2 pathway in neuroprotective effects of pyrroloquinoline quinine against rotenone-induced SH-SY5Y cell injury

Neuroscience 270 (2014) 183–191

INVOLVEMENT OF ERK1/2 PATHWAY IN NEUROPROTECTIVE EFFECTS OF PYRROLOQUINOLINE QUININE AGAINST ROTENONE-INDUCED SH-SY5Y CELL INJURY Q. ZHANG, a,c  J. ZHANG, a,b  C. JIANG, a J. QIN, a,b K. KE b AND F. DING a,c*

cells against rotenone injury by reducing ROS production and maintaining mitochondrial functions through activation of ERK1/2 pathway. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

a Jiangsu Key Laboratory of Neuroregeneration, Nantong University, 19 Qixiu Road, Nantong, JS 226001, PR China b Department of Neurology, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong, JS 226001, PR China c Co-innovation Center of Neuroregeneration, Nantong, JS 226001, PR China

Key words: pyrroloquinoline quinine (PQQ), rotenone, neuroprotection, ERK1/2.

Abstract—Pyrroloquinoline quinone (PQQ), a redox cofactor in the mitochondrial respiratory chain, has been shown to protect neurons against glutamate-induced damage both in vitro and in vivo. In this study, specific inhibitors to each of the mitochondrial complexes were used to find out which reactive oxygen species (ROS)-generating sites could be affected by PQQ. Then we established an in vitro model of Parkinson’s disease (PD) by exposing cultured SH-SY5Y dopaminergic cells to rotenone, a complex I inhibitor. The neuroprotective effects of PQQ were observed by pretreatment of SH-SY5Y cells with PQQ before rotenone injury, and the possible involvement of certain signaling pathways were investigated. PQQ pretreatment prevented SH-SY5Y cells from rotenone-induced apoptosis in a concentrationdependent manner. PQQ neuroprotection was associated with inhibition of intracellular ROS production, modulation of the expression of apoptosis-related Bcl-2 and Bax, and regulation of the level of superoxide dismutase, glutathione, and malondialdehyde. Meanwhile, PQQ up-regulated the gene expression of Ndufs 1, 2, and 4 (complex I subunits), and increased mitochondrial viability and mitochondrial DNA content. Furthermore, PQQ pretreatment activated ERK1/2 phosphorylation in rotenone-injured SH-SY5Y cells, while ERK1/2 inhibition suppressed PQQ neuroprotection. All the results suggested that PQQ could protect SH-SY5Y

INTRODUCTION Pyrroloquinoline quinone (PQQ), a new redox cofactor, has proven to have a strong ability to scavenge reactive oxygen species (ROS) in the cell (Misra et al., 2004, 2012; Zhang et al., 2011). As we know, ROS production is greatly increased under many conditions of toxic stress and plays an important role in the aging and age-related diseases such as Parkinson’s disease (PD) and Alzheimer’s disease (AD). ROS is mainly produced by mitochondrial electron transport chain (ETC), which contains at least four mitochondrial ROS-generating sites (Liu and Schubert, 2009). In order to identify which of the four ROS-generating sites could be affected by PQQ, we applied a range of classical mitochondrial respiratory inhibitors to stimulate ROS production. We noted that ROS produced by the ETC complex I inhibitor (rotenone) or the ETC III inhibitor (antimycin A) could be prevented by PQQ treatment. Interestingly, rotenone, as an inhibitor of ECT complex I, has been extensively adopted to mimic the clinical and pathological features of PD in animal models (Panov et al., 2005; Cannon et al., 2009), because rotenone binds specifically to the complex I, reduces the electron flow to ubiquinone, and increases ROS generation, which in turn damages the complex I itself and other complexes, as well as other proteins and macromolecules in mitochondria (Sanders and Greenamyre, 2013). Since mitochondrial dysfunction and oxidative stress are involved in the pathogenesis of neurodegenerative disorders, some antioxidants with the ability to alleviate mitochondrial dysfunction and inhibit ROS production are predicted to possess potential therapeutic efficacy in such diseases (Chaturvedi and Beal, 2008). For example, coenzyme Q10 (CoQ10) and creatine show promising neuroprotective effects in neurodegenerative disorders, but with limited success in clinical trials (Liu and Wang, 2013). One of the reasons is that many antioxidants cannot easily access the subcellular compartments to exert

