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Dual cell protective mechanisms activated by differing levels of oxidative stress in HT22 murine hippocampal cells a

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Kazunori Sato , Yuki Yamanaka , Masaya Ishii , Kazusa Ishibashi , Yurina Ogura , Ritsuko Ohtani-Kaneko

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, Masugi Nishihara & Taku Nedachi

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Graduate School of Life Sciences, Toyo University, Gunma, Japan

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Faculty of Life Sciences, Toyo University, Gunma, Japan

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Bio-Nano Electronics Research Centre, Toyo University, Saitama, Japan

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Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan Published online: 25 Jul 2014.

To cite this article: Kazunori Sato, Yuki Yamanaka, Masaya Ishii, Kazusa Ishibashi, Yurina Ogura, Ritsuko OhtaniKaneko, Masugi Nishihara & Taku Nedachi (2014) Dual cell protective mechanisms activated by differing levels of oxidative stress in HT22 murine hippocampal cells, Bioscience, Biotechnology, and Biochemistry, 78:9, 1495-1503, DOI: 10.1080/09168451.2014.936343 To link to this article: http://dx.doi.org/10.1080/09168451.2014.936343

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Bioscience, Biotechnology, and Biochemistry, 2014 Vol. 78, No. 9, 1495–1503

Dual cell protective mechanisms activated by differing levels of oxidative stress in HT22 murine hippocampal cells Kazunori Sato1, Yuki Yamanaka2, Masaya Ishii2, Kazusa Ishibashi2, Yurina Ogura1, Ritsuko Ohtani-Kaneko1,2,3, Masugi Nishihara4 and Taku Nedachi1,2,* 1

Graduate School of Life Sciences, Toyo University, Gunma, Japan; 2Faculty of Life Sciences, Toyo University, Gunma, Japan; 3Bio-Nano Electronics Research Centre, Toyo University, Saitama, Japan; 4Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan

Received December 18, 2013; accepted April 14, 2014

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http://dx.doi.org/10.1080/09168451.2014.936343

Oxidative stress is recognized as one of the pathogenic mechanisms involved in neurodegenerative disease. However, recent evidence has suggested that regulation of cellular fate in response to oxidative stress appears to be dependent on the stress levels. In this study, using HT22 cells, we attempted to understand how an alteration in the oxidative stress levels would influence neuronal cell fate. HT22 cell viability was reduced with exposure to high levels of oxidative stress, whereas, low levels of oxidative stress promoted cell survival. Erk1/2 activation induced by a low level of oxidative stress played a role in this cell protective effect. Intriguingly, subtoxic level of H2O2 induced expression of a growth factor, progranulin (PGRN), and exogenous PGRN pretreatment attenuated HT22 cell death induced by high concentrations of H2O2 in Erk1/2-dependent manner. Together, our study indicates that two different cell protection mechanisms are activated by differing levels of oxidative stress in HT22 cells. Key words:

oxidative stress; progranulin; Erk1/2; HT22 cells

Introduction Reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide (H2O2), and hydroxyl radical are involved in a variety of biological responses. The production of ROS is balanced by the production of free radicals, while ROS removal is controlled by the antioxidant defense system inside cells and in the surrounding environment. Oxidative overload or disruption of the antioxidant defense system results in oxidation of proteins, lipids, and DNA ultimately leading to apoptotic cell death, or in extreme cases, necrotic cell

