Microglia activation and associated inflammatory response are involved in the pathogenesis of different neurodegenerative diseases. We have reported that Notch-1 and NF-κB/p65 signalling pathways operate in synergy in regulating the production of proinflammatory mediators in activated microglia. In the latter, there is also evidence by others that glycogen synthase kinase 3β (GSK-3β) mediates the release of proinflammatory cytokines but the interrelationships between the three signalling pathways have not been fully clarified. This is an important issue as activated microglia are potential therapeutic target for amelioration of microglia mediated neuroinflammation. Here we show that blocking of Notch-1 with N-[(3,5-Difluorophenyl) acetyl]-L-alanyl-2-phenylglycine-1,1-dimethylethyl ester (DAPT) in LPS activated BV-2 microglia not only suppressed Notch intracellular domain (NICD) and Hes-1 protein expression, but also that of GSK-3β. Conversely, blocking of the latter with lithium chloride (LiCl) decreased NICD expression in a dose-dependent manner; moreover, Hes-1 immunofluorescence was attenuated. Along with this, the protein expression level of p-GSK-3β and p-AKT protein expression was significantly increased. Furthermore, DAPT and LiCl decreased production of IL-1β, TNF-α, IL-6, iNOS, Cox2 and MCP-1; however, IL-10 expression was increased notably in LiCl treated cells. The effects of DAPT and LiCl on changes of the above-mentioned biomarkers were confirmed by immunofluorescence in both BV-2 and primary microglia. Additionally, NF-κB/p65 immunofluorescence was attenuated by DAPT and LiCl; as opposed to this, IκBα protein expression was increased. Taken together, it is suggested that Notch-1, NF-κB/p65 and GSK-3β operate in synergy to inhibit microglia activation. This may be effected via increased expression of phospho-GSK-3β (p-GSK-3β), phospho-protein kinase B (PKB) (p-AKT) and IκBα. It is concluded that the three signalling pathways are functionally interlinked in regulating microglia activation.
Citation: Cao Q, Karthikeyan A, Dheen ST, Kaur C, Ling E-A (2017) Production of proinflammatory mediators in activated microglia is synergistically regulated by Notch-1, glycogen synthase kinase (GSK-3β) and NF-κB/p65 signalling. PLoS ONE 12(10): e0186764. https://doi.org/10.1371/journal.pone.0186764
Editor: Wenhui Hu, Lewis Katz School of Medicine at Temple University, UNITED STATES
Received: May 31, 2017; Accepted: October 7, 2017; Published: October 19, 2017
Copyright: © 2017 Cao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper.
Funding: This study was supported by grant No. R-181-000-140-592 (E-A L) National University of Singapore. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Microglia are immunocompetent cells in the central nervous system whose activation is implicated in different neurodegenerative diseases or neurological disorders (see Dheen et al. ). It is well documented that microglia activation is regulated by different signaling pathways involved in production of proinflammatory mediators such as TNF-α, IL-1β, nitric oxide and ROS which are deleterious to neurons and oligodendrocytes . Among the various signaling pathways that regulate microglial activation include MAPK p38 , Nuclear Factor- κB /p65 (NF-κB /p65) , glycogen synthase kinase (GSK-3β) [4–6] and Notch-1 [7–8]. In view of this, the various pathways have been considered to be potential therapeutic targets for amelioration of neuroinflammation triggered by activated microglia causing neuronal or oligodendrocyte death .
In our previous studies, we have reported that microglia constitutively express Notch-1 and NF-κB /p65 [3,7]; both pathways play important roles in cellular functions under physiological and pathological conditions. Furthermore, we have demonstrated the functional roles of Notch-1 and NF-κB /p65 in activated microglia in vivo and in vitro [3,7,10–11]. More importantly, we have shown for the first time the interrelation between Notch-1 and NF-κB /p65, emphasizing that both pathways synergistically modulate the proinflammatory function in activated microglia. More specifically, Notch signaling can amplify the proinflammatory response of microglia by enhancing the NF-κB /p65 signaling. It remained to be further determined how the two pathways might work in concert to regulate the microglial cell functions.
Glycogen synthase kinase GSK-3β is a multifunctional kinase whose activity is regulated by serine (inhibitory) and tyrosine (stimulatory) phosphorylation . GSK-3β expression is ubiquitous in eukaryotes and plays multiple roles in various essential physiological and pathological processes including glycogen metabolism, cell cycle control, apoptosis, embryonic development, cell differentiation, cell motility, microtubule function, cell adhesion and inflammation [13–15]. It is part of the molecular machinery regulating the adaptive response to lipopolysaccharide (LPS) stimulation in microglial cells . Furthermore, it regulates toll-like receptor  and controls the production of interleukin-6 after LPS engages with toll-like receptors . GSK-3β mediates the release of interleukin-1β, TNF-α and IL-10 from cortical glia .
