Electron transport chain inhibitors induce microglia activation through enhancing mitochondrial reactive oxygen species production

Author’s Accepted Manuscript Electron transport chain inhibitors induce microglia activation through enhancing mitochondrial reactive oxygen species production Junli Ye, Zhongxin Jiang, Xuehong Chen, Mengyang Liu, Jing Li, Na Liu www.elsevier.com/locate/yexcr

PII: DOI: Reference:

S0014-4827(15)30129-4 http://dx.doi.org/10.1016/j.yexcr.2015.10.026 YEXCR10090

To appear in: Experimental Cell Research Received date: 18 September 2015 Revised date: 21 October 2015 Accepted date: 23 October 2015 Cite this article as: Junli Ye, Zhongxin Jiang, Xuehong Chen, Mengyang Liu, Jing Li and Na Liu, Electron transport chain inhibitors induce microglia activation through enhancing mitochondrial reactive oxygen species production, Experimental Cell Research, http://dx.doi.org/10.1016/j.yexcr.2015.10.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Title page

Electron transport chain inhibitors induce microglia activation through enhancing mitochondrial reactive oxygen species production Junli Ye †*, Zhongxin Jiang ‡*, Xuehong Chen§*, Mengyang Liu‡, Jing Li‡, Na Liu‡ †

Department of Pathophysiology, Medical College, Qingdao University, Qingdao,

Shandong 266071, China; ‡

Department of Clinical Laboratory, the Affiliated Hospital of Medical College Qingdao

University, 266003, Qingdao, China; §

Department of Pharmacology, Medical College, Qingdao University, Qingdao 266071,

China.

*Correspondence to Junli Ye, Department of Pathophysiology, Medical College, Qingdao University, 423 Room, Boya Building, 308 Ningxia Road, Qingdao, 266071, P. R. China. Tel.: +86-532-83780035; Fax: +86-532-83780029; E-mail address: [email protected] *

These authors contributed equally to this work.

Running title: Microglia activation by ETC inhibitors

Lists of Abbreviations: ROS, reactive oxygen species; ETC, electron transport chain; NOX, NADPH oxidase, nicotinamide adenine dinucleotide phosphate-oxidase; LPS, lipopolysaccharide; IFN-γ, interferon-γ ; ROT, rotenone; TTFA, thenoyltrifluoroacetone; AA, antimycin A; NaN3, sodium; APDC, ammonium pyrrolidine dithiocarbamate; IL-1,

interleukin 1;

IL-6,

interleukin 6; IL-12, interleukin 12; TNF-α, tumor necrosis factor α; MAPK, mitogenactivated protein kinase; ERK, extracellular-signal regulated kinase; JNK, c-Jun Nterminal protein kinase; NF-κB, nuclear factor κB; TSPO, translocator protein; DMEM, Dulbecco's-modified Eagle's medium; FBS, fetal bovine serum.

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Abstract Reactive oxygen species ˄ ROS ˅ are believed to be mediators of excessive microglial activation, yet the resources and mechanism are not fully understood. Here we stimulated murine microglial BV-2 cells and primary microglial cells with different inhibitors of electron transport chain (ETC), rotenone, thenoyltrifluoroacetone (TTFA), antimycin A, and NaN3 to induce mitochondrial ROS production and we observed the role of mitochondrial ROS in microglial activation. Our results showed that ETC inhibitors resulted in significant changes in cell viability, microglial morphology, cell cycle arrest and mitochondrial ROS production in a dose-dependent manner in both primary cultural microglia and BV-2 cell lines. Moreover, ETC inhibitors, especially rotenone and antimycin A stimulated secretion of interleukin 1β (IL-1β) , interleukin 6 (IL-6) , interleukin 12 (IL-12) and tumor necrosis factor α (TNF-α) by microglia with marked activation of mitogen-activated proteinkinases (MAPKs) and nuclear factor κB (NF-κB), which could be blocked by specific inhibitors of MAPK and NF-κB and mitochondrial antioxidants, Mito-TEMPO. Taken together, our results demonstrated that inhibition of mitochondrial respiratory chain in microglia led to production of mitochondrial ROS and therefore may activate MAPK/NF-кB dependent inflammatory cytokines release in microglia, which indicated that mitochondrial-derived ROS were contributed to microglial activation.

Key Words˖microglia; ETC; neurodegenerative disease; ROS; mitochondria;

1. Introduction Microglial cells are brain-resident immune cellsˈwhich play a major role in host defense and tissue repair in the central nervous system (CNS) (Prinz and Mildner 2011; Kreutzberg 1996). In pathological conditions including acute brain injury or chronic neurodegenerative diseases such as Parkinson disease and Alzheimer disease (Kreutzberg 1996; Schwarz et al. 2012; Lull and Block 2010), microglial proliferate, migrate, and transform into one or more activated states. Classical M1 activation triggers the

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production of proinflammatory factors such as tumor necrosis factor-α, interleukin-1β (IL-1β) and reactive oxygen species (ROS) and constant activation of microglia and release

of

excess

pro-inflammatory

factors

promoted

the

development

of

neurodegenerative diseases, which can exacerbate brain injury (Horvath and Deleo 2008; Block et al. 2007). However, the mechanisms underlying microglial activation are not fully understood. Recently, many studies have shown that ROS, mainly induced by activation of nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase, NOX), are increasingly implicated as mediators of microglial activation (Park et al. 2008; Yoshioka et al. 2011). NADPH oxidase-dependent ROS may regulate multiple downstream signals such as MAPKs and NF-κB to produce proinflammatory factors and play a vital role in both the initial activation of microglia and their continued activation through reactive microgliosis (Schilling and Eder 2010; Doverhag et al 2008). In addition to NADPH oxidase-dependent formation of ROS, mitochondria, another important resource of intracellular ROS, have been demonstrated to closely relate with the activation of microglia (Banati et al. 2004; Cosenza-Nashat et al. 2009; Choi et al. 2011). By confocal light- and electron microscopic assays, Richard et al. observed that activation of microglia induced by bacterial lipopolysaccharide (LPS) and interferon-γ (IFN-γ) results in a change in the functional organization of mitochondria, i.e., an increase in elongated mitochondrial profiles and ultrastructurally unusual mitochondria and high ATP production (Banati et al. 2004). Some important proteins such as translocator protein (TSPO) and glutamine synthetase, which is required for microglial activation located in mitochondria (Cosenza-Nashat et al. 2009; Choi et al. 2011). Microglial activation is dependent on the normal structure and high energy supply of

