Inhibition of mTOR pathway restrains astrocyte proliferation, migration and production of inflammatory mediators after oxygen–glucose deprivation and reoxygenation

Neurochemistry International 83-84 (2015) 9–18

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Neurochemistry International j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / n c i

Inhibition of mTOR pathway restrains astrocyte proliferation, migration and production of inflammatory mediators after oxygen– glucose deprivation and reoxygenation Chun-Yu Li, Xiao Li, Shuang-Feng Liu, Wen-Sheng Qu, Wei Wang, Dai-Shi Tian * Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China

A R T I C L E

I N F O

Article history: Received 11 November 2014 Received in revised form 2 March 2015 Accepted 5 March 2015 Available online 10 March 2015 Keywords: Mammalian target of rapamycin Reactive astrogliosis Oxygen–glucose deprivation Proliferation Migration Inflammation mediator

A B S T R A C T

Glial scar is a major impediment to axonal regeneration in central nervous system (CNS) disorders. Overcoming this physical and biochemical barrier might be crucial for axonal regeneration and functional compensation during the progression of CNS disorders. The mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase, involved in process of cell proliferation, migration, autophagy and protein synthesis. Rapamycin, an inhibitor of mTOR signaling, can exert neuroprotective effects in several CNS diseases. However, its role in the process of reactive astrogliosis including cell proliferation, migration and cytokine production after cerebral ischemia still remains largely unknown. In this study, we investigated the effects of mTOR blockade in cultured astrocytes exposed to oxygen– glucose deprivation/reoxygenation (OGD/R), a wildly used cellular ischemia model which mimics ideally cerebral ischemia model in vivo. We found that astrocytes became activated after OGD/R, characterized by change of astrocytic morphology, upregulation of GFAP expression, the increase number of Edu positive cells, and accompanied with phosphorylation of mTOR protein and its substrate S6K1. Rapamycin significantly inhibited mTOR signal pathway, suppressed proliferation of astrocytes via modulation of cell cycle progression. Moreover, rapamycin attenuated astrocytic migration and mitigated production of inflammatory factors such as TNF-α and iNOS induced by astrocytes exposed to OGD/R. Taken together, our findings indicated that mTOR blockade by rapamycin attenuates astrocyte migration, proliferation and production of inflammation mediators. We suggest that targeting mTOR pathway in astrocyte activation may represent a potentially new therapeutic strategy against deleterious neurotoxic processes of reactive astrogliosis in CNS disorders such as ischemic stroke. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Astrocytes, the most abundant glial subtype in the central nervous system, undertake a lot of functions, including canvassing neurons and communicating with other neural cell types. In several pathologic conditions, astrocytes proximal to lesion zones respond by a process commonly referred to as reactive astrogliosis (Binder and Steinhauser, 2006; Fuller et al., 2009; Holley et al., 2003; Yang et al., 2011). Reactive astrogliosis that involves cell division and up-regulation of cytoskeletal proteins contributes to formation of

Abbreviations: EdU, 5-ethynyl-29-deoxyuridine; mTOR, mammalian target of rapamycin; p-mTOR, phosphorylated mammalian target of rapamycin; OGD/R, oxygen–glucose deprivation and reoxygenation; TNF-α, tumor necrosis factor-α; iNOS, inducible nitric oxide synthase; GFAP, glial fibrillary acidic protein; DAPI, 4,6diamidino-2-phenylindole; PBS, phosphate-buffered saline. * Corresponding author. Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. Tel.: +86 27 83663337; fax: +86 27 83663337. E-mail address: [email protected] (D.-S. Tian). http://dx.doi.org/10.1016/j.neuint.2015.03.001 0197-0186/© 2015 Elsevier Ltd. All rights reserved.