death.1) Thus, maintaining an appropriate level of ROS is crucial for maintaining cellular homeostasis. Various changes in neurological environments, including inflammation, infection, exposure to radiation, and heavy alcohol consumption results in ROSinduced oxidative stress in the central nervous system (CNS).2,3) In the CNS, oxidative stress is proposed to be involved in the neurological deterioration occurring in several types of neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease, and amyotrophic lateral sclerosis.4–6) When neuronal cells located in the CNS are exposed to strong or continuous oxidative stress, several cellular pathways are activated resulting in mitochondrial dysfunction and the activation of death signaling pathways.1,7) One such signaling cascade activated by ROS accumulation is the mitogenactivated protein (MAP) kinase pathway, which involves the following three major MAP kinases: extracellular signal-related kinase (Erk1/2), c-Jun terminal kinase (JNK), and p38 MAP kinase (p38).8,9) Activation of these MAP kinase family members appears to be related to cellular injury and apoptosis during the development of some neurodegenerative diseases.10) In addition, it was reported that ROS activate phosphatidylinositol 3-kinase (PI3K) cascades via oxidation and inactivation of the phosphatase and tensin homology (PTEN) phosphatase, which negatively regulates PI3K activation.11,12) Moreover, Nrf 2, Ref1, and p66 Shc have also been implicated in the ROS-dependent regulation of cell fate.13) Growth factors such as insulin-like growth factor-1 (IGF-1) and brain-derived growth factor (BDNF) have been reported to prevent the neuronal damage caused by oxidative stress.14) PI3K activation by IGF-1 and BDNF plays an important role in this neuroprotective effect.14) Moreover, it was reported that BDNF-dependent Erk1/2 activation was also important for the

*Corresponding author. Email: [email protected] Abbreviations: BCA, bicinchoninic acid assay; BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; Erk, extracellular signal-related kinase; LDH, lactate dehydrogenase; PBS, phosphate-buffered saline; PGRN, progranulin; PI3K, phosphatidylinositol 3-kinase; RT, room temperature; TBS, Tris-buffered saline. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry

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survival of cerebellar granule cells. Recently, a novel growth factor, progranulin (PGRN) was also reported to protect against oxidative stress.16) However, the intracellular signaling pathway involving PGRN has not been well characterized, although recent efforts have demonstrated that PGRN activates PI3K and Erk1/2 in neuronal and other cell types.17–19) Xu et al. demonstrated that either pharmacological inhibition of Erk1/2 or the PI3K pathway blocked PGRN-dependent neuroprotection against the neurotoxic agent MPP+.20) These data suggest that PGRN activates general growth factor signaling pathways, thereby protecting neuronal cells. Overall, these data indicate ROS accumulation induces neuronal cell death that is reversed by growth factors such as IGF-1, BDNF, and PGRN. On the other hand, different aspects of ROS-dependent cellular responses are also well established. It has been reported that ROS serves as an intracellular second messenger in cells and regulates cell proliferation, differentiation, protein modification, and gene expression.21–29) Sundaresan and colleagues reported that growth factors (including EGF and PDGF), chemokines, and cytokines induced ROS production, thereby activating MAPKs that regulate cell proliferation and matrix production.30) In addition, it has been demonstrated that subtoxic levels of ROS had a proliferation-promoting effect in some cancer cells.31,32) Therefore, small amounts of ROS serve as signaling molecules for stimulating cell proliferation. Overall, the physiological relevance of ROS accumulation is complex. In particular, the details of cellular responses and mechanisms when subjected to different levels of oxidative stress are still not fully understood. In this study, we investigated how different levels of oxidative stress control neuronal cell fate using the HT22 murine hippocampal cell line.33)

Materials and methods Materials. The western blot detection kit (ECL plus or ECL prime detection reagents) was purchased from GE Healthcare Inc. (Rockford, IL, USA). Dulbecco’s Modified Eagle Medium (DMEM), penicillin/ streptomycin, and trypsin-EDTA were purchased from Nakarai Tesque (Kyoto, Japan). Cell culture equipment was purchased from BD Biosciences (San Jose, CA, USA). Calf serum (CS) and fetal bovine serum (FBS) were obtained from BioWest (Nuaille, France). Immobilon-P was purchased from Millipore Corp. (Bedford, MA, USA). Unless otherwise noted, all chemicals were of the purest grade available from Nakarai Tesque, Sigma Chemicals (St. Louis, MO, USA), or Wako Pure Chemical Industries, Ltd (Osaka, Japan). Cell culture. HT22 mouse hippocampal neuronal cells33) were maintained in DMEM supplemented with 10% FBS, 30 g/mL penicillin, and 100 g/mL streptomycin (growth medium) at 37 °C in a 5% CO2 atmosphere. For biochemical studies, cells were grown on 6-well plates (Orange Scientific, Braine-l’Alleud, Belgium) at a density of 1 × 105 cells/well in 2 mL of

