Ionizing radiation induces neuronal differentiation of Neuro-2a cells via PI3-kinase and p53-dependent pathways.

Just Accepted by International Journal of Radiation Biology

Ionizing radiation induces neuronal differentiation of Neuro-2a cells via PI3-kinase and p53-dependent pathways Hyeon Soo Eom, Hae Ran Park, Sung Kee Jo, Young Sang Kim, Changjong Moon, Uhee Jung Doi: 10.3109/09553002.2015.1029595

Int J Radiat Biol Downloaded from informahealthcare.com by Fudan University on 05/08/15 For personal use only.

Abstract Purpose: The influence of ionizing radiation (IR) on neuronal differentiation is not well defined. In this study, we investigated the effects of IR on the differentiation of Neuro-2a mouse neuroblastoma cells and the involvement of tumor protein 53 (p53) and mitogen-activated protein kinases (MAPK) during this process. Materials and methods: The mouse neuroblastoma Neuro-2a cells were exposed to 137Cs γ-rays at 4, 8, or 16 Gy. After incubation for 72 h with or without inhibitors of p53, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and other kinases, the neuronal differentiation of irradiated Neuro-2a cells was examined through analyzing neurite outgrowth and neuronal maker expression and the activation of related signaling proteins by western blotting and immunocytochemistry. Mouse primary neural stem cells (NSC) were exposed to IR at 1Gy. The change of neuronal marker was examined using immunocytochemistry. Results: The irradiation of Neuro-2a cells significantly increased the neurite outgrowth and the expression of neuronal markers (neuronal nuclei [NeuN], microtubule-associated protein 2 [Map2], growth associated protein-43 [GAP-43], and Ras-related protein 13 [Rab13]). Immunocytochemistry revealed that neuronal class III beta-tubulin (Tuj-1) positive cells were increased and nestin positive cells were decreased by IR in Neuro-2a cells, which supported the IR-induced neuronal differentiation. However, the IR-induced neuronal differentiation was significantly attenuated when p53 was inhibited by pifithrin-α (PFT-α) or p53-small interfering RNA (siRNA). The PI3K inhibitor, LY294002, also suppressed the IR-induced neurite outgrowth, the activation of p53, the expression of GAP-43 and Rab13, and the increase of Tuj-1 positive cells. The increase of neurite outgrowth and Tuj-1 positive cells by IR and its suppression by LY294002 were also observed in mouse primary NSC. Conclusion: These results suggest that IR is able to trigger the

neuronal differentiation of Neuro-2a cells and the activation of p53 via PI3K is an important step for the IR-induced differentiation of Neuro-2a cells.

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Ionizing radiation induces neuronal differentiation of Neuro-2a cells via PI3-kinase and p53-dependent pathways


Hyeon Soo Eom1,2, Hae Ran Park1, Sung Kee Jo1, Young Sang Kim2, Changjong Moon3, Uhee Jung1,4* 1

Radiation Biotechnology Research Division, Korea Atomic Energy Research Institute, Korea,

2

Department of Biochemistry, College of Natural Sciences, Chungnam National University, Korea,

3

Department of Veterinary Anatomy, College of Veterinary Medicine, Chonnam National University,

Korea, 4Department of Radiation Biotechnology and Applied Radioisotope, University of Science and Technology (UST), Korea

*Corresponding author: Uhee Jung, Ph.D. Radiation Biotechnology Research Division, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 1266 Sinjeong-dong, Jeongeup-si, Jeonbuk 580-185, South Korea. Tel: +82-63-570-3221. Fax: +82-63-570-3229. E-mail: [email protected]

Short title: IR-induced neuronal differentiation in Neuro-2a Abstract Purpose: The influence of ionizing radiation (IR) on neuronal differentiation is not well defined. In this study, we investigated the effects of IR on the differentiation of Neuro-2a mouse neuroblastoma cells and the involvement of tumor protein 53 (p53) and mitogenactivated protein kinases (MAPK) during this process. Materials and methods: The mouse neuroblastoma Neuro-2a cells were exposed to 137 Cs γ-rays at 4, 8, or 16 Gy. After incubation for 72 h with or without inhibitors of p53, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and other kinases, the neuronal differentiation of irradiated Neuro-2a cells was examined through analyzing neurite outgrowth and neuronal maker expression and the activation of related signaling proteins by western blotting and immunocytochemistry. Mouse primary neural stem cells (NSC) were exposed to IR at 1Gy. The change of neuronal marker was examined using immunocytochemistry. Results: The irradiation of Neuro-2a cells significantly increased the neurite outgrowth and the expression of neuronal markers (neuronal nuclei [NeuN], microtubule-associated protein 2 [Map2], growth associated protein-43 [GAP-43], and Ras-related protein 13 [Rab13]). Immunocytochemistry revealed that neuronal class III beta-tubulin (Tuj-1) positive cells were increased and nestin positive cells were decreased by IR in Neuro-2a cells, which supported the IR-induced neuronal differentiation. However, the IR-induced neuronal differentiation was significantly attenuated when p53 was inhibited by pifithrin-α (PFT-α) or p53-small interfering RNA (siRNA). The PI3K inhibitor, LY294002, also suppressed the IRinduced neurite outgrowth, the activation of p53, the expression of GAP-43 and Rab13, and