a glial scar at lesion site (Mori et al., 2008; Zhu et al., 2007). Astrocytes that have migrated from distal areas to the lesion site may serve as another cellular source of glial scar (Cai et al., 2003; Saadoun et al., 2005). Although glial scar can exert beneficial effects in the acute phase of injury through minimizing the spread of neuroinflammation, protecting nerve from secondary damages, and restoring tissue integrity (Rolls et al., 2009; Silver and Miller, 2004), it also represents a physical and biochemical barrier in chronic phase that inhibits axonal elongation and synaptogenesis, thus hinders functional recovery (Silver and Miller, 2004; Sofroniew, 2009). In this regard, manipulation of glial scar may create a favorable environment for neuronal regeneration and emerge as a promising intervention to attenuate reactive astrogliosis-related damage in neurological disorders, such as ischemic stroke and spinal cord injury. The mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase that controls cell proliferation and metabolism in response to a diverse range of extracellular stimuli such as availability of nutrients, growth factors, mitogens, hormones and stress (Dazert and Hall, 2011; Laplante and Sabatini,

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2012). The best characterized downstream effectors of mTOR include two signaling pathways: the 70-kDa ribosomal protein S6 kinase 1 (p70S6K or S6K1) pathway and the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1, which are key regulators of protein translation in parallel (Fingar et al., 2004). As a central controller of growth and metabolism, mTOR pathway is implicated in many diseases, including cancer, neurodegeneration, and diabetes (Dazert and Hall, 2011). In some cancer types, mTOR signaling is dysregulated by oncogenes and tumor suppressors commonly mutated (Shaw and Cantley, 2006). And mTOR signaling is linked to several neurodegenerative diseases via its role in autophagy such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease (Garelick and Kennedy, 2011; Kruman, 2004). Rapamycin, a macrolide antibiotic, is a potent inhibitor of mTOR as well. It prevents mTOR from further phosphorylation of P70S6K, 4EBP1, thus, indirectly, decreases the proteins involved in transcription and translation of this signaling. Now, a body of studies has found that rapamycin has anti-proliferation and anti-migration properties in many types of cells (Jin et al., 2013; Liu et al., 2010; Molhoek et al., 2005). In this study, we aimed to investigate the effects of mTOR inhibition by rapamycin on astrocyte activation after OGD/R. We used astrocytes exposed to OGD/R to imitate model in vivo ischemia and determined the role of mTOR signaling in astrocytic proliferation, migration and production of inflammation mediators. We found that inhibition of mTOR pathway by rapamycin decreased astrocyte proliferation and migration capacities, which was accompanied by downregulation of TNF-α and iNOS expression. Based on these results, we proposed that targeting mTOR cascades in astrocytes may provide an underlying new therapeutic strategy against formation of glial scar after CNS disorders.

2.2. Immunocytochemistry staining Fixed cells were blocked with 5% BSA/PBS at room temperature for 1 h, incubated simultaneously with mouse anti-GFAP (1:200; Cell Signaling Technology, Beverly, MA, USA) and rabbit anti-p-mTOR (1:50; ser 2448; Cell Signaling Technology, Beverly, MA, USA) at 4 °C overnight. Cells were then washed extensively with phosphatebuffered saline (PBS) and subsequently incubated with corresponding secondary antibody (cyanine-3-conjugated anti-rabbit and FITCconjugated anti-mouse IgG; Jackson Immuno-Research, West Grove, PA, USA) for 1 h at room temperature. Finally, cell nuclei were stained with DAPI for 10 min. Fluorescent images were captured with an Olympus BX-51 fluorescence microscope (Olympus, Tokyo, Japan). 2.3. Cell proliferation and survival assays To assess cell proliferation, cells were plated at a density of 2500 viable cells per well of a 96-well plate and incubated in DMEM containing 10% FBS with 1 nM, 10 nM, 100 nM rapamycin (dissolved in 0.05%, DMSO, Sigma). The culture medium (with or without rapamycin) was replaced every 24 h. At d0, d1, d2, d3 and d4, 10 μL MTT (5 mg/mL; Sigma) was added to each well. Four hours after that, 100 μL/well solution was added to dissolve the sediment. After an overnight incubation, the absorbance of soluble formazan in wells was measured at a 562 nm wavelength with 491 nm as a reference. Each data point was obtained as an average of five values from five wells. The proliferation rate was investigated by a lactate dehydrogenase (LDH) release assay. LDH release was quantified with use of a CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI) according to the manufacturer’s instructions. 2.4. EdU incorporation assay

