MicroRNA-218-5p and Ddx41 reduce brain immune cell inflammation by lowering type I interferon response in a mouse model of Parkinson’s disease

Eight-week-old male C57BL/6J mice (weighing 19–23 g) were obtained from Gempharmatech (Nanjing, China). Mice were maintained under standard conditions (23 ± 2 °C) on a 12-h light/dark cycle, with ad libitum access to food and water in a specific pathogen-free class facility. All animal experiments were approved by the Tongji Hospital Animal Ethics Committee.

A mouse model of PD was generated using MPTP. Ten mice were randomly assigned to either the control or MPTP group. Mice in the MPTP group were injected intraperitoneally with MPTP·HCl (20 mg/kg free base; M0896, Sigma-Aldrich, St. Louis, MO, USA) in phosphate buffer (PBS) at 2-h intervals for a total of four doses in a single day, while the control group received a similar volume of PBS injected intraperitoneally.

We microinjected either miR-218-5p agomir (miR-218) or control agomir (NC) (Ribobio, Guangzhou, China) into the SN using a stereotaxic apparatus to overexpress miR-218-5p. MiRNA agomir is a chemically modified miRNA mimic that has higher stability and activity than conventional miRNA mimics and can effectively simulate the function of endogenous miRNAs. It can be delivered by either systemic or local injection and can exert a lasting effect for up to 6 weeks. Previous studies have demonstrated that miRNA agomirs can effectively regulate gene expression in various tissues such as the brain [19–21]. In this experiment, forty-eight mice were randomly assigned to four groups: NC Control, NC MPTP, miR-218 Control and miR-218 MPTP. Mice were anesthetized with isoflurane (1.5–3% in oxygen) via inhalation and placed in a stereotaxic frame. Then, 0.5 nmol of either miR-218-5p agomir or NC agomir in 2.5 μL of PBS was infused over 10 min into each side of SN at the following coordinates: anteroposterior, − 3.0 mm; mediolateral, ± 1.2 mm; dorsoventral, − 4.7 mm. After each infusion, the needle was left in place for 10 min before being slowly withdrawn. The mice were maintained in a warm environment until they recovered from anesthesia. Mice in the NC Control and miR-218 Control groups were injected with PBS, whereas mice in the NC MPTP and miR-218 MPTP groups were injected with MPTP as described above, 48 h after stereotaxic injection.

Motor function was assessed using the rotarod treadmill (IITC, Woodland Hills, CA, USA). Before the formal test, mice underwent three days of training on the rotarod treadmill at a constant speed of 5 rpm for 10 min each day. Thirteen days after the MPTP injection, mice were subjected to the formal test, in which the rod was programmed to accelerate uniformly from 5 to 40 rpm within 5 min. The test was repeated three times for each mouse, and the average latency to fall off the rod was recorded as a measure of motor function. Mice that did not fall off the rod after 5 min were recorded as 300 s.

Mice were sacrificed under deep anesthesia induced by isoflurane anesthesia. For immunofluorescence, mice were perfused transcardially with 30 mL of precooled PBS, followed by 20–30 mL of precooled 4% paraformaldehyde (PFA). After perfusion, intact brains were harvested, post-fixed in 4% PFA at 4 °C overnight, and then dehydrated in PBS containing 30% sucrose for 3 days. Brains were embedded in optimal cutting temperature material, followed by cutting into 20-µm thick coronal sections with a freezing slicer (Thermo Fisher Scientific, Waltham, MA, USA). For SN tissue, mice were perfused with 30 mL of precooled PBS, then brains were quickly taken out and continuously sliced at 1 mm thickness in the midbrain coronal plane using a pre-cooled mouse brain matrice (RWD Life Science, Shenzhen, China). SN tissue was segmented under a low magnification microscope according to a mouse brain atlas (The Mouse Brain in Stereotaxic Coordinates, third edition), snap-frozen in liquid nitrogen-cooled isopentane, and stored at − 80 °C.

