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

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.

Liquid-Liquid Phase Separation of TDP-43 and FUS in Physiology and Pathology of Neurodegenerative Diseases

“…Aged neurons via prolonged culture times have compromised stress granule assembly and decreased TDP-43 expression level (Khalfallah et al, 2018). Additionally, mutant TDP-43 affects stress granule dynamics in motoneuronlike cells (Ding et al, 2021). Similarly, using iPSC-derived motor neurons, it is shown that ALS-causing FUS mutant FUS P525L can alter neuronal stress granule dynamics (Marrone et al, 2018).…”

DNA Damage and Repair Deficiency in ALS/FTD-Associated Neurodegeneration: From Molecular Mechanisms to Therapeutic Implication

“…The nuclear clearance of FUS and TDP-43 leads to their loss of function in repairing genome damage in addition to their role in RNA processing. Together with the gain of toxicity in the cytosol likely via altered stress granule dynamics, this contributes to neuronal death (Lenzi et al, 2015 ; Wang H. et al, 2018 ; Mitra et al, 2019 ; Wang and Hegde, 2019 ; Zhang et al, 2020 ; Ding et al, 2021 ). A third key genetic factor in ALS/FTD is C9orf72 , which involves an expanded hexanucleotide repeat (GGGGCC) in its non-coding region (DeJesus-Hernandez et al, 2011 ).…”

Role of the Ubiquitin System in Stress Granule Metabolism

“…TDP-43 (Tar DNA-binding protein-43) is frequently present in ubiquitylated inclusions in brain/muscle sections from patients with ALS and/or FTD [ 80 , 95 ]. Disease-linked mutations in TDP-43 increase its nuclear export and association with SGs, thereby presumably inducing their transition into aberrant SGs [ 96 , 97 , 98 ]. The SG-associated protein FUS (fused in sarcoma) is exported from the nucleus to the cytoplasm under stress conditions and associates with SGs.…”