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

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.


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][5][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 selfrenewal 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][12][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 hypoxiainduced 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][22][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][26][27], thereby promoting related protein translation and protecting cells under stressful conditions [28][29][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][32][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.  [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).

Hypoxic Treatment.
Hypoxic conditions were established in a humidified microaerophilic culture system (DWS HypOxystation, UK) using calibrated gases containing 1% O 2 and 5% CO 2 , balanced with N 2 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% O 2 and 5% CO 2 , balanced with N 2 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 (100 μL/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: cell survival rate ð%Þ = ½ðAS − ABÞ/ðAC − ABÞ × 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 Na 2 HPO 4 , 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]. 2.10. 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 594conjugated 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.

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 (Figures 1(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% O 2 ) 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 3 Oxidative Medicine and Cellular Longevity    Oxidative Medicine and Cellular Longevity 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.

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 (Figures 2(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  Oxidative Medicine and Cellular Longevity aforementioned research, hypoxic exposure decreases CIRBP expression.

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   (Figures 4(a) and 4(b)),    indicating that the activation of the PERK-ATF4 signaling pathway may be involved in regulating the hypoxiainduced 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 hypoxiainduced 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)).
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 (Figures 4(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.

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 (Figures 5(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% O 2 , 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% O 2 , and ISRIB intervention can protect neurospheres in 1% O 2 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.  11 Oxidative Medicine and Cellular Longevity 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 (Figures 6(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 siR-NAs 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 1signaling axis.

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][46][47]. Adult NSCs   16 Oxidative Medicine and Cellular Longevity 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% O 2 ) [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 H 2 O 2 -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.

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.

Data Availability
Detailed data are provided in this article to support its conclusions.

Conflicts of Interest
The listed authors declare that the publication of this article does not conflict with their interests.