Intermediate Filament Proteostasis and RNA regulation in Giant Axonal Neuropathy

摘要 中间丝是细胞骨架的重要结构成分。神经丝(NFs)， 神经元的主要IF调节轴突的大小和口径，在许多神经退行性疾病中调节失调 疾病。巨大的轴突肿胀内异常的核因子包涵体是这种罕见和致命疾病的显著特征 巨大轴索神经病(GAN)，由KLHL16基因突变引起。的由来和作用 对GaN中的Nf夹杂物知之甚少。我们第一次观察到，TDP-43是一种必不可少的 RNA结合蛋白，与神经营养因子共定位于人GAN诱导的多能性大轴突包涵体中 干细胞来源的运动神经元(IPSC-MNS)。这些发现揭示了GaN和GAN之间的共同机制 其他TDP-43蛋白病变，最显著的是肌萎缩侧索硬化症(ALS)。此应用程序的目标是 是为了检查GaN神经元是否受到RNA代谢异常的影响。以下是观察到的情况和 可行性研究构成了该提议的基础：(I)类似于RNA应激颗粒的“致密颗粒体” 是体内GaN患者神经元中IF包涵体的显著特征；(Ii)人KLHL16mRNA相关 与TDP-43结合并在应激颗粒中蓄积；(Iii)患者衍生的新型IPSC-MN微流控系统 我们开发了GaN轴突IF病理的镜像，并适用于严格的定量图像分析 (4)GaN IPSC-MNS中的轴突包涵体含有TDP-43，具有一定的药理作用 打乱了。我们在这里要解决的两个主要问题是：1.轴突的NF/TDP-43包涵体 隔离其他应激颗粒成分和KLHL16mRNA(Aim1)，以及2.轴突的存在 核因子/TDP-43包涵体与GAN神经元整体和KLHL16特异性mRNA翻译改变有关？ (AIM2)。我们预计，这些基本问题的答案最终将把我们引向IF包含的起源 在巨大的GaN轴突中，以及这种惊人的病理在GaN疾病进展中的功能后果。

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.