ORIGINAL RESEARCH ARTICLE

Journal of

IGF-1 Alleviates NMDA-Induced Excitotoxicity in Cultured Hippocampal Neurons Against Autophagy via the NR2B/PI3KAKT-mTOR Pathway

Cellular Physiology

YANSONG WANG,1,2 WEI WANG,3 DONGGUO LI,1,4 MI LI,2 PEIPEI WANG,1 JIAN WEN,2 MIN LIANG,2 BO SU,2 AND YANLING YIN1* 1

Department of Neurobiology and Beijing Institute for Brain Disorders, School of Basic Medical Sciences, Capital Medical University, Beijing, P.R. China

2

Department of Spine Surgery, Institute of Hard Tissue Development and Regeneration of Harbin Medical University, Second Affiliated Hospital of Harbin Medical University, Harbin, Hei Long Jiang Province, P.R. China

3

Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Capital Medical University, Beijing, P.R. China

4

Beijing Key Laboratory of Fundamental Research on Biomechanics in Clinical Application, Institute of Biomedical Engineering, Capital Medical University, Beijing, P.R. China

Insulin-like growth factor-1 (IGF-1) is a brain-specific multifunctional protein involved in neuronal polarity and axonal guidance. Mature IGF-1 triggers three enzymes, mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and phosphoinositide phospholipase C-g (PLC-g), which are its predominant downstream regulators. The PI3K-AKT signaling pathway is upstream of the mammalian target of rapamycin (mTOR), which is of great importance in the induction of autophagy. However, whether the neuroprotective effect of IGF-1 against excitotoxicity is mediated by autophagy through the PI3K/AKT/mTOR pathway remains to be elucidated. The induction of autophagy following NMDA treatment was determined by microtubule-associated protein light chain 3 (LC3) conversion and the result of this autophagy was assessed by monitoring the cleavage of caspase 3 in cultured hippocampal neurons. Cell viability was determined using MTT and LDH assay, and PI-staining was used to estimate the fate of autophagy and the protective effect of IGF-1. In addition, IGF-1 was found to decrease autophagy induced by NMDA using transmission electron microscopy and MDC staining. The protective effect of IGF-1 against autophagy was accompanied with up-regulation of phospho-AKT (p-AKT) and phospho-mTOR (p-mTOR), which was blocked by the inhibitor of PI3K. At the same time, the activation of NR2B resulting in the down-regulation of p-AKT and p-mTOR was blocked by IGF-1. Together, these data suggest that NMDA induces the autophagy, followed by apoptosis in cultured hippocampal neurons, and that IGF-1 can block this effect via inhibition of NR2B receptors and activation of the PI3K-AKT-mTOR pathway. J. Cell. Physiol. 229: 1618–1629, 2014. © 2014 Wiley Periodicals, Inc.

Insulin-like growth factor-1 (IGF-1) is a potent neurotrophic and anti-apoptotic factor that occurs naturally in the central nervous system (CNS). As a pleiotropic factor, IGF-1 plays critical roles in the CNS by promoting survival and proliferation of many types of brain cells (Guan, 2008). IGF-1 can be produced by all cell types in the brain and its expression is highest perinatally. In the adult mammalian brain, IGF-1 is produced at much lower levels but expressed with a pattern encompassing the cortex, hippocampus, cerebellum, brainstem, hypothalamus, and spinal cord (Sun et al., 2012). IGF-1 has been shown to act as a neuroprotectant both in in vitro studies and in in vivo animal models of ischemia, hypoxia, trauma in the brain or spinal cord, multiple and amyotrophic lateral sclerosis, and Alzheimer’s and Parkinson’s disease (Miltiadous et al., 2010). IGF-1 has been reported to be capable of crossing the blood–brain barrier (Pan and Kastin, 2000), and, intramuscular IGF-I injection exerts neuroprotective effects against brain ischemic injuries (Chang et al., 2013). Therefore, studies on the mechanism of IGF-1 during neuronal injuries are of great importance, and may provide strategies for neural diseases. Macro-autophagy (hereafter autophagy) has been used to describe the pathways involved in the degradation of intracellular macromolecules. Under normal conditions, © 2 0 1 4 W I L E Y P E R I O D I C A L S , I N C .

Yansong Wang, Wei Wang, and Dongguo Li contributed equally to this work. Contract grant sponsor: National Natural Science Foundation of China; Contract grant numbers: 31200895, 31171105, 81271696. Contract grant sponsor: Beijing Municipal Talents Project; Contract grant number: 2013D005018000010. Contract grant sponsor: Nature Science Foundation of Hei Long Jiang Province; Contract grant numbers: ZD200916, D200902. Contract grant sponsor: WLD Foundation of Harbin Medical University; Contract grant number: QN1115. Contract grant sponsor: Beijing Natural Science Foundation; Contract grant number: 7132025. Contract grant sponsor: Open Project Program of Beijing Key Laboratory of Fundamental Research on Biomechanics in Clinical Application, China. *Correspondence to: Yanling Yin, Department of Neurobiology, Beijing Institute for Brain Disorders, Capital Medical University, #10 You An Men Wai Xi Tou Tiao, Beijing 100069, P.R. China. E-mail: [email protected] Manuscript Received: 28 October 2013 Manuscript Accepted: 5 March 2014 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 7 March 2014. DOI: 10.1002/jcp.24607

