The miR206-JunD Circuit Mediates the Neurotoxic Effect of Methylmercury in Cortical Neurons

Methylmercury (MeHg) causes neuronal death through different pathways. Particularly, we found that in cortical neurons it increased the expression of Repressor Element-1 Silencing Transcription Factor (REST), histone deacetylase (HDAC) 4, Specificity Protein (Sp) 1, Sp4, and reduced the levels of brain-derived neurotrophic factor (BDNF). Herein, in rat cortical neurons we investigated whether microRNA (miR) 206 can modulate MeHg-induced cell death by regulating REST/HDAC4/Sp1/Sp4/BDNF axis. MeHg (1 mu M) reduced miR206 expression after both 12 and 24 h and miR206 transfection prevented MeHg-induced neuronal death. Furthermore, miR206 reverted MeHg-induced REST and Sp4 increase and BDNF reduction at gene and protein level, and reverted HDAC4 protein increase, but not HDAC4 mRNA upregulation. Moreover, since no miR206 seed sequences were identified in the 3'-untranslated regions (3'-UTRs) of REST and SP4, we investigated the role of JunD, that presents a consensus motif on REST, Sp4, and BDNF promoters. Indeed, MeHg increased JunD mRNA and protein levels, and JunD knockdown counteracted MeHg-induced REST, Sp4 increase, but not BDNF reduction. Furthermore, we identified a miR206 binding site in the 3'-UTR of JunD mRNA (miR206/JunD) and mutagenesis of miR206/JunD site reverted JunD luciferase activity reduction induced by miR206. Finally, miR206 prevented MeHg-increased JunD binding to REST and Sp4 promoters, and MeHg-reduced BDNF expression was determined by the increase of HDAC4 binding on BDNF promoter IV. Collectively, these results suggest that miR206 downregulation induced by MeHg exposure determines an upregulation of HDAC4, that in turn downregulated BDNF, and the activation of JunD that, by binding REST and Sp4 gene promoters, increased their expression.