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Research Report
Regulation on Beclin-1 expression by mTOR in CoCl2induced HT22 cell ischemia-reperfusion injury Tao Yanga,b, Dongbo Lia,b, Feng Liua, Lei Qia, Ge Yana, Maode Wanga,n a
Department of Neurosurgery, First Affiliated Hospital of Xi’an Jiaotong University, 277 West Yanta West Road, Xi’an, Shaanxi 710061, PR China a Department of Neurosurgery, Ankang City Central Hospital, Ankang, Shaanxi 725000, PR China
art i cle i nfo
ab st rac t
Article history:
It has been reported that cerebral ischemia/reperfusion (I/R) injury can activate autophagy.
Accepted 9 April 2015
However, the role of autophagy in cerebral I/R injury remains controversy. Two major proteins, mTOR and Beclin-1, govern the formation of autophagosomes to regulate
Keywords:
autophagy activity. However, the cross-talking between Beclin-1 and mTOR in cerebral
Autophagy
I/R injury remains elusive. In this study, global cerebral I/R injury animal model and focal
Beclin-1
cerebral I/R injury animal model were induced to test the variation of Beclin-1 level in vivo.
mTOR
To further confirm the variation of Beclin-1 level and investigate the cross-talking between
Ischemia
Beclin-1 and mammalian target of rapamycin (mTOR) in I/R injury, we used cobalt chloride
Reperfusion
(CoCl2) to develop an I/R injury cell model in HT22 cell line. Our data showed that the levels of Beclin-1 and phosphorylated mammalian target of rapamycin (p-mTOR) were clearly induced by I/R injury in vitro. And the time course studies suggested that the Beclin-1 and mTOR may have coordinated regulation in ischemia stages but not in reperfusion stages. Moreover, inhibitor of mTOR could prevent Beclin-1 decreasing, but this prevention may play opposite roles in different stages of I/R injury. We conclude that this study represents a major advance in our understanding of the cross-talking of two key proteins, Beclin-1 and mTOR, in autophagy and the role of autophagy in cerebral I/R injury. & 2015 Published by Elsevier B.V.
1.
Introduction
Cerebral I/R injury is the third cause of death and results in permanent disability with extensive cerebral tissue damage. Although different mechanisms are involved in the pathogenesis of I/R, increasing evidence showed that autophagy may play a critical role in neuronal survival and death (Fulda, 2012). The role of autophagy in ischemic neurons remains
controversial (Codogno and Meijer, 2005; Rami and Kogel, 2008; Gabryel et al., 2012). Thus, the signaling pathways of autophagy activation would contribute to the understanding of the mechanisms of disease. Autophagy is required for all types of cell to maintain cellular homeostasis, and neurons are particularly sensitive to variation in autophagy activity. Autophagy is up-regulated and promotes cell survival in stress (Lee et al., 2009), which could
n
Corresponding author. E-mail address: [email protected] (M. Wang).
http://dx.doi.org/10.1016/j.brainres.2015.04.016 0006-8993/& 2015 Published by Elsevier B.V.
Please cite this article as: Yang, T., et al., Regulation on Beclin-1 expression by mTOR in CoCl2-induced HT22 cell ischemiareperfusion injury. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.04.016
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also induce cell death directly or indirectly via various physiological processes (Fan et al., 2014). Two major protein complexes, mTOR (Heras-Sandoval et al., 2014; Inoki, 2014) complex 1 and the Beclin-1 (Sun et al., 2009) complex, govern the formation of autophagosomes to regulate autophagy activity. The studies have shown that TOR may be involved in balancing macroautophagic and microautophagic activities (Dubouloz et al., 2005) and signaling pathways that activate mTOR could inhibit autophagy, whereas signaling pathways that inhibit TOR stimulate autophagy (Codogno and Meijer, 2005; Arsham and Neufeld, 2006). In contrast, the Beclin-1 (Atg6) complex, which is involved in the initial step of autophagosome formation, is to stimulate autophagy. Beclin-1 is part of a multiprotein complex, and acts as a platform, recruiting an activator (UVRAG) and a repressor (Bcl-2) of Beclin 1/hVps34-dependent autophagy (Pattingre et al., 2008). However, it is still unknown that how autophagy is regulated in neuronal survival and death and whether the two core complexes of autophagy coordinates the regulations between each other in response to stress. In this study we investigated the variation of Beclin-1 level and crosstalking between Beclin-1 and mTOR, the two core proteins in autophagy formation, in models of in vivo and in vitro of cerebral I/R injury.
2.
Results
2.1.
The level of Beclin-1 in focal/global cerebral I/R injury
The western blotting showed that the level of Beclin-1 was significantly induced and showed significant increase after 24 h of global cerebral I/R injury models compared to the control group (Fig 1A and C, Po0.05). To further conform the finding in global cerebral I/R injury, we performed a middle cerebral artery occlusion (MCAO) model in mice and detected the changes of Beclin-1. The penumbra and contralateral hemisphere were separated 24 h after I/R. The data showed that the levels of Beclin-1 were clearly increased in the penumbra and contralateral hemisphere (Fig 1, B and D, Po0.05).
2.2. The dynamic change of Beclin-1 in CoCl2-induced I/R injury Western blotting was performed to test the expression of Beclin-1 and mTOR/p-mTOR. The analysis showed that Beclin-1 was induced gradually and reached its peak 10 h after CoCl2 treatment. The phosphorylation of mTOR is important for induction of mTOR activity and increasing pmTOR indicated decline in autophagy activity. We examined the p-mTOR level in HT22 cells at different time points of CoCl2 treatment. The results showed that the p-mTOR was induced after treatment for 10 h, while the increasing Beclin1 began to decline (Fig. 2, A and B, Po0.05). We treated HT22 cells for 16 h using 500 mM CoCl2 and then re-incubated the cells with 10% FBS medium to establish the I/R injury cell model. The cells were collected at different time points to test the variation of Beclin-1 and p-mTOR. The data indicated that both Beclin-1 and p-mTOR were induced at early I/R stages and showed the similar expression patterns (Fig. 3, A and B, Po0.05).
2.3. Regulation of rapamycin on Beclin-1 in CoCl2-induced ischemia injury Rapamycin is widely used as inhibitor of mTOR activity and inducer of autophagy. We chose rapamycin to test the coordination between Beclin-1 and mTOR. The treatment of rapamycin clearly induced Beclin-1 under normal condition (Fig. 4, A and B, Po0.05). The HT22 cells were treated by coincubation with 50 nM rapamycin and 500 mM CoCl2 for different time points. The data showed that the expression of Beclin-1 protein was significant increased and maintained the increased level in ischemia injury (Fig. 5, A and B, Po0.05).
