88 Cellular, molecular and developmental neuroscience
SIRT2 is required for lipopolysaccharide-induced activation of BV2 microglia Heyu Chena, Danhong Wuc, Xianting Dinga and Weihai Yinga,b It has been reported that inhibition of sirtuin 2 (SIRT2), a sirtuin family protein, can decrease cellular and tissue injuries in models of Parkinson’s disease (PD) and Huntington’s disease (HD); however, the mechanisms underlying these observations have remained unclear. Because inflammation plays key pathological roles in multiple major neurological disorders including PD and HD, in our current study we tested our hypothesis that SIRT2 plays an important role in microglial activation. We found that treatment of BV2 microglia with lipopolysaccharides led to significant increases in NO and inducible nitric oxide synthase mRNA levels, as well as increases in the levels of tumor necrosis factor-α and interleukin 6 mRNA, which indicated microglial activation. These increases were significantly decreased in the cells with SIRT2 silencingproduced decreases in the SIRT2 level. These observations suggest that SIRT2 is required for lipopolysaccharide-
Sirtuins are NAD+-dependent histone deacetylases [1]. Multiple studies have suggested that sirtuin 2 (SIRT2) plays a critical role in the cellular and tissue injury associated with certain pathological conditions. SIRT2 inhibition has been shown to produce neuroprotective effects in models of neurodegenerative diseases such as Parkinson’s disease (PD) and Huntington’s disease (HD) [2]. In the studies on PD, SIRT2 deletion has been shown to decrease α-synuclein-induced neurotoxicity in the models of PD [3]. In addition, SIRT2 inhibition can reduce nigrostriatal damage induced by 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine through the apoptotic pathway [4]. In the studies on HD, SIRT2 inhibition was found to produce neuroprotective effects in cellular and invertebrate models of HD through downregulation of sterol biosynthesis [5]. Chronic treatment with the brain-permeable SIRT2 inhibitor AK-7 has also been shown to improve motor function, extend survival, and reduce brain atrophy in a mouse model of HD [6]. However, the mechanisms underlying the protective effects of SIRT2 inhibition have remained unclear. Increasing evidence has indicated a key pathological role of inflammation in neurodegenerative diseases, including PD [7] and HD [8]. Because microglial activation is a key event in neuroinflammation in neurodegenerative diseases, which can impair neuronal survival by generating Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website (www.neuroreport.com). 0959-4965 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
induced microglial activation. The findings also suggest that SIRT2 may be a therapeutic target for inhibiting the inflammatory responses in neurological disorders such as PD and HD. NeuroReport 26:88–93 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.. NeuroReport 2015, 26:88–93 Keywords: cytokines, inflammation, microglia, sirtuin 2 a
Med-X Research Institute, School of Biomedical Engineering, bDepartment of Neurology, Ruijin Hospital and cDepartment of Neurology, Third People’s Hospital, Shanghai Jiao Tong University, Shanghai, People’s Republic of China Correspondence to Weihai Ying, PhD, Med-X Research Institute, School of Biomedical Engineering, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, People’s Republic of China Tel: + 86 21 6293 3075; fax: + 86 21 6293 2302; e-mail: [email protected] Received 6 November 2014 accepted 17 November 2014
oxidative stress and cytokines [7,8], we hypothesized that SIRT2 inhibition may produce its protective effects by inhibiting inflammation. It is of both theoretical and therapeutic significance to elucidate the mechanisms underlying the roles of SIRT2 in microglial activation. Using BV2 microglia as a microglial model in this study, we tested whether decreased SIRT2 levels can lead to decreased microglial activation induced by lipopolysaccharides (LPSs). Our study has shown that SIRT2 silencing-produced reductions in SIRT2 levels can lead to a significant decrease in LPS-induced microglial activation, suggesting that SIRT2 is required for LPSinduced microglial activation.
Materials and methods Materials
All of the chemicals were purchased from Sigma (St. Louis, Missouri, USA), except where specified. Cell cultures
The microglial BV2 cells were purchased from the Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were maintained in Dulbecco’s Modified Eagle Medium (containing 4500 mg/l D-glucose, 584 mg/l L-glutamine, and 110 mg/l sodium pyruvate; Thermo Scientific, Waltham, Massachusetts, USA) that contained heated 10% fetal bovine serum (Gibco, Grand Island, New York, USA) and 1% penicillin and streptomycin (Invitrogen, Carlsbad, California, USA) in an incubator with 5% CO2 at 37°C. DOI: 10.1097/WNR.0000000000000305
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SIRT2 is required for microglial activation Chen et al. 89
SIRT2 silencing
When BV2 microglial cells were ∼ 50% confluent, the cells were transfected either with Stealth SIRT2 siRNA (5′AUGAUGAGGAGGUCCACCUUGGAGA-3′; Invitrogen) or with Med GC Stealth RNAi siRNA Negative Control (cat#12935-300; Invitrogen) used as control siRNA. Lipofectamine 2000 (Invitrogen) was used for transfection according to the manufacturer’s instructions. To each well of a 24-well plate, 100 μl of Opti-MEM containing 100 nM Stealth siRNA oligonucleotides and 2 μl lipofectamine 2000 was added to 500 μl of media containing BV2 microglia. The SIRT2 level in the BV2 microglia was determined by western blotting 48 h after transfection. Western blotting
BV2 microglial cells were harvested and lysed in RadioImmunoprecipitation Assay buffer (Millipore, Temecula, California, USA) containing complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and 1 mM Phenylmethanesulfonyl fluoride. After quantification of the protein samples using the BCA Protein Assay Kit (Pierce Biotechnology, Rockford, Illinois, USA), 30 μg of total protein was electrophoresed through a 10% SDS-polyacrylamide gel and then transferred onto a 0.45μm pore-size nitrocellulose membrane. The blots were incubated overnight at 4°C with a rabbit polyclonal antiSIRT2 antibody (1 : 500 dilution; Santa Cruz Biotechnology, Santa Cruz, California, USA); thereafter, they were incubated with horse radish peroxidaseconjugated secondary antibody (Epitomics, Zhejiang Province, China). Protein signals were detected using the ECL detection system (Pierce Biotechnology, Rockford, Illinois, USA). An anti-β-tubulin antibody (Santa Cruz Biotechnology) was used to normalize sample loading and transfer. The intensities of the bands were quantified by densitometry using the Gel-Pro Analyzer (Media Cybernetics, Rockville, Maryland, USA).
