RNA-binding proteins (RBPs) have been shown to play a key role in the pathogenesis of a variety of neurodegenerative disorders. Amyotrophic lateral sclerosis (ALS) is an exemplar neurodegenerative disease characterised by rapid progression and relatively selective motor neuron loss. Nuclear-to-cytoplasmic mislocalisation and accumulation of RBPs have been identified as a pathological hallmark of the disease, yet the spatiotemporal responses of RBPs to different extrinsic stressors in human neurons remain incompletely understood. Here, we used healthy induced pluripotent stem cell (iPSC)-derived motor neurons to model how different types of cellular stress affect the nucleocytoplasmic localisation of key ALS-linked RBPs. We found that osmotic stress robustly induced nuclear loss of TDP-43, SPFQ, FUS, hnRNPA1 and hnRNPK, with characteristic changes in nucleocytoplasmic localisation in an RBP-dependent manner. Interestingly, we found that RBPs displayed stress-dependent characteristics, with unique responses to both heat and oxidative stress. Alongside nucleocytoplasmic protein distribution changes, we identified the formation of stress- and RBP-specific nuclear and cytoplasmic foci. Furthermore, the kinetics of nuclear relocalisation upon recovery from extrinsic stressors was also found to be both stress- and RBP-specific. Importantly, these experiments specifically highlight TDP-43 and FUS, two of the most recognised RBPs in ALS pathogenesis, as exhibiting delayed nuclear relocalisation following stress in healthy human motor neurons as compared to SFPQ, hnRNPA1 and hnRNPK. Notably, ALS-causing valosin containing protein (VCP) mutations did not disrupt the relocalisation dynamics of TDP-43 or FUS in human motor neurons following stress. An increased duration of TDP-43 and FUS within the cytoplasm after stress may render the environment more aggregation-prone, which may be poorly tolerated in the context of ALS and related neurodegenerative disorders. In summary, our study addresses stress-specific spatiotemporal responses of neurodegeneration-related RBPs in human motor neurons. The insights into the nucleocytoplasmic dynamics of RBPs provided here may be informative for future studies examining both disease mechanisms and therapeutic strategy.

Several studies have shown that human induced pluripotent stem cell (iPSC)-derivatives are essentially fetal in terms of their maturational status. Inducing ageing in iPSC-motor neuron (MN) models of amyotrophic lateral sclerosis (ALS) has the potential to capture pathology with higher fidelity and consequently improve translational success. We show here that the telomerase inhibitor BIBR1532, hypothesised to recapitulate the telomere attrition hallmark of ageing in iPSC-MNs, was in fact cytotoxic to feeder-free iPSCs when used at doses previously shown to be effective in iPSCs grown on a layer of mouse embryonic fibroblasts. Toxicity in feeder-free cultures was not rescued by co-treatment with Rho Kinase (ROCK) inhibitor (Y-27632). Moreover, the highest concentration of BIBR1532 compatible with continued iPSC culture proved insufficient to induce detectable telomerase inhibition. Our data suggest that direct toxicity by BIBR1532 is the most likely cause of iPSC death observed, and that culture methods may influence enhanced toxicity. Therefore, recapitulation of ageing hallmarks in iPSC-MNs, which might reveal novel and relevant human disease targets in ALS, is not achievable in feeder-free culture through the use of this small molecule telomerase inhibitor.