# Resetting Proteostasis of CIRBP with ISRIB Suppresses Neural Stem Cell Apoptosis under Hypoxic Exposure

Authors: Yuankang Zou; Ziyan Yuan; Yafei Sun; Maodeng Zhai; Zhice Tan; Ruili Guan; Michael Aschner; Wenjing Luo; Jianbin Zhang
Journal: Oxidative Medicine and Cellular Longevity
 (2022)

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## Abstract

Neurological disorders are often progressive and lead to disabilities with limited available therapies. Epidemiological evidence implicated that prolonged exposure to hypoxia leads to neurological damage and a plethora of complications. Neural stem cells (NSCs) are a promising tool for neurological damage therapy in terms of their unique properties. However, the literature on the outcome of NSCs exposed to severe hypoxia is scarce. In this study, we identified a responsive gene that reacts to multiple cellular stresses, marked cold-inducible RNA-binding protein (CIRBP), which could attenuate NSC apoptosis under hypoxic pressure. Interestingly, ISRIB, a small-molecule modulator of the PERK-ATF4 signaling pathway, could prevent the reduction and apoptosis of NSCs in two steps: enhancing the expression of CIRBP through the protein kinase R- (PKR-) like endoplasmic reticulum kinase (PERK) and activating transcription factor 4 (ATF4) axis. Taken together, CIRBP was found to be a critical factor that could protect NSCs against apoptosis induced by hypoxia, and ISRIB could be acted upstream of the axis and may be recruited as an open potential therapeutic strategy to prevent or treat hypoxia-induced brain hazards.


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## Body

## 1. Introduction

In light of the rapid growth of the global economy and the improvement of people’s quality of life, public health issues are increasingly being prioritized by the public [1]. Changes in the environment can significantly affect public health, except for the pollution and outbreak of environmental epidemics; extreme environmental exposures are concerns by researchers currently [2]. Environmental hypoxia is a common stressor worldwide, particularly at high altitudes, which is associated with a number of health problems and has attracted public attention in recent years [3]. The vast majority of lives on earth depend on oxygen for growth and development, as well as energy metabolism [4–6]. In particular, oxygen consumption by the brain alone accounts for 20% of the total body weight, which accounts for only 2% of the total body mass, and thus, brains are particularly sensitive to oxygen supply [7]. It has been shown that extreme hypoxic exposure can cause fatal diseases such as brain edema, impaired higher brain function such as learning and memory, and increased apoptosis of neurons and altered neuron plasticity [8, 9]. Chronic hypoxic exposure results in impairment of speech and visual working memory, executive control function, and psychomotor function. When organic damage occurs from hypoxic exposure, it is dangerous and effective interventions are lacking. NSCs are cells in the nervous system with self-renewal ability and multidirectional differentiation potential, mainly found in the subventricular zone (SVZ) and subgranular zone (SGZ) in adult mammals [10], and are capable of differentiating into neurons, astrocytes, and oligodendrocytes [11–13]. NSCs therefore are the basis for the proper execution of the function of the mature neural circuits [10, 14]. It has been revealed that low oxygen tension can maintain the undifferentiated state of neural cells and therefore affect cell proliferation and cell fate when the tissue interstitial oxygen concentration in the brain is around 1%-5%. Extreme hypoxic environment exposure can significantly reduce the oxygen partial pressure in the interstitial space of the brain [15]. There is still uncertainty regarding the molecular mechanisms associated with hypoxic oxygen exposure on NSCs.Since its discovery as a neuronal rhythm-regulating molecule, the RNA-binding protein CIRBP has received considerable attention [16] and was recently revealed to be involved in regulating neuronal and tumor cell survival and proliferation under hypoxic exposure in multiple signaling pathways [8, 9, 17, 18]. It is thus a hot point in the research field of hypoxia. Based on previous results from our lab, we found that the expression of CIRBP in mouse hippocampal neurons was reduced under hypoxic pressure [8, 9], and CIRBP was also involved in regulating hypoxia-induced neuronal damage [8, 9, 19]. Therefore, CIRBP acts as a neuroprotector regulating neuron survival in stressful environments. However, it is still little reported whether CIRBP is involved in hypoxia-induced NSC damage.Stimulation from the external environment tends to activate the PERK-ATF4 signaling pathway to maximize cell survival [20]. Cells prefer to inhibit protein translation in chronic hypoxia, thereby reducing the intracellular accumulation of redundant proteins resulting from a specific environment to maximize self-viability. However, prolonged excessive activation of the PERK-ATF4 signaling pathway also inevitably causes increased cell damage and cell death [21–23]. ISRIB, a specific inhibitor for PERK-ATF4, has gained wide focus since its discovery [24]. ISRIB inhibits phosphorylation of the eukaryotic translation initiation factor 2α (eif2α) [25–27], thereby promoting related protein translation and protecting cells under stressful conditions [28–30]. It is found that ISRIB can protect the differentiation level of intermediate precursor cells, regulate neuron survival under stressful environments, enhance the learning and memory ability of brains, etc. [31–33].In this current research, we examined the role of ISRIB in hypoxia-induced NSC apoptosis. A series of investigations reveal that ISRIB can alleviate apoptosis in NSCs via the ATF4-CIRBP axis in a hypoxia environment.


## 2. Materials and Methods

### 2.1. Ethics Statement

There was approval for this study from the Institute of the Fourth Military Medical University’s Laboratory Animal Care and Use Committee (No. IACUC-20190950), and the National Institutes of Health Guide for the Care and Use of Laboratory Animals was followed for all experiments involving animals.


### 2.2. Animal Studies

The experiments were conducted on 8-week-old C57BL/6J male mice [34]. Chow diets were fed to the mice as indicated, and food and water were freely available to the mice in a temperature-controlled environment (22±2°C).


### 2.3. Cell Culture

A mouse NSC line, C17.2, was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Sijiqing Biotechnology, China) and 5% horse serum (Gibco, USA) [18]. From the E12.5 cortex, primary NSCs were isolated and cultured in a NSC medium (NeuroBasal/DMEM/F12 with GlutaMAX, nonessential amino acids, B27 supplement (2%), bFGF (20 ng/mL), and EGF (20 ng/mL)) (Gibco, USA) [35]. Constructed lentivirus was transfected into cells by the addition of polybrene, which also improved the efficacy of infection (OBiO, China). As a preliminary step, the cultures were kept at 37°C in a humidified incubator containing 5% CO2.


### 2.4. Hypoxic Treatment

Hypoxic conditions were established in a humidified microaerophilic culture system (DWS HypOxystation, UK) using calibrated gases containing 1% O2 and 5% CO2, balanced with N2 at 37°C for 6 hrs, 12 hrs, 24 hrs, 36 hrs, and 48 hrs, respectively. Primary NSCs were cultured in the same condition for 7 d [18]. Accordingly, cells were cultured in normoxic conditions (with 21% O2 and 5% CO2, balanced with N2 at 37°C) for identical times as controls. In a similar manner, to mimic hypoxic conditions, mice were placed in simulated sealed chambers (Fenglei Aerial Armament, China) at an altitude of 6000 meters for 3 weeks [8, 9].


### 2.5. Cell Counting Kit-8 Analysis

Inoculate the cell suspension (100μL/well) into a 96-well plate. Add 10 μL CCK8 (Beyotime Biotechnology, China) solution to each well of the plate. Then, incubate the plate in an incubator for 2 hrs [36]. Measure the absorbance at 450 nm using a microplate reader: cellsurvivalrate%=AS−AB/AC−AB×100%, where AS is the absorbance of test wells, AB is the absorbance of blank holes, and AC is the absorbance of control wells.


### 2.6. Cell Cycle Analysis

A total of three cycles of washing were performed in PBS for all treatments; then, the cells were fixed overnight in PBS and ethanol at −20°C. The mixture should be gently spun down for 20 minutes at 37°C in an extraction buffer containing 0.1% Triton X-100, 45 mM Na2HPO4, and 2.5 mM sodium citrate, and then we used the propidium iodide (BD Biosciences, USA) (50 μg/mL) containing 50 μg/mL RNase Ato to stain the pallet for 30 mins at 37°C in the dark. FACS was used for subsequent analysis. CellQuestk and ModFitk software was introduced for data management [18].


