Cocaine-Mediated Downregulation of miR-124 Activates Microglia by Targeting KLF4 and TLR4 Signaling

Abstract

Cocaine is known to activate microglia both in vitro and in vivo. High expression of microglial Toll-like receptors (TLRs) and their downstream signal transducers play critical roles in determining microglial activation status. Emerging reports have also demonstrated that cocaine can enhance the strength of TLR signaling. Detailed molecular mechanisms underlying this phenomenon, however, remain elusive. In this study, we investigated the role(s) of miR-124 in regulating microglial TLR4 signaling in the context of cocaine. Herein, we found a dose- and time-dependent upregulation of KLF4 in cocaine-exposed BV-2 cells and rat primary microglial cells (rPMs). KLF4 also identified as a novel 3'-UTR target directly regulated by miR-124. In parallel, miR-124 regulated multiple TLR4 signaling molecules including TLR4, MyD88, TRAF6, and IRAK1. Repeated doses of cocaine (20 mg/kg; i.p.) administration in mice for 7 days further validated the in vitro key findings. Also, miR-124 overexpression significantly blocked the cocaine-mediated upregulation of pro-inflammatory cytokines. In contrast, miR-124 overexpression notably increased the expression of anti-inflammatory mediators in cocaine-exposed microglial cells. Intriguingly, stereotactic administration of lentivirus-miR-124 in the striatum significantly inhibited cocaine-mediated microglial activation and locomotor hyperactivity in vivo. In summary, these findings implicate the role of miR-124 in regulating TLR4 signaling, thereby indicating a new pathway responsible for cocaine-mediated microglial activation.

Keywords: Cocaine; Microglial cells; MyD88; Neuroinflammation; TLR4; TRAF6; miR-124.

Figure 1

Cocaine increased KLF4 levels in vitro and in vivo. Representative western blots showing the cocaine-mediated dose-dependent upregulation of KLF4 levels in both the BV-2 cells (A) and the rPMs (B). Representative western blots showing the cocaine-mediated time-dependent upregulation of KLF4 levels in both the BV-2 cells (C) and the rPMs (D). (E) Representative western blots showing the increased KLF4 expression levels in the striatal brain region of mice chronically administered with cocaine (20 mg/kg, i.p.) for 7 days. β-actin was used as a protein loading control for all the experiments. The data are presented as mean ± SEM from 6 independent experiments. One-way ANOVA followed by Bonferroni post hoc test was used to determine the statistical significance. *, P < 0.05 vs control.

Figure 2

KLF4 is a novel target regulated by miR-124. (A) Putative miR-124 binding sites in KLF4 gene. The potential complementary residues are shown in red color. (B) Representative western blots showing the overexpression of miR-124 significantly blocked the upregulation of KLF4 in BV-2 cells transfected with miR-124 mimics/control followed with cocaine (10 μM) exposure for 24 h. (C) Representative western blots showing the overexpression of miR-124 significantly blocked the upregulation of KLF4 in rPMs transfected with miR-124 mimics/control followed with cocaine (10 μM) exposure for 24 h. (D) Representative western blots showing the miR-124 inhibitor significantly increased the KLF4 in BV-2 cells transfected with miR-124 inhibitor/control followed with cocaine (10 μM) exposure for 24 h. (E) Representative western blots showing the miR-124 inhibitor significantly increased the KLF4 in rPMs transfected with miR-124 inhibitor/control followed with cocaine (10 μM) exposure for 24 h. (F) Schematic diagram of KLF4-3′-UTR-1 and KLF4-3′-UTR-2 plasm ids. KLF4-3′-UTR-1 plasmid has no miR-124 binding sites while KLF4-3′-UTR-2 plasmid has three miR-124 binding sites. (G) Relative luciferase activity of 3′-UTR-1 and 3′-UTR-2 constructs of KLF4 co-transfected with miR-control, miR-124 mimics, and miR-124 inhibitor in HEK293 cells. Overexpression of miR-124 mimics/inhibitor had no effect on the luciferase activity of KLF4-3′-UTR-1 plasmids while miR-124 mimics significantly inhibited KLF4-3′-UTR-2 luciferase activity. (H) Ago2 IP confirmed the enrichment of the miR-124 target mRNA, KLF4 in Ago2 IP as compared with total RNA isolated from BV-2 cells transfected with miR-124 mimics/control. β-actin was used as a loading control for all the experiments. The data are presented as mean ± SEM from 6 independent experiments. One-way ANOVA followed by Bonferroni post hoc test was used to determine the statistical significance for multiple groups, and a Student's t test was used to determine the statistical significance for between 2 groups. *, P < 0.05 vs. control; #, P < 0.05 vs. cocaine.

