GSK2606414 – Csn Chem

Pharmacological Inhibition of PERK Attenuates Early Brain Injury After Subarachnoid Hemorrhage in Rats Through the Activation of Akt

Abstract

Neuronal apoptosis is a central pathological process in subarachnoid hemorrhage (SAH)-induced early brain inju- ry. Endoplasmic reticulum (ER) stress was reported to have a vital role in the pathophysiology of neuronal apoptosis in the brain. The present study was designed to investigate the po- tential effects of ER stress and its downstream signals in early brain injury after SAH. One hundred thirty-four rats were subjected to an endovascular perforation model of SAH. The RNA-activated protein kinase-like ER kinase (PERK) inhibi- tor GSK2606414 and the Akt inhibitor MK2206 were injected intracerebroventricularly. SAH grade, neurologic scores, and brain water content were measured 72 h after subarachnoid hemorrhage. Expression of PERK and its downstream signals, Akt, Bcl-2, Bax, and cleaved caspase-3, were examined using Western blot analysis. Specific cell types that expressed PERK were detected with double immunofluorescence staining. Neuronal cell death was demonstrated with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). Our results showed that the expression of p-PERK and its downstream targets, p-eIF2α and ATF4, in- creased after SAH and peaked at 72 h after SAH. PERK was expressed mostly in neurons. The inhibition of PERK with GSK2606414 reduced p-PERK, p-eIF2α, and ATF4 expres- sion. Furthermore, GSK2606414 treatment increased p-Akt levels and the Bcl-2/Bax ratio as well as decreased cleaved caspase-3 expression and neuronal death, thereby improving neurological deficits at 72 h after SAH. The selective Akt inhibitor MK2206 abolished the beneficial effects of GSK2606414. PERK, the major transducer of ER stress, is involved in neuronal apoptosis after SAH. The inhibition of PERK reduces early brain injury via Akt-related anti-apopto- sis pathways. PERK may serve as a promising target for future therapeutic intervention.

Keywords : Subarachnoid hemorrhage . Early brain injury . Endoplasmic reticulum stress . PERK . Apoptosis

Introduction

Aneurysmal subarachnoid hemorrhage (SAH) is a fatal cere- brovascular disease with unfavorable outcomes and a high rate of mortality [1]. Past research efforts have traditionally focused on vasospasm, which is considered the most impor- tant determinant of brain injuries and outcomes after SAH. Nevertheless, the failure of anti-vasospastic treatment to im- prove the outcomes of SAH patients in clinical trials has shifted the research interest to SAH-induced early brain injury (EBI). Recent studies have suggested that the pathophysiolog- ical events that occur within 72 h after SAH, which is consid- ered EBI, are the most important factor that determines the prognosis of SAH. The definitive mechanisms of early brain injury after SAH remain unclear, and neuronal apoptosis is believed to have an important role in this process [2, 3].

One of the leading mechanisms that results in apoptosis is endoplasmic reticulum stress (ER stress). The ER is an essen- tial intracellular organelle that is responsible for the synthesis and maturation of proteins. Disturbed ER function results in the accumulation of unfolded proteins and induces ER stress. Subsequently, a signal transduction cascade, the unfolded protein response (UPR), is induced [4]. The UPR is facilitated by three types of ER stress sensor proteins, RNA-activated protein kinase-like ER kinase (PERK), inositol requiring ki- nase 1 (IRE1), and activating transcription factor 6 (ATF6). Activated PERK phosphorylates eukaryotic translation initia- tion factor 2α (elF2α) and results in the upregulation of ER- derived chaperones and cytokines. When mild ER stress occurs, the UPR might improve cell survival by remov- ing misfolded proteins. Studies have shown that mild ER stress inhibits neuronal death by promoting autoph- agy [5]; however, prolonged and severe ER stress re- sults in cell death [6]. Several studies have demonstrat- ed that prolonged ER stress has an important role in the mechanisms that underlies ischemia/reperfusion neuronal damage [7]. Furthermore, the attenuation of ER stress reduces neuronal apoptosis in the experimental models of neurodegenerative disease [8, 9].

