Peroxiredoxin 2 Ameliorates AβO-Mediated Autophagy by Inhibiting ROS via the ROS–NRF2–p62 Pathway in N2a-APP Swedish Cells

In Alzheimer’s disease, reactive oxygen species (ROS) are generated by the deposition of amyloid-beta oligomers (AβOs), which represent one of the important causes of neuronal cell death. Additionally, AβOs are known to induce autophagy via ROS induction. Previous studies have shown that autophagy upregulation aggravates neuronal cell death. In this study, the effects of peroxiredoxin 2 (Prx2), a member of the peroxidase family of antioxidant enzymes, on regulating AβO-mediated autophagy were investigated. Prx2 decreased AβO-mediated oxidative stress and autophagy in N2a-APPswe cells. Further, we examined the relationship between the neuronal protective effect of Prx2 and a decrease in autophagy. Similar to the effects of N-acetyl cysteine, Prx2 decreased AβO-induced ROS and inhibited p62 protein expression levels by downregulating the activation of NRF2 and its translocation to the nucleus. In addition, treatment with 3-methyladenine, an autophagy inhibitor, ameliorates neuronal cell death. Overall, these results demonstrate that the Prx2-induced decrease in autophagy was associated with the inhibition of ROS via the ROS–NRF2–p62 pathway in N2a-APPswe cells. Therefore, our results revealed that Prx2 is a potential therapeutic target in anti-Alzheimer therapy.


Introduction
Alzheimer's disease (AD) is the most common neurodegenerative disease, affecting millions of people worldwide. Typical pathological features of AD include the deposition of β-amyloid plaques due to the accumulation of amyloid-beta oligomers (AβO) [1], which is generated by the sequential proteolytic cleavage of transmembrane amyloid precursor protein (APP) by β-secretases and γ-secretases, which are considered critical initiators of AD [2,3]. Reactive oxygen species (ROS) are small and highly reactive molecules that are generated by the accumulation of AβOs [4] and can oxidize proteins, lipids, and DNA [5]. ROS-mediated oxidative stress leads to cell death [6,7] and induces autophagy [5,7]. Autophagy is an essential, conserved, self-eating process that maintains cellular homeostasis. Autophagy begins at the phagophore and ends with autophagosomal death. Phagophore formation is controlled by multiple signaling events, such as ATG proteins and Beclin-1. Lc3 (microtubule-associated protein 1A/1B-light chain 3) is a soluble protein widely distributed in mammalian tissues and cultured cells. During autophagy, Lc3 is processed and recruited into the extending phagophore [8][9][10]. SQSTM/p62 is an autophagosome cargo

Plasmid Construction
The APP gene and Prx2 gene were obtained from Addgene (Boston, MA, UAS). The APPswe mutation gene was introduced using the Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacture's protocol. The APPswe and Prx2 genes were amplified by polymerase chain reaction (PCR) using LA Taq polymerase (TaKaRa, Shiga, Japan). These genes were cloned into pCR8/GW/TOPO (Thermo Fisher Scientific) and inserted into the pLenti6.3/v5-DEST vector (Thermo Fisher Scientific) using the LR clonase (Thermo Fisher Scientific). Construct sequences were confirmed using DNA sequencing.

Transfection and Preparation of Stable Cell Lines
The N2a cells (1 × 10 5 ) were seeded in 6-well plates and cultured for 24 h. Plenti6.3-APPswe plasmid (1 µg) was transfected into the N2a cells using Effectene (Qiagen, Valencia, CA, USA), according to the manufacturer's instructions. After 9 h, cells were washed with PBS and incubated in fresh medium overnight. After transfection for a day, the N2a-APPswe cells were selected using 8 µg/mL blasticidin (Thermo Fisher Scientific). As for the generation of the N2a-APPswe-Prx2 cells, the Prx2-expressing cells were generated from the N2a-APPswe cells through transfection with the pLenti6.3-Prx2 plasmid (1 µg) using Effectene.

