Pyrroloquinoline quinone against glutamate-induced neurotoxicity in cultured neural stem and progenitor cells.

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Contents lists available at ScienceDirect

International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu

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Pyrroloquinoline quinone against glutamate-induced neurotoxicity in cultured neural stem and progenitor cells Shui Guan a,∗ , Jianqiang Xu b , Yifu Guo a , Dan Ge a , Tianqing Liu a , Xuehu Ma a , Zhanfeng Cui c a

Stem Cell and Tissue Engineering Laboratory, Dalian University of Technology, 116024 Dalian, China School of Life Science and Medicine, Dalian University of Technology, Panjin Campus, 124221 Panjin, China c Department of Engineering Science, Oxford University, OX1 3PJ Oxford, UK b

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Article history: Received 10 December 2014 Received in revised form 26 January 2015 Accepted 17 February 2015 Available online xxx

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Keywords: Pyrroloquinoline quinone Neural stem and progenitor cells Glutamate Reactive oxygen species Neuroprotection

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1. Introduction

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Pyrroloquinoline quinone (PQQ), as a well-known redox enzyme cofactor, has been proven to play important roles in the regulation of cellular growth and development in mammals. Numerous physiological and medicinal functions of PQQ have so far been reported although its effect on neural stem and progenitor cells (NS/PCs) and the potential mechanism were even rarely investigated. In this study, the neuroprotective effects of PQQ were observed by pretreatment of NS/PCs with PQQ before glutamate injury, and the possible mechanisms were examined. PQQ stimulated cell proliferation and markedly attenuated glutamate-induced cell damage in a dose-dependent manner. By observing the nuclear morphological changes and flow cytometric analysis, PQQ pretreatment showed its significant effect on protecting NS/PCs against glutamate-induced apoptosis/necrosis. PQQ neuroprotection was associated with the decrease of intracellular reactive oxygen species (ROS) production, the increase of glutathione (GSH) levels, and the decrease of caspase-3 activity. In addition, pretreatment with PQQ also significantly enhanced the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in the NS/PCs exposed to glutamate. These results suggest that PQQ can protect NS/PCs against glutamate toxicity associated with ROS-mediated mitochondrial pathway, indicating a useful chemical for the clinical application of NS/PCs. © 2015 Published by Elsevier Ltd. on behalf of ISDN.

Neural stem and progenitor cells (NS/PCs) are a dynamic population of cells in the brain that respond not only to injury but also to various physiological stimuli of exercise, nutrients, stress and so on (Péder et al., 2011). The interest in NS/PCs that possess the potential of unlimited self-renewal and the ability to differentiate into neurons, astrocytes and oligodendrocytes has been driven pri-

Abbreviations: AD, Alzheimer’s disease; BrdU, 5-bromo-2 -deoxyuridine; CAT, catalase; CCK-8, cell counting Kit-8; CNS, central nervous system; DCF, dichlorofluorescin; DCF-DA, 2 ,7 -dichlorofluorescin diacetate; DMEM, Dulbecco’s modified Eagle’s medium; FITC, fluorescein isothioncyanate; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, glutathione; LDH, lactate dehydrogenase; NMDA, N-methyl-d-aspartate; NS/PCs, neural stem and progenitor cells; PD, Parkinson’s disease; PI, propidium iodide; pNA, p-nitroanilide; PQQ, pyrroloquinoline quinine; PS, phosphatidylserine; ROS, reactive oxygen species; SOD, superoxide dismutase; TrxR1, thioredoxin reductase 1. ∗ Corresponding author. Tel.: +86 41184706360. E-mail address: [email protected] (S. Guan).

marily by the prospect of using them to treat acute and chronic neurodegenerative diseases of the central nervous system (CNS) (Gage, 2000; Alvarez-Buylla et al., 2001; Lindvall and Kokaia, 2006). Many studies in recent years show that using NS/PCs as donor cells in cell transplantation has great potential and many advantages for the treatment of Parkinson’s disease (PD) and Alzheimer’s disease (AD) (Shihabuddin and Aubert, 2010; Yuan and Shaner, 2013; Dunnett and Rosser, 2014). Nevertheless, for the clinical application of NS/PCs, an enormous challenge is that only a small population of engrafted cells could survive in the host brain after transplantation, which cannot even properly play their physiological functions due to their poor integration into the host neuronal circuits and inadequate release of neurotransmitters (Trzaska et al., 2009). Therefore, it is a pressing need to explore molecular and cellular mechanisms of CNS regeneration and develop novel neuroprotective factors such as the extrinsic soluble proteins, genes and candidate chemicals to promote NS/PCs survival, regulate proliferation and differentiation into specific cellular phenotypes for the prevention of neurodegenerative diseases.

http://dx.doi.org/10.1016/j.ijdevneu.2015.02.008 0736-5748/© 2015 Published by Elsevier Ltd. on behalf of ISDN.