2. Materials and methods 2.1. Primary cell culture and OGD/R model Primary astrocytes were obtained from the neonatal rat’s cerebral cortices following the method described previously (Zhu et al., 2007). In brief, cortical hemispheres of 2-day-old rats were dissociated with 0.25% trypsin in phosphate-buffered saline. Cells were placed on poly-L-lysine-coated plastic flasks and maintained in Dulbecco’s modified Eagle medium: nutrient mixture F-12 (DMEM/F-12) supplemented with 15% fetal bovine serum (HyClone, Logan, UT, USA), at 37 °C in a humidified incubator containing 5% CO2 (Thermo Forma, Marietta, OH, USA). Cells were maintained in complete culture medium for 7–8 days. Prior to experimental treatments, astrocyte cultures were passaged twice. The purity of astrocyte cultures was determined with immunocytochemical staining using antibody against glial fibrillary acidic protein (GFAP, 1:200, Cell Signaling Technology), and 4, 6-diamidino-2-phenylindole (DAPI 10 μg/mL, Sigma), which indicated that more than 98% of cells were GFAP-positive astrocytes. The processing of OGD/R was described in detail by our previous study (Yang et al., 2011). OGD/R model was used to model-ischemia in vitro, without influencing the cell apoptosis in this study. Briefly, the standard culture medium was replaced with a glucose-free DMEM buffer (Gibco, Rockville, MD, USA), and cells were then placed in a hypoxic humidified incubator flushed with a gas mixture of 93% N2/5% CO2/2% O2 (model 3130, with the gas guards for CO2 and LN2 tanks; Thermo Forma, Marietta, OH, USA). Two hours later, cells were cultured in standard medium and normoxia condition for reoxygenation. Rapamycin (10 nM, Tocris) was added to cell cultures 1 h before OGD, whereas control cultures received only 0.05% DMSO. The drug was left in the culture medium during the entire experiment.

Cell proliferation was measured by 5-ethynyl-29-deoxyuridine (EdU) assay using an EdU assay kit (Ribobio, Guangzhou, China) according to the manufacturer’s instructions. EdU (50 μM) was added to the culture medium and then incubated for another 6 h. After that, the cells were fixed with 100% methanol for 15 min at room temperature and then treated with 0.5% Triton X-100 for 20 min at room temperature for permeabilization. After washed with PBS, immunostaining with anti-EdU working solution was performed at room temperature for 30 min. Following a wash with 0.5% TritonX-100 in PBS, the cells were incubated with the Hoechst 33342 dye at room temperature for 30 min. The staining results were photographed with a fluorescence microscope (Olympus, BX51). 2.5. Cell cycle analysis Cell cycle analysis was performed by propidium iodide (PI) staining. Astrocytes were trypsinized and washed with ice-cold PBS (pH 7.4) and fixed in ice-cold 70% ethanol. The cells were then washed with PBS, treated with 500 U/mL RNase (Sigma) at 37 °C for 30 min, and finally stained with propidium iodide (50 μg/mL, Sigma) in PBS. Ten thousand cells were counted for each data point. Cell cycle analysis was performed using a Becton Dickinson (Mountain View, CA, USA). 2.6. Cell migration assay Cell migration assays were exercised using fibronectin-coated polycarbonate filters (8 μm pore size, Transwell; Becton Dickinson, USA). Astrocytes were trypsinized and pre-incubated in suspension in serum-free medium with or without 10 nM rapamycin for 30 min. Two hundred thousand cells/wells containing 1% FBS medium were added to the top compartment. The lower chambers were filled with 600 μL of DMEM supplemented with 1% FBS for control group,

