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: 10274564
• 负责人: Pallavi P. Gopal
• 金额: $ 40.91万
• 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/10274564
• 关键词: ALS patients; Affinity; Amyotrophic Lateral Sclerosis; Animal Model; Axon; Axonal Transport; Biological Assay; Cellular Assay; Clinical; Cytoplasmic Granules; DNA Binding; DNA-Binding Proteins; Data; Dendrites; Disease; Distal; Doctor of Philosophy; Exhibits; Fluorescence Recovery After Photobleaching; Fluorescence Resonance Energy Transfer; Frontotemporal Dementia; Genetic; Genetic Transcription; Genetic Translation; 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; Transact; Transcript; Translations; Work; amyotrophic lateral sclerosis therapy; anterograde transport; base; 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; molecular modeling; mutant; polyglutamine; 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）和额颞叶痴呆症（FTD）是严重的衰弱和致命的 具有重叠的临床、病理和遗传特征的神经退行性疾病。尽管我们的技术取得了进步， 了解ALS/FTD的病理学和遗传学基础， 神经变性仍然知之甚少，目前的治疗方法只能延长几个月的寿命。几乎 所有ALS患者和近一半的FTD患者都有由交互反应组成的病理聚集体， 43 kDa的DNA结合蛋白（TDP-43），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结合蛋白中的突变， 蛋白质引起家族性和散发性ALS/FTD，突出了RNA代谢的改变是常见的致病因素， 神经退行性变的机制最近的研究已经确定了TDP-43和 共济失调蛋白-2是一种RNA结合蛋白，含有一个聚谷氨酰胺（polyQ）区，通常22-23个谷氨酸， 长度Ataxin-2 polyQ束（27-33个谷氨酰胺）扩张增加ALS和ALS-FTD重叠的风险 疾病然而，Ataxin-2 / TDP-43相互作用增加的细胞和分子机制， 疾病风险未知。本提案的目的是确定Ataxin-2 / TDP-1的分子基础。 43相互作用及其对TDP-43依赖性RNA调控的影响，包括RNA剪接和稳定性， 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Ataxin-2是神经元核糖核蛋白颗粒的组分，富含RNA/蛋白质， 这些基因调控mRNA的稳定性、转运和翻译。我们的初步数据显示，Ataxin-2 polyQ扩增破坏TDP-43光漂白后的顺行转运和荧光恢复 含有突变型共济失调蛋白-2的RNA颗粒。总的来说，这些数据支持我们的中心假设： polyQ扩增异常地支架TDP-43 /共济失调蛋白-2相互作用并隔离TDP-43，破坏细胞核 和TDP-43的细胞质功能。我们将使用互补的活细胞 成像和单分子成像方法、单分子FRET、翻译测定和RNA- 测序/转录组学（i）研究共济失调蛋白-2 polyQ扩增对TDP-43转运和后转录的影响。 野生型或共济失调蛋白-2突变神经元中TDP-43靶mRNA的转录调控;以及（ii）鉴定 异常相互作用所需的共济失调蛋白-2和TDP-43结构域以及设计TDP-43的小分子抑制剂 43 /共济失调蛋白-2 polyQ相互作用。这项研究将为以下方面提供新的见解：（1）分子基础 Ataxin-2/TDP-43相互作用增加ALS和ALS-FTD风险，（2）Ataxin-2 polyQ 扩增与TDP-43相互作用，影响转录组和mRNA的时空定位， 神经元，和（3）基于机制的治疗的潜在分子靶点。