Dalian, China), according to the manufacturer’s instructions. Quantitative real-time PCR was performed using SYBR Premix Ex Taq (TaKaRa) and the following primers: interleukin 6 (IL-6; sense 5′-tagtccttcctaccccaatttcc-3′ and anti-sense 5′-ttggtccttagccactccttc-3′), tumor necrosis factor-α (TNF-α; sense 5′-ccctcacactcagatcatcttct-3′ and anti-sense 5′-gctacgacgtgggctacag-3′), and 36B4 (sense 5′-agattcgggatatgctgttggc-3′ and antisense 5′-tcgggtcctagaccagtgttc-3′) as internal control. Quantitative real-time PCR was performed on the ABI 7900HT fast real-time PCR system (Applied Biosystems), equipped with a 384-well reaction block. Data were analyzed using the comparative threshold cycle (Ct) method, and the results were expressed as fold difference normalized to the results obtained from the experiments using 36B4.
Immunocytochemistry
Cell cultures were fixed in 4% paraformaldehyde for 30 min, permeabilized in 0.2% Triton X-100 in PBS for 20 min, and blocked with 10% normal goat serum for 30 min at room temperature. After washing with PBS one time, the cells were incubated with a rabbit monoclonal nuclear factor κ-light chain enhancer of activated B cells (NF-κB) antibody (1 : 500 dilution; Epitomics) in PBS containing 1% goat serum overnight at 4°C and were then incubated with the Alexa Fluor 568 secondary antibody (Molecular Probes, Eugene, Oregon, USA; 1 : 500 dilution) in PBS containing 1% goat serum for 1 h at room temperature. After three washes with PBS, the cells were stained with 5 μg/ml 4′,6-diamidino-2-phenylindole solution (Beyotime) for 5 min, mounted using Fluorescence Mounting Medium (Beyotime), and photographed under a Leica confocal microscope (Leica Microsystems, Wetzlar, Germany).
Nitric oxide assay
Lactate dehydrogenase assay
Nitric oxide (NO) content was determined by assessing the amount of nitrite accumulated in the culture media using Griess Reagent (Beyotime, Jiangsu, China), according to the manufacturer’s instructions. Briefly, 50 μl of culture media or sodium nitrite (as standard) was mixed with 50 μl of Griess reagent I, and then with 50 μl of Griess reagent II. After 5 min at room temperature, A540 nm of the samples and standards was determined using a plate reader (Biotek Instruments Inc., Vermont, USA). The nitrite concentrations of the samples were calculated using the standards and were normalized to the protein concentrations of the samples, which were determined by the BCA assay.
Cell survival was quantified by measuring lactate dehydrogenase (LDH) activity in cell lysates. Briefly, BV2 cells were lysed in a lysing buffer (0.04% Triton X, 2 mM HEPES, 0.2 mM dithiothreitol, 0.01% BSA, pH 7.5) for 20 min. A volume of 50 μl of the cell lysate was mixed with 150 μl of 500 mM potassium phosphate buffer (1.5 mM NADH and 7.5 mM sodium pyruvate, pH 7.5), and the A340 nm change was monitored over 90 s. Cell survival was calculated by normalizing the LDH values of the samples to the LDH activity of the control cultures.
Statistical analyses Quantitative real-time PCR
Total RNA of BV2 microglial cells was isolated using Trizol reagent (Invitrogen) and was reverse-transcribed to cDNA using a PrimeScript RT reagent kit (TaKaRa,
All data are presented as mean ± SE. Data were assessed by one-way ANOVA, followed by the Student–Newman–Keuls post-hoc test. P-values less than 0.05 were considered statistically significant.
