Mechanistic dissection of novel regulators of TDP-43 aggregation identified in a genome-wide CRISPR-Cas9 knockout screen
全基因组 CRISPR-Cas9 敲除筛选中发现的 TDP-43 聚集新型调节因子的机制剖析
基本信息
- 批准号:10404620
- 负责人:
- 金额:$ 3.42万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2020
- 资助国家:美国
- 起止时间:2020-06-01 至 2023-05-30
- 项目状态:已结题
- 来源:
- 关键词:ALS patientsAddressAffectAlzheimer&aposs DiseaseAmyotrophic Lateral SclerosisAstrocytesBindingBiologyCRISPR/Cas technologyCell LineCellsCellular MorphologyClientCoculture TechniquesCollaborationsCoupledCyclic AMP-Dependent Protein KinasesDataDevelopmentDiffuseDiseaseDisease ProgressionDissectionDouble-Stranded RNAEnzyme-Linked Immunosorbent AssayFluorescence-Activated Cell SortingGenerationsGenesGenetic Predisposition to DiseaseGlobal ChangeGoalsHomeostasisImmuneImmune responseImmune signalingImportinsIn VitroIncidenceInterferon-alphaKnock-outLinkMass Spectrum AnalysisMeasuresMediatingMessenger RNAMethyltransferaseMicrogliaMindModelingModificationMolecular ChaperonesMotor NeuronsMutationNeurodegenerative DisordersNuclearNuclear ExportPathogenesisPathologicPathologyPathway interactionsPhasePhysiologic pulsePrevalenceProteinsQuality ControlRNARNA BindingRNA methylationRNA-Binding ProteinsRegulationReporterRibosomal RNARoleSignal PathwaySignal TransductionSmall RNAStainsStructureTestingTherapeuticTimeToxic effectTranslationsWestern BlottingWorkYeast Model SystemYeastsbasecrosslinking and immunoprecipitation sequencingdisease-causing mutationfollow-upfrontotemporal degenerationgenome-widein vitro testinginterestnovelpreventprotein TDP-43protein kinase Rshape analysistherapeutically effective
项目摘要
Project Summary
The mislocalization and aggregation of RNA-binding proteins (RBP) is a pathological hallmark of Amyotrophic
Lateral Sclerosis (ALS). The RBP TAR DNA-binding protein 43 (TDP-43) is of particular interest, as pathological
aggregation of TDP-43 is observed in nearly 97% of ALS patients, despite the fact that disease-causing
mutations in TDP-43 explain fewer than 5% of disease incidence. This observation suggests that misregulation
or mutation of other genes converge on TDP-43 pathology, however it remains poorly understood how the
aggregates arise or whether they can be reversed or prevented. To this end, we have developed Pulse-Shape
Analysis-based aggregation reporters for the TDP-43 that, when coupled to fluorescence activated cell sorting,
quantifies aggregation at the single cell level. This reporter was leveraged against a genome-wide CRISPR-
Cas9 knockout screen to identify regulators of TDP-43 aggregation. Reassuringly, this screen revealed known
interactors and pathways involved in TDP-43 regulation and pathology. Additionally, this work revealed several
novel proteins not previously implicated in TDP-43 biology or aggregation. Initial follow up on one top hit, SRRD,
revealed that this protein reduces TDP-43 aggregation in a mammalian aggregation model, and reduces TDP-
43-associated toxicity in a yeast model, indicating that these genes indeed modify TDP-43 aggregation.
The goal of this proposal is to mechanistically dissect how these novel regulators modify TDP-43
aggregation. Using an induced motor neuron model of TDP-43 aggregation, yeast toxicity models, and in vitro
studies, I will assess the interactors, signaling pathways, and mode of TDP-43 modification for the following four
genes: METTL5, EIF2AK2, XPO4, and SRRD. This work will not only reveal the function of uncharacterized
proteins, but dissect their heretofore unknown mechanisms in regulating TDP-43 aggregation. Through better
understanding of how TDP-43 aggregation is regulated, the field can begin to answer questions regarding the
role of TDP-43 aggregation in disease pathogenesis and progression.
By understanding the regulatory network of underlying TDP-43 aggregation, we can begin to
therapeutically modulate the pathways that contribute to disease.
项目摘要
RNA结合蛋白(RNA binding proteins,RBP)的错误定位和聚集是肌萎缩性侧索硬化的病理标志
侧索硬化症(ALS)。RBP TAR DNA结合蛋白43(TDP-43)是特别感兴趣的,因为病理性
在近97%的ALS患者中观察到TDP-43的聚集,尽管事实上,
TDP-43突变解释了不到5%的疾病发病率。这一观察结果表明,
或其他基因的突变会聚在TDP-43病理学上,然而,仍然不清楚这些基因是如何突变的。
是否会出现聚合物,或者是否可以逆转或防止聚合物。为此,我们开发了脉冲形状
TDP-43的基于分析的聚集报告基因,当与荧光激活细胞分选偶联时,
在单细胞水平上定量聚集。这位记者利用全基因组CRISPR-
Cas9敲除筛选以鉴定TDP-43聚集的调节剂。令人欣慰的是,这个屏幕显示了已知的
参与TDP-43调节和病理学的相互作用物和途径。此外,这项工作还揭示了几个
以前未涉及TDP-43生物学或聚集的新蛋白质。对一个热门案件SRRD的初步跟进,
揭示了这种蛋白质在哺乳动物聚集模型中减少TDP-43聚集,并减少TDP-43聚集。
43-在酵母模型中的相关毒性,表明这些基因确实修饰TDP-43聚集。
这项提案的目标是机械地剖析这些新的调节剂如何修饰TDP-43
聚合来使用TDP-43聚集的诱导运动神经元模型、酵母毒性模型和体外
研究,我将评估的相互作用,信号通路,TDP-43的修改模式,为以下四个
基因:胃L5、EIF 2AK 2、XPO 4和SRRD。这项工作不仅将揭示功能的uncharacterized
蛋白质,但剖析其迄今未知的机制,在调节TDP-43聚集。通过更好
了解TDP-43聚集是如何调节的,该领域可以开始回答有关
TDP-43聚集在疾病发病机制和进展中的作用。
通过了解潜在TDP-43聚集的调控网络,我们可以开始
治疗调节导致疾病的途径。
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)
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Katelyn Sweeney其他文献
Katelyn Sweeney的其他文献
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{{ truncateString('Katelyn Sweeney', 18)}}的其他基金
Mechanistic dissection of novel regulators of TDP-43 aggregation identified in a genome-wide CRISPR-Cas9 knockout screen
全基因组 CRISPR-Cas9 敲除筛选中发现的 TDP-43 聚集新型调节因子的机制剖析
- 批准号:
10268168 - 财政年份:2020
- 资助金额:
$ 3.42万 - 项目类别:
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