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

项目摘要 在神经退行性疾病中，错误折叠的蛋白质堆积是病理特征。的级别 有毒蛋白质，如tau，对应于细胞功能障碍，并导致神经退化。蛋白质水平 通常由合成和降解途径的平衡小心地维持。然而，如何 不同通路之间的稳态是否协调尚不清楚。一种调节失调的蛋白质 在神经退行性疾病中，是微管结合蛋白tau。例如，tau的水平在 阿尔茨海默病的身体大脑，这与病理和临床体征相关。一位候选人 这可能能够协调多条途径，同时控制tau的产生和降解，这是额外的 (蛋白激酶R(PKR)样内质网状激酶)。佩克是内质网(ER)的居民 蛋白质，并在内质网应激时被激活。多发性肌萎缩侧索硬化症的PERK风险变量的识别 GWA的大型研究强调了了解PERK控制蛋白质的方式的重要性 健康和疾病中的动态平衡。使用携带A基因的人诱导多能干细胞(IPSC)系 与肌萎缩侧索硬化症相关的额外风险变量(PERK B)，作者发现内质网应激导致 Tau水平的升高和神经元的死亡。PERK B包含三个编码区SNP，导致 氨基酸第136、166和704位的非同义错义突变。研究发现，福利B是一种 功能性低晶型；其激酶活性因S704A突变而降低。此外，在内质网应激期间 由衣霉素引发的tau蛋白水平在纯合子风险中不是降低，而是增加。 变异(津贴B/B)与对照(津贴A/A)比较。这些数据表明tau蛋白发生了变化。 PERK B引起的动态平衡，但不能排除其他基因的参与，因为 PERK B/B和PERK A/A IPSC系不是等基因的。这项提议试图检验这样一种假设，即 B通过改变蛋白质的产生和/或清除来调节蛋白质的稳态。目标1建议 从携带perk A/A到B/B和B/B到A/A的IPSC产生等基因系。tau的机制 内质网应激期间的动态平衡失调将通过进行蛋白质翻译聚合来研究。 核糖体日落试验和蛋白质降解放线菌素追赶试验。曲唑酮，一项福利 激活剂被建议作为神经退行性变的治疗剂，将进行测试以确定其效果 PERK A/A和B/B两种基因型。这项建议的结果将提供改进的线路和细胞模型 检测内质网应激期间tau和蛋白质的动态平衡调节的系统。这项建议是 适用于R03机制，因为研究结果将为今后R01的编制提供支持 呈件。

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.