c-Abl Tyrosine Kinase-Mediated Neuronal Apoptosis in Subarachnoid Hemorrhage by Modulating the LRP-1-Dependent Akt/GSK3β Survival Pathway

Accumulating evidence suggests that neuronal apoptosis plays a critical role in early brain injury (EBI) after subarachnoid hemorrhage (SAH), and the inhibition of apoptosis can induce neuroprotective effects in SAH animal models. c-Abl has been reported to promote neuronal apoptosis in Alzheimer’s disease and cerebral ischemia, but its role in SAH had not been illuminated until now. In the present study, the effect of c-Abl on neuronal apoptosis induced by SAH was investigated. c-Abl protein levels and neuronal apoptosis were markedly increased 24 h after SAH, and the inhibition of endogenous c-Abl reduced neuronal apoptosis and mortality and ameliorated neurological deficits. Furthermore, c-Abl inhibition decreased the expression of cleaved caspase-3 (CC-3) after SAH. These results demonstrate the proapoptotic effect of c-Abl in EBI after SAH. Additionally, c-Abl inhibition further enhanced the SAH-induced phosphorylation of Akt and glycogen synthase kinase (GSK)3β. LY294002 abrogated the beneficial effects of targeting c-Abl and exacerbated neuronal apoptosis after SAH. SAH decreased LRP-1 levels and downregulated LRP-1 by RAP, and LRP-1 small interfering RNA (siRNA) induced a dramatic decrease in Akt/GSK3β activation in the presence of c-Abl siRNA. This is the first report showing that the c-Abl tyrosine kinase may play a key role in SAH-induced neuronal apoptosis by regulating the LRP-1-dependent Akt/GSK3β survival pathway. Thus, c-Abl has the potential to be a novel target for EBI therapy after SAH.


Introduction
Subarachnoid hemorrhage (SAH) caused by aneurysm rupture is a devastating cerebrovascular disease characterized by high mortality and disability. Nearly 60,000 people worldwide suffer from this disease each year (Feigin et al. 2009). Most survivors also have intractable neurological deficits and cognitive dysfunction (Al-Khindi et al. 2010).
Cerebral vasospasm following SAH is widely accepted as the major cause of poor outcomes (Kassell et al. 1985); however, cumulative data suggest that suppressing vasospasm does not improve patient outcomes (Macdonald et al. 2008). Early brain injury (EBI), defined as a series of pathophysiological changes that occur within the first 72 h after the onset of SAH (Kusaka et al. 2004), has now become a focus of research on alleviating injury from SAH (Fujii et al. 2013). Neuronal apoptosis has been reported to be one of the main pathological mechanisms of EBI after experimental SAH and is observed in human patients with SAH (Peng et al. 2018;Nau et al. 2002). Therefore, resistance against neuron apoptosis in the treatment of EBI after SAH has broad research value and application prospects.
c-Abl is an src-related nonreceptor tyrosine kinase that is widely expressed in the nervous system. Recently, numerous intensive studies have focused on the relationship between c-Abl and central nervous system (CNS) diseases (Schlatterer et al. 2011a). c-Abl has been proven to exert a Cong Yan, Hongwei Yu, and Yao Liu have contributed equally to this work. proapoptotic effect through various mechanisms involved in Parkinson's and Alzheimer's diseases (Brahmachari et al. 2016;Wu et al. 2016). To the best of our knowledge, there has been no report on the role of c-Abl in SAH. The Akt/ glycogen synthase kinase (GSK) 3β signaling pathway was shown to be involved in the neuroprotective mechanism of EBI after SAH, and blockade of the Akt/GSK3β pathway aggravated neuronal apoptosis (Endo et al. 2006). There have been few studies exploring whether c-Abl modulates Akt/GSK3β activity in neurons.
Low-density lipoprotein receptor-related protein 1 (LRP-1), a member of the low-density lipoprotein receptor family, is a scavenger receptor involved in endocytosis and signaling receptor that regulates various cellular processes (Lin and Hu 2014). LRP-1, which regulates the Akt survival pathway and insulin pathway in the brain and promotes the antiapoptotic function of neurons, was previously shown to be highly expressed in neurons. Endogenous LRP-1 knockout led to caspase-3 activation and increased neuronal apoptosis (Fuentealba et al. 2009). In addition, LRP-1 was shown to have a synergistic effect with immunoglobulin on neuronal survival in ischemic stroke, and the LRP-1 antagonist RAP was reported to significantly inhibit the activation of Akt (Lok et al. 2016). A study suggested that the inhibition of c-Abl using imatinib increases basal LRP-1-dependent ERK and Akt activation and maintains pancreatic β cell function and survival (Fred et al. 2015). However, the role of LRP-1 in regulating Akt expression in neuronal survival after SAH is unclear. The current work investigated the role of c-Abl in SAH and verified that the underlying mechanism of this role may be related to the LRP-1-dependent Akt/GSK3β survival pathway for the first time.