Please cite this article in press as: Guan, S., et al., Pyrroloquinoline quinone against glutamate-induced neurotoxicity in cultured neural stem and progenitor cells. Int. J. Dev. Neurosci. (2015), http://dx.doi.org/10.1016/j.ijdevneu.2015.02.008

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Although PQQ has exhibited neuroprotective activity in many cell types and tissues, no studies have been undertaken in relation to its effect on cultured NS/PCs. To our knowledge, exposure of NS/PCs to glutamate at low millimolar concentrations induced a leakage of lactate dehydrogenase (LDH) and caused severe viability loss and apoptotic cell death. We therefore here wished to explore the underlying protective effects of PQQ against glutamate-induced toxicity in cultured NS/PCs. We evaluated the production of ROS, glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), as well as the expression of caspase3, on NS/PCs in vitro, aiming for in-depth understanding of the PQQ neuroprotection and its mechanism. We found that PQQ-pretreated NS/PCs can tolerate 15 mM glutamate and in turn avoid subsequent apoptosis/necrosis.

Fig. 1. The chemical structure of Pyrroloquinline quinone (PQQ), shown here as 4,5-dihydro-4,5-dioxo-1H-pyrrolo(2,3-f) quinoline-2,7,9-tricarboxylic acid.

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Glutamate is one of the most abundant free amino acids in the CNS and is a major excitatory neurotransmitter (Mukhin et al., 1996). It has been well known that neuronal death induced by glutamate toxicity occurs via either necrosis or apoptosis, depending on the intensity and duration of glutamate exposure (Olney, 2003). Excessive accumulation of extracellular glutamate is mediated by glutamate receptors, particularly the activation of N-methyl-daspartate (NMDA) receptor, leading to an intracellular cascade of cytotoxic events including production of reactive oxygen species (ROS) and activation of a sequence of apoptotic cascades, resulting in DNA damage and apoptosis (Reynolds and Hastings, 1995; Kalia et al., 2008). Since the brain is especially vulnerable to oxidative stress because of its high consumption of oxygen and low levels of endogenous antioxidants, some antioxidant agents with the ability to inhibit ROS production and alleviate brain injury may represent an effective therapeutic strategy against glutamate-induced oxidative toxicity. Pyrroloquinoline quinone (PQQ, Fig. 1) is an anionic, watersoluble compound that initially identified as a redox enzyme cofactor, and has been classified as a new B vitamin (Kasahara and Kato, 2003). As an essential nutrient, PQQ has been proven to play important roles in the regulation of cellular growth and development in mammalian system, and is therefore expected to be applied pharmacologically in the near future (He et al., 2003; Misra et al., 2012). Many studies in recent years have shown that PQQ functions as an antioxidant protecting the living cells from oxidative damage in vivo and the biomolecules from artificially produced ROS in vitro for supporting the growth and protection of living cells under stress (Gong et al., 2012; Zhang et al., 2012a,b). As we know, ROS production is greatly increased under many conditions of toxic stress and plays an important role in brain injuries and some neurodegenerative diseases (Rashid et al., 2013). The strong ability of PQQ to scavenge ROS and attenuate oxidative stress in mitochondria may contribute to its protective effects involved in PD and AD (Misra et al., 2004). It has been reported that PQQ could modulate NMDA receptor by directly oxidizing its redox modulatory site and inhibit glutamate-induced ROS production in cultured cortical neurons (Aizenman et al., 1992; Scanlon et al., 1997). One study has been established that PQQ pretreatment may protect human neuroblastoma SH-SY5Y cells against A␤-induced neurotoxicity indicating an effective therapeutic approach to AD (Zhang et al., 2009). In culture primary hippocampal neurons, PQQ also exerts neuroprotective activity to rescue glutamate-induced cell death through inhibition of ROS production, the phosphatidylinositol-3kinase (PI3 K)/Akt-dependent activation of Nrf2 and up-regulation of antioxidant genes (Zhang et al., 2011, 2012b).

