CD2AP在阿尔茨海默病神经元突触异常中的作用及机制研究

Alzheimer’s disease (AD) is the most common neurodegenerative disease with unknown etiology and pathogenesis. Our team found CD2AP is a susceptibility gene for AD in previous studies. CD2AP is a ubiquitously expressed adaptor protein. Previous studies focused its roles in podocyte and found CD2AP knock out leading to abnormal podocyte processes and glomerular filtration membrane which resulted in severe proteinuria. Previous studies have reported that the podocyte major processes share lots of cell biological characteristics such as cytoskeletal elements and molecules working for signal transduction with neural dendrites. But CD2AP function in neuron is far from clear. Recently we found that the expression of CD2AP in peripheral blood of patients with AD was significantly decreased, and the expression level of CD2AP in the hippocampus of AD transgenic mice began to decrease in an early stage. Down regulated CD2AP in cultured neurons resulted in abnormal outgrowth of neural processes and decreased dendritic spine density, which is similar with the phenomenon observed in AD patients and AD transgenic mice. So, we speculated that CD2AP may be implicated in the abnormal synapse of AD. Here we will generate CD2AP neuron conditional knock out mice to further investigate its role in synapse and the specific mechanism. Besides, we will determine the treatment effects of upregulate CD2AP expression in AD transgenic mice. This investigation will be helpful to elucidate the pathologic mechanism of AD and provide new strategy and therapy target for AD.

阿尔茨海默病（AD）是最常见的神经变性病，其病因和发病机制仍不明确。我们研究团队在前期研究中发现CD2AP是AD的易感基因。CD2AP是一种衔接蛋白，既往主要研究其在肾脏足细胞中的作用，敲除后会导致小鼠足细胞突起形成障碍引起严重蛋白尿。足细胞突起和神经元突起在主要组成成分、信号转导分子等方面是相同的，但CD2AP在神经元中的研究甚少。我们近期研究发现AD患者外周血CD2AP表达水平明显下降，并且CD2AP在AD模型鼠海马区表达水平降低，下调神经元中的CD2AP导致突起生长异常和树突棘密度降低，这和以往报道的AD患者和AD模型鼠神经元突触异常的表现相似。综上所述，我们推测CD2AP在AD神经元突触异常中起着重要作用，我们将构建细胞模型和CD2AP条件性敲除小鼠模型研究其在调控突触中的作用及机制，并检测上调CD2AP对AD模型鼠的保护作用，上述研究有助于揭示AD发病机制，为治疗AD提供新策略。

阿尔茨海默病（Alzheimer's disease，AD）是最常见的神经退行性疾病，临床主要表现为进行性的学习和记忆功能障碍以及社会适应能力的下降，患者在晚期完全丧失生活自理能力。AD的发病机制最主要的假说是淀粉样蛋白（Aβ）假说和磷酸化tau蛋白假说。但基于Aβ和磷酸化tau蛋白开发的药物，临床试验均以失败告终。近年来的研究提示AD发病是由多种因素共同参与导致的，越来越多的研究者开始拓宽视野去寻找其他可能在AD发病中起作用的关键分子。本项目应用MassARRAY技术对422名散发性AD患者和1435名正常老年对照进行了75个基因上的95个多肽位点进行高通量筛查，结果发现CD2AP是中国汉族人群散发性AD的易感基因，进一步应用real-time PCR检测了209名散发性AD患者和213名正常老年对照，外周血白细胞中CD2AP的mRNA表达水平，发现其在散发性AD患者外周血中的mRNA表达水平较正常老年对照显著降低，提示CD2AP表达水平下降参与AD的发病。之后，在细胞系、原代培养神经元和胎鼠中观察发现CD2AP参与神经元的迁移和神经突起的生长。为了进一步明确CD2AP在AD发病中的机制，应用Crispr-cas9技术构建了CD2AP神经元条件性敲除和全敲除小鼠，表型评估发现CD2AP神经元条件性敲除小鼠识别记忆出现异常，且全敲除小鼠海马神经元DCX染色较野生型显著降低，电镜检查发现全敲除小鼠的海马神经元突触囊泡的密度和大小与野生型小鼠存在显著差异。蛋白质组学分析结果显示全敲小鼠和野生型小鼠在细胞骨架-内体-溶酶体途径中存在显著差异，提示CD2AP通过影响细胞骨架-内体-溶酶体途径参与AD的发病。本项目为AD的发病机制提供了新的理论依据。