Introduction
Microglial cells are the resident immune effector cells of the central nervous system and are considered the cells critical for inflammation-associated neurotoxicit y[
1]. Under physiological conditions, microglial cells play critical roles in maintaining brain homeostasis through surveillance of the microenvironment of the brain with ramified morphology [
1]. Under neurodegenerative conditions or upon experimental stimulation with lipopolysaccharide (LPS) or interferon (IFN)-γ, microglial cells become activated with amoeboid morphology [
2]. Once activated, microglial cells produce a number of proinflammatory cytokines and factors, such as tumor necrosis factor (TNF)-α, nitric oxide (NO), prostaglandin (PG)E
2, interleukin (IL)-1β, IL-6, and reactive oxygen species (ROS), which in turn contribute to the recruitment of other immune cells and neuronal cell injuries [
2,
3]. There is growing evidence that uncontrolled activation of microglial cells is involved in the progression of several neurodegenerative diseases, including Parkinson’s disease (PD) [
4]. Neuroinflammation mediated by activated microglia was found in both PD patients and rodent models of PD and was positively associated with dopaminergic neuron loss [
4]. Therefore, inhibition of microglial inflammatory activation has become a potential therapeutic strategy against PD.
Glucose metabolism is actively implicated in microglia/macrophage activation-mediated inflammatory responses [
5,
6]. The glucose catabolic metabolism pathway consists of glycolysis and mitochondrial oxidative phosphorylation. Glycolysis is defined as the breakdown of glucose through a series of extramitochondrial biochemical reactions into pyruvic and lactic acids under anaerobic conditions. The process includes the glucose activation phase and the energy extraction phase [
7]. Glycolytic flux is regulated by three rate-limiting steps that are mediated by three key enzymes: hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK) [
7]. In the last step of glycolysis, pyruvate is converted to lactate, which is catalyzed by dehydrogenase (LDH) under anaerobic conditions [
8]. Several lines of evidence have suggested that LPS-activated M1 macrophage cells display metabolic reprogramming, switching from oxidative phosphorylation to aerobic glycolysis, whereas M2 (anti-inflammatory) macrophage/microglial cells switch to oxidative phosphorylation [
9]. LPS stimulation elicits enhanced aerobic glycolysis of macrophages upon the switch from liver type-6-phosphofructo-2-kinase (PFK2) to the ubiquitous type-PFK2 expression [
10]. Similar to macrophages or dendritic cells, activated M1-type microglial cells show a metabolic shift from oxidative phosphorylation to aerobic glycolysis [
11].
Many studies have reported that inhibition of the glycolytic pathway can suppress inflammatory responses. Glucose transporter (Glut)1 was upregulated in activated microglial cells, and the inhibition of Glut-1 suppressed microglial activation [
12]. Hexokinase (HK) 2 was induced in hypoxia-activated microglial cells, and the blockade of HK 2 suppressed ischemic brain injury by inhibiting microglia-mediated neuroinflammation [
13]. 2-Deoxy-
d-glucose (2-DG), a glucose analog, is phosphorylated by hexokinase and thereby competitively inhibits the production of glucose-6-phosphate from glucose and ultimately inhibits glycolysis [
8]. In addition to its inhibitory effect on the proliferation of cancer cells, 2-DG has been recognized as a therapeutic agent for autoimmunity and inflammatory diseases [
14]. The inhibition of glycolysis by 2-DG significantly reduced the production of some proinflammatory factors, such as high mobility group B (HMGB) and IL-1β, whereas it did not affect other proinflammatory cytokines, including TNF-α [
15,
16]. However, in LPS-activated dendritic cells, 2-DG treatment substantially decreased the production of proinflammatory cytokines such as IL-6, IL-12, and TNF-α only at translational levels [
17]. Although the neuroprotective effects of 2-DG in vitro and in vivo have been demonstrated [
18‐
20], the anti-neuroinflammatory activity of glycolysis inhibition has rarely been reported. Recently, we found that 2-DG inhibited the expression of TNF-α, IL-6, iNOS, COX-2, and IL-1β at the transcriptional level [
21]. However, the underlying mechanisms by which 2-DG acts to mitigate the transcriptional expression of proinflammatory genes, particularly in microglial cells, are unclear. Furthermore, the effects of 2-DG on inflammation-mediated dopaminergic neuron cell loss have not yet been investigated. In the present study, we addressed the role of aerobic glycolysis in LPS-activated microglial cells and its potential significance in inflammation-induced dopaminergic neuronal cell death.
