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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.

The disproportionate length of an axon makes its structural and functional maintenance a major task for a neuron. The heterochronic gene lin-14 has previously been implicated in regulating the timing of key developmental events in the nematode C. elegans. Here, we report that LIN-14 is critical for maintaining neuronal integrity. Animals lacking lin-14 display axonal degeneration and guidance errors in both sensory and motor neurons. We demonstrate that LIN-14 functions both cell autonomously within the neuron and non-cell autonomously in the surrounding tissue, and we show that interaction between the axon and its surrounding tissue is essential for the preservation of axonal structure. Furthermore, we demonstrate that lin-14 expression is only required during a short period early in development in order to promote axonal maintenance throughout the animal’s life. Our results identify a crucial role for LIN-14 in preventing axonal degeneration and in maintaining correct interaction between an axon and its surrounding tissue.