Measurement of Intracellular ROS Generation
The production of intracellular ROS was measured using the ROS-specific fluorescent dye CM-H2DCFDA (Thermo Fisher Scientific). The cells were seeded at an initial density of 1 × 10 6 cells in 60 Φ plates and cultured for 24 h. The cells were then washed with phosphate-buffered saline (PBS), followed by the addition of 1 mL Dulbecco's modified Eagle's medium (DMEM; Welgene, Daegu, Korea) (Phenol Red free media) containing 2.5 µM CM-H2DCFDA and incubated for 15 min at 37 • C in the dark. The ROS levels were analyzed using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Measurement of AβO-Induced Cytotoxicity
AβO-induced damage to the plasma membranes of the N2a cells was quantified by the amount of lactate dehydrogenase (LDH) released using the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega Corporation, Madison, WI, USA). The cells were plated at a density of 1 × 10 6 cells in 60 Φ plates and cultured for 24 h. A total of 50 µL of culture medium was added to 96-well plates, followed by 50 µL of assay reagent. The LDH activity in the culture medium was measured by monitoring the absorbance at 490 nm.

Immunocytochemistry
The N2a, N2a-APPswe, and N2a-APPswe-Prx2 cells were seeded on round coverslips for 24 h and then treated with CQ (25 µM) for 24 h. The cells were fixed with 5% paraformaldehyde and permeabilized with 0.25% Triton X-100 in PBS (PBST) for 10 min. The cells were then treated with 1% bovine serum albumin in PBST and incubated overnight at 4 • C with an anti-Lc3 antibody diluted in a blocking solution. After washing three times with PBS, the cells were treated with Alexa Fluor 647 goat anti-rabbit secondary antibody for 4 h at room temperature. The cells were washed three times with PBS, coverslips were mounted on slides using VECTASHIELD medium (Vector Laboratories, Burlingame, CA, USA), and images were obtained using a Carl Zeiss LSM-710 confocal microscope (Carl Zeiss, Oberkochen, Germany).

Isolation of the Nuclear and Cytoplasmic Proteins
The nuclear and cytoplasmic proteins were isolated from the N2a, N2a-APPswe, and N2a-APPswe-Prx2 cells cultured for 24 h using the NE-PER Nuclear and Cytoplasmic Extraction Reagent kit (Thermo Fisher Scientific), following the manufacturer's protocol.

Western Blot Analysis
The cells were harvested using a scraper (SPL Life Science Co., Pocheon, Korea) and lysed using the ice-cold PRO-PREP protein extraction solution (iNtRON Biotechnology Inc., Korea). Protein quantification was performed using the Bradford assay (Bio-Rad, Hercules, CA, USA), and 10-15 µg of protein lysates were separated by electrophoresis on 6-15% SDS-PAGE and then transferred onto nitrocellulose membranes (Pall Corporation, New York, NY, USA). The membranes were blocked with 5% skim milk (BD Biosciences) and incubated with primary antibodies overnight at 4 • C. The primary antibodies used were anti-Aβ (6E10) (1: 1000) and anti-APP The excess secondary antibodies were removed by washing six times with TBST. Specific binding was detected using Clarity Western ECL substrate (Bio-Rad), according to the manufacturer's instructions.

Cell Viability Assay
The cells were plated at a density of 1 × 10 6 cells in 60 Φ plates and cultured for 24 h. A 50 µL/mL CCK cell viability assay kit (Donginbiotech, Seoul, Korea) was added for 30 min at 37 • C. Then, a 200 µL culture medium was added to the 96-well plates. The cell viability was measured by monitoring the absorbance at 450 nm.

Statistical Analysis
Statistical analyses were performed using the GraphPad Prism software (San Diego, CA, USA). Data are presented as the means ± standard errors of the means (SEM) from at least three independent experiments (n ≥ 3). Statistically significant differences were determined using one-way ANOVA. Asterisks indicate calculated p values as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001. The significance threshold was set at p < 0.05.

