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

项目摘要/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，以及这些 疾病缺乏有效的治疗方法。此外，我们缺乏对分子和细胞的了解 控制人类大脑中蛋白质聚集的机制，这将使新的治疗方法 战略。 在许多神经退行性疾病中，tau蛋白是大脑中聚集体的主要成分。 疾病，统称为焦虑症，包括阿尔茨海默氏症。尽管有一些疾病- 相关的tau突变是已知的，我们缺乏对序列控制的全面了解 Tau的聚集和错误折叠。此外，尽管tau在大脑中广泛表达，但它会聚集 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， 提示细胞因子的差异表达在tau错误折叠和 聚合。我假设细胞因素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、不偏不倚的策略，基于CRISPR的功能基因组学和Deep 突变扫描(DMS)，全面剖析序列决定因素和细胞因素 控制tau在ipsc来源的神经元中的错误折叠和聚集。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，这些状态对其 聚合。通过在人类神经元中进行系统研究，我处于独特的地位 确定tau突变的类别和tau聚集和错误折叠背后的细胞因素。 我已经进行了一次有重点的原理验证功能基因组学筛查，并鉴定出了多肽- 脯氨酸异构酶(PPIase)FKBP1a和PIH作为tau齐聚的调节因子。在《目标1》中，我将 描述PPIase FKBP1A和PPIH如何控制tau错误折叠和聚集 我的联合赞助人Bill DeGrado博士和合作者Lukasz Joachimiak博士。AIM 2使用功能基因组学 确定控制tau聚集和错误折叠的细胞因素的方法。后续研究将 确定已确定的因素如何控制tau构象在聚集的途径上。AIM 3使用DMS来 全面确定控制tau聚集和错误折叠的序列因素，并具有特定的 重点剖析了PTM的作用。这些目标的完成将导致一个全面的 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。

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.