| Lead(Pb)pollution affecs the quality and safety of food,and a serious threat to human health.Pb is toxic many organs of the body,of which the central nerous system is the most sensitive.In recent years,a series of progress has been made in the study of the mechanism of lead neurotoxicity at home and abroad.However,the role of epigenetic mechanism in lead neurotoxicity remains unclear.Objective: To explore the mechanism of histone deacetylase 4(HDAC4)in Pbinduced neurotoxicity and provide a powerful therapeutic factor for lead poisoning.Methods and results: 1.Investigate the role of HDAC4 in Pb-induced neurotoxicity.PC12 cells were treated with 10 ?M lead for 24 hours.The expression levels of HDAC4 mRNA and protein were detected by q-PCR and western blot.In vivo,the SpragueDawley(SD)rats(n=6 per group)acquired lead acetate(250 ppm)indirectly from their mothers before weaning,and then directly from drinking water containing lead acetate.The hippocampus was collected after rats were sacrificed at postnatal day 21(PND 21),and detect the expression level of HDAC4.The results shown that HDAC4 was significantly increased in both PC12 cells and rat hippocampal neurons upon Pb exposure.Pharmacological intervention of HDAC4 inhibitor(LMK-235)and shHDAC4(HDAC4-knocking down plasmid)was used to investigate the role of HDAC4 in Pb-induced neurotoxicity by observing the neurite outgrowth of PC12 cells.Blockade of HDAC4 with either LMK-235 or shHDAC4 ameliorated the Pb-induced neurite outgrowth deficits.2.To explore the role of HDAC4 nuclear accumulation in Pb neurotoxicity and its upstream factors.Immunocytochemistry was used to observe the effect of lead on the subcellular distribution of HDAC4.Nuclear and cytoplasmic proteins were extracted to detect the expression level of HDAC4.Interestingly,HDAC4 was aberrantly accumulated in the nucleus upon Pb exposure.HDAC4 plasmid(? NLS2-HDAC4)with deletion of nuclear localization signal was constructed to study the adverse effects of nuclear HDAC4 on lead neurotoxicity,and to detect the effect of Pb expose on PP1,PP2 A and CaMK.We found thatblocking the shuffling from cytosol to nucleus with ?NLS2-HDAC4(the cytosol-localized HDAC4 mutant)was able to rescue the neuronal impairment.In addition,PP1 was identified as the key enzyme responsible for HDAC4 nuclear retention elicited by Pb exposure.Detection of subcellular distribution of HDAC4 by gene knockdown PP1 and observation the neurite outgrowth of PC12 cells.3.The roles of HDAC4 and zeste homolog 2(EZH2)by Pb exposure were studied.Investigated the expression level of HDAC4 in nucleus and cytoplasm proteins by treatment with LMK-235,and observed the subcellular distribution of HDAC4 and its co-localization with EZH2 were detected by immunocytochemistry.The sumoylation level of EZH2 and was detected by adding NEM(N-ethylmaleamide)during the cellular protein extraction process,and the sumoylation level of EZH2 was detected by sumoylation detection kit.Immunofluorescence staining revealed an outstanding interaction that HDAC4 colocalized with the enhancer of EZH2,and this cooperation was enhanced and normalized by the treatment of Pb and LMK-235,respectively,which coincided with the corresponding changes of a novel posttranslational form of EZH2,Sumo-EZH2.The level of sumoylation of EZH2 was enhanced by Pb exposure,and LMK-235 and ?NLS2-HDAC4 plasmids could restore to this change.Conclution: HDAC4 played fundamental roles in regulating Pb neurotoxicity.The nuclear accumulation of HDAC4 was identified as a key molecular process in the Pbtreated neural cells,which was literally mediated by the up-regulation of PP1.The increased presence of HDAC4 enhanced its interaction with a novel functional partner,EZH2,and consequently promoted its sumoylation,a posttranslational modification first assigned to EZH2 by HDAC4.These findings suggested a new PP1-HDAC4-EZH2 pathway in response to Pb neurotoxicity,favoring the understanding and intervention of psychiatric adversities elicited by environmental insults. |
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