Developing and Validating a Novel Tau Toxicity Model in the Budding Yeast

开发并验证芽殖酵母中的新型 Tau 毒性模型

• 批准号: 10574327
• 负责人: Weiwei Dang
• 金额: $ 16万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10574327
• 关键词: Adopted; Alzheimer' s Disease; Antibodies; Biochemical; Biological Assay; Categories; Cells; Clinical; Collaborations; Complex; Data; Databases; Defect; Deletion Mutation; Dementia; Disease; Drosophila genus; Drug Metabolic Detoxication; Eukaryotic Cell; Expression Library; Fluorescence Resonance Energy Transfer; Frontotemporal Dementia; Future; Galactose; Gene Expression; Genes; Genetic; Genetic Screening; Genetic Transcription; Genomics; Growth; Human; Letters; Measures; Microfluidic Microchips; Microfluidics; Modeling; Molecular; Molecular Conformation; Mutate; Nature; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurons; Open Reading Frames; Parkinson Disease; Pathologic; Pathology; Pathway interactions; Phenotype; Physiological; Pilot Projects; Proteins; Repression; Role; Saccharomyces cerevisiae; Saccharomycetales; Stress; TREX2 gene; Tauopathies; Technology Transfer; Testing; Toxic effect; Validation; Variant; Western Blotting; Yeast Model System; Yeasts; alpha synuclein; biological adaptation to stress; cell type; cytotoxic; cytotoxicity; detection sensitivity; differential expression; fluorescence imaging; fly; genetic variant; knock-down; mRNA Export; misfolded protein; model organism; mutant; novel; overexpression; protein aggregation; protein complex; protein expression; protein misfolding; response; stressor; tau Proteins; tau aggregation; tau conformation; tau expression; tau-1; tool; transcriptome; transcriptome sequencing; yeast genetics

PROJECT SUMMARY Many forms of human dementia are caused by aberrantly folded proteins and protein aggregates. Overexpressing these disease proteins, such as α-synuclein and TBD-43, in the budding yeast Saccharomyces cerevisiae, also causes cytotoxicity, allowing unbiased powerful genetic screens for modifiers that enhance or repress toxicity. Hence, this single-celled eukaryotic model has been a powerful genetic tool for discovering molecular pathways regulating human dementia caused by protein misfolding and aggregation, such as the Parkinson’s disease and frontotemporal dementia. Neurofibrillary tangles (NFTs) composed of hyperphosphorylated and aggregated Tau proteins are a major pathological hallmark of Alzheimer’s disease (AD), as well as other neurodegenerative disorders collectively termed Tauopathies. Despite the strong clinical association, how wildtype human Tau proteins become hyperphosphorylated and cytotoxic remains poorly understood. Distinguished from other protein aggregates that cause dementia, overexpressing human Tau is well tolerated in the budding yeast with no apparent growth phenotype, even though human tau proteins are also hyperphosphorylated and aggregated in yeast cells. Hence, no yeast genetic screen for Tau toxicity modifiers has been performed thus far. Yet, tremendous potential remains if Tau-induced cytotoxicity can be reproduced in the simple yeast model, permitting unbiased screens to uncover the molecular pathways involved in tau toxicity and related diseases. In a preliminary screen, we have identified several ORF deletion mutations that show toxicity when human wildtype Tau (2N4R) is expressed. Interestingly, two of these mutant genes, THP1 and SAC3, encoding for subunits of the evolutionarily conserved TREX2 complex, are involved in activating stress response gene expression and mRNA export. Hence, we hypothesize that timely stress response at the transcription level is necessary for yeast cells to antagonize the toxicity induced by misfolded Tau. It is also possible that Tau proteins in thp1∆ and sac3∆ mutants adopt a cytotoxic conformation/state reminiscent of the situation of Tau pathology in neurons. Indeed, our analysis of the NIAGADS database uncovered that AD-associated genetic variants were found in four of the five subunits of the human TREX2 complex, making it one of the top 15 protein complexes enriched for AD variants. For this small pilot project, we will 1) investigate the cause of Tau toxicity in strains mutated for TREX2 subunits using a combination of genetics, genomics and biochemical approaches; 2) develop a set of tools that can help establish a novel Tau toxicity model in the budding yeast, allowing future unbiased screens for genetic modifiers of Tau toxicity.

项目总结 许多形式的人类痴呆症是由异常折叠的蛋白质和蛋白质聚集体引起的。 α-突触核蛋白和TbD-43等病害蛋白在芽殖酵母中的高效表达 酿酒酵母也会引起细胞毒性，允许无偏见的强大的基因筛选，以增强或 抑制毒性。因此，这种单细胞真核细胞模型已经成为发现 蛋白质错误折叠和聚集引起的调节人类痴呆的分子途径，如 帕金森氏症和额颞部痴呆。 由过度磷酸化和聚集的Tau蛋白组成的神经原纤维缠结(NFTs)是一种 阿尔茨海默病(AD)以及其他神经退行性疾病的主要病理特征 统称为牛顿病症。尽管有很强的临床相关性，野生型人Tau蛋白 变得过度磷酸化，细胞毒性仍然鲜为人知。区别于其他蛋白质 导致痴呆的聚集体，过表达人Tau在没有NO的萌芽酵母中很好地耐受 明显的生长表型，即使人类tau蛋白也被过度磷酸化并聚集在 酵母细胞。因此，到目前为止，还没有对Tau毒性修饰剂进行酵母遗传筛选。然而， 如果Tau诱导的细胞毒性能够在简单的酵母模型中复制，那么仍然存在巨大的潜力， 允许无偏见的筛选，以揭示与tau毒性和相关疾病有关的分子途径。 在初步筛查中，我们已经确定了几个ORF缺失突变，当人类 表达了野生型Tau(2N4R)。有趣的是，其中两个突变基因，THP1和SAC3，编码 进化上保守的TREX2复合体的亚基参与激活应激反应基因 表达和信使核糖核酸输出。因此，我们假设在转录水平上及时的应激反应 是酵母细胞对抗错误折叠的Tau的毒性所必需的。也有可能是 TH1∆和Sac3∆突变体中的tau蛋白采用了一种细胞毒性构象/状态，这使人想起 神经元中的tau病理。事实上，我们对NIAGADS数据库的分析发现，与AD相关的基因 在人类TREX2复合体的五个亚基中的四个亚基中发现了变异，使其成为排名前15的蛋白质之一 富含AD变异的复合体。 对于这个小的试点项目，我们将1)调查TREX2突变菌株中Tau毒性的原因 使用遗传学、基因组学和生化方法相结合的亚基；2)开发一套工具， 可以帮助在萌芽酵母中建立新的Tau毒性模型，从而允许未来对基因 Tau毒性的修饰剂。