It has been reported that phosphorylation of GSK-3β down-regulates Notch-1 activity . Both GSK-3β and Notch-1 can modulate production of proinflammatory cytokines in activated microglial cells [19–20]. In consideration of previous study demonstrating the functional relationship between Notch and NF-κB in modulating microglial activation, we speculate that GSK-3β may act as a link between Notch and NF-κB in the modulation of proinflammatory function in microglial cells. In light of this, we sought to determine the functional relationship between GSK-3β, Notch-1 and NF-κB/p65 in activated microglia. We used lithium chloride (LiCl) that is known to directly inhibit GSK-3β through binding to the magnesium-sensitive site of the enzyme [21–22], and indirectly inhibit it by enhancing phosphorylation of specific serine residues. The phosphorylation of GSK-3β by activating upstream AKT and protein kinases A or C can then result in different effects on various processes [23–24]. N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) was used as an inhibitor for Notch via γ-secretase complex in microglial cells challenged with LPS. The treated and untreated microglia were then subjected to real time polymerase chain reaction (RT-PCR) analysis, immunofluorescence labeling, Western blotting and enzyme-linked immunosorbent assay (ELISA).
Materials and methods
All experiments were carried out in accordance with the National Institutes of Health Guide for the care and use of laboratory animals (NIH publications No. 80–23). Use of animals was approved from National University of Singapore (NUS) Institutional Animal Care and Use Committee [IACUC No. R13-5512(A)15]. This study was approved by NUS, Project No. R-181-000-140-592 (E-A L). The animals were housed at room temperature (25°C) and 12h of light and dark cycle. All efforts were made to reduce the number of rats used for primary microglial culture.
Expression of GSK-3β, Notch-1 and proinflammatory mediators in BV-2 cells treated with different doses of LiCl and DAPT and with LPS
Murine BV-2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2% FBS incubated in a humidified incubator with 5% CO2, 95% air at 37°C. The confluent BV-2 cells were plated in a six-well plate at a density of 1.5X106 incubated under different conditions at 37°C in a humidified incubator with 5% CO2/95% air for 24h.
For determination of GSK-3β expression after LiCl treatment, BV-2 cells were divided into five groups. Group 1, BV-2 cells were untreated; group 2, BV-2 cells were stimulated with LPS for 4h; group 3–5, BV-2 cells were treated with LiCl at 15mM, 20 mM and 25mM, respectively, for 0.5h and then challenged with LPS for 4h. In a separate group, BV-2 cells were divided into six groups comprising untreated, LPS and DAPT at 10 μM, 15μM, 20 μM and 25μM pretreatment +LPS to determine the effects on Notch-1 and GSK-3β expression. In both untreated and untreated BV-2 cells, RNA and proteins were harvested for RT-PCR and Western blot analysis, respectively.
BV-2 cells treated with DAPT at 20μM and LiCl at 20mM were selected from the above to investigate their response to changes of Notch pathway, GSK-3β and its upstream AKT
These dosages were selected based the observations that NICD expression was drastically suppressed by LiCl at 20mM, and DAPT at 20μM. In this regard, expression of NF- κB/p65 and IκB a as well as the functional response such as production of proinflammatory cytokines IL-1β, IL-6 and TNF-α, stress mediator iNOS and COX2, anti-inflammatory cytokine IL-10, and the chemokine MCP-1 involved in microglial migration was analyzed. For this, four groups of BV-2 cells were subjected to different treatments. In group 1, the cells were incubated in DMEM with 2% FBS without treatment. In group 2, the cells were treated with LPS for 4 h. In group 3, the cells were treated with DAPT at 20μM (Sigma-Aldrich, MO, USA) for 3 h and then challenged with LPS for 4h. In group 4, the cells were treated with LiCl at 20mM for 0.5h and then challenged with LPS for 4h. Some cell samples were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 15 min and processed for immunofluorescence labeling; others were used for RT-PCR and Western blotting. All experiments were carried out at least in triplicate.
BV-2 cells plated on coverslips coated with poly-L-lysine at a density of 1x106 cells/ml cells per well were treated with LPS, DAPT+LPS and LiCl+LPS, respectively. The cells were fixed in 4% paraformaldehyde in 0.1 M PB for 15 min. After rinsing with PBS, the coverslips with adherent cells were used for double immunofluorescence labeling. BV-2 cells were incubated in goat anti-rabbit activated Notch-1(NICD) polyclonal antibody (dilution 1:500; Abcam, incorporated in the UK), goat polyclonal antibody Hes-1 (dilution 1:200; Santa Cruz Biotechnology), goat anti-rabbit GSK3β (dilution 1:100; Santa Cruz Biotechnology), goat anti-rabbit p-GSK 3β(ser9) (1:250; Cell Signaling Technology), goat anti-rabbit p-AKT(ser473)(1:250; Cell Signaling Technology), goat anti-rabbit IL-1β (dilution 1:500; Chemicon, Temecula, CA), anti-mouse TNF-α antibody (dilution 1:500; Chemicon), anti-mouse IL-10 antibody (dilution 1:200; Santa Cruz Biotechnology) and goat-anti-rabbit MCP-1(1:200; Santa Cruz Biotechnology)(Table 1) Subsequently, the cells were incubated with FITC-conjugated and Cy3-conjugated secondary antibodies for 1 h at room temperature. The coverslips were mounted on the slides with histology Mounti-Fluoroshield with DAPI (Sigma, CA, USA). All images were captured with a confocal microscope (Fluoview1000; Olympus, Tokyo, Japan).
