Intermediate Filament Proteostasis and RNA regulation in Giant Axonal Neuropathy

巨轴突神经病中的中间丝蛋白稳态和 RNA 调节

• 批准号: 10195588
• 负责人: Diane Armao
• 金额: $ 23.33万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10195588
• 关键词: 3' Untranslated Regions; Address; Affect; Amyotrophic Lateral Sclerosis; Axon; Axonal Neuropathy; Binding; Biological; Biology; CRISPR/Cas technology; Caliber; Childhood; Chronic; Computer Analysis; Cytoskeleton; Data; Defect; Disease; Disease Progression; Electron Microscopy; Feasibility Studies; Functional disorder; Gap Junctions; Genes; Genetic Translation; Giant Cells; Human; Image Analysis; Impairment; In Vitro; Intermediate Filament Proteins; Intermediate Filaments; Lead; Link; Measures; Messenger RNA; Microfluidic Microchips; Microfluidics; Molecular; Morphology; Motor Neurons; Mutation; Neurodegenerative Disorders; Neurons; Observational Study; Pathogenesis; Pathologic; Pathology; Patients; Pattern; Peripheral Nerves; Pharmacology; Proteins; Quality Control; RNA; RNA Transport; RNA metabolism; RNA-Binding Proteins; Regulation; Structural Protein; Structure; Swelling; System; Testing; Therapeutic; Time; Translating; Translations; Untranslated RNA; Work; cell component structure; design; frontotemporal lobar dementia-amyotrophic lateral sclerosis; giant axonal neuropathy; gigaxonin; in vivo; induced pluripotent stem cell; inhibitor/antagonist; insight; loss of function mutation; neurofilament; neuronal cell body; novel; protein TDP-43; proteostasis; quantitative imaging; scaffold; stress granule; tool; transcriptomics; ubiquitin-protein ligase

ABSTRACT Intermediate filaments (IFs) are critical structural components of the cell cytoskeleton. Neurofilaments (NFs), the principal IFs of neurons, regulate axon size and caliber and are dysregulated in many neurodegenerative diseases. Abnormal NF inclusions within ‘giant’ axon swellings are hallmark features of the rare and fatal disease Giant Axonal Neuropathy (GAN), which is caused by mutations in the gene KLHL16. The origin and function of NF inclusions in GAN are poorly understood. We have made the first observation that TDP-43, an essential RNA-binding protein, co-localizes with NFs within large axonal inclusions of human GAN induced pluripotent stem cell-derived motor neurons (iPSC-MNs). These findings reveal shared mechanisms between GAN and other TDP-43 proteinopathies, most notably amyotrophic lateral sclerosis (ALS). The objective in this application is to examine if GAN neurons are affected by abnormal RNA metabolism. The following observations and feasibility studies form the basis of the proposal: (i) “dense granular bodies” that resemble RNA stress granules are prominent features of IF inclusions in GAN patient neurons in vivo; (ii) The human KLHL16 mRNA associates with TDP-43 and accumulates in stress granules; (iii) The novel, patient-derived iPSC-MN microfluidic system we developed mirrors the axonal IF pathology of GAN and is amenable to rigorous quantitative image analysis of the inclusions; (iv) Axonal inclusions in GAN iPSC-MNs contain TDP-43, and they can be pharmacologically disrupted. The two main questions we aim to address here, are: 1. Do the axonal NF/TDP-43 inclusions sequester other stress granule components and KLHL16 mRNA (Aim1), and 2. Is the presence of axonal NF/TDP-43 inclusions associated with altered global and KLHL16-specific mRNA translation in GAN neurons? (Aim2). We anticipate that answers to these basic questions will ultimately lead us to the origin of IF inclusions in giant GAN axons and to the functional consequences of this striking pathology in GAN disease progression.

抽象的 中间细丝（IFS）是细胞细胞骨架的关键构件。 神经元的主要IF，调节轴突的大小和口径在许多神经退行性中失调 疾病。 巨大的轴突神经病（GAN），是由基因KLH16中的突变引起的。 我们首先观察到TDP-43，这是必不可少的。 RNA结合蛋白，与NFS在人类GAN诱导多能的大轴突夹杂物中共定位 干细胞衍生的运动神经元（IPSC-MN）。 其他TDP-43蛋白质病，最著名的肌萎缩性外侧扇形（ALS）。 是检查GAN神经元是否受到异常RNA代谢的影响。 盛宴构成了提案的基础：（i）类似于RNA应力颗粒的“密集颗粒体” 是体内GAN患者神经元中的夹杂物的突出特征； TDP-43并积聚在应力颗粒中； 如果GAN的病理和严格的定量图像分析，我们开发了轴突 夹杂物; 我们要在这里解决的两个主要问题是：1。轴突NF/TDP-43 隔离其他应力颗粒成分和KLHL16 mRNA（AIM1），而2。 NF/TDP-43夹杂物与GAN神经元中的全局和KLHL16特异性mRNA翻译有关？ （AIM2）。 在GAN疾病进展中这种惊人的病理学的巨型GAN轴突和功能性调节中。