Tau蛋白异常导致学习记忆损害的分子机制研究

The intracellular accumulation of abnormal tau protein induced learning and memory deficits. Tau is a cytoskeleton protein, however, the underlying mechanisms of memory deficit induced by tau is still unclear. The applicants found that overexpression of tau induced the site-specific phosphorylation of tau accumulated in the nucleus. Moreover, the activities of 10 transcription factors had a significant change, especially, the activity of signal transducer and activator of transcription-1 (STAT1) increased to 7.6 fold compared with the control while tau overexpression. The increased STAT1 activity was also proved by gel shift assay. Recently, studies have reported that STAT1 negatively regulated the cognitive ability, and there was a cognitive correlation between STAT1 protein level and synaptic protein NR1/NR2B levels. Above all, the applicants speculated that the intracellular accumulation of abnormal tau protein may activate STAT1 to inhibit the expression of synaptic/memory-related proteins and impair the learning and memory functions. The purpose of this project is to investigate whether learning and memory impairment induced by the site-specific phosphorylation of tau is through activating STAT1, and its underlying mechanisms in the cell lines and mouse models by gene transfection, behavior tests and electrophysiology, and so on. The results will be helpful to discover the new function of tau, and reveal the molecular mechanisms of the learning and memory deficits induced by the abnormal accumulation of tau protein. The results will also provide new molecular target for protecting against tau-related cognitive deficits.

Tau蛋白在细胞内异常聚积损害学习记忆功能。然而，tau是一种细胞骨架蛋白，对其损伤记忆的机制尚不清楚。申请者最近发现：过表达tau导致胞核中特定位点磷酸化的tau聚积，同时，10个转录因子水平有显著变化，其中，STAT1（信号转导及转录激活蛋白1）的转录活性升高到7.6倍；采用凝胶迁移实验证实了STAT1活性升高。由于STAT1升高与突触蛋白NR1/2B水平负相关，且负性调节学习记忆功能，我们推测，特定位点磷酸化tau蛋白在胞内聚积可能通过上调STAT1活性，抑制突触或/和记忆相关靶基因表达，从而损害学习记忆功能。本项目拟通过基因转染、行为学、电生理等技术，在细胞和小鼠整体水平探讨特定位点磷酸化tau蛋白是否通过上调STAT1的活性而损伤学习记忆，并阐明相关分子机制。研究结果可能发现骨架蛋白tau的新功能，揭示tau损害学习记忆的分子机制，并为保护tau相关疾病的认知功能提供新分子靶标。

背景：阿尔茨海默病(Alzheimer’s disease，AD)是当今神经退行性病变流行病学最为广泛的一种痴呆症，现今仍然没有有效的诊断与治疗手段。AD典型的病理学特征之一是hTau蛋白异常聚集形成的神经原纤维缠结（Neurofibrillary tangels，NFTs）。大量研究表明hTau蛋白异常聚集可以诱发突触毒性与神经退行病变，但是分子机制并不清楚。我们前期实验发现hTau能诱发广泛的基因转录紊乱，追随该线索我们对于hTau模型中转录因子进行筛查，探究其对突触毒性的影响与分子机制。.目的：探讨转录因子STAT1在AD发病中诱发突触毒性作用及分子机制.方法：在hTau异常聚集细胞模型中，进行高通量转录因子活性检测及转录组芯片检测，筛查并验证参与tau病理学改变的分子机制。利用12月龄hTau转基因鼠与海马感染hTau过表达病毒小鼠，AD患者脑组织，验证转录因子STAT1在整体中的蛋白水平与磷酸化水平的改变。进一步探究，转录因子STAT1激活而诱发突触毒性的分子机制。我们利用过表达hTau蛋白腺相关病毒，感染海马CA3区探究细胞内异常聚集的tau蛋白对AD样突触毒性的影响，对小鼠的学习记忆，突触蛋白转录与表达，突触可塑性进行检测。再次，采用生物信息学预测分析STAT1可能结合并调控的靶基因，应用染色质免疫共沉淀，启动子荧光素报告等实验，确认STAT1调控NMDARs基因转录的机制。最终，利用STAT1失活突变病毒与条件性敲除方法，恢复hTau突触毒性，探究干预STAT1转录活性的治疗意义。.结果：在过表达hTau蛋白的HEK293细胞中，我们发现520种mRNA水平有显著性变化；同时在转录因子活性筛查中发现STAT1转录活性显著激活。于12月龄hTau转基因鼠与海马感染hTau过表达病毒小鼠，AD患者脑组织均发现STAT1的激活，包括其核内聚集增多，磷酸化上调等。STAT1的条件性敲除能显著性逆转hTau诱发的学习记忆障碍，突触相关蛋白NMDARs的转录与表达抑制，突触可塑性下降。染色质免疫共沉淀发现STAT1能直接结合NMDARs启动子；荧光素报告基因发现其对NMDARs有转录抑制作用。并在接下来的激酶筛查中发现hTau激活JAK2，而后者介导了STAT1的磷酸化上调与活化。最终利用STAT1-Y701F失活突变病毒抑制其激活，逆转hTau诱发突触毒性作用