MC1568 Inhibits Thimerosal-Induced Apoptotic Cell Death by Preventing HDAC4 Up-Regulation in Neuronal Cells and in Rat Prefrontal Cortex

Abstract

Ethylmercury thiosalicylate (thimerosal) is an organic mercury-based compound commonly used as an antimicrobial preservative that has been found to be neurotoxic. In contrast, histone deacetylases (HDACs) inhibition has been found to be neuroprotective against several environmental contaminants, such as polychlorinated biphenyls, di-2-ethylhexyl phthalate, and methylmercury. The aim of this study was to investigate the effect of HDAC inhibition on thimerosal-induced neurotoxicity in neuroblastoma cells and cortical neurons. Interestingly, we found that thimerosal, at 0.5 μM in SH-SY5Y cells and at 1 μM in neurons, caused cell death by activation of apoptosis, which was prevented by the HDAC class IIA inhibitor MC1568 but not the class I inhibitor MS275. Furthermore, thimerosal specifically increased HDAC4 protein expression but not that of HDACs 5, 6, 7, and 9. Western blot analysis revealed that MC1568 prevented thimerosal-induced HDAC4 increase. In addition, both HDAC4 knocking-down and MC1568 inhibited thimerosal-induced cell death in SH-SY5Y cells and cortical neurons. Importantly, intramuscular injection of 12 μg/kg thimerosal on postnatal days 7, 9, 11, and 15 increased HDAC4 levels in the prefrontal cortex (PFC), which decreased histone H4 acetylation in infant male rats, in parallel increased motor activity changes. In addition, coadministration of 40 mg/kg MC1568 (intraperitoneal injection) moderated the HDAC4 increase which reduced histone H4 deacetylation and caspase-3 cleavage in the PFC. Finally, open-field testing showed that thimerosal-induced motor activity changes are reduced by MC1568. These findings indicate that HDAC4 regulates thimerosal-induced cell death in neurons and that treatment with MC1568 prevents thimerosal-induced activation of caspase-3 in the rat PFC.

p38/Sp1/Sp4/HDAC4/BDNF Axis Is a Novel Molecular Pathway of the Neurotoxic Effect of the Methylmercury

“…In fact, MeHg exposure increased HDAC4 gene and protein expression causing also in the nucleus an increase of its binding to the BDNF gene promoter IV sequence with consequent mRNA and protein reduction of this neuroprotective factor. These results are in accordance with recent in vivo studies demonstrating that intramuscular injection of the organomercury compound thimerosal induces neuronal cell death by increasing HDAC4 expression in rat prefrontal cortex (Guida et al, 2015b, 2016), and that in cortical neurons HDAC4 knockdown improves neuronal survival after oxygen glucose deprivation (OGD, Yuan et al, 2016). Furthermore, we found that of the three main branches of the MAPK cascade (ERK, p38, and JNK), only p38 was involved in regulating HDAC4 gene expression in cells exposed to MeHg.…”

“…In particular, siRNAs against Sp1 reduced Sp1 protein expression respectively of 80 and 68% in SH-SY5Y cells and rat cortical neurons, as already published (Formisano et al, 2015b). Whereas, siRNAs against HDAC4 reduced HDAC4 protein expression respectively of 75 and 71% in SH-SY5Y cells and rat cortical neurons, as already published (Guida et al, 2016). They were also transfected with siRNAs against human and rat Sp4 (siSp4; SI04333588) at 20 nM, both purchased from Quiagen.…”

“…To this aim, we knocked down p38, Sp1, Sp4 and HDAC4, by transfecting neurons with specific siRNAs named sip38, siSp1, siSp4, and siHDAC4 and increased BDNF with a plasmid overexpressing BDNF named pBDNF. Notably, the efficiency of sip38, siSp1, and siHDAC4 to reduce respectively p38, Sp1, and HDAC4 protein expression was previously published (Sirabella et al, 2012; Formisano et al, 2015b; Guida et al, 2015b, 2016). Instead, siSp4 significantly reduced Sp4 expression by 65%, whereas pBDNF increased BDNF expression by 54%, both compared to the respective control (Figures 5G,H).…”

“…For SB239063 experiments cells were pre-treated for 2 h with the drug at 10 μM followed with MeHg 1 μM for 24 h. All transfection experiments in SH-SY5Y and cortical neurons were performed 24 h before MeHg treatment (1 μM/24 h) with HiPerFect Transfection Reagent (Quiagen) in accordance with the manufacture's protocol, as previously reported (Vinciguerra et al, 2014). In SH-SY5Y cells and cortical neurons the synthetic small interfering RNA (siRNA) transfections for Sp1 (siSp1), HDAC4 (siHDAC4), and negative control (siCTL) were performed as already reported (Formisano et al, 2015b; Guida et al, 2016). In particular, siRNAs against Sp1 reduced Sp1 protein expression respectively of 80 and 68% in SH-SY5Y cells and rat cortical neurons, as already published (Formisano et al, 2015b).…”

“…On the other hand a pan HDAC inhibition has been found to be neuroprotective in vitro against the neurotoxicity of bis (2-ethylhexyl) phthalate (DEHP) (Guida et al, 2014), Polychlorinated Biphenyls (PCB) (Formisano et al, 2011, 2015a), and MeHg (Guida et al, 2015b). Recently, it has also been shown that the mercury-containing organic compound Thimerosal induces an increase of the HDAC4 isoform (Guida et al, 2015b, 2016) determining neuronal cell death, but the molecular mechanism of this effect is still unrevealed. On the other hand, 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 also 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).…”