Background
Parkinson’s disease (PD) is one of the most common neurodegenerative diseases worldwide, and its clinical features are characterised by the progressive degeneration of midbrain dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) [
1]. Currently, no effective specialised treatment has been developed for this pathology, and the cause of the neurodegeneration is still unknown. Chronic inflammation in the central nervous system (CNS) plays a critical role in the pathological progression of PD. Microglia, a type of neuroglia, are macrophages in the CNS and are the chief resident immune cells in the brain, where they act as the main active immune defence [
2]. The presence of activated microglial cells within the substantia nigra has been reported in post-mortem studies and in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced animal model of PD [
3]. Impaired or dead midbrain DA neurons can directly induce the activation of microglia, increasing the production of ROS and pro-inflammatory cytokines [
4]. The activated microglia will produce many inflammatory cytokines that contribute to midbrain DA cell apoptosis and death [
5]. Control of microglial activation might help to increase neuronal survival and mitigate PD [
6].
MicroRNAs (miRNAs) are small non-coding RNA molecules that play a complex role in the regulation of transcription of multiple genes via binding to 3′ untranslated regions (3′-UTR) [
7]. Emerging evidence demonstrates that post-transcriptional regulation by miRNA machinery plays an important role in PD pathogenesis [
8,
9]. For example, miR-433 inhibits the translation of FGF20, which has previously been shown to cause PD through both over-expression and point mutations [
10]. In addition, miR-7 protects against MPTP-induced cell death by promoting glycolysis and targeting RelA (p65) [
11,
12]. Specifically, sustained aberrant miRNA expression levels have been described in inflammatory and immune-related neurodegenerative disorders [
13]. Several different microRNAs have been implicated as important regulators and fine-tuners of immune system activation and neuroinflammation. miR-155 is required for α-syn-induced inducible nitric oxide synthase (iNOS) expression in microglia in models of PD [
14], while three of these miRNAs (miR-125b-5p, miR-342-3p, and miR-99a) were specifically expressed in microglia [
15].
MicroRNA-124 (miR-124) is highly expressed in the brain, with an abundance that is (more than 100 times) higher than that in other organs, and it plays a critical role in PD, which regulates apoptosis and autophagy in the MPTP model of PD by targeting Bim and loads nanoparticles to enhance brain repair in PD [
16‐
18]. In fact, miR-124 promotes microglial quiescence, and knockdown of miR-124 in microglia resulted in its activation [
19]. Furthermore, miR-124 mediates cholinergic anti-inflammatory action by inhibiting the production of pro-inflammatory cytokines [
20]. However, whether miR-124 could attenuate microglial activation in the development of PD remains unknown.
MEKK3, a member of the mitogen-activated protein kinase kinase kinase (MAP3K), has also been suggested to play a major role in the inflammation response, including in the process of inducing nuclear factor of kappaB (NF-κB) activation [
21,
22]. To date, only few studies have been conducted about the role of MEKK3 in PD. For example, recent research has shown that HtrA serine peptidase 2 (HtrA2) is phosphorylated upon MEKK3 activation in PD [
23]. In addition, increased colocalisation of NF-κB has been demonstrated in the SNpc of post-mortem PD brains [
24]. Activation of NF-κB plays an important role in the loss of midbrain DA cells in MPTP-intoxicated mice and PD patients [
25,
26]. Even so, little is known about the role of MEKK3 in inflammatory pathogenesis in PD and it remains unclear whether MEKK3 could mediate NF-κB activation in microglia. Hence, in our study, we provide a direct correlation between miR-124 and MEKK3/NF-κB signalling pathways in the inflammatory pathogenesis of PD in vivo and in vitro.
