Lead Exposure Inhibits Expression Of Synaptic Vesicle Glycoprotein 2C Through Neuron-restrictive Silencer Factor

Objective:Lead(Pb)is a kind of toxic heavy metal which is widely existed in nature,and lead exposure can cause damage to multiple systems of the body,especially the damage to the nervous system.The neurotoxicity of lead has been highly concerned,but its toxic mechanism has not been fully clarified.Numerous studies have found that neuron restrictive silencer factor(NRSF)played a very important role in the development of the nervous system.NRSF is a transcription factor containing zinc finger structure,which can be specifically combined with neuron restrictive silencer element(NRSE)to inhibit the transcription of the neuron-specific genes containing NRSE motif,these genes has a major part to play in the formation and maintenance of neuronal phenotype and synaptic plasticity.Synaptic vesicle protein 2C(SV2C)plays a critical role in neurotransmitter release and synaptic transmission,our previous study found that lead exposure can significantly reduce SV2 C m RNA and protein levels in mouse neuro-2a cells(N2a cells),but the lead is how to change the transcriptional level of SV2 C gene remains unclear.However,the promoter region of SV2 C gene containing NRSE motif was found by bioinformatics technique.Therefore,we established the lead exposed rat model to investigate the effects of lead exposure on the expression of NRSF and SV2 C in hippocampus of rats.And the N2 a cell line was selected as an in vitro lead exposure model,to explore the effects of lead exposure on NRSF and SV2 C expression.Finally,on the basis of the N2 a cell model of lead exposure,we interfere the expression of NRSF,to observe whether lead exposure by influencing the expression of NRSF to regulate the transcription level of SV2 C,which may provide theoretical basis to explore the neurotoxic mechanism of lead,and provide new ideas for looking for new drugs for the treatment of lead poisoning.Methods:1.The effects of lead exposure on the expression of NRSF and SV2 C in hippocampus of young male Sprague-Dawley rats.Sexually mature female SpragueDawley rats were randomly divided into three groups: control group,low and high lead-exposed group,treated with lead acetate at the doses of 0,0.5,2.0 g/L respectively through drinking water during the first ten-day of pregnancy until weaning.Yong male rats corresponding to their mother were divided into control,low and high lead-exposed groups.All experiment were conducted at weaning(PNW3).Using Morris water maze system to detect the learning and memory ability of rats.Inductively coupled plasma-atomic emission spectroscopy(ICP-AES)was applied to test the contents of lead in blood and hippocampus.The m RNA level of NRSF and SV2 C in hippocampus were detected with reverse transcription polymerase chain reaction(RT-PCR).Using western blotting(WB)to detect the protein level of NRSF and SV2 C in hippocampus.Immunofluorescent(IF)was employed to detect the protein expression of NRSF in various regions of the hippocampus.2.The effects of lead exposure on the expression of NRSF and SV2 C in N2 a cells.After the N2 a cells steady growth,to add the lead acetate of different concentrations,0μM(control group),1μM and 100μM,were exposed to lead treatment after 48 h.The m RNA level of NRSF and SV2 C in N2 a cells were detected with reverse transcription polymerase chain reaction(RT-PCR).Immunofluorescent(IF)was employed to detect the protein expression of NRSF in N2 a cells.3.Two groups of N2 a cells were treated 100μM lead acetate,silence group was transfected with NRSF-si RNA,the control group was transfected with DDW,the two groups were treated with 100μM lead acetate 48 h at the same time.The m RNA level of NRSF in N2 a cells were detected with reverse transcription polymerase chain reaction(RT-PCR).Using western blotting(WB)to detect the protein level of NRSF in N2 a cells.The m RNA level of SV2 C in N2 a cells were detected with reverse transcription polymerase chain reaction(RT-PCR).Results:1.Lead exposure decreased the ability of learning and memory in rats.Morris water maze test showed that there were significant difference in the escape latency after 4 days’ acquired training experiment,and the escape latency prolonged by the dose-dependent of lead(P<0.05).In the spatial probe test,the number of crossings of rats in LLG and HLG groups were significantly less than in the CON group(P<0.05),while there were also have a statistically significant difference between the LLG and HLG groups(P<0.05).2.Lead exposure significantly elevated the contents of lead in blood and hippocampus.ICP-AES showed that the contents of lead in blood and hippocampus were significantly elevated with the increase of lead exposure dose(P<0.05).3.Lead exposure influenced the expression level of NRSF in hippocampus of young male rat.The RT-PCR analysis showed that the NRSF m RNA level increased in hippocampus of young male rats with lead exposure dose increased(P<0.05).The WB analysis showed that the NRSF protein level also increased in hippocampus of young male rats with lead exposure dose increased(P<0.05).IF manifested that the NRSF protein level increased in hippocampus CA1,CA3 and DG areas of young male rats with lead exposure dose increased(P<0.05).4.Lead exposure influenced the expression level of SV2 C in hippocampus of young male rat.RT-PCR found that the SV2 C m RNA level was down-regulated in hippocampus of young male rats with lead exposure dose increased(P<0.05).The WB analysis showed that the SV2 C protein level also decreased in hippocampus of young male rats with lead exposure dose increased(P<0.05).5.Lead exposure influenced the expression level of NRSF in N2 a cells.The RT-PCR analysis found that the NRSF m RNA level was up-regulated in N2 a cells with lead exposure dose increased(P<0.05).The IF analysis manifested that the NRSF protein level was also up-regulated in N2 a cells with lead exposure dose increased(P<0.05).6.Lead exposure influenced SV2 C m RNA level in N2 a cells.The RT-PCR analysis found that the SV2 C m RNA level was down-regulated in N2 a cells with lead exposure dose increased(P<0.05).7.Under the effect of 100μM lead exposure,compared with the control group,the NRSF expression levels were significantly reduced in NRSF-si RNA group,while the SV2 C m RNA expression level was significantly increased,the differences were statistically significant(P<0.05).Conclusion:Lead exposure may be by increasing the NRSF expression to inhibit the transcriptional level of SV2 C,thus affecting neurotransmitter release and synaptic transmission,thereby affected synaptic plasticity,induced damage of learning and memory.Thus it may provide theoretical basis to explore the neurotoxic mechanism of lead,and provide new ideas for looking for new drugs for the treatment of lead poisoning.