Animals
All animal experiments were approved by the Animal Care and Use Committee of the First Affiliated Hospital of Harbin Medical University and strictly complied with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. A total of 207 male Wistar rats (260-280 g) were purchased from Changchun Yisi Company, which were fed with adequate food and water in a 12/12 h light/dark room with humidity control and a temperature of 25 °C.

SAH Model and Study Design
An SAH rat model was established by endovascular puncture as previously reported (Park et al. 2004). Pentobarbital (40 mg/kg) was injected intraperitoneally to anesthetize the animals. The animals were placed in a supine position, and a median incision was made in the neck to expose the common carotid artery and its branches. A single-stranded nylon thread penetrated the external carotid artery and encountered resistance after entering the internal carotid artery, indicating that it had reached the distal bifurcation of the internal carotid artery. The thread was then advanced approximately 3 mm beyond the resistance point and immediately withdrawn, allowing reperfusion of the internal carotid artery. The same operation was performed on rats in the sham group, except the vessels were not punctured.
Twenty-four rats were used to detect c-abl expression at 24, 48, and 72 h after SAH by western blot (n = 6). To test the role of c-Abl in SAH, 48 rats were randomly divided into four groups as follows: the sham (n = 12), SAH (n = 12), SAH + scramble small interfering RNA (siRNA) (n = 12), and SAH + c-Abl siRNA (n = 12) groups. c-Abl siRNA and scramble siRNA (500 pmol/5 µL) were injected intracerebroventricularly 24 h before SAH. Neuron apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), and levels of the c-Abl, p-Akt, p-GSK3β, and cleaved caspase-3 (CC-3) proteins were measured by Western blotting. The neurological function score and SAH severity were also evaluated. The PI3K/ Akt inhibitor LY294002 was used to explore the underlying mechanism of the role of c-Abl in SAH-induced apoptosis. A total of 36 rats were randomly divided into three groups as follows: the SAH + scramble siRNA + vehicle (n = 12), SAH + c-Abl siRNA + vehicle (n = 12), and SAH + c-Abl siRNA + LY294002 (n = 12) groups. LY294002 (5 mmol/L, 0.5 ml/animal) in a physiological saline solution was injected into the femoral vein 1 h before SAH induction, and rats in the vehicle group were given the same amount of physiological saline. Western blotting was used to detect protein levels. TUNEL was used to test apoptosis. To investigate the role of LRP-1 in the mechanism by which c-Abl regulates Akt/GSK3β, 54 rats were randomly divided into the following groups: the sham, SAH + vehicle (or scramble siRNA) (n = 6), SAH + RAP (or LRP-1 siRNA) (n = 6), SAH + c-Abl siRNA + vehicle (or scramble siRNA) (n = 6), and SAH + c-Abl siRNA + RAP (or LRP-1 siRNA) (n = 6) groups. RAP (0.7 nmol/g body weight) in a physiological saline solution was injected into the femoral vein 1 h before SAH operation, and rats in the vehicle group were given the same amount of physiological saline. Western blotting was conducted to detect protein levels.