The SN slides were fixed with 4% PFA for 10 min and washed three times with PBS for 5 min each time. The tissue area was outlined using a hydrophobic PAP pen. The slices were then blocked with a blocking solution (Beyotime, Shanghai, China) and incubated with primary antibodies overnight at 4 °C. Subsequently, the slices were incubated with secondary antibodies at room temperature in the dark for 1 h. After being washed with PBS, the slices were mounted with The Antifade Mounting Medium with DAPI (Beyotime) and covered with coverslips. The primary antibodies used for immunofluorescence were as follows: anti-tyrosine hydroxylase (TH; rabbit, 1:500; ab137869, Abcam, Cambridge, UK), anti-IBA1 (rabbit, 1:500; 019-19741, Fujifilm Wako Chemical, Osaka, Japan), anti-CD68 (rat, 1:500; MCA341B, Bio-Rad, Hercules, CA) and anti-IRF7 (mouse, 1:200; sc-74471, Santa Cruz Biotechnology, Dallas, TX, USA). The secondary antibodies were labeled with Alexa Fluor 488 or 594 (1:400, Yeasen Biotechnology, Shanghai, China). Confocal microscopy (FV1200; Olympus, Tokyo, Japan) was used to acquire images. To quantify TH⁺ neurons in the SNc, we employed a stereological approach, as previously described [22]. Slices from another 8 sections (140-μm interval) per mouse were collected, and 10 z-stacks (2 μm per stack) confocal images at 10 × magnification were acquired for neuron counting using Fiji 2.9.0 (ImageJ; National Institutes of Health, Bethesda, MD, USA). To quantify IBA1⁺ cells and volumes of CD68⁺IBA1⁺ puncta, images were obtained at 60 × magnification with 10 z-stacks (2-μm per stack). We randomly selected three fields and analyzed three sections per mouse. We performed 3D reconstruction of IBA1⁺ cells and CD68⁺ puncta using the Surface module in Imaris 9.0.3 (Oxford Instrument, Belfast, UK). The volume of IBA1⁺ cells and CD68⁺ puncta was calculated and averaged from 10 to 15 IBA1⁺ cells randomly selected from each mouse. To quantify the average fluorescent intensity of IRF7 + IBA1 + cells, images were captured at 60 × magnification with 6 z-stacks, and three fields were taken at random in each section, and three sections per mouse were analyzed. Microglia were selected by setting a fixed threshold in the IBA1 channel in Fiji, and then the average fluorescent intensity of IRF7 in microglia was calculated.

RNA sequencing (RNA-seq) was performed on SN tissues from mice in the NC control group, NC MPTP group, miR-218 control group, and miR-218 MPTP group (three each). RNA extraction, library construction, sequencing and bioinformatics analysis were performed by Suzhou PANOMIX Biomedical Tech Co. (Suzhou, China). The specific process involved the following steps: firstly, polyA-tailed mRNA was enriched from total RNA using oligo (dT) magnetic beads, and the RNA was then ion-fragmented to produce fragments of around 300 bp. Reverse transcription was performed using RNA as a template, 6-mer random primers and reverse transcriptase to synthesize first-strand cDNA, which was subsequently used to synthesize the second-strand cDNA. After library construction, PCR amplification was used to enrich the library fragments, which were then selected based on size to obtain a final library size of 450 bp. The quality of the library was assessed using the Agilent 2100 Bioanalyzer (Santa Clara, CA, USA), and the total and effective concentrations of the library were determined. The libraries with different index sequences were mixed in proportion to one another based on the effective concentration of the library and the required data amount. The mixed library was diluted to 2 nM and converted to single-stranded DNA using alkaline denaturation. Next-generation sequencing (NGS) was performed on the Illumina sequencing platform (San Diego, CA, USA) using paired-end (PE) sequencing. The libraries were sequenced to a depth of ~ 6 Gbps per sample. The resulting images were converted into raw data in FASTQ format using the sequencing platform's proprietary software. The sequencing data was filtered according to two criteria: (1) removal of 3′ adapter sequences using Cutadapt, and (2) removal of reads with an average quality score below Q20. The resulting high-quality sequences were mapped to the reference genome (Mus musculus.GRCm38.dna.primary_assembly.fa). HTSeq was used to count the number of reads mapped to each gene, which was used as the gene's raw expression level. The expression levels were then normalized using Fragments Per Kilo bases per Million fragments (FPKM).

