Rictor调控actin细胞骨架聚合在雌激素改善学习记忆中的作用与机制研究

It is well established that estrogens and their receptors play important roles in the regulation of learning and memory as well as cognition through action on hippocampal spine density and synapse density, but the detail mechanisms are poorly understood. Rictor is the key component of mammalian target of rapamycin complex 2; limited studies have shown that Rictor controls the polymerization of actin cytoskeleton, which plays pivotal role in the conversion of short-term memory to log-term memory. Knockout of Rictor caused shorter dendritic length and abnormal synaptic transmission, decreased phosphorylation of key molecules related to actin cytoskeletal regulation, increased G-actin/F-actin ratio, and ultimately long-time memory and later LTP disorder. Our previous works have shown that in the hippocampus, steroid receptor coactivator-1 (SRC-1) plays important role in the endogenous estrogen regulation of learning and memory， knockdown of SRC-1 significantly impaired LTP and spatial learning. Recently we found that inhibition of local estrogen synthesis and/or SRC-1 caused significant decrease of hippocampal Rictor level. Meanwhile, these treatments also caused decrease of F-actin/G-actin, spine density and synapse density. Based the above results we hypothesize that Rictor play a crucial role in the mediation of estrogen action on hippocampal synaptic plasticity, learning and memory. Therefore, in this study we will use combination of in vivo and in vitro morphological, molecularincluding RNA inteference and conditional gene knockout, behavior and electrophysiological techniques to investigate the potential roles and mechanisms of Rictor in the estrogen regulation of hippocampal function. We believe these studies will provide new experimental evidence for the elucidation of estrogens action on learning and memory in the hippocampus as well as a novel intervention target for estrogen replacement therapy against neurodegenerative diseases such as Alzheimer’s disease.

雌激素及其受体可调节树突棘密度和突触密度并对学习记忆发挥重要作用，但具体机制不明。Rictor是mTORC2的核心成分，控制着将短时记忆转变为长时记忆所需的actin多聚化，敲除Rictor导致长时记忆和晚期LTP严重受损，但Rictor是否介导了雌激素对学习记忆的调控未见报道。在上一个NSFC项目研究中，我们发现SRC-1的表达受雌激素调节，抑制其表达导致明显的LTP缺陷和学习记忆障碍；近期发现抑制雌激素合成或SRC-1表达导致Rictor水平显著下降，伴随actin聚合、树突棘密度和突触密度显著下降，提示Rictor在雌激素调节学习记忆中可能发挥了重要作用。为此拟采用RNA干扰和条件性基因敲除等手段，于在体和离体研究Rictor调节actin聚合在雌激素改善学习记忆中的作用及其机制，为阐明雌激素调节学习记忆的机理提供新的实验证据，也为雌激素替代预防或治疗老年痴呆提供新的研究靶点。

在本课题中我们利用形态学、分子生物学、行为学技术研究了Rictor在雌激素调节海马突触可塑性和空间学习记忆中的作用和机制，主要有以下发现：1）在雄性小鼠，利用睾丸切除和来曲唑抑制雌激素合成两种方式处理，发现二者均能调节海马Rictor、actin聚合调节蛋白的表达以及下调突触蛋白、树突棘密度和突触密度，但是来曲唑的调节效果更明显且仅来曲唑处理能导致学习记忆行为障碍，表明睾丸雄激素对学习记忆的影响有限而海马雌激素的作用更为重要； 2）在雌性小鼠，利用卵巢切除和注射来曲唑比较了两种处理对节海马Rictor、actin聚合调节蛋白和突触蛋白的表达、树突棘密度和突触密度以及学习记忆的影响，发现来曲唑处理可以导致更严重的Rictor表达下降、actin解聚、突触蛋白表达下降以及树突棘密度、突触密度的下降，来曲唑处理也诱导更严重的学习记忆障碍，表明在调节海马突触可塑性方面海马雌激素的作用可能强于卵巢雌激素； 3）利用雌激素受体的激动剂和拮抗剂，发现雌激素核受体和膜受体均参与了雌激素对海马Rictor、actin聚合调节蛋白、突触蛋白、树突棘密度和突触密度以及学习记忆的调节，Rictor通路激动剂能逆转雌激素受体拮抗剂对actin聚合调节蛋白、突触蛋白、树突棘密度和突触密度以及学习记忆的调节；4）雌激素核受体与膜受体对Rictor、actin聚合调节蛋白、突触蛋白等表达的调节效率相当，雌激素膜受体仅对少数蛋白的表达调节作用稍强；5）用RNA干扰抑制海马Rictor表达可阻断雌激素对卵巢切除小鼠actin聚合、突触密度和树突棘密度以及学习记忆行为的改善作用。以上结果证明了和性腺激素相比，通过芳香化酶途径产生的神经元性雌激素是调节海马Rictor表达、actin细胞骨架聚合以及学习记忆的主要因素，雌激素核受体和膜受体都参与了雌激素对Rictor表达、actin聚合以及学习记忆的调节且调节效率基本相当，Rictor介导的actin细胞骨架聚合状态的变化在上述过程中发挥了关键作用，提示Rictor可能是雌激素调节学习记忆的关键靶点，对雌激素相关性疾病的预防或治疗具有重要的意义。