Accumulation of AβO Increased the Levels of ROS, Cytotoxicity, and Cell Death in N2a APPswe Cells
To analyze the relationship between Prx2 and autophagy during AβO accumulation, we used the N2a cells that stably expressed the APP Swedish mutation (N2a-APPswe), where AβO accumulation was increased due to abnormal βand γ-secretase cleavage. First, we confirmed that the APPswe was stably expressed and accumulation of AβO was higher in the N2a-APPswe cells ( Figure 1A,B). Next, we investigated whether the levels of ROS and cytotoxicity, as well as those of the cleaved poly (ADP-ribose), polymerase (PARP), caspase-3 (cell death markers), and cell viability, increased in a time-dependent manner in the N2a-APPswe cells ( Figure 1C-F). Our results suggest that the AβO accumulation increased oxidative stress and cytotoxicity.
First, we confirmed that the APPswe was stably expressed and accumulation of AβO was higher in the N2a-APPswe cells ( Figure 1A,B). Next, we investigated whether the levels of ROS and cytotoxicity, as well as those of the cleaved poly (ADP-ribose), polymerase (PARP), caspase-3 (cell death markers), and cell viability, increased in a time-dependent manner in the N2a-APPswe cells ( Figure 1C-F). Our results suggest that the AβO accumulation increased oxidative stress and cytotoxicity. (E) Western blot analysis of poly (ADP-ribose), polymerase (PARP), and cleaved caspase-3 expression in N2a-APPswe cells cultured for 6, 12, and 24 h. (F) Cell viability was measured using a CCK cell viability assay kit for N2a-APPswe cells cultured for 6, 12, and 24 h. Data are represented as the means ± SEMs of three independent experiments (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Autophagy Was Increased by APP Swedish Mutation
It has been reported that autophagy can be induced by ROS [5,7] and that an increase in autophagy is observed in the early stages of AD [25]. Therefore, we measured the autophagy marker proteins ATG5, ATG7, Beclin-1 (initiator of autophagy), Lc3 (autophagosome membrane extension and fusion participation protein), and p62 (autophagic degradation target cargo protein) levels. The results showed that the ATG5, ATG7, Beclin-1, and the Lc3-II/Lc3-I ratios increased, while the ratio of p62 decreased, in a time-dependent manner in the N2a-APPswe cells (Figure 2A).
To determine the autophagic flux of the N2a-APPswe cells, we treated the cells with 25 µM CQ, an autophagy inhibitor that suppresses autophagosome-lysosome fusion and decreases autophagic degradation [26]. As shown in Figure 2B, autophagy was increased by APPswe and the levels of Lc3-II/Lc3-I and p62 were higher in the CQ treatment group than in the no-treatment group ( Figure 2B). Confocal microscopy images showed that the number of Lc3 puncta in the N2a-APPswe cells was higher than that in the N2a cells. Furthermore, we observed that the number of Lc3 puncta was increased in the N2a-APPswe cells compared to that in the CQ-treated group ( Figure 2C). Thus, we suggest that autophagy increases in a time-dependent manner in N2a-APPswe cells.

Autophagy Was Increased by APP Swedish Mutation
It has been reported that autophagy can be induced by ROS [5,7] and that an increase in autophagy is observed in the early stages of AD [25]. Therefore, we measured the autophagy marker proteins ATG5, ATG7, Beclin-1 (initiator of autophagy), Lc3 (autophagosome membrane extension and fusion participation protein), and p62 (autophagic degradation target cargo protein) levels. The results showed that the ATG5, ATG7, Beclin-1, and the Lc3-II/Lc3-I ratios increased, while the ratio of p62 decreased, in a time-dependent manner in the N2a-APPswe cells (Figure 2A).
To determine the autophagic flux of the N2a-APPswe cells, we treated the cells with 25 μM CQ, an autophagy inhibitor that suppresses autophagosome-lysosome fusion and decreases autophagic degradation [26]. As shown in Figure 2B, autophagy was increased by APPswe and the levels of Lc3-II/Lc3-I and p62 were higher in the CQ treatment group than in the no-treatment group ( Figure 2B). Confocal microscopy images showed that the number of Lc3 puncta in the N2a-APPswe cells was higher than that in the N2a cells. Furthermore, we observed that the number of Lc3 puncta was increased in the N2a-APPswe cells compared to that in the CQ-treated group ( Figure 2C). Thus, we suggest that autophagy increases in a time-dependent manner in N2a-APPswe cells.