the increase of Tuj-1 positive cells. The increase of neurite outgrowth and Tuj-1 positive cells by IR and its suppression by LY294002 were also observed in mouse primary NSC. Conclusion: These results suggest that IR is able to trigger the neuronal differentiation of Neuro-2a cells and the activation of p53 via PI3K is an important step for the IR-induced differentiation of Neuro-2a cells.


Keywords: Neuro-2a, neuronal differentiation, ionizing radiation, p53, PI3 kinase

INTRODUCTION Ionizing radiation (IR) is one of the general tools for primary and metastatic tumor therapy in the brain. Although radiation therapy is useful for brain tumor treatment because such tumors are difficult to remove through surgery in brain (Bernstein and Gutin 1981) and high-dose IR causes the injury of the DNA, membrane, and cellular


organelles through oxidative stress in normal cells (Oh et al. 2001, Jackson et al. 2011). One of the serious adverse effects of brain radiotherapy is the impairment of memory and cognitive functions (Crossen et al. 1994, Surma-aho et al. 2001). Previous studies have reported that the exposure of a rodent brain to IR results in the inhibition of neurogenesis by the loss of neuronal progenitor cells (NPC) through reactive oxygen species (ROS)induced oxidative stress and apoptosis in the hippocampus and subventricular zone (Jensh et al. 1995, Peissner et al. 1999, Inouye et al. 2000, Raber et al. 2004, Yoshida et al. 2011, Zhang et al. 2011). It was also reported that hippocampal neurogenesis is suppressed by IR-induced chronic inflammatory responses, including the increase of inflammatory cytokines (tumor necrosis factor-α [TNF-α] and interleukin-1β [IL-1β]) and the activation of microglia in cortex and dentate gyrus (Lee et al. 2010, Monje et al. 2002). In mouse models, there is strong evidence that radiation causes a decline of hippocampus-dependent learning and memory by the impairment of hippocampal neurogenesis (Crossen et al. 1994, Abayomi 1996). In addition, it was reported that the exposure of mice to low-dose γ-ray induces alterations of gene expression in the brain in a similar way to aging and Alzheimer’s disease (Lowe et al. 2009). Although some researchers have recently announced that a low-dose X-ray (1 Gy) does not interfere with the proliferation and differentiation of neural stem cells (Isono et al. 2012), the majority

of studies are still focused on the damage of neuronal cells and/or tissues caused by radiation in vitro and in vivo (Jensh et al. 1995, Peissner et al. 1999, Inouye et al. 2000, Raber et al. 2004, Yoshida et al. 2011, Zhang et al. 2011, Lee et al. 2010, Monje et al. 2002, Crossen et al. 1994, Abayomi 1996, Oh et al. 2013, Kanzawa et al. 2006). Interestingly, the latest studies have reported that astrocytic differentiation is accelerated


by X-rays in neural stem cells (NSC) (Ayumi et al. 2012). However, the diverse effects of IR on neuronal differentiation are not well defined. The Neuro-2a neuroblastoma cell line is derived from the mouse neural crest and has been widely used to investigate neuronal differentiation and neurite outgrowth because they have the capacity to differentiate into neurons within a few days by the treatment of several agents, including retinoic acid (RA), growth factors, and dibutyryl cyclic adenosine monophosphate (dbcAMP) (Nakagawara 2004, Tremblay et al. 2010). For example, neuronal differentiation of Neuro-2a cells is induced by cell cycle inhibitors (Inokoshi et al. 1999) and the treatment of dbcAMP can differentiate them into dopamine neurons (Tremblay et al. 2010). In addition, neurite outgrowth of these cells is observed when treated with lactacystin (Fenteany et al. 1994), α-melanocyte-stimulating hormone (Adan et al. 1996) and mevastatin (Evangelopoulos et al. 2009). Generally, tumor protein 53 (p53) regulates the cell cycle, induction of apoptosis, and cellular senescence under various stress conditions (Oren and Rotter 1999, Riley et al. 2008). In neuronal cells, p53 is also involved in neuronal differentiation, neurite outgrowth and axonal regeneration (Arakawa 2005, Tedeschi and Di Giovanni 2009). The latest studies have reported that the level of p53 mRNA and protein is elevated by the inducer of neuronal differentiation, nerve growth factor (NGF), or RA in PC-12

neuron-like pheochromocytoma cells or SH-SY5 human neuroblastoma cells (DiGiovanni et al. 2006, Zhang et al. 2006, Eizenberg et al. 1996). In addition, neurite outgrowth-related proteins such as growth associated protein-43 (GAP-43), Ras-related protein 13 (Rab13), cyclic guanosine monophosphate-dependent protein kinase type I (cGKI) and Coronin 1b were regulated by p53 (DiGiovanni et al. 2006, Zhang et al.


