Mib2 Deficiency Inhibits Microglial Activation and Alleviates Ischemia-Induced Brain Injury

Abstract

Neuroinflammation plays a critical role in ischemia-induced brain injury. Mib2, an E3 ubiquitin ligase, has been reported to regulate Notch signaling and participate in the peripheral immune system. However, the roles of Mib2 in the nervous system are not well characterized. In this study, we show that Mib2 is involved in lipopolysaccharide (LPS)- and oxygen-glucose deprivation (OGD)-induced microglial activation. Mechanistically, Mib2 interacts with the IKK complex and regulates the activation of NF-κB signaling, thus modulating Notch1 transcription in the microglia. Furthermore, we generated a microglia-specific Mib2 knockout mice and found that microglia-specific deletion of Mib2 significantly alleviates ischemia-induced neuroinflammation and brain injury. Taken together, our results reveal a critical role of Mib2 in microglial activation and ischemia-induced brain injury, thus providing a potential target for the treatment of stroke.

Keywords: Mib2; brain injury; ischemia; microglia; neuroinflammation.

Figure 1.

Mib2 knockdown inhibits LPS-induced inflammation. (A), The knockdown efficiency was determined by RT-qPCR analysis after transfected with Mib2 or control siRNA in BV2 cells for 72h. (B-D), The expression levels of IL-6, iNOS and TNFα in control and Mib2 knockdown-BV2 cells were analyzed upon LPS (1 µg/ml) stimulation for indicated times. (E), Western blot analysis of Mib2 and iNOS levels in control and Mib2 knockdown-BV2 cells upon LPS (1 µg/ml) stimulation for indicated times. Data indicate means ± SEM. Data were analyzed using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2.

Mib2 regulates Notch1 signaling in microglia. (A) Western blot analysis of NICD levels (left) and relative band density quantification (right) in control and Mib2 knockdown-BV2 cells upon LPS (1 µg/ml) stimulation for indicated times. (B) Western blot analysis of NICD levels (left) and relative band density quantification (right) in control and Mib2 knockdown-BV2 cells upon OGD treatment (ischemia for 3h and reperfusion for indicated times), I: ischemia, R: reperfusion. (C) Western blot analysis of Notch1 (fl, full length) and NICD levels in control and Mib2 knockdown-BV2 cells upon LPS (1 µg/ml) stimulation for indicated times. (D) RT-qPCR analysis of Notch1 expression levels in control and Mib2 knockdown-BV2 cells upon LPS (1 µg/ml) stimulation for indicated times. Data indicate means ± SEM. Data were analyzed using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.

Notch1 signaling regulates microglial activation. (A) The knockdown efficiency was determined by RT-qPCR analysis after transfected with Notch1 or control siRNA in BV2 cells for 72h. (B-D) The expression levels of IL-6, iNOS and TNFα in control and Notch1 knockdown-BV2 cells were analyzed upon LPS (1 µg/ml) stimulation for indicated times. (E) Western blot analysis of indicated proteins from control and Notch1 knockdown-BV2 cells upon LPS (1 µg/ml) stimulation for indicated times. (F-H) The expression levels of IL-6, iNOS and TNFα in control and Notch1 knockdown-BV2 cells were analyzed upon OGD treatment (ischemia for 3h and reperfusion for indicated times), I: ischemia, R: reperfusion. (I) Western blot analysis of indicated proteins from control and Notch1 knockdown-BV2 cells upon OGD treatment (ischemia for 3h and reperfusion for indicated times), I: ischemia, R: reperfusion. Data indicate means ± SEM. Data were analyzed using Student’s t test and one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.