Methods and materials
Materials
Bacterial lipopolysaccharide (LPS) (Escherichia coli serotype 055:B5), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenylpyridine(MPP+), 2-DG, and 3-bromopyruvic acid (3-BPA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse TNF-α enzyme-linked immunosorbent assay (ELISA) kit and mouse IL-6 ELISA kit were obtained from R&D Systems (Minneapolis, MN). Antibodies used in this study were as follows: anti-phospho-inhibitor of nuclear factor kappa B kinase [p-IKKα (S176)/IKKβ (S177)], anti-p65 subunit of nuclear factor kappa B (NF-κB) (8242S), anti-NF-kappa B inhibitor alpha (IκBα) (4814S), anti-phospho-IκBα (S32) (2859S), anti-cyclooxygenase (COX)-2, anti-jun N-terminal kinase (JNK) (9252S), anti-phospho-JNK (T183/Y185) (4668S), anti-extracellular signal-regulated kinase (ERK) 1/2 (4695S), anti-phospho-ERK 1/2 (9102S), anti-p38 (9212S), anti-p-p38 (T180/Y182) (9215S), anti-phosho-p70-S6K (T421/S132) (9204S), anti-mechanistic target of rapamycin (mTOR) (2972S), anti-phospho-mTOR (S2448) (2971S), and anti-phospho-5′-AMP-activated protein kinase AMPK (T172) (2535S) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-inducible NO synthase (iNOS) (ab15323), anti-acetyl-p65 (acetyl K310) (ab19870), and anti-α-tubulin (ab7219) antibodies were from Abcam (Cambridge, MA, USA); and anti-hexokinase 2 (HK2) antibody was from Bioworld Technology (St. Louis Park, MN, USA). Anti-tyrosine hydroxylase (TH) (AB152) was obtained from Millipore (Billerica, MA, USA), and anti-ionized calcium-binding adaptor molecule 1 (Iba1) (019-19741) was purchased from Wako Chemicals (Chuo-ku, Osaka, Japan). Anti-CD68 (MCA 1957) was obtained from Bio-Red (Hercules, CA, USA).
Cell culture
The BV-2 murine microglial cell line and human embryonic kidney (HEK) 293T cell line were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Carlsbad, USA) containing 10% fetal bovine serum (FBS, PAN Biotech and Lonsera, Aidenbach, Germany), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). MES23.5, a dopaminergic neuroblastoma cells, was derived from hybridization of rat embryonic mesencephalon cells and the murine neuroblastoma cell line N18TG2 [
22]. The cell line was cultured in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F-12, Gibco) with 5% fetal bovine serum (FBS, PAN Biotech, and Lonsera), 100 U/mL penicillin (Gibco), 100 μg/mL streptomycin (Gibco), and 100× insulin-transferrin-selenium (Gibco). All three cell lines were cultured in an incubator at 37°C in a 5% CO
2 atmosphere.
Primary culture of microglia
Primary microglial cells were collected from newborn C57BL/6J mice [
23]. In summary, the newborn mice were washed in 75% alcohol, and the whole brains were isolated and minced in precooled PBS. Then, the cortical tissue was digested for 20 min with 0.25% trypsin. After centrifugation and resuspension, the samples were digested by DNaseI at 37°C and transferred to a single cell suspension. Then, single cells were plated on poly-
d-lysine-coated flasks for 14 days. The microglial cells were obtained from mixed glial cultures on a shaker at 180 rpm for 3 h.
Nitric oxide (NO) measurement
Microglial cells were seeded in a 96-well plate at a density of 2.0×104 cells/well. After LPS stimulation for 24 h, 50 μl of cell culture supernatant was transferred to a new 96-well plate and mixed with 50 μl of Griess reagent. The signals were measured in a microplate reader (Infinite M200 PRO, Tecan, Switzerland) at 550 nm. The data were normalized to a standard curve.