Intracerebroventricular Injection
Intracerebroventricular injection was conducted as previously reported (Suzuki et al. 2010). The rats were anesthetized and placed in stereotactic devices. A 10-μL Hamilton syringe (Hamilton Company, Reno, NV, USA) was inserted into the left ventricle through a burr hole made at the following coordinates: 1.5 mm posterior and 1.0 mm lateral to the bregma and 3.2 mm below the horizontal plane. siRNA in solution was administered at a rate of 0.5 μL/min 24 h before SAH operation. The needle remained in place for 5 min and was then withdrawn slowly. The c-Abl mixed sequences of the sense strands were as follows: 1: 5′-CGG CAG CCU AAA UGA AGA Utt-3′, 2: 5′-CCU AUG GCA UGU CAC CUU Att-3′, 3: 5′-GGU UUA UGA GCU GCU GGA Att-3′. Scramble siRNA sequence: 5′-UUC UCC GAA CGU GUC ACG Utt-3′.

Neurological Scoring
The modified Garcia scale was used to blindly assess neurological deficits in animals 24 h after SAH as previously reported (Yin et al. 2016). The assessment system consisted of six subtests to evaluate the following: spontaneous activity, spontaneous movement of the 4 limbs, forepaw outstretching, climbing, body proprioception, and the response to whisker stimulation. Each subtest was scored from 0 to 3 or 1 to 3, and the total score (from 3 to 18) reflected neurological function ( Table 1). The lower a score was, the worse the neurological function was.

SAH Grade Assessment
SAH severity was quantified with an SAH grading scale based on the amount of bleeding as previously described (Sugawara et al. 2008). The basal cistern of the brain was photographed and divided into six predetermined segments (Fig. 1B), and each segment was scored from 0 to 3, Grade 0: no subarachnoid blood; Grade 1: minimal subarachnoid blood; Grade 2: moderate blood clot with recognizable arteries; Grade3: blood clot obliterating all arteries. The total SAH grade was equal to the sum of all segment scores (maximum SAH grade = 18). Rats in which the SAH grade was < 8 and those for which SAH coexisted with subdural or epidural hemorrhage were excluded.

Western Blot Analysis
Western blotting was conducted as previously reported (Suzuki et al. 2010;Cheng et al. 2010). Briefly, the cerebral cortex from the left hemorrhagic site was collected as a sample 24 h after SAH. After anesthesia with pentobarbital (40 mg/kg), the rats were sacrificed by cervical dislocation and then perfused with glacial PBS transcardially. The soft tissue and skull were separated to obtain the brain tissue. Fresh brain tissue was carefully removed from the basal cortex. The brain tissues were immediately frozen in liquid nitrogen and stored at − 80 °C until used (all operations were performed on ice). Tissues were weighed and homogenized. RIPA lysis buffer and protease inhibitor (PMSF, NaF, etc.) were added to the homogenates, which were lysed on ice for 40-60 min and shaken once every 5-8 min. The samples were centrifuged to collect the supernatants. The protein concentration was detected with a detergent-compatible protein assay kit (Bio-Rad, Hercules, CA, USA). Samples containing the same amount of the target protein (30 μg) were separated by SDS-PAGE, and the proteins were then transferred to a PVDF membrane. The membrane was blocked with 5% nonfat dry milk for 2 h and incubated overnight at 4 °C in the presence of the following primary antibodies: anti β-actin (Abcam, cat#  The elliptic regions were the sampling area of Western blot and staining, in which the coronal section of the specimen used for staining was adopted. C c-Abl inhibition had no effect on the SAH grade after SAH. D Curves of survival rate from every group within 24 h after SAH. There was no significant difference between SAH + scramble siRNA and SAH + c-Abl siRNA groups by log rank (mantel Cox) test. n = 12 per group