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600 μL of DMEM supplemented with 10% FBS and 0.05% DMSO for vehicle-control group, and 600 μL of DMEM supplemented with 10% FBS and 10 nM rapamycin for drug group. The astrocytes were allowed to migrate for 10 h, and then the filters were washed with PBS and the cells from the top surface of the filters were removed with a cotton swab. The filters were then fixed with ice-cold methanol and cell nuclei were stained with DAPI. The filters with nuclei of cells that had migrated to the bottom surface were photographed under an inverted fluorescence microscope. 2.7. Scratch wound assay Astrocytes were grown to confluence in 6 well plates, then were serum starved (1% FBS; 24 h) to be synchronized. The monolayers of astrocytes were wounded by dragging a sterile 200 μL pipette tip across the surface. Immediately, the detached cells and debris were washed out with PBS three times. Cells were then maintained for an additional 24 h in culture medium that was supplemented with 1% FBS and either 10 nM rapamycin or 0.05% DMSO. The images of the closing wound were acquired by inverted microscopy and analyzed using Image-Pro Plus.

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marker GFAP. We found that astrocytes in the control group were quiescent and slim in shape. After OGD/R, astrocytes became activated and presented obvious morphological changes, becoming hypertrophic with high level expression of GFAP. In addition, the hypertrophic astrocytes expressed GFAP immunoreactivity synchronously with p-mTOR expression, indicating that activation of astrocytes was accompanied with mTOR phosphorylation after OGD/R. However, incubation with rapamycin (10 nM) during OGD/R significantly inhibited phosphorylation of mTOR (Fig. 1B). Western blot analysis was also used to detect the phosphorylation of mTOR and its downstream target p70S6K at different time points. The results showed that mTOR was phosphorylated immediately after OGD/R, increased subsequently and reached its peak at 12 h, then decreased mildly at 24 h (Fig. 2A and B). In consistent with mTOR phosphorylation, the phosphorylation of its substrate p70S6K was up-regulated promptly after OGD/R (Fig. 2A and C). Both OGD/Rinduced increases in phosphorylated mTOR and p70S6K could be remarkably reduced by rapamycin, suggesting that rapamycin could effectively inhibit the OGD/R-induced activation of mTOR signaling pathway (Fig. 2A–C). 3.2. mTOR blockade attenuated astrocytic proliferation after OGD/R

2.8. Western blot analysis and ELISA

All data were given as mean values ± SEM. Statistical analysis was evaluated by Student’s t-test or one-way ANOVA followed by Tukey’s post hoc test. Significance was accepted at p < 0.05.

To determine the effect of mTOR inhibition on cell cycle arrest of astrocytes, cytometric cell cycle analysis was used by fluorescenceactivated cell sorting (FACS) technique. At the early stage of OGD/R (3 h), the percentage of astrocytes in S phase in OGD/R group was similar with the control group and there was no significant difference between OGD/R group and rapamycin-treated group. Beginning at 6 h and peaking at 12 h, the percentage of cells in the S stage increased significantly in the OGD/R group and rapamycin treatment markedly decreased the percentage of S stage cells compared with the OGD/R group. At 24 h, the percentage of cells in S stage returned to control levels (Fig. 3A, B). Together, these results showed that pretreatment with rapamycin attenuated the cell number of S stage, indicating that mTOR pathway contributes to astrocyte proliferation. MTT assay was also used to test the role of mTOR pathway in astrocytic proliferation. Compared with the control group, cell numbers of rapamycin-treated group significantly decreased, with 10 nM rapamycin as the optimal dose (Fig. 3C). We then investigated whether the reduction of astrocytic cell number by the LDH release assay was attributable to cell apoptosis or necrosis caused by rapamycin. Astrocyte incubation with rapamycin at 10 nM concentration or DMSO did not significantly differ in mean LDH activity (Fig. 3D), indicating that rapamycin did not exert a cytotoxic effect on astrocytes at low concentration. EdU, a marker of dividing cells, was also used in the present study to detect cell proliferation of astrocytes. At OGD/R 6 h, there was no significant difference in the percentage of EdU (+) astrocytes among the control group, the OGD/R group and rapamycin-treated group. However, at 12 h, EdU (+) cells increased remarkably, and this trend persisted at 24 h. Compared with OGD/R groups, rapamycin showed a significant reduction of Edu (+) cells both at 12 and 24 h (Fig. 4A, B). These results using MTT assay and EdU labeling further confirmed that mTOR pathway is critical for astrocyte proliferation after OGD/R.