### 2.7. Double Staining Annexin V-FITC/PI Assay to Detect Apoptosis

Glutathione-based digestion was carried out on adherent cells using 0.25% trypsin (Beyotime Biotechnology, China), adding 2% BSA to prevent overdigesting. Dilute 10x binding buffer (BD Biosciences, USA) to 1x binding buffer with deionized water. Adherent cells were digested and collected with trypsin without EDTA and centrifuged at 2000 rpm at room temperature for 5-10 mins before collection. The cells were resuspended with precooling 1x PBS (4°C) buffer, then centrifuged at 2000 rpm for 5-10 mins, and the cells were washed; 300μL of 1x binding buffer was added to the cell suspension. For Annexin V-FITC (BD Biosciences, USA) labeling, Annexin V-FITC 5 μL was made and incubated at room temperature for 15 mins, away from light; then, add 5 μL PI staining and 200 μL of 1x binding buffer before loading [37].


### 2.8. Quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

After treatment, the C17.2 cells were rinsed 3 times with PBS buffer and the total RNA was isolated with TRIzol reagent (Invitrogen, USA) and quantified using a NanoDrop ND-100 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). A Reverse Transcription Kit coupled with the ProFlex PCR System (ABI, USA) was used to convert a total of 1000 ng RNAs into cDNAs (PrimeScript RT Master Mix) (TAKARA, Japan). Using TB Green Premix Ex Taq II (TAKARA, Japan) and corresponding primers, the cDNAs were amplified in a 7500 Fast Real-Time PCR System (ABI, USA). An analysis of gene expression was performed using the 2-ΔΔCt method, which involves incubating at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds and 58°C for 30 seconds [38]. Additional File 1 contains primer sequences.


### 2.9. Western Blotting

Homogenized cells were harvested in lysis buffer after being homogenized. BCA assay (Thermo, USA) with an ultraviolet spectrophotometer (SHIMADZU, Japan) was used to quantify total protein extracts. An electrotransfer of proteins to PVDF membranes was carried out using SDS-PAGE gels containing 10% to 15% protein (Hat Biotechnology, China). Following blocking with 5% BSA, primary antibodies were incubated overnight at 4°C as follows: rabbit anti-CIRBP antibody (1 : 500, #10209-2-AP, Proteintech, USA), rabbit anti-aCasp-3 antibody (1 : 400, #9664, Cell Signaling Technology, UK), rabbit anti-ATF4 antibody (1 : 500, #11815, Cell Signaling Technology, UK), mouse anti-CHOP antibody (1 : 500, #2895, Cell Signaling Technology, UK), rabbit anti-Phospho-eif2α (Ser51) antibody (1 : 1000, #3398, Cell Signaling Technology, UK), rabbit anti-eif2α antibody (1 : 1000, #5324, Cell Signaling Technology, UK), rabbit anti-PERK antibody (1 : 500, #3192, Cell Signaling Technology, UK), and mouse anti-β-actin antibody (1 : 1000, mouse, Sigma, USA). Incubation with secondary antibodies was then performed on the blocked membranes (1 : 1000, goat anti-rabbit HRP or goat anti-mouse HRP, CW Biotechnology, China). ECL assay (Bio-Rad, USA) was used to detect bands, and β-actin was used for normalization of signal intensity. ImageJ software was used to calculate the relative protein quantity from bands visualized under an enhanced chemiluminescence system (Bio-Rad, Germany).


### 2.10. Immunohistochemistry Staining

Anesthesia was achieved by intravenous injection of sodium pentobarbital (100 mg/kg) and transcardial perfusion with 0.9% saline in mice. Mice were sacrificed, and brains were fixed in 4% paraformaldehyde overnight. Afterwards, brain tissues were dehydrated with mixed buffer of 30% (w/v) sucrose and 0.1 M phosphate buffer, and then we sectioned the biopsy 20 μm pieces in a longitudinal manner [39]. We washed the obtained sections with PBS (pH 7.4) three times, permeabilized with 0.05% Triton X-100 (Beyotime Biotechnology, China) for 20 mins on an ice bath and blocked with 5% bovine serum albumin (BSA) for 30 mins at room temperature. The sections were incubated at 4°C overnight with primary antibodies, such as rabbit anti-SOX2 antibody (1 : 400, ab97959, Abcam, UK), rabbit anti-KI67 antibody (1 : 500, #9192, Cell Signaling Technology, UK), and rabbit anti-aCasp-3 antibody (1 : 400, #9664, Cell Signaling Technology, UK), and donkey anti-rabbit H&L Alexa Fluor 594-conjugated secondary antibodies (1 : 800, Invitrogen, USA) in PBS were used for immunostaining (red). Nuclei were labeled with DAPI (blue) and then visualized in a ZEISS Axiovert 200 fluorescent microscope, and fluorescence intensity was quantified using ImageJ [40]. While PBS was used as a replacement for primary antibodies in all studies, blanks remained identical and served as negative controls.


### 2.11. Statistical Analysis

All data were shown asmean±SEM, and statistical significance was assessed using the two-tailed unpaired Student’s t-test or one-way ANOVA with Tukey’s multiple comparisons, as applicable. Statistical significance was demonstrated as ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001, and ∗∗∗∗p<0.0001, respectively.


## 2.1. Ethics Statement

There was approval for this study from the Institute of the Fourth Military Medical University’s Laboratory Animal Care and Use Committee (No. IACUC-20190950), and the National Institutes of Health Guide for the Care and Use of Laboratory Animals was followed for all experiments involving animals.


## 2.2. Animal Studies

The experiments were conducted on 8-week-old C57BL/6J male mice [34]. Chow diets were fed to the mice as indicated, and food and water were freely available to the mice in a temperature-controlled environment (22±2°C).


## 2.3. Cell Culture

A mouse NSC line, C17.2, was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Sijiqing Biotechnology, China) and 5% horse serum (Gibco, USA) [18]. From the E12.5 cortex, primary NSCs were isolated and cultured in a NSC medium (NeuroBasal/DMEM/F12 with GlutaMAX, nonessential amino acids, B27 supplement (2%), bFGF (20 ng/mL), and EGF (20 ng/mL)) (Gibco, USA) [35]. Constructed lentivirus was transfected into cells by the addition of polybrene, which also improved the efficacy of infection (OBiO, China). As a preliminary step, the cultures were kept at 37°C in a humidified incubator containing 5% CO2.


## 2.4. Hypoxic Treatment

Hypoxic conditions were established in a humidified microaerophilic culture system (DWS HypOxystation, UK) using calibrated gases containing 1% O2 and 5% CO2, balanced with N2 at 37°C for 6 hrs, 12 hrs, 24 hrs, 36 hrs, and 48 hrs, respectively. Primary NSCs were cultured in the same condition for 7 d [18]. Accordingly, cells were cultured in normoxic conditions (with 21% O2 and 5% CO2, balanced with N2 at 37°C) for identical times as controls. In a similar manner, to mimic hypoxic conditions, mice were placed in simulated sealed chambers (Fenglei Aerial Armament, China) at an altitude of 6000 meters for 3 weeks [8, 9].


## 2.5. Cell Counting Kit-8 Analysis

Inoculate the cell suspension (100μL/well) into a 96-well plate. Add 10 μL CCK8 (Beyotime Biotechnology, China) solution to each well of the plate. Then, incubate the plate in an incubator for 2 hrs [36]. Measure the absorbance at 450 nm using a microplate reader: cellsurvivalrate%=AS−AB/AC−AB×100%, where AS is the absorbance of test wells, AB is the absorbance of blank holes, and AC is the absorbance of control wells.


## 2.6. Cell Cycle Analysis

A total of three cycles of washing were performed in PBS for all treatments; then, the cells were fixed overnight in PBS and ethanol at −20°C. The mixture should be gently spun down for 20 minutes at 37°C in an extraction buffer containing 0.1% Triton X-100, 45 mM Na2HPO4, and 2.5 mM sodium citrate, and then we used the propidium iodide (BD Biosciences, USA) (50 μg/mL) containing 50 μg/mL RNase Ato to stain the pallet for 30 mins at 37°C in the dark. FACS was used for subsequent analysis. CellQuestk and ModFitk software was introduced for data management [18].


## 2.7. Double Staining Annexin V-FITC/PI Assay to Detect Apoptosis

Glutathione-based digestion was carried out on adherent cells using 0.25% trypsin (Beyotime Biotechnology, China), adding 2% BSA to prevent overdigesting. Dilute 10x binding buffer (BD Biosciences, USA) to 1x binding buffer with deionized water. Adherent cells were digested and collected with trypsin without EDTA and centrifuged at 2000 rpm at room temperature for 5-10 mins before collection. The cells were resuspended with precooling 1x PBS (4°C) buffer, then centrifuged at 2000 rpm for 5-10 mins, and the cells were washed; 300μL of 1x binding buffer was added to the cell suspension. For Annexin V-FITC (BD Biosciences, USA) labeling, Annexin V-FITC 5 μL was made and incubated at room temperature for 15 mins, away from light; then, add 5 μL PI staining and 200 μL of 1x binding buffer before loading [37].