Autophagy and Cell Death Were Increased by AβO-Mediated Oxidative Stress
To confirm whether AβO-mediated ROS generation increased autophagy, we used N-acetylcysteine (NAC), which is widely used as an antioxidant reagent [27]. Following treatment with 10 mM NAC, the levels of ROS ( Figure 3A) and autophagy were significantly decreased. Interestingly, p62 protein levels were also decreased ( Figure 3B). To investigate whether NAC treatment decreased p62 protein levels during the autophagic flux, we co-treated cells with CQ. As shown in Figure 3C, the expression levels of the Lc3-II/Lc3-I and p62 proteins were increased following the CQ treatment, while those of p62 were decreased by the NAC treatment. In addition, we discovered that cell death and cytotoxicity were ameliorated by the NAC treatment ( Figure 3D-F). Thus, we suggest that AβO-mediated oxidative stress increases autophagy and cell death.

Autophagy and Cell Death Were Increased by AβO-Mediated Oxidative Stress
To confirm whether AβO-mediated ROS generation increased autophagy, we used N-acetylcysteine (NAC), which is widely used as an antioxidant reagent [27]. Following treatment with 10 mM NAC, the levels of ROS ( Figure 3A) and autophagy were significantly decreased. Interestingly, p62 protein levels were also decreased ( Figure 3B). To investigate whether NAC treatment decreased p62 protein levels during the autophagic flux, we cotreated cells with CQ. As shown in Figure 3C, the expression levels of the Lc3-II/Lc3-I and p62 proteins were increased following the CQ treatment, while those of p62 were decreased by the NAC treatment. In addition, we discovered that cell death and cytotoxicity were ameliorated by the NAC treatment ( Figure 3D-F). Thus, we suggest that AβO-mediated oxidative stress increases autophagy and cell death.

Overexpression of Prx2 Decreased ROS Levels and Autophagy
Prxs have been reported to exert antioxidant effects [17,18]. Furthermore, Prx2 exhibits excellent antioxidant and neuronal protective effects in neurodegenerative diseases [22,23]. To investigate whether Prx2 has a neuroprotective effect in N2a-APPswe cells, we generated N2a-APP-Prx2 cells transfected with the pLenti6.3-Prx2 vector ( Figure 4A). We discovered that the ROS-and AβO-mediated autophagy levels were decreased by Prx2 overexpression in the N2a-APPswe cells ( Figure 4B,C). Interestingly, the p62 protein levels were also decreased ( Figure 4C). Using confocal microscopy, we measured the Lc3 puncta and marked their decrease following Prx2 overexpression in the N2a-APPswe cells ( Figure 4D). Western blot analysis also showed that the Lc3-II/Lc3-I and p62 levels were decreased by Prx2 overexpression, regardless of the presence or absence of CQ ( Figure 4E). Thus, we suggest that Prx2 decreases AβO-mediated ROS production, which ultimately reduces autophagy in N2a-APPswe cells.

Overexpression of Prx2 Downregulated p62 Transcription
According to a previous study, a decrease in autophagy is accompanied by a decrease in LC3-II, as well as an increase in p62 protein, which is less degraded [11]. However, as

Overexpression of Prx2 Downregulated p62 Transcription
According to a previous study, a decrease in autophagy is accompanied by a decrease in LC3-II, as well as an increase in p62 protein, which is less degraded [11]. However, as shown in Figures 3B and 4C, the ROS reduction induced by Prx2 overexpression and NAC treatment did not decrease the expression of p62 protein in autophagosomes. To analyze the transcriptional levels of p62, we used RT-PCR and measured the p62 mRNA levels. First, we confirmed that the NAC treatment downregulated p62 transcription in N2a-APPswe cells ( Figure S2). In N2a-APPswe-Prx2 cells, p62 transcription was also downregulated ( Figure 5A). Therefore, we suggest that ROS reduction due to Prx2 overexpression downregulates p62 transcription. shown in Figures 3B and 4C, the ROS reduction induced by Prx2 overexpression and NAC treatment did not decrease the expression of p62 protein in autophagosomes. To analyze the transcriptional levels of p62, we used RT-PCR and measured the p62 mRNA levels.

