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：
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
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/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

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.

肌萎缩性侧索硬化症（ALS）和额颞痴呆（FTD）令人衰弱和致命神经退行性疾病具有重叠的临床，病理和遗传特征。尽管我们的进步了解ALS/FTD的病理和遗传基础，该基础是细胞机制神经变性仍然很少了解，目前的治疗只会延长几个月的寿命。几乎所有ALS患者和近一半的FTD患者均具有由交易反应组成的病理骨料 43 kDa（TDP-43）的DNA结合蛋白，一种DNA和RNA结合蛋白，在RNA稳定性中具有多个作用，剪接和转录后RNA处理。此外，TDP-43和其他RNA结合中的突变蛋白质引起家族性和零星ALS/FTD，突出了RNA代谢改变为常见的致病作用神经退行性的机理。最近的研究确定了TDP-43和 Ataxin-2，一种RNA结合蛋白，其中包含聚谷氨酰胺（Polyq）裂纹，通常在通常22-23个谷氨酰胺中长度。 Ataxin-2 Polyq道（27-33谷氨酰胺）的膨胀增加了ALS和ALS-FTD重叠的风险疾病。然而，ataxin-2 / tdp-43相互作用增加的细胞和分子机制疾病风险未知。该建议的目的是确定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。在初步和发表的工作中，我们和其他人发现TDP-43和ataxin-2是神经核核蛋白颗粒，富RNA/蛋白质的成分调节mRNA稳定性，运输和翻译的隔室。我们的初步数据表明ataxin-2 TDP-43光漂白后，PolyQ扩展破坏了顺行传输和荧光恢复含有突变型催化剂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扩展异常支架TDP-43 / Ataxin-2相互作用和隔离TDP-43，破坏了核 TDP-43的细胞质功能。我们将使用互补的活细胞检验此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传输和后的影响野生型或ataxin-2突变神经元中TDP-43目标mRNA的转录调节；（ii）确定异常相互作用所需的ataxin-2和TDP-43结构域，并设计了TDP-的小分子抑制剂 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相互作用赋予ALS和ALS-FTD风险，（2）ataxin-2 polyq如何扩展与TDP-43相互作用，以影响mRNA在中的转录组和时空定位神经元和（3）基于机理疗法的潜在分子靶标。