TUNEL Staining and NeuN Double Immunofluorescence Labeling
After anesthesia, the rats were perfused transcardially with 4 °C PBS followed by 4% paraformaldehyde. Then, the brain tissue was taken out immediately and fixed in 4% paraformaldehyde overnight. Sucrose solution was dehydrated in gradient; after rinsed, the brain tissue was quickly frozen on the machine. Finally, the continuous coronal frozen section was made. TUNEL and staining for NeuN, a neuronal marker, were used together to detect neuronal apoptosis. Briefly, frozen sections were rewarmed at room temperature for 20 min, blocked with 5% sheep serum (Equitech-Bio,SS-0100) for 1 h and then incubated with anti-NeuN primary antibody (Cell Signaling Technology, cat# 12,943, 1:200 dilution). A TUNEL kit (Roche, cat# 11,684,795,910) was used to label apoptotic cells after the use of anti-NeuN secondary antibody (Alexa Fluor® Plus 594-conjugated). The sections were incubated with 50 μL of TUNEL reaction mixture (enzyme solution: labeling solution = 1:9), incubated at 37 °C in the dark for 60 min, and washed 3 times with PBS for 5 min each; then, the sections were sealed and observed by fluorescence microscopy (Olympus, Tokyo, Japan). TUNEL-positive cells in five different fields were counted. The results are expressed as cells/mm 2 , and the apoptotic ratio was calculated as the number of apoptotic cells/the total cell number × 100%.

Statistical Analysis
All data are expressed as the mean ± SD. Statistical significance was analyzed using one-way ANOVA followed by Tukey's multiple comparison test. A probability value of p < 0.05 indicated statistical significance. SPSS 13.0 software for Windows was used to perform all statistical analyses.

Mortality, SAH Grade, and Survival Curves
No animals from the sham group died. The mortality rates of the SAH were significantly increased and inhibiting c-Abl with specific siRNA reduced the mortality. The mortality of LY294002 group was higher than that of SAH + c-Abl siRNA + vehicle group. Similarly, the mortality rates of SAH + c-Abl siRNA + RAP (or LRP-1 siRNA) were higher than that of SAH + c-Abl siRNA + vehicle group (Fig. 1A). Figure 1B shows representative images of the brains of rats from the sham and SAH groups. There was no significant difference in SAH severity between the SAH and SAH + c-Abl siRNA groups (p > 0.05; Fig. 1C). There were no significance between the SAH + Scramble siRNA and SAH + c-Abl siRNA groups by Log-rank (Mantel-Cox) test (Fig. 1D).

Inhibition of c-Abl Decreased SAH-Induced Neuronal Apoptosis and Improved Neurologic Deficits
TUNEL and NeuN double staining were performed to determine neuronal apoptosis. As shown in Fig. 2, the number of double-positive cells following TUNEL and NeuN staining was significantly higher in the SAH group than in the sham group (p < 0.05; Fig. 2A, B). However, c-Abl inhibition using specific siRNA significantly decreased neuronal apoptosis compared to that in the SAH and SAH + scramble siRNA groups (p < 0.05; Fig. 2A, B). Neurological deficits were assessed 24 h after SAH. Compared with the sham group, the nerve function scores in the SAH group were decreased significantly. The inhibition of c-Abl improved neurologic deficits (p < 0.05 vs the SAH group; Fig. 2C).

Inhibition of c-Abl Promoted the Phosphorylation of Akt and GSK3β after SAH
Western blotting was conducted to verify the levels of c-Abl, the phosphorylation of Akt, GSK3β, and the apoptosisrelated protein CC-3. The results showed that c-Abl expression increased after SAH and was most significant 24 and 48 h after SAH (p < 0.05; Fig. 3A, B). The phosphorylation of Akt and its downstream protein GSK3β were increased in the SAH group compared to the sham group in our study, which was consistent with the results of a previous study (Endo et al. 2006). When c-Abl expression was blocked by specific siRNA, the phosphorylation of Akt and GSK3β was notably increased. Western blotting also showed that the inhibition of c-Abl downregulated SAH-induced CC-3 expression (p < 0.05; Fig. 3C-G).

LY294002 Abrogated the Effect of c-Abl Inhibition on Neuronal Apoptosis, Neurological Scores, and the CC-3 Protein Level
LY294002, a highly selective inhibitor of the PI3K/Akt signaling pathway, was used to elucidate the role of the Akt/ GSK3β signaling pathway in the neuroprotective mechanism induced by inhibiting c-Abl. The inhibition of c-Abl significantly increased the activity of Akt/GSK3β (Fig. 3) and decreased the number of TUNEL-NeuN double-positive cells after SAH, but LY294002 blocked this beneficial effect (p < 0.05; Fig. 4A, B).The improvement in neurological score in response to c-Abl inhibition was markedly abrogated by LY294002 (p < 0.05; Fig. 4C). LY294002 suppressed the phosphorylation of Akt and GSK3β in response to c-Abl siRNA treatment (p < 0.05; Fig. 5A, C, D), while increasing the expression of CC-3 without influencing c-Abl expression (p > 0.05; Fig. 5A, B, E).