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IGF-1 ALLEVIATES NMDA-EXCITOTOXICITY

autophagy serves to clear long-living cytoplasmic proteins or damaged organelles to maintain normal cell homeostasis (Shehata et al., 2012). Inducible autophagy contributes to survival during several physiological stress conditions such as starvation, tumor suppression, and protection against neurodegenerative diseases and oxidative stress (Kuma et al., 2007; Yue et al., 2009; Lee and Koh, 2010). However, autophagy has also been reported to initiate cell death in response to intracellular damage caused by hypoxia, chemotherapeutic agents, viral infection, or toxins ( Paglin et al., 2001Talloczy et al., 2002; Daido et al., 2004; Kanzawa et al., 2004; Yu et al., 2004). Recently, there has been a resurgence of interest in the relationship between IGF-1 and autophagy. IGF-1 has recently been reported to protect the heart by rescuing mitochondrial metabolism and the energetic state, and thereby reducing cell death and controlling the potentially exacerbate autophagic response to nutritional stress. Nevertheless, little attention has been paid to the role of IGF-1 in promoting cell survival against excitotoxicity through regulation of autophagy, which is always associated with the early stages of injury (Troncoso et al., 2013). N-methyl-D-aspartate receptors (NMDARs) form glutamate-gated ion channels that are widely expressed in the CNS and are highly permeable to calcium ions (Ca2þ) (Liu and Zhao, 2013). NMDARs play central roles in a number of physiological processes, such as long-term potentiation (LTP) and synaptic plasticity. However, the dysfunction of NMDARs is involved in many different neural diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD) (Wardas et al., 2003; Gasparini et al., 2013). In addition, the excitotoxicity during ischemia/hypoxia is mainly due to hyperactivation of NMDARs, which is one of the documented hallmark events that occur following acute brain injury (Jennings et al., 2008; Sadasivan et al., 2010). Therefore, it is important to elucidate the mechanism of NMDAR-related injuries and to explore the effective strategies against it. In this study, we used NMDA to cause acute injury in the hippocampal neurons, and explored the neuroprotective and anti-autophagic effects of IGF-1 on NMDA-induced cytotoxicity. Strikingly, we found that IGF-1 prevented cytotoxicity and autophagy caused by NMDA via the NR2B/PI3K-AKT-mTOR pathway in hippocampal neurons, which suggested that IGF-1 pre-treatment can effectively protect against early excitotoxicity through the suppression of excess autophagy. Methods Hippocampal neuron cultures and treatments

Pregnant Wistar rats were purchased from the animal facility of Capital Medical University or the Second Affiliated Hospital of Harbin Medical University. All experiments involving animals were approved by the Institutional Animal Care and Use Committee (IACUC). Hippocampal neuron cultures were prepared as described previously (Xing et al., 2005). Briefly, pregnant Wistar rats were anesthetized with pentobarbital, and the E18–19 embryos were delivered by cesarean section. The hippocampi were dissected and incubated with 0.25% (w/v) Trypsin–EDTA (Gibco, Inc., Grand Island, NY) for 15 min at 37 °C and mechanically dissociated. The resulting single cell suspension was diluted at a density of 1  105 cells/ml in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Inc.) containing 10% (v/v) fetal bovine serum, 5% (v/v) equine serum, and 2 mM L-glutamine, then plated in 35 mm cell plates coated with poly-L-lysine (Gibco, Inc.) (0.5 mg/ml). Cells were incubated at 37 °C in a humidified incubator with 5% (v/v) CO2. After approximately 20 h, the medium was replaced by serum-free Neurobasal medium (Gibco, Inc.) containing B27 supplement (Gibco, Inc.) and 0.5 mM cytosine arabinoside (Sigma-Aldrich Corp., St. Louis, MO) to inhibit the growth of glial JOURNAL OF CELLULAR PHYSIOLOGY

cells. Every 3–4 days half of the media was replaced and the cultures were used for experiments on 10–14 days after plating. NMDA injury characterization and IGF-1 treatment

After 10–14 days in culture, excitotoxic stress was induced by adding 20, 50, 100, 150, or 200 mM NMDA (Sigma-Aldrich Corp.) for 10 min. The media with NMDA was discarded, and the cells were cultured for 2 more hours. After that, the cells were prepared for whole-cell patch-clamp recording, western blotting, propidium iodide (PI, Sigma-Aldrich Corp.) staining, electron microscopy micrographs and monodansylcadaverine (MDC, Sigma-Aldrich Corp.) staining. To explore the action of IGF-1 against NMDA injury, cultures were pre-treated with IGF-1 (Sigma-Aldrich Corp.) for 24 h prior to exposure to NMDA. Thiazolyl blue tetrazolium bromide (MTT) assay

Ten microliters of MTT (5 mg/ml stock in phosphate-buffered saline [PBS]) was added to each well (96-well plate, 100 ml medium/well), and incubated for 4 h. The insoluble blue formazan was solubilized with 100 ml dimethyl sulfoxide (DMSO), and OD values of the mixture were measured at 550 and 650 nm with a Bio-Rad microplate reader. All MTT assays involved not less than four separate samples, which were measured in triplicate. Survival of vehicle-treated control cells not exposed to NMDA was taken as 100%, with values for the other groups being given as a percentage of the control. Lactate dehydrogenase (LDH) release assay for cell death

The LDH release assay was performed to assess cell death by measuring the release of LDH into the medium from damaged cells due to necrosis and secondary necrosis following apoptosis or autophagic cell death. Culture medium (25 ml) was collected after treatment with NMDA from different groups. An equivalent volume (25 ml) of detection reagent (CytoTox One Reagent, Promega, Madison, WI), was added to each well containing the culture medium and incubated for 30 min in the dark at room temperature. The absorbance was measured (wavelength: 490 nm) using a colorimetric microplate reader (SpectraMax Gemini, Molecular Devices, Sunnyvale, CA). Six replicates for each time point per experiment were assayed and three such experiments were performed. Propidium iodide (PI) Staining