Cytotoxicity assay
Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In brief, after appropriate treatment of BV-2 cells, 30 μl of MTT (Solarbio, Beijing, China) was added to each well and incubated at 37°C for 4 h. The samples were mixed with 100 μl of DMSO and detected at 540 nm by a microplate reader (Infinite M200 PRO).
Enzyme-linked immunosorbent assay (ELISA)
The concentrations of TNF-α and IL-6 in the medium were detected by mouse TNF-α or IL-6 ELISA kits according to the manufacturers’ instructions.
RNA isolation and quantitative real-time PCR
Total RNA from the microglial cells or tissues was isolated by TRIzol reagent and subjected to reverse transfection by Oligo-d(T) and M-MLV reverse transcriptase (Thermo Fisher, USA). Real-time quantitative PCR was performed on an Applied Biosystems 7500 Real-Time PCR system (Foster City, CA, USA) using PrimeScript RT Master Mix (TaKaRa, Dalian, China). The specific primers used in reverse transcription were purchased from GENEWIZ (Suzhou, China) as shown in Table
1. Gapdh was used as a control. The normalized CT values were calculated according to the comparative delta-delta Ct method.
Table 1
Primers used in real-time quantitative PCR
mus-Gapdh | Forwad: | TGTGTCCGTCGTGGATCTGA |
Reverse: | TTGCTGTTGAAGTCGCAGGAG |
mus-iNOS | Forwad: | TAGGCAGAGATTGGAGGCCTTG |
Reverse: | GGGTTGTTGCTGAACTTCCAGTC |
mus-Cox-2 | Forwad: | CAGGCTGAACTTCGAAACA |
Reverse: | GCTCACGAGGCCACTGATACCTA |
mus-Tnf-α | Forwad: | CAGGAGGGAGAACAGAAACTCCA |
Reverse: | CCTGGTTGGCTGCTTGCTT |
mus-Il-1β | Forwad: | TCCAGGATGAGGACATGAGCAC |
Reverse: | GAACGTCACACACCAGCAGGTTA |
mus-Il-6 | Forwad: | GCCAGAGTCCTTCAGAGAGA |
Reverse: | GGTCTTGGTCCTTAGCCACT |
mus-Hk2 | Forwad: | TCATTGTTGGCACTGGAAGC |
Reverse: | TTGCCAGGGTTGAGAGAGAG |
mus-Glut-1 | Forwad: | CAGTTCGGCTATAACACTGGTG |
Reverse: | GCCCCCGACAGAGAAGATG |
mus-Ldha | Forwad: | TGTCTCCAGCAAAGACTACTGT |
Reverse: | GACTGTACTTGACAATGTTGGGA |
mus-G6pdx | Forwad: | CACAGTGGACGACATCCGAAA |
Reverse: | AGCTACATAGGAATTACGGGCAA |
Western blot analysis
BV-2 microglial cells, HEK293T cells, and primary mixed glial cells were lysed on ice for 30 min and shaken every 10 min in denaturing lysis buffer (50 mmol/L Tris [pH 8], 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, and 150 mmol/L NaCl and PMSF), while mouse tissue (the substantia nigra and the striatum) was measured by an ultrasound system before being lysed on ice. After mixing with 5×loading buffer, the proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane, followed by blocking in milk for 2 h. Then, the samples were incubated with the appropriate primary antibodies and HRP-conjugated secondary antibodies. Finally, proteins were detected by enhanced chemiluminescence (ECL) (Millipore) with a ChemiScope 3300 mini (CLINX, Shanghai, China).
Plasmids and siRNA transfection
BV-2 microglial cells or primary mixed glial cells were seeded on a 12-well plate (5.0×10
4 cells/well) 1 day prior to transfection. The cells were transfected with appropriate siRNA or plasmids accompanied by transfection reagents (Lipofectamine®RNAiMAX or Lipofectamine 2000) according to the manufacturer’s instructions. After 24 h, the cells were collected for subsequent experiments. The siRNA sequences are shown in Table
2.