*Corresponding author. Address: 19 Qixiu Road, Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong, JS 226001, PR China. Tel: +86-513-85051800; fax: +86-51385511585. E-mail address: [email protected] (F. Ding).   Contributed equally. Abbreviations: 3-NP, 3-nitropropionic acid; CoQ10, coenzyme Q10; DCFH-DA, 20 ,70 -dichlorofluorescin diacetate; DMEM, Dulbecco’s modified eagle’s medium; DPI, diphenyleneiodonium chloride; ETC, electron transport chain; FBS, fetal bovine serum; FCCP, ptrifluoromethoxyphenylhydrazone; GSH, glutathione; MDA, malondialdehyde; NaN3, sodium azide; PBS, phosphate-buffered saline; PD, Parkinson’s disease; PQQ, pyrroloquinoline quinone; qRTPCR, quantitative real-time reverse transcription PCR; rTdT, terminal deoxynucleotidyl transferase; ROS, reactive oxygen species; RT, reverse transcription; SDS, sodium dodecyl sulfate; SOD, superoxide dismutase; TBS, Tris-buffered saline; TUNEL, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling. http://dx.doi.org/10.1016/j.neuroscience.2014.04.022 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. 183

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protective effects (Sanders and Timothy Greenamyre, 2013). In contrast, mitochondrial-targeted antioxidants, such as mitochondrial CoQ10 (MitoQ), play an important role in modulating ROS-induced mitochondrial permeability transition and cell death in that they combat against ROS at their site of origin in mitochondria (Tauskela, 2007). In this study, an in vitro model of PD was established by exposing cultured SH-SY5Y dopaminergic cells to rotenone, and the effects of pretreatment with PQQ on rotenone-injured SH-SY5Y cells were examined. And we further investigated the possible involvement of certain signaling pathways in the effects of PQQ.

EXPERIMENTAL PROCEDURES Drugs and chemicals PQQ, rotenone, 3-nitropropionic acid (3-NP), antimycin A, sodium azide (NaN3), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), oligomycin, diphenyleneiodonium chloride (DPI), PD98059, trypsin, 20 , 70 -dichlorofluorescin diacetate (DCFH-DA), monoclonal mouse anti-Bcl-2 antibody, monoclonal mouse anti-Bax antibody, monoclonal anti-phospho-MAP kinase1/2 (ERK1/2) (Thr185/Tyr187) antibody, polyclonal anti ERK1/2 (MAPK) antibody, and monoclonal mouse anti-bactin antibody were purchased from Sigma (St Louis, MO, USA). Dulbecco’s modified eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). IRDye 800-conjugated goat anti-mouse IgG and IRDye 800-conjugated donkey anti-rabbit IgG were purchased from Rockland (Gilbertsville, PA, USA). Tissue lysis buffer, protease inhibitor cocktail (100 mM phenylmethanesulfonyl fluoride, 15 lM aprotinin, 100 lM leupeptin, 100 lM bestatin, 100 lM pepstatin and 80 lM E-64 diluted in dimethyl sulfoxide), and BCA-based protein quantification kit were obtained from Biocolor (Shanghai, China). Cell Counting Kit-8 (CCK-8) was obtained from Dojindo (Kumamoto, Japan). Terminal deoxynucleotidyl transferase (rTdT) biotin-dUTP nick end labeling (TUNEL) assay kit was obtained from Promega (Madison, WI, USA). Trizol reagent was from Invitrogen (Carlsbad, CA, USA). Fast EvaGreen qPCR Master Mix was purchased from Biotium (Hayward, CA, USA). Omniscript Reverse Transcription (RT) kit was provided by Qiagen (Valencia, CA, USA). The assay kits for superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) were obtained from the Jiancheng Institute of Biotechnology (Nanjing, Jiangsu, China). Cell culture and treatment Human SH-SY5Y cells, obtained from the American Type Culture Collection (Manassas, VA, USA), were cultured in DMEM supplemented with 10% FBS at 37 °C in a humidified atmosphere of 95% air and 5% CO2. To determine the rotenone concentration required to induce cell injury, SH-SY5Y cells were incubated with different concentrations of rotenone (0.1, 1, 10, 100, 200 and