2.4. The effect of rapamycin on neuronal death in CoCl2induced I/R injury To test the effect of autophagy activity on neuronal survival and death, we carried out MTT assay in CoCl2-induced I/R injury. HT22 cells were treated the same as above. Both ischemia and reperfusion significantly induced cell death. The analyses further showed that co-incubation with 50 nM rapamycin and 500 mM CoCl2 could accelerate cell death in ischemic injury (Fig. 6, A and B, Po0.05), but clearly protect the cells against the injury induced by I/R (Fig. 6, C and D, Po0.05).
3.
Discussion
Autophagy is a self-eating catabolic process to break down and recycle proteins, cytoplasmic components, and damaged organelles in order to maintain cellular homeostasis. Autophagy includes several mechanisms such as macroautophagy, microautophagy, and chaperone-mediated autophagy. Macroautophagy, referred to autophagy, is responsible for the clearance of proteins, organelles, and protein aggregates in response to stress. The autophagy pathway is divided into four distinct steps: initiation/nucleation, autophagosome formation, trafficking/ maturation, and recycling/release of macromolecules. Both the evolutionarily conserved Target of rapamycin kinase complex 1 and the Beclin-1 complex play important roles in the control of autophagy. The TOR complex 1 (TORC1) could inhibit autophagy by regulating the Atg1 complex (Sengupta et al., 2010). In contrast, the Beclin-1 (Atg6) complex, which is involved in the initial step of autophagosome formation, is to stimulate autophagy (Xie and Klionsky, 2007). The studies showed that Beclin-1 induces autophagy by regulation on the lipid kinase Vps-34 protein (Kang et al., 2011). Many other proteins such as Atg14 (Fogel et al., 2013), UVRAG (Liang et al., 2006) and Bif-1/endophilin B1 (Takahashi et al., 2009) induce or block autophagy by interacting with Beclin-1. Some antiapoptotic family members such as Bcl-2 (Ciechomska et al., 2009) and Bcl-XL (Kim et al., 2014) regulate autophagy by interaction with BH3 domain of Beclin-1. And caspase-mediated cleavage of Beclin-1 promotes cross-talk between apoptosis and autophagy. In neurons, autophagy is classified into “basal” and “induced” depending on its roles. In normal conditions, neurons display low levels of autophagosomes. Pathological conditions such as stress or injury could induce autophagy activity. During autophagy induction, neurons undergo a series changes to up-regulate
Please cite this article as: Yang, T., et al., Regulation on Beclin-1 expression by mTOR in CoCl2-induced HT22 cell ischemiareperfusion injury. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.04.016
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Fig. 1 – Western blotting showed that the expression of Beclin-1 protein was significant increased in focal/global cerebral ischemia-reperfusion injury. The bar chart demonstrates the ratio of Beclin-1 relative to actin for each time point. (A) High protein level of Beclin-1 was observed in global cerebral ischemia-reperfusion in rats at the time points of 0 h and 24 h compared to the sham control group respectively. (B) Focal cerebral ischemia-reperfusion mouse models showed that high level of Beclin-1 protein was examined in the penumbra and contralateral hemisphere compared with the control group. (C, D) Semiquantitative analysis (relative optical density) of intensity of staining of Beclin-1 to actin in global cerebral ischemiareperfusion at the time points of 0 h and 24 h and in the penumbra and contralateral hemisphere of focal cerebral ischemiareperfusion injury for each time point. The data are expressed as mean7SEM (n¼ 3, *Po0.05, **Po0.05, compared to the control group). basal level to induced level. Many studies showed that autophagy is controlled by a few key regulators such as Beclin-1, serine-threonine protein and AMP activated kinase (AMPK) (Kelekar, 2005). Beclin-1, the mammalian orthologue of yeast Atg6, has a central role in autophagy and cell survival. During periods of cell stress, Beclin-1 is increased and forms Beclin-1Vps34 complexes by interacting with cofactors, and then induce autophagy (Funderburk et al., 2010). The studies found that Beclin-1 is related to cellular biological functions and many diseases including heart disease (Matsui et al., 2007), development, and neurodegeneration (Winslow and Rubinsztein, 2008). However, the precise functions and mechanisms of Beclin-1 in these physiological processes still have not been elucidated. Binding with Bcl-2 or other autophagy partners provide potential insight into functions of Beclin-1 in different physiological processes in autophagy and in processes independent of autophagy. Cerebral injury caused by I/R are the consequence of a massive cell death. However, the mechanism of I/R cell death are more complicated. In different models of I/R, autophagy activity was induced in cerebral cortex (Moreno et al., 2006), hippocampus (Rami, 2008), and striatum (Zhang et al., 2009) of animals. Whether I/R-induced autophagy contributes to neuronal death or provide a neuroprotection is still controversy. Studies suggested detrimental role of autophagy might be associated with autophagy-necrosis cross-talk (Nikoletopoulou et al., 2013). On the other hand, increasing of Beclin-1 expression and rapamycin had a long-lasting protective effect during ischemic procedure. In current study, we investigated the level
of Beclin-1 during different stages of I/R injury models. Our data showed that Beclin-1 expression was induced earlier than pmTOR and damping of Beclin-1 was accompanied with gradually increasing of p-mTOR during ischemic injury. Inhibition of mTOR could promote Beclin-1 expression and prevent the decreasing of Beclin-1 in later stage of ischemia. Those finding suggested that two key proteins for activating autophagy via different signal pathways may have cross-talking in ischemia stage. Blocking mTOR activity may contribute to Beclin-1 expression which involves in induction of autophagy. In reperfusion injury, our data showed that there was no obvious coordinated regulation between Beclin-1 expression and pmTOR. Furthermore, the analysis on cells survival and death showed activated autophagy could accelerate the cell death in ischemia stage, but promote neurons survival in the reperfusion stage. This indicated that autophagy may play distinct roles in different stages of ischemia or reperfusion injury.
4.
Experimental procedures
4.1.