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2. Materials and methods

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2.1. Chemicals and reagents

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Pyrroloquinoline quinone (PQQ), 5-bromo-2 -deoxyuridine (BrdU), Hoechst 33,258, propidium iodide (PI), 2 ,7 dichlorofluorescin diacetate (DCF-DA), and other chemicals were purchased from Sigma–Aldrich Inc. (St. Louis, Mo, USA). The Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan). Annexin V-fluorescein isothioncyanate (FITC) apoptosis detection kit and IntraPrepTM Permeabilization Reagent were from Backman Coulter Inc. (IM, USA). Caspase3/CPP32Colorimetric Assay Kit and the ApoGSHTM Glutathione Colorimetric Detection Kit were from BioVision Inc. (CA, USA). The assay kits for lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities were from Jiancheng Bioengineering Institute (Nanjing, China). 2.2. Cell culture and treatment All animals used were treated in accordance with the Animal Care and use committee guideline at the United States National Institutes of Health. Cell culture for NS/PCs was performed as previously described (Wilcox et al., 1994; Cummings et al., 1996; Johe et al., 1996; Erlandsson et al., 2001). Briefly, embryonic neural tissue samples were dissected from the hippocampus of Sprague–Dawley (SD) rat on gestation day 13.5 and cells were mechanically isolated by repeated trituration in a serum-free culture media (DF medium) composed of Dulbecco’s modified Eagle’s medium (DMEM) and F12 nutrient (1:1) (Gibco, MD, USA). The dissociated cells were collected by centrifugation at 1000 × g for 5 min and were resuspended in DMEM/F12 medium containing bFGF (10 ng/ml), EGF (20 ng/ml), and N2 supplement (Gibco, MD, USA). The number of live cells was counted by trypan blue exclusion assay in the hemocytometer (Hengtai, Jiangsu, China). Cell cultures were maintained in a watersaturated atmosphere of 5% CO2 at 37 ◦ C and half of the medium was replaced every three days. For monolayer culture, dissociated cells were plated onto poly-l-ornithine (15 ␮g/ml)-coated and laminin (10 ␮g/ml)-coated glass coverslips in well plates for indicated time. 2.3. Cell viability assay Cell viability was measured by using CCK-8 assay, which is based on the conversion of water-soluble tetrazolium salt, WST-8 [2-(2methoxy-4-nitrophenyl)-3- (4-nitrophenyl)-5-(2,4-disulfophenyl) -2H -tetrazolium, monosodium salt] to a water-soluble formazan dye upon reduction in the presence of an electron carrier by dehydrogenases (Ishiyama et al., 1996; Itano et al., 2002). The amount of the formazan dye generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells. Briefly, NS/PCs (1 × 105 cells/ml) were treated with various concentrations


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of PQQ in well plates under monolayer culture conditions for 24 or 48 h. Then the medium was incubated with WST-8 solution in the dark for 3 h at 37 ◦ C. Absorbance was read at 450 nm on a microplate reader (SPECTRAFLUOR, TECAN, Sunrise, Austria).

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The proliferative activity of NS/PCs was evaluated by treating cells with 10 ␮M BrdU, a thymidine analog incorporated into genetic molecules during the S phase of mitotic division, for 24 h with or without PQQ. Cells were fixed with ice-cold 4% paraformaldehyde for 20 min and washed for 10 min × 3 with PBS. Then cells were treated with 2 M HCl for 10 min at 37 ◦ C to denature DNA and with 0.1 M borate buffer (Na2 B4 O7 , pH8.5) for 5 min. After blocked with 4% goat serum in PBS containing 0.3% Triton X-100 for 1 h, the cells were incubated with mouse anti-BrdU monoclonal antibody (dilution 1:1000, Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA) at 4 ◦ C overnight. After washing in PBS, the FITC-conjugated secondary antibody (dilution 1:400, Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA) was added and the cells were incubated for 1 h. Ten random fields were captured in each well, BrdU-incorporated cells were counted and the ratio of BrdU/Total cells was calculated by a fluorescent microscope (Olympus, Tokyo, Japan) equipped with ImagePro Plus 5.0 analysis software.

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2.5. Morphological observation of nuclear change

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The nuclear morphological change was assessed using Hoechst 33,258 staining (Hetman et al., 1999). NS/PCs were fixed in 4% paraformaldehyde and stained with 10 ␮g/ml Hoechst 33,258 (Sigma, St. Louis, Mo, USA) for 10 min at 37 ◦ C and nuclei were visualized using an Olympus Microscope with a WU (near-ultraviolet fluorescence cube) excitation filter (Tokyo, Japan). 2.6. Evaluation of apoptosis and necrosis

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Annexin V-FITC apoptosis detection kit is based on the observation that soon after initiating apoptosis, phosphatidylserine (PS) is translocated from the cytoplasmic face of the plasma membrane to the cell surface. Annexin V has a strong, Ca2+ -dependent affinity for PS and therefore is used as a probe for detecting apoptosis. Cells that have lost membrane integrity will show red staining (PI) throughout the nucleus and therefore will be easily distinguishable from the apoptotic cells. NS/PCs were incubated with a combination of annexin V-FITC and PI at room temperature in the dark for 15 min and quantitatively analyzed by a FACScan flow cytometer (Beckman Coulter, Epics) equipped with the SYSTEM IITM analysis software.

