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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：
10455671
负责人：
Lindsey Renae Hayes
金额：
$ 51.19万
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/10455671
关键词：
Affinity Amyotrophic Lateral Sclerosis Attenuated Autopsy Auxins Axotomy Binding Biological Assay Blood - brain barrier anatomy CRISPR interference CRISPR-mediated transcriptional activation 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 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

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）是一种重要的DNA/RNA结合蛋白，在RNA中起重要作用