400 lM) for 24 h before the cell viability was measured by CCK-8 assay. To observe the protective effects of PQQ, PQQ at different concentrations (0.1, 1, 10 and 100 lM) was added to cell culture for a 24-h incubation before rotenone (100 lM) was added and allowed to incubate for designated times. The cells cultured in plain medium without any treatments were considered as control. ROS assay The intracellular ROS level was determined by the DCFHDA assay. The cells were incubated with DCFH-DA at a final concentration of 20 lM at 37 °C for 30 min in the dark, and then gently rinsed with phosphate-buffered saline (PBS) for three times. The ROS level was measured as the fluorescence of dichlorofluorescin (DCF), an oxidation product of DCFH-DA, using excitation and emission wavelengths of 485 and 528 nm, respectively, on a multi-mode microplate reader (Bio-Tek, Winooski, VT, USA). Cell viability measurement Cell viability was assessed by the CCK-8 method. Briefly, tetrazolium salt-8 (WST-8) solution was added to cultured SH-SY5Y cells in a 96-well plate (10 ll/each well), and allowed to incubate at 37 °C for 2 h. The absorbance (optical density, OD) was measured by spectrophotometry at 450 nm with an ELx-800 microplate reader (Bio-Tek Inc., Winooski, VT). Western blot analysis The SH-SY5Y cells were collected and homogenized in a cell lysis buffer consisting of 62.5 mM Tris, pH 6.8, 10 mM dithiothreitol (DTT), 2% sodium dodecyl sulfate (SDS), 10% glycerol, and a protease inhibitor cocktail. Total protein was quantified with the BCA-based protein quantification kit, and subjected to SDS–polyacrylamide gel electrophoresis (PAGE). The protein sample was transferred to a PVDF membrane (Millipore, Bedford, MA, USA), which was blocked with 5% non-fat dry milk in Tris-buffered saline (TBS, pH 7.4) and incubated with anti-Bcl-2 (1:1000), anti-Bax (1:200), anti-p-ERK1/2 (1:500), anti-ERK1/2 (1:500) and b-actin (1:4000), respectively, at 4 °C overnight. After washing with TBS/ T (TBS with 0.1% Tween 20), IRDye 800-conjugated affinity purified goat anti-mouse IgG (1:5000) or donkey anti-rabbit IgG (1:5000) was applied at room temperature for 30 min. The images were scanned with Odyssey infrared imaging system (Li-COR), and data were analyzed with PDQuest 7.2.0 software (Bio-Rad, Hercules, CA, USA). b-Actin was used as an internal control protein for normalizing protein loading. TUNEL assay The SH-SY5Y cells were plated on a glass coverslip at a density of 1  105 cells/cm2, and fixed in 4% methanolfree formaldehyde in PBS at room temperature for 25 min. After washing with PBS, the cells were permeabilized in 0.2% Triton X-100 in PBS for 5 min,

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and then covered with 100 ll of equilibration buffer at room temperature for 5–10 min. DNA strand breaks were labeled with fluorescein-12-dUTP using rTdT in equilibration buffer at 37 °C for 1 h, avoiding exposure to light. The negative control was incubated in an incubation buffer but without rTdT enzyme. The reaction was terminated with 2  SSC (300 mM sodium chloride and 30 mM sodium citrate, pH 7.4). After washing with PBS, the cells were stained with 1 lg/ml propidium iodide (PI) in PBS at room temperature in the dark for 15 min. TUNEL-positive (apoptotic) cells were detected as localized bright green cells in a red background by using scanning laser confocal microscopy (Leica, Heidelberg, Germany). Data were expressed as the ratio of apoptotic cells to total cells.

