Cocaine-mediated promoter DNA methylation of microRNA-124 promotes microglial activation through TLR4 signaling

Neuroinflammation plays a critical role in the development of reward-related behavior in cocaine self-administration rodents. Cocaine, one of most commonly abused drugs, has been shown to activate microglia both in vitro and in vivo. Detailed molecular mechanisms underlying cocaine-mediated microglial activation remain poorly understood. MicroRNAs (miRs), belonging to a class of small non-coding RNA superfamily have been shown to modulate the activation status of microglia. MiR-124, one of the microglia-enriched miRs functions as an anti-inflammatory regulator that maintains microglia in a quiescent state. To date, the possible effects of cocaine on microglial miR-124 levels and the associated underlying mechanisms have not been explored before. In the current study, we demonstrated that cocaine exposure decreased miR-124 levels in microglia in vitro, and is critical for activation of TLR4 signaling. These findings were further validated in vivo; wherein we demonstrated decreased the abundance of miR-124 in the purified microglia isolated from cocaine-administered mice brains compared with cells from saline administered animals. Molecular mechanisms underlying these effects involved cocainemediated increased mRNA and protein expression of DNMTs in microglia. Consistently, cocaine substantially increased the promoter DNA methylation levels of miR-124 precursors (pri-miR-124-1, and -2) but not that of pri-miR-124-3 both in vitro and in vivo. Krüpple-like factor 4 (KLF4), was identified as a novel substrate of miR-124. Besides KLF4, miR-124 also modulated the levels of several other proteins including TLR4, MyD88, TRAF6, and IRAK1. Reciprocally, overexpression of miR-124 in microglia blocked cocaine-mediated activation both in vitro and in vivo. In summary, our findings demonstrate a yet unexplored mechanism for cocaine-mediated activation of microglia via down-regulation of miR-124 (through promoter DNA hypermethylation), leading ultimately to increased TLR4 activation. Our results thus implicate that epigenetic modulation of miR-124 could be considered as a potential therapeutic approach to ameliorate microglial activation and possibly, the development of cocaine addiction. Support: DA043138, DA033150, DA035203, DA040397, DA041751, MH106425, DA043164, DA036157