Table 2
Sequences of si-RNA used in RNA interference
mus-Hk2 | siHK2 | GGAGAUGCGUAAUGUGGAATT |
mus-Hk2 scramble | | GUAAGGCGUAUGGAAUAGGTT |
mus-Glut-1 | siGLUT-1 | GCUGCCUUGGAUGUCCUAUTT |
mus-Glut-1 scramble | | GUGUUACGUCUGUCGACUCTT |
mus-Ldha | siLDHA | CCACCAUGAUUAAGGGUCUTT |
mus-Ldha scramble | | AGCUACGUCGAUUCUCAAGTT |
Mus-Sirt1-1 | siSIRT1-1 | GCGCAUAGGUCCAUAUACUTT |
Mus-Sirt1 scramble-1 | | GAUUUCCAUUCGCCAGTAGAT |
Mus-Sirt1-2 | siSIRT1-2 | GCGCAUAGGUCCAUAUACUTT |
Mus-Sirt1 scramble-2 | | GGUUUCUACAUCGUCCTACTA |
Mus-Sirt1-3 | siSIRT1-3 | CCGUCUCUGUGUCACAAAUTT |
Mus-Sirt1 scramble-3 | | GAUTUCCAUUCGCCAGUAGAT |
Coimmunoprecipitation (IP)
Protein was extracted in nondenaturing lysis buffer (20 mM Tris HCl [pH 8], 137 mM NaCl, 1% Nonidet P-40 (NP-40), and 2 mM EDTA), followed by incubation with appropriate antibodies overnight. Then, the samples were mixed with protein A/G beads for 4 h, and then, the beads were washed 10 times to remove nonspecifically bound proteins. The beads and protein were mixed with 2x loading buffer and separated in boiling water, followed by Western blotting.
NF-κB luciferase reporter assays
BV-2 microglial cells or HEK293T cells stably expressing the NF-κB reporter were seeded on a 12-well plate one day before compound and LPS (200 ng/mL) stimulation. After 16 h, the cells were lysed in reporter lysis buffer, and luciferase activity was detected with a dual-luciferase assay kit following the manufacturer’s protocol (Promega, USA).
MPTP-induced PD model
Mice (male, C57BL/6J, 6–8 weeks) were obtained from the Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and placed in an SPF laboratory animal room. All the experiments used in this research followed the Guide for the Care and Use of Laboratory Animals (8th edition) and were approved by the Institutional Animal Care and Use Committee of Soochow University. In this experiment, mice were randomly allocated to 3 groups: saline, MPTP alone (30 mg/kg), and 2-DG (400 mg/kg) +MPTP (30 mg/kg). For the 2DG+MPTP group, 2-DG was given 3 days prior to MPTP injections. MPTP was injected for 7 consecutive days, and 2-DG was preadministered 2 h before MPTP injection. The rotarod test and pole test were performed on the 11th day. After behavioral tests, mouse brain tissue was collected for further study. All behavioral tests were performed by an investigator blind to the treatment.
LPS-induced PD model
Mice (male, C57BL/6J, 6–8 weeks) were randomly separated into 3 groups: saline, LPS alone (5 mg/kg), and 2-DG (400 mg/kg) +LPS (5 mg/kg). For the 2DG+LPS group, 2-DG was given 3 days prior to LPS injections. LPS was stereotactically injected into the substantia nigra pars compacta (SNc) on the third day, and 2-DG was administered for 7 consecutive days. On the 11th day, the mice were euthanized, and brain tissue was collected for further study.
Rotarod test
Before the test, mice were trained on the rotarod for 10 min. During testing, each mouse was placed on the rotarod with increasing speed from 4 rpm/min to 40 rpm/min, and the time when mice fell from the rotarod was recorded. The test for each mouse was recorded 3 times. The average of three trials was used as a statistical indicator [
24]. The examiner conducting the rotarod test was blind to the treatment.
Pole test
The pole used in this experiment is a wooden stick with a wooden ball on the top that has a rough surface. During testing, mice were placed on the wooden ball, and the time for the mice to climb from the ball to the base of the stick was recorded. The test for each mouse was recorded 3 times. The average of three trials was used as a statistical indicator. The examiner conducting the pole test was blind to the treatment.