Experimental animals
To confirm the level of Beclin-1 in cerebral I/R injury, two different animal models were established. Adult Sprague– Dawley (S–D) rats (male, 250–280 g) and adult Balb/c mice (male, 20–28 g) were used to establish the global and focal cerebral I/R animal models respectively. All animals were provided by the Experiment Animal Center of the Fourth
Please cite this article as: Yang, T., et al., Regulation on Beclin-1 expression by mTOR in CoCl2-induced HT22 cell ischemiareperfusion injury. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.04.016
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Fig. 2 – Cerebral ischemia injury was simulated by 500 lM CoCl2 in HT22 cells. The bar chart demonstrates the relative ratio of Beclin-1 or p-mTOR to actin at each time point. (A) The expression of Beclin-1 began to increase at 4 h after incubation with 500 lM CoCl2 and a distinct peak was observed 10 h after incubation, then it began to decline. However, the expression of p-mTOR has been induced after treatment for 10 h, while the increased Beclin-1 began to decline. (B) Semiquantitative analysis (relative optical density) of the staining of Beclin-1 and p-mTOR at different ischemia time points. The data are expressed as mean7SEM (n ¼3, *Po0.05, **Po0.05, compared to the control group).
Military Medical University of Chinese PLA and approved by the local experimental ethics committee as well as the institutional animal care and use committee. All of them were kept in the same room under temperature-controlled conditions (humidity of 6075%, 2273 1C) with a 12-h lightdark cycle (the lights were on from 7 a.m. to 7 p.m.) and ad libitum access to water and food. A one-week acclimation period was used before animals were used in the experimental protocol.
4.2.
Global cerebral ischemia-reperfusion animal model
Global cerebral I/R animal model was induced using the fourvessel occlusion method (4-VO) in S–D rats, described by Pulsinelli and Brierley (1979). All rats were divided into three groups randomly: control group, I/R 0-h group, and I/R 24-h group, with eight animals in each group. The animals were anesthetized with 10% chloral hydrate at 300 mg/kg intraperitoneally. The alar foramina of first cervical vertebrae were
Fig. 3 – HT22 cells was cultured with 10% fetal bovine serum after incubation with 500 lM CoCl2 for 16 h to establish ischemia-reperfusion injury in vitro model. The bar chart demonstrates the relative ratio of Beclin-1 or p-mTOR to actin for each time point. (A) It was found that both Beclin-1 and p-mTOR were induced at early ischemia/reperfusion stages and showed the similar expression pattern. (B) Semiquantitative analysis (relative optical density) of the staining of Beclin-1 and p-mTOR at different reperfusion time points. The data are expressed as mean7SEM (n ¼ 3, *Po0.05, **Po0.05, compared to the control group).
exposed and the vertebral arteries were electrocauterized permanently. Then the common carotid arteries (CCAs) were dissected from surrounding tissues and temporarily ligated using the microvascular clamps for 20 min. At the end of the ischemic period, the microvascular clamps were removed and the skin incision was sutured. Body temperature was maintained at 3770.5 1C throughout the surgery by means of a heating blanket and a lamp. Rats were decapitated at 0 h and 24 h after reperfusion respectively and the brains were quickly taken out and stored at 80 1C. In the control group, the same procedures were performed without the 4-VO procedure.
4.3.
Focal cerebral ischemia-reperfusion animal model
Focal cerebral I/R was employed by MCAO in Balb/c mice, described by Longa et al. (1989). The mice were randomly divided into two groups: the control group and the experimental group with eight animals in each group. Briefly, animals were anesthetized with 10% chloral hydrate at
Please cite this article as: Yang, T., et al., Regulation on Beclin-1 expression by mTOR in CoCl2-induced HT22 cell ischemiareperfusion injury. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.04.016
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Fig. 4 – HT22 cells were exposed to the different concentrations of rapamycin (10 nM , 50 nM , 100 nM , 200 nM ). The bar chart demonstrates the relative ratio of Beclin-1 to actin for each time point. (A) Western blotting showed that the expression of Beclin-1 protein was significant increased 24 h after incubation with 50 nM rapamycin. (B) Relative optical density of the staining of Beclin-1 was induced by the different concentration of rampamycin. The data are expressed as mean7SEM (n ¼3, *Po0.05, compared with the control group).
The ischemic core and penumbra were dissected according to well-established protocols in rodent models of unilateral proximal MCAO, as described previously (Ashwal et al., 1998). In brief, each hemisphere was cut longitudinally, from dorsal to ventral 1.5 mm from the midline to exclude medial brain structures that were supplied primarily by the anterior cerebral artery. A transverse diagonal incision at approximately the “2 o’clock” position separated the core from the penumbra. The mice in control group underwent the same procedures except inserting the nylon sutures.
4.4.
Fig. 5 – HT22 cells were co-incubated by 50 nM rapamycin and 500 lM CoCl2. The bar chart demonstrates the relative ratio of Beclin-1 to actin for each time point. (A) Western blotting showed that the expression of Beclin-1 protein was significant increased after co-incubation with 50 nM rapamycin and 500 lM CoCl2 and maintained the increased Beclin-1 level in ischemic injury. (B) Semiquantitative analysis (relative optical density) of intensity of staining of Beclin-1 at different ischemia time points with 50 nM rapamycin. The data are expressed as mean7SEM (n ¼3, *Po0.05, compared with the control group).
300 mg/kg intraperitoneally. The right CCA, the external carotid artery (ECA), and the internal carotid artery (ICA) were exposed through a midline skin incision in the neck. A 4-0 monofilament nylon suture with a rounded tip (Beijing Sunbio Biotech, Beijing, China) was inserted into the ICA via ECA until a slight resistance was felt. One hour later, reperfusion was performed by slowly pulling the nylon suture back, and the wound was sutured thereafter. All the animals were allowed to recover until waken and were returned to their cages with optimum care. 24 h after I/R, the animals were decapitated and the penumbra and contralateral hemisphere brain tissue was collected for further experimentation.
Cell lines, chemicals and antibodies
Mouse hippocampal cell lines (HT22) were contributed from the Laboratory of Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University of Chinese PLA. Cobalt chloride (CoCl2), 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were purchased from Sigma (St Louis, MO, USA). Antibodies against phosphorylated mTOR, mTOR, Beclin-1 and Actin were purchased from Abcam (Cambridge, MA, USA). The following secondary antibodies were used: goat anti-rabbit Alexa 488, goat anti-mouse Alexa 568 (Invitrogen, Carlsbad, CA, USA).
4.5.
Cell cultures
HT22 cells were cultured in DMEM (Hyclone, USA) with 10% fetal bovine serum (Hyclone, USA) and were incubated in a humidified incubator with 5% CO2 at 37 1C, and were seeded on 60-mm culture dishes at 100,000 cells per dish. Cell density was maintained 80% or less confluency to attenuate excessive growth.
