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

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）是一种重要的DNA/RNA结合蛋白，在RNA合成中起重要作用。 加工和稳定性。虽然主要是核定位，超过95%的肌萎缩侧索硬化， 硬化症（ALS）病例和多达一半的额颞叶痴呆（FTD）病例显示TDP-43的错误定位 到细胞质。大量证据将TDP-43错误定位与神经变性的发病机制联系起来 在ALS/FTD中，已在家族性ALS病例亚组中鉴定出TDP-43突变，进一步支持了 TDP-43破坏在疾病中的主要作用。然而，ALS/FTD中TDP-43核清除的原因 仍然未知。一个主要障碍是对TDP-43核出口的机制缺乏了解。 直到最近，TDP-43被认为是核输出受体exportin-1（XPO 1）的货物。然而，在这方面， 最近的多项研究表明，XPO 1不结合TDP-43或介导其输出。替代出口 其机制可能包括从细胞核被动扩散，或与RNA主动共输出，例如通过 NXF 1/TREX mRNA输出复合物。我们的初步数据表明，TDP-43核输出是ATP-和RNA-， 依赖，但可能需要的能量上游的孔，TDP-43核内的流动性，而不是 通过核孔复合体（NPC）的转运。事实上，透化细胞分析表明，一旦 TDP-43从核RNA中被置换，可能扩散到核外。此外，NXF 1生长素的研究- 诱导性降解决定子（AID）细胞系表明，TDP-43输出不需要大量mRNA途径。基于 根据这些数据，我们假设TDP-43的ATP依赖性核束缚，最可能是核RNA， 关键决定了它扩散到细胞质中的速率。在拟议的研究中，我们将执行一项 对靶向文库进行阵列式CRISPRi和CRISPRa筛选，以确定能量依赖性 TDP-43的核束缚命中将在核转运和光漂白试验中得到验证， 在ALS/FTD患者组织中评价。然后我们将联合收割机高含量显微镜与核质 转运试验，包括新开发的透化细胞输出试验，以确认 TDP-43在输出过程中穿过核孔移位。其他AID细胞系和敲减研究 将用于明确排除mRNA输出途径的作用。最后，我们将测试 在我们的初步研究中确定的促进TDP-43核保留的新方法的有效性。 测试将包括TDP-43突变果蝇（与Daniela Zarnescu实验室合作），TDP-43突变果蝇（与Daniela Zarnescu实验室合作）， iNeurons和小鼠坐骨神经轴突切断术，以确定我们是否可以在体内促进TDP-43核保留， 以及该策略是否会减弱TDP-43蛋白病的分子和神经病理学特征。 这些研究将使TDP-43的机制和调节的精确机制的理解。 核出口，并推进候选策略的临床前测试，以促进TDP-43核保留。