Nmnat2改善脑AD样病理和功能变化及机制研究

AD (Alzheimer's disease, AD) is characterized by the formation of extracellular neuritic plaques containing Aβ peptide and intracellular neurofibrillary tangle consisting of abnormal hyperphosphorylated microtubule-associated protein tau. The expression of Nmnat2 significantly decreases in AD patients compared to non-AD controls of the same age. The difference of pathology is conspicuous in different regions of AD brain. Cerebellum suffers from mild damage, but it is severe in hippocampus. Recent data show the expression of Nmnat2 protein is higher in the cerebellum than it in the hippocampus, suggesting Nmnat2 may have neuroprotective effects. Our preliminary results display Nmnat2 levels are significantly decreased in Tg2576 mice compared to non-transgenic littermates control. Interestingly, overexpression of Nmnat2 could alleviate tau proteins phosphorylation and amyloidogenesis, and increase the quantity of dendrites and spines in AD model mice, suggesting that Nmnat2 is closely associated with AD-like lesions, but the mechanism is not clear. Based on this information, we will clarify decrease of Nmnat2 may induce AD-like pathological changes and/or behavioral deficits, and study its mechanisms by using the cell lines (or primary neurons) and Tg2576 mice. Whether it reverses or attenuates the AD-like pathological changes and/or behavioral deficits, we upregulate Nmnat2 levels through virus injection carrying mouse Nmnat2 full-length cDNAs with IRES-enhanced green fluorescent protein (EGFP) or its relevant signaling molecule in Tg2576 mice. This project displays theoretical and practical significance in facilitating understanding the mechanisms of AD and discovering a new target molecules for AD.

神经原纤维缠结和老年斑是阿尔茨海默氏病（AD）特征性病理改变。烟酰胺单核苷酸腺苷酰基转移酶2（Nmnat2）的表达在AD患者明显低于同龄非AD对照，而且Nmnat2高表达的小脑AD样病理变化较轻，提示Nmnat2可能参与AD发病，但机制不清。前期预实验结果显示Nmnat2在AD动物模型转人突变APP小鼠脑明显低于同窝非转基因对照鼠；特异性上调Nmnat2能改善AD模型AD样特征性病理改变如tau蛋白异常磷酸化、Aβ生成以及增加树突分枝与树突棘数量，揭示Nmnat2可能是AD发生的上游重要分子，其作用机制急待阐明。基于此，本课题拟从细胞（细胞系或原代神经元）与整体（转人突变APP小鼠）水平，证明Nmnat2与AD特征性病理改变存在特异性内在联系，并阐明其作用机制；探讨干预Nmnat2或其信号通路是否缓解AD模型鼠AD样病理变化或行为学损伤。通过该课题研究，将为预防和治疗AD提供新思路。

神经原纤维缠结和老年斑是阿尔茨海默氏病（AD）特征性病理改变。烟酰胺单核苷酸腺苷酰基转移酶2（Nmnat2）的表达在AD患者明显低于同龄非AD对照，而且Nmnat2高表达的小脑AD样病理变化较轻，提示Nmnat2可能参与AD发病，但机制不清。本研究将在稳转细胞系和转基因AD鼠上进行。实验将探讨Nmnat2对AD样病理变化Tau蛋白过度磷酸化、Aβ生成以及行为学影响的分子机制。我们构建了人胚肾HEK293稳定表达人全长tau441细胞株（HEK293/tau）以及神经瘤细胞N2a稳定表达人Swedish突变APP695细胞株（N2a/APP），先后构建和制备了Nmnat2的shRNA质粒（siNmnat2）和携带有小鼠Nmnat2基因的高滴度腺相关病毒颗粒（Nmnat2-IRES-EGFP）。研究结果显示：①Nmnat2通过降低PP2A-Tyr307位点的磷酸化，上调了PP2A活性，进而缓解了Tau蛋白多个AD相关位点的磷酸化；② Nmnat2通过提高NAD/NADH比值，增加了AMPK活性，进而上调ADAM10，缓解A生成；③Nmnat2和BDNF相互作用，增加了记忆相关蛋白的表达、树突分枝与树突棘数量，改善了转基因鼠学习记忆。以上研究结果提示Nmnat2在缓解AD样病理变化及转基因鼠行为学损伤中起重要作用，其为Tau病变及AD病人的药物治疗提供理论基础和靶点。