Methods
Animals and treatment
Ten-week-old male C57BL/6 mice were purchased from the Sun Yat-sen University Laboratory Animal Center. The animals were accommodated in a controlled environment and supplied with standard rodent chow and water, and investigators were blinded to the experimental treatment. The mice received one intraperitoneal injection of MPTP-HCl per day (30 mg/kg free base; CAS23007-85-4; Sigma, MO, USA) for five consecutive days, while control mice received saline injections. The mice were decapitated, and once the brain was removed, the ventral midbrain, which contained the SNpc, was dissected and stored at − 80 °C for further study. Regarding the experiment with the exogenous delivery of miR-124 into an animal model, the right lateral ventricle of the mice was surgically implanted with a stereotactic catheter (62004, 62104, and 62204; Woruide, Shenzhen, China). Before the stereotactic intraventricular injection, mice were intraperitoneally anaesthetised by pentobarbital sodium (60 mg/kg). The stereotactic intraventricular injection site was chosen as previously reported (anterior-posterior − 0.5 mm, medial-lateral − 0.7 mm, dorsoventral − 2.9 mm) [
27]. After the injections, the mice were kept warm (37 °C) until they recovered from surgery (1 week). The mice were then administered one dose of agomir (MIMAT0000134; RiboBio, Guangzhou, China) miR-124-3p (20 nM of ribonucleotide in a total volume of 5 μL) through the catheter per day for five consecutive days. Agomir-negative control sequences (MIMAT0000039) were injected into the right lateral ventricle as the negative control. The agomir treatment was performed 2 days prior to the injection of MPTP.
Cell culture and treatment
BV2 microglial cells and SH-SY5Y cells were obtained from the Central Laboratory of Nanfang Hospital (Guangzhou, China). BV2 cells were maintained in DMEM (Gibco, Carlsbad, CA, USA), supplemented with 10% heat-inactivated foetal bovine serum (Gibco) and 0.1% penicillin-streptomycin (Sigma-Aldrich, St. Louis, MI, USA). Human midbrain DA cell line SH-SY5Y cells were maintained in DMEM supplemented with 10% FBS (Gibco) and 0.1% penicillin-streptomycin. Both cells were cultured at 37 °C in a humidified incubator with 95% air/5% CO
2. The reagents for cell culture in our research have been reported in previous studies [
28,
29].
Transfection
The miR-124 mimics (miR10000134), control miRNA mimics (MIMAT0000295), miR-124 inhibitor (miR20000134), and control inhibitors (MIMAT0000039) were synthesised by RiboBio (Guangzhou, China) and were transfected into BV2 cells by using riboFECT™ CP (RiboBio) according to the manufacturer’s protocol. The miRNA-124 mimic sequence was 5′-CCGUAAGUGGCGCACGGAAU-3′. The miR-124 inhibitor sequence was 5′-GGCAUUCACCGCGUGCCUUA-3′. In addition, the small interfering RNA (siRNA) specifically targeting MEKK3 (MEKK3 siRNA) and the control siRNA (siNC) were purchased from Shanghai GenePharma (A10001; Shanghai, China). MEKK3 siRNA and siNC were transfected with Lipofectamine 2000 Reagent (11668019; Invitrogen, USA) according to the manufacturer’s instructions. The sense strand of the MEKK3 siRNA was 5′-GGAGAGACGAAUUAUAGCATT-3′, and the antisense strand was 5′-UGCUAUAAUUCGUCUCUCCTT-3′. The recommended concentrations are as follows: miR-124 mimics (50 nM), miR-124 inhibitor (100 nM), and MEKK3 siRNA (100 nM).
Reverse transcription quantitative real-time polymerase chain reaction
Total RNA was extracted with TRIzol reagent (15596018; Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. For the messenger RNA (mRNA) quantification of the protein-encoding genes, RNA was reverse transcribed to complementary DNA (cDNA) with a random primer (Sangon Biotech, Shanghai, China) using a Reverse Transcription Kit (RR047A; Takara, Dalian, China), and the mRNA levels were determined using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). A primer pair for the detection of mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. RT-qPCR for the detection of miR-124 was performed using miR-124-specific PCR primers (RiboBio) with PrimeScript RT Master Mix (5×) and SYBR Premix Ex Taq™ II (RR047A and RR820A; Takara, Dalian, China) according to the manufacturer’s instructions, normalised to U6 snRNA. The relative expression of each gene was calculated and normalised using the ΔΔCt method. All the sequences of the primers used are as follows:
miR-124 | 5′-GCGAGGATCTGTGAATGCCAAA-3′ |
U6 | 5′-GCTTCGGCAGCACATATACTAAAAT-3′ |
GAPDH | Forward: 5′-GGGAAATTCAACGGCACAGT-3′ |
Reverse: 5′-AGATGGTGATGGGCTTCCC-3′ |
MEKK3 | Forward: 5′-TGTACCTGAGCGACAACAGC-3′ |
Reverse: 5′-CACTGCTGAGGGGATCTAGC-3′ |
TNF-α | Forward: 5′-TATGGCTCAGGGTCCAACTC-3′ |
Reverse: 5′-GGAAAGCCCATTTGAGTCCT-3′ |
IL-6 | Forward: 5′-TTCCATCCAGTTGCCTTCTT-3′ |
Reverse: 5′-CATTTCCACGATTTCCCAGA-3′ |
iNOS | Forward: 5′-GCTTGGGTCTTGTTCACTCC-3′ |
Reverse: 5′-TCCTCTTTCAGGTCACTTTGG-3′ |
TGF-β1 | Forward: 5′-GCACGTGGAGCTGTACCA-3′ |
Reverse: 5′-CAGCCGGTTGCTGAGGTA-3′ |
IL-10 | Forward: 5′-GCCTTATCGGAAATGATCCA-3′ |
Reverse: 5′-AGGGTCTTCAGCTTCTCACC-3′ |
Western blot analysis
A western blotting assay was performed to detect the protein level of MEKK3 and p-p65 in cultured cells and selected mouse midbrains. Total protein was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (P0013B; Beyotime, Jiangsu, China) with protease and phosphatase inhibitors (B14001 and B15001; BioTools, Olathe, KS, USA), following the manufacturer’s protocol. The protein concentration was measured with bicinchoninic acid (BCA) protein assay (Bio-Rad Laboratories, Inc., Berkeley, CA, USA). Equal amounts of protein were isolated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (Beyotime Biotechnology, Shanghai, China) and then transferred to a polyvinylidene fluoride membrane (IPVH00010; Millipore, Bedford, USA). After blocking in 5% Tris-buffered saline-Tween, the membrane was incubated with primary antibody at 4 °C overnight. The antibodies used were as follows: rabbit anti-MEKK3 (NB100-92399; Novus, USA), mouse anti-p-p65 (Ser536; Cell Signaling Technology, USA), rabbit anti-GAPDH (ab8245; Abcam, Cambridge, MA, USA), goat anti-rabbit IgG-HRP (31460; Life Technologies, USA), and rabbit anti-mouse IgG (A-21065; Life Technologies).
Immunohistochemical analysis and quantitative evaluation
Mice were deeply anaesthetised with a pentobarbital and transcardially perfused with saline followed by 4% polyformaldehyde-HCl, and the midbrains were selected. Immunostaining was performed as previously described [
30]. The total number of Iba1-positive cells, tyrosine hydroxylase (TH)-positive neurons, and apoptotic neurons in the SNpc of selected mice was counted as previously described [
31]. The MEKK3 and Iba1
+ integrated optical density (IOD) were determined by using Image-Pro Plus software (Media Cybernetics, Silver Spring, USA). The antibodies used were as follows: rabbit-derived anti-Iba1 (019-19741; 1:500; Wako Chemicals, Japan), rabbit anti-MEKK3 (NB100-92399; 1:100; Novus, USA), rabbit anti-TH (GB11181; 1:100; Servicebio, Wuhan, China), and HRP-labelled goat anti-rabbit IgG (GB23303; 1:500; Servicebio).
Double immunofluorescence staining and confocal laser scanning microscopy
The procedures for double immunofluorescence staining were performed as that in our previous report [
32]. In brief, free-floating 30-μm sections of the midbrain from each group were incubated with rabbit-derived anti-Iba1 (019-19741; 1:500; Wako Chemicals), rabbit anti-MEKK3 (NB100-92399; 1:100; Novus), and rabbit anti-TH (GB11181; 1:100; Servicebio) at 4 °C overnight followed by Cy3-conjugated goat anti-rabbit IgG (GB21303, 1:300; Servicebio) or a goat anti-mouse IgG conjugated with Alexa Fluor 488 (GB25301, 1:400; Servicebio) for 1 h at room temperature (22 ± 2 °C). Viewed under a LSM 880 (Carl Zeiss, Jena, Germany) laser scanning confocal microscopy, immunoreactivity exhibited green or red fluorescence. Confocal images were acquired and analysed using ZEN lite software (Carl Zeiss).
Luciferase reporter assay
According to the TargetScan database, miR-124 potentially binds to MEKK3. To construct luciferase reporter vectors, the 3′-UTR of MEKK3 cDNA fragments containing the predicted potential miR-124 binding sites were subcloned into the XhoI/NotI site of psi-CHECK™-2 Vector (Promega, Madison, WI, USA). The constructs were cotransfected into HEK293 cells along with scramble (50 nM) or miR-124 mimic (50 nM) using riboFECT™ CP as described by the manufacturer. Luciferase activities were measured with the Dual-Luciferase Reporter Assay System (Promega) 48 h after transfection. Renilla luciferase activity was normalised to that of firefly luciferase.