incubated in the medium added with 100 nM MitoTracker Green for 30 min at 37 °C. The fluorescence was recorded on an ELx-800 microplate reader using excitation and emission wavelengths of 490 and 516 nm, respectively. Statistical analysis Data were presented as means ± SEM for three separate experiments (each in triplicate or duplicate). Comparisons were analyzed by a one-way analysis of variance (ANOVA) and subsequent Bartlett’s test. Differences were considered statistically significant at P < 0.05.

RESULTS Measurements of GSH, SOD and MDA The cell protein was quantified by the BCA method as mentioned above, and then subjected to measurements of GSH, SOD, and MDA, respectively, exactly as previously described (Zhang et al., 2011). Quantitative real-time reverse transcription PCR (qRT-PCR) After cell treatment, total RNA was extracted from the cells with a Trizol reagent, and RNA was reverse transcribed into cDNA with an Omniscript RT kit according to the manufacturer’s instructions. The reaction mixture consisted of 10 lL 2  Fast EvaGreen Master Mix, 2 lL ROX, 0.25 lM each primer (Table 1), and 1 lL cDNA. PCR amplifications were performed on the 7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The relative mRNA expression level was calculated by the comparative 2DDCt method, and normalized against b-actin mRNA. Measurement of mitochondrial viability

PQQ inhibited ROS production induced by ETC complex I and III inhibitors As a pilot study, specific inhibitors to each of the ETC complexes were used to screen the effects of PQQ on them. These inhibitors included 20 lM rotenone, 80 lM 3-NP, 4 lM antimycin A, and 10 lM NaN3 for complex I, II, III, and IV, respectively, as well as 100 lM of FCCP (an oxidative phosphorylation uncoupler), 200 lg/ml of oligomycin (an ATP synthase inhibitor), and 20 lM of DPI (a flavoenzyme inhibitor). When these inhibitors alone were added to SH-SY5Y cell culture for a 30-min incubation, DCFH-DA assay indicated that intracellular ROS level was significantly increased by rotenone (a complex I inhibitor), antimycin (a complex III inhibitor), and NaN3 (a complex IV inhibitor), respectively (Fig. 1). When each inhibitor in combination with 100 lM PQQ was added to SH-SY5Y cell culture for a 30-min incubation, DCFH-DA assay indicated that only rotenone (a complex I inhibitor)- or antimycin (a complex III inhibitor)-induced ROS increase could be

The cells were incubated in the medium added with mitochondrial viability stain under sterile conditions at 37 °C for 4 h. The fluorescence was recorded on an ELx-800 microplate reader using excitation and emission wavelengths of 550 and 590 nm, respectively. Measurement of mitochondrial DNA content MitoTracker Green FM staining was performed to measure the mitochondrial DNA content. The cells were Table 1. Oligonucleotide sequences used in qRT-PCR analysis Gene

Primer sequence (50 –30 )

Amplicon size

Ndufs1

Forward-acaacaggcagaggaaa Reverse-gcagtaaaggcataggg Forward-acctacctcttgggcttatg Reverse-tcgccagattctattgttg Forward-cttctttgctaaccaccct Reverse-cacccgcttcacttcat Forward-ttcctgctcgcaataac Reverse-tgcccaacccatcaaag Forward-aggcccctctgaaccctaag Reverse-ccagaggcatacagggacaac

131 bp

Ndufs2 Ndufs3 Ndufs4 b-actin

104 bp 130 bp 103 bp 118 bp

Fig. 1. PQQ inhibited the intracellular level of ROS produced by an inhibitor of ECT complex I and III in SH-SY5Y cells. Cultured SH-SY5Y cells were incubated with 100 lM PQQ and different mitochondrial inhibitors (rotenone: inhibitor of complex I, 3-NP: inhibitor of complex II, antimycin A: inhibitor of complex III, NaN3: inhibitor of complex IV, FCCP: oxidative phosphorylation uncoupler, oligomycin: inhibitor of ATP synthase and DPI: inhibitor of flavoenzymes) respectively. After a 30-min incubation, ROS production was measured by DCFH-DA assay. ⁄P < 0.05 versus the corresponding inhibitor added alone; ##P < 0.01 versus control (no inhibitor added) plus no PQQ added.