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90 NeuroReport 2015, Vol 26 No 2
Fig. 1
Control siRNA LPS (μg/ml)
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To get additional information for determining the role of SIRT2 in LPS-induced microglial activation, we also used the real-time PCR assay to determine the levels of TNF-α and IL-6 mRNA in the cells. We found that SIRT2 silencing significantly (P < 0.001) attenuated TNF-α (76.7 ± 1.57%) and IL-6 mRNA (64.7 ± 8.09%) production induced by 1 μg/ml LPS (Fig. 4). Our study has further investigated the mechanisms underlying the effects of SIRT2 on LPS-induced microglial activation. Our immunostaining assays on NF-κB did not show that
100 SIRT2/β-tubulin (% of control)
silencing on LPS-induced NO production and iNOS mRNA levels. Using Griess reagent to determine the nitrite levels in the media, we assessed NO production by the cells. Our study showed that LPS induced a significant (P < 0.001) increase in NO production (1368.6 ± 49.91% in 0.5 μg/ml LPS-activated microglia, 1724.7 ± 50.75% in 1 μg/ ml LPS-activated microglia) compared with no LPS (100.0 ± 4.74%), which was significantly (P < 0.001) decreased by SIRT2 silencing (794.5 ± 52.82% in 0.5 μg/ml LPS-activated microglia, 1129.5 ± 77.02% in 1 μg/ml LPSactivated microglia; Fig. 2). Our real-time PCR assay also showed that SIRT2 silencing significantly (P < 0.05) attenuated iNOS mRNA production (72.0 ± 3.37%) induced by 1 μg/ml LPS (Fig. 3).
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SIRT2 silencing significantly decreased the SIRT2 level of BV2 microglia. BV2 microglial cells were treated with SIRT2 siRNA or control siRNA for 24 h; thereafter, they were treated with 0.5 or 1 μg/ml LPS for 24 h. The SIRT2 levels were determined by western blotting, which showed that SIRT2 silencing can significantly reduce the SIRT2 levels in both normal cells and LPS-treated cells. N (total number of each group in three experiments) = 11–12. Data were collected from three independent experiments. ***P < 0.001. LPS, lipopolysaccharide; SIRT2, sirtuin 2.
Results We applied SIRT2 siRNA to decrease the SIRT2 level in the microglia. Our western blotting assay showed that treatment of the cells with SIRT2 siRNA for 24 h led to significant (P < 0.001) reductions in the SIRT2 level in both resting microglia (28.5 ± 3.35%) and LPS-activated microglia (33.9 ± 3.38% in 0.5 μg/ml LPS-activated microglia, 39.3 ± 5.41% in 1 μg/ml LPS-activated microglia) compared with control siRNA treatment (Fig. 1). SIRT2 siRNA or/and LPS treatment did not significantly (P > 0.05) change cell survival (Supplementary Fig. 1, Supplemental digital content 1, http://links.lww.com/WNR/ A310), determined by the LDH assay. Because inducible nitric oxide synthase (iNOS) activation and increased NO production are indices of microglial activation, we determined the effects of SIRT2
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SIRT2 silencing significantly decreased LPS-induced NO production by BV2 microglia. BV2 microglial cells were treated with SIRT2 siRNA or control siRNA for 24 h; thereafter, they were treated with 0.5 or 1 μg/ml LPS for 24 h. The nitrite levels in the media were detected using Griess reagent, which showed that SIRT2 silencing can significantly reduce LPS-induced NO production. N = 11–12. Data were collected from three independent experiments. ***P < 0.001. LPS, lipopolysaccharide; NO, nitric oxide; SIRT2, sirtuin 2.
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SIRT2 is required for microglial activation Chen et al. 91
Fig. 3
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iNOS mRNA (% of LPS-treated cells)
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SIRT2 silencing decreased LPS-induced iNOS mRNA levels in BV2 microglia. BV2 microglial cells were treated with SIRT2 siRNA or control siRNA for 24 h; thereafter, they were treated with 1 μg/ml LPS for 12 h. The iNOS mRNA level was determined by real-time PCR, which showed that SIRT2 silencing can significantly reduce the iNOS mRNA level in LPS-treated microglia. N = 9. Data were collected from three independent experiments. *P < 0.05. iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; NO, nitric oxide; SIRT2, sirtuin 2.
SIRT2 silencing can decrease LPS-induced nuclear translocation of NF-κB, thus arguing against the possibility that SIRT2 mediates LPS-induced microglial activation by modulating the nuclear translocation of NFκB (Supplementary Fig. 2, Supplemental digital content 2, http://links.lww.com/WNR/A311).
