Egr-1调控的基因调节网络参与阿尔茨海默病进程中Meynert基底核的早期激活机制

Alzheimer's disease (AD) is the most prevalent form of dementia. The main risk factor is age. The Nucleus Basalis of Meynert (NBM), which is the major cholinergic production area in the brain and is crucial for memory, is strongly affected by AD. However, in the early stages of AD, the NBM is activated, which may play a role of protection or functional compensation since in this stage memory is still intact. NBM cholinergic activity decreases in the late AD stages with neuronal degeneration. Also in the prefrontal cortex (PFC) such activation was found in early AD stages. It is of significant importance to reveal the factors and regulating mechanism of early AD NBM activation for developing new therapeutic targets that may also activate the brain in later AD stages. The change from activation to degeneration of the NBM may improve our understanding of the pathogenesis of AD. We plan to study this issues from a key candidate molecule that is closely associated with synaptic activity and plasticity, i.e. the early growth response factor-1 (Egr-1) which is upregulated in the PFC in the early stages of AD, while down-regulated during later AD stages. Egr-1 has close functional interactions with the typical AD pathological markers Aβ and hyperphosphorylated tau, and it may regulate cholinergic activity. We will use i) postmortem NBM samples of AD patients which cover the complete course of AD, ii) the basal forebrain and the frontal lobe of APPSwe/PS1M146V/TauP301L triple transgenic mice (3xTg-AD), the best AD model at present, of different months of age, and iii) cortical primary cells of 3xTg-AD and SH-SY5Y cell lines, to study with methods of morphology, molecular biology, and pharmacology, Egr-1 expression change in the course of AD, Egr-1 regulation of acetylcholinesterase gene expression and its effect on the generation of hyperphosphorylation of tau and Aβ.

在阿尔茨海默病（AD）的早期阶段，胆碱能生产脑区Meynert基底核（NBM）激活，可能起到功能代偿作用；而NBM于AD晚期活性降低，神经元发生退行性改变。早期生长反应因子-1（Egr-1）与突触活性和可塑性调节密切相关，在AD病程中于前额叶的表达呈早期升高而晚期降低，与Aβ与过度磷酸化tau存在密切相互作用，并可能调节胆碱能系统活性。我们将在1)根据神经病理学分期覆盖AD完整病程的AD患者死亡后NBM样本，2)不同月龄的三重转基因小鼠（3xTg-AD）基底前脑与额叶，3）3xTg-AD皮层原代细胞以及SH-SY5Y等细胞系中，采用形态学、分子生物学、药理学等方法研究Egr-1在AD病程中的改变，Egr-1对乙酰胆碱酯酶（AchE）基因表达的调控及其对过度磷酸化tau和Aβ产生等影响机制。旨在揭示调控AD早期NBM活性升高的因素和作用机制，为寻找激活晚期AD神经元的药物靶点提供科学依据。

脑内胆碱能生产主要区域Meynert基底核（NBM）在阿尔茨海默病（AD）的早期阶段激活，可能起到对大脑的保护或功能代偿作用，使得该阶段记忆功能完整；而NBM活性在AD晚期降低可能导致神经元发生退行性变。揭示调控AD早期NBM活性升高的因素及其作用机制对于开发于AD晚期激活大脑的药物靶点具有重要意义。我们在覆盖AD完整病程（Braak 0-VI期）的死亡后人脑NBM中检测了早期生长反应因子-1（Egr-1）、胆碱乙酰转移酶（ChAT）、过度磷酸化Tau蛋白（p-Tau）和β淀粉样蛋白（Aβ）的表达以及伴随AD病程的改变，发现Egr-1的表达在AD早期保持相对稳定，而于AD晚期下降，且与ChAT表达量呈显著正相关。ChAT与p-Tau表达呈显著负相关，而与Aβ没有相关性。我们还研究了AD病程中前额叶样本的全基因组微阵列结果，分析了Egr-1和乙酰胆碱酯酶（AChE）mRNA在AD进程中的表达改变，并分析了648例覆盖整个AD进程的个体的AD病理分布，发现80岁以上女性AD病理累积较男性更为严重，并发现AD临床前期女性内嗅皮层中p-Tau表达量显著高于男性。我们还对表达人PS1M146V、APPSwe、TauP301L基因的三转基因（3xTg）AD小鼠模型研究了3-4、7-8和11-12月龄期脑中这三类基因以及Aβ、p-Tau、Egr-1、AChE分子的表达，发现7-8月龄开始雌鼠AD病理分子表达显著高于雄鼠，11-12月龄开始出现淀粉样斑块，但是缺乏p-Tau阳性信号。3xTg-AD小鼠也缺乏人脑AD病程进展中的早期激活表现。在SY5Y细胞系采用质粒转染分别上调和下调Egr-1表达、于Egr-1过表达的细胞系中进行双荧光素酶检测并对潜在Egr-1结合位点进行突变、进行电泳迁移率变动分析实验等揭示了Egr-1位于AChE基因启动子上的功能性结合位点，阐明Egr-1直接上调AChE基因表达的分子机制。本项目研究结果已发表于Brain, Brain Pathology，Neurosci Bull等学术期刊，并有后续论文在投稿、准备中。本项目的科学发现揭示了调控AD早期NBM活性升高的因素和作用机制，为进一步揭示AD早期发病机制，包括性别差异机制，并为寻找激活晚期AD神经元的药物靶点提供了科学依据。