Total RNA was extracted from BV-2 cells of all groups using the RNeasy Mini kit (Qiagen, Valencia, CA). Reverse transcription reactions were performed using the reverse transcription system kit (catalog No. 3500; Promega, Singapore). The resultant cDNA was diluted 10 times in double-distilled H20 and kept at -20°C for RT-PCR analysis. Gene sequences for primer design were obtained from the National Centre for Biotechnology Information’s Gene Bank. Primer pairs for GSK-3β, IL- 1β, IL- 6, TNF-α, iNOS and β-actin were designed using the primer design program (Primer 3 software version 1.0). Forward and reverse primer sequences for the genes and their corresponding amplicon size are listed in Table 2. RT-PCR was performed using a 96-well plate run in the ABI machine according to the manufacturer’s instructions. The RT-PCR was briefly carried out in a 10μl final volume containing the following: SYBR Green I master mix (Qiagen); 1μl of 5μM forward primer and reverse primer, respectively, and 3μl of diluted cDNA. After an initial denaturation step at 95°C for 15 min, temperature cycling was initiated. Each cycle consisted of denaturation at 94°C for 15 sec, annealing at 60°C for 25 sec, and elongation at 72°C for 20 sec. In total, 45 cycles were performed. Mouse β-actin was amplified as the control for normalizing the quantities of transcripts of each of the above-mentioned genes. The expression differences for GSK-3β, IL-1β, IL-6, TNF-α and iNOS between the control and treated cells were calculated by normalizing with the β-actin gene expression according to the following formula : Fold change 5 22[Ct (control) gene X–Ct (control) actin]–[Ct (activated) gene X–Ct (activated) actin].
Western blotting analysis and ELISA
The cell supernatant from different treated groups was collected and kept it in a -80°C freezer for ELISA. Culture medium was removed from the culture plate, and BV-2 cells were washed twice with ice-cold PBS. Cells were lysed with lysis buffer, mechanically scraped off with a rubber police- man, and centrifuged at 13,000 rpm for 25 min. Protein concentration of samples was then determined by using a protein assay kit (Bio-Rad, Hercules, CA; catalog No. 500–0002). Next, the protein samples were separated on 4–20% pre-casted sodium dodecyl sulfate-polyacrylamide gels. The proteins embedded in the gel were then transferred to the immobile polyvinylidene difluoride transfer membranes using a semidry electrophoretic transfer cell (Bio-Rad, Hercules, CA). The membranes were washed with TBS+0.1% Tween buffer and then incubated with 5% nonfat dry skim milk for 1h at room temperature. After this, they were incubated with goat anti-rabbit activated Notch-1(NICD) polyclonal antibody (dilution 1:1000; Abcam, incorporated in the UK), goat anti-rabbit deltex dilution 1:1000; Santa Cruz Biotechnology), goat anti-rabbit GSK-3β (dilution 1:1000; Santa Cruz Biotechnology), goat anti-rabbit phospho-GSK-3β (Ser9) (dilution 1:1000; Cell Signaling Technology), goat anti-rabbit phospho-AKT (Ser473) (dilution 1:1000; Cell Signaling Technology), anti-mouse NF-κB /p65 ((dilution 1:1000; Cell Signaling Technology), anti-mouse IκBα (dilution 1:1000; Cell Signaling Technology), anti-mouse iNOS (dilution 1:1000; BD), anti-mouse TNF-α (dilution 1:1000; Sigma-Aldrich), anti-mouse Cox2 (dilution 1:2000; BD), goat anti-rabbit MCP-1(dilution 1:1000; Santa Cruz Biotechnology), anti-rabbit IL-1β (dilution 1:1000; Chemicon) and anti-mouse β-actin (dilution 1:10000; Sigma-Aldrich) (Table 3) overnight on a shaker in a cool room. After three washes with TBS-0.1% Tween, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (dilution 1:5000; Pierce Biotechnology, Rockford, IL) for 1h. The above-mentioned proteins were detected with a chemiluminescence detection system according to the manufacturer’s instructions (Supersignal WestPico Horseradish Peroxidase Detection Kit; Pierce Biotechnology) and developed on the film. The band intensity of these protein expression levels relative to the β-actin was quantified in Image J software (NIH). All experiments were repeated at least in triplicate.
ELISA was carried out to assess some of the cytokines secreted by BV-2 cells in the cell supernatant after different treatments together with groups stimulated with LPS only and control. For this purpose, ELISA kits for mouse TNF-α (Quantikine ELISA, R&D systems), and IL-10 (Mouse IL-10 ELISA Kit, Sigma-Aldrich) were used following the instructions in the manual provided by the respective manufacturer.
