RNA-based regulation of TDP-43 nuclear export in ALS/FTD

ALS/FTD 中基于 RNA 的 TDP-43 核输出调控

基本信息

批准号：
10640898
负责人：
Lindsey Renae Hayes
金额：
$ 52.01万
依托单位：
JOHNS HOPKINS 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：
2021-08-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10640898
关键词：
Affinity Amyotrophic Lateral Sclerosis Attenuated Autopsy Auxins Axotomy Binding Biological Assay CRISPR interference CRISPR-mediated transcriptional activation Cell Line Cell Nucleus Cells Cellular Assay Chemistry Collaborations Complex Custom Cytoplasm DNA Data Defect Diffuse Diffusion Disease Dominant-Negative Mutation Dose Drosophila genus Energy consumption Exclusion Frontotemporal Dementia In Vitro Injections Label Libraries Link Mass Spectrum Analysis Mediating Messenger RNA Microscopy Modeling Molecular Molecular Chaperones Mus Mutation Nerve Crush Nerve Degeneration Neurons Nuclear Nuclear Export Nuclear Pore Nuclear Pore Complex Nuclear RNA Oligonucleotides Pathogenesis Pathway interactions Patients Permeability Photobleaching Pilot Projects Play Preclinical Testing Proteins RNA RNA Binding RNA Processing RNA Splicing RNA Stability RNA-Binding Proteins Regulation Role Small Interfering RNA Spliceosomes Temperature Testing Therapeutic Therapeutic Agents Tissues Treatment Efficacy Uridine Validation analog blood-brain barrier crossing drug development exportin 1 protein familial amyotrophic lateral sclerosis frontotemporal lobar dementia amyotrophic lateral sclerosis helicase in vivo knock-down mRNA Export mutant neuropathology neuroprotection novel strategies nucleocytoplasmic transport nucleoside analog pre-clinical prevent protein TDP-43 receptor sciatic nerve subcutaneous therapeutic evaluation therapeutic target transcriptome sequencing

项目摘要

TAR DNA-binding protein-43 (TDP-43) is an essential DNA/RNA-binding protein that plays a major role in RNA processing and stability. Although predominantly nuclear in localization, over 95% of amyotrophic lateral sclerosis (ALS) cases and up to half of frontotemporal dementia (FTD) cases show mislocalization of TDP-43 to the cytoplasm. Substantial evidence links TDP-43 mislocalization to the pathogenesis of neurodegeneration in ALS/FTD, and TDP-43 mutations have been identified in subset of familial ALS cases, further supporting a primary role for TDP-43 disruption in disease. However, the cause of TDP-43 nuclear clearance in ALS/FTD remains unknown. A major barrier has been lack of understanding of the mechanism of TDP-43 nuclear export. Until recently, TDP-43 was thought to be a cargo of the nuclear export receptor, exportin-1 (XPO1). However, multiple recent studies demonstrate that XPO1 does not bind TDP-43 or mediate its export. Alternative export mechanisms may include passive diffusion from the nucleus, or active co-export with RNA, such as via the NXF1/TREX mRNA export complex. Our preliminary data show that TDP-43 nuclear export is ATP- and RNA- dependent, but the energy may be required upstream of the pore, for TDP-43 intranuclear mobility, rather than translocation across the nuclear pore complex (NPC). Indeed, a permeabilized cell assay suggests that, once displaced from nuclear RNA, TDP-43 likely diffuses out of the nucleus. Moreover, studies in an NXF1 auxin- inducible degron (AID) cell line suggest that the bulk mRNA pathway is not required for TDP-43 export. Based on these data, we hypothesize that ATP-dependent nuclear tethering of TDP-43, most likely to nuclear RNA, critically dictates the rate at which it diffuses into the cytoplasm. In the proposed studies, we will perform an arrayed CRISPRi and CRISPRa screen of a targeted library to determine the mechanism of energy-dependent nuclear tethering of TDP-43. Hits will be validated in nuclear transport and photobleaching assays, and also evaluated in ALS/FTD patient tissue. We will then combine high content microscopy with nucleocytoplasmic transport assays, including a newly developed permeabilized cell export assay, to confirm the mechanism of TDP-43 translocation across the nuclear pore during export. Additional AID cell lines and knockdown studies will be used to definitively exclude a role for the mRNA export pathway. Finally, we will test the therapeutic efficacy of a novel approach to promote TDP-43 nuclear retention, identified during our preliminary studies. Testing will include TDP-43 mutant Drosophila (in collaboration with the Daniela Zarnescu lab), TDP-43 mutant iNeurons, and mouse sciatic nerve axotomy, to determine if we can promote TDP-43 nuclear retention in vivo, and whether this strategy will attenuate molecular and neuropathologic features of TDP-43 proteinopathy. These studies will enable a precise mechanistic understanding of the mechanism and regulation of TDP-43 nuclear export, and advance preclinical testing of a candidate strategy to promote TDP-43 nuclear retention.