We performed principal component analysis (PCA) using the DESeq package in R on each sample based on gene expression levels. The gene expression analysis was conducted using the DESeq package in R, and differentially expressed genes were screened with criteria of |log2FoldChange|> 0.585 and p-value < 0.05. Gene set enrichment analysis (GSEA) was conducted using a local GSEA tool (http://www.broadinstitute.org/gsea/index.jsp↗) and the Gene Ontology (GO) data set of mice. The normalized enrichment score (NES) and false discovery rate (FDR) q-value were calculated using permutation tests to quantify enrichment levels and statistical significance. Pathways with significant enrichment were defined as those meeting the following criteria: |NES|> 1, the nominal (NOM) p-value < 0.05, and FDR q-value < 0.25.

Murine microglia-like BV2 cells, SH-SY5Y cells and human embryonic kidney (HEK) 293 T cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS; Biological Industries, Beit-Haemek, Israel) in a carbon dioxide incubator at 37 °C. The cells were subcultured every three days. For 1-methyl-4-phenylpyridinium (MPP⁺; D048, Sigma-Aldrich) treatment, SH-SY5Y cells were treated with 1 mM MPP⁺ or PBS of equal volume for 24 h. The conditioned media from the SH-SY5Y cells treated with MPP⁺ (MPP⁺-CM) or PBS (PBS-CM) were collected and applied to BV2 cells for 24 h.

BV2 cells were digested and plated into 6-well plates in a high-glucose DMEM medium containing 10% FBS one day before transfection. Transfection was performed when the cells reached ~ 80% confluence. BV2 cells were transfected with miR-218-5p mimic/NC mimic or Ddx41-siRNA/control siRNA (Ribobio) for 6–8 h with LipofectMax transfection reagent (ABP Biosciences, Beltsville, MD, USA), and then treated with MPP⁺-CM or PBS-CM for 24 h.

Samples from SN tissue and BV2 cells were lysed using RIPA lysis buffer (Beyotime) containing a protease inhibitor cocktail and phenylmethylsulfonyl fluoride. Protein concentrations were determined using a bicinchoninic acid kit (Beyotime). Protein content (20 μg) was loaded onto sodium dodecyl sulfate–polyacrylamide gel for gel electrophoresis and transferred to nitrocellulose membranes, which were subsequently blocked with 5% non-fat skim milk and incubated with the primary antibodies at 4 °C overnight. The primary antibody used for WB were as follows: anti-TH (rabbit, 1:1000; ab137869, Abcam), anti-DDX41 (rabbit, 1:1000; 27,500–1-AP, Proteintech, Rosemont, IL, USA), anti-IL-1β (rabbit, 1:1000; #12703, Cell Signaling Technology, Danvers, MA, USA), anti-GAPDH (mouse, 1:10,000; 60,004-1-Ig, Proteintech) and anti-β-actin (mouse, 1:10,000; 66,009-1-Ig, Proteintech). Secondary antibody reactions were performed using goat anti-mouse or goat anti-rabbit IgG-horseradish peroxidase antibodies (1:5000; Proteintech) for 1 h at room temperature. WB protein bands was visualized by enhanced chemiluminescence. Images were collected using the BLT GelView 6000 Pro imaging system (Guangzhou Biolight Biotechnology, Guangzhou, China). To quantify WB protein bands, equally sized region of interest was drawn around each band in Fiji to measure raw integrated density. Each integrated density was normalized to each sample’s control (GAPDH or β-actin).

