Functions of Tau protein in human neural cells

项目摘要/摘要 Tau蛋白由MAPT基因编码，是一种富含神经元的蛋白质，具有公认的 微管结合蛋白。Tau蛋白的异常积聚是几种 神经退行性疾病，包括阿尔茨海默病(AD)和额颞痴呆(FTD)。占优势 遗传性FTD中存在MAPT基因突变，表明tau与神经退行性变有关。 疾病。然而，无论这些突变是导致功能丧失、功能获得还是获得一种新的 函数未知。此外，尽管MAPT中没有导致遗传性AD的突变，但如何 Tau功能的改变导致神经细胞功能障碍和tau蛋白的病理性聚集 仍然不能完全理解。这些问题没有得到解决，很大程度上是因为tau的职能 蛋白质还没有确定下来。虽然tau在神经元中高表达，但在其他神经元中也有表达 中枢神经系统细胞类型，并与从核仁到质膜的细胞定位有关。 此外，tau相互作用的蛋白还表现出一系列的细胞功能，包括基因调节、膜 运输、RNA结合和代谢以及细胞骨架元素。综上所述，这暗示了tau蛋白的作用。 在中枢神经系统细胞中，微管不稳定。许多研究都检验了这种药物的作用 病理性tau，但只有有限数量的研究调查了野生型的细胞功能， 内源性tau。在缺乏tau的情况下检查细胞表型是了解其正常状态的一种方法 功能。到目前为止，大多数研究都是利用啮齿动物模型或转化的人类细胞系来研究tau缺乏。 这些研究显示了各种各样的结果，有时甚至是相互矛盾的。在这项研究中，我们将利用我们的专业知识 人类诱导多能干细胞技术在人类神经培养和皮质中研究tau功能的丧失 有机化合物。我们将使用CRISPR/Cas9来干扰人类tau阅读框架，产生tau-KO细胞系。我们 然后将进行无偏见的转录图谱和基于假设的实验来检验分子 以及缺乏tau表达的神经元和星形胶质细胞的生理后果。我们已经产生了飞行员 数据显示，在tau-ko皮层培养中，参与神经免疫功能的通路强烈上调 与等基因的WT对照相比，我们已经用针对MAPT的shRNA验证了我们的发现。我们的 拟议的实验将使用HiPSC来源的神经元和星形胶质细胞来测试细胞自主和非细胞 对tau缺乏的自主反应。这些tau缺乏症的表型包括粒细胞停滞改变和 我们的初步研究表明，双链RNA的积累。我们还将评估 用野生型星形胶质细胞和野生型神经元培养的tau缺陷型神经元的电生理功能 用缺乏tau的星形胶质细胞培养。据我们所知，在hPSC来源的细胞中还没有tau-KO的报道 神经细胞类型，因此这项研究产生的数据有可能对 了解tau在中枢神经系统中的作用。

Project Summary/Abstract Tau protein, encoded by the MAPT gene, is a neuronally enriched protein with an established role as a microtubule-binding protein. Abnormal accumulation of tau protein is a neuropathological hallmark of several neurodegenerative diseases, including Alzheimer’s disease (AD) and frontotemporal dementia (FTD). Dominant mutations in the MAPT gene are present in inherited FTD, indicating that tau is causal in neurodegenerative disease. However, whether these mutations lead to loss-of-function, gain-of-function or the acquisition of a novel function is unknown. Furthermore, although there are no mutations in MAPT that cause inherited AD, how alterations in tau function contribute to dysfunction in neural cells and pathological aggregation of tau protein are still incompletely understood. These issues have not been resolved, largely because the functions of the tau protein have not been conclusively determined. While highly expressed in neurons, tau is expressed in other CNS cell types and has been linked to cellular localizations ranging from the nucleolus to the plasma membrane. Furthermore tau-interacting proteins display a range of cellular functions including gene regulation, membrane transport, RNA binding and metabolism and cytoskeletal elements. Together this suggests a role for tau protein in central nervous system cells beyond microtubule stabilization. Many studies have examined the effects of pathological tau, but only a limited number of studies have investigated the cellular functions of wild type, endogenous tau. Examining cellular phenotypes in the absence of tau is one approach to understand its normal function. To date, most studies have studied tau deficiency using rodent models or transformed human cell lines and these studies show various and sometimes conflicting results. In this study, we will use our expertise in human induced pluripotent stem cell technology to study loss of tau function in human neural cultures and cortical organoids. We will use CRISPR/Cas9 to disrupt the human tau reading frame, generating tau-KO cell lines. We will then pursue both unbiased transcriptomic profiling and hypothesis-based experiments examining molecular and physiological consequences in neurons and astrocytes deficient in tau expression. We have generated pilot data indicating a strong up-regulation of pathways involved in neuroimmune function in tau-KO cortical cultures compared to isogenic WT controls and we have validated our findings with shRNAs targeting MAPT. Our proposed experiments will use hiPSC-derived neurons and astrocytes to test cell autonomous and non-cell autonomous responses to tau deficiency. These tau deficiency phenotypes include altered granulostasis and the accumulation of double-stranded RNA, as suggested by our preliminary studies. We will also assess electrophysiological function of tau deficient neurons cultured with wild-type astrocytes and wild-type neurons cultured with tau deficient astrocytes. To our knowledge, there are no reports of tau-KO in hiPSC-derived neural cell types, therefore the data generated by this study has the potential to significantly contribute to understanding the role of tau in the central nervous system.