3. Results

3.3. mTOR blockade suppressed astrocyte migration after OGD/R

3.1. Rapamycin inhibited phosphorylation of mTOR signaling in reactive astrocytes after OGD/R

To ascertain the effect of rapamycin on astrocyte migration, a cell migration assay was carried out. We used DMEM/F12 with 1% FBS in the lower chamber as the control group. When the medium was changed to DMEM/F12 with 10% FBS, the number of astrocytes migrating through the film increased approximately two-fold (Fig. 5A2 and B). The increased migration could be partially inhibited when rapamycin was added in the DMEM/F12 with 10% FBS at the same time (Fig. 5A3, B).

Western blot analysis was conducted as previously described (Qu et al., 2012). Cells were washed with ice-cold phosphate-buffered saline (PBS), and the total proteins from the astrocytes were extracted with RIPA buffer supplemented with phosphatase inhibitors. Forty microgram total protein of each sample was loaded on an SDS–PAGE gel. The following primary antibodies were used for western blot analysis: mouse anti-GFAP (1:1000; Cell Signaling Technology), rabbit anti-p-mTOR(ser2448) (1:500; Cell Signaling Technology), rabbit anti-mTOR (1:1000; Cell Signaling Technology), rabbit anti-p-p70S6K (ser389) (1:500; Cell Signaling Technology), rabbit anti-p70S6K (1:1000; Cell Signaling Technology), rabbit anti-iNOS (1:400; Santa Cruz), mouse anti-TNF-α (1:1000; Cell Signaling Technology). Equal protein loading was confirmed with rabbit anti-GAPDH (1:2000; Neomarkers). One night later, the membrane was followed by conjugation with horseradish peroxidase (HRP)-conjugated immunoglobulin G (IgG) for 1 h at room temperature. Lastly, an enhanced chemiluminescence system (ECL kit; Pierce, Rockford, CA, USA) was used to collect digital images, which were then analyzed by Image J (National Institute of Health). Optical density (OD) of the signals was semi-quantified and expressed as the ratio of OD. For the cytokine array, expression of TNF-α in astrocyte lysis was also detected by ELISA and performed according to the manufacturer’s instructions (R&D Systems, USA). Given that iNOS exerts biological effects through NO, we also detected NO level in the supernatants by the Griess reagent kit (Beyotime, Nanjing, China). 2.9. Statistical analysis

To check the purity of astrocyte cultures, immunostaining was determined with GFAP and DAPI, and indicated that more than 98% of cells were GFAP-positive astrocytes (Fig. 1A). To investigate whether mTOR pathway was phosphorylated in astrocytes after OGD/R, we double-immunolabeled with p-mTOR and astrocytic

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Fig. 1. Expression of p-mTOR in astrocyte at 12 h after OGD/R. (a) The purity of astrocyte cultures was determined with GFAP and DAPI immunostaining. (b) mTOR was phosphorylated in astrocyte culture activated by OGD/R and inhibited by rapamycin. p-mTOR (red), GFAP (green) and DAPI (blue). Scale bars = 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

A cell migration assay was also carried out to study the role of mTOR in astrocyte migration (Cory, 2011). Confluent dishes of cells were “wounded” by scraping with a pipette tip, creating a space free of cells. A significant delay in the ability of the astrocytes treated with rapamycin to migrate into the empty space was observed (Fig. 5C and D), with the control scratched-astrocytes decreasing the gap at 24 h, and the astrocytes with rapamycin treatment filling the gap remarkedly became slow at 24 h. Examination of the leading edge of the migrating cells revealed multiple protrusions and extensions in astrocytes in control scratched-group, but a relatively uniform flat edge on the astrocyte in rapamycin group. To exclude the narrow of gap caused by the proliferation of cells, astrocytes were pretreated with DMEM containing 1% FBS for 24 h and treated during all the experiment. Together, these results suggested that mTOR signaling is prominently involved in regulating astrocyte migration after OGD/R. 3.4. mTOR blockade decreased inflammatory cytokine production in astrocytes after OGD/R Astrocyte activation is always accompanied by production of inflammation mediators, such as TNF-α, NO and iNOS, which is