growth medium or on 96-well plates (Orange Scientific) at a density of 3–5 × 103 cells/well in 0.2 mL of growth medium. MTT assay. HT22 cells were seeded on 96-well plates at a density of 3–5 × 103 cells/0.2 mL. After 24 h of incubation, HT22 cells were treated with different concentrations of H2O2 (0–1000 μM) or glutamate (0–10 mM) for 15 h, and then cell viability was measured. The cell viability was evaluated using the MTT cell count kit (Nacalai Tesque, Kyoto, Japan) according to the manufacturer’s protocol. Measurement of cell death. HT22 cells were seeded on 96-well plates and the percentage of dead cells was evaluated using the lactose dehydrogenase (LDH) plus kit (Roche Diagnostics K.K., Basel, Switzerland) according to the manufacturer’s protocol. Western blotting. The expression and phosphorylation of each protein was analyzed by western blot analysis. Briefly, the cells were seeded on 6-well plates at a density of 1 × 105 cells/well for 24 h, and then the cells were treated with different concentrations of H2O2 (0–10 M) or glutamate (0–1 mM) for 5 min. Cell lysates were prepared using lysis buffer (2% sodium dodecyl sulfate (SDS), 1% 2-mercaptoethanol, 10% glycerol, 0.0033% bromophenol blue, and 50 mM Tris–Cl (pH 6.8)). Cell lysates were subjected to 10 or 12% SDSpolyacrylamide gel electrophoresis (1:30, bis:acrylamide). Proteins were transferred to a PVDF membrane (Immobilon-P; Millipore), and the membranes were blocked for 30 min in 3% bovine serum albumin (BSA) in Tris-buffered saline (TBS) containing 0.1% Tween-20. Detection of each protein was achieved with 1-h incubation with a 1:1000 dilution of primary antibody (anti-phospho Erk1/2, anti-Erk1/2, anti-phospho Akt, and anti-Akt (Cell Signaling Technology, Danvers, MA, USA)). Specific total proteins were visualized after subsequent incubation with a 1:5000 dilution of anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase using an ECL plus detection procedure (GE Healthcare Inc.). Protein concentrations were determined using a bicinchoninic acid assay (BCA) (Pierce Biotech. Inc.). At least three independent experiments were performed for each condition. Real-time PCR. HT22 cells were seeded on 6-well plates at a density of 1 × 105 cells/well. After 24-h incubation, the cells were treated with different concentrations of H2O2 (0–250 μM) for 12 h. Total RNA was isolated from cells using the High Pure RNA Isolation Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s protocol. cDNAs were synthesized from total RNA using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan). Fluorescence real-time PCR analysis was performed using the StepOne instrument (Life Technologies Corporation, Grand Island, NY, USA) and the SYBR Green

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detection kit according to the manufacturer’s protocol (Life Technologies or KAPA Biosystems Inc. (Woburn, MA, USA). PCR primers for each gene were as follows: for mouse GAPDH: 5′-TGT GTC CGT CGT CGT CTG A-3′ and 5′-CCT GCT TCA CCA CCT TCT TGA-3′; for mouse PGRN: 5′-GTG CCT ATC CAA GAA CTA CAC CA-3′ and 5′-GCA GCA ATG AAT GTG ATC CTC-3′; for mouse BDNF: 5′-CCC ATG AAA GAA GTA AAC GTC C-3′ and 5′-GTC GTC AGA CCT CTC GAA CC-3′; for mouse IGF-1: 5′-TCA TGT CGT CTT CAC ACC TCT TCT-3′ and 5′-CCA CAC ACG AAC TGA AGA GCA T-3′.

