Elucidating mechanisms of ATXN2 interaction with TDP-43 in human motor neuron models of Amyotrophic Lateral Sclerosis

阐明肌萎缩侧索硬化症人类运动神经元模型中 ATXN2 与 TDP-43 相互作用的机制

• 批准号: 10223698
• 负责人: Stanley Pernell Gill
• 金额: $ 3.96万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10223698
• 关键词: ALS patients; Adult; Affect; Amyotrophic Lateral Sclerosis; Apoptosis; Biological; Biology; Cell Nucleus; Cell model; Cells; Characteristics; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Cytoplasm; DNA-Binding Proteins; Data; Degenerative Disorder; Disease; Dose; Exposure to; Fractionation; Future; Genes; Genetic; Goals; Half-Life; Human; Immunofluorescence Immunologic; Immunoprecipitation; In Vitro; Knock-out; Learning; Lesion; Light; Longevity; Mass Spectrum Analysis; Mediating; Messenger RNA; Metabolic Clearance Rate; Metabolism; Modeling; Motor; Motor Neuron Disease; Motor Neurons; Mus; Nature; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Orthologous Gene; Pathogenicity; Phenotype; Play; Process; Proteins; Publishing; RNA; RNA Binding; RNA Processing; RNA Splicing; RNA metabolism; RNA-Binding Proteins; Reporting; Role; SCA2 protein; Series; Testing; Therapeutic; Toxic effect; Transact; Transgenic Organisms; Yeasts; base; crosslink; excitatory neuron; exhaustion; experimental study; gain of function; in vivo; induced pluripotent stem cell; insight; mRNA Stability; neuroprotection; overexpression; prevent; protein aggregation; recruit; response; screening; stress granule; stressor

Project Summary/Abstract Amyotrophic Lateral Sclerosis is an incurable, rapidly progressing motor neuron degenerative disease with few treatment options. Although the cause of ALS remains poorly understood, one unifying phenotype that arises in approximately 97% of ALS patients, regardless of the presence of an associated genetic lesion, is the formation of insoluble protein aggregates containing Transactive Response DNA-binding protein 43 (TDP-43) in the cytoplasm of degenerating motor neurons. In yeast, primary neurons, and human cell models, TDP-43 overexpression has been shown to be sufficient to recapitulate similar TDP-43 aggregation and subsequent cellular apoptosis. Previous screening studies have identified Ataxin-2 (ATXN2) as a dose-dependent modifier of this TDP-43 overexpression-mediated toxicity. In support of this finding, our preliminary data suggest that knockout of ATXN2 by CRISPR is sufficient to protect iPSCs and differentiating motor neuron cultures from TDP- 43 overexpression-related toxicity. However, no published study to date has deeply examined the mechanism of why this protection occurs upon ATXN2 knockout. This proposal seeks to identify a mechanism by which ATXN2 loss mitigates TDP-43 proteinopathy-mediated toxicity in human motor neurons. Our group and others have reported that both TDP-43 and ATXN2 preferentially interact with proteins involved in mRNA processing and metabolism, including each other. Thus, we firstly hypothesize that ATXN2 loss provides protection from TDP-43 associated toxicity by altering mRNA metabolism and processing of TDP-43. Aim 1 will test how TDP-43 metabolism is changed upon ATXN2 knockout compared to baseline. In particular, we will examine the effect of ATXN2 loss on TDP-43 mRNA levels, mRNA stability, protein levels, and protein stability in the context of baseline TDP-43 levels and TDP-43 overexpression. Secondly, we hypothesize that ATXN2 loss provides protection from TDP-43 associated toxicity by altering interactions between TDP-43 and its target RNAs. In Aim 2, we will explore how ATXN2 loss may provide neuroprotection by changing how TDP-43 interacts with its RNA substrates. We will accomplish this goal in two parts. First (Aim 2.1), we will examine the changes in mRNAs that TDP-43 interacts with in the nucleus and cytoplasm of human motor neurons in the context of ATXN2 loss compared to baseline using Fractionation iCLIP sequencing. Second (Aim 2.2), because both ATXN2 and TDP-43 are recruited to stress granules where mRNAs and RNA binding proteins are transiently sequestered, we will evaluate changes in motor neuron stress granule dynamics and localization that occur in the context of ATXN2 knockout compared to baseline. We predict that these mechanisms shield neurons from detrimental TDP-43 gain of function-related effects. The goal of this proposed project is to provide insight into the role ATXN2 plays in the metabolism of TDP-43 and TDP-43 targets so we may learn more about the underlying biology of TDP-43 associated neurodegeneration that affects nearly all ALS patients. We hope that our findings inform future studies in evaluating actionable therapeutic mechanisms for treating ALS.

项目总结/摘要 肌萎缩侧索硬化症是一种无法治愈的、进展迅速的运动神经元退行性疾病， 治疗方案。虽然ALS的病因仍然知之甚少，但一个统一的表型出现在 大约97%的ALS患者，无论是否存在相关的遗传性病变，都是ALS的形成。 不溶性蛋白质聚集体含有反式反应DNA结合蛋白43（TDP-43）， 退化的运动神经元的细胞质。在酵母、原代神经元和人类细胞模型中，TDP-43 已经显示过表达足以重现类似的TDP-43聚集和随后的 细胞凋亡先前的筛选研究已经将Ataxin-2（ATXN 2）确定为剂量依赖性修饰剂 这种TDP-43过表达介导的毒性。为了支持这一发现，我们的初步数据表明， 通过CRISPR敲除ATXN 2足以保护iPSC和分化运动神经元培养物免于TDP-1。 43过度表达相关毒性。然而，迄今为止还没有发表的研究深入研究了这一机制。 为什么ATXN 2敲除后会发生这种保护。这项建议旨在确定一种机制， ATXN 2损失减轻了人运动神经元中TDP-43蛋白质病介导的毒性。我们集团和其他 已经报道TDP-43和ATXN 2都优先与参与mRNA加工的蛋白质相互作用 和新陈代谢，包括彼此。因此，我们首先假设ATXN 2丢失提供了保护 通过改变TDP-43的mRNA代谢和加工来避免TDP-43相关毒性。目标1将测试 与基线相比，ATXN 2敲除后TDP-43代谢如何变化。特别是，我们将研究 ATXN 2缺失对TDP-43 mRNA水平、mRNA稳定性、蛋白水平和蛋白稳定性的影响， 基线TDP-43水平和TDP-43过表达的背景。其次，我们假设ATXN 2丢失 通过改变TDP-43与其受体之间的相互作用， 靶RNA。在目标2中，我们将探索ATXN 2缺失如何通过改变TDP-43 与其RNA底物相互作用。我们将分两部分实现这一目标。首先（目标2.1），我们将检查 TDP-43与人运动神经元的细胞核和细胞质相互作用的mRNA的变化， 使用分级iCLIP测序，与基线相比，ATXN 2损失的背景。第二（目标2.2），因为 ATXN 2和TDP-43都被募集到应激颗粒中，在应激颗粒中mRNA和RNA结合蛋白被瞬时地 隔离，我们将评估运动神经元应激颗粒动力学的变化和发生在 与基线相比，ATXN 2敲除的背景。我们预测，这些机制保护神经元免受 有害的TDP-43获得功能相关的影响。本项目的目标是提供深入了解 ATXN 2在TDP-43代谢和TDP-43靶点中的作用，因此我们可以更多地了解 TDP-43相关神经变性的潜在生物学影响几乎所有ALS患者。我们希望 我们的发现为将来评估ALS可行治疗机制的研究提供了信息。