## 2.8. Quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

After treatment, the C17.2 cells were rinsed 3 times with PBS buffer and the total RNA was isolated with TRIzol reagent (Invitrogen, USA) and quantified using a NanoDrop ND-100 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). A Reverse Transcription Kit coupled with the ProFlex PCR System (ABI, USA) was used to convert a total of 1000 ng RNAs into cDNAs (PrimeScript RT Master Mix) (TAKARA, Japan). Using TB Green Premix Ex Taq II (TAKARA, Japan) and corresponding primers, the cDNAs were amplified in a 7500 Fast Real-Time PCR System (ABI, USA). An analysis of gene expression was performed using the 2-ΔΔCt method, which involves incubating at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds and 58°C for 30 seconds [38]. Additional File 1 contains primer sequences.


## 2.9. Western Blotting

Homogenized cells were harvested in lysis buffer after being homogenized. BCA assay (Thermo, USA) with an ultraviolet spectrophotometer (SHIMADZU, Japan) was used to quantify total protein extracts. An electrotransfer of proteins to PVDF membranes was carried out using SDS-PAGE gels containing 10% to 15% protein (Hat Biotechnology, China). Following blocking with 5% BSA, primary antibodies were incubated overnight at 4°C as follows: rabbit anti-CIRBP antibody (1 : 500, #10209-2-AP, Proteintech, USA), rabbit anti-aCasp-3 antibody (1 : 400, #9664, Cell Signaling Technology, UK), rabbit anti-ATF4 antibody (1 : 500, #11815, Cell Signaling Technology, UK), mouse anti-CHOP antibody (1 : 500, #2895, Cell Signaling Technology, UK), rabbit anti-Phospho-eif2α (Ser51) antibody (1 : 1000, #3398, Cell Signaling Technology, UK), rabbit anti-eif2α antibody (1 : 1000, #5324, Cell Signaling Technology, UK), rabbit anti-PERK antibody (1 : 500, #3192, Cell Signaling Technology, UK), and mouse anti-β-actin antibody (1 : 1000, mouse, Sigma, USA). Incubation with secondary antibodies was then performed on the blocked membranes (1 : 1000, goat anti-rabbit HRP or goat anti-mouse HRP, CW Biotechnology, China). ECL assay (Bio-Rad, USA) was used to detect bands, and β-actin was used for normalization of signal intensity. ImageJ software was used to calculate the relative protein quantity from bands visualized under an enhanced chemiluminescence system (Bio-Rad, Germany).


## 2.10. Immunohistochemistry Staining

Anesthesia was achieved by intravenous injection of sodium pentobarbital (100 mg/kg) and transcardial perfusion with 0.9% saline in mice. Mice were sacrificed, and brains were fixed in 4% paraformaldehyde overnight. Afterwards, brain tissues were dehydrated with mixed buffer of 30% (w/v) sucrose and 0.1 M phosphate buffer, and then we sectioned the biopsy 20 μm pieces in a longitudinal manner [39]. We washed the obtained sections with PBS (pH 7.4) three times, permeabilized with 0.05% Triton X-100 (Beyotime Biotechnology, China) for 20 mins on an ice bath and blocked with 5% bovine serum albumin (BSA) for 30 mins at room temperature. The sections were incubated at 4°C overnight with primary antibodies, such as rabbit anti-SOX2 antibody (1 : 400, ab97959, Abcam, UK), rabbit anti-KI67 antibody (1 : 500, #9192, Cell Signaling Technology, UK), and rabbit anti-aCasp-3 antibody (1 : 400, #9664, Cell Signaling Technology, UK), and donkey anti-rabbit H&L Alexa Fluor 594-conjugated secondary antibodies (1 : 800, Invitrogen, USA) in PBS were used for immunostaining (red). Nuclei were labeled with DAPI (blue) and then visualized in a ZEISS Axiovert 200 fluorescent microscope, and fluorescence intensity was quantified using ImageJ [40]. While PBS was used as a replacement for primary antibodies in all studies, blanks remained identical and served as negative controls.


## 2.11. Statistical Analysis

All data were shown asmean±SEM, and statistical significance was assessed using the two-tailed unpaired Student’s t-test or one-way ANOVA with Tukey’s multiple comparisons, as applicable. Statistical significance was demonstrated as ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001, and ∗∗∗∗p<0.0001, respectively.


## 3. Results

### 3.1. Hypoxic Exposure Affects the Apoptosis of NSCs

To reveal the phenomenon of damage to NSCs by hypoxic exposure, mice were exposed to a plateau imitating 6000 m in height for 3 weeks. The number of SOX2+ NSCs was found to be significantly reduced in the SGZ and SVZ regions in mice (Figures1(a)–1(c)). The SGZ and SVZ regions had a higher number of apoptotic neurons, according to further investigation (Figures 1(d)–1(f)). To verify this phenomenon and to better understand the molecular mechanisms involved, we conducted in vitro experiment using C17.2-NSCs and simulated the effect of hypoxic exposure (1% O2) on NSCs. With prolonged hypoxic exposure, neuronal cell damage became obvious, causing the number of neuronal cells to significantly decline. Firstly, we examined the changes in NSC viability at different time points under a hypoxia environment via CCK8 assay and found that NSC viability was enhanced and proliferation was accelerated at the early stage of hypoxic exposure. With the prolongation of hypoxic treatment, the NSC viability was weakened and the proliferation was inhibited (Figure 1(g)). Subsequently, we examined the cell cycle change of NSCs after 48 hrs of hypoxic exposure and found that the number of cells in the G2/M mitotic phase was significantly reduced (Figures 1(h) and 1(i)). We further detected that the number of Annexin V+ cells in NSCs increased (Figures 1(j) and 1(k)) significantly via flow cytometry, indicating an increase in apoptosis of NSCs. The evidence above suggests that hypoxic exposure can lead to apoptosis of NSCs, which in turn may impair higher brain functions.Figure 1
Hypoxic exposure affects the apoptosis of NSCs. (a–f) Hypoxic exposure in 8-week-old C57 male mice for 3 weeks. (a) Representative fluorescent immunohistochemical photographs showed SOX2+ NSCs (green) and DAPI (blue) in the SGZ and SVZ regions.Bars=100μm. (b, c) Quantitative analysis of the number of SOX2+ NSCs in the SGZ and SVZ regions for each visual field. The number of SOX2+ NSCs was significantly decreased after hypoxic exposure, n=6. (d) Representative fluorescent immunohistochemical photographs indicated aCasp3+ (green) apoptotic neuronal cells and DAPI (blue) in the SGZ and SVZ regions. Bars=100μm. (e, f) Quantitative analysis of the number of aCasp3+ cells in the SGZ and SVZ regions for each visual field. The number of aCasp3+ cells was significantly increased after hypoxic exposure, n=6. (g) Cell viability was significantly increased at 6 h, 12 h, and 24 h under hypoxic exposure and decreased at 48 h, n=3. (h) Representative graph of the cell cycle under 48 h hypoxic exposure. Those cells that were halted in the G1/G0, S, and G2/M stages are evident in the first gray area (close to the origin), the incubation area, and the second gray area (far away from it), respectively. (i) Percentage of cells at either G1/G0, S, or G2/M phase in the control and hypoxic treatment groups, n=3. (j) Following 48 hours of hypoxia in C17.2-NSCs, apoptosis is detected. (k) The number of apoptotic C17.2-NSCs was significantly decreased after 48 h hypoxic exposure, n=3. Differences between hypoxia and normoxia (21% O2) were indicated with asterisks.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)


### 3.2. Hypoxic Exposure Reduces the Expression of CIRBP

Previous studies have reported that CIRBP, a protective molecule in hypoxia-exposed neural cells, is involved in regulating the apoptotic process of immune cells, while whether CIRBP is involved in regulating hypoxia-induced apoptosis of NSCs has not been claimed. To investigate this, we first examined the expression of CIRBP at different time points in a hypoxia environment. According to Western blot analysis, CIRBP expression decreased with increasing hypoxia duration (Figures2(a) and 2(b)); after that, we used cellular immunofluorescence 48 hrs after hypoxic exposure to detect the expression level of CIRBP, and as a result of hypoxic treatment, CIRBP expression was significantly reduced, which agreed with the outcomes mentioned above (Figures 2(c) and 2(d)). According to the aforementioned research, hypoxic exposure decreases CIRBP expression.Figure 2
Hypoxic exposure reduces the amount of CIRBP. (a) Western blot results indicated CIRBP expression levels in C17.2-NSCs at 24, 36, and 48 hours following hypoxic exposure. (b) Quantification of CIRBP expression levels in C17.2-NSCs showed insignificant change following 24 hours of hypoxic exposure; the CIRBP level was gradually reduced after 36 h and 48 h of hypoxic exposure,n=3. (c) Representative fluorescence photographs suggested the expression level of CIRBP (green) and DAPI (blue) at 48 h hypoxic exposure in C17.2-NSCs. Bars=50μm. (d) Quantitative analysis of the percentage of CIRBP+ C17.2-NSCs in each sight. The percentage of CIRBP+ C17.2-NSCs was significantly reduced 48 h after hypoxic treatment, n=6. Differences between hypoxia and normoxia (21% O2) were indicated with asterisks.
(a)(b)(c)(d)