After NMDA exposure, cells were fixed in 4% (w/v) paraformaldehyde for 10 min. Next, PI at 10 mg/ml was added into the medium for 20 min at 4 °C. Three random 200 images were taken from each coverslip (three per condition) and the percentage of nucleus-condensed cells was determined by a blinded observer. At least, four culture dishes were examined in each condition. Cell death was determined by calculating the ratio of the cells with condensed nuclei over the total number of cells. Autophagosome labeling with MDC staining

The neurons on the coverslips were incubated with a fluorescent dye monodansylcadaverine (MDC; 0.05 mM, Sigma-Aldrich Corp.) in PBS after exposure of NMDA challenges at 37 °C for 10 min, to observe for autophagosomes (Sadasivan et al., 2010). The cells were washed 2 times with PBS, mounted using Antifade solution (Prolong Antifade) and immediately observed by Olympus fluorescence microscope. For their quantification of both average sized autophagosomes and unusually large autophagosomal bodies, for each

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WANG ET AL. experimental condition/well, three randomly selected fields per well (200 magnification) were used and more than 20 cells in each field were analyzed with counted.

(Axon Instruments). Fast and slow capacitances were neutralized and series resistance was always compensated (about 70%).

Transmission electron microscopy

Sample preparation and Western blot analysis

Hippocampal neurons were fixed with 2.5% glutaraldehyde/2% formaldehyde with 0.1 M sodium cacodylate and stored at 4°C until embedding (Lum et al., 2005). Cells were post-fixed with 2% osmium tetroxide followed by an increasing gradient dehydration step using cold graded ethanol (25%, 50%, 70%, 95%, and 100%) and then rinsed in propylene oxide. Cells were then embedded in LX-112 medium (Ladd) and sections were cut ultrathin (90 nm), placed on uncoated copper grids, and stained with 0.2% lead citrate and 1% uranyl acetate. Images were examined with a Hitachi H-7500 electron microscope at 80 kV. For quantitation of autophagosomes, the data obtained from a minimum of 50 independent cells was averaged (mean  SE)

As reported previously (Yin et al., 2013), frozen samples were rapidly thawed and homogenized at 4 °C in Buffer C (50 mM Tris–Cl, pH 7.5, containing 2 mM EDTA, 1 mM EGTA, 100 mM iodoacetamide [SH-group blocker, IAM], 5 mg/ml each of leupeptin, aprotinin, pepstatin A and chymostatin, 50 mM potassium fluoride, 50 nM okadaic acid, 5 mM sodium pyrophosphate). The protein concentration was determined using the BCA kit (Pierce Company, Rockford, IL). Proteins (25 mg) from each sample were loaded for sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (10%, w/v, SDS gel). Protein was then electrophoresed and transferred onto polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Buckinghamshire, UK) at 4 °C. After several rinses with TTBS (20 mM Tris–Cl, pH 7.5, 0.15 M NaCl, and 0.05% Tween-20), the transferred PVDF membrane was blocked with 10% non-fat milk in TTBS for 1 h and then incubated with antiLC3 (Abcam Technology, Cambridge, MA), anti-Beclin-1 (Santa Cruz Biotechnology, Inc., CA), anti-AKT (Cell Signaling Technology (CST), Danvers, MA), anti-mTOR (CST), antiphospho (Thr308) AKT (CST), anti-phospho mTOR (CST), anti-caspase 3 (CST), anti-phospho NR2A (CST), anti-phospho NR2B (Abcam), NR2A (CST), NR2B (CST) antibodies and the corresponding secondary antibodies (Stressgen Biotechnologies Corporation, Victoria, BC, Canada) for 1 h. Following incubation with the primary and secondary antibodies, the Enhanced Chemiluminescence (ECL) kit (GE Healthcare) was employed to detect the signals. To verify equal loading of protein, the blots were reprobed with primary monoclonal antibody against b-actin (Sigma-Aldrich Corp.). 3-Methyladenine (3-MA, Sigma-Aldrich Corp.) and LY294002 (SigmaAldrich Corp.) were added to inhibit microautophagy and PI3K, respectively. Quantitative analysis for immunoblotting was performed after scanning of the X-ray film with Quantitative-One software (Gel Doc 2000 imaging system, Bio-Rad Company, Mississauga, CA).