4.6.
In vitro ischemia and reperfusion treatment
CoCl2, a chemical hypoxia inducer (Ardyanto et al., 2006; Naves et al., 2013), was added into HT22 cells to develop the ischemia model. In the ischemia injury experimental groups, CoCl2 was treated with a final concentration of 500 mM in culture medium and HT22 cells were maintained for 0, 2, 4, 6, 8, 10, 12, 14, and 16 h respectively to simulate ischemic injury cell model. In the I/R injury experiments, HT22 cells were
Please cite this article as: Yang, T., et al., Regulation on Beclin-1 expression by mTOR in CoCl2-induced HT22 cell ischemiareperfusion injury. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.04.016
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Fig. 6 – Cell viability was investigated by MTT assay. (A, B) HT22 cells were exposed to 500 lM CoCl2 in culture medium to simulate ischemic injury cell model. The analysis showed that co-incubation with 50 nM rapamycin and 500 lM CoCl2 could accelerate the cell death in ischemic injury compared with the control group (*Po0.05, **Po0.05). C D HT22 cells were pretreated with DMSO, 50 nM rapamycin, 500 lM CoCl2 and co-incubation with 50 nM rapamycin and 500 lM CoCl2 for 16 h respectively and then re-incubated with 10% FBS medium for 10 and 14 h respectively to establish the ischemia-reperfusion injury cell models. The results showed that pre-treatment with rapamycin could protect HT22 cells from reperfusion injury compared with control group (*Po0.05, **Po0.05).
incubated with 500 mM CoCl2 for 16 h and removed the culture medium thereafter. Meanwhile, DMEM with 10% fetal bovine serum were added and maintained for 0, 2, 4, 6, 8, 10, 12, and 14 h respectively. While the cells in control group were treated with DMEM with 10% fetal bovine serum and were maintained in the same condition.
4.7.
MTT assay
Cell viability was investigated by measuring metabolism of MTT. MTT solution (5 mg/mL) was added to the culture medium, the pretreated HT22 cells were maintained for 4 h at 37 1C, and MTT solution were removed there. The solubilization solution containing 20% sodium dodecyl sulfate (pH
4.8) and 50% dimethylformamide was added and absorptions at 570 nM were measured. Cell viability was determined as percentage of living cells.
4.8.
Western blot analysis
The pretreated HT22 cells were washed once with ice-cold PBS and then were suspended in a lysis buffer (20 mM Tris– HCl, pH 7.5, 150 mM sodium chloride, 1% Triton, 1 mM EGTA, 1 mM EDTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 μg/ml leupeptin, 1 mM PMSF). After centrifugation at 10,000 g for 15 min, equal amounts of cellular protein lysates, which were determined by bicinchoninic acid protein assay (Pierce, Rockford, IL, USA),
Please cite this article as: Yang, T., et al., Regulation on Beclin-1 expression by mTOR in CoCl2-induced HT22 cell ischemiareperfusion injury. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.04.016
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were separated by SDS-PAGE and electrophoretically transferred to PVDF membranes (Millipore). Following treatment with 5% BSA at room temperature for 2 h, the membranes were probed with each antibody at 4 1C overnight followed by horseradish peroxidase conjugated anti-rabbit or mouse IgG secondary antibodies (Invitrogen, USA). Bound antibodies were visualized by chemiluminescence detection on autoradiographic films. Blots were stripped and reprobed with actin. Image J was used for Western blotting quantification.
4.9.
Statistical analysis
The quantitative data were expressed as the mean7SEM and statistically analyzed with one-way ANOVA followed by Q2 Tukey's post-hoc test using SPSS18.0. A P-value of less than 0.05 was considered significant.
Acknowledgments Q3 This work was sponsored by National Natural Science Foun-
dation of China (No. 81371349). The authors would like to thank Dr. Qian Yang (The Laboratory of Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University of Chinese PLA) for continuous scientific support to our work and all members of the Laboratory for helpful discussions. We declare that there is no conflict of interest.
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Heras-Sandoval, D., Perez-Rojas, J.M., Hernandez-Damian, J., PedrazaChaverri, J., 2014. The role of PI3K/AKT/mTOR pathway in the modulation of autophagy and the clearance of protein aggregates in neurodegeneration. Cell Signal. 26, 2694–2701. Inoki, K., 2014. mTOR signaling in autophagy regulation in the kidney. Semin. Nephrol. 34, 2–8. Kang, R., Zeh, H.J., Lotze, M.T., Tang, D., 2011. The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ. 18, 571–580. Kim, S.Y., Lee, D.H., Song, X., Bartlett, D.L., Kwon, Y.T., Lee, Y.J., 2014. Role of Bcl-xL/Beclin-1 in synergistic apoptotic effects of secretory TRAIL-armed adenovirus in combination with mitomycin C and hyperthermia on colon cancer cells. Apoptosis 19, 1603–1615. Kelekar, A., 2005. Autophagy. Ann. N. Y. Acad. Sci. 1066, 259–271. Lee, J.Y., He, Y., Sagher, O., Keep, R., Hua, Y., Xi, G., 2009. Activated autophagy pathway in experimental subarachnoid hemorrhage. Brain Res. 1287, 126–135. Liang, C., Feng, P., Ku, B., Dotan, I., Canaani, D., Oh, B.H., Jung, J.U., 2006. Autophagic and tumour suppressor activity of a novel Beclin1-binding protein UVRAG. Nat. Cell Biol. 8, 688–699. Longa, E.Z., Weinstein, P.R., Carlson, S., Cummins, R., 1989. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20, 84–91. Matsui, Y., Takagi, H., Qu, X., Abdellatif, M., Sakoda, H., Asano, T., Levine, B., Sadoshima, J., 2007. Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMPactivated protein kinase and Beclin 1 in mediating autophagy. Circ. Res. 100, 914–922. Moreno, S., Imbroglini, V., Ferraro, E., Bernardi, C., Romagnoli, A., Berrebi, A.S., Cecconi, F., 2006. Apoptosome impairment during development results in activation of an autophagy program in cerebral cortex. Apoptosis 11, 1595–1602. Nikoletopoulou, V., Markaki, M., Palikaras, K., Tavernarakis, N., 2013. Crosstalk between apoptosis, necrosis and autophagy. Biochim. Biophys. Acta 1833, 3448–3459. Naves, T., Jawhari, S., Jauberteau, M.O., Ratinaud, M.H., Verdier, M., 2013. Autophagy takes place in mutated p53 neuroblastoma cells in response to hypoxia mimetic CoCl(2). Biochem. Pharmacol. 