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

项目总结/摘要 神经元是高度极化的细胞，其过程长达一米，依赖于mRNA的运输 和局部平移以快速响应远端刺激。mRNA转运的缺陷已经在一个 一些神经退行性疾病，包括肌萎缩侧索硬化症和额颞叶痴呆症 (ALS/FTD）。此外，在RNA结合蛋白（RBP）中发现了许多ALS/FTD相关突变， 参与mRNA到局部翻译位点的轴突运输。这些ALS/FTD相关的RBP 经历相分离，即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mRNA转运颗粒形成的基础 疾病突变改变了这些RBP进行相分离的能力，导致病理性的 在患者组织中观察到夹杂物。使用来自诱导多能干细胞的神经元， 固定细胞成像，转录组学和蛋白质组学，我将确定转录物是否被共转运， 疾病突变对mRNA包装的影响，并开发反义寡核苷酸， 改善疾病相关包装缺陷。目的1将确定ALS/FTD相关蛋白是否 FUS、TDP-43和MATR 3转运三种转录本，目的地为不同的轴突隔室，SHANK 1， ATP 5 B和NTM，单独或共同包装成颗粒，如果蛋白质共定位于相同的 颗粒。然后，我将确定ALS/FTD突变对RBP蛋白质和mRNA包装的影响 转化为运输颗粒。目的2将鉴定FUS、TDP-43和MATR 3的蛋白和mRNA组分。 含有运输颗粒，以及阐明规则mRNA包装使用生物信息学和 实验技术目标3将确定先前显示的改变RBP相位分离的模式是否 能够挽救ALS/FTD突变株系中的包装缺陷。然后，我将使用CRISPR抑制来确定 互补靶点，其敲低在发育前达到与过表达相似的效果， 验证针对这些靶标的反义寡核苷酸。我假设ALS/FTD相关的RBP将 共同包装转录本，目的是同一轴突室，并改变相分离，由于 ALS/FTD突变改变了这种包装。拟议的工作将解决目前在这一作用方面的知识差距， ALS/FTD的mRNA转运，这对于理解mRNA转运的基本机制至关重要 以及开发有效的治疗方法来治疗或预防神经变性。结果 从这个建议将提供关键的理解机制的颗粒包装，他们是如何 在神经退行性疾病中被破坏，并将确定和验证治疗方法，以挽救ALS/FTD相关的 mRNA转运颗粒包装缺陷。该奖学金将围绕技术技能培训 需要完成拟议的项目以及专业发展活动，以准备申请人 作为一个独立的研究者。

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.