Discussion The major observations from our current study include the following: first, SIRT2 silencing-produced reductions in the SIRT2 level of BV2 microglia can significantly attenuate the LPS-induced increases in NO and iNOS mRNA levels, and second, SIRT2 silencing can also significantly attenuate LPS-induced increases in TNF-α and IL-6 mRNA levels. Collectively, these observations suggest that SIRT2 is required for LPS-induced microglial activation. A number of studies have indicated that SIRT2 plays a significant role in multiple biological processes, including cell motility, cell mitosis, and oligodendrocyte differentiation [9–12]. Previous studies have also indicated that SIRT2 is a mediator of cell death. In particular, SIRT2 inhibition was shown to decrease the injury in cellular and animal models of PD and HD [2]. As inflammation plays a critical pathological role in PD [7] and HD [8], we
have hypothesized that SIRT2 may be important for microglial activation – a key factor in neuroinflammation. The observations from our current study suggest that decreased SIRT2 levels can lead to inhibition of microglial activation. Thus, our study suggests that SIRT2 inhibition-produced decreases in injury in models of PD may result from the capacity of SIRT2 inhibition to lead to a decrease in inflammation. Although nuclear translocation of NF-κB is a key factor in microglial activation, our observations from the current study argue against the possibility that SIRT2 mediates LPS-induced microglial activation by modulating NF-κB. Future studies are warranted to further investigate the mechanisms underlying the effects of decreased SIRT2 levels on LPS-induced microglial activation. It is also of significance to use animal models to determine whether decreased SIRT2 levels can lead to a decrease in LPSinduced microglial activation. We conducted a NO assay at 24 h after LPS treatment and real-time PCR assays of TNF-α and IL-6 at 6 h after LPS treatment for the following reasons: multiple previous studies have shown that LPS can rapidly increase the transcription of inflammatory cytokines at 6 h after LPS treatment [13]; therefore, we conducted real-time PCR assays of TNF-α and IL-6 at 6 h after LPS treatment. Indeed, we found that LPS induced significant increases in TNF-α and IL-6 mRNA expression levels at 6 h after the LPS treatment. Previous studies have also suggested that nitrite – a widely used marker of iNOS activation – is detected in the media at relatively late time points after LPS treatment, such as 24 h after LPS treatment [13]. Indeed, we found only a mild increase in the nitrite level in the cell medium at 18 h after LPS treatment, whereas we found a significant increase in the nitrite levels in the cell medium at 24 h after LPS treatment. There is one study that has shown that 50 ng/ml LPS can induce increases in cytokine levels in N9 microglial cell lines 20 h after LPS treatment, which was significantly enhanced by stable silencing of SIRT2 [14]. This observation seems to be contradictory to the results of our current study. However, our experimental conditions are significantly different from those in the study by Pais and colleagues. The concentrations of LPS in our study were 500–1000 ng/ml, which are 20–40-fold higher than the LPS concentrations used in their study. It is not surprising that SIRT2 could play differential roles in microglial activation when microglia are exposed to markedly different concentrations of LPS. Previous studies have also suggested that poly-ADP-ribose polymerase plays a detrimental role in a model of severe ischemia/reperfusion [15,16], whereas it plays a beneficial role in a model of mild ischemia [17,18]. Therefore, our study has suggested that, at least for relatively strong microglial activation, decreased SIRT2 levels can lead to
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92 NeuroReport 2015, Vol 26 No 2
Fig. 4
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(b) ∗∗∗
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TNF-α mRNA (% of LPS-treated cells)
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SIRT2 silencing attenuated the LPS-induced increases in the levels of TNF-α and IL-6 mRNA. BV2 microglial cells were treated with SIRT2 siRNA or control siRNA for 24 h; thereafter, they were treated with 1 μg/ml LPS for 6 h. The TNF-α and IL-6 mRNA levels were determined by real-time PCR, which showed that SIRT2 silencing can significantly attenuate LPS-induced increases in TNF-α and IL-6 mRNA levels. N = 9. Data were collected from three independent experiments. ***P < 0.001. IL-6, interleukin 6; LPS, lipopolysaccharide; NO, nitric oxide; SIRT2, sirtuin 2; TNF-α, tumor necrosis factor-α.
decreased microglial activation. In other words, SIRT2 activity is required for the microglial activation induced by relatively high levels of microglial activators. Considering the observations from the previous study [14] and our current study, it is possible that SIRT2 plays contrasting roles in microglial activation under different conditions. Complex roles of SIRT2 in biological processes have also been suggested by previous studies. Multiple studies have suggested that SIRT2 produces either detrimental [19, 20] or beneficial effects [19,20] on cell survival under different conditions. The complex roles of SIRT2 in cell death are not surprising, as SIRT2 can affect the activity of transcriptional factors such as forkhead box O3A (FOXO3a) [21], which can increase the expression of both proapoptotic and antiapoptotic proteins. Future studies are warranted to further investigate the mechanisms underlying the differential roles of SIRT2 in inflammation and cell survival. We found that a 70% decline in SIRT2 expression led to only an ∼ 20–30% change in the levels of nitrite and the mRNA of the cytokines. Therefore, our study suggests that SIRT2 is only one of the important factors in LPS-induced microglial activation. Certainly there are other factors or pathways involved in LPS-induced microglial activation. It is necessary to further investigate the mechanisms underlying LPS-induced microglial activation.
Previous studies have shown that BV2 microglial cells retain most of the morphological, phenotypical, and functional properties of freshly isolated microglial cells. The findings from studies using BV2 microglia are also reproducible in those using primary microglial cultures [22]. Therefore, BV2 cells have been widely used as a microglial model [23], as this model has been considered a suitable model system for primary microglial cultures [24]. Future studies are warranted to further determine whether our findings using BV2 cells are reproducible under in-vivo conditions. Conclusion
Our results have shown that reduction in the SIRT2 level by siRNA can lead to a significant decrease in LPSinduced microglial activation, including NO production and production of the inflammatory cytokines TNF-α and IL-6, suggesting that SIRT2 is critical for microglial activation induced by LPS.
Acknowledgements This study was supported by Chinese National Science Foundation Grants #81171098 and #81271305 (to W. Y.), a National Science and Technology Major Project Grant for ‘Major New Drugs Innovation and Development’ #2014ZX09507008 (to X. D.), Shanghai Jiao Tong
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SIRT2 is required for microglial activation Chen et al. 93
University Grants for Interdisciplinary Research on Medicine and Engineering (to W. Y.), and a Research Fund (#11-E-3) of the Science and Technology Committee, Baoshan District of Shanghai (to D. W.). The authors acknowledge Jiaxiang Shao, Hui Nie, and Yexin Li for their technical assistance.
12 13
14
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Conflicts of interest
There are no conflicts of interest.
