ApoE4通过影响NMDA受体功能加剧5XFAD小鼠认知功能障碍

发布时间：2018-05-05 07:04

本文选题：阿尔茨海默病ApoE + N-甲基-D-门冬氨酸受体　；参考：《福建医科大学》2013年博士论文

【摘要】：【目的】ApoEε4作为晚发型家族性AD和散发性AD的易感基因已被国内外学者普遍接受。本论文旨在原有的理论和实验研究的基础上，利用5xFAD小鼠和不同ApoE基因背景的转基因小鼠研究内源性ApoE异构体介导脑内NMDA受体功能失调在早期AD发病机制中的作用，并同时利用体内和体外研究，从整体、细胞和分子等不同水平深入探讨可能的机制。【方法】1.SPF级2、4、6月龄雌性ApoE-TR/5XFAD小鼠，随机分组，每组9～12只，分为ApoE2/5XFAD组、ApoE3/5XFAD组, ApoE4/5XFAD组，ApoEko/5XFAD组，应用Y迷宫、Morris水迷宫等行为学实验检测ApoE基因型对5XFAD小鼠学习记忆影响的情况。通过免疫组化技术检测海马CA1区NMDAR1、NMDAR2B表达情况，并采用Westernblot方法，检测突触相关蛋白的表达水平变化。 2.通过体外原代培养P0海马神经元并应用重组人ApoE2/3/4进行干预，通过MTT实验确立ApoE安全剂量范围，分为ApoE2干预组（200nm）、ApoE3（200nm）干预组、ApoE4（200nm）干预组、溶媒对照组。通过流式细胞术、激光共聚焦显微镜检测海马神经元细胞钙的情况，并借助Westernblot、激光共聚焦显微镜、免疫共沉淀等方法，从NMDA受体→Ca~(2+)→钙调蛋白（CaMK-II、DAPK1）→CREB磷酸化→BDNF通路探讨ApoE4对海马神经元钙失衡的影响及可能的分子机制。【结果】 1. ApoE4能明显加剧5XFAD小鼠的学习记忆能力的下降。 2. ApoE4能明显下调5XFAD小鼠海马区突触相关蛋白PSD-95、NMDA受体亚基、Drebin、p－CREB/CREB以及BDNF表达水平，并通过NMDAR/p－CaMKII/CREB途径下调了BDNF的表达，从而加剧5XFAD小鼠认知功能的损害。 3. ApoE4能导致海马神经元细胞内钙通道异常，并通过调节NMDA受体→Ca~(2+)→钙调蛋白（CaMK-II、DAPK1）→CREB磷酸化→BDNF通路相关蛋白的表达水平，引起细胞内钙超载，进一步探讨ApoE4经由NMDA受体影响海马神经元钙稳态以及NMDA受体后信号的可能机制。【结论】ApoE4可通过NMDAR/p－CaMKII/CREB途径下调了BDNF的表达，影响突触可塑性，进而加剧5XFAD小鼠认知功能障碍。ApoE4能导致海马神经元细胞内钙通道异常，引起细胞内钙超载，并通过调节NMDA受体→Ca~(2+)→钙调蛋白（CaMK-II、DAPK1）→CREB磷酸化→BDNF通路相关蛋白的表达水平，，进一步证实了ApoE4经由NMDA受体影响海马神经元钙稳态以及NMDA受体后信号的机制。
[Abstract]:[objective] ApoE 蔚 4 as a susceptibility gene of late onset familial AD and sporadic AD has been widely accepted by scholars at home and abroad. On the basis of previous theoretical and experimental studies, this thesis aims to investigate the role of endogenous ApoE isomers in the pathogenesis of early AD by using 5xFAD mice and transgenic mice with different ApoE gene backgrounds. At the same time, in vivo and in vitro studies, from the whole, cell and molecular levels to explore the possible mechanisms. [methods] female ApoE-TR/5XFAD mice of 1.SPF grade 2 and 4 and 6 months of age were randomly divided into ApoE2/5XFAD group (ApoE3 / 5XFAD group) and ApoE4/5XFAD group (ApoEko / 5XFAD group). The influence of ApoE genotype on learning and memory of 5XFAD mice was investigated by using Y-maze Morris water maze and other behavioral experiments. The expression of NMDAR1 and NMDAR2B in hippocampal CA1 was detected by immunohistochemical technique, and the expression level of synaptophysin was detected by Westernblot method. 2. The P0 hippocampal neurons were cultured in vitro and intervened with recombinant human ApoE2/3/4. The safe dose range of ApoE was established by MTT experiment. The P0 hippocampal neurons were divided into two groups: ApoE2 intervention group (ApoE3 + 200nm) intervention group (ApoE4 + 200nm) intervention group and solvent control group (control group). Calcium in hippocampal neurons was detected by flow cytometry, laser confocal microscopy, Western blot, laser confocal microscopy and immunoprecipitation. The effect of ApoE4 on calcium imbalance in hippocampal neurons and its possible molecular mechanism were investigated by NMDA receptor Ca~(2) calmodulin (CaMK-IIP DAPK1) and CREB phosphorylated BDNF pathway. [results] 1. ApoE4 significantly increased the ability of learning and memory in 5XFAD mice. 2. ApoE4 could significantly down-regulate the expression of synaptic associated protein (PSD-95) and BDNF in the hippocampus of 5XFAD mice. It also down-regulated the expression of BDNF through the NMDAR/p-CaMKII/CREB pathway, thus exacerbating the impairment of cognitive function in 5XFAD mice. 3. ApoE4 can induce abnormal calcium channel in hippocampal neurons and induce intracellular calcium overload by regulating the expression of CREB phosphorylation and BDNF pathway related proteins by regulating NMDA receptor Ca~(2) calmodulin (CaMK-IIP DAPK1). To explore the possible mechanism of ApoE4 affecting calcium homeostasis and postreceptor signal of NMDA receptor in hippocampal neurons via NMDA receptor. [conclusion] ApoE4 can down-regulate the expression of BDNF through the NMDAR/p-CaMKII/CREB pathway, affect synaptic plasticity, and then aggravate the cognitive dysfunction of 5XFAD mice. ApoE4 can lead to abnormal calcium channels in hippocampal neurons and induce intracellular calcium overload. By regulating the expression level of CREB phosphorylated BDNF pathway associated with calmodulin (CaMK-IIP DAPK1) of NMDA receptor Ca~(2, the mechanism of ApoE4 affecting calcium homeostasis and NMDA postreceptor signal in hippocampal neurons via NMDA receptor was further demonstrated.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R749.16

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