### 3.3. CIRBP Attenuates Hypoxia-Induced Apoptosis in NSCs

In view of the negative association between the expressions of CIRBP and apoptosis of NSCs under a hypoxia environment, we hypothesized that CIRBP expression may somewhat reduce the apoptosis brought by hypoxia in NSCs. To prove this, we constructed NSCs with lentivirus overexpressing CIRBP (LV-CIRBP) and LV-NC as a negative control. First, significant overexpression of CIRBP was found in C17.2 cells by RT-PCR and Western blot, as analyzed by RT-PCR and Western blot, respectively (Figures3(a)–3(c)). After all, flow cytometric analysis confirmed that there were significantly fewer apoptotic cells (Figures 3(d) and 3(e)), and the expression of proapoptosis (Figures 3(f) and 3(g)) in the LV-CIRBP group changed in the same direction under hypoxic conditions. Further confirmation of CIRBP’s protective effect on NSCs was obtained by cultivating primary neural stem cells (LV-NC/LV-CIRBP) in normoxia and hypoxia. In comparison to those grown in normoxia with mean neurosphere diameters, primary neural stem cells produced smaller neurospheres in 1% O2 (Figures 3(h) and 3(i)). The finding above implied that CIRBP is involved in the apoptosis of NSCs brought by hypoxic exposure.Figure 3
CIRBP attenuates hypoxia-induced apoptosis in NSCs. (a) Constructed lentivirus overexpressing CIRBP in C17.2-NSCs (LV-CIRBP), and as shown by RT-PCR, the expression of CIRBP mRNA has increased significantly,n=3. (b) Western blot results indicated CIRBP expression levels in C17.2-NSCs in the LV-CIRBP group and LV-NC group. (c) The LV-CIRBP group had significantly increased expression levels of CIRBP protein compared to the LV-NC group, n=3. (d) The detection of apoptosis in C17.2-NSCs after 48 h hypoxic exposure. (e) In hypoxia, LV-CIRBP cells showed significantly less apoptosis than LV-NC cells, n=3. (f) The expression level of aCasp-3 in C17.2-NSCs after 48 h hypoxic exposure. (g) In hypoxia, LV-CIRBP group expression of aCasp-3 was significantly lower than LV-NC group expression, n=3. (h) Neurosphere formation by primary neural stem cells (LV-NC/LV-CIRBP) in response to oxygen concentration. (i) The measurement of the diameters of neurospheres on day 7. Differences between hypoxia and normoxia (21% O2) were indicated with asterisks.
(a)(b)(c)(d)(e)(f)(g)(h)(i)


### 3.4. PERK-ATF4 Signaling Pathway Is Involved in Hypoxia-Induced Apoptosis of NSCs and Regulates the Expression of CIRBP

Overstimulation from the external environment tends to activate the integrative stress response of cells to maximize cell survival. Under prolonged hypoxic pressure, cells prefer to inhibit protein translation, thereby reducing the intracellular accumulation of redundant proteins caused by the specific environmental pressure and thus maximizing their viability. Inevitably, those intracellular changes are unable to counteract the damage to the cells. There was a significant increase in PERK-ATF4 signaling pathway expression by Western blotting (Figures4(a) and 4(b)), indicating that the activation of the PERK-ATF4 signaling pathway may be involved in regulating the hypoxia-induced apoptosis of NSCs. In addition, we treated NSCs with GSK2606414, a selective inhibitor of PERK, and Western blot assays revealed that the ratio of p-PERK/PERK and p-eif2α/eif2α was significantly reduced in the hypoxia-induced pressure-and-GSK2606414 treatment group, and the expressions of ATF4, C/EBP homologous protein (CHOP), and other related molecules were also significantly reduced, indicating that GSK2606414 is capable in inhibiting well the activation of the PERK-ATF4 signaling pathway under hypoxic pressure (Figures 4(c)–4(f)). Later, we conducted flow assay and showed that the hypoxia-induced apoptosis of NSCs was significantly reduced after GSK2606414 treatment (Figures 4(g) and 4(h)).Figure 4
PERK-ATF4 signaling pathway is involved in hypoxia-induced apoptosis of NSCs and regulates the expression of CIRBP. (a) Changes in expression of PERK-ATF4 signaling pathway-related protein levels in C17.2-NSCs following 48-hour hypoxic exposure. (b) PERK and eif2α levels did not change significantly, while p-PERK, p-eif2α, CHOP, and ATF4 levels increased apparently in C17.2-NSCs after hypoxic exposure after 48 h, n=3. (c) Detection of the changes in the expression of PERK-ATF4 signaling pathway-related molecular proteins in C17.2-NSCs after hypoxic exposure after 48 h combined with 10 nM GSK2606414 or DMSO treatment. (d) The ratio of p-PERK/PERK protein levels. This ratio was significantly reduced in the hypoxic exposure-and-GSK2606414 treatment group compared to the hypoxic exposure, n=3. (e) The ratio of p-eif2α/eif2α protein levels. This ratio was significantly decreased in the hypoxic exposure-and-GSK2606414 treatment group compared to the hypoxic exposure, n=3. (f) Changes in CHOP and ATF4 protein levels. Those levels were both significantly reduced in the hypoxic exposure-and-GSK2606414 treatment group compared to the hypoxic exposure-and-DMSO treatment group, n=3. (g) Detection of the apoptosis in C17.2-NSCs under hypoxic exposure and 10 nM GSK2606414 or DMSO treatment for 48 h. (h) Compared with hypoxic exposure and DMSO, the GSK2606414 treatment group showed a decrease in apoptotic cells, n=3. (i) Detection of changes in CIRBP and aCasp3 protein levels in C17.2-NSCs exposed to hypoxia and 10 nM GSK2606414 or DMSO treatment for 48 h. (j) Hypoxia and GSK2606414 treatment significantly increased CIRBP protein levels compared to hypoxia and DMSO treatment, n=3. (k) The protein level of aCasp3 was significantly increased in the hypoxic exposure-and-GSK2606414 treatment group compared to the hypoxic exposure-and-DMSO treatment group, n=3. (l) Detection of changes in CIRBP and aCasp3 protein levels in C17.2-NSCs treated with 2 μg/mL Tunicamycin or DMSO for 0 h, 6 h, 12 h, 24 h, 36 h, and 48 h. (m) Tunicamycin treatment prolonged for 12 hours, 24 hours, 36 hours, and 48 hours gradually reduced CIRBP levels, n=3. (n) Incubation with Tunicamycin for 12 hours, 24 hours, 36 hours, and 48 hours significantly increased the level of aCasp3 protein, n=3. Comparison of exploring time on novel objects is indicated by asterisks.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)(n)In combination with the previous findings we made that CIRBP is a protective molecule for neuronal cell survival under hypoxic pressure, we are interested in whether the PERK-ATF4 signaling pathway regulates the expression of CIRBP involved in hypoxia-induced apoptosis of NSCs. Further, we found that hypoxic exposure coupled with GSK2606414 treatment resulted in an upregulation of CIRBP and a downregulation of aCasp3 in Western blot analyses (Figures4(i)–4(k)). In addition, we administered the PERK-ATF4 signaling pathway activator Tunicamycin for different time points. The Western blot showed that as the duration of the Tunicamycin treatment increased, the expression of CIRBP steadily reduced, and the expression of aCasp3 was significantly increased by Tunicamycin treatment for 48 hrs, indicating that apoptosis was significantly increased (Figures 4(l)–4(n)). The above results suggest that the PERK-ATF4 signaling pathway is involved in hypoxia-induced apoptosis of NSCs and regulates the expression of CIRBP.


