Background
Parkinson’s disease (PD) is the second most common neurodegenerative disorder and characterized pathologically by the loss of pigmented dopaminergic (DA) neurons in the substantial nigra pars compacta (SNc) [
1]. The precise reasons for the selective loss of SNc DA neurons are not entirely clear. Despite intensive researches on DA neurons, recent studies show that glial cells, especially microglia, may be involved in the progressive degeneration of DA neurons [
2-
4]. Studies of post-mortem brain tissue of PD patients show the presence of activated microglia in the affected brain regions, strongly supporting that neuroinflammatory process is associated with neuron loss [
5]. Furthermore, the activated microglia has been shown to contribute to DA neuronal death not only by releasing pro-inflammation factors such as tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) [
6,
7] but also by producing hyperoxides and peroxidases including reactive oxygen species (ROS) and NADPH oxidase [
8-
10].
As an important innate immunity in the central nervous system (CNS), modest activated microglia is necessary and beneficial for brain health. But controlling over vigorous immune responses is critical for preventing this reactivity from being over-activated and damaging the brain. A lot of such controlling mechanisms may contribute to the chronic pathogenesis in neurodegenerative diseases [
11,
12]. Interleukin-10 (IL-10) is an anti-inflammation cytokine with important roles in preventing inflammation [
13]. IL-10 regulates exuberant immune response of microglia by inhibiting their release of pro-inflammatory mediators such as TNF-α and IL-1β [
14] and increasing the release of anti-inflammatory mediators such as IL-1 receptor antagonist (IL-1ra) and soluble TNF-α receptors [
15,
16]. Given the critical role of IL-10 in regulating the course of microglia-mediated inflammatory response, elucidating how IL-10 is regulated may provide valuable information for understanding the pathogenesis of PD. One important aspect of controlling of IL-10 is the level of its gene transcription. The structure of
IL-10 gene promoter contains AT-rich putative transcription factor myocyte enhancer factor 2 (MEF2) binding site. Recent evidence from lymphocytes identifies
IL-10 gene as a potential MEF2 transcriptional target [
17,
18].
MEF2s, initially identified as a nuclear factor important for muscle cell differentiation [
19], have four mammalian isoforms, MEF2, A to D. The N-terminus of MEF2 mediates dimerization and DNA binding, while the C-terminus of MEF2 functions as transcriptional activation domains. MEF2s have been found to play a central role in the activation of the genetic programs that regulate cell proliferation, differentiation, and apoptosis in increasing types of cells [
20]. Our previous study demonstrates that MEF2D promotes the survival of DA neurons in the SNc under stress conditions. Negative regulation of MEF2D by toxic signals contributes to DA neuronal death [
21].
In spite of the studies of MEF2D in neurons, its function and regulation in microglia are entirely unknown. In the present study, we examined the function of MEF2D in activated microglia. Our data showed that the expression and activity of MEF2D were significantly induced in activated microglia. MEF2D regulated the expression for IL-10 in microglia. Silencing MEF2D expression led to a decrease in IL-10 mRNA and protein. This contributed to an increase in inflammation-induced and microglia-mediated toxicity to DA neuronal cells. These results establish a direct link between MEF2D and IL-10 activity in microglia-mediated inflammatory response, suggesting that MEF2D may play a critical role in preventing over-exuberant immune responses and protecting neurons from microglia-mediated neurotoxicity in PD.
Methods
Animal and tissue preparations
C57bl/6 male mice (25 ~ 30 g), purchased from the Experimental Animal Center of the Fourth Military Medical University, were used according to the Guidelines for Animal Care and Use of the Fourth Military Medical University (Xi’an, People’s Republic of China). All efforts were made to minimize animal suffering and to reduce the number of animals used. Mice received four intraperitoneal (i.p.) injections of 20 mg/kg free base 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Sigma-Aldrich, St. Louis, MO, USA) at 2-h intervals. Control mice were injected with phosphate buffer solution (PBS) alone at the same frequency. At 1 day, animals were anesthetized (10% chloralhydrate, i.p.) and transcardially perfused with PBS. The brains were fixed with cold 4% paraformaldehyde. Serial brain sections (30 μm thick) containing the SNc were collected for further analysis.
Cell culture and treatment
BV2 cells were cultured in Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12 containing 2.8 mM L-glutamine, 15 mM HEPES) (Gibco, Grand Island, NY, USA) supplemented with 5% fetal bovine serum (FBS) (Gibco), and incubated with 5% CO2 at 37°C.
