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.

项目摘要在神经退行性疾病中，错误折叠蛋白的积累是病理标志。水平有毒蛋白（例如tau）对应于细胞功能障碍并引起神经退行性。蛋白质水平通常通过合成和降解途径的平衡仔细维护。但是，如何稳态在不同的途径之间进行了精心策划。一种失调的蛋白质在神经退行性疾病中，是微管结合蛋白tau。例如，tau级别升高验尸大脑后阿尔茨海默氏病，这与病理和临床体征相关。一个候选人这可能能够协调多个途径，控制tau的生产和降解，这是PERK （蛋白激酶R（PKR） - 样性内质网激酶）。 PERK是内质网（ER）居民蛋白质，并在ER应力期间被激活。鉴定多个tauopathy的PERK风险变体大型GWA研究强调了理解振兴蛋白质方式的重要性健康和疾病方面的体内平衡。使用人类诱导的多能干细胞（IPSC）线携带 PERK风险变异（PERK B）与陶氏病有关，作者发现ER应力导致达到tau水平和神经元死亡的升高。 PERK B包含三个编码区SNP，导致氨基酸位置的非同义错义突变136、166和704。发现PERK B是一个功能性低对形；其激酶活性通过S704A突变降低。另外，在ER应力期间由纯合风险增加，由穿刺霉素触发的tau蛋白水平而不是降低与对照（PERK A/A）相比，变体（PERK B/B）。这些数据表明tau蛋白发生了改变由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系列不是等源性的。该提案旨在检验振兴的假设 b通过蛋白质产生和/或清除率的变化使蛋白质稳态失调。目标1提案从IPSC产生携带PERK A/A到B/B和B/B到A/A的同源线。 tau的机制 ER应力期间的稳态失调将通过进行蛋白质翻译来研究核糖体和日落测定和蛋白质降解环己酰亚胺追逐测定法。曲唑酮，振作将作为神经变性的治疗剂提出的激活剂进行测试以确定其效果在PERK A/A和B/B基因型上。该提案的结果将提供改进的线路和细胞模型在ER应力期间，总体上检查TAU和蛋白质稳态的调节系统。该提议是适用于R03机制，结果将为准备未来R01的准备提供支持提交。