Immunofluorescence labeling of activated Notch-1 (NICD), p-GSK-3β, NF-κB/p65 and CD11 in primary microglial cells after different treatments
To confirm that the results in BV-2 cell line was reproducible we have used the primary microglial cells in freshly prepared microglia derived from the newborn rat brain. The primary microglia would represent better the microglia in the developing brain. For this purpose, postnatal (day 3–5) rats were anaesthetized with Ketamine 75mg/kg and Xylazine 10mg/Kg cocktail and primary glia cell cultures were prepared. The brains were dissected under sterilized condition and were collected into a dish with chilled 1XPBS containing penicillin and streptomycin to wash away blood two times. The dish was kept on ice cubes. After this, the cerebral cortex was dissected out, and the adherent meninges and blood vessels on the brain surface were removed with the fine tip of a pair of sterilized forceps. The brain tissue samples derived from 3 pups were transferred into a 50 ml centrifuge tube with 10ml dissociated medium and aspirated up and down with a disposable serological pipette. The smashed tissues with the dissociated medium were transferred to a 75 cm culture flask and placed on the shaker at 37°C at 180 rpm for 25 min. The dissociated tissue mixture was transferred through 70μM of cell strainer and collected into a 50-ml centrifuge tube placed up-straight for 3 min. The upper portion of mixture was transferred into a 50-ml tube and spun down at 1000 rpm for 10 min. After this, the supernatant was discarded and the cell pellets were reseeded into cell culture chambers. The medium was changed the next day and thereafter every 3–4 days. The cells were allowed to grow in the chamber for 10–14 days until full confluency. The mild trypsin method  was adopted to purify the mixture microglial cells. The upper intact layer was discarded, while the bottom layer was mainly microglial cells. The chambers with containing cells were grouped as untreated and treated with LPS, DAPT+LPS and LiCl+LPS. The above treated cells and untreated cells were fixed in 4% paraformaldehyde for p-GSK-3β, NICD, NFKB/p65, and CD11b immunofluorescence double labeling.
The statistical significance of differences between control and treatment groups was calculated using one-way analysis of variance (ANOVA). When two groups were compared, Student's t-test was used. Statistical significance was determined by p<0.05 (*) and p<0.01 (**). The values represent the mean ± SD at least in triplicate.
Effects of LiCl on GSK-3β mRNA and DAPT on GSK-3β /NICD protein expression
The mRNA level for GSK-3β was moderately increased in BV-2 cells when stimulated with LPS when compared with the control cells (Fig 1A). However, GSK-3β mRNA expression was significantly suppressed in BV-2 cells when treated with LiCl at 10, 15, 20 and 25 mM followed by LPS, when compared with cells treated with LPS alone (Fig 1A). Western blot shows NICD and GSK-3β protein expression was significantly decreased in BV-2 cells treated with DAPT at 10, 15, 20 and 25μM with LPS, when compared with cells treated with the control or LPS alone (Fig 1B). The suppression of NICD expression by DAPT was most evident at 20μM compared with other treatments. LiCl at 20mM, and DAPT at 20μM were used for all subsequent experimental analysis because both dosages appeared to exert a significant effect on GSK-3β mRNA and NICD/ GSK-3β protein expression, respectively.
GSK-3β mRNA (A) and NICD/ GSK-3β protein (B) expression in BV-2 microglia after pre-treatment with different doses of LiCl and DAPT, respectively, followed by LPS. RT-PCR results show GSK-3β mRNA level was moderately increased after LPS but was suppressed significantly with LiCl at different doses compared with LPS group (A). NICD protein expression was suppressed more substantially by DAPT at 20μM; likewise, GSK-3β protein was decreased drastically by DAPT at different doses (B). The values represent the mean ± SD in triplicate.
LiCl decreases NICD-1, Deltex, GSK-3β protein expression along with that of proinflammatory mediators
The protein expression changes of Notch-1(NICD), Deltex, and GSK-3β in BV-2 microglial cells treated with different doses of LiCl and stimulated with LPS were analyzed. By Western blotting, the specific protein size of NICD, Deltex and GSK-3β was 80kDa, 67kDa and 46kDa, respectively, when probed with their respective antibody. The density for the respective bands was significantly decreased in BV-2 cells treated with different doses of LiCl and challenged with LPS when compared with cells treated with LPS alone (Fig 2A–2C). Concomitantly, expression of MCP-1, TNF-α, and iNOS, with their specific size band at 12kDa, 26kDa,130kDa, respectively, in BV-2 cells showed a drastic decrease when treated with different doses of LiCl and stimulated with LPS comparing with cells treated with LPS alone (Fig 2D).
A shows specific bands of NICD (80kDa), Deltex (67kDa) and GSK-3β (46kDa). Bar graphs (B, C) show ratio of density changes by Image J; Western blotting (D) shows specific bands of MCP-1(12kDa), TNF-α (26kDa) and iNOS (130kDa). Note the decrease of these proteins by LiCl treatment. The values represent the mean ± SD in triplicate.