Primary mixed glial cell cultures were derived from post-natal days 0 to 3 (P0-P3) Sprague-Dawley rat brains. Shortly, the whole brains were dissociated in trypsin without EDTA for 10 min at 37°C and then were cultured in DMEM/F12 supplemented with 10% FBS. Mixed glial cell cultures, which were cultured in poly-D-lysine-coated (0.1 mg/ml) (Sigma-Aldrich) T75 flasks and on cell slides, were incubated at 37°C and 5% CO2 for 13 to 16 days. When the cells in T75 flasks became confluent, the flasks were shaken at 250 rpm/min for 2 h to detach microglia. The microglia after purification were cultured in DMEM/F12 with 10% FBS for 3 days before their use for the following experiments.
SN4741, a mouse embryonic substantial nigra-derived cell line [
22], was cultured at 33°C with 5% CO
2 in RF medium (DMEM with 10% FBS, 1%
D-glucose, 1% penicillin-streptomycin, and 140 mM
L-glutamine). Experiments were usually done when cells reached 50% to 60% confluence.
When reaching 60% to 70% confluence, the BV2 cells and primary microglia cells were exposed to lipopolysaccharide (LPS, 1.0 μg/ml) (Sigma-Aldrich) for a designed time point, respectively. Then, the cells were enriched for the following experiments.
For neutralizing antibody experiments, BV2 cells were treated with 1.0 μg/ml LPS in the presence or absence of anti-IL-10 neutralizing antibody (1.0 μg/ml, Abcam, Cambridge, UK) for 24 h.
Lentivirus infection
Recombinant lentivirus vector expressing shRNA-MEF2D to silence MEF2D gene expression was obtained commercially from Hanbio, Shanghai, China. BV2 cells were seeded in a six-well plate and were infected with lentivirus in 1 ml medium for 4 h. The medium was added to 2 ml, and cells were continuously infected for another 24 h. After being replaced with fresh medium, the cells were cultured and used for studies.
Immunofluorescence
Mixed glia cell slides and brain sections were fixed with 4% formaldehyde solution, incubated with 0.1% Triton X-100 for 30 min, and blocked with 5% bovine serum albumin (BSA) (Sigma-Aldrich) in PBS for 30 min. Samples were incubated at 4°C overnight with primary anti-Iba-1 (1:50; Abcam, Cambridge, UK) combined with anti-MEF2D (1:200; BD Transduction Laboratories, San Jose, CA, USA); then they were washed three times with PBS and incubated with secondary antibody conjugated with FITC in room temperature for 2 h. Samples were counterstained with DAPI (1:500; Sigma) for 10 min and photographed using a confocal microscope (C2 Si, Nikon, Minato, Japan).
Immunoblotting
Cell protein was harvested with ice-cold lysis buffer containing protease inhibitors. Protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then the separated protein was transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore Corporation, Bedford, MA, USA). After 2-h blocking with 5% fat-extracted milk at room temperature, the membranes were incubated overnight at 4°C with primary antibodies against MEF2D (1:3,000; BD) and β-actin (1:4,000; Abcam). Then the membranes were washed with TBST three times and then were treated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Protein bands were visualized by chemiluminescence detection.
Quantitative real-time PCR
Total RNA was extracted from each sample according to the manufacturer’s protocol for TRIzol reagent (Invitrogen, Paisley, Scotland, UK). Complementary DNA (cDNA) was generated from total RNA using random primer and MMLV reverse transcriptase (Invitrogen), using a final volume of 20 μl. Quantitative real-time PCR (qPCR) analysis was performed in triplicate using QuantiFast™ SYBR® Green PCR Kit (Qiagen, Hilden, Germany). All gene-specific mRNA expression values were compared to β-actin mRNA levels as a standard. The primer sequences for each gene are listed as follows:
-
MEF2D forward, 5′-CGTTGGGAATGGCTATGTC-3′;
-
MEF2D reverse, 5′-GAGGCCCTGGCTGAGTAA-3′;
-
IL-10 forward, 5′-GCTCTTACTGACTGGCATGAG-3′;
-
IL-10 reverse, 5′-CGCAGCTCTAGGAGCATGTG-3′;
-
β-actin forward, 5′-AAGGACTCCTATAGTGGGTGACGA-3′;
-
β-actin reverse, 5′-ATCTTCTCCATGTCGTCCCAGTTG-3′.
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation assay (ChIP) was done following a procedure provided by a ChIP Assay Kit (Millipore). The product of DNA was analyzed by using qPCR and semiquantitative PCR with
IL-10 gene promoter specific primers as follows:
-
forward, 5′-CTGTCTGCCTCAGGAAAT-3′;
-
reverse, 5′-CTAAAGAACTGGTCGGAAT-3′.