ER stress has dual roles in cell fate, and the potential con- tribution of ER stress to SAH-induced early brain injury is not completely understood. The present study was designed to investigate the role of ER stress in early brain injury following SAH and its influence on cell apoptosis. A small molecule GSK2606414, a potent selective inhibitor of PERK [10], was used as an experimental tool for translational studies in a rat model of SAH.

Materials and Methods

Animals

All procedures were approved by the ethics committee of Zhejiang University and followed the NIH guidelines for the Care and Use of Laboratory Animals. One hundred forty-eight male Sprague–Dawley (SD) rats (280–330 g) were obtained from SLAC Laboratory Animal Co., Ltd. (Shanghai, China). The rats were housed in a humidity-controlled room (25 ± 1 °C, 12-h light/dark cycle) and were raised with free access to water and food.

SAH Model

The endovascular perforation model of SAH was performed, as previously described [11]. Briefly, with 3 % isoflurane an- esthesia, the left carotid artery and its branches were dissected. A sharpened, 4–0 monofilament nylon suture was inserted rostrally into the left internal carotid artery from the external carotid artery and perforated the bifurcation of the anterior and middle cerebral arteries. Sham-operated rats underwent the same procedures, with the exception that the suture was with- drawn without puncture.

Experimental Design

In experiment 1, the time course of PERK, elF2α, and ATF4 was determined after SAH. Rats were assigned to six groups: sham (n = 8), SAH 3 h (n = 6), SAH 6 h (n = 6), SAH 12 h (n = 6), SAH 24 h (n = 6), or SAH 72 h (n = 8). The brain samples of six rats in each group were used for Western blot analysis. Double immunohistochemistry staining of PERK with neuronal nuclei (NeuN) was performed in sham (n = 2) and 72 h after SAH (n = 2).

In experiment 2, wherein we studied the outcomes of treat- ment, 64 rats were randomly divided into four groups: sham (n = 16), SAH + vehicle (5 μl sterile saline, n = 16), SAH + GSK2606414 (30 μg in 5 μl sterile saline, n = 16), or SAH + GSK2606414 (90 μg in 5 μl in sterile saline, n = 16). SAH grade, neurologic score, and brain water content were measured at 72 h after SAH in all groups (n = 6). The expres- sion of PERK, elF2α, Bcl-2, Bax, Akt, and cleaved caspase-3 was analyzed by Western blot at 72 h after SAH (n = 6). Double immunohistochemistry staining of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) and NeuN was also performed in each group at 72 h after SAH (n = 4).

In experiment 3, in which we studied the mechanism of action of the therapy, 30 rats were randomly divided into five groups: sham (n = 6), SAH+ vehicle (n = 6), SAH+ GSK2606414 (90 μg in 5 μl in sterile saline, n = 6), SAH+ MK2206 (100 μg in 5 μl in sterile saline, n = 6), or SAH + GSK2606414 (90 μg) + MK2206 (100 μg, n = 6). MK2206 (100 μg, Selleck Chemicals, Houston, TX) was injected intracerebroventricularly at 1 h after SAH. PERK, Akt, Bcl- 2, Bax, and cleaved caspase-3 were determined at 72 h after SAH by Western blot analysis in all groups.

Intracerebroventricular Drug Administration

Intracerebroventricular administration was performed as re- ported previously [12]. Briefly, rats were placed in a stereo- taxic apparatus under 2.5 % isoflurane anesthesia. A small burr hole was drilled into the skull according to the following coordinates relative to bregma: 1.5 mm poste- rior and 1.0 mm lateral. The needle of a 10-μl Hamilton sy- ringe (Microliter701; Hamilton Company, Reno, NV) was stereotactically inserted into the left lateral ventricle through the burr hole, 3.5 mm below the horizontal plane of the breg- ma. The PERK inhibitor GSK2606414 (Selleck Chemicals, Houston, TX) or Akt inhibitor MK2206 (100 μg, Selleck Chemicals, Houston, TX) was dissolved in DMSO and further diluted in saline to a final DMSO concentration of 0.5 %. Five microliters of GSK 2606414 (30 μg, 90 μg), 5 μl MK2206 (100 μg), or vehicle was infused at a rate of 0.5 μl/min 1 h after SAH induction. The syringe was left in situ for an addi- tional 10 min before slowly removing. In experiment 1, the sham rats were subjected to the same procedure without inserting the needle.