Whole-cell patch-clamp recording

The whole-cell voltage-clamp technique was used to record currents. The patch electrodes of thick-walled boro-silicate glass (VWR Scientific, West Chester, PA) were pulled on a PP-83 micropipette puller (Narishige, Japan). The patch-pipette solution contained (in mM): 140 KCl, 10 HEPES, 10 EGTA, 2 MgCl2, 2 Na2ATP, 1 CaCl2, pH 7.3. The typical resistance of glass electrodes was 3–5 MV when filled with intracellular pipette solution. During experiments, culture dishes were rinsed and perfused with extracellular solution containing (in mM): 140 NaCl, 5 KCl, 1 MgCl2, 0.5 CaCl2, 10 glucose, 10 HEPES, pH 7.4. Miniature excitatory post-synaptic currents (mEPSCs) were recorded using the GABAA receptor antagonist bicuculline (50 mM, Sigma-Aldrich Corp.), the AMPA receptor antagonist DNQX (20 mM, Tocris) and TTX (1 mM, Sigma-Aldrich Corp.) added to the extracellular solution. mEPSCs were recorded without synaptic stimulation at a holding potential of 70 mV from cultured hippocampal neurons for at least 5 min. Cells were chosen for recording based on their morphology and density of surrounding cells. The criterion was that they were similar to hippocampal pyramidal neurons. Relatively isolated or spherically shaped cells were avoided. To record synaptically evoked NMDAR-mediated EPSCs (eEPSCs), randomly selected pairs of neurons (50–150 mm apart) were recorded using perforated patch clamp at room temperature (Gerkin et al., 2007). Pre-synaptic stimulation was given by step depolarization (100 mV, 1–2 msec), either single or paired (100 msec IPI) delivered to a single neuron at 30 sec intervals. Initial stimuli to record eEPSCs did not begin until 20 min after a seal was obtained on the post-synaptic neuron, to allow for perforation of the membrane by amphotericin B (Sigma-Aldrich Corp.). eEPSCs were recorded at a holding potential of 20 mV in extracellular solution containing bicuculline and the AMPA receptor antagonist DNQX (20 mM, Tocris). D,L-APV (50 mM) was applied at the end of a subset of experiments to confirm NMDAR-mediated EPSCs. The NR2B-denpendent component of NMDAR EPSCs was evaluated by application of extracellular solution containing additional RO25-6981 (0.5 mM, Sigma-Aldrich Corp.). The RO25-6981-insensitive current was subtracted from NMDARmediated EPSCs to calculate the current mediated by NR2Bcontaining receptors. Only monosynaptic connections, defined as producing an evoked EPSC (eEPSC) with a rising phase beginning less than 5 msec after the onset of the pre-synaptic stimulus, were considered in eEPSCs analysis. Data were collected with an Axopatch 200B amplifier (Axon Instruments, Forster City, CA) and acquired and analyzed using pCLAMP 9 JOURNAL OF CELLULAR PHYSIOLOGY

Statistical analysis

Data are presented as the mean  SE. Statistical analysis was conducted by one-way analysis of variance followed by all pairwise multiple comparison procedures using the Bonferroni test (sigmastat 10.0). A value of P < 0.05 was considered statistically significant. Results NMDA-induced Beclin-1-dependent autophagy in a dose-dependent manner

To assess the possible induction of autophagy following acute NMDA exposure, we sought to study protein levels of the autophagy protein marker Beclin-1 (Atg-6). We performed immunoblots on cell lysates obtained from cultures following treatment with or without NMDA at different concentrations for 10 min. Medium containing NMDA was discarded, and the neurons were cultured for 2 h in normal medium. The neurons were collected for Western blotting. Beclin-1 levels appeared to be increased in NMDA-treated neurons when compared with controls at concentrations ranging from 20 to 100 mM post-treatment (Fig. 1A,B). Microtubule- associated light chain-3 II protein (LC3-II), also called Atg-8, is the only known autophagy protein marker that specifically associates with autophagosomes and not with other vesicular structures (Kuma et al., 2007). In parallel, we also

IGF-1 ALLEVIATES NMDA-EXCITOTOXICITY

Autophagic cell death usually involves a caspase-dependent mechanism (Baehrecke, 2005). As shown in Figure 1A,D, E, in this study, increased proteolytic cleavage of pro-caspase 3 into a 17 kDa active caspase 3 fragments was observed, which was induced by NMDA at 20, 50, or 100 mM. Nevertheless, when the concentration of NMDA was increased to 150 or 200 mM, caspase 3 was activated without the involvement of autophagy. Together these results indicate that a certain concentration of NMDA could induce autophagy accompanied by the induction of apoptosis. Therefore, when a specific concentration of NMDA is reached, it may elicit the occurrence of apoptosis without the involvement of autophagy. NMDA-induced autophagy-triggered cell death of hippocampal neurons

Macroautophagy is considered as a self-limiting survival strategy, which is critical for cell survival following some physiological and pathological conditions. Nevertheless, there are some contradictory results about whether autophagy is beneficial for the survival of cells. Autophagy may degrade excess or abnormal macromolecules in favor of survival. However, excessive autophagy can initiate cell death, so autophagy is also known as type II programmed cell death, which is a novel way of causing cell death in addition to apoptosis and necrosis (Shi et al., 2012; Zhang et al., 2013). In this study, hippocampal neurons were exposed to 20, 50, or 100 mM NMDA, and the extent of autophagy and caspase 3 activation was investigated. To verify the fate of NMDAinduced autophagy, the activation of caspase 3 and cell viability were observed by Western blotting, MTT and LDH assay. To confirm the role of autophagy, 3-MA, an inhibitor of macroautophagy, was added into the medium before the NMDA treatment. NMDA increased the activation of caspase 3 (Fig. 2A–C) and decreased cell viability significantly (Fig. 2D,E), which was abolished by 3-MA. These results showed that NMDA at 50 mM elicited autophagy-dependent cell death. Fig. 1. NMDA treatment increased the expression of Beclin-1, LC3-II and induced the cleavage of caspase 3 in cultured hippocampal neurons. A: Representative results. B: Quantitative analysis revealed that NMDA can increase the expression of Beclin-1 at 20, 50, and 100 mM (*P < 0.05, **P < 0.01 vs. that of control, n ¼ 6 per group). C: Quantitative analysis revealed that NMDA could increase the ratio of LC3-II/LC3-I at 20, 50, and 100 mM versus that of the control, similar to the expression of Beclin-1 (*P < 0.05, **P < 0.01 vs. that of control, n ¼ 6 per group), which indicated the increase in LC3-II levels by NMDA is Beclin-1 dependent. D,E: Expression of pro-caspase 3 and cleavage of caspase 3. Different from the upper results, NMDA at 20, 50, 100, 150, and 200 mM all induced the cleavage of caspase 3 (**P < 0.01 vs. that of control, n ¼ 6 per group). These results suggest apoptosis induced by 20, 50, and 100 mM NMDA was autophagy dependent. NMDA at a certain concentration caused apoptosis without the involvement of autophagy.