TAR DNA结合蛋白-43（TDP-43）是必不可少的DNA/RNA结合蛋白，在RNA中起主要作用处理和稳定性。虽然主要是核定核的，但超过95％的肌萎缩性侧面硬化病（ALS）病例和多达一半的额颞痴呆（FTD）病例显示TDP-43的静置错误到细胞质。大量证据将TDP-43错误定位与神经退行性的发病机理联系起来在ALS/FTD中，在家族性ALS病例的子集中已经确定了TDP-43突变，进一步支持A TDP-43破坏在疾病中的主要作用。但是，ALS/FTD中TDP-43核清除率的原因仍然未知。主要的障碍是缺乏对TDP-43核出口机理的理解。直到最近，TDP-43被认为是核出口受体Exportin-1（XPO1）的货物。然而，最近的多项研究表明，XPO1不结合TDP-43或介导其出口。替代出口机制可能包括从细胞核中被动扩散，或与RNA共同出口，例如通过 NXF1/Trex mRNA导出复合物。我们的初步数据表明，TDP-43核输出是ATP和RNA- 依赖性，但可能需要孔的上游，对于TDP-43核内迁移率，而不是跨核孔复合物（NPC）的易位。确实，透化细胞测定法表明，一次 TDP-43从核RNA中移位，可能会从细胞核中扩散。此外，对NXF1生长素的研究可诱导的degron（AID）细胞系表明TDP-43导出不需要大量mRNA途径。基于在这些数据上，我们假设TDP-43的ATP依赖性核束缚，最有可能是核RNA，批判性地决定了它扩散到细胞质中的速率。在拟议的研究中，我们将执行有针对性库的Arryed Crispri和Crispra屏幕，以确定能量依赖的机理 TDP-43的核束缚。点击将在核运输和光漂白测定中得到验证，也将在ALS/FTD患者组织中进行评估。然后，我们将将高含量显微镜与核细胞质结合在一起运输分析，包括新开发的通透性细胞出口测定法，以确认出口期间，TDP-43跨核孔的易位。其他援助细胞线和敲低研究将用于明确排除mRNA输出途径的作用。最后，我们将测试治疗性在我们的初步研究中确定的一种新型方法来促进TDP-43核保留率的功效。测试将包括TDP-43突变果蝇（与Daniela Zarnescu实验室合作），TDP-43突变体无神经元和小鼠坐骨神经轴切开术，以确定我们是否可以促进TDP-43体内核保留率，以及该策略是否会减弱TDP-43蛋白质病的分子和神经病理特征。这些研究将使对TDP-43的机制和调节的机理和调节有了精确的机械理解核出口，并预先对促进TDP-43核保留的候选策略进行临床前测试。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Nuclear RNA binding regulates TDP-43 nuclear localization and passive nuclear export.

DOI：
10.1016/j.celrep.2022.111106
发表时间：
2022-07-19
期刊：
CELL REPORTS
影响因子：
8.8
作者：
Duan, Lauren;Zaepfel, Benjamin L.;Aksenova, Vasilisa;Dasso, Mary;Rothstein, Jeffrey D.;Kalab, Petr;Hayes, Lindsey R.
通讯作者：
Hayes, Lindsey R.

RNA-mediated ribonucleoprotein assembly controls TDP-43 nuclear retention.

RNA 介导的核糖核蛋白组装控制 TDP-43 核保留。

DOI：
10.1371/journal.pbio.3002527
发表时间：
2024
期刊：
PLoS biology
影响因子：
9.8
作者：
DosPassos,PatriciaM;Hemamali,ErandikaH;Mamede,LohanyD;Hayes,LindseyR;Ayala,YunaM
通讯作者：
Ayala,YunaM

Order from chaos: Using CSF proteomics to predict ALS progression.