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mitochondria. High oxygen consumption of mitochondrial is inevitably accompanied by the formation of ROS, which may be another source of activated microglial. Moreover, including increased metabolic rates, other various stress condition such as hypoxia or membrane damage all markedly induce mitochondrial ROS production (Brookes et al. 2004). However, the exact mechanism of mitochondria-derived ROS in microglial activation is not fully understood.  To explore the possible role of mitochondria in ROS-dependent microglial activation, here we stimulated murine microglial BV-2 cells and primary microglial cells with different inhibitors of respiratory chain to induce ROS production in mitochondria. Four different mitochondrial electron transport chain (ETC) inhibitors used to block key enzymes of the respiratory chain (complex ĉ,Ċ,ċ and Č respectively) were tested in this study: rotenone (ROT), which blocks the complex I ubiquinone (UQ) pathway; thenoyltrifluoroacetone (TTFA), which blocks the complex II UQ pathway; antimycin A (AA), which blocks complex III; and sodium azide (NaN3 ), which blocks complex IV. Rotenone, thenoyltrifluoroacetone (TTFA), antimycin A, or NaN3 were added to the microglial cells cultural medium and cell morphology, viability and cell cycle of microglial cells were observed. Also, mitochondrial profile including mitochondrial ROS production, mitochondrial membrane potential, mitochondrial ultra-structure and proinflammatory cytokine release, MAPK and NF-кB activation of microglia were investigated.

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2. Materials and methods 2.1 Chemicals and reagents Propidium iodide (PI), rotenone, TTFA, antimycin A, NaN3 and mitochondriatargeted SOD (Mito-TEMPO) were purchased from Sigma (St. Louis, MO, USA). Mitotracker deep red, Mitotracker green and MitoSOX RED were purchased from Invitrogen. The phosphor-specific ERK1/2 (Thr202/Tyr204), JNK (Thr183/Tyr185) and p38 (Thr180/Tyr182) antibodies, total (unphosphorylated) ERK1/2, JNK1/2 and p38 antibodies, anti-phospho-NF-кB/p65 antibodies, anti-β-actin antibody, anti-mouse IgG HRP-linked and anti-rabbit IgG HRP-linked secondary antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). PD98059, SB203580, SP600125 and NF-кB inhibitor ammonium pyrrolidine dithiocarbamate (APDC) were purchased from Calbiochem (Darmstadt, Germany). Dulbecco's-modified Eagle's medium (DMEM) containing L-arginine (200 mg/L), fetal bovine serum (FBS), and other tissue culture reagents were purchased from Gibco (Grand Island, NY). 2.2 Cell culture Mouse microglial cells in primary cultures were prepared as described previously with some modifications (Giulian and Baker 1986). Briefly, 1-3 day old C57Bl/6 mice were decapitated according to the guidelines of the Chancellor’s Animal Research Committee at the Medical College of Qingdao University and were in compliance with institutional guidelines (Permit Number: 2010-0025), in accordance with The Chinese Ministry of Science and Technology Guidelines on the Humane Treatment of Laboratory Animals (vGKFCZ-2006-398) and National Institutes of Health Guide for Care and Use

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of Laboratory Animals (Publication No.85-23, revised 1985). Meninges were removed from the brains. Neopallia were dissected and enzymatically (1% trypsin, Invitrogen, 0.05% DNAse, Worthington, 2 min) and mechanically dissociated. The resulting cells were centrifuged (200 × g, 10 min), suspended in culture medium (DMEM, Invitrogen) supplemented with penicillin (100 U/ml), streptomycin (100 ­g/ml) (Invitrogen) and heat-inactivated fetal bovine serum (10% FBS, Gibco); and plated into 75-cm2 flasks (BD Falcon) precoated with poly-L-lysine (0.1 mg/ml; Sigma) at a density of 5 × 107 cells per flask. Media were replaced the next day with DMEM/F12 containing 20% FCS. Cells were grown for 7 d without changing the medium to allow microglial proliferation. Microglia was harvested by shaking for 30 min on a rotary shaker at 120 rpm. The enriched microglia were >95% pure as determined by OX-42-IR (a marker for microglia; Serotec, USA) and GFAP-IR (a marker for astrocyte; Sigma, USA). Cells were cultured in DMEM, with 20% fetal bovine serum (FBS), penicillin (100 units/ml), and streptomycin (100 μg/ml) in 5% CO2, 95% air at 37°C in a humidified incubator. BV-2 immortalized murine microglial cells were provided by the Cell Culture Center of the Chinese Academy of Medical Sciences (China). Cells were cultured in DMEM with 15% heat-inactivated fetal calf serum (FCS), penicillin (100 units/ml), and streptomycin (100 μg/ml) in 5% CO2, 95% air at 37°C in a humidified incubator. 2.3 Cell treatment Microglial cells were stimulated with different doses of rotenone, TTFA, antimycin A or NaN3 (0.01-1000μM) alone respectively, or cotreated with Mito-TEMPO˄200μM˅, and thereafter further analysis were examined as described below. For the study on the activity of MAPK/NF-кB, cells were pretreated with MAPK or NF-кB inhibitors,