Immunohistochemistry
The mice were euthanized, and normal saline was perfused into the left ventricle, followed by paraformaldehyde (4%) perfusion. After perfusion, the mouse brains were collected and fixed in phosphate-buffered paraformaldehyde for 3 days at 4°C and then dehydrated in 30% sucrose solutions until they sank. The brain was frozen and cut into 20-μm slices with a freezing microtome. For immunofluorescence staining, the brain slices were washed in PBS and blocked with fetal bovine serum (FBS). Then, the samples were soaked with the appropriate antibody overnight. The next day, brain slices were washed in PBST for 30 min before or after incubation with secondary fluorescent antibodies for 2 h in dark. The samples were placed on a microscope slide and observed by confocal microscopy (Zeiss LSM710 META, Germany).
For histological quantification, every fifth 20-μm-thick section of the region spanning bregma −2.92 to −3.64 mm was analyzed. Five sections of clear Substantia Nigra region per mouse were first delineated using a 4× objective. The number of TH positive cells was quantified manually using 10× objective. It ensured that we selected the same regions of interest among the different groups, and three mice of each group were used for statistical analysis. Similarly, the optical density (OD) of Iba1or CD68 immunoreactivity in the same sections was measured using ImageJ analysis software with 10× objective. Scorings of TH-positive cells and optical density of Iba1 or CD68 immunostaining were performed by a researcher blind to experiment treatment.
ADP/ATP ratio assay
An ADP/ATP assay kit (Sigma-Aldrich) was used in the ADP/ATP ratio assay. In brief, BV-2 cells were seeded in a 12-well plate (1×105 cells/well). After LPS stimulation for 16 h, the cells were digested and collected in a centrifuge tube. Then, the cells were seeded in a 96-well plate with a white background (1×104 cells/well). The ATP reagent was prepared and added to each well. The samples were placed at room temperature for 1 min and measured by a luminescence reporter assay system (Promega, Madison, WI, USA). The value was recorded as [(RLU)A]. After 10 min, the samples were measured again as [(RLU)B]. After recording [(RLU)B], 5 mL of ADP reagent was prepared and added to samples without delay. Then, the samples were incubated at room temperature for 1 min before the luminescence was detected as [(RLU)C]. The ADP/ATP ratio was calculated following the manufacturer’s instructions.
NAD+/NADH assay
After LPS stimulation for 16 h, BV-2 microglial cells were digested and resuspended into two precooled tubes. Then, 100 μL of NAD extraction buffer or NADH extraction buffer was prepared and added to each tube. After heating at 60°C for 5 min, 20 μL of assay buffer and 100 μL of the opposite extraction buffer were added to each sample, followed by centrifugation at 14,000 rpm for 5 min. Finally, 80 μL of working reagent was added to each sample, and the optical density was detected immediately (OD0) and 15 min later (OD15). NAD+/NADH was calculated according to the manufacturer’s instructions.
Extracellular flux assays (Seahorse Bioscience, Chicopee, MA) were used to measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of BV-2 microglial cells. BV-2 microglial cells were plated in a Seahorse XF microplate at a density of 2.0×104 cells/well and cultured overnight. Then, the plate was balanced in a non-CO2 incubator at 37°C for 30 min. Meanwhile, Seahorse XF Glycolysis Stress Test Kit assay medium was prepared, and glutamine was added to Seahorse XF Base Medium. Finally, the compounds were diluted in an assay medium and added to the microplate. The ECAR and OCR were measured in an Agilent Seahorse XFe/XF96 or 24 Analyzer.
Coculture
Microglia cells were seeded in triplicate at a density of 5x104 in a 24-well plate. Microglia cells were pretreated with 2-DG for 30 min before LPS addition. After stimulation with LPS for 6h, the cell culture supernatant was discarded and fresh media were added. After cultured for 24h, the condition media (CM) from compound or vehicle-treated microglia cells were collected. MES23.5 dopaminergic neural cells were cultured in a 96-well plate (2.0×104 cells/well), and after 12 h, the conditioned medium was added to MES23.5 for 24 h. Then, cell viability was measured by MTT assay and flow cytometry.
