MicroRNA-30e regulates neuroinflammation in MPTP model of Parkinson’s disease by targeting Nlrp3
Accumulating evidences suggest that neuroinflammation is a pathological hallmark of Parkinson’s disease (PD), a neurodegenerative disorder characterized by loss of dopaminergic neurons in substantia nigra pars compacta (SNpc). MicroRNAs have been recently recognized as crucial regulators of inflammatory responses. Here, we found significant downregulation of microRNA-30e (miR-30e) in SNpc of MPTP-induced PD mice. Next, we employed miR-30e agomir to upregulate miR-30e expression in MPTP-treated mice. Our results showed that delivery of miR-30e agomir remarkably improved motor behavioral deficits and neuronal activity, and inhibited the loss of dopamine neurons. Moreover, the increased α-synuclein protein expression in SNpc of MPTP-PD mice was alleviated by the upregulation of miR-30e. Further, miR-30e agomir administration also attenuated the marked increase of inflammatory cytokines, such as TNF-α, COX-2, iNOS, and restored the decreased secretion of BDNF in SNpc. In addition, we demonstrated for the first time that miR-30e directly targeted to Nlrp3, thus suppressing Nlrp3 mRNA and protein expression. Finally, miR-30e upregulation significantly inhibited the activation of Nlrp3 inflammasome as evident from the decreased Nlrp3, Caspase-1 and ASC expressions and IL-18 and IL-1β secretions. Taken together, our study demonstrates that miR-30e ameliorates neuroinflammation in the MPTP model of PD by decreasing Nlrp3 inflammasome activity. These findings suggesting that miR30e may be a key inflammation-mediated molecule that could be a potential target for PD therapeutics.
KeywordsParkinson’s disease Neuroinflammation Neurodegeneration Nlrp3 inflammasome miR-30e
Parkinson’s disease (PD) is the second only to Alzheimer’s disease (AD) as the most common neurodegenerative movement disorder, which is characterized with progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and accumulation of α-synuclein (α-syn) in Lewy bodies [1, 2]. The clinically used drugs, including L-DOPA, monoamine oxidase type B inhibitors and catechol-O-methyltransferase inhibitors, could only ameliorate the symptoms through supplementing the absent dopamine, but fail in delaying the process of dopamine neuronal degeneration because it could not protect against neurons injury [3, 4, 5]. Thus, developing a more effective agent remains the top priority in prevention and treatment of PD.
Several lines of researches have suggested that neuroinflammation is considered as the major central event in the process of dopaminergic neuronal cell death in PD [6, 7, 8]. Enhanced levels of proinflammatory cytokines such as TNF-α, COX-2, IL-1β and IL-18 can be found in the analysis of postmortem brain of PD patients [9, 10]. Moreover, the activity of IL-1β and IL-18 is critical controlled by a cytoplasmic multiprotein, called “inflammasome”, which contains nod-like receptor protein 3 (Nlrp3), adaptor protein ASC and proinflammatory mediators Caspase-1, IL-18 and IL-1β . Activation of Nlrp3 inflammasome has been observed in a variety of neurodegenerative diseases, including AD and amyotrophic lateral sclerosis (ALS) . Importantly, Nlrp3 might be associated with the development of PD and be a potential target for the treatment of PD [6, 13]. However, the mechanisms underlying the regulation of Nlrp3 inflammasome activity in PD are poorly understood.
Accumulating evidences indicate that post-transcriptional regulation by microRNAs (miRs) is important for the regulation of gene expression and inflammatory responses [6, 14]. Sustained aberrant expression levels of several different miRs have been described in inflammation-related neurodegenerative disorders, including multiple sclerosis (MS), AD, ALS and PD [15, 16, 17, 18]. Therefore, identification of novel miR machinery that modulates neuroinflammation not only helps to understand the development of PD, but also provides a new approach for the treatment of PD. In our study, we found that exogenous delivery of miR-30e ameliorated neuronal injury, neuroinflammaiton and dyskinesia in MPTP-induced PD mice. Furthermore, miR-30e directly targeted to Nlrp3, which in turn mediated Nlrp3 inflammsome activity and inflammation.