2006, Tedeschi et al. 2009a, 2009b). The aim of this study was to determine whether IR could induce the neuronal differentiation in Neuro-2a cells and the activation of p53 is involved in this process. Our results showed that IR induced the neurite outgrowth and the increase of neuronal markers in Neuro-2a cells. Furthermore, this IR-induced neuronal differentiation was blocked by inactivators of p53 and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), which suggests that IR is able to trigger neuronal differentiation, and that the activation of p53 via PI3K is an important step of IR-induced differentiation of Neuro-2a cells. MATERIALS AND METHODS Cell culture Mouse neuroblastoma Neuro-2a cells were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s Medium (DMEM; Hyclone, Logan, UT, USA) supplemented with 10 % (volume/volume) fetal bovine serum (Gibco, Grand Island, NY, USA), 100 U/ml penicillin (Sigma, St. Louis, MO, USA), and 100 µg/ml streptomycin (Sigma) at 37 °C in a 5 % CO2-humidified atmosphere. The cells were subcultured every 2-3 days.

For a primary neural stem cell culture, the hippocampus tissues of the brains were isolated in newborn C57BL/6 mice. After washing twice in Hank’s Balanced Salt Solution (HBSS; Sigma), the hippocampus tissues were digested in a digestion buffer containing 10 units/ml papain (Sigma), 0.45 mg/ml cysteine (Sigma) and 1000 units/ml DNase I (Sigma) in HBSS, pH 7.6, at 37 °C for 30 min, and were shaken gently every 5


min. The digested tissues were washed in HBSS three times and then triturated in DMEM/F12 (Gibco) using a pipette. The cells were cultured in a growth media (1× N2 supplement (Gibco), 20 ng/ml epidermal growth factor (EGF; Gibco), 20 ng/ml basic fibroblast growth factors (bFGF; Gibco), and 2 μg/ml gentamycin (Gibco) in DMEM/F12) in a multi-well chamber slide (Corning, New York, NY, USA) coated with poly-D-lysine (5mg/ml) (Sigma) at 37 °C in a 5 % CO2-humidified atmosphere (Sen et al. 2002). The growth media were changed every other day. Irradiation and treatment of cells 3-5 × 105 cells were plated in a 100 mm dish or multi-well chamber slide and incubated for 24 hr before irradiation. The cells in a culture dish were exposed to 137Cs γ-rays (0.95 Gy/min) at 1, 4, 8, and 16 Gy with a Gamma Cell 40 Exactor (Nordion International Inc., Ottawa, Ontario, Canada) and the culture media were replaced with fresh media within 30 minutes. For p53 inhibition, PFT-α (Sigma) was added to the culture media at the final concentration of 20 or 40 µM 2 hr prior to the irradiation or p53-siRNA (Trp53 Silencer Select Pre-designed siRNA, ID: s75472; Invitrogen, Carlsbad, CA, USA) was transfected to Neuro-2a cells using Lipofectamine RNAiMAX (Invitrogen) using the manufacturer’s protocol at 24hr prior to the irradiation. After incubation for 72hr, the effect of PFT-α and p53-siRNA was identified through western blotting. For the experiments using

inhibitors on mitogen-activated protein kinase kinase (MEK) (PD98059; Cell Signaling Technologies, Boston, MA, USA), protein kinase A (PKA) (H-89; Cell Signaling Technologies), p38 (SB203580; Cell Signaling Technologies), PI3k (LY294002; Cell Signaling Technologies), tropomyosin receptor kinase A (Trk A) (AG879; Tocris, Moorend Farm Avenue, Bristol, UK), tropomyosin receptor kinase B (Trk B) (ANA12;