85, 1153–1161. Pattingre, S., Espert, L., Biard-Piechaczyk, M., Codogno, P., 2008. Regulation of macroautophagy by mTOR and Beclin 1 complexes. Biochimie 90, 313–323. Pulsinelli, W.A., Brierley, J.B., 1979. A new model of bilateral hemispheric ischemia in the unanesthetized rat. Stroke 10, 267–272. Rami, A., Kogel, D., 2008. Apoptosis meets autophagy-like cell death in the ischemic penumbra: Two sides of the same coin? Autophagy 4, 422–426. Rami, A., 2008. Upregulation of Beclin 1 in the ischemic penumbra. Autophagy 4, 227–229. Sun, Q., Fan, W., Zhong, Q., 2009. Regulation of Beclin 1 in autophagy. Autophagy 5, 713–716. Sengupta, S., Peterson, T.R., Sabatini, D.M., 2010. Regulation of the mTOR complex 1 pathway by nutrients, growth factors, and stress. Mol. Cell. 40, 310–322. Takahashi, Y., Meyerkord, C.L., Wang, H.G., 2009. Bif-1/endophilin B1: a candidate for crescent driving force in autophagy. Cell Death Differ. 16, 947–955. Winslow, A.R., Rubinsztein, D.C., 2008. Autophagy in neurodegeneration and development. Biochim. Biophys. Acta 1782, 723–729. Xie, Z., Klionsky, D.J., 2007. Autophagosome formation: core machinery and adaptations. Nat. Cell Biol. 9, 1102–1109. Zhang, X.D., Wang, Y., Wu, J.C., Lin, F., Han, R., Han, F., Fukunaga, K., Qin, Z.H., 2009. Down-regulation of Bcl-2 enhances autophagy activation and cell death induced by mitochondrial dysfunction in rat striatum. J. Neurosci. Res. 87, 3600–3610.
Please cite this article as: Yang, T., et al., Regulation on Beclin-1 expression by mTOR in CoCl2-induced HT22 cell ischemiareperfusion injury. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.04.016
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Research Report
Regulation on Beclin-1 expression by mTOR in CoCl2induced HT22 cell ischemia-reperfusion injury Tao Yanga,b, Dongbo Lia,b, Feng Liua, Lei Qia, Ge Yana, Maode Wanga,n a
Department of Neurosurgery, First Affiliated Hospital of Xi’an Jiaotong University, 277 West Yanta West Road, Xi’an, Shaanxi 710061, PR China a Department of Neurosurgery, Ankang City Central Hospital, Ankang, Shaanxi 725000, PR China
art i cle i nfo
ab st rac t
Article history:
It has been reported that cerebral ischemia/reperfusion (I/R) injury can activate autophagy.
Accepted 9 April 2015
However, the role of autophagy in cerebral I/R injury remains controversy. Two major proteins, mTOR and Beclin-1, govern the formation of autophagosomes to regulate
Keywords:
autophagy activity. However, the cross-talking between Beclin-1 and mTOR in cerebral
Autophagy
I/R injury remains elusive. In this study, global cerebral I/R injury animal model and focal
Beclin-1
cerebral I/R injury animal model were induced to test the variation of Beclin-1 level in vivo.
mTOR
To further confirm the variation of Beclin-1 level and investigate the cross-talking between
Ischemia
Beclin-1 and mammalian target of rapamycin (mTOR) in I/R injury, we used cobalt chloride
Reperfusion
(CoCl2) to develop an I/R injury cell model in HT22 cell line. Our data showed that the levels of Beclin-1 and phosphorylated mammalian target of rapamycin (p-mTOR) were clearly induced by I/R injury in vitro. And the time course studies suggested that the Beclin-1 and mTOR may have coordinated regulation in ischemia stages but not in reperfusion stages. Moreover, inhibitor of mTOR could prevent Beclin-1 decreasing, but this prevention may play opposite roles in different stages of I/R injury. We conclude that this study represents a major advance in our understanding of the cross-talking of two key proteins, Beclin-1 and mTOR, in autophagy and the role of autophagy in cerebral I/R injury. & 2015 Published by Elsevier B.V.
1.
Introduction
Cerebral I/R injury is the third cause of death and results in permanent disability with extensive cerebral tissue damage. Although different mechanisms are involved in the pathogenesis of I/R, increasing evidence showed that autophagy may play a critical role in neuronal survival and death (Fulda, 2012). The role of autophagy in ischemic neurons remains
controversial (Codogno and Meijer, 2005; Rami and Kogel, 2008; Gabryel et al., 2012). Thus, the signaling pathways of autophagy activation would contribute to the understanding of the mechanisms of disease. Autophagy is required for all types of cell to maintain cellular homeostasis, and neurons are particularly sensitive to variation in autophagy activity. Autophagy is up-regulated and promotes cell survival in stress (Lee et al., 2009), which could
n
Corresponding author. E-mail address: [email protected] (M. Wang).
http://dx.doi.org/10.1016/j.brainres.2015.04.016 0006-8993/& 2015 Published by Elsevier B.V.
Please cite this article as: Yang, T., et al., Regulation on Beclin-1 expression by mTOR in CoCl2-induced HT22 cell ischemiareperfusion injury. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.04.016
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also induce cell death directly or indirectly via various physiological processes (Fan et al., 2014). Two major protein complexes, mTOR (Heras-Sandoval et al., 2014; Inoki, 2014) complex 1 and the Beclin-1 (Sun et al., 2009) complex, govern the formation of autophagosomes to regulate autophagy activity. The studies have shown that TOR may be involved in balancing macroautophagic and microautophagic activities (Dubouloz et al., 2005) and signaling pathways that activate mTOR could inhibit autophagy, whereas signaling pathways that inhibit TOR stimulate autophagy (Codogno and Meijer, 2005; Arsham and Neufeld, 2006). In contrast, the Beclin-1 (Atg6) complex, which is involved in the initial step of autophagosome formation, is to stimulate autophagy. Beclin-1 is part of a multiprotein complex, and acts as a platform, recruiting an activator (UVRAG) and a repressor (Bcl-2) of Beclin 1/hVps34-dependent autophagy (Pattingre et al., 2008). However, it is still unknown that how autophagy is regulated in neuronal survival and death and whether the two core complexes of autophagy coordinates the regulations between each other in response to stress. In this study we investigated the variation of Beclin-1 level and crosstalking between Beclin-1 and mTOR, the two core proteins in autophagy formation, in models of in vivo and in vitro of cerebral I/R injury.