SAH Grade

The severity of SAH was blindly evaluated using the SAH grading scale at the time of euthanasia as previously reported [13]. Briefly, the basal cistern was divided into six segments that were scored from 0 to 3: grade 0, no subarachnoid blood; grade 1, minimal subarachnoid blood; grade 2, moderate blood with visible arteries; and grade 3, blood clot covering all arteries within the segment. A total score that ranged from 0 to 18 was obtained after adding the scores from all six segments. Animals that received a score <8 were excluded from the study. Neurologic Scores Neurological deficits were evaluated at 72 h after SAH with an 18-point scoring system named the Modified Garcia Scale [14]. Briefly, spontaneous activity, symme- try in the movement of four limbs, forepaw outstretching, climbing, body proprioception, and re- sponse to vibrissae touch were tested (3–18 points). Brain Water Content Brains were removed at 72 h after surgery and quickly sepa- rated into the left hemisphere, right hemisphere, cerebellum, and brain stem. Each part was weighed immediately after removal (wet weight) and then dried at 105 °C for 72 h. The percentage of the water content was calculated as ([wet weight − dry weight]/wet weight) × 100 % [15]. Immunohistochemistry and Quantification of Neuronal Cell Death Rats were anesthetized and transcardially perfused with 0.1 M PBS followed by 4 % paraformaldehyde (pH 7.4). Brains were collected and post-fixed (4 % PFA, 4 °C, 24 h) and then transferred into a 30 % sucrose solution for 2 days. Coronal frozen sections (10 μm) were placed onto slides for fluores- cence staining. Brain slices were washed with 0.1 M PBS and then incubated with blocking solution (10 % normal goat se- rum, 0.1 % Triton X-100 in 0.1 M PBS) for 2 h at room temperature. Sections were incubated overnight at 4 °C with rabbit anti-PERK (1:400, Santa Cruz Biotechnology), mouse anti-NeuN (1:400, EMD Millipore). Sections were then washed with 0.1 M PBS and incubated for 2 h at room tem- perature with secondary antibodies. The sections were visual- ized by a fluorescence microscope. Photomicrographs were saved and merged by Image Pro Plus software (Olympus, Melville, NY). Terminal deoxynucleotide transferase-deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) staining was performed to detect apoptotic cell death according to the manufacturer’s protocol (Roche Inc., Basel, Switzerland). The extent of neuronal damage was eval- uated by an apoptotic index, which was calculated as the av- erage number of TUNEL-positive neurons in six sections per brain at ×400 magnification. The data were expressed as cells per square millimeter. Western Blot Analysis Rats were euthanized at 72 h after SAH induction, and left- brain hemispheres were harvested and processed as previously described [7]. Equal amounts of protein (50 μg) were loaded onto sodium dodecyl sulfate–polyacrylamide gels. The pro- teins were electrophoresed at 100 V for 1 h and then trans- ferred to polyvinylidene fluoride membranes at 100 V for 1 h. The membranes were incubated overnight at 4 °C with prima- ry antibodies for rabbit anti-PERK (1:1000, Cell Signaling), rabbit anti-p-PERK (1:1000, Cell Signaling), rabbit anti- eIF2α (1:1000, Cell Signaling), rabbit anti-p-eIF2α (1:1000, Cell Signaling), rabbit ATF4 (1:1000, Santa Cruz Biotechnology), rabbit anti-Akt (1:1000, Cell Signaling), rab- bit anti-p-Akt (1:1000, Cell Signaling), rabbit anti-Bax (1:1000, Abcam), rabbit anti-Bcl-2 (1:1000, Abcam), and rab- bit anti-Caspase-3 (1:1000, Santa Cruz Biotechnology). The membranes were processed with horseradish-peroxidase- conjugated secondary antibodies at room temperature for 1 h. Bands were visualized using the ECL Plus chemilumines- cence reagent kit (Amersham Bioscience, Arlington Heights, IL). The band densities were quantified with the Image J soft- ware (NIH). The results were expressed as the relative density