sought to examine if there were increased levels of the autophagosomes associated lipidated LC3-II form. Immunoblotting analysis of cell lysates from NMDA-exposed culture was performed using the anti-LC3 antibody that detects both LC3-I and LC3-II. There was also an increase in LC3-II levels or the ratio of LC3-II/LC3-I (from about 1.00 to 1.40, 1.60) (Fig. 1A,C) following NMDA exposure. This indicated that there may be a pool of LC3-II being generated following NMDA exposure that was translocated to the outer membrane of the autophagosomes (Mizushima and Yoshimori, 2007; Sadasivan et al., 2010). In this experiment, the change in Beclin-1 and LC3II levels was the same, which suggested NMDA-induced autophagy was Beclin-1 dependent. JOURNAL OF CELLULAR PHYSIOLOGY

IGF-1 decreased NMDA-induced autophagy, and decreased the cell death

Previous results from this study revealed that NMDA-induced autophagy played a role in NMDA-induced cell death. Because autophagy is vital in this process, it was of great importance to explore the protective effect of IGF-1 at different concentrations (1, 5, 10, and 100 ng/ml) on NMDA-induced autophagy (Fig. 3 A). The ratio of LC3-II/LC3-I was assessed by Western blotting. The result showed that IGF-1-pre-treatment at 100 ng/ml decreased the ratio of LC3-II/LC3-I significantly when compared with the naïve NMDA exposure group, while the effect of 1, 5, or 10 ng/ml IGF-1 against NMDA-induced autophagy was not significant (Fig. 3B), which indicated that the protective effect of IGF-1 was concentration-dependent. Furthermore, the results of PI-staining revealed IGF-1 decreased the number of neurons with condensation nuclei following NMDA exposure (Fig. 3 C,D), suggesting IGF-1 suppressed autophagy and then decreased cell death. To further test the effect of NMDA and IGF-1 on autophagy, the number of autophagosomes was investigated with transmission electron microscopy and MDC staining. We examined the ultrastructure of the hippocampal neurons treated with naïve NMDA (50 mg/ml) or IGF-1 (100 ng/ml) pretreatment before the exposure of NMDA. At this dose, NMDA-induced autophagy and then apoptosis, which was suppressed by IGF-1. Transmission electron microscopy showed autophagosomes in cells containing recognizable cellular material or a late autophagosome fusing with a lysosome (A). The number of autophagosomes was

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Porter, 1994). Synaptic NMDA receptor activation may help regulate dendritic outgrowth and establish final synaptic connections in the developing CNS (Kalb, 1994). To examine whether IGF-1 influenced NMDA-induced receptor activation, we recorded the mEPSCs in hippocampal neurons (A–C), which may be mediated by the action potential-independent spontaneous release of glutamate from pre-synaptic terminals (Hori and Endo, 1992). It was found there was no significant difference in the frequency of mEPSCs between the experimental groups, suggesting that pre-synaptic glutamate release was at a similar level (Fig. 5D). However, it was observed that NMDA increased the amplitude of mEPSCs, indicating higher post-synaptic reactivity. This effect of NMDA could be blocked by IGF-1 pre-treatment (Fig. 5E). IGF-1 may have an effect on NMDA receptors; however, the mechanism remains unclear. Although the synaptic receptor of NMDA will not be the sole effective site, these electrophysiological results could reflect both the transformation of the NMDA receptor and the feedback of glutamate release from pre-synaptic terminals. IGF-1 decreased NMDA-induced hyperphosphorylation of NR2B and NR2B-dependent eEPSC

Fig. 2. Exposure of hippocampal cells to 50 mM NMDA caused autophagy and then cell death. A: Representative results of the expression of pro-caspase 3 and cleaved caspase 3. B,C: Quantitative analysis of the expression of pro-caspase 3 and cleavage of caspase 3. NMDA increased the cleavage of caspase 3 (*P < 0.05 vs. that of control, n ¼ 6 per group), which was abolished by 3-MA. D: Quantitative analysis of the MTT assay showed that NMDA decreased cell viability (**P < 0.01 vs. that of control, n ¼ 6 per group), which may be abolished by 3-MA. E: Quantitative analysis of LDH release showed that NMDA increased LDH release, which may be abolished by 3-MA. All these data indicated that apoptosis and cell death induced by NMDA at 50 mM induced were autophagy dependent.

significantly higher in the NMDA-treated cells and was markedly lower in the control and IGF-1-pre-treated group (Fig. 4B). The fluorescence intensity in MDC-positive autophagosomes were relatively sparse and weak in hippocampal neurons of the control or IGF-1 pre-treatment group when compared with cultures treated with naïve NMDA (Fig. 4C). Consistent with the immunoblotting results, the suppression of NMDA-induced autophagy by IGF-1 was further confirmed. IGF-1 decreased NMDA-induced post-synaptic hyper activation

Glutamate is a major excitatory neurotransmitter in the CNS, and plays an important role in functions such as synaptic plasticity, learning, and memory (Greenamyre and JOURNAL OF CELLULAR PHYSIOLOGY