Tocris), ataxia telangiectasia mutated (ATM) (KU55933; Selleckchem, Houston, TX, USA) and ataxia telangiectasia and Rad3-related protein (ATR) (VE821; Selleckchem), Neuro-2a cells were treated with these inhibitors at 5 or 10 µM, 2 hr prior to the irradiation. Assay of neurite outgrowth Microscopic images of cells were captured using a DFC500 R2 digital camera (Leica, Wetzlar, Germany) at 72 hr after irradiation of Neuro-2a cells. To determine the rate of neurite-bearing cells and the neurite length, approximately 200 cells from three randomly taken images were analyzed each using Image J software (ver. 1.46r; National Institutes of Health, Bethesda, MD, USA). The cells with neurite longer than the cell diameter were regarded as neurite-bearing cells, and only neurite-bearing cells were included in calculating the neurite length (DiAnna et al. 2003). Western blot The cells were lysed in a lysis buffer (50 mM Tris-Cl, pH 7.6, 150 mM NaCl, 1% NP-40, 25 mM NaF, 1 mM Na3VO4, 2 mM ethylene glycol tetraacetic acid (EGTA; Sigma), protease inhibitor cocktail (Sigma)) for 30 min on ice. After centrifugation at 16000 × g for 10 min, and 4 °C for 15 min, the supernatant was collected, and its protein concentration was then measured using a bicinchoninic acid (BCA) protein assay kit (Thermo, Rockford, IL, USA). Proteins (25-30 μg) were resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (GE Healthcare, Little Chalfont, Bucks, UK). Membranes were blocked in 5 % skim milk

(Sigma) or bovine serum albumin (BSA; Sigma), and incubated overnight at 4 °C with antibodies of microtubule-associated protein 2 (Map2) (1:1000 dilution; Sigma), neuronal nuclei (NeuN) (1:1000 dilution; Millipore, Temecula, CA, USA), neuronal class III beta-tubulin (Tuj-1) (1:1000 dilution; Cell Signaling Technologies), p53 (1:1000 dilution; Cell Signaling Technologies), phospho-p53 (p-p53) (1:1000 dilution; Cell Signaling Technologies), protein kinase B (AKT) (1:1000 dilition; Cell Signaling Technologies), phospho-AKT (p-AKT) (1:1000 dilution; Cell signaling Technologies), glial fibrillary acidic protein (GFAP) (1:1500 dilution; Cell signaling Technologies) and β-actin (1:2000 dilution; Cell Int J Radiat Biol Downloaded from informahealthcare.com by Fudan University on 05/08/15 For personal use only.

Signaling Technologies). After washing with Tris-buffered saline-Tween 20 (TBST), the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G (IgG) or antirabbit IgG antibody (1:2500 dilution; Cell Signaling Technologies) for 1 hr. The membranes were washed in TBST, and the immunoreactives were then detected using an enhanced chemiluminescence detection kit (Millipore). The results were visualized and analyzed using a digital gel imaging system (EDAS290; Eastman Kodak Co., Rochester, NY, USA).

Total RNA isolation and Real-time PCR The total RNA was isolated using an easy-BLUE total RNA extraction kit (iNtRON Biotechnology, Seongnam-Si, Gyeonggi-do, Korea). A quantification and purity check of the RNA was performed based on an absorbance rate of A260/A280. 5 µg aliquots of total RNA were reverse transcribed into cDNA using moloney-murine leukemia virus (M-MLV) reverse transcriptase (Promega, Madison, WI, USA). The cDNA (1 µl RT product) was analyzed through real-time PCR using a StepOne Real-Time PCR system (Applied Biosystems; Grand Island, NY, USA) with SYBER Green TOPrealTM qPCR 2X PreMIX (Enzynomics, Daejeon, Korea). The primers were as follows: GAP43 F: 5’AACGGAGACTGCAGAAAGCA-3’ and R: 5’-CTCATCCTGTCGGGCACTTT-3’; Rab13 F: 5’-CTTCAACAGCACTTACATCT-3’ and R: 5’CGGTAATAGGCGGTAGTTAT-3’; β-actin F: 5’-GCAAGCAGGAGTACGATGAG-3’ and R: 5’-AGGGTGTAAAACGCAGCTCA-3’. The amplification conditions of GAP43,

Rab13 and β-actin were as follows: 95°C for 15 min, followed by 40 cycles at 95°C for 15 s, 60°C for 10 s, and 72°C for 15 s. The β-actin was used as a control for normalization. Immunocytochemistry Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15


min and permeabilized in 0.5 % Triton X-100 in PBS for 10 min at room temperature (RT). The cells were washed in PBS for 5 min and incubated with a blocking buffer containing 10% normal goat serum (Thermo sci., Grand Island, NY, USA), 5% BSA and 0.5 % Tween 20 in PBS at RT for 2 hr. The cells were then incubated for 2hr at RT with primary antibodies of Nestin (1:100 dilution, conjugated Cy3; Millipore) and Tuj-1 (1:400 dilution, conjugated Alexa; Millipore). The cells were washed for 10 min in PBS, and Hoechst 33258 (50 nM) (Sigma) was used for the counterstaining of nuclei. Fluorescent images were captured using a DFC500 R2 digital camera (Leica). To determine the ratio of Tuj-1 positive or Nestin positive cells, approximately 500 Neuro2a cells or 300 neurospheres of NSCs from three to five randomly taken images were each analyzed by Image J software (ver. 1.46r; National Institutes of Health). Statistical Analysis All data were presented as the mean ± standard deviation (SD) of triplicated experiments. The statistical significance was analyzed using an analysis of variance (ANOVA) followed by a Tukey’s test for multiple comparisons and 2-way ANOVA for the synergistic effects using SPSS statistic 22 (IBM, Armonk, NY, USA). p < 0.05 was considered significant. RESULTS