of the band as compared β-actin, after being normalized to the sham-operated group. Statistical Analysis Data were expressed as the mean ± SEM. One-way ANOVA followed by Tukey multiple comparisons test was used for comparison between groups. p < 0.05 was considered statisti- cally difference. All statistical analyses were performed using GraphPad Prism for Windows (LaJolla, CA, USA). Results Physiological Data All physiological parameters were monitored during the sur- gical procedure. The mean arterial pressure (80–120 mm Hg), arterial pH (7.35–7.45), PO2 (85–95 mm Hg), PCO2 (35– 45 mm Hg), and blood glucose levels (95–125 mg/dl) remained within normal ranges, and no significant changes of these physiological variables were observed between the different groups (data not shown). Expression of PERK, eIF2α, ATF4, and Akt after SAH The expression of p-PERK was significantly elevated 3 h after SAH (p < 0.05) and peaked at 72 h after SAH (p < 0.05, Fig. 1a). A similar tendency was observed in the expression of p-eIF2α, and the downstream kinase ATF4. Both started to increase at 12 h and peaked at 72 h after SAH (p < 0.05, Fig. 1b, c). p-Akt expression decreased significantly at 12 h and reached a minimum at 72 h after SAH (p < 0.05, Fig. 1d). PERK Distribution in Cells in the Left Hemisphere After SAH Double immunostaining of PERK with NeuN in both sham and vehicle groups suggested that PERK was mainly expressed in neurons (Fig. 2) and PERK expression was in- creased at 72 h after SAH. Inhibition of PERK Reduced Brain Edema and Alleviated Neurological Deficits at 72 h After SAH Two dosages of the PERK inhibitor GSK2606414 (30 μg/ 5 μl, 90 μg/5 μl) were administered intracerebroventricularly at 1 h after SAH. Brain edema and neurological scores were measured at 72 h after SAH. When rats were euthanized, subarachnoid blood clots were mainly found around the circle of Willis and the ventral brainstem. The SAH grade scores were not significantly different among the groups (p > 0.05, Fig. 3a). Remarkable neurobehavioral function impairment was observed in the SAH group when compared with the sham group at 72 h after SAH (p < 0.05, n = 6, Fig. 3b). Post-SAH treatment of high-dosage GSK2606414 significant- ly ameliorated neurobehavioral deficits at 72 h after SAH (p < 0.05 vs SAH + vehicle, n = 6, Fig. 3b). Accordingly,experimental SAH evoked a significant increase in brain water content in the left and right hemisphere at 72 h after SAH (p < 0.05 vs sham, n = 6, Fig. 3c). A high dose of GSK2606414 significantly decreased the water content in both hemispheres at 72 h (p < 0.05 vs SAH + vehicle, n = 6, Fig. 3c). Fig. 1 Time course of PERK, phosphorylated PERK, eIF2α, phosphorylated eIF2α, ATF4, Akt, and phosphorylated Akt expression in the left hemisphere after subarachnoid hemorrhage (SAH). The expression of p- PERK was significantly elevated at 3 h after SAH and peaked at 72 h after SAH (a). p-eIF2α (b) and ATF4 (c) expression significantly increased at 12 h and peaked at 72 h after SAH. p-Akt (d) expression decreased significantly at 12 h and reached a minimum at 72 h after SAH. Relative densities of each protein have been normalized against the sham group. n = 6 for each group.*p < 0.05 versus sham, @p < 0.05 versus SAH 72 h. Fig. 2 Representative microphotographs of immunofluorescence staining showing localization of PERK (green) with NeuN (red) in sham and vehicle groups. PERK exhibited predominantly neuronal cytoplasmic expression in rats brain at 72 h after SAH. N = 2 for each group. Scale bar = 50 μm (Color figure online). Inhibition of PERK Promotes Neuronal Survival at 72 h After SAH Administering GSK2606414 significantly lowered the expression of p-PERK (Fig. 4a) and p-eIF2α (Fig. 4b) when compared to the SAH + vehicle group (p < 0.05). The ratio of Bcl-2/Bax (Fig. 4c) was significantly re- duced in the SAH + vehicle group, while the expression level of cleaved caspase-3 (Fig. 4d) was significantly increased in the SAH + vehicle group (p < 0.05 vs sham). The administration of GSK2606414 significantly re- versed these processes (p < 0.05 vs SAH + vehicle), and there was a significant difference between the high- dosage group and low-dosage group (p < 0.05). TUNEL staining localized with the neuronal marker NeuN. The total number of TUNEL and NeuN double-stained cells significantly increased at 72 h after SAH (p < 0.05 vs sham, Fig. 5). Treatment of GSK2606414 significantly reduced the number of TUNEL-positive neurons (p < 0.05 vs SAH + vehicle), and high dosage was more effective (p < 0.05 vs SAH + GSK2606414 30 μg/5 μl). Fig. 3 GSK2606414 improved neurologic function and decreased brain water content at 72 h after subarachnoid hemorrhage (SAH). There was no significant difference in SAH grading among all experimental groups at 72 h after SAH (a). High dosage of GSK2606414 improved neurobehavioral deficits at 72 h after SAH (b). High dosage of GSK2606414 reduced brain water content at 72 h after SAH (c). N =6 for each group. *p < 0.05 versus sham; #p < 0.05 versus SAH + vehicle. Fig. 4 GSK2606414 inhibited phosphorylated PERK (p-PERK) and phosphorylated eIF2α (p- eIF2α) expression, increased the Bcl-2 level, and reduced Bax and cleaved caspase-3 levels at 72 h after subarachnoid hemorrhage (SAH). Changes in p-PERK (a), p-eIF2α(b), Bcl-2, Bax(c), and cleaved caspase-3(d) expression in different groups. N = 6 for each group. *p < 0.05 versus sham;#p < 0.05 versus SAH + vehicle; ξp < 0.05 versus SAH + GSK2606414 (30 μg). Role of Downstream Akt in PERK-Mediated Injury 72 h After SAH To further determine the role of Akt in the PERK signaling pathway, MK 2206, a highly selective inhibitor of Akt, was injected intracerebroventricularly at 1 h after SAH. The Akt inhibitor MK2206 had no effect on PERK expression that was induced by SAH (Fig. 6a). The increased expression of p-Akt found with the GSK2606414 treatment was significantly sup- pressed after MK 2206 administration (p < 0.05 vs SAH + GSK2606414, Fig. 6b). The inhibition of PERK by GSK2606414 significantly promoted cell survival by increas- ing the Bcl-2/Bax ratio and reducing cleaved caspase-3 ex- pression (p < 0.05 vs SAH + vehicle, Fig. 6c D); however, this beneficial effect was significantly reversed by MK 2206 in- jection (p < 0.05 vs SAH + GSK2606414, Fig. 6c, d). Discussion In the present study, we explored the role of PERK in the early brain injury after SAH. The endovascular perforation model of SAH enhanced the expression of PERK and its downstream effectors, eIF2α and ATF4, which peaked at 72 h after SAH. PERK was expressed mostly in neurons. PERK aggravated neuronal apoptosis and neurologic deficits, whereas inhibition of PERK by GSK2606414 remarkably decreased neuronal apoptosis, reduced brain edema, and improved neurological functions. The potent PERK inhibitor GSK2606414 exerted its neuroprotective effects by promoting Akt activation, in- creasing Bcl-2 expression, and reducing Bax and caspase-3 levels. Furthermore, selective inhibition of Akt signaling abolished the anti-apoptotic effects of GSK2606414. Fig. 5 GSK2606414 decreased the number of TUNEL-positive neurons at 72 h after SAH. Representative microphotographs showed the co- localization of NeuN (red) with TUNEL (green)-positive cells in the bilateral basal cerebral cortex at 72 h after SAH (a). Quantitative analysis of TUNEL-positive neurons showed that GSK2606414 decreased the number of apoptotic cells after SAH (b). Scale bar = 100 μm, *p < 0.05 versus sham; #p < 0.05 versus SAH + vehicle; ξp < 0.05 versus SAH + GSK2606414 (30 μg). Within 72 h, early brain injury results in many neurological injuries within after SAH, including the disturbance of ion hemostasis and the generation of oxidative stress and gluta- mate [16]. The endoplasmic reticulum (ER) is an organelle in which proteins are synthesized and