Materials and methods
Materials and reagents
8-week-old male C57BL/6 mice were purchased from the SLAC Laboratory (Shanghai, China) and were maintained in cages with constant temperature (21 ± 1 °C), relative humidity (60%), a strict 12 h/12 h light–dark cycle, and free access to water and food. The experimental protocol was approved by Institutional Animal Care and Use Committee of Henan Provincial People Hospital and carried out in accordance with the guidelines for the Care and Use of Laboratory Animals.
MiR-30e agomir, miR-30e mimics, and corresponding negative control miRNA were obtained from GenePharm (Shanghai, China). SuperScriptIII First-Strand Synthesis system, fetal bovine serum (FBS) Dulbecco’s modified Eagle’s medium, penicillin, streptomycin and lipofectamine 2000 were purchased from Invitrogen (CA, USA). Antibodies against α-syn, Nlrp3, ASC, Caspase-1 and β-actin were from Cell Signaling Technology (MA, USA). Tyrosine hydroxylase (TH) antibody, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody, HRP-donkey anti-goat IgG antibody and the Enhanced Chemiluminesecence Kit were from Millipore (MA, USA). Nissl staining solution, diaminobenzidine (DAB) and RIPA buffer were purchased from Beyotime (Jiangsu, China). All chemicals and reagents unless otherwise indicated were obtained from Sigma (MO, USA).
Animal model and miR-30e agomir delivery
The mice were received 3 times of intraperitoneally (i.p.) injection of MPTP (20 mg/kg) at days 1, 7, and 14. Control mice were administrated with saline only. Mice were killed at different times after the first MPTP injection: 1, 3, 7, 10, and 14 days. For the delivery of miR-30e in MPTP mice, a stereotactic catheter was surgically implanted into the right lateral ventricle of mice (Bregma: −2 mm, Lateral: 2 mm, Dorsoventral: 3 mm). 5 μL of saline containing 20 nmol/L of miR-30e agomir or a scramble sequence control miRNA (negative control) was injected through the catheter per day for 7 consecutive days. The first treatment of agomir was performed 2 h after the last injection of MPTP. The schematic diagram of miR-30e administration is illustrated in Figure S1. Mice were killed immediately after behavioral assessments on day 21 by decapitation. The ventral midbrain containing the SNpc was dissected and stored at −80 °C for further experiments.
Quantitative reverse transcription (qRT-PCR) analysis
Total RNA from SNpc tissues or BV-2 cells were extracted using RNAiso Plus Reagent (Takara, Dalian, China) and reverse transcription was performed with the SuperScriptIII First-Strand Synthesis system. Quantitative assay of genes expressions was performed using a SYBR QPCR Kit (Toyobo, Osaka, Japan) and ABI 7500 real-time PCR system (Applied Biosystems, CA, USA). The gene expression was normalized to the GAPDH and calculated using the ΔCT method. The specific primer sequences used were listed in Table S1.
The mice were evaluated for motor balance and coordination using a rotary rod apparatus (Harvard Apparatus, MA, USA) at different times as indicated. All animals were pretrained before staring the experiment. Each mouse was placed in the apparatus (diameter: 7 cm, length: 30 cm) and operates at a constant speed of 30 rpm. The three latencies to fall recorded by magnetic trip plates were averaged to yield a final value, and the maximum cutoff time was set as 180 s.
A wooden pole of ~ 50 cm in length and ~ 1 cm in diameter was wrapped in gauze and a cork ball of 2.5 cm was glued on top of the pole. Each mouse was placed on top of the ball and the time required for the mouse to climb down the pole was recorded. The test was repeated three times to evaluate the average. The cutoff time was 250 s.
The mice were suspended by their forepaws to a horizontal wire. The mouse was scored as 3 if grasped the wire with two hind paws, 2 if grasped the wire with one hind paw, and 1 if grasped the wire with any of the hind paws.
Each mouse was placed at one end of a 100 cm long and 2 cm wide beam, which was elevated 1 cm above the ground. The time required for the mouse to cross the beam was measured. The cutoff time was set as 120 s.
