The molecular pathology of the ALS-related G376D TARDBP mutation in iPSC-derived motor neurons

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective degeneration of upper and lower motor neurons. One of the principal pathological hallmarks of ALS is the abnormal cytoplasmic mislocalization and aggregation of TAR DNA-binding protein 43 (TDP-43), a multifunctional RNA-binding protein involved in RNA metabolism and stress granule dynamics. Mutations in the TARDBP gene have been associated with both familial and sporadic ALS and are thought to contribute to altered cellular stress responses, defective RNA processing, and impaired proteostasis. Among these variants, the TARDBP p.G376D mutation is associated with a particularly aggressive familial form of ALS identified in a large Apulian pedigree from Southern Italy.Aim of the StudyThe aim of this study was to investigate stress-induced cellular alterations associated with the TARDBP p.G376D mutation using complementary experimental models.In particular, the study aimed to:Characterize TDP-43 localization and stress granule dynamics under acute and chronic oxidative stress conditions;Analyze cellular stress responses in patient-derived fibroblasts and iPSC-derived motor neurons carrying the TARDBP p.G376D mutation;Establish and optimize a differentiation protocol for the generation of motor neurons from patient-derived induced pluripotent stem cells (iPSCs);Investigate the behavior of stress-related ribonucleoprotein granules in vivo using a Drosophila melanogaster model expressing Me31B-GFP.ResultsAcute and chronic oxidative stress conditions were induced using sodium arsenite, and TDP-43 localization together with stress granule formation were analyzed by immunofluorescence using TDP-43 and TIAR markers.Under acute stress conditions, healthy control motor neurons showed a transient formation of TIAR-positive stress granules followed by an efficient recovery after stress removal. In contrast, motor neurons carrying the TARDBP p.G376D mutation exhibited a pronounced stress response, characterized by increased cytoplasmic redistribution of TDP-43, abundant TIAR-positive granules, and incomplete recovery following stress exposure. Under chronic stress conditions, G376D fibroblasts showed a reduced proportion of cells containing detectable TIAR-positive stress granules compared with control and other TARDBP-mutant lines, together with persistent alterations in TDP-43 localization during recovery. In iPSC-derived motor neurons, chronic stress induced relatively few clearly detectable stress granules compared with acute stress, while mutant cells continued to exhibit alterations in TDP-43 subcellular distribution, suggesting a possible dissociation between stress granule formation and TDP-43 redistribution.To complement the human-derived cellular models, stress granule dynamics were also investigated in Drosophila melanogaster larvae expressing the Me31B-GFP reporter. Qualitative fluorescence analysis of salivary glands showed that both starvation and heat stress promoted the formation of Me31B-positive cytoplasmic granules, with a more pronounced effect observed under heat stress conditions, supporting the concept that environmental stress modulates ribonucleoprotein granule dynamics also in vivo.ConclusionsOverall, the findings obtained in this study support the involvement of altered stress granule dynamics, abnormal stress responses, and TDP-43 mislocalization in ALS pathogenesis associated with the TARDBP p.G376D mutation. The results suggest that mutant cells may exhibit impaired recovery following stress exposure and altered cytoplasmic redistribution of TDP-43, potentially contributing to motor neuron vulnerability.Furthermore, this work highlights the importance of integrating different experimental systems, including patient-derived fibroblasts, iPSC-derived motor neurons, and in vivo Drosophila models, to investigate the molecular mechanisms underlying TDP-43-related neurodegeneration and cellular stress responses in ALS.