The role of neuronal mRNA transport granule packaging in ALS/FTD

神经元 mRNA 转运颗粒包装在 ALS/FTD 中的作用

• 批准号: 10271529
• 负责人: Veronica Hanley Ryan
• 金额: --
• 依托单位: U.S. NATIONAL INST/NEURO/DS/STROKE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10271529
• 关键词: role; neuronal; mRNA; transport; granule

PROJECT SUMMARY/ABSTRACT Neurons are highly polarized cells with processes reaching up to one meter in length that rely on mRNA transport and local translation to rapidly respond to distal stimuli. Defects in mRNA transport have been identified in a number of neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). Additionally, many ALS/FTD-associated mutations are found in RNA binding proteins (RBPs), which participate in the axonal transport of mRNA to sites of local translation. These ALS/FTD-associated RBPs undergo phase separation, a phenomenon where proteins and nucleic acids demix from the surrounding solution, forming membraneless organelles. Phase separation likely underlies mRNA transport granule formation and disease mutations alter the ability of these RBPs to undergo phase separation, leading to the pathological inclusions observed in patient tissue. Using neurons derived from induced pluripotent stem cells, live and fixed cell imaging, transcriptomics, and proteomics, I will determine if transcripts are co-transported, the effects of disease mutations on mRNA packaging, and develop antisense oligonucleotides to improve disease-associated packaging defects. Aim 1 will determine if the ALS/FTD-associated proteins FUS, TDP-43, and MATR3 transport three transcripts destined for different axonal compartments, SHANK1, ATP5B, and NTM, separately or co-package them into granules and if the proteins co-localize to the same granule. Then, I will determine the effect of ALS/FTD mutations to the RBPs on protein and mRNA packaging into transport granules. Aim 2 will identify the protein and mRNA components of FUS, TDP-43, and MATR3- containing transport granules, as well as elucidate the rules for mRNA packaging using bioinformatic and experimental techniques. Aim 3 will determine if modalities previously shown to alter RBP phase separation are able to rescue packaging defects in ALS/FTD mutant lines. Then, I will use CRISPR inhibition to determine complementary targets whose knockdown achieves a similar effect as overexpression before developing and validating antisense oligonucleotides against these targets. I hypothesize that ALS/FTD-associated RBPs will co-package transcripts destined for the same axonal compartment and that changes in phase separation due to ALS/FTD mutations alter this packaging. The proposed work will address the current knowledge gap on the role of mRNA transport in ALS/FTD, which is crucial for both understanding the basic mechanisms of mRNA transport in neurons as well as the development of effective therapeutics to treat or prevent neurodegeneration. Results from this proposal will provide critical understanding of the mechanisms of granule packaging, how they are disrupted in neurodegenerative disease, and will identify and validate therapies to rescue ALS/FTD-associated mRNA transport granule packaging defects. This fellowship will be centered around training in the technical skills required to complete the proposed project as well as professional development activities to prepare the applicant for success as an independent researcher.

项目摘要/摘要 神经元是高度极化的细胞，其突起长达一米，依赖于mRNA的运输 和局部翻译，以对远端刺激快速反应。在mRNA转运方面的缺陷已经在一种 一些神经退行性疾病，包括肌萎缩侧索硬化症和额颞痴呆 (ALS/FTD)。此外，在RNA结合蛋白(RBPs)中发现了许多与ALS/FTD相关的突变，其中 参与信使核糖核酸的轴突转运到局部翻译部位。这些与ALS/FTD相关的限制性商业惯例 经历相分离，这是一种蛋白质和核酸与周围环境分离的现象 溶液，形成无膜细胞器。相分离可能是形成信使核糖核酸转运颗粒的基础 疾病突变改变了这些限制性商业惯例经历时相分离的能力，导致了病理上的 在患者组织中观察到的包裹体。使用来自诱导多能干细胞的神经元，活的和 固定的细胞成像，转录组，和蛋白质组学，我会确定转录本是否共运， 疾病突变对mRNA包装的影响，并开发反义寡核苷酸来 改善与疾病相关的包装缺陷。AIM 1将确定ALS/FTD相关蛋白 FUS、TDP-43和MATR3运输三个转录本，目的地是不同的轴突室，SHANK1， ATP5B和NTM，单独或共同包装成颗粒，如果蛋白质共定位到相同的 颗粒剂。然后，我将确定ALS/FTD突变对限制性商业惯例在蛋白质和mRNA包装上的影响 变成运输颗粒。目的2鉴定FUS、TDP-43和MATR3的蛋白质和mRNA组分。 包含转运颗粒，以及阐明使用生物信息学和 实验技术。目标3将确定之前显示的改变RBP相分离的模式是否 能够挽救ALS/FTD突变系的包装缺陷。然后，我将使用CRISPR抑制来确定 互补靶点，其敲除在形成和发展之前达到与过度表达相似的效果 针对这些靶点验证反义寡核苷酸。我假设ALS/FTD相关的限制性商业惯例 共同打包的转录本将被送往相同的轴突间隔室，并且由于 ALS/FTD突变改变了这种包装。拟议的工作将解决目前关于这一角色的知识差距 在ALS/FTD中的信使核糖核酸转运，这对于理解信使核糖核酸转运的基本机制是至关重要的 以及开发有效的治疗方法来治疗或防止神经变性。结果 将提供对颗粒包装机制的关键理解，以及它们是如何 在神经退行性疾病中被破坏，并将识别和验证拯救ALS/FTD相关的治疗方法 MRNA转运颗粒包装缺陷。该奖学金将以技术技能培训为中心。 需要完成建议的项目以及专业发展活动，为申请者做好准备 作为一名独立研究人员取得成功。