To observe the NF-κB activity, luciferase assay was performed as previously described [
33]. The cells in 12-well plates were cotransfected with 0.5 μg NF-κB-responsive luciferase reporter plasmid containing four κB sites (pNF-κB-Luc; Clontech) and 0.2 μg pSV-β-galactosidase expression plasmid (Promega). After 24 h, cells were treated with different experimental conditions, and luciferase activities were analysed using a luminometer and then normalised with β-galactosidase activity.
Microglial culture supernatant transfer model
To test the neurotoxic effects of activated microglia, confluent SH-SY5Y cells were cultured in complete medium and treated with the supernatants of lipopolysaccharide (LPS)-stimulated BV2 cells. To study the effects of different transfection materials, the BV2 cells were exposed in the presence or absence of various materials for 48 h and then stimulated with the 1 μg/mL LPS for another 12 h. The resulting culture supernatants were collected, centrifuged to eliminate cell debris, and transmitted to SH-SY5Y cells for 12 h to induce cell apoptosis and death. The cells were then harvested and washed three times with PBS buffer.
Flow cytometry analysis
The apoptosis of the SH-SY5Y cells that were harvested in the microglial culture supernatant (MCS) transfer model was quantified using an annexin V-fluorescein isothiocyanate/propidium iodide (PI) apoptosis detection kit (Dojindo, Tokyo, Japan). The cells were pelleted and resuspended in 5 μL fluorescein isothiocyanate-labelled annexin V (V-FITC), and 5 μL propidium iodide staining solution was added to the cells, followed by incubation at room temperature (shielded from light) for 10 min. The number of apoptotic cells was assayed with flow cytometry (FACSVerse; Becton Dickinson, CA, CT, USA).
Statistical analysis
All data were presented as the mean ± SD from three independent experiments. Statistical analysis was carried out using one-way ANOVA and two-tailed Student’s t test. P < 0.05 was considered to indicate a statistically significant difference.
Discussion
Increasing evidence has demonstrated that miRNAs play pivotal roles in neuron biology and that miR-124 is highly expressed in the brain and during CNS development [
39,
40]. Studies have demonstrated that miR-124 may alleviate neuron death in diverse types of neurodegeneration diseases, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and multiple sclerosis [
16,
41]. In addition, miR-124 expression is downregulated in neurons from the MPTP-induced PD model [
42]. Evidence shows that miR-124 is a critical mediator for the peripheral and CNS inflammatory process by inhibiting the activation of microglia/macrophages and reducing the production of pro-inflammatory cytokine [
20,
43]. However, the underlying mechanism is still unclear. Notably, we would demonstrate the potential neuroprotective role of miR-124 in both cell lines and a pre-clinical mouse model for PD. In our study, we found that miR-124 was downregulated in the LPS-stimulated BV2 cells in a dose- and time-dependent manner, which is consistent with previous reports that miR-124 is a key regulator of microglial cell quiescence in the CNS [
19]. We then suggested that decreasing miR-124 levels may be necessary for the progression of microglial cell activation and the production of inflammatory mediators. An increased level of miR-124 following transfection with miR-124 mimics in BV2 cells resulted in a significant reduction in the expression levels of the neurotoxic cytokines iNOS, IL-6, and TNF-α and in a high increase in the levels of the anti-inflammatory cytokines TGF-β1 and IL-10. Meanwhile, the expression levels of IL-6 and TNF-α were highly increased while the neuroprotective factor IL-10 was decreased after intraperitoneal injection of MPTP. Specifically, our data for exogenous delivery of miR-124 showed the same trend in MPTP-induced inflammatory response in vivo, which was consistent with in vitro experiments. However, we obtained the opposite result when transfecting with the miR-124 inhibitor in comparison with the miR-124 mimics. Consequently, these data indicate that the over-expression of miR-124 could effectively attenuate LPS- or MPTP-induced microglial activation in vitro or in vivo.