processed. Insults that per- turb ER function result in ER stress [17]. Several studies have shown that ER stress was induced by oxidative stress [18], ionic cellular derangement [19], mitochondrial calcium overloading [20], and toxic glutamate release [21] in various diseases. As a central regulator of ER stress, PERK/eIF2α signaling has a central role in neuronal cell fate. The PERK/ eIF2α branch of ER stress mediates the transient shutdown of translation in response to rising levels of misfolded proteins in the endoplasmic reticulum. The increased activation of PERK/eIF2α signaling results in the sustained reduction of global protein synthesis, thereby resulting in neuronal loss and clinical disease [22, 23]. In a cerebral ischemia model, the expression of PERK/eIF2α signaling was significantly increased [24]. PERK and eIF2α levels were observed to be elevated in a mouse model of traumatic brain injury [25]. In the present study, we determined the time course of the ex- pression of PERK and its downstream signals, elF2α and ATF4, after SAH. Consistent with the above mentioned ob- servations, the ratio of p-PERK/PERK was significantly ele- vated at 3 h after SAH and peaked at 72 h. The ratio of p- elF2α/elF2α and p-ATF4/ATF4 were both significantly in- creased at 12 h and peaked at 72 h after SAH. We also deter- mined the time course expression of pro-survival factor Akt after SAH. The p-Akt expression decreased significantly at 12 h and reached a minimum at 72 h after SAH. Based upon these results, PERK activity was strongly associated with de- creased pro-survival markers. In this study, PERK was found to be expressed mostly in neurons by immunofluorescence staining. This observation is consistent with most previous studies that PERK expression increased in neurons after CNS injury [25, 26]. We further investigated the effects of PERK inhibition by GSK2606414 on early brain injury after SAH. GSK2606414 administration significantly inhibited p-PERK and p-eIF2α expression, improved neurobehavioral deficits, and reduced brain edema at 72 h after SAH. Additionally, PERK inhibition increased the expression of Bcl-2 as well as decreased Bax and cleaved caspase-3 expression. Double immunofluores- cence staining demonstrated that neuronal apoptosis was induced by SAH, while PERK inhibition significantly pro- moted neuronal survival. These results support a neuroprotec- tive role of PERK inhibition following SAH. This result is consistent with previous studies in which severe ER stress resulted in cell apoptosis, while the suppression of ER stress promoted cell survival and improved neurological function [8, 9, 25]; however, there were also some reports that demonstrat- ed that PERK activation reduced cell apoptosis in several dis- ease models [5, 27]. ER stress may have dual roles in cell fate, and the mechanism by which ER stress mediates pro- apoptotic or pro-survival effects remains unclear. The ability of cells to respond to perturbations in ER function is critical for cell survival. Under certain physiological or pathological conditions, if the stimuli are mild, ER stress might be introduced as a host defense mechanism to promote cell sur- vival through autophagy activation by providing nutrients via the auto-digestion of damaged organelles and proteins [5, 28]. If the stimuli are severe and prolonged, ER stress is unable to compensate for these stimuli and cell apoptosis would be in- duced [8, 9]. In experimental SAH studies, there is an acute increase in intracranial pressure (ICP), extracellular glutamate, oxidative stress, inflammation, calcium overloading, and a resultant decrease in cerebral blood flow (CBF) after the onset of subarachnoid hemorrhage [16]. Following SAH, blood im- mediately spreads through the subarachnoid space, thereby covering the cerebral cortex with a thick blood clot. The blood clot splintered over the next 3 days, while these breakdown products