LiCl and DAPT increase p-GSK-3β and p-AKT but decrease GSK-3β, NICD and Deltex protein expression
To ascertain the interrelationship between Notch signaling and GSK-3β, DAPT (20μM) and LiCl (20mM) were used as inhibitor for Notch and GSK-3β, respectively, to pre-treat BV-2 cells followed by LPS. The protein expression for p-GSK-3β, GSK-3β, p-AKT, NICD and Deltex was then analysed. Western blotting showed p-GSK-3β, GSK-3β and p-AKT appeared at specific bands at 46kDa, 46kDa and 60kDA, respectively (Fig 3A). The band density for p-GSK-3β and p-AKT in DAPT+LPS and LiCl+LPS (Fig 3A–3C) was significantly increased when compared to LPS group; on the other hand, the band density of GSK-3β was decreased (Fig 3A). Meanwhile, the Western blotting results (Fig 3D), showed that besides reducing NICD levels (as already shown in Figs 1B and 2A), DAPT+LPS significantly reduced Deltex levels compared with LPS group. A comparable effect was observed in LiCl+LPS, as also shown in Fig 2A.
Western blotting (A) shows the specific bands of p-GSK-3β (46kDa), GSK-3β (46kDa) and p-AKT (60kDa). Bar graphs (B, C) show ratio of density change of specific bands for p-GSK-3β and p-AKT by Image J. (D). Western blotting shows the specific bands of NICD (80kDa) and Deltex (67kDa). The values represent the mean ± SD in triplicate.
DAPT and LiCl decrease NICD, Hes-1 and GSK-3β but enhance p-GSK-3β and p-AKT immunofluorescence in BV-2 cells
NICD immunofluorescence was markedly enhanced both in the cytoplasm and nucleus in LPS treated cells (Fig 4A and 4B); however, the fluorescence intensity in the cytoplasm, especially in the nucleus in DAPT+LPS and LiCl+LPS treated cells was noticeably attenuated (Fig 4A and 4B). In parallel to NICD, Hes-1 immunofluorescence that was augmented in LPS treated cells was decreased in DAPT+LPS and LiCl+LPS treated cells (Fig 4C). GSK-3β immunofluorescence was moderately attenuated in LPS, DAPT+LPS and LiCl+LPS compared with the control cells (Fig 4C) which is consistent with the Western blot results (Fig 3A).
A shows immunofluorescence double labelling NICD and DAPI. Bar graphs in B show the immunofluorescence intensity of NICD by image J. C shows Hes-1 and GSK-3β immunofluorescence double labelling. Immunofluorescence double labelling for p-GSK3β/ CD11, and p-AKT/CD11 is shown in D and E. DAPI staining of nucleus is also shown. Scale bars in A, C, D and E = 40μm.
Immunofluorescence signaling for p-GSK-3β and p-AKT was noticeably increased both in the cytoplasm and nucleus in DAPT +LPS and LiCl+LPS treated cells, when compared with cells treated with LPS (Fig 4D and 4E).
DAPT and LiCl increase p-GSK-3β but decrease NICD immunofluorescence in primary microglia
Immunofluorescence labeling of primary microglial cells showed that p-GSK-3β (green) and NICD (green) double labeled with CD11b (red) was noticeably increased following treatment with DAPT+LPS and LiCl+LPS in comparison with those treated with LPS alone (Fig 5A and 5B). Conversely, NICD immunofluorescence was decreased after DAPT and LiCl treatment (Fig 5B). The results confirm that in BV-2 cells (see Fig 4).
DAPT and LiCl decrease NF-κB /p65 expression but increase that of IκBα
The protein expression level for NF-κB/p65 was increased in LPS group compared to control (Fig 6A) and decreased after DAPT+LPS and LiCl+LPS treatment comparing with LPS only (Fig 6A), while that of IκBα was upregulated in DAPT+LPS and LiCl+LPS groups compared with the LPS group (Fig 6A). Note that NF-κB/p65 immunofluorescence in primary microglia was increased after treatment with LPS; the immunofluorescence intensity was attenuated in DAPT+LPS and LiCl+LPS groups (Fig 6B).
Western blotting (A) shows decrease in NF-κB /p65 but increase in IκBα expression in BV-2 cells treated with DAPT (20μM) +LPS and LiCl (20mM)+LPS. B shows NF-κB and lectin staining in primary microglia treated with LPS, DAPT+LPS and LiCl+LPS. Note the increase in NF-κB immunofluorescence by LPS which is attenuated by DAPT and LiCl. Scar bars in B = 40μm.
DAPT and LiCl decrease expression of proinflammatory mediators
By RT-PCR, the mRNA level of IL-1β, IL-6, iNOS and TNF-α was significantly decreased in BV-2 cells treated with DAPT+LPS and LiCl+LPS compared with the LPS treated group (Fig 7A and 7D). By Western blot, the protein expression level of COX2, iNOS and IL-1β was significantly decreased in the above-mentioned groups (Fig 7E). Additionally, ELISA results showed that TNF-α production was drastically reduced in BV-2 cells culture supernatant in DAPT+LPS and LiCl+LPS groups compared with the LPS treated group (Fig 7F).