### 3.5. ISRIB Alleviates Hypoxia-Induced Apoptosis of NSCs

Since the discovery of the PERK-ATF4 specific inhibitor, ISRIB has gained considerable attention because of its ability to protect cell survival and function by inhibiting the phosphorylation of the translation initiation factor eif2α and thereby promoting the translation of related proteins under stressful conditions. So, we wonder if ISRIB can alleviate hypoxia-induced apoptosis of NSCs. To examine this, we gave mice hypoxic exposure and daily intraperitoneal injection of ISRIB for 3 weeks.According to immunohistochemical data, SGZ and SVZ regions of the mouse gained more SOX2+ NSCs under the hypoxic exposure-and-ISRIB administration group (Figures5(a)–5(c)), suggesting that ISRIB can well protect the survival of NSCs under hypoxic pressure. We further verified the protective effect of ISRIB in in vitro studies. The administration of ISRIB significantly inhibited the apoptosis of NSCs, and the flow results were consistent with the in vivo results (Figures 5(d) and 5(e)). As determined by cell cycle analysis, ISRIB treatment did not result in a greater number of G2/M-phase NSCs (Figures 5(f) and 5(g)), suggesting that ISRIB could well alleviate the apoptosis of NSCs induced by hypoxic exposure but not promote the division and proliferation of NSCs exposed to hypoxia in this in vitro experiment. To further confirm the protective effect of ISRIB on NSCs in 1% O2, primary neural stem cells (DMSO/ISRIB) in both normoxia and hypoxia were cultured and the size of neurospheres was examined. In comparison to those grown in normoxia with mean neurosphere diameters, primary neural stem cells (DMSO) produced smaller neurospheres in 1% O2, and ISRIB intervention can protect neurospheres in 1% O2 to rise bigger (Figures 5(h) and 5(i)). The above results suggest that ISRIB administration alleviates the apoptosis of NSCs induced by hypoxic exposure.Figure 5
ISRIB alleviates hypoxia-induced apoptosis of NSCs. (a) Representative fluorescence immunohistochemistry photographs of 8-week-old C57 male mice treated with hypoxic exposure+0.25 mg/kg ISRIB daily intraperitoneal injection for 3 weeks showed SOX2+ NSCs (green) and DAPI (blue) in SGZ and SVZ regions.Bars=100μm. (b, c) Quantitative analyses of the number of SOX2+ NSCs in SGZ and SVZ regions. The number of SOX2+ NSCs was significantly increased in the hypoxic exposure-and-ISRIB treatment group compared to the hypoxic exposure-and-DMSO treatment group, n=6. (d) Detection of the apoptosis in C17.2-NSCs under hypoxic exposure and 50 nM ISRIB or DMSO treatment for 48 h. (e) The number of apoptotic cells was significantly decreased in the hypoxic exposure-and-ISRIB treatment group compared with the hypoxic exposure-and-DMSO treatment group, n=3. (f) The representative graph of the cell cycle in C17.2-NSCs under hypoxic exposure and 50 nM ISRIB or DMSO treatment for 48 h. (g) Cell percentages at phases G1/G0, S, and G2/M in the normoxia-and-DMSO treatment group, normoxia-and-ISRIB treatment group, hypoxia-and-DMSO treatment group, and hypoxia-and-ISRIB treatment group, n=3. (h) A 7-day culture of primary neural stem cells (DMSO/ISRIB) was conducted in normoxia or 1% oxygen for the formation of neurospheres. (i) Measurement of the diameters of neurospheres on day 7. Asterisks represent differences compared with exploring time on novel objects.
(a)(b)(c)(d)(e)(f)(g)(h)(i)


### 3.6. ISRIB Ameliorates Apoptosis of NSCs Induced by Hypoxic Exposure through the ATF4-CIRBP Signaling Axis

Based on the above, ISRIB as a specific PERK-ATF4 inhibitor can significantly improve NSC apoptosis induced by hypoxic exposure, and the PERK-ATF4 signaling pathway is involved in regulating the expression of CIRBP; we wanted to investigate whether ISRIB ameliorates hypoxia-induced apoptosis of NSCs through the ATF4-CIRBP signaling axis. Firstly, we found that the administration of 50 nM ISRIB significantly increased the expression of CIRBP after hypoxic exposure (Figures6(a)–6(c)), indicating that ISRIB can regulate the expression of CIRBP. After that, we used the constructed LV-CIRBP NSC line and administered Tunicamycin, the activator of the PERK-ATF4 signaling pathway, and found that the expression of aCasp-3 was significantly decreased in the LV-CIRBP group relative to the LV-NC group, indicating that CIRBP can reduce apoptosis in NSCs induced by activation of the PERK-ATF4 signaling pathway (Figures 6(d)–6(f)). Further, in the successful knockdown CIRBP NSC line (sh-CIRBP), after exposure to hypoxia, the expression of aCasp-3 was considerably higher in the sh-CIRBP group than in the sh-NC group, suggesting that CIRBP plays an important role in apoptosis induced by activation of the PERK-ATF4 signaling pathway. In an additional study, we revealed that related molecules did not significantly change in the LV-CIRBP group relative to the LV-NC group and after hypoxic exposure of NSCs (Figures 6(j) and 6(k)), indicating that CIRBP is located downstream of the PERK-ATF4 signaling pathway and that increasing the expression of CIRBP does not negatively regulate the expression of related molecules of the upstream PERK-ATF4 signaling pathway. We then constructed siRNAs targeting ATF4 and verified the interference efficiency via RT-PCR. The results suggested that all three types of constructed siRNAs could significantly inhibit the expression of ATF4 (Figure 6(l)). Further investigations revealed that inhibition of ATF4 expression could significantly increase the expression of CIRBP in NSCs (Figures 6(m) and 6(n)). These results suggest that ISRIB ameliorates apoptosis of NSCs induced by hypoxic exposure through the ATF4-CIRBP signaling axis.Figure 6
ISRIB ameliorates apoptosis of NSCs induced by hypoxic exposure through the ATF4-CIRBP signaling axis. (a) Detection of changes in the CIRBP level in C17.2-NSCs after injection of 20 nM, 50 nM, and 100 nM ISRIB. (b) Changes in the CIRBP level in C17.2-NSCs after 20 nM, 50 nM, and 100 nM ISRIB treatment. The protein level expression of CIRBP was significantly increased in the hypoxic exposure-and-ISRIB treatment group compared to the hypoxic exposure-and-DMSO treatment group,n=3. (c) Changes in the CIRBP level in C17.2-NSCs following 50 nM ISRIB treatment. Hypoxic exposure and ISRIB treatment significantly increased the expression of CIRBP mRNA compared with hypoxic exposure and DMSO treatment, n=3. (d) Changes in the CIRBP and aCasp3 levels in C17.2-NSCs after 2 μg/mL Tunicamycin treatment for 48 h in the LV-NC group and LV-CIRBP group. (e) A significant increase in CIRBP protein expression was observed in LV-CIRBP compared to LV-NC, n=3. (f) The expression of the aCasp3 protein level was significantly reduced in the LV-CIRBP group compared with the LV-NC group after Tunicamycin treatment for 48 h, n=3. (g) Changes in the CIRBP and aCasp3 levels in C17.2-NSCs after hypoxic exposure and 50 nM ISRIB treatment for 48 h in the sh-NC group and sh-CIRBP group. (h) A significant decrease in CIRBP expression was observed in the sh-CIRBP group when compared with the sh-NC group, n=3. (i) The expression of the aCasp-3 protein was significantly increased in the sh-CIRBP group compared to the sh-NC group after hypoxic exposure and 50 nM ISRIB treatment for 48 h, n=3. (j) Changes in the expression of PERK-ATF4 signaling pathway-related protein levels in C17.2-NSCs after hypoxic exposure for 48 h in the LV-NC group and LV-CIRBP group. (k) No significant differences were found in the expression level of eif2α, p-eif2α, CHOP, and ATF4 protein molecules in C17.2-NSCs after hypoxic exposure for 48 h in the LV-NC group and LV-CIRBP group, n=3. (l) Detection of changes in the expression of ATF4 mRNA and verification of the efficiency of ATF4 interference, n=3. (m) Changes in the expression of the CIRBP protein level in the siATF4-1 group, siATF4-2 group, and siNC group. (n) The expression of the CIRBP protein level was significantly increased in the siATF4-1 group and siATF4-2 group relative to the siNC group, n=3. Comparison of exploring time on novel objects is indicated by asterisks.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)(n)


