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: 10400026
• 负责人: Stanley Pernell Gill
• 金额: $ 3.45万
• 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 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10400026
• 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.

项目摘要/摘要 肌萎缩性侧索硬化症是一种无法治愈的，迅速发展的运动神经元退化性疾病，很少 治疗选择。尽管ALS的原因仍然很熟悉，但在 无论存在相关遗传病变如何，大约97％的ALS患者是形成 含有交易反应DNA结合蛋白43（TDP-43）的不溶性蛋白聚集体的 退化运动神经元的细胞质。在酵母中，原发性神经元和人类细胞模型TDP-43 已证明过表达足以概括类似的TDP-43聚集，然后 细胞凋亡。先前的筛查研究已将ataxin-2（ATXN2）鉴定为剂量依赖性修饰符 在TDP-43过表达介导的毒性中。为了支持这一发现，我们的初步数据表明 CRISPR敲除ATXN2的敲除足以保护IPSC，并将运动神经元培养物与TDP-区分开 43与过表达相关的毒性。但是，迄今为止尚未发表的研究深入研究了该机制 为什么这种保护在ATXN2敲除时进行。该建议旨在确定一种机制 ATXN2损失减轻了TDP-43蛋白质病介导的人类运动神经元的毒性。我们的小组和其他人 已经报道TDP-43和ATXN2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ATXN2损失提供了保护 通过改变mRNA代谢和TDP-43的加工，从TDP-43相关的毒性。 AIM 1将测试 与基线相比，ATXN2敲除时TDP-43代谢如何改变。特别是，我们将检查 ATXN2损失对TDP-43 mRNA水平，mRNA稳定性，蛋白质水平和蛋白质稳定性的影响 基线TDP-43级别和TDP-43过表达的上下文。其次，我们假设ATXN2损失 通过改变TDP-43及其ITS之间的相互作用，提供了TDP-43相关毒性的保护 目标RNA。在AIM 2中，我们将探讨ATXN2损失如何通过更改TDP-43的方式提供神经保护作用 与其RNA底物相互作用。我们将分为两个部分实现这一目标。首先（AIM 2.1），我们将检查 TDP-43与人类运动神经元的细胞核和细胞质相互作用的mRNA变化 与基线相比，使用分级ICLIP测序与基线相比，ATXN2损失的上下文。第二（AIM 2.2），因为 ATXN2和TDP-43均被募集到瞬时mRNA和RNA结合蛋白的应力颗粒 隔离，我们将评估运动神经元应力颗粒动力学和本地化的变化 与基线相比，ATXN2敲除的上下文。我们预测这些机制使神经元免受 有害的TDP-43功能相关效应的增益。这个拟议项目的目的是提供有关 ATXN2在TDP-43和TDP-43目标的代谢中扮演的角色，因此我们可以了解更多有关 TDP-43相关神经退行性的潜在生物学影响几乎所有ALS患者。我们希望那样 我们的发现为未来的研究提供了评估治疗ALS的可操作治疗机制的研究。