Bar graphs (A, B, C, D) show expression changes of IL-1β, IL-6, iNOS and TNF-α in different treatments. Western blotting (E) shows specific bands for IL-1β (31 and 17kDa), COX2 (72kDa) and iNOS (130kDa). ELISA (F) shows significant decrease in TNF-α production in the supernatant of BV-2 cells after DAPT+LPS and LiCl+LPS. ELISA (G) shows significant increase in IL-10 production after DAPT+LPS and LiCl+LPS, being more pronounced in the latter. The values represent the mean ± SD in triplicate.
DAPT and LiCl attenuate expression of proinflammatory mediators but increase that of IL-10
Double labeling showed that IL-1β (green), TNF-α (red), and MCP-1 (green) immunofluorescence intensity was noticeably decreased in DAPT+LPS and LiCl+LPS treated BV-2 cells compared with LPS treated group (Fig 8A and 8B). On the other hand, interleukin-10 expression was increased in microglia treated with LPS, DAPT+LPS, and LiCl+LPS compared with the control cells (Fig 8A and 8B). The increase was confirmed by ELISA for IL-10 being more substantial in LiCl+LPS in comparison with the LPS group (Fig 7G).
A shows immunofluorescence for IL-1β (green), TNF-α (red), MCP-1 (green) and IL-10 (green) in BV-2 cells treated with LPS, DAPT+LPS, LiCl+LPS, respectively. Bar graphs in B show the immunofluorescence intensity changes for IL-1β, TNF-α, MCP-1 and IL-10 by Image J. Scale bars in A = 30μm. The values represent the mean ± SD in triplicate.
The present results have shown that following the inhibition of proteolysis of Notch-1 with DAPT in LPS activated BV-2 microglia, GSK-3β expression was decreased at mRNA and protein levels indicating that Notch-1 can modulate GSK-3β. Associated with this was the significant increase in phosphorylated GSK-3β (Ser9) (p-GSK-3β) in cells treated with DAPT+LPS. Very interestingly, there was also significant increase in p-AKT (ser473). Meanwhile, NF- κB /p65 protein expression was decreased, whereas that of IκBα was increased. The mRNA and protein expression levels of proinflammatory cytokines IL-1β, IL-6 and TNF-α were decreased along with that of iNOS. The results therefore support the hypothesis that Notch-1 can regulate GSK-3β and NF-κB /p65 concurrently in microglial activation in production of proinflammatory mediators.
A possible explanation for the decrease in GSK-β in microglia treated with DAPT +LPS may be due to the reduction in phosphorylated NICD caused by a decrease of NICD. Microglial cells may have to maintain an optimal level of GSK-3β for cell survival through increase in phosphorylation of GSK-3β as well as negatively feedback to promote its upstream AKT phosphorylation. This may then compensate for the decrease in AKT activity following inhibition of γ-secretase by DAPT. In this connection, it is relevant to note that Notch signalling can activate the PI3K-AKT pathway in a variety of cell lines .
We next explored whether GSK-3β can reciprocally regulate Notch-1 as well as NF-κB taking into consideration of the fact that the last two mentioned had earlier been reported to operate synergistically [3,7]. To ascertain this, we treated the BV-2 cells with LiCl. Remarkably, NICD and Hes-1 expression was decreased at protein level in BV-2 cells and primary microglia treated with LiCl+LPS. Furthermore, Deltex protein expression was decreased in BV-2 cells given the same treatment. Meanwhile, NF-κB/p65 protein expression level was decreased as opposed to IκBa expression that was increased in BV-2 cells and primary microglia treated with LPS+LiCl. Moreover, the mRNA expression levels of pro-inflammatory cytokines IL-1β, IL-6, TNF-α and iNOS were decreased; on the other hand, the anti-inflammatory cytokine IL-10, a marker for M2 phenotype  was increased. It would appear therefore that IL-10 may be differentially regulated compared with the proinflammatory mediators and that LiCl promotes polarization of microglia from M1 to M2 . The present results are consistent with Foltz et al  who reported that GSK-3β was able to phosphorylate Notch-1 in vitro, and that NICD has to be phosphorylated by GSK-3β for its translocation to the nucleus . In light of the above, it is suggested that Notch-1 signalling interacts with GSK-3β in regulating the production proinflammatory mediators and anti-inflammatory cytokine IL-10 in activated microglia. Meanwhile, Notch-1 is reciprocally regulated by GSK-3β as evident by the decrease in NICD and Hes-1 expression by LiCl coupled with increase in p-GSK-3β and p-AKT. This is consistent with a previous report that phosphorylation of GSK-3β down-regulates Notch-1 activity .
