CIB1调控树突棘消长泛素化/磷酸化双通路在低剂量重金属联合暴露致神经行为异常的分子机制

Low level heavy metal mixture pollution has become a severe issue of environmental pollution, among which lead (Pb), cadmium (Cd), arsenic (As) and mercury (Hg) are typical neurotoxins that can be detected simultaneously in the blood of the general population. However, the combined impacts of these heavy metals on human health as well as the mechanism are still unclear. Our previous studies had found that Pb, Cd, As and Hg combined exposure evoked synergistic neurotoxicity when their individual metals were below no observed adverse effect levels (NOAELs) and at the related levels of these metals in the blood of population, and induced dendritic spines injuries and finally caused neural behavior abnormalities, and both dendritic spine related ubiquitination pathway Snk-SPAR and phosphorylation pathway PAK1-LIMK1-Cofilin played some part in that process. Studies have confirmed that both of these two pathways can be regulated by calcium binding protein CIB1. Therefore, we hypothesized that Pb2+, Cd2+, As3+ and Hg2+ mimics Ca2+ binding to CIB1 to regulate the ubiquitination and phosphorylation binary pathways of dendritic spines, leading to neural behavior abnormalities. In our study, we explore the binding effect of these four heavy metal ions with CIB1, then excavate the influence of CIB1 expression changes on neural behavior abnormalities caused by combined exposure of low-level heavy metals, and further to elucidate the molecular mechanisms of CIB1 regulated dendritic spines related ubiquitination and phosphorylation binary pathways in low level heavy metal combined exposure induced abnormal neural behavior. This study will provide new intervention strategies for the prevention and treatment of nervous system diseases caused by heavy metal pollution.

重金属低剂量复合污染是当今环境污染面临的重大难题，其中铅、镉、砷和汞是能在一般人群血液中检测到的典型神经毒物。但其联合暴露所致健康损害及机制尚不明确。我们前期发现当单个重金属浓度为一般人群血液负荷相关水平（低于NOAELs）时，这四种重金属联合暴露可产生协同神经毒性而引起树突棘损伤致神经行为异常，且树突棘消长泛素化通路Snk-SPAR和磷酸化通路PAK1-LIMK1-Cofilin在其中均起一定作用。研究证实这两条通路均可被钙结合蛋白CIB1调控。因此我们假设铅、镉、砷和汞模拟钙与CIB1结合进而调控树突棘消长泛素化和磷酸化双通路致神经行为异常。本项目拟从动物、细胞及分子水平，探究重金属离子与CIB1的结合作用，挖掘CIB1在低剂量重金属联合暴露致神经行为异常中的作用，并阐明CIB1调控树突棘消长泛素化/磷酸化双通路在其中的分子机制。研究将为防治重金属污染所致神经系统疾病提供新的干预靶点。

低剂量重金属复合污染已成为威胁人类生存和发展的重大公共卫生问题。深入研究环境低剂量重金属联合暴露所致健康损害效应及其相关机制对于重金属污染防治具有十分重要的理论价值和实际意义。因此，本研究从钙信号调控的角度出发，在整体动物、细胞和分子水平研究钙结合蛋白CIB1调控树突棘消长泛素化/磷酸化双通路在低剂量重金属联合暴露所致神经行为异常的作用机制。首先，在不同年龄段动物模型，通过一系列神经行为功能测试发现，一般人群血液负荷相关水平的低剂量重金属联合暴露引起大鼠学习记忆和感知觉能力损害，并伴随大鼠海马组织和原代培养的海马神经元CIB1表达水平降低。其次，采用高尔基染色、鬼笔环肽染色、激光共聚焦等技术，发现低剂量重金属联合暴露损伤了神经行为的结构基础树突棘的可塑性，引起大鼠海马神经元树突棘密度减少，蘑菇型树突棘比例降低。而应用慢病毒感染、质粒转染、蛋白免疫印记、免疫荧光等技术，发现低剂量重金属联合暴露通过CIB1分别介导树突棘消长磷酸化通路PAK1-LIMK1-Cofilin和泛素化通路Snk-SPAR改变树突棘动态变化，进而导致神经行为损伤。随后，应用质粒转染结合激动剂、抑制剂进行双通路同时调控，发现CIB1可以同时调节树突棘磷酸化和泛素化通路介导低剂量重金属联合暴露致树突棘损害，且树突棘磷酸化的贡献作用较泛素化通路更显著。最后，采用蛋白重组纯化、圆二色光谱技术，在体外明确了低剂量重金属离子及其联合暴露与蛋白质CIB1的结合作用，发现低剂量重金属联合暴露改变了CIB1的二级空间结构，进而影响其功能作用。此外，基于树突棘损伤调控机制进一步探索了环境丰富早期干预有效改善了低剂量重金属联合暴露所致神经行为异常。本项目的研究成果为进一步明确重金属联合暴露导致神经行为异常的分子机制提供了科学依据，并为防治环境重金属污染所致神经系统疾病提供可能的干预靶点和策略。