Results

However, when cells were exposed to lower concentrations of H2O2 (10 M) or glutamate (0.5 mM), cell viability was significantly increased (Fig. 1(B) and (D)). These results demonstrated that low levels of oxidative stress may not necessarily be toxic to cells; the effect of H2O2 and glutamate was biphasic in HT22 cells. To investigate the mechanism underlying the increase in cell viability when exposed to a low level of oxidative stress, we studied the intracellular signaling events that were activated by low concentrations of H2O2 or glutamate. Since MAP kinases are activated by oxidative stress,8,9) we initially tested whether these molecules were activated by low concentrations of H2O2 or glutamate. Phosphorylation of Erk1/2 was slightly but significantly increased by ~1.5-fold in response to a low concentration of H2O2 or glutamate (Fig. 2(A) and (C)). Erk1/2 activation appeared to be important for cell protection when exposed to a low level of oxidative stress, as the administration of 10 M U0126, a specific MEK1/2 inhibitor, completely abrogated this effect (Fig. 2(B) and (D)).

Low-level oxidative stress increases HT22 cell survival Numerous studies have shown that neuronal cell death is induced by strong oxidative stress.1) We investigated the effect of various concentrations of H2O2 or glutamate on HT22 hippocampal cells, followed by evaluation of cell death using the MTT assay. As expected, when cells were exposed to ≥100 M H2O2 or ≥2 mM glutamate, cell viability gradually decreased in a concentration-dependent manner (Fig. 1(A) and (C)).

High-level oxidative stress induces PGRN expression As shown in Fig. 1, when the cells were exposed to a high concentration of H2O2 or glutamate, cell viability was reduced. It has been demonstrated that cell protective mechanism(s) can be activated by deathpromoting stimuli34); we investigated the activation of these neuroprotective mechanism(s) in response to a high level of oxidative stress. Results from several

Statistical analysis. Comparisons among treatment groups were tested using one-way ANOVA or the Student’s t-test. Differences where the p-value was less than 0.05 were considered statistically significant.

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Fig. 1. Effects of differing levels of oxidative stress on HT22 cell survival. Notes: ((A)–(D)) HT22 cells were treated with different concentrations of H2O2 (0–1000 μM) ((A), (B)) or glutamate (0–10 mM) ((C), (D)) for 15 h, and cell viability was measured using the MTT assay. At least three independent experiments were performed. Statistical analysis was performed as described in the materials and methods (n = 3–6, *p < 0.05).

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experiments indicated that expression of the growth factor PGRN was significantly induced by a high concentration of H2O2 (100 µM) (Fig. 3(A)) in HT22 cells, which is the same concentration that initiated cell death (Fig. 1(A)). In contrast, the expression of BDNF was significantly reduced by 250-M H2O2 treatment (Fig. 3(B)). Similarly, IGF-1 expression was reduced by 100-M and 250-M H2O2 treatment (Fig. 3(C)). Potential role of PGRN induction by high-level oxidative stress Next, we examined the potential role of PGRN induction by high-level oxidative stress. HT22 cells were pretreated with different concentrations of PGRN for 24 h, followed by stimulation with 250-M H2O2 for 15 h. LDH release from cells during the final 15 h was measured to evaluate cell toxicity. As expected, 250-M H2O2 treatment increased HT22 cell death (Fig. 4); however, 1 ng/mL PGRN pretreatment completely attenuated the H2O2-induced cell death (Fig. 4). Intriguingly, pretreatment with 10 or 100 ng/mL PGRN did not reduce the H2O2-dependent cell death, suggesting that an appropriate concentration of PGRN (~1 ng/mL) was required to elicit this protective effect. In the

