Early glycolytic reprogramming controls microglial inflammatory activation

Abstract

Background: Microglial activation-mediated neuroinflammation plays an important role in the progression of neurodegenerative diseases. Inflammatory activation of microglial cells is often accompanied by a metabolic switch from oxidative phosphorylation to aerobic glycolysis. However, the roles and molecular mechanisms of glycolysis in microglial activation and neuroinflammation are not yet fully understood.

Methods: The anti-inflammatory effects and its underlying mechanisms of glycolytic inhibition in vitro were examined in lipopolysaccharide (LPS) activated BV-2 microglial cells or primary microglial cells by enzyme-linked immunosorbent assay (ELISA), quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, immunoprecipitation, flow cytometry, and nuclear factor kappa B (NF-κB) luciferase reporter assays. The anti-inflammatory and neuroprotective effects of glycolytic inhibitor, 2-deoxoy-D-glucose (2-DG) in vivo were measured in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-or LPS-induced Parkinson's disease (PD) models by immunofluorescence staining, behavior tests, and Western blot analysis.

Results: We found that LPS rapidly increased glycolysis in microglial cells, and glycolysis inhibitors (2-DG and 3-bromopyruvic acid (3-BPA)), siRNA glucose transporter type 1 (Glut-1), and siRNA hexokinase (HK) 2 abolished LPS-induced microglial cell activation. Mechanistic studies demonstrated that glycolysis inhibitors significantly inhibited LPS-induced phosphorylation of mechanistic target of rapamycin (mTOR), an inhibitor of nuclear factor-kappa B kinase subunit beta (IKKβ), and NF-kappa-B inhibitor alpha (IκB-α), degradation of IκBα, nuclear translocation of p65 subunit of NF-κB, and NF-κB transcriptional activity. In addition, 2-DG significantly inhibited LPS-induced acetylation of p65/RelA on lysine 310, which is mediated by NAD-dependent protein deacetylase sirtuin-1 (SIRT1) and is critical for NF-κB activation. A coculture study revealed that 2-DG reduced the cytotoxicity of activated microglia toward MES23.5 dopaminergic neuron cells with no direct protective effect. In an LPS-induced PD model, 2-DG significantly ameliorated neuroinflammation and subsequent tyrosine hydroxylase (TH)-positive cell loss. Furthermore, 2-DG also reduced dopaminergic cell death and microglial activation in the MPTP-induced PD model.

Conclusions: Collectively, our results suggest that glycolysis is actively involved in microglial activation. Inhibition of glycolysis can ameliorate microglial activation-related neuroinflammatory diseases.

Keywords: 2-DG; Glycolytic inhibitors; Microglial cells; NF-κB; Neuroinflammation.

Fig. 1

LPS rapidly induces microglial aerobic glycolysis. a Real-time changes in the ECAR of the BV-2 microglial cells treated with LPS (200 ng/mL) (arrow indicates initiation time of LPS treatment). BV-2 microglial cells were treated with LPS for the indicated times, and then, the expression of Glut-1 (b), HK-2 (c), and LDHA (d) was determined by quantitative real-time PCR (Q-PCR). Data are presented as mean ± S.D. (n=3) and are representative of results obtained from three independent experiments. *p<0.05, **p<0.01, compared to the control group

Fig. 2

Aerobic glycolysis is required for microglial inflammatory activation. BV-2 microglial cells or primary microglial cells were pretreated with 2-DG (600-1000 μM) for 30 min, followed by LPS (200 ng/mL) treatment for 24 h. The nitrite in the cell culture medium was determined using the Griess reaction (a BV-2 microglial cells; c primary microglia). Cell viability was determined by MTT assay (b BV-2 microglial cells; d primary microglia). The concentrations of IL-6 (e) and TNF-α (f) in the primary microglial cell culture media were analyzed by ELISAs. The expression of iNOS, COX-2, and IL-1β (g) in BV-2 microglial cells was determined by Western blotting (upper), and the relative protein levels were quantified by densitometry analysis (lower). Data are presented as mean ± S.D. (n=3) and are representative of results obtained from three independent experiments. *p < 0.05, **p <0.01, compared to the NONE group or LPS alone group