Flow cytometry
BV-2 microglial cells were treated with 2-DG for 30 min prior to lipopolysaccharide (LPS)-Alexa Fluor® 488 (L23351, Invitrogen, CA, USA) stimulation. After 2 h, cells were resuspended and washed in PBS for 3 times. The cell-associated fluorescence was measured by flow cytometry analysis (Beckman Coulter, Brea, CA, USA).
Statistical analysis
All data are presented as the mean ± standard deviation (S.D.) from at least three independent experiments and analyzed with GraphPad Prism software (version 8.0.1; San Diego, CA, USA). Comparisons between multiple groups were analyzed by one-way analysis of variance (ANOVA) or two-way analysis of variance (ANOVA) with Tukey’s test. p< 0.05 was considered statistically significant.
Discussion
In the present study, it was found that inhibition of aerobic glycolysis suppressed LPS-induced microglial proinflammatory gene expression at both mRNA and protein levels via the inhibition of NF-κB transcriptional activity. The mechanistic studies indicated regulation of AMPK/mTOR/IKKβ and NAD+/SIRT1/p65 signaling pathways may be involved in the inhibitory activity of glycolytic inhibitors on LPS-induced NF-κB transcriptional activation. Furthermore, 2-DG significantly attenuated microglial activation and dopaminergic cell loss in both LPS- and MPTP-induced PD models.
The metabolic shift from oxidative phosphorylation to aerobic glycolysis has emerged as a hallmark of the proinflammatory activation of microglial cells. However, it is largely unknown whether this reprogramming is required for the expression of proinflammatory genes in LPS-activated microglial cells. We found that the knockdown of glycolytic enzymes and application of small-molecule inhibitors both blocked LPS-induced microglial activation, suggesting that glycolysis is essential for microglial inflammatory activation. Previously, it was reported that aerobic glycolysis controlled IFN-gamma production upon the binding of GAPDH to the AU-rich elements of the 3′-untranslated regions of IFN-gamma mRNA in T cells [
33]. It was also reported that GAPDH-ARE binding is critical for posttranslational control of TNF-α production in LPS-primed monocytes [
34]. However, in dendritic cells, 2-DG suppressed the production of TNF-α, IL-6, and IL-12 at posttranscriptional levels through GAPDH-ARE binding independent mechanisms [
17]. In our results, we found that glycolytic inhibition reduced the expression of proinflammatory genes at both mRNA and protein levels, suggesting that transcriptional mechanisms are also involved in the regulation of microglial activation by glycolysis.
During microglial activation, the expression of proinflammatory genes is mainly controlled by NF-κB and AP-1, which are key transcriptional factors that regulate the expression of a number of genes critical for inflammatory responses. After binding with LPS, TLR4 elicits TAK1 activation by recruiting adaptor molecules and protein kinases such as MyD88, IRAKs, and TRAF6, resulting in phosphorylation of IKKβ and MAPK s[
35]. The activation of MAPKs, including JNK, p38, and ERK1/2, elicits transcriptional factor AP-1 activation, ultimately leading to the expression of proinflammatory genes. In the present study, we found that glycolytic inhibitors did not reduce LPS-induced MAPK phosphorylation but suppressed IKKβ phosphorylation, suggesting that TAK1 is not involved in the anti-inflammatory activity of glycolytic inhibitors in microglial cells. In addition, we also found that glycolytic inhibitors blocked both IKKβ and p65 overexpression—but not TAK1 overexpression-induced NF-κB luciferase activity. Furthermore, inhibition of glycolysis suppressed LPS-induced the phosphorylation and nuclear translocation of p65, and the phosphorylation and degradation of IκBα in microglial cells. Taken together, these results strongly suggest that IKKβ and p65 may be targets of glycolytic inhibition.