2.
Results
2.1.
The level of Beclin-1 in focal/global cerebral I/R injury
The western blotting showed that the level of Beclin-1 was significantly induced and showed significant increase after 24 h of global cerebral I/R injury models compared to the control group (Fig 1A and C, Po0.05). To further conform the finding in global cerebral I/R injury, we performed a middle cerebral artery occlusion (MCAO) model in mice and detected the changes of Beclin-1. The penumbra and contralateral hemisphere were separated 24 h after I/R. The data showed that the levels of Beclin-1 were clearly increased in the penumbra and contralateral hemisphere (Fig 1, B and D, Po0.05).
2.2. The dynamic change of Beclin-1 in CoCl2-induced I/R injury Western blotting was performed to test the expression of Beclin-1 and mTOR/p-mTOR. The analysis showed that Beclin-1 was induced gradually and reached its peak 10 h after CoCl2 treatment. The phosphorylation of mTOR is important for induction of mTOR activity and increasing pmTOR indicated decline in autophagy activity. We examined the p-mTOR level in HT22 cells at different time points of CoCl2 treatment. The results showed that the p-mTOR was induced after treatment for 10 h, while the increasing Beclin1 began to decline (Fig. 2, A and B, Po0.05). We treated HT22 cells for 16 h using 500 mM CoCl2 and then re-incubated the cells with 10% FBS medium to establish the I/R injury cell model. The cells were collected at different time points to test the variation of Beclin-1 and p-mTOR. The data indicated that both Beclin-1 and p-mTOR were induced at early I/R stages and showed the similar expression patterns (Fig. 3, A and B, Po0.05).
2.3. Regulation of rapamycin on Beclin-1 in CoCl2-induced ischemia injury Rapamycin is widely used as inhibitor of mTOR activity and inducer of autophagy. We chose rapamycin to test the coordination between Beclin-1 and mTOR. The treatment of rapamycin clearly induced Beclin-1 under normal condition (Fig. 4, A and B, Po0.05). The HT22 cells were treated by coincubation with 50 nM rapamycin and 500 mM CoCl2 for different time points. The data showed that the expression of Beclin-1 protein was significant increased and maintained the increased level in ischemia injury (Fig. 5, A and B, Po0.05).
2.4. The effect of rapamycin on neuronal death in CoCl2induced I/R injury To test the effect of autophagy activity on neuronal survival and death, we carried out MTT assay in CoCl2-induced I/R injury. HT22 cells were treated the same as above. Both ischemia and reperfusion significantly induced cell death. The analyses further showed that co-incubation with 50 nM rapamycin and 500 mM CoCl2 could accelerate cell death in ischemic injury (Fig. 6, A and B, Po0.05), but clearly protect the cells against the injury induced by I/R (Fig. 6, C and D, Po0.05).
3.
Discussion
Autophagy is a self-eating catabolic process to break down and recycle proteins, cytoplasmic components, and damaged organelles in order to maintain cellular homeostasis. Autophagy includes several mechanisms such as macroautophagy, microautophagy, and chaperone-mediated autophagy. Macroautophagy, referred to autophagy, is responsible for the clearance of proteins, organelles, and protein aggregates in response to stress. The autophagy pathway is divided into four distinct steps: initiation/nucleation, autophagosome formation, trafficking/ maturation, and recycling/release of macromolecules. Both the evolutionarily conserved Target of rapamycin kinase complex 1 and the Beclin-1 complex play important roles in the control of autophagy. The TOR complex 1 (TORC1) could inhibit autophagy by regulating the Atg1 complex (Sengupta et al., 2010). In contrast, the Beclin-1 (Atg6) complex, which is involved in the initial step of autophagosome formation, is to stimulate autophagy (Xie and Klionsky, 2007). The studies showed that Beclin-1 induces autophagy by regulation on the lipid kinase Vps-34 protein (Kang et al., 2011). Many other proteins such as Atg14 (Fogel et al., 2013), UVRAG (Liang et al., 2006) and Bif-1/endophilin B1 (Takahashi et al., 2009) induce or block autophagy by interacting with Beclin-1. Some antiapoptotic family members such as Bcl-2 (Ciechomska et al., 2009) and Bcl-XL (Kim et al., 2014) regulate autophagy by interaction with BH3 domain of Beclin-1. And caspase-mediated cleavage of Beclin-1 promotes cross-talk between apoptosis and autophagy. In neurons, autophagy is classified into “basal” and “induced” depending on its roles. In normal conditions, neurons display low levels of autophagosomes. Pathological conditions such as stress or injury could induce autophagy activity. During autophagy induction, neurons undergo a series changes to up-regulate
Please cite this article as: Yang, T., et al., Regulation on Beclin-1 expression by mTOR in CoCl2-induced HT22 cell ischemiareperfusion injury. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.04.016
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Fig. 1 – Western blotting showed that the expression of Beclin-1 protein was significant increased in focal/global cerebral ischemia-reperfusion injury. The bar chart demonstrates the ratio of Beclin-1 relative to actin for each time point. (A) High protein level of Beclin-1 was observed in global cerebral ischemia-reperfusion in rats at the time points of 0 h and 24 h compared to the sham control group respectively. (B) Focal cerebral ischemia-reperfusion mouse models showed that high level of Beclin-1 protein was examined in the penumbra and contralateral hemisphere compared with the control group. (C, D) Semiquantitative analysis (relative optical density) of intensity of staining of Beclin-1 to actin in global cerebral ischemiareperfusion at the time points of 0 h and 24 h and in the penumbra and contralateral hemisphere of focal cerebral ischemiareperfusion injury for each time point. The data are expressed as mean7SEM (n¼ 3, *Po0.05, **Po0.05, compared to the control group). basal level to induced level. Many studies showed that autophagy is controlled by a few key regulators such as Beclin-1, serine-threonine protein and AMP activated kinase (AMPK) (Kelekar, 2005). Beclin-1, the mammalian orthologue of yeast Atg6, has a central role in autophagy and cell survival. During periods of cell stress, Beclin-1 is increased and forms Beclin-1Vps34 complexes by interacting with cofactors, and then induce autophagy (Funderburk et al., 2010). The studies found that Beclin-1 is related to cellular biological functions and many diseases including heart disease (Matsui et al., 2007), development, and neurodegeneration (Winslow and Rubinsztein, 2008). However, the precise functions and mechanisms of Beclin-1 in these physiological processes still have not been elucidated. Binding with Bcl-2 or other autophagy partners provide potential insight into functions of Beclin-1 in different physiological processes in autophagy and in processes independent of autophagy. Cerebral injury caused by I/R are the consequence of a massive cell death. However, the mechanism of I/R cell death are more complicated. In different models of I/R, autophagy activity was induced in cerebral cortex (Moreno et al., 2006), hippocampus (Rami, 2008), and striatum (Zhang et al., 2009) of animals. Whether I/R-induced autophagy contributes to neuronal death or provide a neuroprotection is still controversy. Studies suggested detrimental role of autophagy might be associated with autophagy-necrosis cross-talk (Nikoletopoulou et al., 2013). On the other hand, increasing of Beclin-1 expression and rapamycin had a long-lasting protective effect during ischemic procedure. In current study, we investigated the level
of Beclin-1 during different stages of I/R injury models. Our data showed that Beclin-1 expression was induced earlier than pmTOR and damping of Beclin-1 was accompanied with gradually increasing of p-mTOR during ischemic injury. Inhibition of mTOR could promote Beclin-1 expression and prevent the decreasing of Beclin-1 in later stage of ischemia. Those finding suggested that two key proteins for activating autophagy via different signal pathways may have cross-talking in ischemia stage. Blocking mTOR activity may contribute to Beclin-1 expression which involves in induction of autophagy. In reperfusion injury, our data showed that there was no obvious coordinated regulation between Beclin-1 expression and pmTOR. Furthermore, the analysis on cells survival and death showed activated autophagy could accelerate the cell death in ischemia stage, but promote neurons survival in the reperfusion stage. This indicated that autophagy may play distinct roles in different stages of ischemia or reperfusion injury.
