Role of Ataxin-2 polyglutamine expansion on TDP-43 transport and post-transcriptional RNA regulation in neurons

Ataxin-2 聚谷氨酰胺扩增对神经元 TDP-43 转运和转录后 RNA 调节的作用

• 批准号: 10653704
• 负责人: Pallavi P. Gopal
• 金额: $ 41.44万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-15 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10653704
• 关键词: ALS patients; Affinity; Amyotrophic Lateral Sclerosis; Animal Model; Axon; Axonal Transport; Binding; Biological Assay; Cellular Assay; Clinical; Cytoplasm; Cytoplasmic Granules; Cytoskeleton; DNA Binding; DNA-Binding Proteins; Data; Dendrites; Disease; Distal; Doctor of Philosophy; Exhibits; Fluorescence Recovery After Photobleaching; Fluorescence Resonance Energy Transfer; Frontotemporal Dementia; Genetic; Glutamine; Goals; Human; Immunofluorescence Immunologic; Immunoprecipitation; Knock-in Mouse; Knock-out; Knowledge; Label; Length; Life; Link; Maintenance; Measures; Messenger RNA; Molecular; Molecular Target; Motor Neuron Disease; Motor Neurons; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nuclear; Oligonucleotides; Pathogenesis; Pathogenicity; Pathologic; Pathology; Patients; Photobleaching; Post-Transcriptional RNA Processing; Post-Transcriptional Regulation; Property; Proteins; Publishing; Puromycin; RNA; RNA Binding; RNA Recognition Motif; RNA Splicing; RNA Stability; RNA metabolism; RNA-Binding Proteins; Regulation; Research; Ribonucleoproteins; Risk; Role; SCA2 protein; Solubility; Techniques; Testing; Toxic effect; Transcript; Translations; Work; amyotrophic lateral sclerosis therapy; anterograde transport; biophysical properties; crosslink; dementia risk; design; disorder risk; frontotemporal lobar dementia amyotrophic lateral sclerosis; imaging approach; in vivo; induced pluripotent stem cell; insight; interdisciplinary approach; knock-down; live cell imaging; mRNA Stability; mRNA Translation; molecular dynamics; molecular modeling; mutant; neuron component; polyglutamine; posttranscriptional; protein TDP-43; response; scaffold; single molecule; single-molecule FRET; small molecule; small molecule inhibitor; spatiotemporal; sporadic amyotrophic lateral sclerosis; transcriptome; transcriptome sequencing; transcriptomics; translation assay

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are profoundly debilitating and fatal neurodegenerative diseases with overlapping clinical, pathologic and genetic features. Despite advances in our understanding of the pathology and genetic basis of ALS/FTD, the cellular mechanisms underlying neurodegeneration remain poorly understood, and current treatments extend life for only a few months. Almost all ALS patients and nearly half of FTD patients have pathologic aggregates composed of transactive response DNA-binding protein of 43 kDa (TDP-43), a DNA and RNA-binding protein with multiple roles in RNA stability, splicing and post-transcriptional RNA processing. Moreover, mutations in TDP-43 and other RNA-binding proteins cause familial and sporadic ALS/FTD, highlighting altered RNA metabolism as a common pathogenic mechanism of neurodegeneration. Recent studies have identified genetic interactions between TDP-43 and Ataxin-2, an RNA-binding protein that contains a polyglutamine (polyQ) tract normally 22-23 glutamines in length. Expansions of the Ataxin-2 polyQ tract (27-33 glutamines) increase risk for ALS and ALS-FTD overlap disease. However, the cellular and molecular mechanisms by which Ataxin-2 / TDP-43 interactions increase disease risk are unknown. The objective of this proposal is to determine the molecular basis of Ataxin-2 / TDP- 43 interactions and their impact on TDP-43 dependent RNA regulation, including RNA splicing and stability as well as spatiotemporal localization and translation of mRNA. In preliminary and published work, we and others find that TDP-43 and Ataxin-2 are components of neuronal ribonucleoprotein granules, RNA/protein-rich compartments that regulate mRNA stability, transport and translation. Our preliminary data show that Ataxin-2 polyQ expansions disrupt anterograde transport and fluorescence recovery after photobleaching of TDP-43 RNA granules that contain mutant Ataxin-2. Collectively, these data support our central hypothesis: Ataxin-2 polyQ expansions aberrantly scaffold TDP-43 / Ataxin-2 interactions and sequester TDP-43, disrupting nuclear and cytoplasmic functions of TDP-43. We will test this central hypothesis using complementary live-cell imaging and single-molecule imaging approaches, single-molecule FRET, translation assays, and RNA- sequencing/transcriptomics (i) to study the effect of Ataxin-2 polyQ expansions on TDP-43 transport and post- transcriptional regulation of TDP-43 target mRNAs in wild-type or Ataxin-2 mutant neurons; and (ii) to identify Ataxin-2 and TDP-43 domains required for aberrant interaction and to design small molecule inhibitors of TDP- 43 / Ataxin-2 polyQ interactions. The proposed research will provide new insights into (1) the molecular basis of Ataxin-2/TDP-43 interactions that confer increased ALS and ALS-FTD risk, (2) how Ataxin-2 polyQ expansions interact with TDP-43 to impact the transcriptome and spatiotemporal localization of mRNA in neurons, and (3) potential molecular targets for mechanism-based therapies.