## 3.1. Hypoxic Exposure Affects the Apoptosis of NSCs

To reveal the phenomenon of damage to NSCs by hypoxic exposure, mice were exposed to a plateau imitating 6000 m in height for 3 weeks. The number of SOX2+ NSCs was found to be significantly reduced in the SGZ and SVZ regions in mice (Figures1(a)–1(c)). The SGZ and SVZ regions had a higher number of apoptotic neurons, according to further investigation (Figures 1(d)–1(f)). To verify this phenomenon and to better understand the molecular mechanisms involved, we conducted in vitro experiment using C17.2-NSCs and simulated the effect of hypoxic exposure (1% O2) on NSCs. With prolonged hypoxic exposure, neuronal cell damage became obvious, causing the number of neuronal cells to significantly decline. Firstly, we examined the changes in NSC viability at different time points under a hypoxia environment via CCK8 assay and found that NSC viability was enhanced and proliferation was accelerated at the early stage of hypoxic exposure. With the prolongation of hypoxic treatment, the NSC viability was weakened and the proliferation was inhibited (Figure 1(g)). Subsequently, we examined the cell cycle change of NSCs after 48 hrs of hypoxic exposure and found that the number of cells in the G2/M mitotic phase was significantly reduced (Figures 1(h) and 1(i)). We further detected that the number of Annexin V+ cells in NSCs increased (Figures 1(j) and 1(k)) significantly via flow cytometry, indicating an increase in apoptosis of NSCs. The evidence above suggests that hypoxic exposure can lead to apoptosis of NSCs, which in turn may impair higher brain functions.Figure 1
Hypoxic exposure affects the apoptosis of NSCs. (a–f) Hypoxic exposure in 8-week-old C57 male mice for 3 weeks. (a) Representative fluorescent immunohistochemical photographs showed SOX2+ NSCs (green) and DAPI (blue) in the SGZ and SVZ regions.Bars=100μm. (b, c) Quantitative analysis of the number of SOX2+ NSCs in the SGZ and SVZ regions for each visual field. The number of SOX2+ NSCs was significantly decreased after hypoxic exposure, n=6. (d) Representative fluorescent immunohistochemical photographs indicated aCasp3+ (green) apoptotic neuronal cells and DAPI (blue) in the SGZ and SVZ regions. Bars=100μm. (e, f) Quantitative analysis of the number of aCasp3+ cells in the SGZ and SVZ regions for each visual field. The number of aCasp3+ cells was significantly increased after hypoxic exposure, n=6. (g) Cell viability was significantly increased at 6 h, 12 h, and 24 h under hypoxic exposure and decreased at 48 h, n=3. (h) Representative graph of the cell cycle under 48 h hypoxic exposure. Those cells that were halted in the G1/G0, S, and G2/M stages are evident in the first gray area (close to the origin), the incubation area, and the second gray area (far away from it), respectively. (i) Percentage of cells at either G1/G0, S, or G2/M phase in the control and hypoxic treatment groups, n=3. (j) Following 48 hours of hypoxia in C17.2-NSCs, apoptosis is detected. (k) The number of apoptotic C17.2-NSCs was significantly decreased after 48 h hypoxic exposure, n=3. Differences between hypoxia and normoxia (21% O2) were indicated with asterisks.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)


## 3.2. Hypoxic Exposure Reduces the Expression of CIRBP

Previous studies have reported that CIRBP, a protective molecule in hypoxia-exposed neural cells, is involved in regulating the apoptotic process of immune cells, while whether CIRBP is involved in regulating hypoxia-induced apoptosis of NSCs has not been claimed. To investigate this, we first examined the expression of CIRBP at different time points in a hypoxia environment. According to Western blot analysis, CIRBP expression decreased with increasing hypoxia duration (Figures2(a) and 2(b)); after that, we used cellular immunofluorescence 48 hrs after hypoxic exposure to detect the expression level of CIRBP, and as a result of hypoxic treatment, CIRBP expression was significantly reduced, which agreed with the outcomes mentioned above (Figures 2(c) and 2(d)). According to the aforementioned research, hypoxic exposure decreases CIRBP expression.Figure 2
Hypoxic exposure reduces the amount of CIRBP. (a) Western blot results indicated CIRBP expression levels in C17.2-NSCs at 24, 36, and 48 hours following hypoxic exposure. (b) Quantification of CIRBP expression levels in C17.2-NSCs showed insignificant change following 24 hours of hypoxic exposure; the CIRBP level was gradually reduced after 36 h and 48 h of hypoxic exposure,n=3. (c) Representative fluorescence photographs suggested the expression level of CIRBP (green) and DAPI (blue) at 48 h hypoxic exposure in C17.2-NSCs. Bars=50μm. (d) Quantitative analysis of the percentage of CIRBP+ C17.2-NSCs in each sight. The percentage of CIRBP+ C17.2-NSCs was significantly reduced 48 h after hypoxic treatment, n=6. Differences between hypoxia and normoxia (21% O2) were indicated with asterisks.
(a)(b)(c)(d)


## 3.3. CIRBP Attenuates Hypoxia-Induced Apoptosis in NSCs

In view of the negative association between the expressions of CIRBP and apoptosis of NSCs under a hypoxia environment, we hypothesized that CIRBP expression may somewhat reduce the apoptosis brought by hypoxia in NSCs. To prove this, we constructed NSCs with lentivirus overexpressing CIRBP (LV-CIRBP) and LV-NC as a negative control. First, significant overexpression of CIRBP was found in C17.2 cells by RT-PCR and Western blot, as analyzed by RT-PCR and Western blot, respectively (Figures3(a)–3(c)). After all, flow cytometric analysis confirmed that there were significantly fewer apoptotic cells (Figures 3(d) and 3(e)), and the expression of proapoptosis (Figures 3(f) and 3(g)) in the LV-CIRBP group changed in the same direction under hypoxic conditions. Further confirmation of CIRBP’s protective effect on NSCs was obtained by cultivating primary neural stem cells (LV-NC/LV-CIRBP) in normoxia and hypoxia. In comparison to those grown in normoxia with mean neurosphere diameters, primary neural stem cells produced smaller neurospheres in 1% O2 (Figures 3(h) and 3(i)). The finding above implied that CIRBP is involved in the apoptosis of NSCs brought by hypoxic exposure.Figure 3
CIRBP attenuates hypoxia-induced apoptosis in NSCs. (a) Constructed lentivirus overexpressing CIRBP in C17.2-NSCs (LV-CIRBP), and as shown by RT-PCR, the expression of CIRBP mRNA has increased significantly,n=3. (b) Western blot results indicated CIRBP expression levels in C17.2-NSCs in the LV-CIRBP group and LV-NC group. (c) The LV-CIRBP group had significantly increased expression levels of CIRBP protein compared to the LV-NC group, n=3. (d) The detection of apoptosis in C17.2-NSCs after 48 h hypoxic exposure. (e) In hypoxia, LV-CIRBP cells showed significantly less apoptosis than LV-NC cells, n=3. (f) The expression level of aCasp-3 in C17.2-NSCs after 48 h hypoxic exposure. (g) In hypoxia, LV-CIRBP group expression of aCasp-3 was significantly lower than LV-NC group expression, n=3. (h) Neurosphere formation by primary neural stem cells (LV-NC/LV-CIRBP) in response to oxygen concentration. (i) The measurement of the diameters of neurospheres on day 7. Differences between hypoxia and normoxia (21% O2) were indicated with asterisks.
(a)(b)(c)(d)(e)(f)(g)(h)(i)


## 3.4. PERK-ATF4 Signaling Pathway Is Involved in Hypoxia-Induced Apoptosis of NSCs and Regulates the Expression of CIRBP