Induction of neuronal differentiation by ionizing radiation in Neuro-2a cells We evaluated the effect of ionizing radiation (IR) on neuronal differentiation in Neuro-2a cells. The cells were exposed to 4-16 Gy of IR, and incubated for 72 hr. The viability of the cells was decreased, and cell cycle G2 arrest and apoptosis were increased by IR in a dose-dependent manner (data not shown). Neurite outgrowth was measured as described


in the materials and methods. As shown in Figure 1A and 1B, the rate of neurite-bearing cells was significantly increased by 4 Gy (34 %, p < 0.05), 8 Gy (89 %, p < 0.01) and 16 Gy (88 %, p < 0.01) of IR in a dose-dependent manner. In addition, the neurite length of neurite-bearing cells was significantly increased by IR in a dose-dependent manner (1.6 fold at 4 Gy, p < 0.05; 4.0 fold at 8 Gy, p < 0.01; 4.2 fold at 16 Gy, p < 0.01). We then confirmed the change in the expression of neuronal maker proteins, Map2, and NeuN in Neuro-2a cells. IR at 4 Gy and 8 Gy markedly increased the expressions of Map2 (p < 0.01) and NeuN (p < 0.05) (Figure 1C and 1D). Inhibition of IR-induced neuronal differentiation by inactivation of p53 in Neuro-2a cells To investigate the role of p53 in IR-induced differentiation, we first examined the phosphorylation of p53 by IR. As shown in Figure 1C and 1D, IR significantly induced the phosphorylation of p53 at 4 Gy and 8 Gy. Next, we investigated whether the activation of p53 is required for IR-induced neuronal differentiation. The Neuro-2a cells were treated with PFT-α, an inactivator of p53, at 20 or 40 μM for 2 hr, and then exposed to IR at 8 Gy and incubated for 72 hr. The PFT-α treatment abolished the IR-induced phophorylation of p53, showing that PFT-α efficiently inhibited the activation of p53 by IR (Figure 2A and 2B). The IR-induced increases of neuronal markers, Map2 and NeuN,

were also significantly abolished by PFT-α treatment (Figure 2A and 2B). As shown in Figure 2C, the increase of neurite length induced by IR was also significantly ameliorated by 40 μM PFT-α (1.8 fold increase) compared to IR control (3.1 fold increase). To further confirm the role of p53, we also examined the effect of p53-siRNA on the neurite length. Similar to the result of PFT-α, p53-siRNA significantly inhibited the IR-induced neurite


outgrowth (Figure 2D). These results suggest that the activation of p53 is a critical step for IR-induced neuronal differentiation in Neuro-2a cells. Inhibition of IR-induced neurite outgrowth by selective inhibition of PI3-Kinase We evaluated the involvement of protein kinases known to be associated with neurite outgrowth signaling. The Neuro-2a cells were treated with inhibitors of MEK (PD98059), p38 (SB203580), PKA (H-89) and PI3K (LY294002) prior to the exposure to IR at 8 Gy. As shown in Figure 3A, the IR-induced neurite outgrowth was not affected by inhibitors of MEK, PKA and p38, but was decreased by the inhibitor of PI3K in a dose-dependent manner (2.8 fold at 5 μM, p < 0.05; 1.9 fold at 10 μM, p < 0.01) compared to IR control (3.8 fold at 8 Gy), showing that PI3K activation is essential for IR-induced neurite outgrowth. In addition, we investigated the relation between IR-induced neurite outgrowth and DNA damage and neurotrophin signaling. The Neuro-2a cells were treated with inhibitors of neurotrophin receptors, Trk A (AG879) and Trk B (ANA12), and DNA damage recognition factors, ATM (KU55933) and ATR (VE821), before the exposure to IR. However, the inhibitors of Trk A, TrkB, ATM and ATR did not block the neurite outgrowth by IR in Neuro-2a cells (Figure 3B).

