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:
- 金额:$ 15.5万
- 依托单位:
- 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
- 来源:
- 关键词:Alzheimer&aposs DiseaseAmericanAmino AcidsApoptosisAutophagocytosisAutopsyBinding ProteinsBiogenesisBiological AssayBiological ModelsBrainCRISPR/Cas technologyCell LineCell modelCellsClinicalCodeCombined Modality TherapyCyclic AMP-Dependent Protein KinasesCycloheximideDataDegradation PathwayDementiaDevelopmentDideoxy Chain Termination DNA SequencingDiseaseDisease PathwayEndoplasmic ReticulumEquilibriumFailureFosteringFunctional disorderFutureGeneral PopulationGenesGenotypeHaplotypesHealthHomeostasisHumanHuman GeneticsLeadLysosomesMaintenanceMeasuresMicrotubulesMissense MutationMissionMutationNational Institute on AgingNerve DegenerationNeurodegenerative DisordersNeuronsPathologicPathologyPathway interactionsPatientsPersonal SatisfactionPharmaceutical PreparationsPhosphotransferasesPositioning AttributePreparationProblem SolvingProcessProductionProtein BiosynthesisProteinsPublic HealthReagentRegulationResearchRibosomesStressSynapsesTauopathiesTestingTherapeutic AgentsTranslationsTrazodoneTunicamycinbrain cellcompleted suicideeffective therapyendoplasmic reticulum stressgenome wide association studyimprovedinduced pluripotent stem cellmisfolded proteinneuron lossnoveloff-target siteprotein degradationprotein kinase Rproteostasisresponserisk varianttargeted treatmenttau Proteinstau aggregationtherapy developmenttrial design
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,错误折叠蛋白质的积累是病理标志。水平
毒性蛋白质如tau对应于细胞功能障碍并引起神经变性。蛋白水平
通常通过合成和降解途径的平衡小心地维持。但如何
在不同的途径之间协调的稳定状态尚不清楚。一种失调的蛋白质
是微管结合蛋白tau。例如,tau水平升高,
阿尔茨海默氏病死后大脑,这与病理学和临床症状相关。一个候选
能够协调多种途径,控制tau蛋白的产生和降解的是PERK
(蛋白激酶R(PKR)样内质网激酶)。PERK是内质网(ER)的居民,
蛋白质,并在ER应激期间被激活。多发性硬化症患者tau蛋白病的PERK风险变异的鉴定
大型GWA研究强调了理解PERK控制蛋白质的方式的重要性
在健康和疾病中的体内平衡。使用携带人诱导多能干细胞(iPSC)的人诱导多能干细胞系,
PERK风险变体(PERK B)与tau蛋白病相关,作者发现ER应激导致
导致tau蛋白水平升高和神经元死亡PERK B包含三个编码区SNP,导致
在氨基酸位置136、166和704处的非同义错义突变。发现PERK B是一种
功能性低晶型;其激酶活性通过S704 A突变降低。此外,在ER应激期间,
由衣霉素触发,tau蛋白水平,而不是被降低,在纯合子风险增加,
变体(PERK B/B)与对照(PERK A/A)相比。这些数据表明tau蛋白的改变
内稳态由PERK B引起,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。这项建议旨在测试假设,PERK
B通过蛋白质产生和/或清除的变化来失调蛋白质稳态。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
在内质网应激过程中的稳态失调将通过进行蛋白质翻译多聚-
核糖体和SUnSET测定和蛋白降解放线菌酮追踪测定。曲唑酮,一种额外的好处
激活剂,被提议作为神经变性的治疗剂,将被测试以确定其效果
关于PERK A/A和B/B基因型。本建议的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和蛋白质的体内平衡的调节。这项建议是
研究结果将为R 01的制备提供支持
成绩.
项目成果
期刊论文数量(1)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
The Role of PERK in Understanding Development of Neurodegenerative Diseases.
- DOI:10.3390/ijms22158146
- 发表时间:2021-07-29
- 期刊:
- 影响因子:5.6
- 作者:Smedley GD;Walker KE;Yuan SH
- 通讯作者:Yuan SH
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