Downregulation of LRP-1 Suppressed the Increases in p-Akt and p-GSK3β Induced by c-Abl Inhibition after SAH
To investigate the effects of LRP-1 downregulation on the activity of Akt/GSK3β induced by inhibiting c-Abl after SAH, LRP-1 was downregulated with a specific siRNA and the inhibitor RAP, and Akt/GSK3β activity was detected by Western blotting. The results showed that LRP-1 levels were significantly decreased after SAH in the SAH group in comparison with the sham group and that the specific siRNA and inhibitor (RAP) further reduced LRP-1 levels (p < 0.05; Fig. 6A-C). In the control groups (SAH + vehicle and SAH + scramble siRNA groups), we observed increased Akt and GSK3β phosphorylation in response to c-Abl siRNA treatment. This effect was not observed in groups in which LRP-1 downregulation was induced by systemic siRNA delivery and RAP (p < 0.05; Fig. 6D-H).

Discussion
Our results in an SAH rat model demonstrated for the first time that c-Abl plays a proapoptotic role and confirmed that its mechanism may be related to the LRP-1-dependent Akt/GSK3β signaling pathway (Fig. 7). In this study, we observed that the suppression of c-Abl by siRNA significantly improved neurological deficits, downregulated CC-3, and decreased neuronal apoptosis in response to SAH. These results showed that c-Abl was involved in apoptotic mechanisms in EBI after SAH and that inhibiting c-Abl can ameliorate poor outcomes. In addition, Western blotting results suggested that the suppression of c-Abl further enhanced the expression of p-Akt and p-GSK3β induced by EBI following SAH. We used LY294002, a selective inhibitor of PI3K/Akt, to treat SAH rats that had been administered c-Abl siRNA. LY294002 abolished the beneficial effects of c-Abl blockade on neurological outcomes and apoptosis. LRP-1 was decreased after SAH, and LRP-1 expression was downregulated by RAP and LRP-1 siRNA in the presence of c-Abl siRNA, resulting in a significant decrease in Akt/GSK3β activation. These results suggest that c-Abl contributes to SAH-mediated neuronal apoptosis via suppressing the LRP-1-dependent Akt/GSK3β pathway. c-Abl is an src-associated nonreceptor tyrosine kinase that is widely expressed in the nuclei and cytoplasm of nerve cells. Nuclear c-Abl mainly regulates the cell cycle, determines cell fate, and participates in the development and morphogenesis of neurons (Leonberg and Chai 2007;Nagar et al. 2003;Smith and Mayer 2002). c-Abl has little known function in fully differentiated neurons. c-Abl has been reported to be active in diseases such as prion disease, cerebral ischemia, Parkinson's disease, and Alzheimer's disease (Schlatterer et al. 2011a;Brahmachari et al. 2016;Wu et al. 2016;Sun et al. 2018). Suppression of the c-Abl/p73 pathway was reported to inhibit neuronal apoptosis and improve neuronal dysfunction in an Niemann-Pick Type C disease mouse model (Alvarez et al. 2008), but c-Abl overexpression induced neuronal death and increased neuronal inflammation in the mouse forebrain (Schlatterer et al. 2011b). Oxidative stress activates c-Abl in neurons, which then activates p53 or p73 to initiate neuronal apoptosis (Klein et al. 2011;Lee et al. 2008). Reactive oxygen free radicals are one of the main mediators of SAH pathology. Accumulating evidence indicates that reactive oxygen species (ROS) production and oxidative stress begin to emerge early after SAH (Chen et al. 2014).
Despite these advances, whether c-Abl is involved in the apoptotic process in response to SAH has not been reported. In the present study, we determined that c-Abl expression and neuronal apoptosis were notably increased 24 h after SAH and that c-Abl inhibition decreased neuronal apoptosis and improved neurological deficits. Our results showed that c-Abl also had a proapoptotic effect in the pathogenesis of SAH.
Akt, also called protein kinase B, plays an essential role in a variety of important cellular processes, including cell survival, proliferation, and apoptosis (Manning and Cantley 2007). GSK3β, a downstream protein of Akt, is a survival pathway protein that mediates cell survival and apoptosis in numerous pathological states (Endo et al. 2006). It has been Fig. 3 Effects of inhibiting c-Abl on the expression of p-Akt/p-GSK3β/CC-3 in the ipsilateral cortex 24 h after SAH. c-Abl expression increased after SAH, and arrived at the climax at 24 and 48 h. Inhibition of c-Abl promoted the phosphorylation of Akt and GSK3β after SAH, but downregulated CC-3 expression. A, C Representative western blots. B, D, E, F, G Quantitative analysis of c-Abl, p-Akt, p-GSK3β, and CC-3. n = 6 per group. * p < 0.05 versus sham, # p < 0.05 versus SAH + vehicle/SAH + scramble siRNA reported that GSK3β activity may be regulated by its phosphorylation at tyrosine-216 (irritant) and serine-9 (inhibitory) (Bhat et al. 2000;Grimes and Jope 2001). Activated Akt (serine-473) phosphorylates GSK3β on serine-9 to inhibit its activity and reduce apoptosis (Hetman et al. 2000). Accumulated data in an SAH experimental model suggest that the Akt pathway is involved in the biological process of neuron survival. The phosphorylation of Akt and GSK3β has been correlated with EBI after SAH (Endo et al. 2006). There have been some hypotheses on the downstream mechanisms of the Akt/GSK3β pathway, although these mechanisms are not yet understood. For instance, the Akt pathway inhibits activity of the proapoptotic kinase GSK3β, which inhibits neuronal apoptosis dependent on the apoptosis-related protein caspase pathway or the mitochondrial pathway by regulating Bcl2/Bax levels (Si et al. 2018). Further study of the effectors downstream of Akt/ GSK3β is necessary to elucidate the mechanism of acute brain injury after SAH. Therefore, we determined levels of the c-Abl and p-Akt/p-GSK3β proteins in SAH rats. The c-Abl and p-Akt/p-GSK3β protein levels were markedly increased in the SAH group, which was consistent with a previous report (Endo et al. 2006). Nevertheless, c-Abl siRNA administration significantly reduced c-Abl expression but increased Akt/GSK3β phosphorylation. These results seem contradictory because of the interaction between the apoptotic and survival mechanisms after SAH. Accordingly, we make two assumptions. First, medium is necessary for c-Abl to regulate the activity of the downstream products of Akt/GSK3β after SAH. Second, other mechanisms regulate Akt/GSK3β activity after SAH. The present results showed that blockade of the Akt/ GSK3β pathway with LY294002 abolished the reduction in neuronal apoptosis and improvement in neurological score induced by c-Abl inhibition. Thus, we deduce that c-Abl exerts a proapoptotic effect on neurons by inhibiting the Akt/ GSK3β survival pathway after SAH. However, the detailed mechanism of the interaction of c-Abl with Akt/GSK3β requires further research.
LRP-1, a multifunctional multiligand receptor involved in the regulation of endocytosis and many cellular processes (Lin and Hu 2014), is abundantly expressed in cortical and hippocampal neurons in the brain (Fuentealba et al. 2009). LRP-1 has a variety of biological neuronal functions closely related to the maintenance of synapses, metabolism of lipoproteins, and clearance of amyloid-β (Aβ) in the brain and participate in various mechanisms of neuron survival (Fuentealba et al. 2009). Previous studies have shown that LRP-1 regulates the downstream insulin receptor and Akt pathway to inhibit neuronal apoptosis in the pathological state of Alzheimer's disease (Fuentealba et al. 2009). According to previous data, many important cytoplasmic adaptors in signal transduction can bind the tail of LRP-1. For example, an experiment in yeast confirmed that Dab1 specifically binds the cytoplasmic tail of LRP-1 at the second NPXY motif (Howell et al. 1999;Trommsdorff et al. 1998). Activation of the Akt pathway by α2M through LRP-1 has been well characterized in neurons. LRP-1 was Fig. 6 Downregulation of LRP-1 with RAP and siRNA abolished the effects of suppressing c-Abl, reversed the changes in p-Akt, p-GSK3β levels. RAP and LRP-1 siRNA decreased the levels of LRP-1 after SAH. The increases of p-Akt and p-GSK3β expression induced by inhibiting c-Abl were abolished by downregulation of LRP-1 in response to RAP and siRNA, respectively. A, D Representative western blots. B, C, E, F, G, H Quantitative analysis of LRP-1, p-Akt, and p-GSK3β protein levels. n = 6 per group. * p < 0.05 versus sham, # p < 0.05 versus SAH + vehicle/scramble siRNA, & p < 0.05 versus SAH + c-Abl siRNA + vehicle/scramble siRNA found to bind α2M in Schwann and PC12 cells, promoting the binding of LRP-1 to Dab1 and then activating the Akt pathway (Mantuano et al. 2008). However, c-Abl inhibition by imatinib increased LRP-1-dependent ERK and Akt activation and contributed to pancreatic β cell function and survival. It was reported that the LRP-1 was a regulator between c-Abl and Akt, and LRP-1 was indispensable for the effects of c-Abl in inhibiting Akt expression (Fred et al. 2015). We administrated RAP 1 h and specific siRNA 24 h before SAH induction to inhibit LRP-1 and further disturbed the interaction between c-Abl and Akt/GSK3β. The objective of this experiment further illustrated the role of Akt/GSK3β in the effects of c-Abl on apoptosis. Our work provides the first evidence suggesting a link between c-Abl function and LRP-1-dependent Akt/GSK3β signaling in SAH. In the present work, we observed increased Akt and GSK3β phosphorylation in response to c-Abl siRNA after SAH, but LRP-1 downregulation with an siRNA or inhibitor suppressed the increase in p-Akt and p-GSK3β induced by c-Abl siRNA. This finding indicates that LRP-1 is necessary for c-Abl-mediated regulation of Akt/GSK3β activity in SAH.