absence of H2O2 treatment, 1 ng/mL PGRN had no apparent effect on cell viability (Fig. 4). On the other hand, significant effects of PGRN on H2O2-induced apoptosis, assessed by TUNEL assay, were not observed (data not shown), thus, PGRN pretreatment appeared to be important for attenuating necrotic cell death induced by H2O2. Erk1/2 activation was involved in PGRN-dependent neuroprotection in HT22 cells Finally, we studied the mechanism by which PGRN protects HT22 cells from high concentrations of H2O2. Previous finding has suggested that PI3K and MAP kinase cascades are activated by PGRN20); therefore, we investigated the activation of these pathways in HT22 cells. HT22 cells were treated with different concentrations of PGRN for 5 min, and phosphorylation of each signaling molecule was assessed by western blot analysis. As shown in Fig. 5(A), phosphorylation of Erk1/2 was slightly but significantly increased by 1–10 ng/mL PGRN treatment (Fig. 5(A)), while PGRN did not significantly affect Akt phosphorylation (Fig. 5(B)). To investigate the physiological significance of this PGRN-dependent Erk1/2 activation, 10-M U0126 was

Fig. 2. Erk1/2 activation induced by low-level oxidative stress was important for increasing HT22 cell viability. Notes: ((A), (C)) HT22 cells were treated with different concentrations of H2O2 (0–10 μM) (A) and glutamate (0–1 mM) (C) for 5 min. The phosphorylation of MAP kinases was analyzed by western blot analysis. The intensity of each band was quantified using Image J software and normalized against the control. The graph represents the average intensity from at least three independent experiments (n = 3–4, *p < 0.05). ((B), (D)) HT22 cells were treated with different concentration of H2O2 (B) and glutamate (D) for 15 h in the presence of MEK1/2 inhibitor U0126 (10 M). The cell viability was evaluated using the MTT assay (n = 4–5).

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neuronal cells. In particular, chronic exposure of neurons to oxidative stress was proposed to play an important role in neurodegenerative disorders characterized by a progressive loss of cortical neurons.35,36) In addition, the hippocampus, which plays a central role in memory and is one of the most vulnerable areas at early stages of AD,37) is also affected by oxidative stress.38,39) On the other hand, a challenging aspect of evaluating oxidative stress is that low-level oxidative stress can display a beneficial effect.40–42) Therefore, an important question has been raised as to how cells monitor the stress level and consequently activate distinct intracellular signaling to produce protective cellular responses. In the present study, we investigated how HT22 hippocampal neuronal cells responded to different levels of oxidative stress. Because HT22 cells do not possess ionotropic glutamate receptors, this line has often been used for analyzing oxidative glutamate toxicity.43) We used glutamate and H2O2 for inducing oxidative stress to confirm whether the cellular responses are equally observed. When HT22 cells were exposed to a low level of oxidative stress, Erk1/2 was activated, which is likely important for increasing cell viability (Figs. 1 and 2). On the other hand, high-level oxidative stress initiated HT22 cell death and significantly enhanced PGRN expression (Fig. 3). Although the impact of increased PGRN expression in response to a high level of oxidative stress on cell survival still needs to be elucidated, addition of exogenous PGRN attenuated cell death induced by a high concentration of H2O2 in HT22 cells (Figs. 4 and 5).

Fig. 3. High-level oxidative stress induced PGRN gene expression in HT22 cells. Notes: (A–C) HT22 cells were treated with different concentrations of H2O2 (0–250 μM) for 15 h, and the gene expression of each growth factor was evaluated by real-time PCR analysis ((A): PGRN, (B): BDNF, (C): IGF-1). Three independent experiments were performed (n = 3, *p < 0.05).