Overexpression of Prx2 Decreased Autophagy via the ROS-NRF2-p62 Pathway
NRF2, which belongs to the basic leucine zipper (bZIP) family of transcription factors, responds to oxidative stress. Under oxidative stress, NRF2 is isolated from Keap1 and translocated to the nucleus to activate the antioxidant response elements (ARE), such as heme oxygenase (HO-1), located in the p62 promoter region, which ultimately upregulates the expression of p62 mRNA [7,28,29]. Therefore, we investigated whether Prx2 decreases autophagy via the ROS-NRF2-p62 pathway. We discovered that NRF2 and HO-1 were increased, while Keap1 expression was decreased, in N2a-APPswe cells. In

Overexpression of Prx2 Decreased Autophagy via the ROS-NRF2-p62 Pathway
NRF2, which belongs to the basic leucine zipper (bZIP) family of transcription factors, responds to oxidative stress. Under oxidative stress, NRF2 is isolated from Keap1 and translocated to the nucleus to activate the antioxidant response elements (ARE), such as heme oxygenase (HO-1), located in the p62 promoter region, which ultimately upregulates the expression of p62 mRNA [7,28,29]. Therefore, we investigated whether Prx2 decreases autophagy via the ROS-NRF2-p62 pathway. We discovered that NRF2 and HO-1 were increased, while Keap1 expression was decreased, in N2a-APPswe cells. In contrast, Prx2 overexpression decreased NRF2, as well as HO-1, and increased Keap1 ( Figure 5B). In addition, as shown in Figure 5C, in N2a-APPswe cells, NRF2 was significantly increased in the nucleus, while Prx2 overexpression decreased its translocation. Therefore, these results indicate that Prx2 reduces AβO-mediated oxidative stress by suppressing NRF2 activation and translocation to the nucleus. Prx2 decreases ROS levels to inhibit the ROS-NRF2-p62 pathway, which plays an important role in the oxidative stress pathway associated with the autophagy process.

Overexpression of Prx2 Ameliorated Cytotoxicity and Cell Death in N2a-APPswe Cells
Finally, we investigated the neuroprotective effect of Prx2 overexpression against AβO accumulation in N2a-APPswe cells. The results showed that Prx2 overexpression decreased cytotoxicity and cell death in N2a-APPswe cells ( Figure 6). In addition, to determine whether AβO-mediated autophagy can lead to cell death, we used 3-MA, which inhibits autophagy by targeting PI3K [30]. In the N2a-APPswe cells, autophagy was inhibited following treatment with 1 mM 3-MA for 4 h ( Figure S3A). Furthermore, we observed that cell death was attenuated ( Figure S3B,C). In contrast, we found that treatment with 50 nM rapamycin for 24 h, which is an autophagy inducer, aggravated cell death during autophagy induction in the N2a-APPswe cells ( Figure S3D-F). Taken together, these results suggest that AβO-mediated autophagy can lead to cell death in N2a-APPswe cells. contrast, Prx2 overexpression decreased NRF2, as well as HO-1, and increased Keap1 (Figure 5B). In addition, as shown in Figure 5C, in N2a-APPswe cells, NRF2 was significantly increased in the nucleus, while Prx2 overexpression decreased its translocation. Therefore, these results indicate that Prx2 reduces AβO-mediated oxidative stress by suppressing NRF2 activation and translocation to the nucleus. Prx2 decreases ROS levels to inhibit the ROS-NRF2-p62 pathway, which plays an important role in the oxidative stress pathway associated with the autophagy process.

Overexpression of Prx2 Ameliorated Cytotoxicity and Cell Death in N2a-APPswe Cells
Finally, we investigated the neuroprotective effect of Prx2 overexpression against AβO accumulation in N2a-APPswe cells. The results showed that Prx2 overexpression decreased cytotoxicity and cell death in N2a-APPswe cells ( Figure 6). In addition, to determine whether AβO-mediated autophagy can lead to cell death, we used 3-MA, which inhibits autophagy by targeting PI3K [30]. In the N2a-APPswe cells, autophagy was inhibited following treatment with 1 mM 3-MA for 4 h ( Figure S3A). Furthermore, we observed that cell death was attenuated ( Figure S3B,C). In contrast, we found that treatment with 50 nM rapamycin for 24 h, which is an autophagy inducer, aggravated cell death during autophagy induction in the N2a-APPswe cells ( Figure S3D-F). Taken together, these results suggest that AβO-mediated autophagy can lead to cell death in N2a-APPswe cells. Western blot analysis of PARP and cleaved caspase-3 protein expression levels in N2a, N2a-APPswe, and N2a-APPswe-Prx2 cells cultured for 24 h. (C) Cell viability was measured using a CCK cell viability assay kit for N2a, N2a-APPswe and N2a-APPswe-Prx2 cells cultured for 24 h. Data are represented as the means ± SEMs of three independent experiments (** p < 0.01, and *** p < 0.001).