Figure 3

Overexpression of miR-124 inhibited the cocaine-mediated TLR4 upregulation. (A) Representative western blots showing the cocaine-mediated dose-dependent upregulation of TLR4 levels in BV-2 cells. (B) Representative western blots showing the cocaine-mediated time-dependent upregulation of TLR4 levels in BV-2 cells (C) Representative western blots showing the increased TLR4 expression levels in the striatal brain region of mice chronically administered with cocaine (20 mg/kg, i.p.) for 7 days. (D) Putative miR-124 binding sites in TLR4 gene. The potential complementary residues are shown in red color. (E) Representative western blots showing the overexpression of miR-124 significantly blocked the upregulation of TLR4 in BV-2 cells transfected with miR-124 mimics/control followed with cocaine (10 μM) exposure for 24 h. (F) Representative western blots showing the miR-124 inhibitors significantly increased the TLR4 in rPMs transfected with miR-124 inhibitor/control for 24 h. (G) Representative western blots showing the overexpression of miR-124 significantly blocked the upregulation of TLR4 in rPMs transfected with miR-124 mimics while inhibition of miR-124 increased the TLR4 upregulation in rPMs transfected with the miR-124 inhibitor for 24 h. (H) Ago2 IP confirmed the enrichment of the miR-124 target mRNA, TLR4 in Ago2 IP as compared with total RNA isolated from BV-2 cells transfected with miR-124 mimics/control. β-actin was used as a loading control for all the experiments. The data are presented as mean ± SEM from 6 independent experiments. One-way ANOVA followed by Bonferroni post hoc test was used to determine the statistical significance for multiple groups and a Student's t test was used to determine the statistical significance for between 2 groups. *, P < 0.05 vs. control; #, P < 0.05 vs. cocaine.

Figure 4

Overexpression of miR-124 blocked the cocaine-mediated upregulation of MyD88, IRAK1, and TRAF6. (A) Putative miR-124 binding sites in MyD88, IRAK1, and TRAF6 genes. The potential complementary residues are shown in red color. Representative western blots showing the overexpression of miR-124 significantly blocked the upregulation of MyD88 (B), IRAK1 (D), and TRAF6 (F) in BV-2 cells transfected with miR-124 mimics/control followed with cocaine (10 μM) exposure for 24 h. Representative western blots showing the inhibition of miR-124 significantly increased the upregulation of MyD88 (C), IRAK1 (E), and TRAF6 (G) in BV-2 cells transfected with miR-124 inhibitor/control followed with cocaine (10 μM) exposure for 24 h. (H, I) Representative western blots showing the overexpression of miR-124 significantly blocked the upregulation of MyD88, IRAK1, and TRAF6 in rPMs transfected with miR-124 mimics while inhibition of miR-124 increased the upregulation of MyD88, IRAK1, and TRAF6 in rPMs transfected with the miR-124 inhibitor for 24 h. (J) Ago2 IP confirmed the enrichment of the miR-124 target mRNAs, MyD88, IRAK1, and TRAF6 in Ago2 IP as compared with total RNA isolated from BV-2 cells transfected with miR-124 mimics/control. β-actin was used as a loading control for all the experiments. The data are presented as mean ± SEM from 6 independent experiments. One-way ANOVA followed by Bonferroni post hoc test was used to determine the statistical significance for multiple groups, and a Student's t test was used to determine the statistical significance for between 2 groups. *, P < 0.05 vs. control; #, P < 0.05 vs. cocaine.

Figure 5

Overexpression of miR-124 inhibited cocaine-mediated microglial activation in vitro. qPCR analysis showing the overexpression of miR-124 significantly inhibited the increased mRNA expressions of various proinflammatory mediators such as TNFα (A), CCL2 (B), and NOS2 (C), in BV-2 cells transfected with miR-124 mimics/control followed with cocaine (10 μM) exposure for 24 h. In contrast, qPCR analysis showing the overexpression of miR-124 significantly increased mRNA expressions of various anti-inflammatory mediators such as TGFβ (D), IL4 (E), and IL10 (F), in BV-2 cells transfected with miR-124 mimics/control followed with cocaine (10 μM) exposure for 24 h. (G and H) qPCR analysis showing the overexpression of miR-124 significantly inhibited the mRNA expressions of c/EBP and ARG1 in BV-2 cells transfected with miR-124 mimics/control followed with cocaine (10 μM) exposure for 24 h. β-actin was used as an internal housekeeping gene for all the experiments. The data are presented as mean ± SEM from 6 independent experiments. One-way ANOVA followed by Bonferroni post hoc test was used to determine the statistical significance for multiple groups, and a Student's t test was used to determine the statistical significance for between 2 groups. *, P < 0.05 vs. control; #, P < 0.05 vs. cocaine.

Figure 6

Overexpression of miR-124 inhibited cocaine-mediated microglial activation in vivo. (A) Schematic showing the stereotactic injection of lentivirus (LV)-miR-124 followed by chronic cocaine administration (20 mg/kg; i.p. for 7 days) in mice. (B) Representative fluorescent-microscopic image showing the overexpression of miR-124 colocalized with microglia in the striatum region of mice stereotactically injected with LV-miR-124. (C) qPCR analysis showing the expression levels of miR-124 in the striatal region of mice stereotactically injected with LV-miR-124 followed by cocaine administration. (D-G) Representative western blots and quantifications showing the expression levels of KLF4, TLR4, and Iba-1 in the striatal region of mice stereotactically injected with LV-miR-124 followed by cocaine administration. β-actin was used as a protein loading control for all the experiments. The data are presented as mean ± SEM from 8 independent experiments. Two-way ANOVA followed by Tukey post hoc test was used to determine the statistical significance for multiple groups, and a Student's t test was used to determine the statistical significance for between 2 groups. *, P < 0.05 vs. saline; #, P < 0.05 vs. cocaine.

Figure 7

Schematic diagram representing the miR-124-mediated regulation of TLR4 signaling in the context of cocaine.