TDP-43 Mutation Affects Stress Granule Dynamics in Differentiated NSC-34 Motoneuron-Like Cells.

Qiao Ding,Ernst Wolvetang,Dominic C. H. Ng,Massimo A. Hilliard,Peter G. Noakes,Justin Chaplin,Matthew J. Morris

doi:10.3389/fcell.2021.611601

Qiao Ding, Ernst Wolvetang + Show 5 more

Open Access

https://doi.org/10.3389/fcell.2021.611601

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Abstract

Amyotrophic Lateral Sclerosis (ALS) is characterized by degeneration of motor neurons in the brain and spinal cord. Cytoplasmic inclusions of TDP-43 are frequently reported in motor neurons of ALS patients. TDP-43 has also been shown to associate with stress granules (SGs), a complex of proteins and mRNAs formed in response to stress stimuli that temporarily sequester mRNA translation. The effect of pathogenic TDP-43 mutations within glycine-rich regions (where the majority of ALS-causing TDP-43 mutations occur) on SG dynamics in motor neurons is poorly understood. To address this issue, we generated murine NSC-34 cell lines that stably over-express wild type TDP-43 (TDP-43WT) or mutant forms (ALS-causing TDP-43 mutations TDP-43A315T or TDP-43M337V). We then differentiated these NSC-34 lines into motoneuron-like cells and evaluated SG formation and disassembly kinetics in response to oxidative or osmotic stress treatment. Wild type and mutant TDP-43 appeared to be largely retained in the nucleus following exposure to arsenite-induced oxidative stress. Upon arsenite removal, mutant TDP-43 clearly accumulated within HuR positive SGs in the cytoplasm, whereas TDP-43WT remained mostly within the nucleus. 24 h following arsenite removal, all SGs were disassembled in both wild type and mutant TDP-43 expressing cells. By contrast, we observed significant differences in the dynamics of mutant TDP-43 association with SGs in response to hyperosmotic stress. Specifically, in response to sorbitol treatment, TDP-43WT remained in the nucleus, whereas mutant TDP-43 relocalized to HuR positive SGs in the cytoplasm following exposure to sorbitol stress, resulting in a significant increase in TDP-43 SG numbers. These SGs remained assembled for 24 h following removal of sorbitol. Our data reveal that under certain stress conditions the rates of SG formation and disassembly is modulated by TDP-43 mutations associated with ALS, and suggest that this may be an early event in the seeding of insoluble cytoplasmic inclusions observed in ALS.

Highlights

Amyotrophic lateral sclerosis (ALS) is the most common adultonset motor neuron disease
In order to determine if Amyotrophic Lateral Sclerosis (ALS)-causing mutations of TDP-43 enhance the association with stress granules (SGs) in motoneuron-like cells, we first stably transfected NSC-34 cells with TDP-43WT, familial ALS patients (fALS)-causing mutations TDP43A315T, and TDP-43M337V, that were N-terminally tagged with Enhanced Green Fluorescent Protein (EGFP), or transfected with EGFP alone
We have shown that both wild type (WT) and mutant TDP-43 were present in the nuclei, which was reminiscent of ALS patients who harbor TDP-43 mutations, but do not show symptoms until a later age (Mackenzie et al, 2007; Wegorzewska et al, 2009; Deng et al, 2010)

Summary

Introduction

Amyotrophic lateral sclerosis (ALS) is the most common adultonset motor neuron disease. About 90% of patients exhibit sporadic ALS (sALS), whereas in ∼10% of familial ALS patients (fALS) a genetic cause has been identified, including mutations in the TDP-43 gene (trans-active response (TAR)-DNA binding protein 43 kDa) (Nowicka et al, 2019). Both sporadic and familial ALS patients present with TDP-43-containing insoluble cytoplasmic inclusions (Arai et al, 2006; Neumann et al, 2006), suggesting that TDP-43 plays a central role in ALS pathogenesis. We investigate whether ALScausing mutations in TDP-43 affect the kinetics of SG formation or disassembly in motoneuron-like cells derived from the murine NSC-34 cell line, a neuroblastoma-spinal cord hybrid cell line developed by Cashman et al (1992)

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