Conclusion
These results associate SAH-induced c-Abl overexpression with neuronal apoptosis in EBI and suggest that inhibiting c-Abl improves neurological deficits. In addition, the proapoptotic effect of c-Abl following SAH might be mediated via the LRP-1-dependent Akt/GSK3β signaling pathway.
• Inhibition of c-Abl reduced neuronal apoptosis and improved neurological deficits in SAH • c-Abl played pro-apoptotic role by inhibiting Akt/GSK3β survival pathway in EBI after SAH • LRP-1 was essential for c-Abl to regulate Akt/GSK3β pathway in SA Author Contribution Professor Gao is the corresponding author, responsible for the design, supervision, and communication of the whole research. Cong Yan, Hongwei Yu, and Yao Liu contributed equally to Fig. 7 Schematic diagram of c-Abl-mediated neuron apoptosis in subarachnoid hemorrhage this study and were responsible for the establishment of animal models, data analysis, and article writing. Hongbo Zhao, Kongbin Yang, and Qi Shao were responsible for neurological function score and apoptosis detection. Yingqiang Zhong, Pei Wu, Chunlei Wang, and Wenyang Zhao are responsible for the related contents of WB. Jingwei Li and Nan Liu were responsible for lateral ventricular injection of siRNA and drug injection. Jinglong Di, Chen Li, and Luhao Bao were responsible for assessing the severity of SAH and calculate animal mortality.
Funding This study was supported by the National Natural Science Foundation of China which belong to Cheng Gao (Nos. 81070944, 31372268, 82071317).

Declarations
Ethics Approval All procedures about animal were approved by the Animal Care and Use Committee of the First Affiliated Hospital of Harbin Medical University.
Research Involving Human Participants and/or Animals All procedures involving animals were in accordance with the ethical standards of the First Affiliated Hospital of Harbin Medical University.
Informed Consent This research group uses animals for experimental research and passes the ethical review without involving informed consent.

Conflict of Interest
The authors declare no competing interests.