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significantly attenuated by PQQ (Fig. 1). Based on these observations, rotenone-induced neurotoxicity via ROS elevation was chosen to be investigated in the following experiments. Rotenone caused cell apoptosis in cultured SHSY5Ycells Rotenone, a complex I inhibitor, has been widely used to develop animal models of PD. In this study, an in vitro model of PD was established by exposing cultured SH-SY5Y cells to different concentrations (0.1, 1, 10, 100, 200 and 400 lM) of rotenone for 24 h. CCK-8 assay showed that rotenone significantly decreased cell viability in a concentration-dependent manner (Fig. 2A). For example, cell viability was reduced by about 40% and 50% after exposure to 100 and 200 lM rotenone for 24 h, respectively. In the following experiments, therefore, 100 lM rotenone was used to induce neurotoxicity. After the cells were exposed to 100 lM rotenone for 12, 24, or 48 h, the morphological change of injured cells was observed under phase contrast microscopy, respectively. The control cells (no exposure to rotenone) exhibited a normal cell shape with a round cell body and intact cell membrane, but rotenone-injured cells showed an early apoptotic cell death morphology, such as plasma membrane blebbing and cell shrinkage (Fig. 2B). Western blot analysis was performed to detect the protein expression of Bcl-2 and Bax, two apoptoticrelated genes, in SH-SY5Y cells after exposure to different concentrations of rotenone. The expression ratio of Bcl-2 to Bax was decreased by rotenone in a dose-dependent manner (Fig. 2C, D).

PQQ pretreatment rescued SH-SY5Y cells from rotenone injury Cultured SH-SY5Y cells were pretreated with 100 lM PQQ for 24 h, and then exposed to 100 lM rotenone for an additional 24 h. TUNEL assay showed that apoptotic (TUNEL positive, green-stained) cells occupied approximately 30% of total cells following rotenone exposure, but pretreatment with PQQ significantly decreased the percentage of apoptotic cells in total cells to approximately 10% (Fig. 3A, B), suggesting that PQQ inhibited rotenone-induced cell apoptosis in SH-SY5Y cells. The expression ratio of Bcl-2 to Bax began to decrease at 3 h of rotenone exposure and maintained a low level until 24 h of rotenone exposure, while pretreatment with PQQ alleviated rotenone-induced decrease in the Bcl-2/Bax ratio during 3–24 h of rotenone exposure, showing a time-dependent pattern, with a significant alleviation appearing at 24 h of rotenone exposure (Fig. 3C, D).

ERK1/2 activation was involved in PQQ neuroprotection To investigate the possible involvement of signaling pathways in the neuroprotective effects of PQQ, Western blot analysis was performed to determine ERK1/2 phosphorylation in cultured SH-SY5Y cells under different conditions. When the cells were exposed to 100 lM rotenone for different times (15, 30, 60, and 180 min respectively), the ratio of phosphorylated ERK1/ 2 (p-ERK1/2) to total ERK (t-ERK1/2) in the cells was hardly changed. During rotenone exposure, however, the ratio of p-ERK1/2/t-PEK1/2 in the cells that had

Fig. 2. Rotenone (Rot) induced apoptosis in cultured SH-SY5Y cells. Cultured SH-SY5Y cells were exposed to different concentrations (0.1, 1, 10, 100, 200, 400 lM) of Rot for 24 h, followed by a CCK-8 assay to measure cell viability (A) and images captured under phase contrast microscopy to observe cell morphology (B). Western blot analysis was applied to detect the expression of Bcl-2 and Bax in SH-SY5Y cells exposed to 0.1–100 lM Rot for 24 h. The results are shown in a representative Western blot image (C) and a histogram of the change of Bcl-2/Bax ratio with the concentration of Rot used (D). b-Actin was used for equal protein loading. ##P < 0.01 versus control (without Rot exposure).

