Development of isogenic iPSC cell lines expressing tauopathy-related PERK risk variants to study homeostatic control of protein levels during ER stress

开发表达 tau 蛋白病相关 PERK 风险变异的同基因 iPSC 细胞系，以研究 ER 应激期间蛋白质水平的稳态控制

基本信息

批准号：
10323680
负责人：
Shauna H Yuan
金额：
$ 15.5万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-01-15 至 2022-12-31
项目状态：
已结题

项目摘要

Project Summary In neurodegenerative diseases, accumulation of misfolded proteins is the pathological hallmark. Levels of the toxic proteins, such as tau, correspond to cellular dysfunctions and cause neurodegeneration. Protein levels are normally carefully maintained by the balance of the synthetic and degradative pathways. However, how the steady state is orchestrated between the different pathways is not clear. One protein that is dysregulated in neurodegenerative diseases is the microtubule-binding protein tau. For example, tau levels are elevated in the Alzheimer’s disease post-mortem brain, and this correlate with pathology and clinical signs. One candidate that may be able to coordinate multiple pathways, controlling both production and degradation of tau, is PERK (protein kinase R (PKR)-like endoplasmic reticulum kinase). PERK is an endoplasmic reticulum (ER) resident protein, and is activated during ER stress. The identification of a PERK risk variant for tauopathy in multiple large GWA studies underscores the importance of understanding the ways that PERK controls protein homeostasis in health and in disease. Using human induced pluripotent stem cell (iPSC) lines carrying a PERK risk variant (PERK B) associated with tauopathies, the authors made the discovery that ER stress leads to an elevation in tau levels and neuronal death. PERK B contains three coding-region SNPs resulting in nonsynonymous missense mutations at amino acid positions 136, 166 and 704. It was found that PERK B is a functional hypomorph; its kinase activity is reduced via the S704A mutation. Additionally, during ER stress triggered by tunicamycin, tau protein levels, instead of being reduced, are increased in the homozygous risk variant (PERK B/B) compared to the control (PERK A/A). These data suggest an alteration in tau protein homeostasis caused by PERK B, but the involvement of other genes could not be ruled out, because the PERK B/B and PERK A/A iPSC lines were not isogenic. This proposal seeks to test the hypothesis that PERK B dysregulates protein homeostasis, via changes in protein production and/or clearance. Aim 1 proposes to generate isogenic lines from iPSC carrying PERK A/A to B/B and B/B to A/A. The mechanisms of tau homeostatic dysregulation during ER stress will be investigated by performing protein translation poly- ribosome and SUnSET assay and protein degradation cycloheximide chase assay. Trazodone, a PERK activator which is proposed as a therapeutic agent for neurodegeneration, will be tested to determine its effect on PERK A/A and B/B genotypes. The results of this proposal will provide improved lines and cell model system to examine regulation of homeostasis of tau and proteins in general during ER stress. This proposal is suitable for the R03 mechanism, for the results will provide the support for the preparation of a future R01 submission.

项目概要在神经退行性疾病中，错误折叠蛋白质的积累是其病理标志。有毒蛋白质，例如 tau，会导致细胞功能障碍并导致神经变性。通常是通过合成途径和降解途径的平衡来小心维持的。不同途径之间协调的稳态尚不清楚。神经退行性疾病中的关键因素是微管结合蛋白 tau，例如，tau 蛋白水平升高。阿尔茨海默病死后大脑，这与病理学和临床症状相关。 PERK 可能能够协调多种途径，控制 tau 的产生和降解（蛋白激酶 R (PKR) 样内质网激酶）。蛋白，并在 ER 应激过程中被激活鉴定多种 tau 蛋白病的 PERK 风险变异。大型 GWA 研究强调了解 PERK 控制蛋白质方式的重要性使用携带a的人类诱导多能干细胞（iPSC）系来维持健康和疾病中的稳态。 PERK 风险变异 (PERK B) 与 tau蛋白病相关，作者发现 ER 应激会导致 PERK B 包含三个编码区 SNP，导致 tau 水平升高和神经死亡。 136、166和704位氨基酸的非同义错义突变。发现PERK B是一个功能性亚型；在 ER 应激过程中，其激酶活性也会通过 S704A 突变而降低。由衣霉素触发，tau 蛋白水平非但没有降低，反而增加了纯合风险与对照 (PERK A/A) 相比，变体 (PERK B/B) 这些数据表明 tau 蛋白发生了变化。 PERK B 引起的稳态，但不能排除其他基因的参与，因为 PERK B/B 和 PERK A/A iPSC 系不是同基因的。该提案旨在检验 PERK 的假设。 B 通过改变蛋白质产生和/或清除来调节蛋白质稳态。从携带 PERK A/A 到 B/B 和 B/B 到 A/A 的 iPSC 生成同基因系 tau 的机制。内质网应激期间的稳态失调将通过进行蛋白质翻译多聚来研究核糖体和 SUnSET 测定以及蛋白质降解放线菌酮追踪测定（PERK）。被提议作为神经退行性疾病治疗剂的激活剂将进行测试以确定其效果关于 PERK A/A 和 B/B 基因型的研究结果将提供改进的细胞系和细胞模型。系统来检查 ER 应激期间 tau 蛋白和蛋白质的稳态调节。适合R03机制，该结果将为未来R01的准备工作提供支持提交。