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PD98059, SB203580, SP600125 and APDC, for 3h, and then treated with ETC inhibitors. Cells for viability assays were performed using 96-well plates (seeded at 4 × 104 cells/well) while other assays were grown in 6-well plates (6h105 cells/well) or onto coverslips (15 × 104 cells/ coverslip). 2.4 Cell proliferation and viability analysis Cells were seeded in a 24-well plate at a density of 5h104 cells/well or 96-well plates at a density of 4 h 104 cells/well and incubated with different inhibitors for 24h. Cell morphology was observed under the phase contrast microscope. Cell viability was determined using LDH Cytotoxicity Assay Kit according to the supplier’s recommendation (Bi Yuntian Biological Technology Institution, Shanghai, China). All experiments were performed in triplicate. 2.5 Cell apoptosis and cell cycle Cell apoptosis and cell cycle distribution was monitored by flow cytometry using a flow cytometric viability probe, Propidium iodide (PI) staining (Sigma). Briefly, BV-2 cells were harvested, washed, and fixed in 70% ethanol overnight at 4°C. Prior to flow cytometry, cells were washed and stained with 1 ml of PI (5 µg/ml) containing 0.1 mg/ml RNase A. DNA content was determined with a FACScan flow cytometer (BectonDickinson, Franklin Lakes, NJ, USA) and the proportion of cells in a particular phase of cell cycle was determined with CellQuest software (Becton-Dickinson, San Jose, CA, USA ). 2.6 Measurement of mitochondrial potential and mitochondrial ROS accumulation Mitochondrial ROS production and mitochondrial potential was measured using three types of mitochondrial-specific labels that distinguish ROS-generating mitochondria (

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MitoSOX RED, which can be selectively oxidized by superoxide anion in the mitochondria), respiring mitochondria (Mitotracker deep red) and total mitochondria (Mitotracker green). First, microglial cells were plated in six-well plates and treated with inhibitors for 24 h. After a brief wash, cells were rinsed and incubated with MitoSOX RED, Mitotracker deep red or Mitotracker green for 10 minutes in the dark at 37°C. Next, cells were washed twice with PBS, trypsinized. Mitochondrial ROS and mitochondrial potential were observed by FACS analysis with 488 nm excitation/515-545 nm emission filters. Data analysis was performed with CELLQuest software and the mean fluorescence intensity was used to quantify the responses. A minimum of 10,000 cells were acquired for each sample. 2.7 Transmission electron microscopy Cultured microglial cells were initially plated in triplicate at a density of 5 ×105 cells /well in 6 well plates and then treated with different inhibitors for 24h. Cells were trypsinized, collected into eppendorff tube after washing and then fixed by 2.5% glutaraldehyde at 4ć. 24h later, microglia were washed by PBS, fixed by osmic acid, then washed by distilled water, and dehydrated by dimethylketone. After embedment in Epon-812, the sample was cut into ultrathin sections (70 nm). The ultrathin sections were dyed with uranium acetate and plumbum citrate and examined with JEM-1200EX electron microscopy (Chen et al. 2000). 2.8 ELISAs for TNF- a, IL-1β, IL-6 and IL-12 The amount of TNF-a, IL-1β, IL-6 and IL-12 was measured using ELISA kits purchased from R&D Systems (Minneapolis, MN, USA) following the manufacturer’s instruction. Briefly, standards and samples were added to a 96-well ELISA plate

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precoated with biotinylated anti-TNF-α, anti-IL-1β, anti-IL-6 or anti-IL-12 antibody. After washing away unbound substances, enzyme-linked polyclonal antibody respectively specific for TNF-a, IL-1β, IL-6 or IL-12 was added to the wells and incubated for two hours. The wells were then washed four times and filled with the substrate solution for an incubation of 30 minutes. The reaction was terminated by the stop solution. Absorbance was read at 450 nm in a microplate reader. The concentration of each sample was calculated from the standard curve prepared using the cytokine standards. Three wells were treated per experiment. The concentrations of the cytokines and growth factor were calculated in pg/ml protein. 2.9 Western blotting For the quantification of protein expression, Western blot analysis was used. Microglial cells in six-well plates were incubated and stimulated as described above. Cells were washed with ice cold PBS and lysed with RIPA-Buffer (150 mM NaCl, 10mM Tris, 0.1% SDS, 1% Triton-X-100, 1% Deoxycholate, 5 mM EDTA, pH 7.4) with protease inhibitors (Roche, 04693159001). Nuclear and cytosolic extracts of microglial cells were prepared by lysing cells with Igepal CA-630 followed by differential centrifugation as before (Woo et al. 2003). Whole cell lysates were electrophoresed (SDS/PAGE), transferred to polyacrylamide gels, and immunoblotted using antibodies directed against appropriate antibodies: for ERK (1:200, Santa Cruz)ˈp-ERK (1:200, Santa Cruz)ˈp38 (1:200, Santa Cruz)ˈp-p38 (1:200, Santa Cruz)ˈJNK(1:200, Santa Cruz)ˈp-JNK (1:200, Santa Cruz)ˈIкB (1:1000, Santa Cruz), p-IкB (1:1000, Santa Cruz), p65(1:1000, Santa Cruz) and β-actin (1:1,000, Santa Cruz). Immunoblot analysis was performed with horseradish peroxidase–conjugated anti-mouse and anti-rabbit IgG