NMDARs consist of three different subtypes: NR1, NR2, and NR3. Of the subtypes, most studies have focused on NR2. Some studies report that NR2A and NR2B play different roles in the CNS. The synaptic NR2A subpopulation activates the downstream neuronal and survival-signaling complex, which is primarily found in synapses and leads to neuronal survival. The extrasynaptic NR2B subpopulation activates the downstream neuronal death-signaling complex, which is primarily found in the extrasynaptic sites, which are involved in neuronal death under pathological conditions (Lai et al., 2011; Liu and Zhao, 2013). In this study, we found IGF-1 blocked NMDAmediated post-synaptic hyperactivation. To explore this further, the phosphorylation of NR2A and NR2B was observed by western blotting. It was found that NMDA exposure increased the hyperphosphorylation of NR2B at Ser 1303 (A, C), which is consistent with a previous report (Liu and Zhao, 2013). IGF-1 pre-treatment reduced elevated NR2B subunit phosphorylation. At the same time, there was no significant difference between the different experimental groups in the phosphorylation of NR2A (Fig. 6A,B). Combined with our mEPSCs statistical results, we confirmed that NMDA exposure at 50 mM played its role through NR2B activation, which is the effective site of IGF-1. However, to provide a direct proof of the role of IGF-1 in the regulation of NR2B, eEPSCs were recorded before and after application of RO25-6981 (RO) at a concentration shown to block NR2B-containing NMDARs selectively (0.5 mM). We calculated the percentage change in charge transfer after RO application, which reflects the proportion of current carried by NR2B-containing receptors (Snyder et al., 2011). These results confirmed that IGF-1 decreased NMDAR-mediated EPSC amplitude primarily, if not entirely, through a decrease in currents mediated by NR2B-containing receptors (Fig. 6D,E). IGF-1 alleviated NMDA-induced excitotoxicity via the NR2B/PI3K-AKT pathway

The signaling pathway composed of PI3K, AKT, and mTOR plays a vital role in the regulation of cell proliferation, growth, differentiation, and survival (Oldham and Hafen, 2003). The protective effect of IGF-1 has been reported to occur via the PI3K-AKT pathway. In this study, we explored whether the blockage of IGF-1 against NMDA-induced autophagy was through PI3K-AKT pathway. We found that NMDA suppressed the phosphorylation of AKT at Ser308 (A,B) and mTOR (Fig. 7A,C), and then increased the expression of

IGF-1 ALLEVIATES NMDA-EXCITOTOXICITY

Fig. 3. IGF-1 decreased NMDA-induced autophagy in a concentration-dependent manner. A: Representative results of Western blots. B: Quantitative analysis showed that NMDA could increase the ratio of LC3-II/LC3-I (*P < 0.05, vs. that of control, n ¼ 6 per group), which was blocked by IGF-1 pre-treatment at 100 ng/ml (#P < 0.05, that of naïve NMDA treatment group, n ¼ 6 per group). IGF-1 at 1, 5, and 10 ng/ml had no effect on the increase in the ratio of LC3-II/LC3-I induced by NMDA, indicating the effect of IGF-1 is concentration dependent. C: Representative results of PI staining. D: Quantitative analysis showed that NMDA could increase the ratio of cells with condensed nuclei (*P < 0.01, vs. that of control, n ¼ 6 per group), which could be blocked by IGF-1 pre-treatment at 100 ng/ml (*P < 0.05, that of naïve NMDA treatment group, n ¼ 6 per group).

Beclin-1 and the ratio of LC3-II/LC3-I (Fig. 7A,D,E), which suggested that NMDA may play a role by inhibiting PI3K-AKT pathway. The effect of NMDA can be blocked by RO or IGF-1 pre-treatment (Fig. 7A–E). Although RO is a selective inhibitor of NR2B, it can up-regulate the NMDA-induced hypophosphorylation of AKT and mTOR, which may act as a proof that NMDA dose induce its effect via NR2B/PI3K-AKT-mTOR pathway. With respect to IGF-1, it can block the activation of NR2B and reverse NMDA-triggered hypo-phosphorylation of AKT and mTOR in this experiment. We also treated cells with the PI3K inhibitor, LY294002, before IGF-1 pre-treatment and found LY294002 reduced the phosphorylation of AKT and JOURNAL OF CELLULAR PHYSIOLOGY

mTOR increased by IGF-1, indicating that IGF-1 activated PI3KAKT-mTOR in addition to suppressing NR2B (Fig. 7A–E). Discussion

Autophagy is a major protein degradation pathway in which isolated membranes sequester part of the cytoplasm to form a double-membrane vesicle, called an autophagosome (AP) (Sadasivan et al., 2010). Under basal conditions, autophagy is considered a critical cytoprotective pathway, involving in the formation of double membrane autophagosomes that capture portions of cytoplasm before fusing with lysosomes where

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Fig. 4. IGF-1 decreased the number of autophagosomes increased by NMDA. A: Representative results of transmission electron microscopy. Arrowheads depicted representative autophagosomes quantitated in cells containing recognizable cellular material or a late autophagosome fusing with a lysosome B. Quantitative analysis showed that NMDA could increase the number of autophagosomes (**P < 0.01, vs. that of control), which could be blocked by IGF-1 pre-treatment at 100ng/ml (**P < 0.01, vs. that of naïve NMDA treatment group). C: Representative results of MDC staining. NMDA exposure induced the formation of MDC-positive autophagosomes, which could be reduced by IGF-1 pre-treatment. White arrows indicate dense autophagosome staining in cell bodies following prolonged exposure to NMDA.