Discussion
Autophagy is the process of maintaining cellular homeostasis in response to diverse stress conditions, such as starvation, brain injury, and genotoxic stress [7]. Recently, several studies have reported that oxidative stress acts as the converging point of these stimuli with ROS, which induces autophagy among the main intracellular signal transducers

Discussion
Autophagy is the process of maintaining cellular homeostasis in response to diverse stress conditions, such as starvation, brain injury, and genotoxic stress [7]. Recently, several studies have reported that oxidative stress acts as the converging point of these stimuli with ROS, which induces autophagy among the main intracellular signal transducers [5,7,15]. ROS are small and highly reactive molecules that can be generated by AβO accumulation [4].
Numerous studies have shown that autophagy is decreased in AD models due to dysfunctional autophagy [31][32][33]. However, an increase in autophagy has also been reported in the early stages of AD [25]. Therefore, we investigated whether AβO-induced autophagy played a role in neuronal cell death. In our study, we first confirmed that autophagy was increased in a time-dependent manner by the accumulation of AβO in the N2a-APPswe cells (Figure 2A). To determine whether the increased autophagy and cell death were due to an APPswe mutation, we generated N2a-Mock cells as a sham control group ( Figure S4). As shown in Figure 2B,C, the autophagic flux of the N2a-APPswe cells was blocked by the CQ treatment. In contrast, the number of Lc3 puncta in the N2a-APPswe cells was higher than that in the N2a cells. Furthermore, the NAC treatments decreased AβO-mediated autophagy and apoptosis ( Figure 3B-F). These results indicate that NAC, an antioxidant reagent, decreases autophagy and apoptosis following AβO-induced oxidative stress.
The Prx family of antioxidant enzymes scavenges peroxides via the cysteine residue in its active site. In particular, our previous studies have shown that Prxs exhibit antitoxic and antioxidant effects on AβO in neuronal cells [34,35]. Furthermore, it has been reported that Prx2 also exhibits antioxidant and neuroprotective effects [22,36]. Therefore, we investigated the relationship between Prx2 and AβO-mediated autophagy. Upon the overexpression of Prx2, we observed a decrease in the ROS levels and AβO-mediated autophagy ( Figure 4B,C). Furthermore, Prx2 overexpression attenuated AβO-mediated cell death and cytotoxicity ( Figure 6), indicating that Prx2 has a protective effect against AβO-mediated oxidative stress and toxicity.
According to recent studies, ROS-induced autophagy is meant to clear ROS and maintain cellular homeostasis, whereas excess ROS-induced autophagy also increases the generation of ROS and aggravates cell death [6,15,37]. In this study, we observed that a decrease in cell death was accompanied by a reduction in autophagy ( Figures 3B,D, 4C and 6B,C). To confirm whether AβO-mediated autophagy could lead to cell death, we treated cells with 3-MA, an autophagy inhibitor [30], and rapamycin, an autophagy inducer. Our results showed that cell death was attenuated in the N2a-APPswe cells when autophagy was inhibited and that it was aggravated during autophagy induction ( Figure S3). Furthermore, it also has been reported that decreased autophagy can protect neurons from Aβ [38][39][40]. Thus, we suggest that AβO-mediated autophagy aggravates cell death in N2a-APPswe cells. The regulation of autophagy may delay the onset of AD, which makes it a viable pathway that can be pharmacologically targeted.
In autophagy-related studies, decreased levels of autophagy are accompanied by an increase in the level of p62, which is a cargo receptor protein usually degraded by autophagosomes that plays a transfer role in autophagy [10,11]. Interestingly, we discovered that decreased autophagy was accompanied by a decrease in p62 expression levels following the NAC treatment ( Figure 3B). Prx2 overexpression also supported this finding ( Figure 4C). Under stressed conditions, increased NRF2 protein can enter the nucleus to induce the transcription of p62 through the NRF2 non-canonical pathway [28,41]. Therefore, we conducted further research on p62-related pathways. The NRF2-p62 pathway has been revealed as part of the relationship between the antioxidant response and autophagy [12,42]. In normal cellular conditions, NRF2, an antioxidant transcription factor, is bound to Keap1, an E3 ubiquitin ligase adaptor protein, and it leads to NRF2 ubiquitination and degradation by the proteasome. Under oxidative stress, Keap1 is challenged to undergo critical cysteine modifications and blocks the ubiquitylation of NRF2, which allows Keap1 to bind p62 and degrade it via autophagy (called the NRF2 non-canonical pathway) [12]. Our results showed that the AβO-mediated ROS production increased NRF2 protein levels and decreased Keap1 protein levels ( Figures 4B and 5B). Further, our results revealed that the NRF2 nucleoprotein levels were much higher than those in the cytoplasm in the N2a-APPswe cells ( Figure 5C). Alternatively, other studies have shown that p62 can also induce NRF2 [28,42], which is in line with our analysis of NRF2 mRNA levels in the N2a-APPswe cells. We confirmed that NRF2 mRNA levels were also increased compared to those in the control group ( Figure S5). Further, we showed that Prx2 over-expression induced a decrease in ROS levels, which reduced the accumulation of NRF2 protein and downregulated the transcription of p62 ( Figures 4A-C and 5). These results indicate that AβO-mediated autophagy is induced by the ROS-induced increase in p62, the transcription of which is induced by the nucleoprotein NRF2. Here, we showed that Prx2 ameliorated AβO-mediated autophagy by inhibiting ROS via the ROS-NRF2-P62 pathway in the N2a-APPswe cells.