Erk1/2 activation is involved in cell protection in response to a low level of oxidative stress in HT22 cells A low level of oxidative stress slightly but significantly increased HT22 cell survival (Fig. 1). Similarly, there have been several reports demonstrating the beneficial effects of a low concentration of ROS. First, it was reported that preconditioning with low-level oxidative stress resulted in cellular resistance when the cells

added in the presence of 1 ng/mL PGRN for 24 h, followed by stimulation with 250-M H2O2; cell death was then measured using the LDH assay. In the presence of the Erk1/2 inhibitor, the protective effect of PGRN on H2O2-dependent cell death was completely abolished (Fig. 5(C)), which indicated that PGRN-dependent Erk1/2 activation was crucial for attenuating the oxidative stress-dependent HT22 cell death.

Discussion Accumulation of ROS results in oxidative stress, which directly and indirectly modulates the fate of

Fig. 4. PGRN attenuated oxidative stress-induced HT22 cell death. Notes: HT22 cells were pretreated with different concentrations of PGRN (0–100 ng/mL) for 24 h, and the medium was exchanged with DMEM containing different concentrations of H2O2 (0–250 μM) for 15 h. Cell toxicity was evaluated using the LDH assay (n = 4–5, **p < 0.01, *p < 0.05).

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Fig. 6. Two distinct cell protective mechanisms were observed in HT22 cells in response to different levels of oxidative stress. Notes: (1) Low-level oxidative stress directly increased cell viability via Erk1/2 activation. (2) High-level oxidative stress-induced cell death, whereas, it also exerted cell protective mechanisms perhaps mediated by the autocrine/paracrine effects of PGRN.

were subsequently exposed to a high level of oxidative stress by overexpressing Bcl-2 in PC12 cells.40) Additionally, Ding et al. reported that neuronal cells increased the expression of proteasome subunits following exposure to low levels of oxidative stress and thus reduced neurotoxicity.41) During cellular injury and apoptosis induced by oxidative stress, several types of MAP kinase family are activated.42) In particular, oxidative stress in neuronal cells is often accompanied by activation of MAP kinases such as Erk1/2 and p38.44,45) Furthermore, it has been reported that a pharmacological inhibition of Erk1/2 suppressed oxidative stress-induced cell apoptosis42); these data suggest that the cell death induced by oxidative stress was at least partially mediated by Erk1/2. Meanwhile, several reports have suggested that Erk1/2 activation plays a central role in neuroprotection against oxidative stress.46,47) The diversity of the role of Erk1/2 in neuroprotection may be dependent on the changing kinetics of Erk1/2, such as the amplitude and duration of activation and/or subcellular localization. The diverse bio-effects of Erk1/2 are now under investigation in our laboratory.

Fig. 5. PGRN-dependent Erk1/2 activation was important for cell protection against high-level oxidative stress. Notes: ((A), (B)) HT22 cells were treated with different concentrations of PGRN (0–100 ng/mL) for 5 min. Phosphorylation of Erk1/2 (A) Akt (B) was monitored by western blot analysis. The graph represents the average intensity from five independent experiments (n = 5, *p < 0.05). (C) HT22 cells were pretreated with PGRN in the presence or absence of U0126 (10 M) for 24 h, then stimulated with 250 M H2O2 for 15 h. Cell toxicity was evaluated using the LDH assay (n = 3–6, **p < 0.01).

PGRN expression is enhanced by a high level of oxidative stress An important observation made in the present study was that expression of the PGRN gene was induced by a high level of oxidative stress at the same concentration of H2O2 that initiated cell death (Fig. 3). PGRN (also known as granulin/epithelin precursor (GEP), proepithelin, PC cell-derived growth factor, or acrogranin) is a 67.5-kDa cysteine-rich protein expressed in most cell types.48) Importantly, numerous reports have suggested an association between mutations of the PGRN gene and a certain type of familial frontotemporal lobar degeneration (FTLD) accompanied by the accumulation of ubiquitin-positive inclusion bodies.49,50) In most cases, PGRN mutants encoded incomplete or inactive peptides resulting in a 50% reduction of functional PGRN (haploinsufficiency), which led to neuronal cell death in the CNS.51–53)

