Functions of Tau protein in human neural cells

Tau蛋白在人类神经细胞中的功能

基本信息

批准号：
10658624
负责人：
Jessica Elaine Young
金额：
$ 26.99万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-15 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10658624
关键词：
Affect Alzheimer&apos s Disease Alzheimer&apos s disease related dementia Arsenites Astrocytes Binding Binding Proteins CRISPR/Cas technology Cell Cycle Cell Line Cell Nucleolus Cell Nucleus Cell membrane Cell physiology Cells Cellular Stress Central Nervous System Coculture Techniques Conditioned Culture Media Cytoskeleton DNA Damage DNA Transposable Elements Data Deposition Double-Stranded RNA Electrophysiology (science)Elements Etoposide Frontotemporal Dementia Functional disorder Gene Expression Regulation Genetic Transcription Genotype Goals Heat shock proteins Hippocampus Human Human Cell Line Immunoprecipitation Inflammatory Inherited Knock-out Knockout Mice Laboratories Lead Link Long-Term Potentiation MAPT gene Measures Microtubule Stabilization Microtubules Molecular Mus Mutation Nerve Degeneration Neurodegenerative Disorders Neurofibrillary Tangles Neuroglia Neuroimmune Neurons Organoids Pathologic Pathway interactions Phase Phenotype Physical condensation Physiological Play Proteins Quantitative Reverse Transcriptase PCR RNA Binding RNA metabolism Rattus Reading Frames Regulation Reporting Reproducibility Rodent Model Role Techniques Testing Transformed Cell Line Transmembrane Transport Up-Regulation Work biological adaptation to stress cell type differentiation protocol dominant genetic mutation experimental study gain of function genome editing hyperphosphorylated tau immunocytochemistry induced pluripotent stem cell induced pluripotent stem cell technology knock-down lentiviral-mediated loss of function multi-electrode arrays neural neuroblastoma cell neurodegenerative phenotype neuropathology novel protein function response single-cell RNA sequencing small hairpin RNA stress granule tau Proteins tau aggregation tau expression tau function tau interaction transcriptome transcriptomic profiling

项目摘要

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.

项目摘要/摘要由MAPT基因编码的Tau蛋白是一种神经元富集的蛋白微管结合蛋白。 tau蛋白的异常积累是几种神经病理学标志神经退行性疾病，包括阿尔茨海默氏病（AD）和额叶痴呆症（FTD）。主导的 MAPT基因中的突变存在于遗传的FTD中，表明TAU是神经退行性的因果关系疾病。但是，这些突变是导致功能丧失，功能获得或获得新颖功能未知。尽管MAPT中没有引起遗传AD的突变，但是如何 tau功能的改变导致神经细胞功能障碍和tau蛋白的病理聚集是仍然不完全理解。这些问题尚未得到解决，主要是因为tau的功能蛋白质尚未得出结论。虽然在神经元中高度表达，但tau在其他中枢神经系统细胞类型已与从核仁到质膜的细胞局部定位有关。此外转运，RNA结合和代谢和细胞骨架元素。这共同表明了tau蛋白的作用在中枢神经系统细胞中，微管稳定。许多研究检查了病理tau，但只有有限的研究研究了野生型的细胞功能，内源性tau。在没有TAU的情况下检查细胞表型是一种理解其正常的方法功能。迄今为止，大多数研究都使用啮齿动物模型研究了Tau缺乏症或转化的人类细胞系这些研究表明了各种矛盾的结果。在这项研究中，我们将使用我们的专业知识人类诱导的多能干细胞技术研究人类神经培养和皮质中Tau功能的丧失器官。我们将使用CRISPR/CAS9破坏人类Tau阅读框架，从而产生tau-ko细胞系。我们然后将纯化无偏的转录组分析和基于假设的实验检查分子神经元和星形胶质细胞缺乏Tau表达的身体后果。我们已经产生了飞行员数据表明在tau-ko皮质培养物中涉及神经免疫功能的途径的强烈上调与等源性WT对照相比，我们已经用靶向MAPT的SHRNA验证了我们的发现。我们的提出的实验将使用HIPSC衍生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缺乏表型包括改变的肉芽肿和正如我们的初步研究所建议的，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缺乏神经元的电生理功能用tau缺乏的星形胶质细胞培养。据我们所知，没有关于HIPSC衍生的Tau-Ko的报道神经细胞类型，因此本研究产生的数据有可能显着贡献了解Tau在中枢神经系统中的作用。