Conclusions
ROS induces autophagy by isolating NRF2 from Keap1 and translocating NRF2 to the nucleus, which ultimately promotes p62 transcription. In contrast, the overexpression of Prx2 decreased AβO-mediated autophagy by inhibiting ROS generation to suppress NRF2 translation and p62 transcription. Therefore, for the first time, we showed that Prx2 overexpression decreased AβO-mediated autophagy and reduced cell death via the ROS-NRF2-p62 pathway in N2a-APPswe cells (Figure 7). We believe that autophagy regulation may delay the onset of AD, and that Prx2 might represent an efficient antioxidant for neuronal protection, which makes it a viable pharmacological target. APPswe cells. We confirmed that NRF2 mRNA levels were also increased compared to those in the control group ( Figure S5). Further, we showed that Prx2 overexpression induced a decrease in ROS levels, which reduced the accumulation of NRF2 protein and downregulated the transcription of p62 ( Figures 4A-C and 5). These results indicate that AβO-mediated autophagy is induced by the ROS-induced increase in p62, the transcription of which is induced by the nucleoprotein NRF2. Here, we showed that Prx2 ameliorated AβO-mediated autophagy by inhibiting ROS via the ROS-NRF2-P62 pathway in the N2a-APPswe cells.

Conclusions
ROS induces autophagy by isolating NRF2 from Keap1 and translocating NRF2 to the nucleus, which ultimately promotes p62 transcription. In contrast, the overexpression of Prx2 decreased AβO-mediated autophagy by inhibiting ROS generation to suppress NRF2 translation and p62 transcription. Therefore, for the first time, we showed that Prx2 overexpression decreased AβO-mediated autophagy and reduced cell death via the ROS-NRF2-p62 pathway in N2a-APPswe cells (Figure 7). We believe that autophagy regulation may delay the onset of AD, and that Prx2 might represent an efficient antioxidant for neuronal protection, which makes it a viable pharmacological target. Figure 7. A schematic diagram demonstrates that Prx2 decreases AβO-mediated autophagy by inhibiting ROS via the ROS-NRF2-p62 pathway and ameliorates AβO-mediated autophagic cell death.