involved in secondary neuronal cell death after CNS injuries (Iadecola et al., 1995; Zhang et al., 2009). To examine the role of mTOR pathway in the production of inflammatory cytokine from activated astrocyte after OGD/R, the expression of TNF-α and iNOS was measured by Western blots analysis. In the present study, we found that TNF-α level was weak in the control group. However, its expression was increased shortly after OGD/R and sustained for at least 24 h (Fig. 6A and B). The increase of TNF-α expression after OGD/R was suppressed in those cells pretreated with rapamycin in contrast to untreated cells (Fig. 6A and B). Similarly, the expression level of iNOS protein was elevated as early as 3 h after OGD/R, peaked at 6 h and 12 h, and then returned to control levels at 24 h. At the different time points, iNOS levels were markedly decreased in the rapamycin-treated group vs. the untreated group (Fig. 6A and C). These findings demonstrated that OGD/R could induce astrocytic intracellular inflammatory mediators’ production, whereas mTOR blockade could suppress their production after OGD/R. In order to test the inflammatory activation of astrocytes, the synthesis of inflammatory cytokine measured by Western blot was also confirmed by ELISA. Astrocytes were cultured in normal and

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Fig. 2. Inhibition of rapamycin on mTOR pathway. (a) The representative western blot of p-mTOR/t-mTOR and its downstream target p-p70S6K/t-p70S6K expression. (b) Statistical analysis of p-mTOR/t-mTOR in control, OGD/R and rapamycin-treated group. (c) Statistical analysis of values of p-p70S6K/t-p70S6K in OGD/R and rapamycin-treated group. Values are expressed as the mean ± SEM (n = 6, **p < 0.01 vs. the OGD/R group).

OGD/R condition without or with treatment of rapamycin. After 12 and 24 hr of incubation, the concentration of TNF-α in astrocyte lysis was measured by ELISA. Given that iNOS exerts biological effects through NO, we detected NO level by the Griess reagent kit. The supernatants were collected to measure the NO production through the accumulated level of nitrite. We also found the similar inhibitory effects of rapamycin on inflammatory cytokine production including TNF-α and NO (Fig. 6D, E). 4. Discussion The molecule of mTOR phosphorylates at least three distinct classes of substrates, the eIF4E-binding proteins (4EBP-1 to 3), the ribosomal protein S6 kinases (S6K1 and S6K2) and the serumand glucocorticoid-inducible kinase 1. As a central controller of metabolism, mTOR can control cell growth, proliferation, migration, translation, transcription and autophagy through the phosphorylation of its two major downstream substrates P70S6K and 4E-BP1 (Capdevila et al., 2009; Martin and Hall, 2005). Previous studies have shown that the PI3K/AKT/mTOR signaling pathway is activated and involved in many cancers and carcinogenesis (Fulda, 2009; Hennessy et al., 2005). Activation of mTOR cascades in cytomegalic neurons and astrocytes was also observed and considered a possible pathogenetic mechanism in cortical dysplasia (Ljungberg et al., 2006). mTOR pathway activation in acutely reactive astrocytes and microglial cells in early stages following CNS insult has also been described (Codeluppi et al., 2009; Park et al., 2012). To our knowledge, there are little data about the role of mTOR pathway in cerebral ischemia and cultured astrocytes in the OGD/R model. In the present study, we found that the mTOR pathway was also upregulated significantly in reactive astrocytes after OGD/R. In addition, phosphorylation of p70S6K persisted longer than that for mTOR after OGD/R, perhaps because p70S6K is a downstream target. The expression of p-mTOR (at ser2448) and p-p70S6K (ser389) could be significantly inhibited by rapamycin. These results indicated

