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

• 批准号: 10285495
• 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
• 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/10285495
• 关键词: Affinity; Amyotrophic Lateral Sclerosis; Attenuated; Autopsy; Auxins; Axotomy; Binding; Biological Assay; Blood - brain barrier anatomy; CRISPR interference; Cell Line; Cell Nucleus; Cells; Cellular Assay; Chemistry; Collaborations; Complex; Consumption; Custom; Cytoplasm; DNA; Data; Defect; Diffuse; Diffusion; Disease; Dominant-Negative Mutation; Dose; Drosophila genus; 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; Structure; Temperature; Testing; Therapeutic; Therapeutic Agents; Tissues; Treatment Efficacy; Uridine; Validation; analog; base; 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; Affinity; Amyotrophic Lateral Sclerosis; Attenuated; Autopsy; Auxins; Axotomy; Binding; Biological Assay; Blood - brain barrier anatomy; CRISPR interference; Cell Line; Cell Nucleus; Cells; Cellular Assay; Chemistry; Collaborations; Complex; Consumption; Custom; Cytoplasm; DNA; Data; Defect; Diffuse; Diffusion; Disease; Dominant-Negative Mutation; Dose; Drosophila genus; 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; Structure; Temperature; Testing; Therapeutic; Therapeutic Agents; Tissues; Treatment Efficacy; Uridine; Validation; analog; base; 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

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体内TDP-43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。