The midbrain tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm thick sections. For Nissl staining, the sections were incubated with nissl staining solution at 50 °C for 20 min. After rinsing with distilled water, sections were dehydrated with 95% ethyl alcohol, 70% ethyl alcohol in secession. The number of staining cells in SNpc was counted using a BX51 light microscope (Olympus, Tokyo, Japan) at higher magnification (× 400).
Sections of brain tissues were permeabilized with Triton X-100 and blocking with 1% goat serum in saline at room temperature, and incubated with a primary antibody to TH at 1:200 dilution at 4 °C overnight. After washing with saline, sections were incubated with secondary goat–rabbit IgG antibody for 1 h at room temperature and washed three times. DAB solution was added to incubate for 3 min. Images were captured with a BX51 light microscope.
Total protein was extracted from selected mouse midbrain or BV-2 cells using RIPA lysis buffer, and quantified using a bicinchoninic acid protein assay kit (Thermo, MA, USA). Western blotting was performed as previously described . Different primary antibodies used were as following: TH, α-syn, β-actin, ASC (diluted 1:1000), Nlrp3 and Caspase-1 (diluted 1:500). After incubation with corresponding secondary antibody (HRP-conjugated goat anti-rabbit or donkey anti-goat IgG antibody, diluted 1:1000), bands were visualized with the Enhanced Chemiluminescence Kit and determined with a densitometry software (Image J, NIH, Maryland, USA).
Enzyme-linked immunosorbent assay (ELISA)
The level of TNF-α, COX-2, iNOS, BDNF, IL-18 and IL-1β in SNpc was determined using immunoassay kits with the instructions provided by manufacturer (R&D System, MN, USA). All samples were assayed and absorbance was read using a microplate reader (Multiskan Spectrum, Thermo, MA, USA).
Murine BV-2 microglial cells were obtained from the Cell Bank of Chinese Academy of Medical Science (Shanghai, China) and were maintained in Dulbecco’s modified Eagle’s medium with 10% heat-inactivated FBS, 100 U/mL penicillin and 100 mg/mL streptomycin at 37 °C in a humidified incubator under 5% CO2 condition.
In vitro miR-30e mimics transfection
For overexpression of miR-30e in BV-2 cells, the cells were transfected with miR-30 mimics or negative control miRNA using Lipofectamine 2000 according to the manufacturer’s protocol. After 48-h transfection, cells lysis was used for luciferase assay or western blotting analysis.
Luciferase reporter assay
The binding of miR-30e to the target gene Nlrp3 was assayed by luciferase experiment. A wild-type murine Nlrp3 mRNA 3′UTR luciferase reporter construct was amplified by PCR from the Nlrp3 mRNA (NM_145827) 3′-UTR sequence and then cloned into the psiCHECK2-3′UTR vector (Ambio Inc., TX, USA). For mutant construct of Nlrp3 3′UTR, deletion mutagenesis and fusion-PCR were performed. BV-2 cells were co-transfected with either wild-type or mutant Nlrp3 3′UTR, plus miR-30e mimics or negative control for 48 h. Luciferase activity was assessed using Dual-Luciferase Reporter Assay System (Promega, WI, USA) according to the manufacturer’s instructions.
Data were presented as mean ± SEM. The statistical significance of differences between two groups was analyzed by one-way analysis of variance (ANOVA) or the unpaired two-tailed Student’s t test using SPSS 10.0 statistical software (SPSS Inc., IL, USA). P < 0.05 was considered to be statistically significant.