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using enhanced chemiluminescence Western blotting detection reagents (Amersham Bioscience, Piscataway, NJ, USA). The bands were scanned and densitometrically analyzed using an automatic image analysis system (Alpha Innotech Corporation, San Leandro, CA, USA). These quantitative analyses were normalized to β-actin (after stripping). 2.10 Statistical analysis All quantitative data and experiments described in this study were repeated at least three times. All analyses were performed blinded such that experimenters performing data analysis were unaware of the treatments. Data were expressed as means ± S.E.M. The criterion for statistical significance was P<0.05. Bartlett’s tests showed no significant differences in group variances; therefore, data were evaluated using parametric statistics. Comparisons between the different groups were performed by either Student’s t-test or one-way ANOVA followed by post hoc Bonferroni tests for comparison among means. All data were analyzed using GRAPHPAD PRISM (GraphPad Software Inc., La Jolla, CA, USA) data analysis software. 3. Results 3.1 Inhibition of mitochondrial respiratory chain changed microglial cell viability and microglial morphology Studies have observed the effects of mitochondrial toxins like rotenone and 3-NP on microglial activation mostly at low concentrations range (1-500nM) (Ferger et al. 2010; Yuan et al. 2013; Gao et al. 2013). According to the existing literatures and our preliminary tests, here we first observed the cytotoxity of different concentrations of ETC inhibitors (0.01–1000μM) on murine microglial BV-2 cells and primary microglial cells. LDH assays showed that treatment BV-2 cells or primary microglial cells with high

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concentrations (100-1000μM) of rotenone, TTFA, antimycin A, or NaN3 for 24h resulted in significantly loss of cell viability in a dose-dependent manner, respectively (Fig.1A). At ranges of 0.01-1μM, no obvious cell cytotoxity but mild proliferation could be observed in BV-2 cells or primary microglial cells. 10μM ETC inhibitors treatment, especially rotenone and antimycin A showed slightly inhibition on cell growth, but the differences were not significant compared with non-treated cells. Under phase contrast microscopy, the morphology of microglial cells treated with ETC inhibitors (1-10μM) for 24 h changed in varying degrees and most cells became round or amoeboid and the cell processes become shorter. Whereas in control non-treated cells, microglial cells were characterized by long bipolar or unipolar processes and elongated cell bodies (Fig.1B). Microglial cells began to undergo cell death characters such as cell refraction and bleaching in high levels (100-1000μM) ETC inhibitors-treated groups. These results indicated that ETC inhibitors could activate microglial cells at certain ranges of concentrations. 3.2 Inhibition of mitochondrial respiratory chain resulted in changes of cell cycle distribution and cell apoptosis in microglial cells Suppression of microglial cell growth by ETC inhibitors can be explained by cell cycle arrest and cell death. To determine the mechanism responsible for ETC inhibitorsmediated inhibition of cell growth, BV-2 cells were treated with different concentrations of ETC inhibitors for 24h and the cell growth and the cell cycle phase distribution was then analyzed by flow cytometry. As shown in Fig. 2, no significant differences were found in cell cycle distribution between control non-treated cells and cells treated with 0.01-1μM ETC inhibitors (P>0.05). Treatment with high concentrations of ETC inhibitors (10-1000μM) resulted in significant changes in cell cycle distribution and cell apoptosis, especially in rotenone and antimycin A groups. Rotenone arrested microglial cells at the G2/M phase and the number of sub-G1 cells (apoptotic cells) gradually increased in a dose-dependent manner. Cells challenged with low concentrations of

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antimycin A or NaN3 showed slow accumulation at the G1 phases, and S phase arrest could be observed in a subset of TTFA-treated cells (Fig. 2). Similar to rotenone, treatment with TTFA, antimycin A and NaN3 for 24 h resulted in a concentrationdependent increase of cells in the sub-G1 stage of the cell cycle. At 1000μM levels, subG1 phase populations in rotenone, TTFA, antimycin A and NaN3 group were 29.8%, 21.3%, 35.8% and 32.7%, respectively, which were more than 2.8% in control nontreated cells. Although different ETC inhibitors showed similar effects on cell viability, the results from cell cycle analysis seems to suggest that different ETC inhibitors might affect microglial cell growth via cell cycle progression through distinct mechanisms and different signaling pathways. 3.3 Inhibition of mitochondrial respiratory chain increased the production of mitochondrial ROS and decreased mitochondrial membrane potential The main source of cellular ROS is mitochondria and mitochondrial depolarization (decrease in mitochondrial membrane potential) is another major event in mitochondrial dysfunction (Zhang et al. 2014; Li et al. 2003). To further investigate possible implication of mitochondria in microglial activation, we artificially induced ROS in mitochondria by different ETC inhibitors. We observed mitochondrial ROS production and mitochondrial membrane potential by using three types of mitochondria-specific labels that distinguish ROS-generating mitochondria (MitoSOX), respiring (Mitotracker deep red) and total (Mitotracker green) (Fig.3). In agreement with some previous reports (Li et al. 2003; Indo et al. 2007), addition of the ETC inhibitor resulted in mitochondrial ROS production in microglial cells in a dose-dependent manner (Fig. 3A). ROS-generating mitochondria were observed more significant when complex I or complex III of microglial cells were inhibited by rotenone or antimycin A, respectively, whereas the complex II inhibitor TTFA and the complex Č inhibitor NaN3 had less effects than rotenone and antimycin A (Fig. 3A). Meanwhile, determined by Mitotracker deep red and Mitotracker green staining, microglial cells treated with ETC inhibitors showed the loss of mitochondrial

