circ100783作为miR-34b分子海绵在铅暴露海马SNARE 复合体形成和突触囊泡释放中的机制研究

Exposure of Lead(Pb), a known neurotoxicant, whether occupational or environmental exposure, is a very serious problem all over the world. Recent population studies have suggested that exposures to lead are at an increased risk of cognitive impairment. Lead poisoning, can impair spatial learning and memory probably via impairing the hippocampal long-term potentiation as well as hippocampal neuronal injury. .Based on the results of preliminary experiments，we raised the hypothesis that circ100783 and miR-34b in neurons of hippocampus is involved in the Pb exposure induced hippocampal LTP impairment and neuronal injury; at the same time, acts as miR-34b molecular sponges, circ100783 may involve the SNAREs(soluble N-ethylmaleimide sensitive factor attachment protein receptors) complex protein and the formation of synaptic vesicles. .Membrane trafficking and exocytosis of the eukaryotic cell appear to be regulated through a complex assembly of a set of evolutionary conserved proteins called SNARE proteins. The SNARE super family has three core members, VAMP, SNAP-25 and Syntaxin, which are distinctively distributed on vesicle membrane (v-SNARE: VAMP) and the target plasma membrane (t-SNARE: SNAP-25 and Syntaxin). Evolutionarily conserved SNARE proteins form a complex that drives membrane fusion in eukaryotes. According to the preliminary experiment， neurons were treated with a series of concentrations of lead， the expression of SNARE complex gene and protein， and the number of active synaptic vesicles were determined．.To test this hypothesis and clarify its underlying mechanisms, To explore the molecular mechanism of neurotransmitter release disturbed by lead，especially its effect on SNARE complex protein and synaptic vesicles in neurons．Thus, we investigated the Pb-exposure on the learning and memory of rats, cytokine release, hippocampal LTP and the SNAREs complex protein and the formation of synaptic vesicles as well as neuronal injury in vivo or in vitro model. Based on the technology of knockout、knockdown and protein analysis, The changes of these parameters were also observed. In vitro Lead-exposure also induced significantly change of key circ100783 and miR-34b molecules in neurons. Inhibiting the circ100783 and miR-34b activation may play the role in the above-mentioned lead-exposure induced changes. Our results showed that lead can change the key circRNA and microRNA, such as circ100783 and miR-34b, which regulates VAMP2 and SNARE, may cause neurotransmitter release, maybe cause the action of synaptic vesicles in neurons. Key circRNA and microRNA and its target molecules will be tested in our labs with our samples. These results may lay the preliminary foundation for the further study of lead and signal transduction pathways, and these results maybe throw light on preventation and treatment of injury induced by lead.

流行病学调查发现铅暴露可引起儿童学习记忆降低。研究发现铅暴露可对海马结构功能产生一定影响，但确切机制尚不清楚。结合文献和我们最新的实验结果，海马circ100783作为miR-34b分子海绵可能在铅诱导海马神经元SNARE复合体形成和突触囊泡释放中的发挥作用。在此基础上，本项目应用基因敲除模型动物和分子克隆等方法，研究：1.建立铅暴露动物模型，研究铅对大鼠的神经毒性作用；检测铅对大鼠海马突触囊泡超微结构及SNARE复合体蛋白表达的影响；2.研究海马circ100783和miR-34b分子的表达谱及指纹图谱、表达调控及功能研究；3.研究circ100783和miR-34b分子及备选分子和靶分子在铅暴露海马突触囊泡形成和释放过程中的作用。所获关键circRNA和miRNA及其靶分子将用特殊环境标本库样本进行验证。本课题将为深入探讨铅神经毒性的防治，为最终阐明海马损伤机制提供基础数据。

流行病学调查发现铅暴露可引起儿童学习记忆降低。研究发现铅暴露可对海马结构功能产生一定影响，但确切机制尚不清楚。结合文献和我们最新的实验结果，海马circRNA作为miRNA分子海绵可能在铅诱导海马神经元SNARE复合体形成和突触囊泡释放中的发挥作用。在此基础上，本项目研究：1. 建立铅暴露体内和体外模型，动物行为学实验等表明铅暴露损伤小鼠学习记忆的模型建立成功；2. 明确铅暴露后海马突触囊泡的变化，发现铅暴露组的海马区组织松散，突触间隙模糊，突触前膜边界不清晰；3. 检测circ100783/miR-34b在铅暴露突触囊泡释放中的作用，发现现有条件下，VAMP2/miR-34b/circ100783的表达未检测出显著性变化；4. 检测铅暴露后全转录组表达谱变化，发现DE环状RNA、DE miRNAs和DE mRNA的生物过程主要富集于转录和DNA模板化的调控、信号转导等；5. 探索影响突触囊泡释放的关键circRNA分子-miRNA分子及其功能研究，目前研究集中在circ0002647-miR128和snap25轴。本课题将为深入探讨铅神经毒性的防治，为最终阐明海马损伤机制提供基础数据。