Additionally, we have shown that LiCl suppressed the expression of NF-κB/p65 and increased IκBα expression in activated microglia indicating a role of GSK-3β in modulating NF-κB /p65. We show here that DAPT and LiCl treatment had resulted in substantial increase in p-GSK-3β and p-AKT expression along with reduced production of proinflammatory mediators. It is therefore suggested that increase in p-GSK-3β and p-AKT may be related to regulating NF-κB/p65 in production of proinflammatory mediators in activated microglia, although the underlying mechanism remains to be ascertained. We have previously reported that inhibition of Notch signaling would exert its influence through TRAF6 on NF-kB/p65 . However, as NF-kB activity is controlled at different levels by positive and negative regulatory elements, multiple targets may exist for the action of Notch signaling in NF-kB activity. For example, activation of phosphatidylinositol-3 (PI-3) kinase and AKT by RNS60 has been reported to rapidly upregulate IκBα via CREB that is anti-inflammatory . Interestingly, we have shown that both DAPT and LiCl upregulated IκBα suggesting that suppression of NF-κB/p65 by both inhibitors may be mediated via IκBα. All in all, it is obvious that the mechanistic links between the three pathways in microglial activation are complex and remain to be fully elucidated.
In conclusion, using DAPT and LiCl as inhibitor for Notch- 1 and GSK-3β, respectively, the present results have demonstrated a complex interplay between Notch-1, GSK-3β and NF-κB/p65 signalling pathways. It is suggested that the resulting increase in p-GSK-3β, p-AKT and IκBα is involved in regulating microglia activation in production of proinflammatory mediators and/or anti-inflammatory cytokine, IL-10. It is well documented that robust microglia activation can lead to perturbation of the microenvironment especially when there is excess production of TNF-α, IL-1β, nitric oxide, glutamate etc. over a prolonged duration. This would exacerbate neuroinflammation that is implicated in different neurological diseases and disorders. Therefore, in designing of an effective therapeutic strategy aiming at amelioration of neuroinflammation mediated by activated microglia [1, 30–33], all three signalling pathways and their interrelationship among others should be addressed; indeed, targeting only one mechanism is far from sufficient.
This study was supported by grant No. R-181-000-140-592 (E-A L) National University of Singapore.
- 1. Dheen ST, Kaur C, Ling E-A (2007). Microglial activation and its implications in the brain diseases. Curr Med Chem 14: 1189–1197 pmid:17504139
- 2. Zhou Y, Ling E-A and Dheen ST (2007). Dexamethasone suppresses MCP-1 production via MAP kinase phosphatase-1 dependent inhibition of JNK and p38 MAPK in activated rat microglia. J Neurochem 102: 667–678. pmid:17403137
- 3. Cao Q, Li P, Lu J, Dheen ST, Kaur C, Ling E-A (2010) Nuclear factor-κB/p65 responds to changes in the Notch signalling pathway in murine BV-2 cells and in amoeboid microglia in postnatal rats treated with the γ-secretase complex blocker DAPT. J Neurosci Res. 88(12):2701–2714. pmid:20648656
- 4. Ajmone-Cat MA1, D'Urso MC1, di Blasio G1, Brignone MS1, De Simone R1, Minghetti L2 (2016) Glycogen synthase kinase 3 is part of the molecular machinery regulating the adaptive response to LPS stimulation in microglial cells. Brain Behav Immun. 55:225–235. pmid:26593276
- 5. Cortés-Vieyra R, Bravo-Patiño A, Valdez-Alarcón JJ, Juárez MC, Finlay BB, Baizabal-Aguirre VM (2012) Role of glycogen synthase kinase-3 beta in the inflammatory response caused by bacterial pathogens. J Inflamm. 9:23.
- 6. Maixner DW, Weng H-R (2014) The role of glycogen synthase kinase 3 beta in neuroinflammation and pain. J Pharm Pharmacol (Los Angel) 1(1): 001– pmid:25309941
- 7. Cao Q, Lu J, Kaur C, Sivakumar V, Dheen ST, Ling E-A, et al. (2008) Expression of Notch-1 receptor and its ligands Jagged-1 and Delta-1 in amoeboid microglia in postnatal rat brain and murine BV-2 cells. Glia. 56 (11):1224–1137. pmid:18449946
- 8. Zeng WX, Han YL, Zhu GF, Huang LQ, Deng YY, Wang QS, et al. (2017) Hypertonic saline attenuates expression of Notch signaling and proinflammatory mediators in activated microglia in experimentally induced cerebral ischemia and hypoxic BV-2 microglia. BMC Neurosci. 18(1):32. pmid:28288585
- 9. Yuan Y, Fang M, Wu C-Y, Ling E-A (2016) Scutellarin as a potential therapeutic agent for microglia-mediated neuroinflammation in cerebral ischemia. Neuromol Med. 18: 264–273.
- 10. Yao L, Cao Q, Wu C, Kaur C, Hao A, Ling E-A (2013) Notch signalling in the central nervous system with special reference to its expression in microglia. CNS Neurol Disord Drug Targets. 12(6):807–814. pmid:24047525
- 11. Yao L, Kaur C, Dheen ST, Hao A, Lu J, Ling E-A, et al. (2013) Notch-1 signalling regulates microglia activation via NF-κB pathway after hypoxic exposure in vivo and in vitro. PLoS One. 8(11):e78439. pmid:24223152
- 12. Grimes CA1, RS Jope (2001) The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling. Prog Neurobiol. 65(4):391–426. pmid:11527574
- 13. Doble BW1, Woodgett JR (2003) GSK-3: tricks of the trade for a multi-tasking kinase. J Cell Sci. 16 (Pt 7):1175–1186.