Accordingly, MEKK3, an inflammation-associated protein that has been identified as a target of miR-124 in our study, is upregulated in the MPTP-induced PD model. However, the same trend is found for the expression of MEKK3 in activated microglial cells that were stimulated by the Toll-like receptor 4 ligand LPS. Specifically, we then found that MEKK3 was mainly expressed in microglial cells rather than in neuronal cells and that its expression is significantly elevated with microglial activation in vivo. To further clarify the role of MEKK3 in the microglia inflammatory processes, we transfected MEKK3 siRNA into the BV2 cells. The results reflected that the expression levels of TNF-α, iNOS, and IL-6 were significantly decreased. Moreover, the knockdown of MEKK3 suppressed the activity of NF-κB in microglia, which agrees with previous studies that MEKK3 can mediate NF-κB activation [
21]. Nevertheless, we observed that the transfection of miR-124 mimic to microglial cells could counter-regulate the MEKK3 upregulation induced by LPS in vitro, as miR-124 targeting MEKK3 was demonstrated by the luciferase reporter assay in our study. We further treated mice with MPTP and found that an increasing expression level of MEKK3 was evidenced in the SNpc, whereas we observed a similar result in a silencing MEKK3 gene study where MEKK3 expression level was also reduced by upregulation of miR-124 in vivo. Therefore, our data identified MEKK3 as a target of miR-124. Correspondingly, we observed a parallel alteration of MEKK3 and Iba1
+ in both MPTP-induced and agomir-treated mice, and we considered that MEKK3 was a sign of the neuroinflammation.
The NF-κB, which is a ubiquitous transcription factor that regulates immune and cell survival signalling pathways, plays a pivotal role in both inflammatory response and cell survival [
44]. NF-κB consists of a group of seven transcription factors and is known for its crucial transcription factors that regulate the expression levels of pro-inflammatory cytokines such as TNF-α, iNOS, and IL-6 [
45,
46]. Currently, the activation of NF-κB has been reported in the CNS of neurodegenerative diseases in animal models and human patients [
47]. Several studies support the hypothesis that the activation of NF-κB p65 plays an important role in PD pathogenesis and that the selective inhibition of NF-κB protected dopaminergic neurons from MPTP toxicity [
46]. NF-κB has been found to be activated in the SNpc in a hemiparkinsonian monkey model of PD and that the inhibition of NF-κB could supress the secretion of pro-inflammatory molecules, protect the midbrain DA cells from death, and improve locomotor activity [
48]. In fact, treatment with LPS can significantly increase the NF-κB activity in microglial cells, and morphine alters the LPS-induced activation of NF-κB through PKCε-Akt-ERK1/2 signalling [
49]. In our study, we determined that the expression of p-p65 was obviously increased in LPS-stimulated microglial cells and in MPTP-induced PD model. In addition, several miRNAs have been reported to modify cell behaviour by regulating the NF-κB pathway. MicroRNA-26b suppresses NF-κB signalling by targeting TAK1 and TAB3, which is a potent inhibitor of the NF-κB pathway [
50]. The miR-146 levels were downregulated, and the upregulation of miR-146 expression may be of neuroprotective value in AD, whereas the levels of its target proteins IL-1 receptor-associated kinase-1 and NF-κB increased in the microglial cells of PS-2 knockout mice [
51]. Clearly, the identification of miRNAs that target NF-κB signalling may provide novel molecular targets for disease therapy. On the basis of the above-mentioned studies, we examined the anti-inflammatory properties of miR-124 and further studied the potential mechanisms of its neuroprotective effect. Our results showed that over-expression of miR-124 significantly suppressed the expression of LPS- or MPTP-induced upregulation and the activation of p-p65 in protein level. In particular, we considered that miR-124 could mediate the NF-κB signalling pathway by regulating the expression of MEKK3, which might be a therapeutic target in inflammatory responses. Meanwhile, we found no significant difference in the p-p65 levels of the pro-inflammatory cytokines regardless of transfection of miR-124 mimic or miR-124 inhibitor when the cells were pre-treated with MEKK3 siRNA, demonstrating that miR-124 regulates the LPS-induced secretion of pro-inflammatory cytokines and NF-κB in microglial cells, at least partly, through the regulation of MEKK3.
Finally, we found that the apoptosis and death of SH-SY5Y cells could be suppressed when the microglial activation was inhibited by the upregulation of miR-124 or the knockdown of MEKK3 in MCS. Meanwhile, the exogenous delivery of miR-124 could attenuate the activation of microglia in MPTP-treated mice and prevent MPTP-dependent apoptotic midbrain DA cell death in vivo. The observed increase in the neuronal apoptosis rate most likely results from the decreased levels of the inflammatory cytokines present in the conditioned medium because direct treatment of the neuron with LPS did not affect cell apoptosis. Thus, it was reasonable that neuroinflammation induced by activated microglia might play an important role in the PD pathogenic process.