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2.7. Measurement of caspase-3 activity

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The activation of caspase-3 was determined using the Caspase-3/CPP32Colorimetric Assay Kit. The assay is based on spectrophotometric detection of the chromophore p-nitroanilide (pNA) after cleavage from the labeled substrate DEVD (Asp-GluVal-Asp)-pNA. Comparison of the absorbance of pNA from an apoptotic sample with an uninduced control allows determination of the fold increase in CPP32 activity. NS/PCs were harvested and caspase-3 activity was determined according to the manufacturer instructions. Absorbance of the chromophore pNA produced was measured using a microplate reader (SPECTRAFLUOR, TECAN, Sunrise, Austria) at 405 nm. Activity of caspase-3 was expressed relative to the amount of protein in the cell extracts determined using a Bradford Assay Kit (Bio-Rad, USA).

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2.8. Measurement of intracellular ROS formation DCF-DA, which is intracellularly oxidized to the fluorescent dichlorofluorescin (DCF) in the presence of oxidants, was used to measure relative levels of cellular peroxides (Wang and Joseph, 1999; Lautraite et al., 2003). NS/PCs were incubated with DCF-DA (1 mM) at 37 ◦ C for 1 h in the dark, and then gently rinsed with PBS three times. After centrifugation at 1000 × g for 5 min, the supernatants were removed and the pellets were resolved with 1% Triton X-100, and ROS levels were measured as the fluorescence of oxidation product of DCF-DA, DCF, at an excitation wavelength of 480 nm and an emission wavelength of 540 nm using a fluorescence microplate reader (JASCO, FP-6500, Japan). 2.9. Measurement of total GSH levels Intracellular total GSH (reduced form GSH + oxidized form GSSG) levels were determined with the ApoGSHTM Glutathione Colorimetric Detection Kit according to manufacturer instruction. NS/PCs were centrifuged at 700 × g for 5 min at 4 ◦ C and the supernatants were removed. The pellets were washed with ice-cold PBS, lysed in 80 ␮l ice-cold Glutathione Buffer and incubated on ice for 10 min. Then the samples were dissolved with 5% 5-sulfosalicylic acid (SSA, 20 ␮l) and centrifuged at 8000 × g for 10 min. The supernatants (20 ␮l) were incubated in 160 ␮l of the reaction mix buffer at room temperature for 10 min and then substrate solution (20 ␮l) was added and the mixture was incubated for a further 10 min. Absorbance at 415 nm was measured by using a microplate reader (SPECTRAFLUOR, TECAN, Sunrise, Austria). The standard curve was obtained from absorbance of the diluted GSH Standard that was incubated in the mixture as in samples. 2.10. Assays for LDH, SOD, CAT, and GPx activities Enzyme activity assays completely complied with the manufacture’s instructions. LDH is a stable cytoplasmic enzyme that is present in all cells and rapidly released from the cytosol of damaged cells into the surrounding medium following loss of membrane integrity resulting from either apoptosis or necrosis. SOD, CAT and GPx played pivotal roles as enzymatic defense mechanisms against oxidative stress-induced cell damage. A low steady-state level of intracellular superoxide is maintained by SOD, and H2 O2 , generated by SOD is removed by CAT or GPx. NS/PCs were washed with icecold PBS, harvested by centrifugation at 1000 × g for 5 min, pooled in 0.5 ml of 0.1 M phosphate buffer (pH 7.4) and homogenized. The homogenate was centrifuged at 3000 × g for 20 min at 4 ◦ C, and the supernatant was used for the activity assay according to the manufacturer instructions. LDH leakage was calculated as the percentage of LDH in the medium versus total LDH activity in the cells. Activities of SOD, CAT, and GSH-Px were expressed relative to the amount of protein in the cell extracts determined using a Bradford Assay Kit (Bio-Rad, USA). 2.11. Statistical analysis Data were expressed as mean ± S.E.M. from three independent experiments and evaluated using one-way ANOVA followed by Student’s t-test. Significant differences were established at P < 0.05.