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Fig. 3. PQQ inhibited rotenone (Rot)-induced cell damage in cultured SH-SY5Y cells. Cultured SH-SY5Y cells were pretreated with 100 lM PQQ for 24 h and then exposed to 100 lM Rot for an additional 24 h. TUNEL assay was applied to detect the percentage of apoptotic (TUNEL-positive) cells in total cell population (A, B). SH-SY5Y cells not pretreated or pretreated with 100 lM PQQ for 24 h were exposed to 100 lM Rot for additional 3, 6, 12, and 24 h, respectively, followed by Western blot analysis to detect the expression of Bcl-2 and Bax (C, D). SH-SY5Y cells not pretreated or pretreated with 100 lM PQQ for 24 h were exposed to 100 lM Rot for additional 15, 30, 60, and 180 min, respectively, followed by Western blot analysis to detect the expression of p-ERK1/2 and t-ERK1/2 (E, F). The results are shown in representative Western blot images (C, E) and curve lines (D, F). b-Actin was used for equal protein loading. ##P < 0.01 versus control. ⁄⁄P < 0.01 versus no PQQ pretreatment at the same time point.

been pretreated with PQQ was significantly increased as compared to that in the cells without PQQ pretreatment. This increase started from 15 min of rotenone exposure and persisted until 180 min of rotenone exposure with the highest increase occurring at 30 min of rotenone exposure (Fig. 3D, E), suggesting PQQ activation of ERK1/2 pathway. PD98059, an inhibitor of MAPK kinase, was added to cell culture to verify whether activation of ERK1/2 was required for the neuroprotective effects of PQQ. SH-SY5Y cells were pretreated with 100 lM PQQ for 24 h, and then exposed to 100 lM rotenone for an additional 24 h in the presence or absence of 10 lM PD98059, followed by CCK-8 or TUNEL assay. PD98059 reversed PQQ attenuation of cell viability loss in rotenone-injured cells (Fig. 4A), and blocked PQQ

inhibition of rotenone-induced cell apoptosis (Fig. 4B), providing counter-evidence that ERK1/2 activation was required for protective effects of PQQ. In addition, PD98059 also attenuated PQQ-induced increase in SOD activity or GSH concentration in rotenone-injured SHSY5Y cells (Fig. 4C, E). On the contrary, MDA production was increased by rotenone exposure, and this increase could be attenuated by PQQ pretreatment, while in turn, PQQ-induced attenuation was inhibited in the presence of PD98059 (Fig. 4E). PQQ protected rotenone injured-SH-SY5Y cells by maintaining mitochondrial functions Mitochondrial functions play important roles in the survival of SH-SY5Y cells. According to qRT-PCR data, the

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Fig. 4. Activation of ERK1/2 signaling was involved in neuroprotective effects of PQQ. SH-SY5Y cells were incubated with 100 lM PQQ for 24 h and then exposed to 100 lM rotenone (Rot) for additional 24 h in the presence or absence of 10 lM PD98059, followed by CCK-8 assay to measure cell viability (A), followed by TUNEL staining to detect cell apoptosis (B), or followed by determination of SOD activity (C), GSH concentration (D), and MDA concentration (E), respectively. ##P < 0.01 versus control (without any treatment); ⁄P < 0.05 and ⁄⁄P < 0.01 versus Rot exposure;   P < 0.05 and   P < 0.01 versus PQQ pretreatment followed by Rot exposure.

mRNA expression of Ndufs1, Ndufs 2, or Ndufs 4 (ECT complex I subunits) in the cells was decreased by rotenone injury, while PQQ pretreatment reversed rotenone-induced decrease in the mRNA expression of Ndufs1 and Ndufs4 to some extent. Interestingly, PQQ reversion was blocked by ERK1/2 inhibition (Fig. 5A–D). In addition, mitochondrial viability or mitochondria DNA content in the cells were both enhanced by PQQ in a concentration-dependent manner, and the PQQ effects were inhibited by PD98059 (Fig. 5E, F).