Electrophoretic mobility shift assay
Electrophoretic mobility shift assay (EMSA) was performed as described [
23]. The Binding reactions were incubated with MEF2 specific probe or mutant probe, and for super shift assay, reactions were incubated with MEF2D antibody for 1 h at 4°C after incubation with labeled probe. The sequences of the probes used for EMSA are as follows:
Enzyme-linked immunosorbent assay
Detection of IL-10 in the supernatant of treated and untreated BV2 cells cultures was determined with a mouse IL-10 enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Wiesbaden, Germany) according to manufacturer procedures, and results were raised as picogram per milliliter.
MTT assay
Cells were seeded in 96-well plates (5 × 103 cells/well) and incubated at 37°C. After treatment, 20 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/ml) (Millipore) were added to each well, and the plate was incubated for 4 h. Then, after removing supernatant, 150 μl of dimethylsulfoxide (DMSO) (Millipore) was added to each well and mixed thoroughly for 10 min. The optical density (OD) was measured at 490 nm.
TUNEL staining
For terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, FragEL™ DNA Fragmentation Detection Kit (Millipore) was used according to manufacturer’s instructions.
Statistical analyses
Data were expressed as mean ± standard error of the mean (SEM) from at least three independent experiments. Data were analyzed by either one-way ANOVA or two-way ANOVA as appropriate. Statistical analyses were carried out using SPSS 19.0. A value of P < 0.05 was considered statistically significant.
Discussion
Transcription factor MEF2s play important roles in neuronal development, synaptic plasticity, and survival [
28-
30]. Our previous studies show regulation of MEF2D activity by autophagy pathway is related to DA neuronal homeostasis and viability under stress conditions related to PD [
23]. A few studies have reported a link between MEF2s and immune system. For example, MEF2s have been found to participate in immune responses of T and B lymphocytes. In addition, MEF2C level is found to increase in the activated microglia around the hippocampus after ischemia [
31-
33]. In the current study, we provide the first evidence demonstrating the presence of MEF2D in microglia and its function in microglia-mediated inflammation response. Our data indicate that in quiescent microglia, the levels of both MEF2D mRNA and protein are low. Stimulation by neurotoxin or LPS significantly induces MEF2D level and activity. Further, we demonstrate that the increase in MEF2D level is necessary for the transcriptional activation of
IL-10 gene expression and for damping the level of pro-inflammatory cytokines such as TNF-α. Thus, our data establish MEF2D as a critical negative regulator of neuroinflammation.
Our previous findings indicate that MEF2D functions in the nucleus and mitochondria in SNc DA neurons to promote neuronal survival [
23,
27]. Our current data have revealed that MEF2D-mediated regulation of IL-10 in microglia protects a cell line derived from DA neuronal progenitor against the cytotoxicity exerted by prolonged activation of microglia. Together, they indicate control of the over-activation of microglia as a novel mechanism by which MEF2D protects cells from inflammation-induced toxicity. It is important to investigate the temporal change of MEF2D in microglia in PD and determine if loss of MEF2D function in microglia may contribute inflammation-related injury in the pathogenic process of the disease.
Our data indicate that MEF2D mRNA level is robustly induced in microglia in response to LPS. The precise mechanisms underlying this induction remain unknown. It may involve either activation of
mef2d gene transcription or increase in MEF2D mRNA stability. Robust increase in MEF2D transcript has been reported during hepatic stellate cells activation, a stimulation analogous to LPS-induced microglial response [
34]. Since it is currently unclear how
mef2d gene promoter is regulated, further studies are needed to clarify the regulatory mechanism(s) involved.
As an anti-inflammatory cytokine, IL-10 plays a critical role in controlling the intensity of inflammatory response by inhibiting production of various pro-inflammatory cytokines such as IL-1, IL-6, and TNF-α [
35,
36]. Expression of
IL-10 gene is highly inducible, which represents a major mechanism of regulation. Several transcription factors have been implicated in IL-10 regulation including C/EBPβ, SP1, NF-κB, and CREB under various conditions [
37-
40]. But how IL-10 expression is regulated in microglia has not been elucidated. Our data show that the kinetics of MEF2D expression and activity parallels closely with the induction of IL-10. MEF2D binds directly to a region in
IL-10 gene promoter that contains a well-conserved MEF2D binding site. Moreover, MEF2D activity is required for LPS-induced IL-10 mRNA expression. Together, our findings provide strong evidence supporting MEF2D as a primary transcription factor that directly regulates
IL-10 gene transcription in microglia.
Acknowledgements
The authors thank Zixu Mao from the Department of Pharmacology and Neurology, Emory University, USA, for providing plasmids. This work was supported by the Chinese National 973 Projects Grant 2011CB510000 (QY) and the National Natural Science Foundation of China, Grant No. 31371400 (QY).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SY and QY designed the study. SY, LG, FL, FG, GZ, ZC, and JL performed the experiments. SY and QY analyzed the data and wrote the manuscript. SY, BW, and QY revised the manuscript. All authors have read and approved the final version of the manuscript.