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3.1. Effects of PQQ on the proliferation of cultured NS/PCs

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NS/PCs were treated with different concentrations of PQQ (3, 30, 300, 3000 and 30,000 nM) for indicated times (24 and 48 h), and the effect of PQQ on cell viability was evaluated by CCK-8


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Fig. 2. Effects of PQQ on the proliferation of cultured NS/PCs. (A) Cells (1 × 105 cells/ml) were treated with various concentrations (30–30000 nM) of PQQ for 24 h and 48 h at 37 ◦ C. Cell viability was assessed by the CCK-8 method as described in Materials and methods. Data are mean ± S.E.M. values obtained from four culture wells per experiment, determined in three independent experiments. (*) P < 0.05, and (**) P < 0.01, compared with the control group. (B) Quantification of BrdU-immunoreactive cells in control (a) and PQQ (b, 30 nM; c, 300 nM; d, 3000 nM) groups during a first 24 h period. Data are mean ± S.E.M. values obtained from three independent experiments. Ten random fields were counted in each culture. (*) P < 0.05, compared with the control group.

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assay. As shown in Fig. 2A, at concentrations from 30 to 3000 nM, PQQ showed a dose-dependent proliferative effect on the growth of NS/PCs and did not induce changes in cell morphology. The treatment with 3000 nM concentration of PQQ caused 147.5 ± 4.8% and 152.3 ± 2.9% increase in cell viability compared with the control, respectively. Thus, it was decided to use a concentration at range of 30–3000 nM for all subsequent experiments. On the other hand, the presence of proliferating cells in S phase was examined by BrdU labeling. BrdU was added to the NS/PCs culture during the first 24 h period, and BrdU incorporated into the cells was visualized immunocytochemically. As shown in Fig. 2B, the number of BrdU-immunoreactive cells was significantly increased after treatment with PQQ. 3.2. Effects of PQQ on glutamate-induced cell viability loss and apoptotic death in cultured NS/PCs To choose appropriate concentration of glutamate, NS/PCs were exposed to various concentrations of glutamate for 24 h. A doseand time-dependent increase in LDH leakage was demonstrated in NS/PCs exposed to different concentrations of glutamate. At a concentration greater than 10 mM, LDH leakage was significantly increased compared with that of untreated cells. Similarly, CCK8 assay also showed a dose-dependent decrease in cell viability.

Significant reduction in viability was observed in the cells treated with greater than 15 mM glutamate (data not shown). Therefore, it was decided to use a concentration of 15 mM for the subsequent assays. In order to determine whether PQQ protects NS/PCs from glutamate-induced cytotoxicity, NS/PCs were pretreated with different concentrations of PQQ (30-3000 nM) for 24 h, following exposure to 15 mM glutamate for another 24 h. The results of Fig. 3A showed that pretreatment of NS/PCs with PQQ dose-dependently suppressed glutamate-induced leakage of LDH, and these findings were further verified by CCK-8 assay (Fig. 3B). To clarify the inhibitory effect of PQQ against cytotoxicity of glutamate, we investigated the effect on the nuclear morphological changes observed in the glutamate-treated cells. Nuclear staining with Hoechst 33,258 by fluorescence microscopy demonstrated that after exposure to 15 mM glutamate for 24 h, the condensation and fragmentation of nuclei characteristic of apoptotic cells were evident in non-treated control group. By contrast, in the presence of 3000 nM PQQ, a dramatic reduction in nuclear fragmentation was observed (Fig. 4A). In addition to the morphological evaluation, we performed flow cytometric AnnexinV-FITC/PI staining analysis to determine the type of glutamate-induced cell death of NS/PCs: apoptosis versus necrosis. As shown in Fig. 4B, compared with the control group, the early apoptosis and late apoptosis/necrosis rate increased remarkably from 1.8 ± 1.1% and 10.4 ± 1.6% to 74.9 ± 1.9%


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LDH leakage (% of total)

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GSH is one of the most important molecules in mammalian cells for scavenging ROS. To clarify the changes in intracellular oxidative stress induced by glutamate and/or PQQ treatment, the effect of PQQ on the level of intracellular total GSH was assessed. As shown in Fig. 5B, the GSH content was significantly depleted in NS/PCs after exposure to 15 mM glutamate for 24 h. Although treatment of the cells with PQQ alone did not affect the cellular total GSH levels, it was effective to attenuate the reduction of GSH levels in glutamate-induced cells. Furthermore, we examined intracellular SOD, CAT and GPx activities. As shown in Table 1, the activities of SOD, CAT and GPx all tended to decrease in the cells exposed to 15 mM glutamate, whereas pretreatment with PQQ significantly augmented their activities.