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membrane potential in a dose-dependent manner, especially in rotenone or antimycin A treated cells. At 10μM levels, rotenone or antimycin A treatment led to significant loss of mitochondrial membrane potential, whereas TTFA and NaN3 had only minor effects (Fig. 3B). Transmission electron microscopy showed the ultrastructure changes of microglial cells treated with ETC inhibitors (Fig.3C). In control untreated microglia, the mitochondria of cells was characterized by folded cristae and voluminous intracristal spaces embedded in an electron dense, homogeneous matrix. The increased numbers of elongated and swelling mitochondrial profiles were observed in subcytotoxic rotenone or antimycin A (10μM) treated microglia, which is consistent with the characteristics of activated microglia. Also, some autophagic vacuoles could be observed in rotenone or antimycin A treated microglia. 3.4 Inhibition of mitochondrial respiratory chain increased the production of proinflammatory cytokines in microglial cells Upon activation, microglia release high levels of interleukins and other cytokines which are part of the inflammatory response contributed to neuronal injury (Kim et al. 2004). To evaluate the impact of ETC inhibitors on microglial activation, we analyzed the release of four pro-inflammatory cytokines, TNF-α, IL-1β, IL-6 or IL-12 in the media. BV2 cells or primary microglial cells were treated with four above-mentioned ETC inhibitors for 24 hours. ELISA assays showed that rotenone or antimycin A stimulation significantly elevated the levels of TNF-α, IL-1β, IL-6 or IL-12 in a dose-dependent manner at the range of 0.01-1000μM (Figure 4, P < 0.05). Conversely, little of these cytokines were detectable in the media of microglia cultures after TTFA or NaN3 treatment. These data suggest that rotenone and antimycin A may induce an inflammatory response in primary cultured microglia and BV-2 cells and TTFA or NaN3 may be not directly act on microglia to induce inflammation.

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3.5 Inhibitory effects of mitochondrial antioxidants Mito-TEMPO on the ETCinduced activation of microglia To confirm the role of mitochondrial ROS in microglial activation, we cotreated microglia with mitochondrion-specific antioxidant Mito-TEMPO˄200µM˅and ETC inhibitors˄10µM˅ for 24h to reduce mitochondrial ROS levels (Fig. 5A and Fig.5B). MitoSOX staining showed that cotreatment with Mito-TEMPO could effectively reduced ETC-induced mitochondrial ROS production both in BV-2 and primary cultured microglial cells (P<0.05). Given that activation and inflammatory responses of microglia are consequences of mitochondrial ROS production, the inhibitory effects of MitoTEMPO on cytokines release induced by ETC inhibitors was further examined. As shown in Fig.6, cotreatment with Mito-TEMPO could significantly inhibit the production of TNF-α, IL-1β, IL-6 or IL-12 in rotenone or antimycin A treated BV-2 cells and primary cultured microglia (P<0.05), which indicated that mitochondrial-derived ROS production may be involved in microglial inflammation response. 3.6 Activation of microglia induced by ETC inhibitors was related to MAPK/NF-кB signals Current researches have demonstrated that MAPKs and NF-κB play important roles in microglia activation by modulating the expression of pro-inflammatory cytokines (Yuan et al. 2013; Gao et al. 2013). Therefore, we investigated the effects of rotenone, TTFA, antimycin A and NaN3 on the activity of p38, JNK, ERK, and NF-κB in BV-2 cells. We observed the time-course changes of MAPKs and NF-κB phosphorylation by Western blot analysis at 15, 30, 60 min after ETC inhibitors treatment (Fig. 7). The results showed that inhibition of

mitochondrial respiratory chain by rotenone and

antimycin A indeed induced marked activation of p38, ERK, JNK, and NF-κB in microglial cells in a time-dependent manner (P<0.05), which stimulatory effects were slightly more than TTFA and NaN3 (P<0.05). The peak of activation for each kinase 14

varied within different ETC inhibitors. In rotenone-treated cells, p38, ERK and JNK activation peaked at 30 minutes post stimulation, and IκB activation and p65 translocation to nuclear peaked at one hour. In TTFA, antimycin A or NaN3-treated cells, the time course experiments showed the activation of p38 and JNK peaked at 30 min after stimulation and the activation of ERK and IκB and p65 translocation to nuclear were sustained up to 60 min (Fig. 6A). To investigate the role of MAPK and NF-κB signaling pathway on the expression of pro-inflammatory cytokines induced by ETC inhibitors, we investigated the effect of specific p38 inhibitor SB203580, specific ERK1/2 inhibitor PD98059, specific JNK inhibitor SP600125 and NF-кB inhibitor ADPC on 10µM ETC inhibitors-induced proinflammatory cytokines in BV2 cells (Fig. 8). ELISA assays showed that pharmacological inhibitors of p38, JNK and NF-кB pathways could significantly reduce the production of pro-inflammatory cytokines induced by rotenone and antimycin A (P<0.05), whereas PD98059 showed less inhibitory effects on the cytokines production compared with SB203580, SP600125 and ADPC (P<0.05). Although TTFA and NaN3treated cells produced very little cytokines, it can be blocked by specific inhibitors MAPK and NF-κB signaling pathway (data not shown). Altogether, these results suggested that MAPK-NF-кB pathways, especially p38, JNK and NF-кB mediated mitochondrial ROS-induced inflammatory responses in microglia. 4. Discussion Studies have demonstrated that ROS play a vital role in both the initial activation of microglia and their continued effects on neuronal injury (Bordt and Polster 2014; Block et al. 2007). Mitochondria are another major intracellular source of ROS apart from NADPH oxidase (Indo et al. 2007; Bordt and Polster 2014). However, the production of ROS in microglia in microglia and how mitochondrial ROS contributed to microglial activation are still unclear. To investigate the possible implication of mitochondria in

