Intermediate Filament Proteostasis and RNA regulation in Giant Axonal Neuropathy

摘要 中间丝（IF）是细胞骨架的关键结构成分。神经丝（NFs） 神经元的主要IF调节轴突大小和口径，并且在许多神经退行性疾病中失调。 疾病在“巨大”轴突鞘内的异常NF包涵体是罕见和致命疾病的标志性特征 巨轴突神经病（GAN），由KLHL 16基因突变引起。的起源和作用 GAN中的NF包涵体知之甚少。我们已经做出了第一个观察，TDP-43，一个重要的 RNA结合蛋白，与人GAN诱导的多能干细胞的大轴突内含物内的NF共定位 干细胞衍生的运动神经元（iPSC-MN）。这些发现揭示了GAN和 其他TDP-43蛋白病，最显著的是肌萎缩侧索硬化症（ALS）。本申请的目的是 是检查GAN神经元是否受到异常RNA代谢的影响。下列意见和 可行性研究形成了该提议的基础：（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包涵体的突出特征;（ii）人KLHL 16 mRNA相关 与TDP-43和积累在应激颗粒;（iii）新的，患者来源的iPSC-MN微流体系统 我们的研究反映了GAN的轴突IF病理学，并适用于严格的定量图像分析。 （iv）GAN iPSC-MN中的轴突内含物含有TDP-43，并且它们可以被抑制; 被打乱了我们在这里要解决的两个主要问题是：1。轴突NF/TDP-43包涵体 隔离其他应激颗粒组分和KLHL 16 mRNA（Aim 1），和2.是轴突的存在 NF/TDP-43包涵体与GAN神经元中改变的全局和KLHL 16特异性mRNA翻译相关？ （目标2）。我们期望这些基本问题的答案将最终引导我们找到IF包体的起源 在巨大的GAN轴突和功能的后果，这种惊人的病理学在GAN疾病的进展。

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.