RyRs调控细胞内钙信号介导突触可塑在铅所致认知障碍中的作用

Neurotoxicity caused by ubiquitous and lingering lead has been showed to have a broad spectrum of toxic effects from developmental neurotoxicity of globally focused concern across to neurodegenerative diseases with a serious threat to the health. Extensive and intensive studies have been being urgently eager to elucidate its mechanism and discover a new target for its preventive and treatment. Synaptic plasticity and its regulation mechanisms provide the neurobiological basis for exploring the lead neurotoxicity mechanism intensively. On the basis of our feasibility investigations and the latest researches with signal pathway in synaptic plasticity. The hypothesis is proposed that RyRs as the hub of intracellular Ca2+ signals mediated synaptic plasticity may play a key role in the lead- induced cognitive impairment. In this project, applying Confocal Laser Scanning Microscope, Patch Clamp Amplifier System, Radioligand receptor binding assay and morphologic quantitative analysis, as well as cellular and molecular techniques of siRNA, DNA recombination and so on, we will try to elucidate the fundamental role of RYRs-regulated calcium signal mediated synaptic plasticity in lead- induced cognitive impairment from reverse and forward as well as the model of action between Pb2+ and RyRs in integral, cellular and molecular level from experimental and population-based studies, and the correlation between RyRs in peripheral blood lymphocytes of Pb2+ exposure human and lead- induced cognitive impairment. Our study will give additional information to reveal the lead neurotoxicity mechanism, provide new clues to discover the new target for lead intoxication prevention and treatment and offer new ideas to seek the emerging biomarker for lead hazards.

挥之不去的环境铅污染所致神经毒性从备受全球关注的发育毒性效应跨越到严重威胁健康的神经退行性病变，其机制不明确，期待防治新靶点的广泛深入探究。突触可塑及其调节机制的研究为深入探讨铅神经毒性机制提供了神经生物学基础。基于前期研究基础，结合突触可塑信号通道最新研究，提出突触可塑信号通路中RyRs作为细胞内Ca2+信号枢纽介导突触可塑性可能是铅神经毒性作用的关键环节。拟采用激光共聚焦技术、多导离子通道检测技术、放射性配体结合技术、细胞定量分析技术以及siRNA、DNA重组等分子生物学技术，从实验和人群研究两方面，在整体、细胞和分子水平，从反向和正向以及Pb2+与RyRs作用方式上探讨RyRs调控细胞内钙信号介导突触可塑性在铅所致认知障碍中的作用及铅暴露人群外周血淋巴细胞RyRs等与铅所致认知障碍的关联。深入揭示铅神经毒性的分子机制，为发现新的防治靶标提供新线索和寻找铅所致危害的新兴标志提供新思路。

环境铅污染所致的神经系统损伤从备受全球关注的发育毒性效应跨越到严重威胁健康的神经退行性病变，均以学习记忆损害、认知障碍等为主要临床表现。因而，铅暴露对学习记忆、认知生物学基础—突触可塑性的影响以及分子机制的深入研究势必有助于铅神经毒性损害的防治。于是，本项目围绕“铅暴露可通过内质网RyRs调控细胞内钙信号，影响突触可塑性诱导其认知障碍”这一科学假说展开研究工作。首先，采用流式细胞技术、激光共聚焦技术和放射性配体受体结合技术和多导离子通道检测技术，分别在整体动物水平、细胞和分子水平上，发现环境铅暴露通过内质网钙释放通道—ryanodine 受体（RyRs）引起大鼠海马组织及神经元细胞内游离钙增加，钙稳态失调，并抑制RyRs开放导致钙信号传递下降，证实了RyRs在铅暴露所致细胞内钙稳态和信号系统异常中的调节作用；其次，通过Weston blot、RT-PCR、Golgi-Cox染色、免疫荧光双染共定位、siRNA干扰、细胞定量分析等，发现了铅暴露导致RyRs调控细胞内钙信号系统异常会使钙依赖性认知信号通路蛋白CaMKⅡ和CREB的磷酸化比值下降、钙依赖性凋亡信号通路蛋白ErK1/2的磷酸化激活和bcl2抗凋亡蛋白表达降低，以及介导钙信号刺激依赖性大鼠海马突触回路的树突棘密度下降、不同功能的树突棘类型比例发生改变、突触中AMPA受体减少使得突触传递效率下降，且铅暴露引起各年龄段大鼠海马突触数量减少、突触后致密质PSD厚度变薄、突触间隙宽度增宽和突触界面曲率下降；明确了铅暴露导致RyRs调控细胞内钙信号系统异常对学习记忆和认知生物学基础突触及有关信号通路的影响。最后，再回到整体动物水平和海马脑片水平上，判辨和阐述了铅暴露环境下，铅离子可通过内质网RyRs调控细胞内钙信号诱导认知障碍。研究还发现从怀孕到断乳之前的生命早期铅暴露，在停止铅暴露后，随着时间的延长有一定的自我修复能力，但是损害难以恢复到正常状态。.本项目研究阐明的不同水平、时间和方式的铅暴露所致认知障碍和突触可塑性改变的RyRs分子机制，为铅暴露所致发育神经毒性和神经退行性病变的预防和治疗提供了新线索和潜在的新靶点。