16
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SIRT2 is required for lipopolysaccharide-induced activation of BV2 microglia Heyu Chena, Danhong Wuc, Xianting Dinga and Weihai Yinga,b It has been reported that inhibition of sirtuin 2 (SIRT2), a sirtuin family protein, can decrease cellular and tissue injuries in models of Parkinson’s disease (PD) and Huntington’s disease (HD); however, the mechanisms underlying these observations have remained unclear. Because inflammation plays key pathological roles in multiple major neurological disorders including PD and HD, in our current study we tested our hypothesis that SIRT2 plays an important role in microglial activation. We found that treatment of BV2 microglia with lipopolysaccharides led to significant increases in NO and inducible nitric oxide synthase mRNA levels, as well as increases in the levels of tumor necrosis factor-α and interleukin 6 mRNA, which indicated microglial activation. These increases were significantly decreased in the cells with SIRT2 silencingproduced decreases in the SIRT2 level. These observations suggest that SIRT2 is required for lipopolysaccharide-
Sirtuins are NAD+-dependent histone deacetylases [1]. Multiple studies have suggested that sirtuin 2 (SIRT2) plays a critical role in the cellular and tissue injury associated with certain pathological conditions. SIRT2 inhibition has been shown to produce neuroprotective effects in models of neurodegenerative diseases such as Parkinson’s disease (PD) and Huntington’s disease (HD) [2]. In the studies on PD, SIRT2 deletion has been shown to decrease α-synuclein-induced neurotoxicity in the models of PD [3]. In addition, SIRT2 inhibition can reduce nigrostriatal damage induced by 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine through the apoptotic pathway [4]. In the studies on HD, SIRT2 inhibition was found to produce neuroprotective effects in cellular and invertebrate models of HD through downregulation of sterol biosynthesis [5]. Chronic treatment with the brain-permeable SIRT2 inhibitor AK-7 has also been shown to improve motor function, extend survival, and reduce brain atrophy in a mouse model of HD [6]. However, the mechanisms underlying the protective effects of SIRT2 inhibition have remained unclear. Increasing evidence has indicated a key pathological role of inflammation in neurodegenerative diseases, including PD [7] and HD [8]. Because microglial activation is a key event in neuroinflammation in neurodegenerative diseases, which can impair neuronal survival by generating Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website (www.neuroreport.com). 0959-4965 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
induced microglial activation. The findings also suggest that SIRT2 may be a therapeutic target for inhibiting the inflammatory responses in neurological disorders such as PD and HD. NeuroReport 26:88–93 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.. NeuroReport 2015, 26:88–93 Keywords: cytokines, inflammation, microglia, sirtuin 2 a
Med-X Research Institute, School of Biomedical Engineering, bDepartment of Neurology, Ruijin Hospital and cDepartment of Neurology, Third People’s Hospital, Shanghai Jiao Tong University, Shanghai, People’s Republic of China Correspondence to Weihai Ying, PhD, Med-X Research Institute, School of Biomedical Engineering, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, People’s Republic of China Tel: + 86 21 6293 3075; fax: + 86 21 6293 2302; e-mail: [email protected] Received 6 November 2014 accepted 17 November 2014
oxidative stress and cytokines [7,8], we hypothesized that SIRT2 inhibition may produce its protective effects by inhibiting inflammation. It is of both theoretical and therapeutic significance to elucidate the mechanisms underlying the roles of SIRT2 in microglial activation. Using BV2 microglia as a microglial model in this study, we tested whether decreased SIRT2 levels can lead to decreased microglial activation induced by lipopolysaccharides (LPSs). Our study has shown that SIRT2 silencing-produced reductions in SIRT2 levels can lead to a significant decrease in LPS-induced microglial activation, suggesting that SIRT2 is required for LPSinduced microglial activation.
Materials and methods Materials
All of the chemicals were purchased from Sigma (St. Louis, Missouri, USA), except where specified. Cell cultures
The microglial BV2 cells were purchased from the Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were maintained in Dulbecco’s Modified Eagle Medium (containing 4500 mg/l D-glucose, 584 mg/l L-glutamine, and 110 mg/l sodium pyruvate; Thermo Scientific, Waltham, Massachusetts, USA) that contained heated 10% fetal bovine serum (Gibco, Grand Island, New York, USA) and 1% penicillin and streptomycin (Invitrogen, Carlsbad, California, USA) in an incubator with 5% CO2 at 37°C. DOI: 10.1097/WNR.0000000000000305
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SIRT2 is required for microglial activation Chen et al. 89
SIRT2 silencing
When BV2 microglial cells were ∼ 50% confluent, the cells were transfected either with Stealth SIRT2 siRNA (5′AUGAUGAGGAGGUCCACCUUGGAGA-3′; Invitrogen) or with Med GC Stealth RNAi siRNA Negative Control (cat#12935-300; Invitrogen) used as control siRNA. Lipofectamine 2000 (Invitrogen) was used for transfection according to the manufacturer’s instructions. To each well of a 24-well plate, 100 μl of Opti-MEM containing 100 nM Stealth siRNA oligonucleotides and 2 μl lipofectamine 2000 was added to 500 μl of media containing BV2 microglia. The SIRT2 level in the BV2 microglia was determined by western blotting 48 h after transfection. Western blotting
BV2 microglial cells were harvested and lysed in RadioImmunoprecipitation Assay buffer (Millipore, Temecula, California, USA) containing complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and 1 mM Phenylmethanesulfonyl fluoride. After quantification of the protein samples using the BCA Protein Assay Kit (Pierce Biotechnology, Rockford, Illinois, USA), 30 μg of total protein was electrophoresed through a 10% SDS-polyacrylamide gel and then transferred onto a 0.45μm pore-size nitrocellulose membrane. The blots were incubated overnight at 4°C with a rabbit polyclonal antiSIRT2 antibody (1 : 500 dilution; Santa Cruz Biotechnology, Santa Cruz, California, USA); thereafter, they were incubated with horse radish peroxidaseconjugated secondary antibody (Epitomics, Zhejiang Province, China). Protein signals were detected using the ECL detection system (Pierce Biotechnology, Rockford, Illinois, USA). An anti-β-tubulin antibody (Santa Cruz Biotechnology) was used to normalize sample loading and transfer. The intensities of the bands were quantified by densitometry using the Gel-Pro Analyzer (Media Cybernetics, Rockville, Maryland, USA).