that rapamycin administration could effectively block the activation of mTOR signaling pathway in activated astrocytes after OGD/R in vitro. Reactive astrogliosis is a complex process that can be provoked by many types of CNS insults, including cerebral ischemia (Yang et al., 2011; Zhu et al., 2007), spinal cord injury (Tian et al., 2006; Wanner et al., 2013), and other CNS diseases (Silver and Miller, 2004). Although reactive astrogliosis encircling the injury core may carry out critical protective functions (Pekny et al., 2007), excessive astrogliosis does lead to detrimental glial scar formation, which serve as a local biochemical and physical barrier for axonal recovery (Silver and Miller, 2004; Zhu et al., 2007). The serine/threonine kinase mTOR is a key regulator of cell proliferation downstream of growth factor receptors, in addition to its role in mediating cell responses to nutrients (Chiang and Abraham, 2007). This pathway was elucidated using cultured astrocytes isolated from the adult spinal cord and appears to also be functional in reactive astrocytes in vivo (Codeluppi et al., 2009). Our study showed a similar result in cultured astrocytes from rat cerebral cortex. In our ischemia-reperfusion model in vitro, OGD/R triggered a higher cell percentage of S stage, increased the number of EdU (+) proliferating cells, and enhanced expression of GFAP, indicated astrocyte activation and proliferation. Rapamycin administration could effectively block astrocytic cell cycle progression, and accordingly reduce the number of EdU (+) cells, but had no impact on GFAP expression (data were not shown). To exclude the possibility that the reduction of astrocyte cell number is attributable to the toxic effect of rapamycin, we detected the LDH released level in culture and found that there was no significant difference of cell survival between rapamycin group and control group. In fact, the concentration of rapamycin at 10 nM applied in cultured astrocytes has been reported in several articles (Codeluppi et al., 2009; Wu et al., 2010), and proved to be no cytotoxic. In the mean time, the astrocytes growth curve measured by the MTT assay revealed that the rapamycin-treated group reached maximal levels earlier than that in the control group.

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Fig. 3. Rapamycin attenuates cell cycle progression of reactive astrocytes after OGD/R. (a) Representative pictures of flow cytometric analysis of control, OGD/R, and rapamycintreated group at 12 h. (b) Statistical analysis of percentage of astrocytes in S phase. Values are expressed as the mean ± SEM (n = 6, *p < 0.05, vs. OGD/R group). (c) Rapamycin decreased astrocytes numbers by MTT assay. Values are expressed as the mean ± SEM (n = 6, *p < 0.01, vs. control group). (d) Statistical analysis of LDH release level.

Fig. 4. Rapamycin attenuates proliferation of astrocytes after OGD/R. (a) Immunostaining of EdU (red) and DAPI (blue) at the OGD/R12h. Scale bar = 100 μm. (b) Statistical analysis of percentage of Edu-positive astrocytes in each group. Data were evaluated by Student’s t test and values are expressed as the mean ± SEM (n = 6, *p < 0.05, vs. the OGD/R group). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Astrocytes are known to migrate toward the site of brain injury (Auguste et al., 2007). As the key step of glial scar formation, astrocyte migration is regulated by various factors (Auguste et al., 2007; Miao et al., 2010; Zuidema et al., 2014), such as TGF-β and MMP-9

(Lin et al., 2014; Wang et al., 2011). Besides, it also has been reported (Kalderon, 2005; Papadopoulos and Verkman, 2008; Saadoun et al., 2005) that cultured astrocytes from AQP4 knockout mice are with weaker migratory activity without affecting proliferation. The

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Fig. 5. Rapamycin inhibits astrocytes migration. (a) Representative picture of transwell. A1, 1% FBS group; A2, 10% FBS group; A3, rapamycin-treated group. Scale bar = 100 μm. (b) Statistical analysis of relative migration rate of astrocytes by one-way ANONA followed by Tukey’s post hoc test in each group. (n = 6, **p < 0.01 vs. control group, *p < 0.05 vs. EGF group). (c) Representative figures about the scratch wound assay. C1, the control group when scratch was just finished; C2 the control group at 24 h. C3, the rapamycin-treated group at 24 h. (d) Statistical analysis of width of scratch healing. Values are expressed as the mean ± SEM (n = 6, *p < 0.05 vs. the control group).