Given that TAK1 was not involved in the anti-inflammatory effects of the glycolytic inhibitors in microglial cells, another signaling molecule may be upstream of IKKβ and p65. There are several lines of evidence showing that mTOR, a downstream molecule of AMPK, is involved in the activation of NF-κB signaling pathways [
28,
36,
37]. mTOR promotes NF-κB transcriptional activity by directly interacting with IKK, and rapamycin, an inhibitor of mTOR reduces proinflammatory gene expression by blocking NF-κB activation in microglial cells [
38]. Our previous study also demonstrated that rapamycin significantly suppressed IKKβ phosphorylation in LPS-activated BV-2 microglial cells [
28]. In the present study, we found that inhibition of glycolysis suppressed LPS-induced mTOR phosphorylation, which was consistent with the findings from previous report which indicated that 2-DG was shown to inhibit mTOR in some cancer cell lines [
39]. Phosphorylation of mTOR is mainly regulated by the upstream molecule AMPK [
38]. AMPK is a highly conserved protein kinase complex that is a key component of cellular energy metabolism in eukaryotes [
38]. Activation of AMPK is inhibited by high levels of fructose-1,6-bisphosphate (FBP), an intermediate metabolite of glycolysis [
40]. AMPK induces rapid inhibition of mTOR signaling by directly phosphorylating tuberous sclerosis complex subunit 2 (TSC2) and rapto r[
38]. There is growing evidence showing that activation of AMPK significantly inhibits microglial inflammatory activation by blocking the NF-κB signaling pathway [
41,
42]. AMPK is activated by the phosphorylation of Thr172 at a high ADP/ATP rati o[
41]. In the present study, glycolytic inhibitors activated AMPK by increasing the ADP/ATP ratio. Activation of AMPK resulted in the inhibition of mTOR phosphorylation and IKKβ activation in microglial cells, suggesting that the AMPK/mTOR/IKKβ signaling pathway is at least partly involved in the anti-inflammatory mechanisms of glycolytic inhibition. In addition to phosphorylation, the acetylation of p65 is also crucial in regulating NF-κB transcriptional activation under inflammatory conditions [
43]. The acetylation of p65 is mainly mediated by different histone acetyltransferases, including CREB-binding protein and p300 [
43]. The acetylation status of p65 is also negatively controlled by histone deacetylases, including SIRT1 [
29]. SIRT1, an NAD
+-dependent protein deacetylase, can deacetylate p65 at lysine 310, thereby inhibiting NF-κB transcriptional activation [
29]. SIRT1 activator resveratrol or overexpression of SIRT1 can significantly inhibit microglial inflammatory activation by suppressing NF-κB transcriptional activity [
44]. In the present study, 2-DG blocked the LPS-induced decrease in the NAD
+/NADH ratio and p65 acetylation, resulting in the inhibition of LPS-induced NF-κB activation. These results suggest that inhibition of the NAD
+-dependent SIRT1/NF-κB pathway might contribute to the anti-inflammatory effects of glycolytic inhibition.
Although neuronal cell death is the hallmark of neurodegenerative diseases, microglial cell-mediated neuroinflammation was considered important in the pathogenesis and progression of neurodegenerative diseases, including PD [
45]. Overactivated microglial cells create an inflammatory microenvironment by releasing various proinflammatory factors, ultimately contributing to neurodegenerative progression [
46]. Many studies have demonstrated that inhibition of microglial activation by anti-inflammatory compounds or modulation of the expression of genes attenuated neuronal cell injuries under neurodegenerative conditions, suggesting that suppression of microglial activation may be considered a potential therapeutic approach to neurodegenerative diseases [
47‐
49]. In the present study, we found that the glycolytic inhibitor 2-DG significantly inhibited neuronal cell death induced by conditioned media from activated microglial cells. We also found that pretreatment with 2-DG significantly attenuated dopaminergic cell death and microglial cell activation. Consistent with our study, a previous report demonstrated that 2-DG reduced MPTP-induced TH-positive cell death by increasing the expression of the stress-responsive proteins GRP78 and HSP70 in neuronal cells [
20]. However, we did not find a protective effect of 2-DG on MPP
+-induced neuronal death in vitro. On the other hand, many studies have suggested that 2-DG may inhibit cell growth or promote cell apoptosis in various tumor cells [
50], suggesting that the effect of 2-DG on cell survival is dependent on the cell type or nature of the stimuli. It is suggested that stereotactic injection of LPS is able to induce loss of DA neurons. We found that 2-DG significantly ameliorated LPS-induced DA loss in vivo. Thus, it is reasonable to conclude that 2-DG ameliorates DA neuron loss in vivo by inhibiting microglia-mediated neuroinflammation.
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