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Cell viability (% of control)

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300 nM

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PQQ + Glu Fig. 3. Effects of PQQ on glutamate-induced cell viability loss in cultured NS/PCs. Cells (1 × 105 cells/ml) were treated with PQQ at different concentrations (30–3000 nM) for 24 h, following exposure to 15 mM glutamate for another 24 h, and then LDH leakage (A) and viability of the cells (B) were determined by the method as described in Section 2. Data are mean ± S.E.M. values obtained from four culture wells per experiment, determined in three independent experiments, (*) P < 0.05, and (**) P < 0.01 statistically different from the glutamate-induced group.

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and 21.7 ± 3.1% in glutamate treatment alone, respectively. However, pretreatment with 3000 nM PQQ decreased the apoptosis rate to 66.4 ± 3.7% and 18.2 ± 4.8%, respectively. The cellular pathway of glutamate-induced NS/PCs death was examined by assessing caspase-3 activity, which plays a critical role in apoptosis. As shown in Fig. 4C, observation at 24 h following glutamate stimulation demonstrated that caspase-3 activity was increased to over 3-fold of the non-treated control group. Pretreatment with 3000 nM PQQ showed a significant decrease in caspase-3 activity, indicating that PQQ attenuated apoptosis of NS/PCs induced by glutamate via suppression of the caspase cascade.

3.3. Effects of PQQ on glutamate-induced change of intracellular ROS, total GSH levels, the activities of SOD, CAT and GPx in cultured NS/PCs The initial stimulation of ROS has been implicated in the pathogenesis of several disease states. In this study, the level of ROS was detected to explore the role of oxidative stress in the effect of PQQ against glutamate-induced cytotoxicity. As shown in Fig. 5A, 15 mM glutamate dramatically increased ROS production compared to untreated cells, and pretreatment with 3000 nM PQQ significantly suppressed this induction at 24 h. Interestingly, PQQ treatment alone also decreased the level of intracellular ROS significantly in NS/PCs, indicating that PQQ may play a critical role on depressing ROS production.

NS/PCs, residing in both fetal and adult brain, are known to differentiate into various kinds of neurons and astrocytes therefore play important roles in the development and injuries repair of the brain (Temple, 2001; Alvarez-Buylla et al., 2002; Kim, 2004). Many factors have been shown to exert their effects on NS/PCs, influencing their proliferation, apoptosis and differentiation (Gao and Gao, 2007; Anthony et al., 2008). There is growing evidence to support the fact that NS/PCs migrate toward injured lesions and then appear to participate in the active recovery of the damaged area under the reactivation by oxidative and several other stresses (Ro et al., 2013; Tseng et al., 2013). However, NS/PCs can also be damaged by hypoxia and other stresses and it has been reported that oxidative stress inhibits survival signals and activates death signals in NS/PCs as well as neurons (Jin et al., 2003; Zhao et al., 2007). It is well known that glutamate toxicity is an important mechanism of neuronal injury, including oxidative stress-mediated neurotoxicity (Kanki et al., 2004; Hirata et al., 2011; Chen et al., 2011). Excessive sustained glutamate receptor stimulation with persistent depolarization can produce metabolic and functional exhaustion inducing neuronal damages (Gill, 2000; Mattson, 2008). Several recent studies have shown that elevated levels of extracellular glutamate induced depletion of cellular antioxidants such as GSH and ascorbate, thereby inducing oxidative stress in the cell (Shih et al., 2006; Tojo et al., 2002; Conrad and Sato, 2012; Ma et al., 2012). In agreement with previous studies, our results revealed that exposure to glutamate for 24 h led to cell viability loss and typical apoptotic nuclei formation in a dose-dependent manner in cultured NS/PCs, accompanied by the activation of caspase-3 involved in the mitochondrial pathway of apoptosis. We also observed that after glutamate exposure, intracellular ROS level was increased, and GSH level was decreased. Meanwhile, as intracellular enzymatic defense mechanisms, SOD, CAT, and GPx, three potent ROS scavengers were also decreased. These results showed that both ROS generation and mitochondrial dysfunction contributed to glutamate-induced neurotoxicity in cultured NS/PCs. In recent years, PQQ has been receiving much attention owing to its nutritional importance and physiological functions (Rucker et al., 2009). PQQ is able to scavenge ROS and attenuate oxidative stress in mitochondria, thus exerting protective effects against oxidative stress-induced cell damage in the heart, liver and brain (Hobara et al., 1988; Zhu et al., 2004; Tao et al., 2007; Ohwada et al., 2008). Like other quinone-based antioxidants, PQQ also works in concentration-dependent manner. Up to 10 ␮M, it works predominantly as antioxidant, while beyond 50 ␮M it acts as pro-oxidant. Both the antioxidant and pro-oxidant nature of PQQ have functional significance in biology (He et al., 2003). PQQ at low micromolar concentrations modulates the kinetic properties of the mammalian selenoprotein thioredoxin reductase 1 (TrxR1) and also inhibits glutathione reductase (GR) in a non-covalent reversible manner