DISCUSSION PQQ was initially found as a redox cofactor in bacteria (Hauge, 1964; Salisbury et al., 1979), and later proved to be essential for various plants, animals and humans (Cline and Tsien, 1991; Stites et al., 2000; Kasahara and Kato, 2003; Felton and Anthony, 2005; Rucker et al., 2005, 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). Our previous studies have revealed that PQQ could antagonize glutamate-induced neuronal injury both in vitro and in vivo (Zhang et al., 2011, 2012, 2013). ROS generation from the ETC complex I, II and III is modulated in an insult-specific manner and the

ETC sites are differentially accessible to common antioxidants (Liu and Schubert, 2009). In this study, therefore, we first intended to find out the exact ROS-generating sites that could be affected by PQQ treatment. The results showed that ROS produced by complex I and III inhibitors could be attenuated by PQQ, suggesting these two sites might be accessible to PQQ. Previous studies report that PQQ inhibits the amyloid fibril formation and cytotoxicity of the C-truncated asynuclein variants (Giordano et al., 2012), and PQQ-modified a-Syn (36–46) peptide, a partial sequence of a-synuclein, prevents a-synuclein amyloid fibril formation (Park et al., 2009). These findings suggest that PQQ may be a potential candidate therapeutic agent for PD. Considering that rotenone, a complex I inhibitor, has been widely used in the PD model, we aimed to investigate the possible protective effects of PQQ in the rotenone-induced PD model. Rotenone is used worldwide as an insecticide and fish poison, and can induce PD-like symptoms in neurons through disrupting ATP supply (Sherer et al., 2003). It also has the ability to cross biological membranes and pass the blood–brain barrier, and thus inhibits mitochondrial complex I to increase ROS release and cause cell apoptosis. Exposure of animals to rotenone triggered a range of responses associated with PD, including dopaminergic cell death and ROS production (Martins et al., 2013). Dopaminergic neurons appear to be particularly susceptible to rotenone-induced degeneration (Giordano

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Fig. 5. PQQ maintained mitochondrial functions of SH-SY5Y cells injured by rotenone (Rot) with an involvement of ERK1/2 pathway. SH-SY5Y cells were pretreated with 100 lM PQQ for 24 h and then exposed to 100 lM Rot for additional 24 h in the presence or absence of 10 lM PD98059, followed by qRT-PCR to determine the mRNA expression of Ndufs1, 2, 3, and 4 (A–D), or followed by measurements of mitochondrial viability (E) and mitochondria DNA content (F), respectively. #P < 0.05, ##P < 0.01 versus control (without any treatment); ⁄P < 0.05 and ⁄⁄P < 0.01 versus Rot exposure;  P < 0.05 and   P < 0.01 versus PQQ pretreatment followed by Rot exposure.

et al., 2012). SH-SY5Y is a human neuroblastoma cell line that produces dopamine and expresses dopamine transporters, and so it has been extensively used as an in vitro cell model for dopaminergic neuron injury and death. Some research demonstrated that rotenoneinduced toxicity is dependent on the concentration and time duration of exposure (Martins et al., 2013), but other research showed that rotenone induced a biphasic pattern of neurotoxicity in SH-SY5Y cells (Sherer et al., 2003). In this study, we found that exposure to rotenone for 24 h led to cell apoptosis in SH-SY5Ycells in a concentration-dependent manner, accompanied by the destroyed balance between anti-apoptotic proteins and

pro-apoptotic proteins. We also observed that after rotenone exposure, the activity of SOD and GSH, two potent ROS scavengers, in the cells were both decreased, and the production of MDA, an indicator of oxidative stress, in the cells was increased. In addition, mitochondrial function was also damaged by rotenone exposure. Collectively, the results suggested that both ROS generation and mitochondrial dysfunction contributed to rotenone-induced neurotoxicity. Although the mechanisms underlying neurodegeneration in PD are multifactorial, mitochondrial dysfunction and oxidative stress are widely considered to be central to the pathogenesis of PD (Ge et al., 2013). Recently,

