Mechanisms of tau misfolding in neurons elucidated by deep mutational scanning and CRISPR screening

通过深度突变扫描和 CRISPR 筛选阐明神经元中 tau 蛋白错误折叠的机制

• 批准号: 9761340
• 负责人: Avi Jacob Samelson
• 金额: $ 6.12万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-17 至 2022-06-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9761340
• 关键词: Alzheimer' s Disease; Antibodies; Binding Sites; Biology; Biophysics; Brain; CRISPR screen; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Disease; Environment; Follow-Up Studies; Future; Genes; Genomic approach; Goals; Human; In Vitro; Institution; Isomerase; Kinetics; Mass Spectrum Analysis; Measures; Molecular; Molecular Conformation; Mutation; Neurodegenerative Disorders; Neurons; Onset of illness; Pathway interactions; Patients; Play; Positioning Attribute; Proline-Rich Domain; Property; Proteins; Proteolysis; Refractory; Research; Research Personnel; Role; Structure; Tauopathies; Techniques; Time; Toxic effect; Training; Treatment Efficacy; Work; base; career; crosslink; cytotoxicity; data integration; differential expression; experimental study; follow-up; functional genomics; functional plasticity; genome-wide; in vivo; induced pluripotent stem cell; insight; knock-down; loss of function; mutation screening; new therapeutic target; novel therapeutic intervention; prevent; protein aggregation; protein folding; tau Proteins; tau aggregation; tau conformation; tau mutation; therapeutic target

PROJECT SUMMARY/ABSTRACT Protein aggregation is a hallmark of neurodegenerative diseases, including Alzheimer’s, and these diseases lack effective therapeutics. Moreover, we lack an understanding of the molecular and cellular mechanisms controlling protein aggregation in the human brain, which would enable new therapeutic strategies. The protein tau is the major constituent of aggregates in the brain in a number of neurodegenerative diseases, collectively called tauopathies, including Alzheimer’s Disease. Although a number of disease- associated tau mutations are known, we lack a comprehensive understanding of how sequence controls tau aggregation and misfolding. Furthermore, tau, despite being widely expressed in the brain, aggregates at disease-onset in only specific neuronal subtypes. This phenomenon, called selective vulnerability, suggests that differential expression of cellular factors plays a key role in tau misfolding and aggregation. I hypothesize that cellular factors and sequence changes control specific tau states that determine its aggregation and misfolding in human neurons. Here, I propose two systematic, unbiased strategies, CRISPR-based functional genomics and Deep Mutational Scanning (DMS), to comprehensively dissect how both sequence determinants and cellular factors control tau misfolding and aggregation in iPSC-derived neurons. Integration of results from these studies with biophysical and structural follow-up experiments will determine specific tau states that are essential for its aggregation. By performing systematic studies in human neurons, I am uniquely positioned to determine classes of tau mutations and cellular factors that underlie tau aggregation and misfolding. I have performed a focused proof-of-principle functional genomics screen and identified the peptidyl- proline isomerases (PPIases) FKBP1A and PPIH as regulators of tau oligomerization. In Aim 1, I will characterize how the PPIases FKBP1A and PPIH control tau misfolding and aggregation in collaboration with my co-sponsor Dr. Bill DeGrado and collaborator Dr. Lukasz Joachimiak. Aim 2 uses a functional genomics approach to identify cellular factors that control tau aggregation and misfolding. Follow-up studies will determine how identified factors control tau conformations on-pathway to aggregation. Aim 3 uses DMS to comprehensively determine the sequence factors that control tau aggregation and misfolding, with a specific emphasis on dissecting the role of PTMs. Completion of these aims will result in a comprehensive understanding of the underlying cellular factors and sequence determinants that control tau misfolding and aggregation, and reveal potential therapeutic targets.

项目总结/摘要 蛋白质聚集是神经退行性疾病的标志，包括阿尔茨海默氏症，这些 疾病缺乏有效的治疗方法。此外，我们缺乏对分子和细胞的理解， 控制蛋白质聚集在人脑中的机制，这将使新的治疗 战略布局 tau蛋白是许多神经退行性疾病中脑中聚集体的主要成分。 疾病，统称为tau蛋白病，包括阿尔茨海默病。虽然有一些疾病- 相关的tau突变是已知的，我们缺乏对序列如何控制 tau聚集和错误折叠。此外，尽管tau蛋白在大脑中广泛表达， 仅在特定的神经元亚型中发病。这种现象被称为选择性脆弱性， 表明细胞因子的差异表达在tau蛋白错误折叠中起关键作用， 聚合来我假设细胞因子和序列变化控制特定的tau状态， 确定其在人类神经元中的聚集和错误折叠。 在这里，我提出了两个系统的，无偏见的策略，CRISPR为基础的功能基因组学和深度 突变扫描（DMS），全面剖析序列决定簇和细胞因子 在iPSC衍生的神经元中控制tau错误折叠和聚集。将这些研究的结果与 生物物理和结构后续实验将确定特定的tau状态，这是必不可少的， 聚合来通过对人类神经元进行系统的研究，我处于独特的地位， 确定tau蛋白突变的类别和导致tau蛋白聚集和错误折叠的细胞因子。 我已经进行了一个集中的原理验证功能基因组学筛选，并确定了肽基- 脯氨酸异构酶（PPIases）FKBP 1A和PPIH作为tau寡聚化的调节剂。在目标1中，我将 表征PPIases FKBP 1A和PPIH如何与以下协同控制tau错误折叠和聚集： 我的共同赞助人比尔·德格拉多博士和合作者卢卡斯·约奇米亚克博士。Aim 2使用功能基因组学 鉴定控制tau聚集和错误折叠的细胞因子的方法。后续研究将 确定所鉴定的因子如何控制tau构象在聚集的途径上。目标3使用DMS， 全面确定控制tau聚集和错误折叠的序列因素， 强调剖析PTM的作用。这些目标的实现将导致一个全面的 了解控制tau错误折叠的潜在细胞因子和序列决定因素， 聚集，并揭示潜在的治疗靶点。