MiR-30e was downregulated in SNpc of MPTP-PD mice model
Effect of miR-30e agomir on body weight in MPTP-administrated mice
MiR-30e upregulation improved the dyskinesia induced by MPTP
MiR-30e attenuated dopaminergic neuronal loss and α-syn expression in SNpc of MPTP-PD mice
Effect of MiR-30e on inflammatory markers and BDNF levels in MPTP-PD mice
Nlrp3 is a target gene of miR-30e
MiR-30e suppressed Nlrp3 inflammasome activation in SNpc of MPTP-PD mice
This study uncovers a link between miR-30e and Nlrp3 inflammasome-mediated neuroinflammation in the pathogenesis of PD. We provide convinced evidence that miR-30e improves neuronal damage, neuroinflammaiton and dyskinesia via negatively regulating Nlrp3 expression and inhibiting NLRP3 inflammasome activation in MPTP-induced PD mice model. MPTP is the most valuable neurotoxin for inducing animal PD model that producing many features of the biological and pathological changes similar to human PD . After rapidly crossing the blood–brain barrier by systemic injection, MPTP is taken up by the astrocytes and catalyzed into the toxic moiety that can be transported into dopaminergic neurons, leading to neuronal damage and dyskinesia [21, 22]. Thus, here, we used MPTP to stimulate dopaminergic neuron loss in vivo to induce PD.
MiRs have been shown to act at the post-transcriptional level by binding the 3′UTR of their target mRNA, leading to degradation of the target gene expression . To date, there are a few studies revealing the critical role of miRs in the pathogenesis of PD. For example, miR-133b expression was found to be decreased in the midbrain of PD patients as well as in mouse models . Moreover, miR-124 targeted to bim and in turn inhibited dopaminergic neurons loss, a key event during the development of PD . In addition, miR-7, miR-153 and miR-155 negatively regulated α-syn expression, which is a crucial regulator for neuroinflammation in PD [15, 24]. In the present study, we investigated the alteration of miR-30e in SNpc by qRT-PCR and the results showed that the expression of miR-30e was downregulated gradually after MPTP injection, suggesting miR-30 might also have a role in the pathogenesis of PD.
Although miR-30e has been shown to be involved in the regulation of glioma cells differentiation and invasion [25, 26], the exact role of miR-30e in PD has not been shown previously. As mentioned in the evidence cited above, MPTP administration is known to decrease neuronal activity and the density of TH-positive neurons, indicating degeneration of the dopaminergic neurons in SNpc [4, 8, 22]. The deficiency of dopamine level makes patients suffer from different degree of behavioral motor deficit . In the current study, our results showed that MPTP injection produced behavior disorder, as evidenced by rota-rod test, pole test, traction test and beam-crossing task. However, delivery of miR-30e agomir in midbrain effectively prolonged the duration time of mice on rotating-stick, decreased the latency to cross straight run way on narrow beam, and increased the grasping force as well as the rate of climbing pole. Furthermore, we investigated whether miR-30e upregulation improves motor function through protecting against MPTP-induced neuronal damage. Nissl staining showed that restoration of miR-30e in PD mice could increase the neuronal activity. In addition, the loss of TH activity as well as a decrease in TH protein expression is thought to contribute to dopamine deficiency, which is the most prominent at media levels of SNpc . Immunohistochemistry and western blotting analysis for TH expression revealed that the loss of dopamine neuron in PD mice was dramatically less pronounced after miR-30e agomir delivery. These results indicate that miR-30e can protect against neuronal injury in MPTP-induced PD mice model.
It has been reported that excessive accumulation of α-syn is a pathological hallmark of PD patients, especially in SNpc [19, 24]. Here, we demonstrated that miR-30e overexpression could effectively attenuate MPTP-induced the increase of α-syn expression in SNpc. Considering that α-syn-triggered neuroinflammation has an important in the pathogenesis of PD , we also examined the effect of miR-30e on inflammatory cytokines secretion in SNpc. The results showed that miR-30e upregulation almost abolished the increase of TNF-α, COX-2 and iNOS secretion. Moreover, aberrant alterations in BDNF expression or signaling may contribute to neurodegeneration and sustained decreased BDNF mRNA expression can be observed in SNpc of PD patients . In this study, we found that the reduction of BNDF secretion in SNpc was markedly reversed by miR-30e agomir treatment.