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research advances in PD treatment have been gradually translated into the clinic with promising results, the pathogenesis-targeted therapies are still lacking (Jankovic and Poewe, 2012). Clinical applications of antioxidants have not achieved complete success (Choi et al., 2011) mainly because they cannot easily access the subcellular compartments where ROS is generated. In contrast, PQQ shows a unique advantage due to its neuroprotective potential through modulation of ROS production, as evidenced by our observation that PQQ pretreatment rescued SH-SY5Y cells from apoptotic cell death that was induced by rotenone via ROS generation. Apart from PQQ amelioration of ROS generation, PQQ also increased the activity of SOD and GSH, two antioxidant enzymes, and decreased the level of MDA, a product of lipid peroxidation. Our findings provided further evidence for the antioxidative property of PQQ. It has been known 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). In this study, Western blotting showed that ERK1/2 pathway was activated in PQQ neuroprotection, and MEK (MAP kinase kinase) inhibitor PD98059 was able to block the neuroprotective action of PQQ. Meanwhile, we noted that the effects of PQQ on oxidative stress biomarkers (SOD, GSH, and MDA) could also be counteracted by PD98059. Taking into account that MEK is the immediate upstream activator of ERK, we deduce that MEK/ERK pathway might participate in the protective effect of PQQ against rotenone-induced cell death in SH-SY5Y cells. This deduction is consistent with the previous results that a rapid activation of ERK1/2 in SH-SY5Y cells serves as a self-protective response, and ERK1/2 pathway plays an important role in protection against rotenone toxicity (Park et al., 2009). PQQ stimulates mitochondrial biogenesis in mouse hepatocytes, which might be beneficial in diseases associated with mitochondrial dysfunction. (Chowanadisai et al., 2009). In this study, we verified that rotenone-induced decrease in mitochondrial viability could be blocked by PQQ. Similarly, mitochondrial DNA content was also significantly increased by PQQ. Ndufs1–Ndufs4 are the genes encoding one of the subunits of mitochondrial complex I, and they are required for complete assembly and function of the complex I (Scacco et al., 2003; Alvarez-Fischer et al., 2011; Parsons et al., 2011). Deletion of the Ndufs4 gene would abolish complex I activity in midbrain mesencephalic neurons cultured from embryonic mice, and Ndufs4/ dopamine neurons were more sensitive than Ndufs4+/+ neurons to rotenone toxicity. Ndufs4 inactivation potentiates rotenone toxicity in dopamine neurons (Choi et al., 2008, 2011). In this study, we observed that PQQ pretreatment up-regulated the mRNA expression of Ndufs1, 2, and 4, suggesting that rotenone-induced mitochondrial dysfunction could be ameliorated by PQQ, and we further confirmed that PQQ amelioration was also dependent on ERK1/2 activation. These results, for the first time,

demonstrated that PQQ acted as a mitochondrial stimulant to improve mitochondrial functions in dopaminergic cell line damaged by rotenone, a mitochondrial toxin.

CONCLUSION PQQ could protect rotenone injured-SH-SY5Y cells by reducing ROS production, inhibiting oxidative stress, and maintaining mitochondrial functions, and MEK/ERK pathway played an important role in PQQ-conferred protection of SH-SY5Y cells against rotenone-induced neurotoxicity. Certainly, it is possible that multiple signaling pathways are involved in the proctective effects of PQQ, which deserves to be further investigated. Our findings suggest that PQQ may act as a promising neuroprotective agent for treating neurodegenerative diseases. Acknowledgments—This study was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) of Jiangsu Province, China, National Natural Science Foundation of China (Grant Nos. 81171180 and 81201017) and Natural Science Funding from the Education Department of Jiangsu Province, China (Grant No. 12KJB310011). We thank Professor Jie Liu for assistance in manuscript preparation.

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(Accepted 8 April 2014) (Available online 19 April 2014)