role of mTOR in cell migration has been reported in only a few cell types, such as endothelial cells (Pan et al., 2014), smooth muscle cells (Kerr and Patterson, 2004) and fibroblasts (Berven et al., 2004). Several studies (Codeluppi et al., 2009) observed that the EGFdependent chemotactic migration can be inhibited by rapamycin in cultured astrocytes from adult rat spinal cord. However, whether mTOR could partly regulate the cortex astrocyte migration after OGD/D remains to be elucidated. In the present study, we demonstrated that migration ability of primary reactive astrocytes after OGD/R in a cell migration and scratch wound assay could be suppressed in incubation with the mTOR specific inhibitor rapamycin, indicating that mTOR pathway is involved in astrocyte migration. In fact, since rapamycin could inhibit both proliferation and migration in some cell types, this has been suggested for therapeutic strategies, such as atherosclerosis (Dzau et al., 2002). Neurons, astrocytes, microglia and oligodendrocytes can produce inflammatory mediators in the physiological condition, albeit at low levels. The constitutive expression of genes encoding cytokines in the brain suggests that cytokines may contribute to normal functions of the CNS (Lucas et al., 2006). However, the inflammatory response gives rise to the pathogenesis of neurodegeneration in common neurological diseases such as stroke and Alzheimer’s disease (Saleh et al., 2004). Modulation of neuroinflammation can reduce neuronal apoptosis, diminish infarct volume and improve neurological scores after rat MCAO (Zhang et al., 2009). Suppression of

proinflammatory cytokines also decreased myelin loss and production of axonal outgrowth inhibitors, and contribute to improved functional outcome after rat SCI (Tian et al., 2009). It is acknowledged that the activity of iNOS can result in increased production of nitric oxide, a common toxic factor during ischemia (Iadecola et al., 1995), while TNF-α is an important proinflammatory factor that enhances neuronal damage (Chen et al., 2011; McKerracher et al., 1994). Both of iNOS and TNF-α were reported to inhibit apoptotic cell death via attenuation of expression (Lee et al., 2004; Yune et al., 2003). Single inflammatory processes without obvious physical damage are detrimental to CNS tissue and directly lead to secondary damage, progressive cavitation, and upregulation of glial scar–associated inhibitory molecules (Fitch et al., 1999). Administration of rapamycin significantly decreased levels of TNF-α after SCI and contributed to functional recovery from the injury (Chen et al., 2013). Our current study showed that rapamycin reduced the expression of these inflammatory cytokines in astrocytes after OGD/R. These results further suggest that rapamycin may be a promising therapeutic strategy in CNS disorders for its reduction of inflammation induced CNS damage. In summary, it is encouraging that our study indicated that rapamycin inhibited proliferation and migration of astrocytes, and reduced the inflammatory mediator profile induced by OGD/R in vitro in which mTOR is hyper-activated in reactive astrocytes. Recently, several studies have demonstrated that mTOR blockade can improve neuronal survival following OGD (Fletcher et al., 2013). In

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Fig. 6. Rapamycin decreases inflammatory cytokine production from astrocyte after OGD/R. (a) TNF-α and iNOS in the astrocytes exposed to OGD/R were measured by western blot. (b) Statistical analysis of the relative values of TNF-α in the control, OGD/R and OGD/R + Rapa group. (c) Statistical analysis of the relative values of iNOS in the control, OGD/R and OGD/R + Rapa group. (d) Statistical analysis of TNF-α production of astrocyte lysis detected by ELISA in the control, OGD/R and OGD/R + Rapa group. (e) Statistical analysis of NO production of the supernatants detected by NO Griess reagent kit in the control, OGD/R and OGD/R + Rapa group. Values are expressed as the mean ± SEM (n = 6, *p < 0.05 vs. the OGD/R group).

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