Dalian, China), according to the manufacturer’s instructions. Quantitative real-time PCR was performed using SYBR Premix Ex Taq (TaKaRa) and the following primers: interleukin 6 (IL-6; sense 5′-tagtccttcctaccccaatttcc-3′ and anti-sense 5′-ttggtccttagccactccttc-3′), tumor necrosis factor-α (TNF-α; sense 5′-ccctcacactcagatcatcttct-3′ and anti-sense 5′-gctacgacgtgggctacag-3′), and 36B4 (sense 5′-agattcgggatatgctgttggc-3′ and antisense 5′-tcgggtcctagaccagtgttc-3′) as internal control. Quantitative real-time PCR was performed on the ABI 7900HT fast real-time PCR system (Applied Biosystems), equipped with a 384-well reaction block. Data were analyzed using the comparative threshold cycle (Ct) method, and the results were expressed as fold difference normalized to the results obtained from the experiments using 36B4.
Immunocytochemistry
Cell cultures were fixed in 4% paraformaldehyde for 30 min, permeabilized in 0.2% Triton X-100 in PBS for 20 min, and blocked with 10% normal goat serum for 30 min at room temperature. After washing with PBS one time, the cells were incubated with a rabbit monoclonal nuclear factor κ-light chain enhancer of activated B cells (NF-κB) antibody (1 : 500 dilution; Epitomics) in PBS containing 1% goat serum overnight at 4°C and were then incubated with the Alexa Fluor 568 secondary antibody (Molecular Probes, Eugene, Oregon, USA; 1 : 500 dilution) in PBS containing 1% goat serum for 1 h at room temperature. After three washes with PBS, the cells were stained with 5 μg/ml 4′,6-diamidino-2-phenylindole solution (Beyotime) for 5 min, mounted using Fluorescence Mounting Medium (Beyotime), and photographed under a Leica confocal microscope (Leica Microsystems, Wetzlar, Germany).
Nitric oxide assay
Lactate dehydrogenase assay
Nitric oxide (NO) content was determined by assessing the amount of nitrite accumulated in the culture media using Griess Reagent (Beyotime, Jiangsu, China), according to the manufacturer’s instructions. Briefly, 50 μl of culture media or sodium nitrite (as standard) was mixed with 50 μl of Griess reagent I, and then with 50 μl of Griess reagent II. After 5 min at room temperature, A540 nm of the samples and standards was determined using a plate reader (Biotek Instruments Inc., Vermont, USA). The nitrite concentrations of the samples were calculated using the standards and were normalized to the protein concentrations of the samples, which were determined by the BCA assay.
Cell survival was quantified by measuring lactate dehydrogenase (LDH) activity in cell lysates. Briefly, BV2 cells were lysed in a lysing buffer (0.04% Triton X, 2 mM HEPES, 0.2 mM dithiothreitol, 0.01% BSA, pH 7.5) for 20 min. A volume of 50 μl of the cell lysate was mixed with 150 μl of 500 mM potassium phosphate buffer (1.5 mM NADH and 7.5 mM sodium pyruvate, pH 7.5), and the A340 nm change was monitored over 90 s. Cell survival was calculated by normalizing the LDH values of the samples to the LDH activity of the control cultures.
Statistical analyses Quantitative real-time PCR
Total RNA of BV2 microglial cells was isolated using Trizol reagent (Invitrogen) and was reverse-transcribed to cDNA using a PrimeScript RT reagent kit (TaKaRa,
All data are presented as mean ± SE. Data were assessed by one-way ANOVA, followed by the Student–Newman–Keuls post-hoc test. P-values less than 0.05 were considered statistically significant.
