铅诱导AMPA受体调控蛋白GRIP改变在其损伤学习记忆中的作用和机制研究

Developmental exposure to Pb results in irreversible impairment to learning and memory. It has been reported that Pb disturbs the NMDA receptor-mediated LTP. Recent studies indicated that GluR1 and GluR2, two major subunits of the AMPA receptor (AMPAR), play a key role in the LTP generation and learning and memory processes. GRIP is a regulatory protein which involve in the generation, transport and anchor of the AMPAR. Our previous experiments showed that GluR2 expressed in homogeneously in the dentate gyrus, Hilus, CA1 and CA3 of the Hippocampus after Pb exposure, which was accompanied with the markedly increased expression of GRIP and moderate increase of NEEP21. The aim of this study is to investigate the effect of lead on cerebral GluR2, APMAR expression in newborn rats, and to explore the relationship between hippocampal LTP impairment and the functions of APMAR in vivo and in vitro. With advanced molecular biological and electricphysiological technologies, we will prove our hypothesis that the elevated expression of GRIPs results in a translocation of AMPAR to the basement membrane and accelerated endocytosis cycle, further influence the remove of GluR2 AMPA receptor from postsynaptic membrane during LTP. This study will be helpful to clarify the mechanisms of Pb-induced impairment of learning and memory.

发育期铅损伤学习记忆是一个不可逆难治性问题，公认损伤机制为铅干扰NMDA谷氨酸离子型通道影响海马LTP产生。新近发现AMPAR亚基GluR1、GluR2在海马LTP及学习记忆产生与维持中起重要作用，而GRIPs是AMPAR运输、锚定调控蛋白，本项目预实验表明铅中毒后能导致GluR2在齿状回、海马门区CA1、CA3区呈斑块状分布；GRIP以及NEEP21均表达升高，尤以GRIP表达增加最为明显。基于此本项目拟在大鼠在体、海马脑片、细胞培养结合形态学、分子生物学、电生理技术研究新生大鼠铅中毒对GluR2调控蛋白、AMPAR亚基表达影响，确立海马LTP损伤与AMPA受体功能变化之间关系，阐明关键蛋白GRIPs表达升高是导致GluR2亚基膜向掺入、内吞循环加速，从而影响LTP时含GluR2 AMPA受体在突触后膜区移除，进而导致LTP损伤的假说，为全面阐明铅损伤学习记忆机制提供新的实验依据。

针对铅损伤学习记忆功能的机制与防治研究一直是环境神经毒理学研究领域的热点与难点。公认其损伤机制是铅干扰NMDA谷氨酸离子型通道从而影响海马LTP的产生。而新近研究表明AMPA受体亚基GluR2在海马LTP产生及学习记忆产生与维持中起重要作用；GRIP是AMPAR产生、运输和锚定的调控蛋白，本课题拟通过利用大鼠在体内、海马脑片、细胞培养结合形态学、分子生物学和电生理等技术手段来研究新生大鼠铅中毒对GluR2、GRIP和AMPA受体各自表达影响，阐明GluR2调控蛋白关键蛋白GRIPs在铅暴露导致LTP损伤中的作用机制。研究结果显示：大鼠围产期染铅模型（Perinatal lead exposure）能较好模拟儿童染铅情况，3-4W龄大鼠是铅干扰动物神经系统发育最关键的时期；孕期染铅后铅可在皮质、海马等脑区内蓄积，尤其是海马，导致学习记忆功能下降，而与血铅数值无关；蓄积于脑内的铅，造成大鼠海马区神经元树突棘密度降低，且thin、filopodium和mushroom形态的树突棘数量减少，同时引起突触结构改变，突触间隙变窄, 突触后致密区增大，导致海马神经元的结构可塑性改变；影响了海马神经元的功能可塑性，造成兴奋性突触传递的变化，表现为mEPSC的幅度以及频率均显著性减少，表明突触后神经元胞质内的AMPA受体转位插入到突触后膜上减少，是导致AMPA受体在膜上的功能发生改变的原因；铅中毒后神经细胞内GRIP以及NEEP21分子表达增加，使内吞的AMPA受体GluR2大量聚集在细胞内，延迟了GluR2再利用，导致了GluR2在细胞膜上数量减少，相应突触后膜区含GluR2的AMPA受体移除发生障碍，进而导致LTP损伤，引起学习记忆功能的下降。本研究初步明确了铅中毒损伤海马LTP引起学习记忆能力下降中AMPA受体的机制，为阐明铅损伤学习记忆机制提供了新的思路和实验基础，同时也为铅中毒的防治提供了新的干预靶点。