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microglial activation, here we first artificially induced ROS in mitochondria by different inhibitors of mitochondrial respiratory chain, rotenone, TTFA, antimycin A and NaN3 in microglia and our results found that inhibitors of mitochondrial respiratory chain by ETC inhibitors may induce mitochondrial ROS production and lead to microglial morphology changes and pro-inflammatory cytokines release, which indicated the possible role of mitochondrial dysfunction in ROS-dependent microglial activation. Some researchers have reported microglial activation upon exposure to chronic or low concentrations of mitochondrial toxins such as rotenone or 3-NP in vivo and in vitro (Ferger et al. 2010; Yuan et al. 2013; Gao et al. 2013; Zhang et al. 2014), however little information available about mitochondrial dysfunction by different ETC inhibitors treatment on microglial activation. To achieve the excessive ROS production and microglial activation, here we observed the dose-effect relationship of inhibition of mitochondrial respiratory chain by rotenone, TTFA, antimycin A and NaN3 in BV-2 cells or primary microglial cells. LDH assay showed cytotoxity of high concentrations ETC inhibitors treatment (100-1000μM) on microglia and the mild proliferatory effects of low concentrations of rotenone (0.01-1μM), which were in line with the above mentioned studies (Ferger et al. 2010; Yuan et al. 2013; Gao et al. 2013; Zhang et al. 2014). At the subcytotoxic dose, 10μM level, the activated morphological changes, characterized by round or ameboid cell body and shorter cell processes could be observed in most ETC inhibitors groups. At high levels (100-1000μM), ETC inhibitors-treated microglia began to undergo cell death characters such as cell refraction and bleaching, which is consistent with the cell viability results. The effects of mitochondrial complexes defects on cellular growth may be controlled by different cell cycle distribution. Diverse effects of ETC inhibitors on cell cycle and cell apoptosis have been reported in other non-microglia cell lines (Armstrong et al. 2001;

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Byun et al. 2008). Byun et al. have reported that rotenone induced cell cycle arrest at G2/M, TTFA induced overall delay in cell cycle progression, antimycin A induced slow accumulation at G1 and S phases, and complex Č inhibitor, oligomycin resulted in substantial accumulation of cells at G1 in asynchronous cells (Byun et al. 2008). In line with these finding, our data in BV-2 cells found G2/M arrest in 0.01-10μM rotenone group, G1 and S arrest in 0.01-10μM antimycin A and NaN3 group. However, the cell cycle profile in TTFA-treated BV-2 cells showed different distributions with S phase arrest and concentration-dependent increase of cells in the sub-G1 stage were observed in all ETC inhibitor groups. Meanwhile, microglial activation always begin with microglial cells proliferate, which is highly related to cell cycle changes and studies have found that cell cycle inhibition could attenuate microglia induced inflammatory response and alleviates neuronal cell death in the CNS trauma (Kabadi SV et al., 2014; Zhang Q et al., 2009; Cernak I et al., 2005). Combined with the above existing literature, the different effects of ETC inhibitors on cell cycle appeared to depend on mitochondrial complexspecific inhibition, cell-type specific and the kind of stimuli and length of time. The exact molecular mechanism should be identified in our further study. Mitochondrial superoxide, which is generated in the electron transport systems of mitochondria in vivo and could convert to H2O2 and hydroxyl radicals, is the main source of cellular ROS (Chance et al. 1979; Bandy and Davison 1990; Guidot et al. 1993). Superoxide is mainly generated from complex ĉ, complex ċ and complex Ċ (Chen et al. 2003; Koopman et al. 2010; Jezek and Hlavata 2005). Under the damage to electron transport machinery, overwhelming superoxide resulted in mitochondrial depolarization (decrease in mitochondrial membrane potential), which constitute the major event in mitochondrial dysfunction (Zhang et al. 2014; Li et al. 2003). In agreement with some

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previous reports (Koopman et al. 2010; Chen et al. 2003; Jezek and Hlavata 2005), our present results confirmed the excessive dose-dependent mitochondrial ROS production and loss of mitochondrial membrane potential after different ETC inhibitors stimulation by three mitochondrial specific probe, MitoSOX, Mitotracker deep red and Mitotracker green staining. ROS-generating mitochondria and loss of mitochondrial membrane potential were observed more significant when complex ĉ or complex ċ of microglial cells were inhibited by rotenone or antimycin A, whereas the complex Ċ inhibitor TTFA and the complex Č inhibitor NaN3 had less effect. Moreover, the ultrastructure changes of microglial cells observed by transmission electron microscopy showed more increased number of elongated and swelling mitochondrial profiles in rotenone or antimycin A treated microglia. The dose-dependent mitochondrial injury are basically in line with the results observed by LDH assays, which showed dose-dependent increase in cell cytotoxity of microglial cells induced by ETC inhibitors. As microglial cells response to oxidative stress stimulation first involves activation of ROS-related signalling pathway thus may lead to activation of microglia and microglial inflammatory responses. Overwhelming ROS production could result in cellular damage and subsequent cell death. Our results clearly suggested mitochondria are a major source of intracellular ROS and the cytotoxic effects of ETC inhibitors. Also, our findings about different effects of ETC inhibitors on ROS production suggested the Complex Ϩand Ϫ are more important main sites for superoxide production than complex II and the complex Č, which have been reported by others (Koopman et al. 2010; Chen et al. 2003). The response intensity depend on cell type, the kind and strength of stimuli and length of stimulation time. We inferred that the different effects of low concentration of rotenone and antimycin on mitochondria and cell death related to different intracellular signalling pathway activated by moderate production of mitochondrial ROS and the direct evidences should be explored in the future.