Finally, we explored the mechanisms by which miR-30e inhibited neuroinflammation in SNpc of PD mice. Intriguing, although MPTP-induced α-syn expression was inhibited by miR-30e agomir, we found that the luciferase activity of α-syn was not affected by miR-30e (data not shown), suggesting α-syn is not the direct target of miR-30e. Notably, α-syn has been recognized to induce the IL-1β production in a process that depends, at least partially, on Nlrp3 inflammasome [6, 28]. In the current study, we demonstrated for the first time that Nlrp3 was a potential target of miR-30e. The luciferase assay indicated that miR-30e targeted the 3′UTR region of Nlrp3 to negatively regulate Nlrp3 mRNA and protein expression. In response to a variety of inflammatory stimuli, the Nlrp3 inflammasome, along with the adaptor protein ASC, induces the activation of Caspase-1 and the maturation of proinflammatory cytokines IL-18 and IL-1β, leading to trigger inflammation [11, 29]. Accordingly, our results showed that Nlrp3, ASC and Caspase-1 expressions, and IL-18 and IL-1β secretions were all increased in SNpc of PD mice. However, miR-30e restoration abolished the above elevations. Consistent with the protein expressions in SNpc, the mRNA levels of the Nlrp3 inflammasome were also decreased after miR-30e agomir treatment. These data suggest that the activation of Nlrp3 inflammasome may contribute to MPTP-induced neuroinflammation in SNpc, whereas miR-30e inhibits this process by targeting Nlrp3. Furthermore, considering the critical role of Nlrp3 inflammasome in the development of neurodegenerative diseases [6, 12, 13], our study also indicate that miR-30e induces neuron regeneration at least partially via inhibition Nlrp3 inflammasome-mediated inflammation.
In conclusion, our study demonstrates that miR-30e negatively regulates Nlrp3 expression, which in turn attenuates neuroinflammation in SNpc of PD mice through inhibiting Nlrp3 inflammasome activity. These findings indicate that targeting miR-30e by a genetic approach may provide a novel strategy for the treatment of PD.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no potential conflicts of interest.
- 6.Zhou Y, Lu M, Du RH, Qiao C, Jiang CY, Zhang KZ, Ding JH, Hu G. MicroRNA-7 targets Nod-like receptor protein 3 inflammasome to modulate neuroinflammation in the pathogenesis of Parkinson’s disease. Mol Neurodegener. 2016;11:28. doi: 10.1186/s13024-016-0094-3.CrossRefPubMedPubMedCentralGoogle Scholar
- 13.Marcellino D, Suarez-Boomgaard D, Sanchez-Reina MD, Aguirre JA, Yoshitake T, Yoshitake S, Hagman B, Kehr J, Agnati LF, Fuxe K, Rivera A. On the role of P2X(7) receptors in dopamine nerve cell degeneration in a rat model of Parkinson’s disease: studies with the P2X(7) receptor antagonist A-438079. J Neural Transm. 2010;117(6):681–7. doi: 10.1007/s00702-010-0400-0.CrossRefPubMedGoogle Scholar
- 19.Nuber S, Tadros D, Fields J, Overk CR, Ettle B, Kosberg K, Mante M, Rockenstein E, Trejo M, Masliah E. Environmental neurotoxic challenge of conditional alpha-synuclein transgenic mice predicts a dopaminergic olfactory-striatal interplay in early PD. Acta Neuropathol. 2014;127(4):477–94. doi: 10.1007/s00401-014-1255-5.CrossRefPubMedPubMedCentralGoogle Scholar
- 22.Reksidler AB, Lima MM, Dombrowski P, Andersen ML, Zanata SM, Andreatini R, Tufik S, Vital MA. Repeated intranigral MPTP administration: a new protocol of prolonged locomotor impairment mimicking Parkinson’s disease. J Neurosci Methods. 2008;167(2):268–77. doi: 10.1016/j.jneumeth.2007.08.024.CrossRefPubMedGoogle Scholar
- 26.Jiang L, Lin C, Song L, Wu J, Chen B, Ying Z, Fang L, Yan X, He M, Li J, Li M. MicroRNA-30e* promotes human glioma cell invasiveness in an orthotopic xenotransplantation model by disrupting the NF-kappaB/IkappaBalpha negative feedback loop. J Clin Investig. 2012;122(1):33–47. doi: 10.1172/JCI58849.CrossRefPubMedGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.