Overstimulation from the external environment tends to activate the integrative stress response of cells to maximize cell survival. Under prolonged hypoxic pressure, cells prefer to inhibit protein translation, thereby reducing the intracellular accumulation of redundant proteins caused by the specific environmental pressure and thus maximizing their viability. Inevitably, those intracellular changes are unable to counteract the damage to the cells. There was a significant increase in PERK-ATF4 signaling pathway expression by Western blotting (Figures4(a) and 4(b)), indicating that the activation of the PERK-ATF4 signaling pathway may be involved in regulating the hypoxia-induced apoptosis of NSCs. In addition, we treated NSCs with GSK2606414, a selective inhibitor of PERK, and Western blot assays revealed that the ratio of p-PERK/PERK and p-eif2α/eif2α was significantly reduced in the hypoxia-induced pressure-and-GSK2606414 treatment group, and the expressions of ATF4, C/EBP homologous protein (CHOP), and other related molecules were also significantly reduced, indicating that GSK2606414 is capable in inhibiting well the activation of the PERK-ATF4 signaling pathway under hypoxic pressure (Figures 4(c)–4(f)). Later, we conducted flow assay and showed that the hypoxia-induced apoptosis of NSCs was significantly reduced after GSK2606414 treatment (Figures 4(g) and 4(h)).Figure 4
PERK-ATF4 signaling pathway is involved in hypoxia-induced apoptosis of NSCs and regulates the expression of CIRBP. (a) Changes in expression of PERK-ATF4 signaling pathway-related protein levels in C17.2-NSCs following 48-hour hypoxic exposure. (b) PERK and eif2α levels did not change significantly, while p-PERK, p-eif2α, CHOP, and ATF4 levels increased apparently in C17.2-NSCs after hypoxic exposure after 48 h, n=3. (c) Detection of the changes in the expression of PERK-ATF4 signaling pathway-related molecular proteins in C17.2-NSCs after hypoxic exposure after 48 h combined with 10 nM GSK2606414 or DMSO treatment. (d) The ratio of p-PERK/PERK protein levels. This ratio was significantly reduced in the hypoxic exposure-and-GSK2606414 treatment group compared to the hypoxic exposure, n=3. (e) The ratio of p-eif2α/eif2α protein levels. This ratio was significantly decreased in the hypoxic exposure-and-GSK2606414 treatment group compared to the hypoxic exposure, n=3. (f) Changes in CHOP and ATF4 protein levels. Those levels were both significantly reduced in the hypoxic exposure-and-GSK2606414 treatment group compared to the hypoxic exposure-and-DMSO treatment group, n=3. (g) Detection of the apoptosis in C17.2-NSCs under hypoxic exposure and 10 nM GSK2606414 or DMSO treatment for 48 h. (h) Compared with hypoxic exposure and DMSO, the GSK2606414 treatment group showed a decrease in apoptotic cells, n=3. (i) Detection of changes in CIRBP and aCasp3 protein levels in C17.2-NSCs exposed to hypoxia and 10 nM GSK2606414 or DMSO treatment for 48 h. (j) Hypoxia and GSK2606414 treatment significantly increased CIRBP protein levels compared to hypoxia and DMSO treatment, n=3. (k) The protein level of aCasp3 was significantly increased in the hypoxic exposure-and-GSK2606414 treatment group compared to the hypoxic exposure-and-DMSO treatment group, n=3. (l) Detection of changes in CIRBP and aCasp3 protein levels in C17.2-NSCs treated with 2 μg/mL Tunicamycin or DMSO for 0 h, 6 h, 12 h, 24 h, 36 h, and 48 h. (m) Tunicamycin treatment prolonged for 12 hours, 24 hours, 36 hours, and 48 hours gradually reduced CIRBP levels, n=3. (n) Incubation with Tunicamycin for 12 hours, 24 hours, 36 hours, and 48 hours significantly increased the level of aCasp3 protein, n=3. Comparison of exploring time on novel objects is indicated by asterisks.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)(n)In combination with the previous findings we made that CIRBP is a protective molecule for neuronal cell survival under hypoxic pressure, we are interested in whether the PERK-ATF4 signaling pathway regulates the expression of CIRBP involved in hypoxia-induced apoptosis of NSCs. Further, we found that hypoxic exposure coupled with GSK2606414 treatment resulted in an upregulation of CIRBP and a downregulation of aCasp3 in Western blot analyses (Figures4(i)–4(k)). In addition, we administered the PERK-ATF4 signaling pathway activator Tunicamycin for different time points. The Western blot showed that as the duration of the Tunicamycin treatment increased, the expression of CIRBP steadily reduced, and the expression of aCasp3 was significantly increased by Tunicamycin treatment for 48 hrs, indicating that apoptosis was significantly increased (Figures 4(l)–4(n)). The above results suggest that the PERK-ATF4 signaling pathway is involved in hypoxia-induced apoptosis of NSCs and regulates the expression of CIRBP.


## 3.5. ISRIB Alleviates Hypoxia-Induced Apoptosis of NSCs

Since the discovery of the PERK-ATF4 specific inhibitor, ISRIB has gained considerable attention because of its ability to protect cell survival and function by inhibiting the phosphorylation of the translation initiation factor eif2α and thereby promoting the translation of related proteins under stressful conditions. So, we wonder if ISRIB can alleviate hypoxia-induced apoptosis of NSCs. To examine this, we gave mice hypoxic exposure and daily intraperitoneal injection of ISRIB for 3 weeks.According to immunohistochemical data, SGZ and SVZ regions of the mouse gained more SOX2+ NSCs under the hypoxic exposure-and-ISRIB administration group (Figures5(a)–5(c)), suggesting that ISRIB can well protect the survival of NSCs under hypoxic pressure. We further verified the protective effect of ISRIB in in vitro studies. The administration of ISRIB significantly inhibited the apoptosis of NSCs, and the flow results were consistent with the in vivo results (Figures 5(d) and 5(e)). As determined by cell cycle analysis, ISRIB treatment did not result in a greater number of G2/M-phase NSCs (Figures 5(f) and 5(g)), suggesting that ISRIB could well alleviate the apoptosis of NSCs induced by hypoxic exposure but not promote the division and proliferation of NSCs exposed to hypoxia in this in vitro experiment. To further confirm the protective effect of ISRIB on NSCs in 1% O2, primary neural stem cells (DMSO/ISRIB) in both normoxia and hypoxia were cultured and the size of neurospheres was examined. In comparison to those grown in normoxia with mean neurosphere diameters, primary neural stem cells (DMSO) produced smaller neurospheres in 1% O2, and ISRIB intervention can protect neurospheres in 1% O2 to rise bigger (Figures 5(h) and 5(i)). The above results suggest that ISRIB administration alleviates the apoptosis of NSCs induced by hypoxic exposure.Figure 5
ISRIB alleviates hypoxia-induced apoptosis of NSCs. (a) Representative fluorescence immunohistochemistry photographs of 8-week-old C57 male mice treated with hypoxic exposure+0.25 mg/kg ISRIB daily intraperitoneal injection for 3 weeks showed SOX2+ NSCs (green) and DAPI (blue) in SGZ and SVZ regions.Bars=100μm. (b, c) Quantitative analyses of the number of SOX2+ NSCs in SGZ and SVZ regions. The number of SOX2+ NSCs was significantly increased in the hypoxic exposure-and-ISRIB treatment group compared to the hypoxic exposure-and-DMSO treatment group, n=6. (d) Detection of the apoptosis in C17.2-NSCs under hypoxic exposure and 50 nM ISRIB or DMSO treatment for 48 h. (e) The number of apoptotic cells was significantly decreased in the hypoxic exposure-and-ISRIB treatment group compared with the hypoxic exposure-and-DMSO treatment group, n=3. (f) The representative graph of the cell cycle in C17.2-NSCs under hypoxic exposure and 50 nM ISRIB or DMSO treatment for 48 h. (g) Cell percentages at phases G1/G0, S, and G2/M in the normoxia-and-DMSO treatment group, normoxia-and-ISRIB treatment group, hypoxia-and-DMSO treatment group, and hypoxia-and-ISRIB treatment group, n=3. (h) A 7-day culture of primary neural stem cells (DMSO/ISRIB) was conducted in normoxia or 1% oxygen for the formation of neurospheres. (i) Measurement of the diameters of neurospheres on day 7. Asterisks represent differences compared with exploring time on novel objects.
(a)(b)(c)(d)(e)(f)(g)(h)(i)