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Fig. 4. Effects of PQQ on glutamate-induced apoptotic cell death in cultured NS/PCs. Cells (1 × 105 cells/ml) were treated with 3000 nM PQQ for 24 h at 37 ◦ C, following exposure to 15 mM glutamate for another 24 h, and then (A) were observed by fluorescence microscopy (200×) after nuclei staining with Hoechst 33,258, (B) were determined by flow cytometric Annexin V-FITC/PI staining analysis, and (C) the activation of caspase-3 was determined by detecting fluorescence of the chromophore p-nitroanilide (pNA). The figures are representative for three different experiments, and white arrows show the nucleus of apoptotic cells. Data are mean ± S.E.M. values obtained from three independent experiments, (***) P < 0.001, (**) P < 0.01, statistically different from the control group, and (#) P < 0.05, (##) P < 0.01 statistically different from the glutamate-induced group.


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Table 1 Effects of PQQ on the activities of SOD, CAT and GPx in glutamate-induced NS/PCs. SOD (U/mg protein) Control Glutamate-induced PQQ PQQ + glutamate

4.73 3.86 4.58 4.67

± ± ± ±

0.88 0.76 0.62 0.54*

CAT (␮mol/mg protein) 2.28 1.79 2.23 3.12

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GPx (␮mol/mg protein)

0.24 0.82 0.67 0.47*

88.95 78.56 82.44 85.72

± ± ± ±

2.46 5.22 3.32 2.41*

Cells (1 × 105 cells/ml) were treated with 3000 nM PQQ for 24 h at 37 ◦ C, following exposure to 15 mM glutamate for another 24 h. Data are shown as mean ± S.E.M. values obtained from four wells per experiment, determined in three independent experiments. * P < 0.05, statistically different from the glutamate-induced group.

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(Xu and Arnér, 2012). PQQ may stimulate epithelial cell proliferation by activating epidermal growth factor receptor by oxidation and subsequent inactivation of protein tyrosine phosphatase 1B via its redox cycling (Kimura et al., 2012). In cultured fibroblasts, PQQ also acts as a potential growth factor to enhance cell proliferation when added to cell cultures (Naito et al., 1993; Kumazawa et al., 2007). The most promising application of PQQ has potentially been in neuroprotection, which could be implicated either the functioning of PQQ as a redox sensor to oxidative stress or as a signaling molecule helping in the growth of neurons (Misra et al., 2012). It has been demonstrated that PQQ could produce more mature and high-density regenerated nerves cells in sciaticnerve-deficit model created in rats, suggesting that PQQ is a potent enhancer for the regeneration of peripheral nerves (Liu et al., 2005). Although PQQ is a potent growth factor in both in vitro and in vivo models, its detailed mechanism of action still remains unclear. In the present study, we noted that PQQ (over 30 nM in concentration) significantly increased the cellular viability of NS/PCs and stimulated cell proliferation in a dose-dependent manner. PQQ significantly increased BrdU incorporation into NS/PCs during the first 24 h, indicating that the compound could have a positive effect on the development of CNS. Furthermore, the neuroprotective effects of PQQ were observed by pretreatment of NS/PCs with PQQ before glutamate injury. PQQ has been reported to display a strong antiapoptotic activity in cortical neurons and activate multiple cell survival pathways that are essential to cell growth, survival and differentiation (Mebratu and Tesfaigzi, 2009). The present experiments demonstrated that increased LDH leakage and decreased viability in NS/PCs exposed to glutamate were significantly attenuated by pretreatment with PQQ, and glutamate-induced apoptotic death in NS/PCs was also reduced by PQQ through observing the nuclear morphological changes and flow cytometric analysis. Apart from PQQ amelioration of ROS generation, PQQ also increased intracellular antioxidant defense mechanisms, including the activities of SOD, CAT and GPx and the cellular total GSH levels. Intracellular accumulation of ROS is believed to be an important indicator for understanding glutamate-induced neuronal cell death. Clinical applications of antioxidants have not achieved complete success mainly because they cannot easily access the subcellular compartments where ROS is generated (Choi et al., 2011). Our findings indicate that PQQ may act as a growth factor or potent neuroprotective agent to contribute to mammalian growth and treatment of neurodegenerative diseases. There is accumulating evidence indicating that PQQ affects intracellular signaling pathways, such as DJ-1/c-Jun N-terminal protein kinase (JNK)/caspase and Ras/Raf/ERK/STAT pathways, which are correlated with cell proliferation, cell death, mitochondriogenesis and oxidative metabolism (Kumazawa et al., 2007; Nunome et al., 2008; Chowanadisai et al., 2009). PQQ has been reported to influence the activity of DJ-1 which is capable of inactivating JNK/caspase pathway to modulate oxidative stress-induced neuronal apoptosis (Nunome et al., 2008; Rucker et al., 2009). As a major compartment of energy metabolism, mitochondrial plays a pivotal role in the progress of caspase-denpendent apoptosis (Forbes-Hernández et al., 2013). ROS from mitochondrial may