Suppressed expression of neurite outgrowth-related genes and neuronal markers by PI3K inhibition in irradiated Neuro-2a cells To investigate the role of PI3K in IR-induced neurite outgrowth and neuronal differentiation, we examined the expression levels of neurite outgrowth related genes (GAP43 and Rab13) and neuronal marker proteins (GFAP, Tuj-1, Map2 and NeuN) in


irradiated Neuro-2a cells. The mRNA levels of GAP43 and Rab13 were increased by IR, but the pretreatment of LY294002 ameliorated these increases (Figure 4A and 4B). The expressions of neuronal marker proteins were then examined. As shown in Figure 5, the astrocyte marker, GFAP, was not affected by IR or LY294002. However, the neuronal markers (Tuj-1, Map2 and NeuN) were induced by IR, and this induction was abolished by LY294002. In addition, we examined the expression of Tuj-1 (neuronal marker) and Nestin (neural stem/progenitor cell marker) using immunocytochemistry in irradiated Neuro-2a cells. IR increased the Tuj-1 positive cells and decreased the Nestin positive cells, and LY294002 almost completely suppressed the effects of IR (Figure 6). These results suggest that PI3K is required for IR-induced neuronal differentiation in Neuro-2a cells. Inhibition of PI3K-AKT and p53 activation by PI3K inhibition in irradiated Neuro2a cells To confirm the effects of PI3K inhibition on PI3K-AKT, p53 signaling, and neuronal differentiation, we examined the protein levels of phosphorylated AKT and p53, and neuronal marker proteins in Neuro-2a cells. The PI3K inhibitor, LY294002, decreased the level of phophorylated AKT in irradiated cells (Figure 5), showing that LY294002 efficiently blocked the PI3K-AKT signal. In addition, LY294002 suppressed the

phosphorylation of p53 in irradiated cells (Figure 5), which suggests that the PI3K-AKT signal is an upstream regulator of p53 activation in IR-induced neuronal differentiation. The IR-induced increase of neuronal marker proteins (Tuj-1, MAP2, NeuN) were also suppressed by LY294002. Induction of neuronal differentiation by IR and its suppression by PI3K inhibition


in mouse primary neural stem cells We evaluated the effect of IR on neuronal differentiation in primary neural stem cells (NSC). Because the radiation doses of 2Gy or higher sharply decreased the viability of the cells (data not shown), we selected a radiation dose of 1Gy in this experiment. NSCs were treated with a vehicle or PI3K inhibitor (LY294002) prior to exposure to IR at 1 Gy, and incubated for 72 hr. In accordance with the results of Neuro-2a cells, the increase of neurite outgrowth and Tuj-1-positive cells, and the decrease of Nestin-positive cells were observed in irradiated primary NSCs by immunocytochemistry analysis, and these effects were reversed using a PI3K inhibitor (Figure 7). DISCUSSION In general, ionizing radiation (IR) induces apoptosis and/or premature senescence to cell and tissue through DNA damage and oxidative stress (Oh et al. 2001, Jackson et al. 2011). An exposure of brain to radiation in animal models causes chronic inflammation as manifested by an abnormal increase of cytokines and activated microglias, and subsequent cognitive loss resulting from an injury of the hippocampus, which is a key part for learning and memory (Crossen et al. 1994, Abayomi 1996). In addition, radiation affects the neuronal stem cells (NSC), a decisive factor of neurogenesis and neuronal repair. An exposure of the C17.2 neural progenitor cell line to γ-ray at 16 Gy induces the

cell death and generation of cellular ROS (Oh et al. 2013). In NSCs isolated from E16 rat embryos, irradiation at 10 Gy leads to an increase of apoptosis and an inhibition of neuronal differentiation (Kanzawa et al. 2006). Contrastively, some studies have reported that irradiation at 8 Gy to neuronal precursor cells isolated from newborn mouse causes no significant changes in neuronal differentiation (Chen et al. 2012) and NSCs prepared


from mouse ES cells retained the ability of proliferation and differentiation when exposed to 1 Gy of X-ray (Isono et al. 2012). Although a number of studies have focused on the damage of neuronal cells and/or tissues caused by radiation in vitro and in vivo, several studies reported the influence of IR on neuronal differentiation showing that the exposure of X-ray induced the neurite outgrowth in PC-12 cells (Abeyama et al. 1995) and the astrocyte-specific differentiation in NSCs (Ayumi et al. 2012). In addition, an increase of astrocytes is observed in the whole brain of irradiated mice between 120 and 180 days after a single dose of 20-45 Gy radiation. However, the influences of IR on neuronal differentiation are largely unknown (Chiang et al. 1993). Thus, we investigated the effect of radiation on neuronal differentiation in Neuro-2a neuroblastoma cells as an in vitro model. As expected, the radiation at 4, 8, and 16 Gy significantly decreased the viability of the cells, and increased the cell cycle G2 arrest and apoptotsis in a dose-dependent manner (data not shown). However, it was observed that neurite outgrowth was significantly induced in the surviving cells by IR in a dosedependent manner (Figure 1A and 1B). Although neurite outgrowth is a marker of neuronal differentiation, it can be induced by several factors without neuronal differentiation (Min et al. 2006). Therefore, we further examined the effects of IR on