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90 NeuroReport 2015, Vol 26 No 2
Fig. 1
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To get additional information for determining the role of SIRT2 in LPS-induced microglial activation, we also used the real-time PCR assay to determine the levels of TNF-α and IL-6 mRNA in the cells. We found that SIRT2 silencing significantly (P < 0.001) attenuated TNF-α (76.7 ± 1.57%) and IL-6 mRNA (64.7 ± 8.09%) production induced by 1 μg/ml LPS (Fig. 4). Our study has further investigated the mechanisms underlying the effects of SIRT2 on LPS-induced microglial activation. Our immunostaining assays on NF-κB did not show that
100 SIRT2/β-tubulin (% of control)
silencing on LPS-induced NO production and iNOS mRNA levels. Using Griess reagent to determine the nitrite levels in the media, we assessed NO production by the cells. Our study showed that LPS induced a significant (P < 0.001) increase in NO production (1368.6 ± 49.91% in 0.5 μg/ml LPS-activated microglia, 1724.7 ± 50.75% in 1 μg/ ml LPS-activated microglia) compared with no LPS (100.0 ± 4.74%), which was significantly (P < 0.001) decreased by SIRT2 silencing (794.5 ± 52.82% in 0.5 μg/ml LPS-activated microglia, 1129.5 ± 77.02% in 1 μg/ml LPSactivated microglia; Fig. 2). Our real-time PCR assay also showed that SIRT2 silencing significantly (P < 0.05) attenuated iNOS mRNA production (72.0 ± 3.37%) induced by 1 μg/ml LPS (Fig. 3).
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SIRT2 silencing significantly decreased the SIRT2 level of BV2 microglia. BV2 microglial cells were treated with SIRT2 siRNA or control siRNA for 24 h; thereafter, they were treated with 0.5 or 1 μg/ml LPS for 24 h. The SIRT2 levels were determined by western blotting, which showed that SIRT2 silencing can significantly reduce the SIRT2 levels in both normal cells and LPS-treated cells. N (total number of each group in three experiments) = 11–12. Data were collected from three independent experiments. ***P < 0.001. LPS, lipopolysaccharide; SIRT2, sirtuin 2.
Results We applied SIRT2 siRNA to decrease the SIRT2 level in the microglia. Our western blotting assay showed that treatment of the cells with SIRT2 siRNA for 24 h led to significant (P < 0.001) reductions in the SIRT2 level in both resting microglia (28.5 ± 3.35%) and LPS-activated microglia (33.9 ± 3.38% in 0.5 μg/ml LPS-activated microglia, 39.3 ± 5.41% in 1 μg/ml LPS-activated microglia) compared with control siRNA treatment (Fig. 1). SIRT2 siRNA or/and LPS treatment did not significantly (P > 0.05) change cell survival (Supplementary Fig. 1, Supplemental digital content 1, http://links.lww.com/WNR/ A310), determined by the LDH assay. Because inducible nitric oxide synthase (iNOS) activation and increased NO production are indices of microglial activation, we determined the effects of SIRT2
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SIRT2 silencing significantly decreased LPS-induced NO production by BV2 microglia. BV2 microglial cells were treated with SIRT2 siRNA or control siRNA for 24 h; thereafter, they were treated with 0.5 or 1 μg/ml LPS for 24 h. The nitrite levels in the media were detected using Griess reagent, which showed that SIRT2 silencing can significantly reduce LPS-induced NO production. N = 11–12. Data were collected from three independent experiments. ***P < 0.001. LPS, lipopolysaccharide; NO, nitric oxide; SIRT2, sirtuin 2.
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SIRT2 is required for microglial activation Chen et al. 91
Fig. 3
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iNOS mRNA (% of LPS-treated cells)
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SIRT2 silencing decreased LPS-induced iNOS mRNA levels in BV2 microglia. BV2 microglial cells were treated with SIRT2 siRNA or control siRNA for 24 h; thereafter, they were treated with 1 μg/ml LPS for 12 h. The iNOS mRNA level was determined by real-time PCR, which showed that SIRT2 silencing can significantly reduce the iNOS mRNA level in LPS-treated microglia. N = 9. Data were collected from three independent experiments. *P < 0.05. iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; NO, nitric oxide; SIRT2, sirtuin 2.
SIRT2 silencing can decrease LPS-induced nuclear translocation of NF-κB, thus arguing against the possibility that SIRT2 mediates LPS-induced microglial activation by modulating the nuclear translocation of NFκB (Supplementary Fig. 2, Supplemental digital content 2, http://links.lww.com/WNR/A311).