16

(A)

**

14

Relative DCF Fluorescence

403

12 ##

10 8

*

6 4 2 0 control

(B) GSH contents (% of control)

402

PQQ

Glu

PQQ+Glu

120 #

100 80

*

60 40 20 0 control

PQQ

Glu

PQQ+Glu

Fig. 5. Effects of PQQ on glutamate-induced change of intracellular ROS and total glutathione levels in cultured NS/PCs. Cells (1 × 105 cells/ml) were treated with 3000 nM PQQ for 24 h at 37 ◦ C, following exposure to 15 mM glutamate for another 24 h. Each value represents mean ± S.E.M. obtained from four culture wells per experiment, determined in three independent experiments. (*) P < 0.05, (**) P < 0.01 in comparison with the control group, (#) P < 0.05 and (##) P < 0.01 in comparison with the glutamate-induced group.

oxidize membrane proteins of mitochondrial, change mitochondrial outer membrane permeabilization, and lead to disruption of mitochondrial membrane potential, which contribute to the release of cytochrome c and apoptosis (Petrosillo et al., 2003). In this study, we observed that PQQ pretreatment significantly inhibited the activation of caspase-3 in glutamate-injured NS/PCs, suggesting the potential neuroprotective mechanism that PQQ against glutamateinduced oxidative stress and mitochondrial dysfunction in cultured NS/PCs. Further experiments will be designed to explore signal transduction pathways of PQQ on its antioxidant and antiapoptotic activities. In summary, the present studies demonstrated that PQQ significantly stimulated cellular proliferation and exerted potent neuroprotective activity against glutamate-induced cell damage in cultured NS/PCs. Moreover, the ability of PQQ to scavenge ROS and inhibit apoptosis, as indicated by the decreased ROS production, increased GSH level, increased SOD, CAT and GPx activities, and suppression of caspase-3, may be involved in ROS-mediated mitochondrial pathway.


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ARTICLE IN PRESS

Acknowledgements This work was subsidized by grants from the National Natural Science Foundation of China (NSFC. 30800288/31300809), and the Fundamental Research Funds for the Central Universities(DUT12JB04/DUT14RC(3)145). We also express our sincere thanks to the Ministry of Science and Technology (MOST) Major International Collaboration Project(2011DFA31960).

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The cofactor pyrroloquinoline quinone.

progenitor cells in Alzheimer's disease.

Sequential Differentiation of Embryonic Stem Cells into Neural Epithelial-Like Stem Cells and Oligodendrocyte Progenitor Cells.

progenitor cells.

Biochemistry and physiology of pyrroloquinoline quinone and quinoprotein dehydrogenases.

progenitor cells.

Redox-regulated fate of neural stem progenitor cells.

progenitor cells.

progenitor cells react to non-glial cns neoplasms.

Pyrroloquinoline quinone protects mouse brain endothelial cells from high glucose-induced damage in vitro.

In Vitro Neurotoxicity Resulting from Exposure of Cultured Neural Cells to Several Types of Nanoparticles.

Progenitor Cells.

Extractions of pyrroloquinoline quinone from crude biological samples.

Progenitor Cells, Oligodendrocyte Precursor Cells, and Endothelial Progenitor Cells.

Landscape of monoallelic DNA accessibility in mouse embryonic stem cells and neural progenitor cells.

progenitor cells.

Induced neural stem cells protect neuronal cells against apoptosis.

The pyrroloquinoline quinone (PQQ) coenzymes: a case of mistaken identity.

Huperzine A protects neural stem cells against Aβ-induced apoptosis in a neural stem cells and microglia co-culture system.

Comparative neurotoxicity screening in human iPSC-derived neural stem cells, neurons and astrocytes.

Differentiation of oligodendrocyte progenitor cells from dissociated monolayer and feeder-free cultured pluripotent stem cells.

Neural Stem and Progenitor Cells in Nervous System Function and Therapy.

Bone Morphogenetic Protein 4 Signalling in Neural Stem and Progenitor Cells during Development and after Injury.

Cholecystokinin-induced protection of cultured cortical neurons against glutamate neurotoxicity.