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Constant activation of microglia could produce large amounts of neurotoxic factors, such as TNF-α, IL-1β, IL-6, IL-12, which are highly related to the pathogenesis of brain injury, neurodegenerative disorders such as Alzheimer’s disease and Parkinson disease (Tambuyzer et al. 2009) and complex central nervous system dysfunctions such as cognition, sleep and depression (Dantzer et al. 2008; McAfoose and Baune 2009; Menza et al. 2010). The stimulatory effects of rotenone on the production of pro-inflammatory factors in microglia have been confirmed by some researchers (Yuan et al. 2013; Gao et al. 2013; Zhou et al. 2008; Chang et al. 2011), although some other results are a bit different (Klintworth et al. 2009). In line with most reports, our present study found the dose-dependent effects of rotenone or antimycin A stimulation on the TNF-α, IL-1β, IL-6 or IL-12 production in both BV-2 cells and primary culture microglia by ELISA assays and these effects could be partly blocked by antioxidants Mito-TEMPO. Conversely, little cytokines levels were observed in TTFA or NaN3 treated microglia. These data suggested that rotenone and antimycin A may induce an inflammatory response in primary cultured microglia and BV-2 cells and TTFA or NaN3 may be not directly act on microglia to induce inflammation, and mitochondrial-derived ROS production may be involved in microglial inflammation response. MAPKs and NF-κB are two important downstream regulators of ROS and their signaling is critical in modulating the expression of pro-inflammatory cytokines in microglia activation (Yuan et al. 2013; Gao et al. 2013; Zhou et al. 2008). In our present BV-2 cell model, we observed that inhibition of mitochondrial respiratory chain by rotenone or antimycin A indeed induced marked activation of p38, ERK, JNK and NF-κB in microglial cells in a time-dependent manner, which stimulatory effects were slightly more than TTFA and NaN3. And ELISA assay confirmed that inhibition of the MAPKs and NF-κB pathway with respective inhibitors decreases the production of proinflammatory cytokines of BV-2 cells induced by ETC inhibitors. Although the peak of activation for each kinase varied within different ETC inhibitors and the effects of the

19

inhibitors of MAPKs and NF-κB pathway are in general rather modest in our present study, our result are basically consistent with some exsiting reports on rotenone (Yuan et al. 2013; Gao et al. 2013; Zhou et al. 2008) and we speculated that the differences in activity of MAPK and NF-κB may be related with the different cell model, ETC inhibitors and the concentrations of stimuli, which need our further study. In addition, considering SB203580 and SP600125 may have off-target effects, our future work will focus on inhibition of MAPK and NF-κB signaling pathway components by RNAimediated downregulation or using combinations of more inhibitors. 5. Conclusion In conclusion, our present study first induced mitochondrial ROS production by blocking key enzymes of the mitochondrial respiratory chain and observed their stimulatory effects on microglial activation. We provided evidences that the microglia could be activated by different ETC inhibitors, through inhibiting mitochondrial complex ĉ, Ċ, ċ or Č respectively, induction of mitochondrial ROS, activation of MAPK and NF-κB and resulting in the release of the pro-inflammatory cytokines. Our study provided useful information for understanding the role of mitochondria in microglial activation and effective protections against mitochondrial ROS in excessive microglial activation. In fact, inhibition of the mitochondrial respiratory chain diminishes aerobic energy metabolism, which may interfer with microglial inflammatory responses. Further experiments should be carried out to explore the molecular mechanisms of different ETC inhibitors on the signaling pathways of mitochondrial ROS-mediated microglial activation and the studies on the connection of mitochondria homeostasis including mitochondrial energy metabolism with microglial inflammatory responses should be performed.

20

Acknowledgments We are grateful to Dr. Junsheng YE for providing us with kind advices and for thoughtful discussions. We also wish to thank Dr Shuhong Qiao for providing us with antibodies and advice. The work was supported by the National Natural Science Foundation

of

China

(Grant

No.

Research Award Fund for Outstanding

31100824

and

Middle-aged

81473384), and

the Young

Scientist of Shandong Province (Grant No. BS2012YY004) and the Shandong Provincial Natural Science Foundation, China (Grant No. ZR2010HL068).

Authorship: Junli Ye carried out the whole studies, participated in cell treatment, cell viability analysis, measurement of mitochondrial potential and mitochondrial ROS accumulation, ELISAs for pro-inflammatory cytokines and drafted the manuscript. Zhongxin Jiang carried out cell culcure and ELISAs for pro-inflammatory cytokines and drafted the manuscript. Xuehong Chen participated in the cell proliferation and viability analysis, cell apoptosis and cell cycle analysis, transmission electron microscopy and drafted the manuscript. Mengyang Liu participated in ELISAs for pro-inflammatory cytokines and performed the statistical analysis. Jing Li participated in surgical procedures and helped to draft the manuscript. Na Liu participated in Western blotting and helped to cell culture. All authors read and approved the final manuscript.

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Declaration of Interest All authors in this paper declare that there are no conflicts of interest in this research.

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Figure Legends Fig. 1. Effects of mitochondrial ETC inhibitors on cell viability and cellular morphology of microglial cells. BV-2 cells and primary cultural microglia were stimulated with the indicated final concentration (0.01-1000μM) of rotenone (Rot), 2-theonyltrifluoroacetone (TTFA), antimycin A (AA), and NaN3 for 24 h. (A) Cells viability was measured with LDH assay. Each bar represents the mean ±S.E.M of triplicate microcultures. *p < 0.05 compared to untreated cells by Student's t test. (B) Cellular morphology was visualized

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under the phase contrast microscope following ETC inhibitors treatment at 24h. Scale bar=20 μM.