several neuronal differentiation markers in Neuro-2a cells and primary NSCs. We demonstrated in this study that IR increased the expressions of neuronal marker proteins, Map2, NeuN, and Tuj-1 (Figure 1C and 1D, Figure 6), and decreased the expression of Nestin, a neural stem cell marker, in Neuro-2a cells (Figure 6). Although it was reported that astrocytic differentiation is accelerated by X-rays (Ayumi et al. 2012), our results


showed that the IR did not induce any change of the level of astrocyte marker, GFAP (Figure 5). In addition, GFAP positive cells were not detected by ICC in irradiated Neuro-2a cells or NSCs (data not shown). These results suggested that IR induces neuronal differentiation without pharmacological stimulation in Neuro-2a cells. Interestingly, we observed that IR-induced neuronal differentiation was related with p53. Other groups have reported that neurite outgrowth and axonal regeneration are induced by p53 over-expression in primary cortical neurons prepared from rat embryonic cortices (DiGiovanni et al. 2006) and similar results have been reported for neuronal-like PC12 and neuroblastoma cells (Hughes et al. 2000, Montano 1997, Poluha et al. 1996, 1997). Our study confirmed the necessity of p53 for IR-induced neurite outgrowth and neuronal differentiation by IR. IR at 4 and 8Gy significantly led to the phosphorylation of p53 (Figure 1C and 1D), and the treatment of a p53 inactivator, PFT-α, suppressed the neurite outgrowth and expressions of Map2 and NeuN in Neuro-2a cells exposed to IR (Figure 2). In addition, the suppression of p53 expression by p53-siRNA decreased the neurite outgrowth (Figure 2D). These results indicate that the activation of p53 is required for neurite outgrowth and neuronal differentiation by IR in Neuro-2a cells. The p53-dependent neurite outgrowth is known to be associated with neurotrophin or similarly to neurotrophin, retinoic acid (RA) signaling. Neurotophins such as nerve

growth factor (NGF), brain-derived neurotrophin factor (BDNF), and RA induces neurite outgrowth and neuronal differentiation through Trk or other receptors, causing the activation of p53 by its downstream signaling cascades, including MAPK, cAMP and PI3K pathways ((DiGiovanni et al. 2006, Zhang et al. 2006, Patapoutian and Reichardt 2001, Lane and Bailey 2005, Canon et al. 2004, Poongodi et al. 2002, Moore and


Goldberg 2011, Jingbo et al. 2012, Gracia et al. 2002). In addition, the p53-dependent transcription of cytoskeleton remodeling-related genes including GAP-43, cGK1, Coronin 1b and Rab13 is important for neurite outgrowth (DiGiovanni et al. 2006, Zhang et al. 2006, Tedeschi et al. 2009a, 2009b). Thus, we examined the upstream mediators of p53 activation in IR-induced neurite outgrowth using inhibitors of MEK, p38, PKA and PI3K. Only the PI3K inhibitor, LY294002, inhibited the IR-induced neurite outgrowth, while the inhibitors of MEK, PKA and p38 had no effect (Figure 3A). The elevation of neurite outgrowth-related genes, GAP-43 and Rab13, by IR was attenuated by LY294002 (Figure 4). In addition, the inhibition of PI3K resulted in a decrease of p53 and AKT phosphorylation, and the neuronal marker expression (Tuj-1, Map2 and NeuN) (Figure 5). In addition, the increase of Tuj-1 positive cells and the decrease of Nestin positive cells were abolished by the inhibition of PI3K in irradiated Neuro-2a cells (Figure 6). These results show that the neurite outgrowth and neuronal differentiation by IR required the activation of p53 through PI3K in Neuro-2a cells. Because Neuro-2a cells are derived from neuroblastoma, their responses to IR can be different from those of NSCs in the brain. Therefore, to investigate the in vivo relevance of the results from Neuro-2a cells, we examined the effect of IR on neuronal differentiation in mouse primary NSCs. IR at 1Gy significantly increased neurite

outgrowth and Tuj-1 positive cells and decreased Nestin positive cells in primary NSCs (Figure 7). The treatment of LY294002 abolished the IR-induced differentiation of primary NSCs (Figure 7). These results suggest that the neuronal differentiation is induced by IR and this process requires the activation of PI3K in primary NSCs as well as Neuro-2a cells.


