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

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

项目摘要/摘要蛋白质聚集是包括阿尔茨海默氏症在内的神经退行性疾病的标志，这些疾病缺乏有效的治疗。此外，我们对分子和细胞缺乏了解控制人脑中蛋白质聚集的机制，这将使新的治疗策略。蛋白质tau是许多神经退行性的大脑聚集体的主要成分包括阿尔茨海默氏病在内的综合称为tauopathies的疾病。虽然许多疾病 - 相关的tau突变是已知的，我们缺乏对序列如何控制的全面理解 tau聚集和错误折叠。此外，tau，dospite在大脑中广泛表达，聚集仅在特定神经元亚型中发作。这种现象称为选择性脆弱性，表明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改变了特定的tau表明确定其在人类神经元中的聚集和错误折叠。在这里，我提出了两种系统的，公正的策略，基于CRISPR的功能基因组学和深度突变扫描（DMS），以全面剖析两个序列如何确定和细胞因子控制tau在IPSC衍生的神经元中的折叠和聚集。这些研究的结果与生物物理和结构性后续实验将确定特定的TAU状态，这些状态对其至关重要聚合。通过在人类神经元中进行系统研究，我独特地定位于确定tau突变和细胞因子的类别，这些因素是tau聚集和错误折叠的基础。我已经进行了重点的原始功能基因组学筛查，并确定了肽基 - 脯氨酸异构酶（PPIASES）FKBP1A和PPIH作为TAU低聚的调节剂。在AIM 1中，我会表征PPIAS FKBP1A和PPIH控制如何与Tau折叠和聚合合作我的共同赞助者比尔·德格拉多（Bill Degrado）博士和合作者卢卡斯·约阿希玛克（Lukasz Joachimak）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。后续研究将确定识别因子如何控制tau构象在聚集道上。 AIM 3使用DMS 全面确定控制tau聚集和错误折叠的序列因子，并具有特定的强调解剖PTM的作用。这些目标的完成将导致全面了解控制tau错误折叠和聚集并揭示了潜在的治疗靶标。