04% puromycin (5 mg/ml) to screen cells stably expressing APP695swe. Cells were cultured at 5% CO 2 , 37°C.

Glucose uptake and measurement of ATP level in cells
Glucose uptake by cells and the content of intracellular ATP were measured with Glucose Kit and ATP Assay Kit (Nanjing Jian Cheng, Nanjing, China). Brie y, after cells were treated as indicated for each experiment, cell samples preparation and detection procedures were done following the kit instruction.

Flow cytometry ROS level assays
According to the ROS Assay Kit (Beyotime) instruction, cells were loaded by an in-situ probe with DCFH-DA, and then cells were collected by centrifugation at 1000 rpm for 5 min. DCF uorescence was detected by ow cytometry, which was the intracellular ROS level.

Enzyme-linked immunosorbent assay (ELISA)
The cell culture medium of each group was collected, and the content of Aβ 1−40 and Aβ 1−42 were detected according to the instruction of ELISA Kit (SAB, Maryland, USA).

Statistical analysis
All experiments were performed at least three times and statistical analysis was done using the SPSS13.0 package (SPSS Inc., Chicago, USA). The values for each group are expressed as the means ± S.E.M. The ANOVA test was used whenever more than two groups were compared, and the signi cance level was set at p<0.05. A repeated measurement variance test was used to analyze time points comparisons. P values of less than 0.05 (p<0.05) were considered to be statistically signi cant.