- 14. Li X1, Zhu W, Roh MS, Friedman AB, Rosborough K, Jope RS (2004) In vivo regulation of glycogen synthase kinase-3β (GSK3β) by serotonergic activity in mouse brain. Neuropsychopharmacology. 29(8):1426–1431. pmid:15039769
- 15. Wang H, Brown J, Gu Z, Garcia CA, Liang R, Alard P, et al. (2011) Convergence of the mammalian target of rapamycin complex 1- and glycogen synthase kinase 3-β–signaling pathways regulates the innate inflammatory response. J Immunol. 186 (9) 5217–5226. pmid:21422248
- 16. Martin M, Rehani K, Jope RS, Michalek SM (2005) Toll-like receptor-mediated cytokine production is differentially regulated by glycogen synthase kinase. Nat Immunol. 6(8):777–784. pmid:16007092
- 17. Beurel E1, Jope RS (2009) Lipopolysaccharide-induced interleukin-6 production is controlled by glycogen synthase kinase-3 and STAT3 in the brain. J Neuroinflammation. 6:9. pmid:19284588
- 18. Espinosa L, Inglés-Esteve J, Aguilera C, Bigas A (2003) Phosphorylation by glycogen synthase kinase-3 beta down-regulates Notch activity, a link for Notch and Wnt pathways. J Biol Chem. 278(34):32227–32235. pmid:12794074
- 19. Koistinaho J, Malm T, Goldsteins G (2011) Glycogen synthase kinase-3β: a mediator of inflammation in Alzheimer's disease? Int J Alzheimers Dis. 2011:129753. pmid:21629736
- 20. Gentle ME, Rose A, Bugeon L, Dallman MJ (2012) Noncanonical Notch signalling modulates cytokine responses of dendritic cells to inflammatory stimuli. J Immunol. 189 (3):1274–1284. pmid:22753939
- 21. Stambolic V, Ruel L, Woodgett JR (1997) Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells. Curr Biol. 7(3):196.
- 22. Shahal B, Piel E, Mecz L, Kremer I, Klein E (1996) Lack of advantage for imipramine combined with lithium versus imipramine alone in the treatment of major depression—a double-blind controlled study. Biol Psychiatry. 40(11):1181–1183. pmid:8931923
- 23. Jope RS (2003) Lithium and GSK-3: one inhibitor, two inhibitory actions, multiple outcomes. Trends Pharmacol Sci. 24(9):441–443. pmid:12967765
- 24. Meijer L, Flajolet M, Greengard P (2004) Pharmacological inhibitors of glycogen synthase kinase 3. Trends Pharmacol Sci. 25(9):471–480. pmid:15559249
- 25. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 25:402–408 pmid:11846609
- 26. Donat CK, Scott G, Gentleman SM, Sastre M (2017) Microglial activation in traumatic brain injury. Frontiers in Aging Neuroscience. Volume 9: article 208 p. 1–20
- 27. Foltz DR, Santiago MC, Berechid BE, Nye JS (2002) Glycogen synthase kinase-3 beta modulates notch signalling and stability. Curr Biol. 12:1006–1011. pmid:12123574
- 28. Matsuno K, Diederich RJ, Go MJ, Blaumueller CM, Artavanis-Tsakonas S (1995) Deltex acts as a positive regulator of Notch signalling through interactions with the Notch ankyrin repeats. Development. 121(8):2633–2644. pmid:7671825
- 29. Khasnavis S, Arundhati Jana A, Roy A, Wood T, Ghosh S, Watson R, et al. (2012) Suppression of nuclear factor-kappa B activation and inflammation in microglia by a physically-modified saline. J Biol Chem See comment in PubMed Commons below 287(35): 29529–29542 pmid:22753407
- 30. Yuan Y, Fang M, Wu C-Y, Ling E-A (2016) Scutellarin as a potential therapeutic agent for microglia-mediated neuroinflammation in cerebral ischemia. Neuromolecular Medicine 18:264–273. pmid:27103430
- 31. Karthikeyan A, Patnala R, Jadhav SP, Ling E-A, Dheen ST. (2016) MicroRNAs: Key players in microglia and astrocyte mediated inflammation in CNS pathologies. Current Medicinal Chemistry 23(30):3528–3546. pmid:27528056
- 32. Ziemka-Nalecz M, Jaworska J, Zalewska T (2017) Insights into the neuroinflammatory responses after neonatal hypoxia-ischemia J Neuropathol Exp Neurol 76 (8): 644–654. pmid:28789477
- 33. Kaur C, Rathnasamy G, Ling E-A (2017) Biology of microglia in the developing brain J Neuropathol Exp Neurol pp. 1–18 (In press).