Fig. 3

Glycolytic inhibitors suppress the inflammatory response by negatively regulating NF-κB signaling pathways. a BV-2 microglial cells stably expressing NF-κB luciferase reporter construct were pretreated with 2-DG (600–1000 μM) for 30 min, followed by LPS (200 ng/mL) stimulation for 16 h. The transcriptional activity of NF-κB was determined by luciferase reporter assay. b BV-2 microglial cells were pretreated with 2-DG for 30 min, followed by treatment of lipopolysaccharide (LPS)-Alexa Fluor® 488 for 2 h. The binding efficiency was analyzed using flow cytometry. c HEK293T cells stably expressing NF-κB luciferase reporter construct were transfected with indicated plasmid. After a 16-h transfection, the cells were treated with 2-DG for 8 h. The NF-κB transcriptional activity was determined by luciferase reporter assay. d BV-2 microglial cells were pretreated with 2-DG (1000 μM) for 30 min, followed by LPS (200 ng/mL) treatment for 30 min. The expression of p-JNK, JNK, p-ERK, ERK, p-p38, and p38 was detected by Western blotting (left), and the relative protein levels were quantified by densitometric analysis (right). e BV-2 microglial cells were treated with 2-DG (1000 μM) for 30 min prior to LPS stimulation for 30 min. The expression of p-TBK1 was analyzed by Western blotting (upper), and the relative protein levels were quantified by densitometric analysis (lower). Data are presented as mean ± S.D. (n=3) and are representative of results obtained from three independent experiments. *p < 0.05, **p <0.01 compared to the LPS alone group or control group

Fig. 4

Glycolytic inhibitors suppress LPS-induced IKKβ activation by modulating AMPK/mTOR signaling in microglial cells. a BV-2 microglial cells were pretreated with 2-DG (600–1000 μM) for 30 min, followed by LPS (200 ng/mL) treatment for 15 min. The expression of p-IKKα/β, p-IκB-α, and IκB-α was analyzed by Western blotting (upper), and the relative protein levels were quantified by densitometric analysis (lower). b BV-2 microglial cells were transfected with si-Scramble or si-HK2. After 48 h, the cells were stimulated with LPS (200 ng/ml) for the indicated time (0, 5, 10, 20, 30, 60 min), and then, the expression of HK2, p-IκB-α, and IκB-α was determined by Western blotting (upper), and the relative protein levels were quantified by densitometric analysis (lower). c BV-2 microglial cells were pretreated with 2-DG (600–1000 μM) for 30 min, followed by LPS (200 ng/mL) treatment for the indicated time. After 1 h, the expression of p-AMPK and p-mTOR was determined by Western blotting (upper), and the relative protein levels were quantified by densitometric analysis (lower). d BV-2 microglial cells were pretreated with 2-DG (600–1000 μM) for 30 min, followed by LPS (200 ng/mL) treatment for 16 h. The ADP/ATP ratio was calculated as described in the methods and materials section. Data are presented as mean ± S.D. (n=3) and are representative of results obtained from three independent experiments. *p < 0.05, **p <0.01, compared to the LPS group

Fig. 5

Glycolytic inhibitors suppress LPS-induced transcriptional activity of NF-κB via inhibition of NAD+/SIRT1-mediated p65 acetylation. a BV-2 microglial cells were pretreated with 2-DG (1000 μM) or 3-BPA (100 μM) for 30 min, followed by LPS (200 ng/mL) treatment for 1 h. The expression of p65 subunit of NF-κB p65 and p-p65 subunit of NF-κB in the total, cytosolic, and nucleus lysates was determined by Western blotting (left), and the relative protein levels were quantified by densitometric analysis (right). b BV-2 microglial cells were pretreated with 2-DG (1000 μM) for 30 min followed by LPS (200 ng/mL) treatment for 1 h. The extracts were subjected to immunoprecipitation (IP) with anti-p65 antibody or anti-IgG followed by immunoblotting analysis with anti-acetylated-p65 antibody and anti-p65 antibody. The expression of ac-p65 was determined by Western blotting (upper), and the relative protein levels were quantified by densitometric analysis (lower). c HEK293T cells were cotransfected with p300 and p65 plasmids for 24 h, followed by 2-DG (1000 μM) treatment for 30 min and then treated with TNF-α (10 ng/mL) for 1 h. The extracts were subjected to immunoprecipitation (IP) with anti-p65 or anti-IgG followed by immunoblotting analysis with anti-acetylated-p65 antibody and anti-p65 antibody. The expression of ac-p65 was determined by western blotting (upper) and the relative protein levels were quantified by densitometric analysis (lower). d BV-2 microglial cells were pretreated with 2-DG (600–1000 μM) for 30 min, followed by LPS (200 ng/mL) treatment for 16 h. The NAD⁺/NADH ratio was measured as described in the methods and materials section. e–g BV-2 microglial cells were transfected with si-SIRT1 (40 nM) or si-Scramble for 48 h, followed by LPS (200 ng/mL) treatment for 6 h. The mRNA expression of SIRT1, TNF-α, and IL-1β was measured by qPCR. Data are presented as mean ± S.D. (n=3) and are representative of results obtained from three independent experiments. *p<0.05, **p <0.01 compared to the LPS, si-Scramble or si-Scramble + LPS group, respectively