Increased Aβ and ROS levels in N2a/APP695swe cells
Compared with N2a/WT cells, Aβ 1−40 and Aβ 1−42 contents were increased in N2a/APP695swe cells ( Fig. 1, a + b). Previous studies have reported that hypometabolism of glucose was most likely associated with reduced expression and function of key proteins in the glucose metabolic reaction pathway, which decreased with high levels of ROS [15,14] . Thus, we measured ROS levels by Flow Cytometry techniques, and the results showed that ROS concentration in N2a/APP695swe cells was strongly higher than that in N2a/WT cells (Fig. 1c). (a + b) N2a/WT and N2a/APP695swe cells culture medium were collected to analyze Aβ levels. The contents of Aβ 1−40 and Aβ 1−42 in the APP group were signi cantly higher than that in the WT group. (c) Intracellular ROS levels were detected by Flow Cytometry. As shown in the column, the ROS contents (P1 value) in the APP group were higher than that in the WT group. *P≤0.05, **P≤0.01, and ***P≤0.001, **** P≤0.0001.
3.2 Decreased GLUT1 expression, the glucose uptake and ATP contents in N2a/APP695swe cells To detect whether the glucose metabolism of N2a/APP695swe cells was impaired, the glucose uptake and ATP contents of N2a/WT and N2a/APP695swe cells were detected respectively. Our results showed that glucose uptake and ATP contents of N2a/APP695swe cells were signi cantly lower than that of N2a/WT cells (Fig. 2, a + b). Considering that GLUT1 and GLUT3 are the main transporters responsible for the acquisition of glucose by nerve cells, we tested these two proteins to analyze the possible reasons for the impaired cellular glucose uptake. Western blots results indicated that compared with the WT group, the expression of GLUT1 in the APP group was decreased, and the difference was statistically signi cant ( Fig. 2c), while the expression of GLUT3 was not changed, and the difference was not statistically signi cant (Fig. 2d). 3.3 GLUT1 enhanced the glucose uptake and ATP contents, reduced Aβ levels in N2a/APP695swe cells To verify that the disordered cellular glucose metabolism was caused by low GLUT1 expression, we transfected N2a/WT and N2a/APP695swe cells with GLUT1-overexpressed plasmids, and western blot results showed the protein expression of GLUT1 after transfection (Fig. 3a). As we expected that after upregulated GLUT1, the glucose uptake and ATP contents were increased in both the WT and APP groups, with statistically signi cant differences (Fig. 3, b + c). Then we proceeded to test the Aβ levels and the ELASA results showed that both Aβ 1−40 and Aβ 1−42 contents were reduced (Fig. 3, d + e). overexpression plasmid was transfected into N2a/WT and N2a/APP695swe cells for 24 h and the GLUT1 protein level was detected by Western blot. (b + c) Measurement of intracellular glucose uptake and ATP level. After upregulated GLUT1, the glucose uptake and ATP contents were increased in both two groups.
(d + e) N2a/WT and N2a/APP695swe cells culture medium were collected to analyze Aβ levels. After overexpression of GLUT1, the contents of Aβ 1−40 and Aβ 1−42 were markedly reduced in the APP group.
3.4 ROS scavenger N-acetyl-L-Cysteine (NAC) increased GLUT1 expression, the glucose uptake and ATP contents, decreased Aβ levels in N2a/APP695swe cells Next, we treated the cells with the N-acetyl-L-Cysteine (NAC), which was commonly used as a scavenger of ROS. We explored four concentration gradients (3, 5, 10, 15 mM) according to the drug instruction of NAC and relevant literature [16] . The CCK-8 results showed that cells incubated with 5 mM were the most active, and almost all cells died after incubation with NAC concentration greater than that (Fig. 4a). After NAC treatment for 24 h, ROS concentrations decreased in N2a/APP695swe cells (Fig. 4b). Western blot results showed that GLUT1 expression was signi cantly upregulated, compared with the control group (Fig. 4c), the glucose uptake and ATP contents also increased (Fig. 4, d + e). Meanwhile, the contents of  3.5 Phosphorylation of PI3K/Akt pathway decreased in N2a/APP695swe cells To clarify the mechanism by which ROS regulated GLUT1, we further tested the PI3K/Akt signal pathway, an important upstream pathway that controlled GLUT1. Our results suggested that the phosphorylation levels of PI3K and Akt in the APP group were lower than that in the WT group, and the difference was statistically signi cant. The total protein expressions of PI3K and Akt were not a signi cant variety (Fig. 5,  a + b). 3.6 NAC upregulated PI3K/Akt/GLUT1 pathway levels, increased the glucose uptake and ATP contents, decreased Aβ levels Then we treated N2a/APP695swe cells with NAC and/or PI3K inhibitor LY294002. LY294002 is a potent inhibitor of PI 3-kinase activity based on the quercetin structure, which inhibits PI3K by reversibly binding to the ATP site of PI3K [17] . According to the drug instructions and literature [18] , we selected four concentration gradients (5, 10, 20, 40 µM) to screen the optimal concentration of LY294002 for N2a/APP695swe cells. The CCK-8 results showed that the cell viability was signi cantly reduced at 20 µM LY294002 concentration (Fig. 6a), whereas cells became shrunken when the concentration was 40 µM after 24 h treatment, thus we chose 20 µM as the drug concentration. After NAC treatment we found that p-PI3K, p-Akt, and GLUT1 expression were increased, while decreased after LY294002 addition (Fig. 6b). Similarly, the glucose uptake and ATP contents were reduced after the cells were co-incubated with LY294002 in the medium containing NAC, with statistically signi cant differences (Fig. 6, c + d).