4.
Experimental procedures
4.1.
Experimental animals
To confirm the level of Beclin-1 in cerebral I/R injury, two different animal models were established. Adult Sprague– Dawley (S–D) rats (male, 250–280 g) and adult Balb/c mice (male, 20–28 g) were used to establish the global and focal cerebral I/R animal models respectively. All animals were provided by the Experiment Animal Center of the Fourth
Please cite this article as: Yang, T., et al., Regulation on Beclin-1 expression by mTOR in CoCl2-induced HT22 cell ischemiareperfusion injury. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.04.016
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Fig. 2 – Cerebral ischemia injury was simulated by 500 lM CoCl2 in HT22 cells. The bar chart demonstrates the relative ratio of Beclin-1 or p-mTOR to actin at each time point. (A) The expression of Beclin-1 began to increase at 4 h after incubation with 500 lM CoCl2 and a distinct peak was observed 10 h after incubation, then it began to decline. However, the expression of p-mTOR has been induced after treatment for 10 h, while the increased Beclin-1 began to decline. (B) Semiquantitative analysis (relative optical density) of the staining of Beclin-1 and p-mTOR at different ischemia time points. The data are expressed as mean7SEM (n ¼3, *Po0.05, **Po0.05, compared to the control group).
Military Medical University of Chinese PLA and approved by the local experimental ethics committee as well as the institutional animal care and use committee. All of them were kept in the same room under temperature-controlled conditions (humidity of 6075%, 2273 1C) with a 12-h lightdark cycle (the lights were on from 7 a.m. to 7 p.m.) and ad libitum access to water and food. A one-week acclimation period was used before animals were used in the experimental protocol.
4.2.
Global cerebral ischemia-reperfusion animal model
Global cerebral I/R animal model was induced using the fourvessel occlusion method (4-VO) in S–D rats, described by Pulsinelli and Brierley (1979). All rats were divided into three groups randomly: control group, I/R 0-h group, and I/R 24-h group, with eight animals in each group. The animals were anesthetized with 10% chloral hydrate at 300 mg/kg intraperitoneally. The alar foramina of first cervical vertebrae were
Fig. 3 – HT22 cells was cultured with 10% fetal bovine serum after incubation with 500 lM CoCl2 for 16 h to establish ischemia-reperfusion injury in vitro model. The bar chart demonstrates the relative ratio of Beclin-1 or p-mTOR to actin for each time point. (A) It was found that both Beclin-1 and p-mTOR were induced at early ischemia/reperfusion stages and showed the similar expression pattern. (B) Semiquantitative analysis (relative optical density) of the staining of Beclin-1 and p-mTOR at different reperfusion time points. The data are expressed as mean7SEM (n ¼ 3, *Po0.05, **Po0.05, compared to the control group).
exposed and the vertebral arteries were electrocauterized permanently. Then the common carotid arteries (CCAs) were dissected from surrounding tissues and temporarily ligated using the microvascular clamps for 20 min. At the end of the ischemic period, the microvascular clamps were removed and the skin incision was sutured. Body temperature was maintained at 3770.5 1C throughout the surgery by means of a heating blanket and a lamp. Rats were decapitated at 0 h and 24 h after reperfusion respectively and the brains were quickly taken out and stored at 80 1C. In the control group, the same procedures were performed without the 4-VO procedure.
4.3.
Focal cerebral ischemia-reperfusion animal model
Focal cerebral I/R was employed by MCAO in Balb/c mice, described by Longa et al. (1989). The mice were randomly divided into two groups: the control group and the experimental group with eight animals in each group. Briefly, animals were anesthetized with 10% chloral hydrate at
Please cite this article as: Yang, T., et al., Regulation on Beclin-1 expression by mTOR in CoCl2-induced HT22 cell ischemiareperfusion injury. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.04.016
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Fig. 4 – HT22 cells were exposed to the different concentrations of rapamycin (10 nM , 50 nM , 100 nM , 200 nM ). The bar chart demonstrates the relative ratio of Beclin-1 to actin for each time point. (A) Western blotting showed that the expression of Beclin-1 protein was significant increased 24 h after incubation with 50 nM rapamycin. (B) Relative optical density of the staining of Beclin-1 was induced by the different concentration of rampamycin. The data are expressed as mean7SEM (n ¼3, *Po0.05, compared with the control group).
The ischemic core and penumbra were dissected according to well-established protocols in rodent models of unilateral proximal MCAO, as described previously (Ashwal et al., 1998). In brief, each hemisphere was cut longitudinally, from dorsal to ventral 1.5 mm from the midline to exclude medial brain structures that were supplied primarily by the anterior cerebral artery. A transverse diagonal incision at approximately the “2 o’clock” position separated the core from the penumbra. The mice in control group underwent the same procedures except inserting the nylon sutures.