Fig. 6

Glycolytic inhibitors reduce CM of activated microglial cell-induced MES23.5 neuroblastoma cell death. a BV-2 microglial cells were pretreated with 2-DG (600–1000 μM) for 30 min, followed by LPS (200 ng/mL) treatment for 6 h. The culture media was removed, and fresh medium was added. After 24 h of incubation, culture medium was added to the MES23.5 cells. MES23.5 cells were incubated with CM for 24 hours, and then, the cell viability was accessed by MTT assay. Cell viability was determined by MTT assay. b Primary microglial cells were pretreated with 2-DG (600–1000 μM) for 30 min, followed by LPS (200 ng/mL) treatment for 6 h. The culture media were removed, and fresh medium was added. After 24 h of incubation, culture medium was added to the MES23.5 cells. MES23.5 cells were incubated with CM for 24 h, and then, the cell viability was accessed by MTT assay. c MES23.5 cells were pretreated with 2-DG (125–1000 μM) before stimulation of MPP⁺ (1000 μM) for 24 h. Cell viability was determined by MTT assay. Data are presented as mean ± S.D. (n=3) and are representative of results obtained from three independent experiments. *p <0.05, **p <0.01, compared to the LPS or MPP⁺-alone group

Fig. 7

Glycolytic inhibitor 2-DG ameliorates neuroinflammation and inflammation-mediated dopaminergic cell loss in an LPS-induced mouse PD model. a Schematic diagram of drug administration. Mice were injected intraperitoneally with 2-DG (400 mg/kg/day) or saline (NS) for sequential 10 days. On the fourth day, mice were injected stereotactically with LPS (5 mg/kg) or saline (NS). After 10 days, the mice were euthanized for IHC analysis. b Double immunostaining for TH and Iba1 in the SN. c, d The number of TH positive cells was quantified manually, and the optical density of Iba1 immunoreactivity in same sections was measured using ImageJ analysis software (n=3 mice per group). Magnification, ×10, scale bar, 100 μm (left). Boxed rectangular regions were enlarged (right). Magnification, ×40, scale bar, 25 μm. e Double immunostaining for TH and CD68 in the SN. f The optical density of CD 68 immunoreactivity was measured using ImageJ analysis software (n=3 mice per group). Magnification, ×10, scale bar, 100 μm. Boxed rectangular regions were enlarged (right). Magnification, ×40, scale bar, 25 μm. Data are presented as the means ± S.E.M. *p <0.05, **p <0.01, compared to the LPS group. g The expression IL-1β in the SN was determined by Western blotting (left), and the relative protein levels were quantified by densitometric analysis (right, n=3 mice per group). Data are presented as the means ± S.D. *p <0.05, **p <0.01, compared to the LPS group

Fig. 8

Glycolytic inhibitor 2-DG significantly ameliorates MPTP-induced behavioral deficits in mice. a Schematic diagram of drug administration. Mice were injected intraperitoneally with 2-DG (400 mg/kg/day) or saline (NS) for sequential 10 days. On the fourth day, mice were injected intraperitoneally with MPTP (30 mg/kg/day) or saline (NS) for 7 days. Pole climbing test (b) and rotarod test (c) were conducted on the 11th day (n=10 mice per group). d Double immunostaining for TH and Iba-1 in the SN. Magnification, ×10, scale bar, 100 μm (left). Boxed rectangular regions were enlarged (right). Magnification, ×40, scale bar, 25 μm. e–f The number of TH-positive cells was quantified manually, and the optical density of Iba1 immunoreactivity in same sections were measured using ImageJ analysis software (n=5 mice per group). The data are presented as the means ± S.E.M. *p <0.05, **p <0.01, compared to the MPTP group. g TH protein expression in the striatum (STR) and SN was determined by Western blotting (left), and the relative protein levels were quantified by densitometric analysis (right, n=3 mice per group). The data are presented as the means ± S.D. *p <0.05, **p <0.01, compared to the MPTP group

Fig. 9

Proposed anti-neuroinflammatory mechanisms of glycolytic inhibition. Glycolytic inhibition suppressed LPS-induced activation of NF-κB signaling pathways by modulating NAD⁺/SIRT1/p65 and AMPK/mTOR/IKK/p65 signaling pathways. Therefore, inhibition of glycolysis reduced the death of DA neurons by suppressing microglial inflammatory activation