Mib2 regulates NF-κB signaling by targeting IKK complex. (A) Western blot analysis of iNOS and the phosphorylated and total IKKα, IKKβ, Erk, P38 levels upon LPS stimulation for indicated times in control and Mib2 knockdown BV2 cells. (B) 293T cells were transfected with Myc-tagged Mib2 and Flag-tagged IKKα. Cell lysates were immunoprecipitation with Flag antibody and co-immunoprecipitation of Myc-tagged Mib2 was detected by Western blot analysis. (C) 293T cells were transfected with Myc-tagged Mib2 and HA-tagged IKKβ. Cell lysates were immunoprecipitation with HA antibody and co-immunoprecipitation of Myc-tagged Mib2 was detected by Western blot. (D) 293T cells were transfected with Myc-tagged Mib2 and Flag-tagged IKKγ. Cell lysates were immunoprecipitation with Flag antibody and co-immunoprecipitation of Myc-tagged Mib2 was detected by Western blot analysis. (E-F) 293T cells were transfected with Flag-tagged Mib2 WT or ΔR (Ring domain deletion) or vector plasmid together with HA-tagged ubiquitin, Myc-tagged IKKα or IKKγ. Cell lysates were immunoprecipitated with Myc antibody and immunoblotted with HA antibody. (G) 293T cells were co-transfected with NF-κB luciferase reporter plasmids and Flag-Mib2 WT or ΔR (Ring domain deletion) or vector plasmid, 16 h after transfection, cells were lysed, and the fluorescence values were measured. (H-I) 293T cells were co-transfected with Notch1 or Hes1 luciferase reporter plasmids and Myc-Mib2, P65 or vector plasmid, 16 h after transfection, cells were lysed, and the fluorescence values were measured. Data indicate means ± SEM. Data were analyzed using Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5.

Microglial Mib2 knockout alleviates ischemia induced brain injury. (A) Gene strategy for Mib2^f/f mice and Mib2 conditional knockout (Mib2 cKO) mice: The loxP elements were inserted upstream and downstream of exon 5 by using the CRISPR/Cas 9 method. The recombination between the loxP sites was occurred and the target sequence was deleted when Tamoxifen-inducible Cre-recombinase was expressed. (B) Identification of mouse genotypes by agarose gel electrophoresis. The upper panel is for the identification of loxP site, the lower panel s for the identification of wild-type allele. WT for wild-type; f/+ for Mib2 heterozygous mice; f/f for Mib2 homozygous mice. (C) Schematic of the experimental design: Mib2^f/f mice were crossed with Cx3Cr1-creER mice to obtain Mib2^f/f and Mib2^f/f Cx3Cr1-creER littermates. These littermates were then given tamoxifen (200 μg/g by intragastric administration) for three consecutive days at one-month-old to induce Mib2^f/f contrast and Mib2 conditional knockout (Mib2 cKO) mice. tMCAo was performed after 30-days-tamoxifen administration, mice were then sacrificed after 24 or 72 h reperfusion. (D) Microglia was isolated by flow cytometry from normal 2-month-old mice brain in Mib2^f/f and Mib2 cKO group (CD11b⁺CD45^low cells), (n = 3 mice per group). (E) The Mib2 knockdown efficiency was determined by RT-qPCR analysis. (F) Relative images of TTC stained brain slices were shown at different time points. (G) Brain infarction volume was quantified at 24 h after tMCAo (n=5 and 7). (H), Brain infarction volume was quantified at 72 h after tMCAo (n=10 and 13). Data indicate means ± SEM. Data were analyzed using Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6.

Microglial Mib2 knockout reduces microglial activation and inflammation. (A) The contralateral and ipsilateral of brain slices from mice underwent tMCAo and the location of ischemic penumbra. (B) Immunofluorescence staining representation of microglia in ischemic penumbra and the corresponding position on contralateral of mice underwent tMCAo after 72h reperfusion. Scale bars, 20 μm. (C) Quantification of microglial numbers in ischemic penumbra and the corresponding position on contralateral. (D) Quantification of microglial soma area in ischemic penumbra and the corresponding position on contralateral. (E-H), RT-qPCR analysis of Notch1, Hes1, IL-6 and iNOS expression levels in ischemic penumbra regions of brain tissues. (I) Schematic model of Mib2-regulated-inflammation in microglia. Data indicate means ± SEM. Data were analyzed using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.