These data indicated that maintaining the activity of the PI3K/Akt/GLUT1 pathway is essential for Aβ clearance. 3.7 Activated PI3K/Akt/GLUT1 increased the glucose uptake and ATP contents, decreased Aβ levels in N2a/APP695swe cells To further demonstrate that the PI3K/Akt pathway was dependent on GLUT1 to improve glucose metabolism levels, we co-treated cells with SC79, an activator of Akt, and WZB117, a GLUT1-speci c inhibitor. WZB117 binds to amino acid residues in the central channel region of GLUT1 to form three hydrogen bonds thereby inhibiting GLUT1 [19] . Likewise, we selected four concentration gradients (1, 2, 4, 8 µg/mL) based on drug speci cation and literature [20] to screen the optimal concentration of SC79 for cells. The CCK-8 results showed that the cell viability was increased at 4 µg/mL SC79 concentration (Fig. 7a). We also determined the best concentration of WZB117 was 10 µmol/L based on the instruction and CCK-8 assay results because most of the cells died when the WZB117 concentration was 15 µmol/L (Fig. 7b). After SC79 and WZB117 incubated cells together for 24 h, glucose uptake and ATP contents were markedly decreased, and the differences were statistically signi cant (Fig. 7, c + d). The contents of Aβ 1−40 and Aβ 1−42 were reduced after SC79 treatment for 24 h, while increased after WZB117 addition ( Fig. 7, e + f).  The pathogenesis of AD is complex, and many mechanisms are involved in the pathogenesis of AD in a synergistic manner, such as Aβ toxicity, oxidative stress, and impaired glucose metabolism [21] [22] . During the stage of AD development, the ability of the brain to utilize glucose is progressively impaired, even to the point of decreasing ATP production by 50% [7] . Glucose is the main source of energy for the nervous system and only relies on speci c transporter to cross the phospholipid bilayer into cells [23] . Glucose uptake in the brain is mainly reliant on GLUT1 and GLUT3, and is closely related to their levels [24,25] [26] . Studies have reported that the accumulation of ROS, the reduction of GLUT1/GLUT3, and the impaired glucose metabolism all occurred in brain regions associated with memory and cognitive function [1] [5] . A previous study found that 4-Hydroxynonenal (HNE, an aldehyde product of membrane lipid peroxidation) impaired the glucose transport process, however, GLUT3 protein expression was not altered [27] . In the present study, we found that high levels of ROS and Aβ, GLUT1 expression was signi cantly reduced, while GLUT3 showed no signi cant change. We further showed by NAC treatment and overexpression experiments that in vitro ROS blocked glucose metabolism by decreasing GLUT1 expression. Therefore, it possible that GLUT1 is vulnerable to ROS attack during the shuttling and transport of glucose across the cytoplasm and cytosol, and eventually glucose uptake and glucose metabolism were impaired.
To investigate the mechanism of ROS-induced GLUT1 de ciency, we examined PI3K/Akt pathway levels in N2a/APP695swe cells. Our data showed that ROS reduced GLUT1 expression by suppressing PI3K/Akt pathway activity resulting in impaired glucose metabolism. The latest review summarized the role of PI3K/Akt pathway in AD and supported our results [28] . The article pointed out that abnormalities of the PI3K/Akt pathway, especially a decrease in Akt phosphorylation, prevented GLUT from transporting glucose into the cell and reduced ATP. Additional studies have also supported our results. Tahir Ali et al. investigated the antioxidant effects of anthocyanins in vivo and in vitro, and found that the reduction in p-PI3K/Akt/GSK3β pathway levels was caused by ROS induced by amyloid β oligomers (AβO) [10] . ROS could affect energy metabolism via the PI3K/Akt pathway, particularly Akt, which is a metabolically critical sensor [29] . Therefore, we believed that maintaining PI3K/Akt signaling pathway levels was helpful in protecting neuronal cells from damage by AD risk factors.
Aβ is produced by sequential cleavage of amyloid precursor protein (APP) by β-secretase and γ-secretase [30] . ROS could activate γ-secretase to trigger Aβ production [31] . Under normal conditions, the production, degradation and clearance of Aβ are in a dynamic equilibrium. However, various abnormalities lead to impaired Aβ degradation or clearance, and total Aβ production increased, while Aβ generation did not increase [32][33] [34] . It is suggested that Aβ deposition in the brain is mainly due to abnormal clearance. Oxidative stress could produce interference in the clearance of Aβ by oxidizing the low-density lipoprotein receptor-related protein 1 (LRP1), a key Aβ clearance transporter in the brain [35] . Interestingly, Winkler, E.A. et al. found that GLUT1 de ciency decreased Aβ clearance by reducing LRP1 expression in vivo, thereby accelerating Aβ deposition [36] . Here, we found that Aβ levels were negatively correlated with PI3K/Akt/GLUT1 pathway levels and ATP. Thus, in the present study, scavenging ROS to reduce Aβ was likely due to a certain degree of restored cellular energy supply and clearance of Aβ being enhanced. Meanwhile, clearance of Aβ required the PI3K/Akt/GLUT1 pathway to maintain ATP levels and that Aβ accumulation correlated with GLUT1 levels. The speci c molecular mechanisms require further investigation. Our data suggested that GLUT1 could work as a therapeutic target for AD, maintaining neuronal glucose metabolism and promoting the clearance of Aβ.
In conclusion, the present study found that ROS reduced GLUT1 expression by inhibiting PI3K/Akt pathway activity, which results in impaired glucose metabolism in N2a/APP695swe cells. Our results suggest that the PI3K/Akt/GLUT1 pathway might play a bridge role between Aβ, ROS, and neurometabolic disorders in AD. In future studies, we will continue to explore the function of GLUT1 (including GLUT3) in transporting glucose and the mechanisms regulating its movement between plasma membranes. Decreased GLUT1 expression, the glucose uptake and ATP contents in N2a/APP695swe cells The N2a/WT and N2a/APP695swe cells were collected separately from three different culture asks and the proteins were extracted for the Western blot analysis. (a + b) Measurement of intracellular glucose uptake and ATP contents. (c + d) Western blot analysis of GLUT1 and GLUT3 proteins. GLUT1 expression was decreased in the APP group and GLUT3 was not signi cantly changed. Numbers 1, 2, and 3 represent proteins extracted at three different dates. *P≤0.05, **P≤0.01, and ***P≤0.001.  ROS scavenger N-acetyl-L-Cysteine (NAC) increased GLUT1 expression, the glucose uptake and ATP contents, decreased Aβ levels in N2a/APP695swe cells Phosphorylation of PI3K/Akt pathway decreased in N2a/APP695swe cells Figure 6 NAC upregulated PI3K/Akt/GLUT1 pathway levels, increased glucose uptake and ATP contents, decreased Aβ levels Activated PI3K/Akt/GLUT1 increased glucose uptake and ATP contents, decreased Aβ levels in N2a/APP695swe cells Schematic: ROS reduces PI3K/Akt pathway phosphorylation levels leading to cellular GLUT1 de ciency and impaired glucose uptake, NAC improves this state