Fig.2. Effects of mitochondrial ETC inhibitors on cell apoptosis and cell cycle of microglial cells. BV-2 cells were incubated with DMEM containing different ETC inhibitors (0.01-1000μM) for 24 h. Cell cycle analysis was determined by FACS analysis of PI stained cells as described under Materials and Methods. Apoptosis was measured as percentage of cells containing hypodiploid amounts of DNA (sub G1).

Fig. 3. Effects of mitochondrial ETC inhibitors on mitochondrial ROS production, mitochondrial membrane potential and mitochondrial morphorlogy in microglia. BV-2 cells and primary cultural microglia were stimulated with the indicated final concentration of rotenone (Rot), 2-theonyltrifluoroacetone (TTFA), antimycin A (AA), and NaN3 for 24 h. (A) Cells were stainned with MitoSOX for 30min and the levels of the mitochondrial ROS production were evaluted by quantitatively analyses of MitoSOX Red fluorescence signal intensity by flow cytometry. The data were represented as intensity of ROS production of cells. Each bar represents the mean ±S.E.M of triplicate microcultures. *p < 0.05 compared to non-treated cells by Student's t test. (B) Cells were stainned with Mitotracker green and Mitotracker deep red for 30min and the mitochondrial membrane potential were evaluted by quantitatively analyses of fluorescence signal intensity by flow cytometry. (C) The morphological mitochondrial ultrastructural changes in BV-2 cells were observed under transmission electric microscopy. The mitochondria of control cells were characterized by folded cristae and

28

voluminous intracristal spaces embedded in an electron dense, homogeneous matrix. The increased numbers of elongated and swelling mitochondrial profiles and some autophagic vacuoles were observed in rotenone or antimycin A treated microglia. Scale bars=1μm.

Fig. 4. Effects of mitochondrial ETC inhibitors on the proinflammatory cytokine secretion by cultured microglia. BV-2 cells and primary cultural microglia were stimulated

with

the

indicated

final

concentration

of

rotenone

(Rot),

2-

theonyltrifluoroacetone (TTFA), antimycin A (AA), and NaN3 for 24 h. The mean concentration of TNF-a, IL-1β, IL-6 and IL-12 in the culture medium of microglia was measured by ELISA. Each bar represents the mean ±S.E.M of triplicate microcultures. The concentration of each sample was calculated from the standard curve prepared using the cytokine standards and growth factor were calculated in pg/ml protein. *p < 0.05 compared to non-treated cells by Student's t test.

Fig.5. Effects of antioxidant Mito-TEMPO on the mitochondrial ETC inhibitors-induced mitochondrial ROS production in microglia. (A) BV-2 cells and (B) primary cultural microglia were treated with rotenone, TTFA, antimycin A or NaN3 (10μM) alone or combined with Mito-TEMPO˄200μM˅for 24h. Cells were stainned with MitoSOX for 30min and the levels of the mitochondrial ROS production were evaluated by quantitatively analyses of MitoSOX Red fluorescence signal intensity by flow cytometry. The data were represented as intensity of ROS production of cells. Each bar represents the mean ±S.E.M of triplicate microcultures. *p < 0.05, **p < 0.01, compared to non-

29

treated control cells by Student's t test. #p < 0.05 compared to corresponding control cells by Student's t test.

Fig.6.. Effects of antioxidant Mito-TEMPO on the mitochondrial ETC inhibitors-induced proinflammatory cytokine secretion by cultured microglia. BV-2 cells and primary cultural microglia were treated with rotenone, TTFA, antimycin A or NaN3 (10μM) alone or combined with Mito-TEMPO˄200μM˅for 24h. The mean concentration of TNF-a, IL-1β, IL-6 and IL-12 in the culture medium of microglia was measured by ELISA. Each bar represents the mean ±S.E.M of triplicate microcultures. The concentration of each sample was calculated from the standard curve prepared using the cytokine standards and growth factor were calculated in pg/ml protein. *p < 0.05, **p < 0.01, compared to nontreated control cells by Student's t test. #p < 0.05 compared to corresponding basal cells by Student's t test.

Fig.7. Effects of mitochondrial ETC inhibitors on the activity of MAPKs and NF-κB in microglia. BV-2 cells were treated with rotenone, TTFA, antimycin A or NaN3 (10μM) for indicated time respectively. The cytoplasmic and nuclear fractions from the BV2 cells were harvested for Western blot analysis. The activation of p38, ERK and JNK at different time points was assessed by measuring the respective phosphorylated form of MAPKs. Total amount of ERK, JNK, and p38 MAPK was measured as MAPK controls. The activation of NF-κB at different time points was assessed by measuring the phosphorylated form of IκB and nuclear and cytosolic p65. β-Actin served as the marker for the cytoplasmic fraction, and Max served as the marker for the nuclear fraction.

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Fig.8. Inhibition of MAPK or NF-κB signaling on ETC inhibitors-mediated proinflammatory cytokine secretion by cultured microglia. BV-2 cells were pretreated with MAPK or NF-кB inhibitors, PD98059, SB203580, SP600125 and APDC, for 3h, and then treated with ETC inhibitors (10μM) for 24h. The mean concentration of TNF-a, IL-1β, IL-6 and IL-12 in the culture medium of microglia was measured by ELISA. Each bar represents the mean ±S.E.M of triplicate microcultures. The concentration of each sample was calculated from the standard curve prepared using the cytokine standards and growth factor were calculated in pg/ml protein. *p < 0.05, **p < 0.01, compared to nontreated control cells by Student's t test. #p < 0.05 compared to corresponding basal cells by Student's t test.

Highlights 

l ETC inhibitors induce mitochondrial ROS production in microglia l ETC inhibitors stimulated secretion of pro-inflammatory cytokines by micriglia l MAPK/NF-κB pathways maybe related to the microglial activation by ETC inhibitors

31

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8