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 (NF) 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。 “巨大”轴突肿胀内的异常 NF 包涵体是这种罕见且致命疾病的标志性特征 巨轴突神经病 (GAN)，由 KLHL16 基因突变引起。的起源和作用 GAN 中的 NF 包含物人们知之甚少。我们首次观察到 TDP-43，一种重要的 RNA 结合蛋白，与 NF 共定位于人类 GAN 诱导的多能性的大轴突包涵体中 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，最明显的是肌萎缩侧索硬化症 (ALS)。本申请的目标 目的是检查 GAN 神经元是否受到异常 RNA 代谢的影响。以下观察和 可行性研究构成了该提案的基础：(i) 类似于 RNA 应激颗粒的“致密颗粒体” 是 GAN 患者神经元体内 IF 内含物的显着特征； (ii) 人类 KLHL16 mRNA 相关者 与 TDP-43 一起并在应激颗粒中积累； (iii) 新型、源自患者的 iPSC-MN 微流体系统 我们开发的模型反映了 GAN 的轴突 IF 病理学，并且适合严格的定量图像分析 内含物； (iv) GAN iPSC-MN 中的轴突包含物含有 TDP-43，它们可以在药理学上发挥作用 扰乱了。我们在此要解决的两个主要问题是： 1. 轴突 NF/TDP-43 包涵体是否存在 隔离其他应激颗粒成分和 KLHL16 mRNA (Aim1)，以及 2. 是否存在轴突 NF/TDP-43 内含物与 GAN 神经元中全局和 KLHL16 特异性 mRNA 翻译的改变相关？ （目标2）。我们预计这些基本问题的答案将最终引导我们了解 IF 内含物的起源 巨大的 GAN 轴突以及这种引人注目的病理学在 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.