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

基本信息

批准号：
10450875
负责人：
Pallavi P. Gopal
金额：
$ 40.97万
依托单位：
YALE UNIVERSITY
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：
2021-07-15 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10450875
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

项目摘要

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) 会导致严重衰弱和致命具有重叠临床、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 的病理学和遗传基础以及潜在的细胞机制人们对神经退行性疾病仍知之甚少，目前的治疗方法只能延长生命几个月。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 DNA 和 RNA 结合蛋白，在 RNA 稳定性中具有多种作用，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 ALS/FTD，突显 RNA 代谢改变是一种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 Ataxin-2，一种 RNA 结合蛋白，含有聚谷氨酰胺 (polyQ) 束，通常含有 22-23 个谷氨酰胺长度。 Ataxin-2 polyQ 束（27-33 谷氨酰胺）的扩展增加了 ALS 和 ALS-FTD 重叠的风险疾病。然而，Ataxin-2 / TDP-43 相互作用增强的细胞和分子机制疾病风险未知。该提案的目的是确定 Ataxin-2 / TDP- 的分子基础 43 相互作用及其对 TDP-43 依赖性 RNA 调节的影响，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成部分，富含RNA/蛋白质调节 mRNA 稳定性、运输和翻译的区室。我们的初步数据表明 Ataxin-2 PolyQ 扩展破坏 TDP-43 光漂白后的顺行运输和荧光恢复含有突变 Ataxin-2 的 RNA 颗粒。总的来说，这些数据支持我们的中心假设：Ataxin-2 PolyQ 扩增异常地支架 TDP-43/Ataxin-2 相互作用并隔离 TDP-43，破坏核 TDP-43 和细胞质功能。我们将使用互补的活细胞来测试这个中心假设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 Ataxin-2 polyQ 扩展对 TDP-43 转运和后处理的影响野生型或 Ataxin-2 突变神经元中 TDP-43 靶标 mRNA 的转录调控； (ii) 确定 Ataxin-2 和 TDP-43 结构域是异常相互作用和设计 TDP-小分子抑制剂所需的 43/Ataxin-2 polyQ 相互作用。拟议的研究将为（1）分子基础提供新的见解 Ataxin-2/TDP-43 相互作用导致 ALS 和 ALS-FTD 风险增加，(2) Ataxin-2 polyQ 如何扩展与 TDP-43 相互作用，影响 mRNA 的转录组和时空定位神经元，以及（3）基于机制的治疗的潜在分子靶点。