4.4.
Fig. 5 – HT22 cells were co-incubated by 50 nM rapamycin and 500 lM CoCl2. The bar chart demonstrates the relative ratio of Beclin-1 to actin for each time point. (A) Western blotting showed that the expression of Beclin-1 protein was significant increased after co-incubation with 50 nM rapamycin and 500 lM CoCl2 and maintained the increased Beclin-1 level in ischemic injury. (B) Semiquantitative analysis (relative optical density) of intensity of staining of Beclin-1 at different ischemia time points with 50 nM rapamycin. The data are expressed as mean7SEM (n ¼3, *Po0.05, compared with the control group).
300 mg/kg intraperitoneally. The right CCA, the external carotid artery (ECA), and the internal carotid artery (ICA) were exposed through a midline skin incision in the neck. A 4-0 monofilament nylon suture with a rounded tip (Beijing Sunbio Biotech, Beijing, China) was inserted into the ICA via ECA until a slight resistance was felt. One hour later, reperfusion was performed by slowly pulling the nylon suture back, and the wound was sutured thereafter. All the animals were allowed to recover until waken and were returned to their cages with optimum care. 24 h after I/R, the animals were decapitated and the penumbra and contralateral hemisphere brain tissue was collected for further experimentation.
Cell lines, chemicals and antibodies
Mouse hippocampal cell lines (HT22) were contributed from the Laboratory of Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University of Chinese PLA. Cobalt chloride (CoCl2), 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were purchased from Sigma (St Louis, MO, USA). Antibodies against phosphorylated mTOR, mTOR, Beclin-1 and Actin were purchased from Abcam (Cambridge, MA, USA). The following secondary antibodies were used: goat anti-rabbit Alexa 488, goat anti-mouse Alexa 568 (Invitrogen, Carlsbad, CA, USA).
4.5.
Cell cultures
HT22 cells were cultured in DMEM (Hyclone, USA) with 10% fetal bovine serum (Hyclone, USA) and were incubated in a humidified incubator with 5% CO2 at 37 1C, and were seeded on 60-mm culture dishes at 100,000 cells per dish. Cell density was maintained 80% or less confluency to attenuate excessive growth.
4.6.
In vitro ischemia and reperfusion treatment
CoCl2, a chemical hypoxia inducer (Ardyanto et al., 2006; Naves et al., 2013), was added into HT22 cells to develop the ischemia model. In the ischemia injury experimental groups, CoCl2 was treated with a final concentration of 500 mM in culture medium and HT22 cells were maintained for 0, 2, 4, 6, 8, 10, 12, 14, and 16 h respectively to simulate ischemic injury cell model. In the I/R injury experiments, HT22 cells were
Please cite this article as: Yang, T., et al., Regulation on Beclin-1 expression by mTOR in CoCl2-induced HT22 cell ischemiareperfusion injury. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.04.016
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Fig. 6 – Cell viability was investigated by MTT assay. (A, B) HT22 cells were exposed to 500 lM CoCl2 in culture medium to simulate ischemic injury cell model. The analysis showed that co-incubation with 50 nM rapamycin and 500 lM CoCl2 could accelerate the cell death in ischemic injury compared with the control group (*Po0.05, **Po0.05). C D HT22 cells were pretreated with DMSO, 50 nM rapamycin, 500 lM CoCl2 and co-incubation with 50 nM rapamycin and 500 lM CoCl2 for 16 h respectively and then re-incubated with 10% FBS medium for 10 and 14 h respectively to establish the ischemia-reperfusion injury cell models. The results showed that pre-treatment with rapamycin could protect HT22 cells from reperfusion injury compared with control group (*Po0.05, **Po0.05).
incubated with 500 mM CoCl2 for 16 h and removed the culture medium thereafter. Meanwhile, DMEM with 10% fetal bovine serum were added and maintained for 0, 2, 4, 6, 8, 10, 12, and 14 h respectively. While the cells in control group were treated with DMEM with 10% fetal bovine serum and were maintained in the same condition.
4.7.
MTT assay
Cell viability was investigated by measuring metabolism of MTT. MTT solution (5 mg/mL) was added to the culture medium, the pretreated HT22 cells were maintained for 4 h at 37 1C, and MTT solution were removed there. The solubilization solution containing 20% sodium dodecyl sulfate (pH
4.8) and 50% dimethylformamide was added and absorptions at 570 nM were measured. Cell viability was determined as percentage of living cells.
4.8.
Western blot analysis
The pretreated HT22 cells were washed once with ice-cold PBS and then were suspended in a lysis buffer (20 mM Tris– HCl, pH 7.5, 150 mM sodium chloride, 1% Triton, 1 mM EGTA, 1 mM EDTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 μg/ml leupeptin, 1 mM PMSF). After centrifugation at 10,000 g for 15 min, equal amounts of cellular protein lysates, which were determined by bicinchoninic acid protein assay (Pierce, Rockford, IL, USA),
Please cite this article as: Yang, T., et al., Regulation on Beclin-1 expression by mTOR in CoCl2-induced HT22 cell ischemiareperfusion injury. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.04.016
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were separated by SDS-PAGE and electrophoretically transferred to PVDF membranes (Millipore). Following treatment with 5% BSA at room temperature for 2 h, the membranes were probed with each antibody at 4 1C overnight followed by horseradish peroxidase conjugated anti-rabbit or mouse IgG secondary antibodies (Invitrogen, USA). Bound antibodies were visualized by chemiluminescence detection on autoradiographic films. Blots were stripped and reprobed with actin. Image J was used for Western blotting quantification.
4.9.
Statistical analysis
The quantitative data were expressed as the mean7SEM and statistically analyzed with one-way ANOVA followed by Q2 Tukey's post-hoc test using SPSS18.0. A P-value of less than 0.05 was considered significant.
Acknowledgments Q3 This work was sponsored by National Natural Science Foun-
dation of China (No. 81371349). The authors would like to thank Dr. Qian Yang (The Laboratory of Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University of Chinese PLA) for continuous scientific support to our work and all members of the Laboratory for helpful discussions. We declare that there is no conflict of interest.
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Please cite this article as: Yang, T., et al., Regulation on Beclin-1 expression by mTOR in CoCl2-induced HT22 cell ischemiareperfusion injury. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.04.016
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