肌萎缩侧索硬化症(ALS)和额颞叶痴呆(FTD)是严重的致命疾病 具有重叠的临床、病理和遗传特征的神经退行性疾病。尽管我们的 对ALS/FTD的病理和遗传学基础、潜在的细胞机制的理解 神经退行性变仍然知之甚少，目前的治疗方法只能延长几个月的生命。差不多了 所有ALS患者和近一半的FTD患者都有由反式反应组成的病理集合体 43 kDa的DNA结合蛋白(TDP-43)是一种在RNA稳定性中具有多种作用的DNA和RNA结合蛋白， 剪接和转录后RNA加工。此外，TDP-43和其他RNA结合的突变 蛋白质导致家族性和散发性的ALS/FTD，突出了RNA代谢改变是一种常见的致病因素 神经退变的机制。最近的研究已经确定了TDP-43和TDP-43之间的遗传相互作用 Aaxin-2是一种RNA结合蛋白，它含有一个多聚谷氨酰胺(PolyQ)通路，通常含有22-23个谷氨酸。 长度。Aaxin-2多聚Q区(27-33个谷氨酸)的扩展增加了ALS和ALS-FTD重叠的风险 疾病。然而，Aaxin-2/TDP-43相互作用增加的细胞和分子机制 疾病风险是未知的。这项建议的目的是确定Aaxin-2/TDP-2的分子基础。 43相互作用及其对依赖TDP-43的RNA调节的影响，包括RNA剪接和稳定性，如 以及mRNA的时空定位和翻译。在初步和已发表的工作中，我们和其他人 发现TDP-43和Aaxin-2是富含RNA/蛋白质的神经元核糖核蛋白颗粒的组成部分 调节信使核糖核酸稳定性、运输和翻译的间隔室。我们的初步数据显示，Aaxin-2 多聚Q扩张破坏TDP-43光漂白后的顺行转运和荧光恢复 含有突变型Aaxin-2的RNA颗粒。总的来说，这些数据支持我们的中心假设：Aaxin-2 多聚Q扩张异常地支架TDP-43/Aaxin-2相互作用和隔离TDP-43，破坏核 以及TDP-43的细胞质功能。我们将使用互补的活细胞来验证这一中心假设 成像和单分子成像方法、单分子FRET、翻译分析和RNA- 测序/转录组学(I)研究Aaxin-2多聚体的扩增对TDP-43转运和后转录的影响 野生型或Aaxin-2突变型神经元中TDP-43靶向mRNAs的转录调控；以及(Ii)鉴定 Aaxin-2和TDP-43结构域是异常相互作用和设计TDP-2小分子抑制剂所必需的 43/Aaxin-2多聚Q相互作用。这项拟议的研究将为(1)分子基础提供新的见解 Aaxin-2/TDP-43相互作用导致ALS和ALS-FTD风险增加，(2)Aaxin-2 PolyQ如何 扩增产物与TDP-43相互作用影响大鼠心肌细胞转录组学和时空定位 神经元，以及(3)基于机制的治疗的潜在分子靶点。

项目成果

Sequence Determinants of TDP-43 Ribonucleoprotein Condensate Formation and Axonal Transport in Neurons.

• DOI: 10.3389/fcell.2022.876893
• 发表时间: 2022
• 期刊: Frontiers in cell and developmental biology
• 影响因子: 5.5

Ataxin-2 polyglutamine expansions aberrantly sequester TDP-43, drive ribonucleoprotein condensate transport dysfunction and suppress local translation.

Ataxin-2 聚谷氨酰胺扩增异常地隔离 TDP-43，导致核糖核蛋白凝聚物转运功能障碍并抑制局部翻译。

• DOI: 10.1101/2023.01.30.526372
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Wijegunawardana,Denethi;Vishal,SonaliS;Venkatesh,Neha;Gopal,PallaviP
• 通讯作者: Gopal,PallaviP