Discussion The major observations from our current study include the following: first, SIRT2 silencing-produced reductions in the SIRT2 level of BV2 microglia can significantly attenuate the LPS-induced increases in NO and iNOS mRNA levels, and second, SIRT2 silencing can also significantly attenuate LPS-induced increases in TNF-α and IL-6 mRNA levels. Collectively, these observations suggest that SIRT2 is required for LPS-induced microglial activation. A number of studies have indicated that SIRT2 plays a significant role in multiple biological processes, including cell motility, cell mitosis, and oligodendrocyte differentiation [9–12]. Previous studies have also indicated that SIRT2 is a mediator of cell death. In particular, SIRT2 inhibition was shown to decrease the injury in cellular and animal models of PD and HD [2]. As inflammation plays a critical pathological role in PD [7] and HD [8], we
have hypothesized that SIRT2 may be important for microglial activation – a key factor in neuroinflammation. The observations from our current study suggest that decreased SIRT2 levels can lead to inhibition of microglial activation. Thus, our study suggests that SIRT2 inhibition-produced decreases in injury in models of PD may result from the capacity of SIRT2 inhibition to lead to a decrease in inflammation. Although nuclear translocation of NF-κB is a key factor in microglial activation, our observations from the current study argue against the possibility that SIRT2 mediates LPS-induced microglial activation by modulating NF-κB. Future studies are warranted to further investigate the mechanisms underlying the effects of decreased SIRT2 levels on LPS-induced microglial activation. It is also of significance to use animal models to determine whether decreased SIRT2 levels can lead to a decrease in LPSinduced microglial activation. We conducted a NO assay at 24 h after LPS treatment and real-time PCR assays of TNF-α and IL-6 at 6 h after LPS treatment for the following reasons: multiple previous studies have shown that LPS can rapidly increase the transcription of inflammatory cytokines at 6 h after LPS treatment [13]; therefore, we conducted real-time PCR assays of TNF-α and IL-6 at 6 h after LPS treatment. Indeed, we found that LPS induced significant increases in TNF-α and IL-6 mRNA expression levels at 6 h after the LPS treatment. Previous studies have also suggested that nitrite – a widely used marker of iNOS activation – is detected in the media at relatively late time points after LPS treatment, such as 24 h after LPS treatment [13]. Indeed, we found only a mild increase in the nitrite level in the cell medium at 18 h after LPS treatment, whereas we found a significant increase in the nitrite levels in the cell medium at 24 h after LPS treatment. There is one study that has shown that 50 ng/ml LPS can induce increases in cytokine levels in N9 microglial cell lines 20 h after LPS treatment, which was significantly enhanced by stable silencing of SIRT2 [14]. This observation seems to be contradictory to the results of our current study. However, our experimental conditions are significantly different from those in the study by Pais and colleagues. The concentrations of LPS in our study were 500–1000 ng/ml, which are 20–40-fold higher than the LPS concentrations used in their study. It is not surprising that SIRT2 could play differential roles in microglial activation when microglia are exposed to markedly different concentrations of LPS. Previous studies have also suggested that poly-ADP-ribose polymerase plays a detrimental role in a model of severe ischemia/reperfusion [15,16], whereas it plays a beneficial role in a model of mild ischemia [17,18]. Therefore, our study has suggested that, at least for relatively strong microglial activation, decreased SIRT2 levels can lead to
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92 NeuroReport 2015, Vol 26 No 2
Fig. 4
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TNF-α mRNA (% of LPS-treated cells)
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1 LPS (μg/ml)
LPS (μg/ml)
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SIRT2 siRNA
SIRT2 silencing attenuated the LPS-induced increases in the levels of TNF-α and IL-6 mRNA. BV2 microglial cells were treated with SIRT2 siRNA or control siRNA for 24 h; thereafter, they were treated with 1 μg/ml LPS for 6 h. The TNF-α and IL-6 mRNA levels were determined by real-time PCR, which showed that SIRT2 silencing can significantly attenuate LPS-induced increases in TNF-α and IL-6 mRNA levels. N = 9. Data were collected from three independent experiments. ***P < 0.001. IL-6, interleukin 6; LPS, lipopolysaccharide; NO, nitric oxide; SIRT2, sirtuin 2; TNF-α, tumor necrosis factor-α.
decreased microglial activation. In other words, SIRT2 activity is required for the microglial activation induced by relatively high levels of microglial activators. Considering the observations from the previous study [14] and our current study, it is possible that SIRT2 plays contrasting roles in microglial activation under different conditions. Complex roles of SIRT2 in biological processes have also been suggested by previous studies. Multiple studies have suggested that SIRT2 produces either detrimental [19, 20] or beneficial effects [19,20] on cell survival under different conditions. The complex roles of SIRT2 in cell death are not surprising, as SIRT2 can affect the activity of transcriptional factors such as forkhead box O3A (FOXO3a) [21], which can increase the expression of both proapoptotic and antiapoptotic proteins. Future studies are warranted to further investigate the mechanisms underlying the differential roles of SIRT2 in inflammation and cell survival. We found that a 70% decline in SIRT2 expression led to only an ∼ 20–30% change in the levels of nitrite and the mRNA of the cytokines. Therefore, our study suggests that SIRT2 is only one of the important factors in LPS-induced microglial activation. Certainly there are other factors or pathways involved in LPS-induced microglial activation. It is necessary to further investigate the mechanisms underlying LPS-induced microglial activation.
Previous studies have shown that BV2 microglial cells retain most of the morphological, phenotypical, and functional properties of freshly isolated microglial cells. The findings from studies using BV2 microglia are also reproducible in those using primary microglial cultures [22]. Therefore, BV2 cells have been widely used as a microglial model [23], as this model has been considered a suitable model system for primary microglial cultures [24]. Future studies are warranted to further determine whether our findings using BV2 cells are reproducible under in-vivo conditions. Conclusion
Our results have shown that reduction in the SIRT2 level by siRNA can lead to a significant decrease in LPSinduced microglial activation, including NO production and production of the inflammatory cytokines TNF-α and IL-6, suggesting that SIRT2 is critical for microglial activation induced by LPS.
Acknowledgements This study was supported by Chinese National Science Foundation Grants #81171098 and #81271305 (to W. Y.), a National Science and Technology Major Project Grant for ‘Major New Drugs Innovation and Development’ #2014ZX09507008 (to X. D.), Shanghai Jiao Tong
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SIRT2 is required for microglial activation Chen et al. 93
University Grants for Interdisciplinary Research on Medicine and Engineering (to W. Y.), and a Research Fund (#11-E-3) of the Science and Technology Committee, Baoshan District of Shanghai (to D. W.). The authors acknowledge Jiaxiang Shao, Hui Nie, and Yexin Li for their technical assistance.
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Conflicts of interest
There are no conflicts of interest.
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