Intermediate Filament Proteostasis and RNA regulation in Giant Axonal Neuropathy

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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.