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It has been demonstrated that PGRN is expressed in both neurons and microglia in CNS,54,55) and several groups have investigated the regulation of PGRN expression. In non-neural cells, Guerra et al. demonstrated that PGRN expression in fibroblasts was enhanced by physiologically and clinically relevant microenvironmental stresses including hypoxia and acidosis.56) In addition, liposoluble vitamins or steroid hormones were also involved in the control of PGRN expression.57,58) The detailed molecular mechanism of PGRN gene expression regulation also remains elusive. Jiao et al. recently demonstrated that the evolutionarily conserved binding site for microRNA-29b (miR-29b) was in the 3′ untranslated region of human PGRN (hPGRN) mRNA.59) They also demonstrated that ectopic expression of miR-29b decreased hPGRN expression. A recent report suggested that PKC signaling was involved in the regulation of PGRN in human ovarian cancer cell lines.60) In HT22 cells, the molecular machinery regulating PGRN expression in response to a high level of oxidative stress is currently unknown. However, because PGRN mutations associated with FTLD appear to be those of haploinsufficiency, further investigations are important to identify novel mechanisms controlling PGRN expression by the other wildtype PGRN allele. Intriguingly, expression of other neurotropic factors (i.e. IGF-1 and BDNF) was decreased (Fig. 3). These results suggested that induction of PGRN by a high level of oxidative stress was a specific cellular response regulated by specific intracellular signals. Because many reports verified IGF-1 and BDNF as neurotropic factors,61,62) further studies are required to understand why PGRN expression was induced when HT22 cells were exposed to toxic levels of oxidative stress. Exogenous PGRN ameliorated oxidative stressdependent cell death We showed that exogenous addition of PGRN ameliorated HT22 cell death induced by a high level of oxidative stress (Fig. 4). Likewise, there are several reports showing that PGRN exerts neuroprotective effects. For example, Xu et al. showed that extracellular PGRN rescued cortical neurons from cell death induced by glutamate and MPP+.20) In addition, it was also reported that the cell viability in serum-free medium was increased by PGRN overexpression in NSC34 neuron-like cells.16) Moreover, in the mouse brain, PGRN overexpression prevented ischemia-induced brain injury, and PGRN overexpression in glial cells protected cells from LPS-mediated cytotoxicity.63) We found Erk1/2 activation was required for PGRN-dependent cell protective effect (Fig. 5(C)). It has been reported that TNFα-dependent activation of Nrf 2/ARE system, regulating the expression of anti-oxidative enzymes, was mediated by Erk1/2 activation.64) So far, however, we observed little effects of PGRN on intracellular ROS level, assessed by protein carbonylation (data not shown). The detail mechanism(s) how this PGRN-dependent Erk1/2 activation protects HT22 cells are under investigation. The multifunctional characteristics of PGRN should also be noted. We previously reported that neural

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progenitor cell (NPC) proliferation was induced by PGRN.65) Thus, it is possible that PGRN secretion in response to a high level of oxidative stress also controls NPC proliferation. Together, in the CNS, disruption of this oxidative stress-dependent PGRN inducible system may be directly involved in the induction of neuronal vulnerability and the reduction of NPC proliferation. Conclusion In conclusion, our results suggest that HT22 cells possessed at least two distinct cellular protective mechanisms that were modulated by the level of oxidative stress experienced (Fig. 6). Importantly, these two distinct cell protective mechanisms could be categorized as (1) an endogenous mechanism and (2) an autocrine/ paracrine mechanism. The difference between these two systems may allow a cell to respond to oxidative stress independently or cooperatively with other cells. In other words, the first mechanism is completed within a single cell, while the second mechanism may be important for the homeostatic maintenance of multicellular systems such as the CNS.

Acknowledgment The authors would like to gratefully acknowledge Dr. Jerome Lamartine (Université Claude Bernard Lyon 1) for many helpful comments.

Funding This work was supported by Grants-in-Aid for Scientific Research (S) [23228004], (C) [24580147] from the Japan Society for the Promotion of Science.

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