## 3.6. ISRIB Ameliorates Apoptosis of NSCs Induced by Hypoxic Exposure through the ATF4-CIRBP Signaling Axis

Based on the above, ISRIB as a specific PERK-ATF4 inhibitor can significantly improve NSC apoptosis induced by hypoxic exposure, and the PERK-ATF4 signaling pathway is involved in regulating the expression of CIRBP; we wanted to investigate whether ISRIB ameliorates hypoxia-induced apoptosis of NSCs through the ATF4-CIRBP signaling axis. Firstly, we found that the administration of 50 nM ISRIB significantly increased the expression of CIRBP after hypoxic exposure (Figures6(a)–6(c)), indicating that ISRIB can regulate the expression of CIRBP. After that, we used the constructed LV-CIRBP NSC line and administered Tunicamycin, the activator of the PERK-ATF4 signaling pathway, and found that the expression of aCasp-3 was significantly decreased in the LV-CIRBP group relative to the LV-NC group, indicating that CIRBP can reduce apoptosis in NSCs induced by activation of the PERK-ATF4 signaling pathway (Figures 6(d)–6(f)). Further, in the successful knockdown CIRBP NSC line (sh-CIRBP), after exposure to hypoxia, the expression of aCasp-3 was considerably higher in the sh-CIRBP group than in the sh-NC group, suggesting that CIRBP plays an important role in apoptosis induced by activation of the PERK-ATF4 signaling pathway. In an additional study, we revealed that related molecules did not significantly change in the LV-CIRBP group relative to the LV-NC group and after hypoxic exposure of NSCs (Figures 6(j) and 6(k)), indicating that CIRBP is located downstream of the PERK-ATF4 signaling pathway and that increasing the expression of CIRBP does not negatively regulate the expression of related molecules of the upstream PERK-ATF4 signaling pathway. We then constructed siRNAs targeting ATF4 and verified the interference efficiency via RT-PCR. The results suggested that all three types of constructed siRNAs could significantly inhibit the expression of ATF4 (Figure 6(l)). Further investigations revealed that inhibition of ATF4 expression could significantly increase the expression of CIRBP in NSCs (Figures 6(m) and 6(n)). These results suggest that ISRIB ameliorates apoptosis of NSCs induced by hypoxic exposure through the ATF4-CIRBP signaling axis.Figure 6
ISRIB ameliorates apoptosis of NSCs induced by hypoxic exposure through the ATF4-CIRBP signaling axis. (a) Detection of changes in the CIRBP level in C17.2-NSCs after injection of 20 nM, 50 nM, and 100 nM ISRIB. (b) Changes in the CIRBP level in C17.2-NSCs after 20 nM, 50 nM, and 100 nM ISRIB treatment. The protein level expression of CIRBP was significantly increased in the hypoxic exposure-and-ISRIB treatment group compared to the hypoxic exposure-and-DMSO treatment group,n=3. (c) Changes in the CIRBP level in C17.2-NSCs following 50 nM ISRIB treatment. Hypoxic exposure and ISRIB treatment significantly increased the expression of CIRBP mRNA compared with hypoxic exposure and DMSO treatment, n=3. (d) Changes in the CIRBP and aCasp3 levels in C17.2-NSCs after 2 μg/mL Tunicamycin treatment for 48 h in the LV-NC group and LV-CIRBP group. (e) A significant increase in CIRBP protein expression was observed in LV-CIRBP compared to LV-NC, n=3. (f) The expression of the aCasp3 protein level was significantly reduced in the LV-CIRBP group compared with the LV-NC group after Tunicamycin treatment for 48 h, n=3. (g) Changes in the CIRBP and aCasp3 levels in C17.2-NSCs after hypoxic exposure and 50 nM ISRIB treatment for 48 h in the sh-NC group and sh-CIRBP group. (h) A significant decrease in CIRBP expression was observed in the sh-CIRBP group when compared with the sh-NC group, n=3. (i) The expression of the aCasp-3 protein was significantly increased in the sh-CIRBP group compared to the sh-NC group after hypoxic exposure and 50 nM ISRIB treatment for 48 h, n=3. (j) Changes in the expression of PERK-ATF4 signaling pathway-related protein levels in C17.2-NSCs after hypoxic exposure for 48 h in the LV-NC group and LV-CIRBP group. (k) No significant differences were found in the expression level of eif2α, p-eif2α, CHOP, and ATF4 protein molecules in C17.2-NSCs after hypoxic exposure for 48 h in the LV-NC group and LV-CIRBP group, n=3. (l) Detection of changes in the expression of ATF4 mRNA and verification of the efficiency of ATF4 interference, n=3. (m) Changes in the expression of the CIRBP protein level in the siATF4-1 group, siATF4-2 group, and siNC group. (n) The expression of the CIRBP protein level was significantly increased in the siATF4-1 group and siATF4-2 group relative to the siNC group, n=3. Comparison of exploring time on novel objects is indicated by asterisks.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)(n)


## 4. Discussion

The brain, the organ with the highest oxygen consumption, is very sensitive to hypoxia. Hypoxic exposure may cause a range of neurological disorders and impairment of higher brain functions, particularly learning and memory capacity [41, 42]. It has been found that learning and memory capacity in mice is significantly impaired after high-altitude exposure [43, 44]. However, the molecular mechanisms of learning and memory loss induced by hypoxia remain uncertain. NSCs are fundamental to the proper execution of mature neural circuit function, and researchers are currently focusing on the effects of hypoxia on NSC proliferation, differentiation, and apoptosis [45–47]. Adult NSCs are mainly found in the SGZ and SVZ regions, and they can repair damaged neural circuits by differentiating into daughter neurons and glial cells [48, 49]. It has been investigated that the activation of proapoptotic genes in NSCs is earlier than that of proproliferative genes and that the number of NSCs is significantly decreased when the oxygen concentration is low (3% O2) [50, 51]. Therefore, the degree of damage under the hypoxia environment is tightly associated with the oxygen concentration in NSCs [18]. Oxygen concentration may occur to be below 1% in the onset area of pathological hypoxic diseases, such as acute plateau edema, stroke and cerebral embolism, and brain tumors, and in even severe cases [52, 53], anoxia may happen. According to early studies, 1% oxygen concentration significantly impeded the speed of proliferation and reduced the amount of NSCs [18], and the prolonged excessive hypoxia caused a considerable rise in NSC apoptosis and therefore could lead to irreversible damage to the nervous system [54, 55]. Our studies revealed that prolonged hypoxic exposure could significantly induce apoptosis of NSCs and decrease the number of those cells. According to the existing experimental results, we believe that long-term hypoxic exposure can significantly damage cells, leading to the occurrence of NSC apoptosis. As NSCs have been damaged, the cell cycle of NSCs is bound to be affected. Therefore, the effect of hypoxic exposure on the cell cycle of NSCs also reflects apoptosis to a certain extent. Thereby, clarifying the mechanisms of hypoxia-induced damage in NSCs is important for understanding the brain damage resulting from high-altitude exposure and preventing the induced pathological brain diseases.The expression of CIRBP, a protective stress response protein, can be regulated by hypoxia, UV radiation, glucose deprivation, and osmolality [56]. As part of the stress response, CIRBP binds to the 3-untranslated region (UTR) of target mRNAs to regulate their stability [9, 56]. Recently, the investigation suggested that CIRBP is neuroprotective against H2O2-induced apoptosis via the protein kinase B (Akt) and extracellular signal-regulated protein kinase (ERK) pathways in rat primary cortical neurons and neuro2a (N2a) cells [57]. In addition, CIRBP could also alleviate apoptosis of neuronal cells by inhibiting ROS in mitochondria and thus act as a neuroprotector [19]. Sakurai et al. observed that a mild hypothermia-induced increase in the CIRBP level inhibited the apoptosis induced by tumor necrosis factor α via caspase-8 activation and ERK phosphorylation [58]. Zhang et al. revealed the neuroprotective effect of CIRBP in inhibiting neuron apoptosis by inhibiting the apoptosis of mitochondria in the subfreezing environment [59]. As expected, CIRBP significantly protects NSCs from hypoxia-induced apoptosis, although the molecular mechanisms underlying this role remain unknown.ISRIB, a highly selective PERK inhibitor with good characteristics in pharmacokinetic experiments, can inhibit the expression of endogenous ATF4, while X-Box Binding Protein 1 (XBP1) mRNA splicing and XBP1 expression remain unchanged, and it has no extensive effect on translation, transcription, or stability of mRNA in unstressed cells. ISRIB inhibits endoplasmic reticulum balance by inhibiting the balance of estrogen receptors and the PERK signaling pathway, reducing the survival rate of cells exposed to endoplasmic reticulum stress. Neuronal cell damage induced by hypoxia is often accompanied by sustained activation of the PERK-ATF4 signaling pathway. Research studies suggested that under sustained stress response, eif2α phosphorylation was inhibited via the reduction of PERK phosphorylation, and a wide range of downstream mRNA translations was therefore inhibited [60]. Meanwhile, ATF4 molecule expression was remarkably upregulated, prompting the production of molecules like CHOPs and triggering the coding of genes associated with apoptosis [61]. As the specific inhibitor of PERK-ATF4, ISRIB could enhance cell survival and function by promoting the phosphorylation of the translation initiation factor eif2α and therefore upregulating the translation of relevant molecules under stressful conditions [25]. We found that the PERK-ATF4 signal was significantly activated and that the expression of CIRBP was gradually decreased after hypoxic exposure, indicating that through the PERK-ATF4 signaling pathway, ISRIB controls the expression of CIRBP in a neuroprotective manner.


## 5. Conclusions

In conclusion, our study suggested that (1) hypoxic exposure decreased the expression of CIRBP, significantly increased the apoptosis of NSCs, and significantly activated the PERK-ATF4 signaling pathway; (2) CIRBP as a neuroprotective molecule could significantly decrease the apoptosis of NSCs under hypoxic pressure; and (3) ISRIB as a neuroprotective agent could regulate the expression of CIRBP via the PERK-ATF4 signaling pathway. Our findings provide insights into mechanisms of cognition and memory impairment and offer potential therapeutic targets and pharmacological strategies for injury protection in diseases associated with hypoxic exposure.



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