Next, we investigated the upstream signaling of PI3K in irradiated Neuro-2a cells. A neurotrophin, such as NGF and BDNF, requires receptors including Trk A and Trk B, for the trigger of neurite outgrowth or neuronal differentiation-related signal transduction (Patapoutian and Reichardt 2001). The key sensors of DNA lesions, such as ATM and ATR, which are activated by DNA damage from IR are implicated with DNA break repair, cell cycle and apoptosis (Caporali et al. 2004, Yoshida et al. 2008, Myers and Cortez 2006). Thus, we examined the association of IR-induced neurite outgrowth with the neurotrophin receptors, Trk A and Trk B, and the DNA damage response proteins, ATM and ATR. However, the inhibitors of Trk A, Trk B, ATM and ATR all failed to affect the IR-induced neurite outgrowth (Figure 3B). These results suggest that the signaling of neurite outgrowth by IR is not mediated by the neurotrophin receptors and the DNA damage signaling in Neuro-2a cells. Some studies have reported that the neurite outgrowth or neuronal differentiation is associated with reactive oxygen species (ROS) (Min et al. 2006, Xiaohui et al. 2011, Yona et al. 2001), and the retinoic acid receptors are required for neurogenesis (Shuiliang et al 2012). Further studies should investigate how IR-induced neuronal differentiation associate with ROS and other receptor signaling. In this study, we did not examine the neural function of the differentiated Neuro-2a cells, and it is not clear whether IR-induced neuronal differentiation could bring a beneficial

effect such as the replenishment of damaged neuronal cells or the adverse effect resulting in the depletion of neural stem cells in vivo. Accordingly, future studies should investigate the neural functional verification of differentiated Neuro-2a cells such as the secretion of neurotransmitters, the expression of neurotransmitter receptors and the influence of IR on differentiation in animal models to verify the in vivo relevance of our


results. These studies will contribute to an understanding of the acute and chronic effects of IR on neuronal cells and to the development of strategies to reduce the degenerative damages of the brain during brain radiotherapy. In conclusion, our results suggest that IR is able to trigger the neurite outgrowth and neuronal differentiation in Neuro-2a cells, and the activation of p53 through PI3K may be an important step for IR-induced neuronal differentiation in these cells. Acknowledgments This work was supported by National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2012M2A2A7035656). Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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Figure legends Figure 1. Neuronal differentiation induced by IR in Neuro-2a cells. At 24 hr after plating, the cells were exposed to IR at 4-16 Gy and incubated at 37 ˚C for 72 hr. The microscopic images showed that the neurite outgrowth was induced by IR (A) (× 200 magnification). Each 200 cells in three randomly taken images were analyzed for the rate


of neurite-bearing cells, and the neurite length was analyzed by Image J software (B). Subsequently, cells were lysed and analyzed by a western blot for neuronal markers, Map2 and NeuN (D), and phosphorylated p53 (D). The results represent the mean ± SD from triplicate data. *p < 0.05, **p < 0.01 vs 0 Gy group.


Figure 2. Effect of p53 inhibition on neuronal differentiation in irradiated Neuro-2a cells. At 24 hr after plating, the cells were treated with PFT-α at 40 μM for 2 hr and p53siRNA for 24hr, and then exposed to IR at 8 Gy and incubated at 37 ˚C for 72 hr. The cells were lysed and analyzed by western blot for phosphorylated p53 and neuronal markers, Map2 and NeuN (A), and the expression level of each protein was quantified by


densitometry (B). In addition, each 200 cells in three randomly taken images were analyzed for the neurite length on the treatment of PFT-α (C) and p53-siRNA (D) using Image J software. The results represent the mean ± SD from triplicate data. *p < 0.05, **p < 0.01 vs non-treated group, †p < 0.05, ††p < 0.01 vs radiation only group.


Figure 3. Effect of inhibitors on neurite outgrowth signal-related proteins in irradiated Neuro-2a cells. At 24 hr after plating, the cells were treated with inhibitors of MEK (PD98059), PKA (H-89), p38 (SB203580) and PI3K (LY294002) (A), and Trk A (AG879), Trk B (ANA12), ATM (KU55933) or ATR (VE821) (B) at 5 or 10 μM for 2 hr, and then exposed to IR at 8 Gy and incubated at 37 ˚C for 72 hr. Each 200 cells in


three randomly taken images were analyzed for the neurite bearing cells by Image J software. The IR-induced neurite outgrowth is not affected by the inhibitors of MEK, PKA, p38, Trk A, Trk B, ATM, and ATR but was decreased by the inhibitor of PI3K (A). The results represent the mean ± SD from triplicate data. *p < 0.05, **p < 0.01 vs radiation only group.


Figure 4. Inhibition of neurite outgrowth-related genes by PI3K inhibitor (LY294002) in irradiated Neuro-2a cells. At 24 hr after plating, the cells were treated with LY294002 at 10 μM for 2 hr, and then exposed to IR at 8 Gy and incubated at 37 ˚C for 72 hr. Subsequently, cells were lysed and analyzed by real-time PCR for the level of GAP43 (A) and Rab13 mRNA (B). The results represent the mean ± SD from triplicate data. *p

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