P38/Sp1/Sp4/HDAC4/BDNF Axis Is a Novel Molecular Pathway of the Neurotoxic Effect of the Methylmercury

Natascia Guida,Giusy Laudati,Gianfranco Di Renzo,Valeria Valsecchi,Carmine Selleri,Rossana Sirabella,Luigi Formisano,Lorella M T Canzoniero,Luigi Mascolo

doi:10.3389/fnins.2017.00008

Abstract

The molecular pathways involved in methylmercury (MeHg)-induced neurotoxicity are not fully understood. Since pan-Histone deacetylases (HDACs) inhibition has been found to revert the neurodetrimental effect of MeHg, it appeared of interest to investigate whether the pattern of HDACs isoform protein expression is modified during MeHg-induced neurotoxicity and the transcriptional/transductional mechanisms involved. SH-SY5Y neuroblastoma cells treated with MeHg 1 μM for 12 and 24 h showed a significant increase of HDAC4 protein and gene expression, whereas the HDACs isoforms 1–3, 5, and 6 were unmodified. Furthermore, MeHg-induced HDAC4 increase was reverted when cells were transfected with siRNAs against specificity protein 1 (Sp1) and Sp4, that were both increased during MeHg exposure. Next we studied the role of extracellular-signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK), and p38 mitogen-activated protein kinases (MAPKs) in MeHg—induced increase of Sp1, Sp4, and HDAC4 expression. As shown by Western Blot analysis MeHg exposure increased the phosphorylation of p38, but not of ERK and JNK. Notably, when p38 was pharmacologically blocked, MeHg-induced Sp1, Sp4 protein expression, and HDAC4 protein and gene expression was reverted. In addition, MeHg exposure increased the binding of HDAC4 to the promoter IV of the Brain-derived neurotrophic factor (BDNF) gene, determining its mRNA reduction, that was significantly counteracted by HDAC4 knocking down. Furthermore, rat cortical neurons exposed to MeHg (1 μM/24 h) showed an increased phosphorylation of p38, in parallel with an up-regulation of Sp1, Sp4, and HDAC4 and a down-regulation of BDNF proteins. Importantly, transfection of siRNAs against p38, Sp1, Sp4, and HDAC4 or transfection of vector overexpressing BDNF significantly blocked MeHg-induced cell death in cortical neurons. All these results suggest that p38/Sp1-Sp4/HDAC4/BDNF may represent a new pathway involved in MeHg-induced neurotoxicity.

Highlights

Exposure to MeHg is detrimental, for the developing brain (Ceccatelli et al, 2013)
Since HDAC4 expression can be regulated by Sp family transcription factors (Liu et al, 2006), we evaluated the effect of MeHg exposure on the expression of specificity protein 1 (Sp1), Sp3, and Sp4 in SHSY5Y cells
The results of the present study showed that MeHg exposure increased the expression of the HDAC4 isoform in SH-SY5Y cells and that the stimulation of the P-p38/Sp1/Sp4 pathway was the mechanism by which HDAC4 mRNA and protein are up-regulated during MeHg exposure

Summary

Introduction

Exposure to MeHg is detrimental, for the developing brain (Ceccatelli et al, 2013). It is known that HDAC4 mRNA expression is regulated by the transcription factors specificity proteins 1 (Sp1) and 3 (Sp3) (Liu et al, 2006), that have been associated in vivo to neuronal cell death after stroke (Formisano et al, 2015b), and in vitro to cell death after PCB exposure, through the up-regulation of RE1-Silencing Transcription factor (REST) (Formisano et al, 2015c) These findings prompted us to investigate whether HDAC4 is the only isoform involved in MeHg-induced neuronal cell death and the molecular mechanisms responsible for its increase. At last in the present study we found that MeHg increases HDAC4 expression in SH-SY5Y nuroblastoma cells and in rat cortical neurons and its toxic effect is due to the triggering of the p38/Sp1-Sp4/HDAC4/BDNF pathway

Methods

Results

Conclusion