their contents are ultimately degraded (Mizushima and Komatsu, 2011). Physiologically, autophagy is a process in which cytoplasmic components such as damaged organelles and long-lived proteins are cleared and delivered to the lysosomal compartment for maintaining normal cellular homeostasis. Autophagy has been reported to be of great importance in the development of the CNS (Komatsu et al., 2006). However, the function of autophagy in mature neurons remains controversial. Autophagy is a tightly regulated pathway that can be stimulated by multiple forms of cellular stress induced by nutrient or growth factor deprivation, hypoxia, reactive oxygen species (ROS), DNA damage, protein aggregation, organelle damage, or intracellular pathogens (Kroemer et al., 2010; Liu and Zhao, 2013). JOURNAL OF CELLULAR PHYSIOLOGY

In addition, autophagy is increasingly recognized as having key functions in human diseases, such as degenerative diseases and cancers. Therefore, it represents a new area for the development of therapeutics (Nencioni et al., 2013). In the treatment of cancer, autophagy can either act as a chemoresistant process or have a tumor suppressive function. Finally, because autophagy is now a candidate for the treatment of neurodegenerative diseases, autophagic inducers may be applied clinically for this purpose (Garcia-Arencibia et al., 2010). Although autophagy has been shown to occur during reperfusion after ischemia/hypoxia and even during ischemic/hypoxic pre-conditioning, contradictory results have emerged and require further investigation (Gao et al., 2012; Shi et al., 2012; Zhang et al., 2013). For example, in some studies,

IGF-1 ALLEVIATES NMDA-EXCITOTOXICITY

Fig. 5. IGF-1 pre-treatment could block NMDA-induced hyperactivation of post-synaptic receptors. A–C: Representative results of mEPSCs in different experimental groups. D,E: Quantitative analysis showed the frequency and mean amplitude of mEPSCs in the control (n ¼ 15), naïve NMDA treatment (n ¼ 12) and IGF-1 pre-treatment (n ¼ 11) groups. There was no significant difference in the frequency of mEPSCs between the different experimental groups (P > 0.05). However, a significant difference in the mean amplitude of mEPSCs between the NMDA treatment and control group was observed (**P < 0.01), suggesting higher post-synaptic reactivity. In addition, there was a significant difference in the mean amplitude of mEPSCs between the naïve NMDA treatment and IGF-1 (100 ng/ml) pre-treatment groups, indicating the effect of NMDA could be blocked by IGF-1 effectively (**P < 0.01).

the induction of autophagy seems vital in the protection against ischemia/hypoxia, while in others, inhibition of autophagy is beneficial for protection. Even the effect of autophagy during reperfusion is inconsistent. Excitotoxicity is a major contributor to ischemic/hypoxic injury. In this study, we investigated NMDA-induced autophagy in hippocampal neurons prone to injuries, which may be a further proof that autophagy is deleterious after the reperfusion. Beclin-1 is regarded as a marker of the autophagosome membrane, which is involved in the early stages of autophagy by JOURNAL OF CELLULAR PHYSIOLOGY

promoting the nucleation of the autophagic vesicle and recruiting proteins from the cytosol (Ferraro and Cecconi, 2007). In this study, there was an increase in LC3-II levels or the ratio of LC3-II/LC3-I following NMDA exposure, as measured by quantitative immunoblotting. This result suggests that there may be a pool of LC3-II being generated following NMDA exposure that is translocated to the outer membrane of the autophagosome (Kuma et al., 2007; Mizushima and Yoshimori, 2007; Sadasivan et al., 2010). At the same time, an increased expression of Beclin-1 was observed

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Fig. 6. IGF-1 decreased NMDA-induced hyperphosphorylation of NR2B at Ser1303 and the amplitude of eEPSCs. A: Representative results of Western blots. B: There was no significant difference between the phosphorylation of NR2A at Tyr1246 (P > 0.05, vs. that of control, n ¼ 6 per group). C: There was a significant difference in the phosphorylation of NR2B at Ser1303 between the control and naïve NMDA treatment group (**P < 0.01, n ¼ 6 per group), and between the naïve NMDA treatment and IGF-1 pre-treatment groups (**P < 0.01, n ¼ 6 per group). D: Representative whole-cell voltage clamp recordings of eEPSCs in hippocampal neurons from the control and IGF-1-treated groups, each before and after bath application of RO25-6981 (0.5 mM). E: Bar graph representing the RO25-6981-sensitive component of NMDAR-mediated EPSCs. Charge transfer was reduced by IGF-1 (**P < 0.01, vs. that of control, n ¼ 6 per group).

and the blockage of Beclin-1 reduced LC3-II levels or the ratio of LC3-II/LC3-I, suggesting NMDA-induced autophagy is Beclin-1 dependent. When the autophagy was inhibited by 3-MA, apoptosis, and neuronal injury decreased. These results suggest that autophagy and caspase 3 activation may be interlinked. From these observations, we conclude that NMDA-induced autophagy is deleterious to neurons. JOURNAL OF CELLULAR PHYSIOLOGY

Christopher et al. found IGF-1 pre-treatment could increase the susceptibility of retinal neurons and Müller glia to excitotoxic damage by stimulating non-astrocytic inner retinal glial to proliferate, migrate distally into the retina, and become reactive (Zelinka et al., 2012). Guan et al. reported that IGF-I can decrease ischemic brain injury and promote the exerciseinduced protective effects against brain insults (Guan, 2008;