continue to generate toxic agents [29]. As a result, these insults after SAH induce severe and prolong ER stress, which subsequently results in neuronal apoptosis. The present study showed that increased ER stress was observed at 72 h after SAH and that the inhibition of PERK expression promot- ed neuronal survival. Fig. 6 Akt inhibition by MK2206 abolished the beneficial effects of GSK2606414 at 72 h after subarachnoid hemorrhage (SAH). Akt inhibitor MK2206 had no effect on PERK expression that was induced by SAH (a). Increased expression of p-Akt by GSK2606414 was suppressed after MK 2206 administration (b). GSK2606414 treatment increased Bcl-2 level as well as reduced Bax and cleaved caspase-3 expression; however, this protective effect was reversed by MK2206 administration (c, d). N =6 for each group. *p < 0.05 versus sham; #p < 0.05 versus SAH + vehicle; &p < 0.05 versus SAH + GSK2606414. We further investigated the role of Akt in PERK-mediated neuronal apoptosis. Akt is a serine/threonine kinase and has a critical role in the modulation of cell development, growth, and survival. When Akt is activated, it phosphorylates a di- verse number of protein substrates, including GSK-3β and nuclear factor-kappa B (NF-κB). Many studies have demon- strated that Akt signaling has a crucial role under physical and pathological conditions to promote neuronal survival [30, 31]. In the present study, the time course demonstrated that the ratio of p-Akt/Akt decreased significantly at 12 h after SAH and reached a minimum at 72 h. PERK inhibition by GSK2606414 treatment significantly increased p-Akt expres- sion. Furthermore, GSK2606414 treatment increased Bcl-2 levels as well as decreased Bax and caspase-3 expression, which were all blocked by the co-administration of the selec- tive Akt inhibitor MK2206. These results suggest that GSK2606414 administration suppresses PERK activity and enhances the consequent Akt-related neuronal survival after SAH. Similar studies have shown that GSK2606414 exerts its neuroprotective effects in Alzheimer’s disease and Parkinson’ disease as a highly selective inhibitor of PERK [22, 32]. Yet, some reports have also shown that GSK2606414-treated mice experience weight loss and mild hyperglycemia during a long- term study [9]. These effects are likely due to the systemic effects of this compound; however, the effects on glucose metabolism due to pharmacological inhibition of PERK by GSK2606414 are relatively mild [9]. Given the increase in neuronal survival with PERK inhibition, it would be predicted that, in the absence of toxic side effects, generalized pharma- cological PERK inhibition would have a beneficial effect in early brain injury after SAH. Although the immediate translational value of GSK2606414 may be limited by its systemic effects, it has important translational implications for future studies. There are a few limitations to this study. First, several studies of cerebral ischemia have suggested that ER stress is also related to neuroinflammation [33, 34]. In the present study, we focused on the potential apoptotic effect of PERK and its downstream signals without an investigation of neuroinflammation. Further studies are required to explore the role of PERK in neuroinflamma- tion after SAH. Second, PERK was reported to increase the enzymatic activity of calcineurin by direct interaction [35]. Calcineurin then facilitated calcium overloading in the cytosol, which was considered to be the possible mechanism of cell apoptosis. Therefore, the connections between PERK and other downstream signals in neuronal apoptosis after SAH require further studies. In conclusion, we suggested that PERK, the major trans- ducer of ER stress, has an important role in neuronal apoptosis after SAH. The inhibition of PERK may have the potential to reduce early brain injury via Akt-related anti-apoptosis pathways. 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