Total RNA was extracted from the SN tissue of mice and cells using RNAiso Plus reagent (Takara, Kusatsu, Japan). RNA reverse transcription and qPCR amplification were performed using PrimeScript RT Reagent Kits (RR036A and RR037A; Takara) and ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China), respectively. The primers for miR-218-5p and U6 were obtained from the Bulge-Loop miRNA qPCR Primer Set (Ribobio). The primers for mRNA provided by Tsingke Biotechnology (Beijing, China) are listed in Additional file 1: Table S1. qPCR amplifications were performed using CFX Connect Detection System (Bio-Rad) as follows: 3 min at 95 °C, 10 s at 95 °C for 40 cycles, 30 s at 55 °C. qPCR was performed using the 2 ^−ΔΔCt method with Actb (for mRNA) or U6 (for miR-218-5p) as controls.

HEK 293 T cells were seeded one day before transfection. miR-218-5p mimic or NC mimic were co-transfected with pmirGLO vectors, including wild-type (WT) or mutated (MUT) 3′UTR of Ddx41. Luciferase activity was measured 48 h after transfection, according to the manufacturer’s protocol (GeneCopoeia, Rockville, MD).

GraphPad Prism (v8.0.2; GraphPad Software, San Diego, CA) was applied for the statistical analysis. Two-group comparisons were performed using a two-tailed unpaired Student’s t-test. For multi-group comparisons, two-way analysis of variance (ANOVA) and Tukey’s multiple comparison test were used. Statistical significance was established at p < 0.05.

To evaluate the effect of miR-218-5p on the loss of DA neurons in MPTP-induced mice, we first examined miR-218-5p levels in the SN using qPCR. We found that miR-218-5p expression was downregulated 14 days post MPTP administration (Fig. 1a). Next, mice were stereotaxically infused with miR-218-5p agomir (or NC agomir) into the SN and then injected intraperitoneally with MPTP 3 days after stereotaxic injection (Fig. 1b). miR-218-5p agomir treatment significantly increased miR-218-5p expression after 17 days (Fig. 1c). The number of TH⁺ DA neurons and TH expression were reduced in MPTP- and NC agomir-treated mice, as determined by immunofluorescence and WB analyses. However, miR-218-5p agomir treatment alleviated the reduction in TH⁺ neurons and TH expression (Fig. 1d–g). Moreover, miR-218-5p overexpression significantly attenuated the MPTP-induced behavioral deficits in the rotarod test 13 days post MPTP injection (Fig. 1h). These data indicate that miR-218-5p overexpression alleviates the loss of DA neurons and motor deficits in MPTP-induced mice.

To explore whether miR-218-5p overexpression affects microglial phenotypes in MPTP-induced mice, double immunofluorescence staining with anti-IBA1 (a marker for microglia) and anti-CD68 (a marker for phagocytosis) was performed to identify microglial inflammation in the SN (Fig. 2a). The number and volume of IBA1⁺ microglia, as well as CD68 puncta in IBA1⁺ microglia were significantly increased in the SN of MPTP-induced mice, whereas these effects were attenuated in mice with miR-218-5p overexpression (Fig. 2a, b). Additionally, MPTP administration increased the level of pro-inflammatory cytokine IL-1β, whereas overexpression of miR-218-5p inhibited it (Fig. 5b, c). This suggests that miR-218-5p overexpression inhibits MPTP-induced microglia-mediated neuroinflammation.

To determine the mechanism by which miR-218-5p affects microglial inflammation, we performed RNA-seq on the SN tissue of mice. Volcano plots of DEG analysis showed that 59 genes were upregulated and 33 genes were downregulated in the SN of MPTP-induced mice (Fig. 3a). However, miR-218-5p overexpression upregulated 119 genes and downregulated 51 genes in MPTP-induced mice (Fig. 3b). Notably, the expression of several IFN-I-related genes (Irf7, Ddx60, Nlrc5) was upregulated in MPTP-treated mice (Fig. 3a, c), while miR-218-5p overexpression downregulated the expression of these genes (Irf7, Ddx60, Nlrc5) (Fig. 3b, c). We verified the expression of Irf7, Ddx60, and Nlrc5 by qPCR (Fig. 3d). Moreover, miR-218-5p overexpression led to decreased expression of several genes associated with IFN-I and inflammatory responses in the baseline (Additional file 1: Fig. S1). The top differential GO terms with p-value in GSEA are shown in Fig. 4a, b. We found that MPTP treatment upregulated several IFN-I response-related terms, including “cellular response to interferon-beta”, “response to interferon-beta, and “regulation of type I interferon-mediated signaling pathway” (Fig. 4a, c; Additional file 1: Fig. S2). Yet, miR-218 overexpression downregulated IFN-I terms including “cellular response to interferon-beta” and “response to interferon-beta” (Fig. 4b, c; Additional file 1: Fig. S2). Our data suggest that the IFN-I response is upregulated in the MPTP mouse model of PD, which may be due to the downregulation of miR-218-5p.