IGF-1 ALLEVIATES NMDA-EXCITOTOXICITY

Fig. 7. IGF-1 played a role in the suppression of NMDA-induced autophagy via the PI3K-AKT-mTOR signaling pathway. A: Representative results. B,C: Quantitative analysis showed that NMDA could decrease the phosphorylation of AKT at Ser308 and mTOR (*P < 0.05 vs. that of control, n ¼ 6 per group), which may be reversed by RO25-6981 or IGF-1 (100 ng/ml) pre-treatment (#P < 0.05 vs. that of naïve NMDA group, n ¼ 6 per group). The PI3K inhibitor, LY294002 (10 mM) could decrease the IGF-1-induced hyperphosphorylation of AKT and mTOR (*P < 0.05 vs. that of control, &P < 0.05 vs. that of IGF-1 pre-treatment, n ¼ 6 per group). D,E: Quantitative analysis showed that NMDA could increase the expression of Beclin-1 and the ratio of LC3-II/LC3-I (*P < 0.05 vs. that of control, n ¼ 6 per group), which was reversed by RO25-6981 or IGF-1 pre-treatment (#P < 0.05 vs. that of naïve NMDA group, n ¼ 6 per group). LY294002 (10 mM) decreased IGF-1-reduced Beclin-1 expression and the ratio of LC3-II/LC3-I (*P < 0.05 vs. that of control, &P < 0.05 vs. that of IGF-1 pre-treatment, n ¼ 6 per group).

Guan et al., 2003). Recently, much attention has been placed on the relationship between IGF-1 and autophagy, which revealed paradoxical results, too. Luo et al. found that up-regulation of IGF-1 increased autophagy activity (Luo et al., 2013), while other studies have shown that IGF-1 decreased autophagy (Bitto et al., 2010; Zhang et al., 2010; Troncoso et al., 2012). This contradiction may be a result of from different experimental conditions, such as different tissues and animal ages. In this study, pre-treatment with IGF-1 at 100 ng/ml decreased the ratio of LC3-II/LC3-I induced by NMDA, and increased cell viability, indicating the protective role of IGF-1 JOURNAL OF CELLULAR PHYSIOLOGY

against NMDA through the inhibition of autophagy. IGF-1 has been suggested to play an important role in regulating cell survival and neuronal plasticity through the PI3K/AKT pathway (Chang et al., 2013). As reported, mTOR, the downstream molecule of PI3K/AKT can downregulate autophagy activity. In this study, we found the protection of IGF-1 against NMDAinduced autophagy and apoptosis were blocked by the inhibitor of PI3K, which suggested that IGF-1 played a role in the protection against excitotoxicity through PI3K/AKT pathway. Previously, Jia et al. (2006) reported IGF-1 inhibited autophagy through AKT, which is partly analogical with our result. NMDARs play a key physiological role in the nervous system during development as well as in the formation of the neuronal circuit. NMDARs have recently attracted considerable attention because of their role in synaptic plasticity and excitotoxicity (Liu and Zhao, 2013). In this experiment, we found that NMDA increased the amplitude of mEPSCs, but had no effect on the frequency of sEPSC. IGF-1 played a role in decreasing the amplitude, suggesting its effect is via postsynaptic reactivity. Combined with our other results, it can be concluded that IGF-1 is protective against NMDA-induced excitotoxicity through suppression of autophagy and reactivity of NMDARs. Nevertheless, proof that sole extrasynaptic NMDARs are activated is required. When the PI3K-AKT pathway was inhibited, the protective effect of IGF-1 was blocked, suggesting the effect of IGF-1 is via this signaling pathway (Fig. 8). Moreover, the hyperphosphorylation of NR2B was accompanied with the hypo-phosphorylation of AKT and mTOR, indicating NR2B may be associated with the regulation of the PI3K-AKT pathway. In this study, the influence of IGF-1 on NR2B was represented by the synaptic currents. However, the relationship between synaptic plasticity and autophagy remains to be investigated. Sun et al. (1991) reported that the cellular effects of insulin and IGF-I were mediated principally by the insulin receptor substrate (IRS) proteins. Irs2 deficiency impairs activation of the NR2B subunit of NMDA receptors, indicating that IGF-1 may be involved in the activation of NR2B through IRS2 (Martin et al., 2012). It has been reported that IGF-1 regulates cerebellar NMDA receptors via AKT phosphorylation of NR2C at S1096 (Chen and Roche, 2009). Although IGF-1 was shown to play a role via the dephosphorylation of NR2B in this article, the precise mechanism remains to be clarified. From this study, we draw the following conclusions: (1) NMDA may elicit autophagy of hippocampal neurons in a dosedependent manner, and then triggers the apoptosis; (2) IGF-1 pre-treatment can rescue hippocampal neurons against NMDA-induced autophagy and increase the viability of neurons after NMDA exposure; (3) The effect of IGF-1 is dependent on the PI3K-AKT-mTOR signaling pathway; (4) IGF-1 could play a role in NMDA-induced injuries by suppressing the phosphorylation of NR2B, and activating the PI3K-AKT-mTOR pathway, which can be blocked by NMDA. Based on these findings, we propose that IGF-1 alleviates NMDA-induced injury by reducing autophagy, which may be important in NMDA-induced excitotoxicity and the early phase of ischemic injury, AD, and PD. The characteristics of IGF-I as a neuroprotectant both in in vitro and in vivo studies, and its ability to cross the blood–brain barrier, make it a potent agent for related diseases. Thus, our findings represent a novel approach that may be potentially useful in managing clinical excitotoxic-related diseases. Acknowledgments

This work was supported by the following grants: National Natural Science Foundation of China (31200895, 31171105, 81271696), Beijing Municipal Talents Project (2013D005018000010), the Nature Science Foundation of Hei

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Fig. 8. Proposed model for the role of IGF-1 against NMDA-induced autophagy. The activation of NR2B dephosphorylates AKT and mTOR, which is suppressed by IGF-1. Green arrows represent the activation of the downstream signaling pathway, while red arrows represent the inhibition of the downstream signaling pathway. The dashed lines represent the possible or indirect relationship.

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