To investigate if miR-218-5p inhibits IFN-I response in microglia, we performed double immunofluorescence staining with anti-IBA1 and anti-IRF7 antibodies in the SN (Fig. 2c). The IRF7 protein was predominantly distributed in IBA1⁺ microglia (Fig. 2c), and the fluorescent intensity of IRF7 in microglia was elevated in MPTP-induced mice, while miR-218-5p overexpression suppressed its expression (Fig. 2d). This suggests that miR-218-5p regulates the IFN-I response of microglia and may modulate neuroinflammation through this pathway.

We next investigated the possible mechanisms of miR-218-5p regulating IFN-I response. TargetScan 7.2 was used to predict target genes of miR-218-5p. We found that the 3′UTR of the IFN-I-related gene Ddx41 (encoding DEAD-box helicase 41 [DDX41]) is complementary to miR-218-5p (Fig. 5a). DDX41 is a DNA sensor that recognizes viral DNA [23] and cytosolic DNA [24] in the cytoplasm, and subsequently induces IFN-I responses by interacting with STING [25, 26]. DDX41 protein levels were increased in the SN of MPTP-injected mice, as analyzed by WB analysis, while DDX41 expression was reduced following miR-218-5p overexpression (Fig. 5b, c). Dual luciferase reporter assays confirmed that miR-218-5p targets Ddx41 3′UTR (Fig. 5d). These results suggest that miR-218-5p targets Ddx41.

To elucidate whether miR-218-5p regulates the microglial IFN-I response and affects microglia-mediated inflammation, we established SH-SY5Y-BV2 conditioned culture systems (Fig. 6a), due to the fact that previous studies have suggested that the inflammation of microglia caused by MPTP is secondary to the MPP⁺ induced neurotoxicity [27]. We treated BV2 cells with MPP⁺ conditioned media (MPP⁺-CM) from SH-SY5Y cells, and found that miR-218-5p expression was downregulated in BV2 cells treated with MPP⁺-CM (Fig. 6b). Furthermore, the expression of IFN-I response related genes Ifnb1 and Irf7 (Fig. 6d, e) and the protein level of DDX41 (Fig. 6i, j) were upregulated in BV2 cells after MPP⁺-CM stimulation. In contrast, miR-218-5p overexpression (Fig. 6b) downregulated Ifnb1 and Irf7 expression (Fig. 6d, e) and DDX41 level (Fig. 6i, j) induced by MPP⁺-CM. Consistent with this, MPP⁺-CM stimulation upregulated the expression of pro-inflammatory cytokines Il1b, Il6 and Tnf, whereas miR-218-5p overexpression attenuated their expression (Fig. 6f–h).

Next, we investigated whether miR-218-5p regulates microglial IFN-I response and inflammation by inhibiting the expression of Ddx41. BV2 cells were transfected with Ddx41 siRNA (si-Ddx41) to knock down Ddx41. The expression of Ifnb1 and Irf7 and the level of DDX41 were increased in BV2 cells after MPP⁺-CM stimulation, while Ddx41 knockdown suppressed their expression (Fig. 7a, b, f, g). In addition, the expression of Il6, Il1b and Tnf was elevated in BV2 cells induced by MPP⁺-CM, while Ddx41 knockdown prevented the elevation of these pro-inflammatory cytokines (Fig. 7c–g).

All research involving animals was approved by Tongji Hospital Animal Ethics Committee, China.

Not applicable.

The authors declare that they have no competing interests.

The data that support the findings of this study are available from the corresponding author on reasonable request.