Ruolo della TDP-43 nella formazione dei granuli da stress nella Sclerosi Laterale Amiotrofica

Amyotrophic Lateral Sclerosis (ALS) is a late-onset neurodegenerative disease characterized by the selective loss of upper and lower motor neurons; most ALS cases are sporadic, and only 5-10% are familial. About 4% of familial cases, are due to mutations in TARDBP, the gene encoding TDP-43, that is an ubiquitous nuclear protein that regulates mRNA functions and metabolism. Recent studies suggest that TDP-43 may regulates stress granules dynamics that are cytoplasmic structures composed of non-translating messenger ribonucleoproteins (mRNPs) that rapidly aggregate in cells exposed to adverse environmental conditions. Stress granules function in part to triage RNA and sequester transcripts not needed for coping with the stress. We evaluated stress granule dynamics in primary fibroblast cultures from skin of ALS patients carriying TARDBPA382T mutation, ALS patients without any TARDBP mutation and healthy controls. After treatment with sodium arsenite (0,5 mM), for 30 and 60 minutes, we observed a significantly higher number of cells exhibiting stress granules, identified by immunostaining for specific markers (TIA-1 and HuR), in fibroblasts from healthy controls compare with those from ALS patients carriying TARDBPA382T mutation. Moreover, fibroblasts from healthy controls showed more stress granules per cell compare with those from ALS patients, while no differences were observed in stress granule size between groups. Fibroblasts from ALS patients without any TARDBP mutation, showed the same ability to form stress granules as cells from healthy controls, confirming that the decrease was associated to TARDBPA382T mutation rather than other factors attributable to ALS. In all samples analyzed TDP-43 immunostaining was always observed into the nucleus of all the cells and even after sodium arsenite treatment TDP-43 was never localized in stress granules. The involvement of TDP-43 in stress granule assembly was confirmed by silencing TARDBP gene in fibroblasts from healthy controls. After sodium arsenite treatment, fibroblasts in wich TARDBP gene was silenced, showed a significantly lower number of cells exhibiting stress granules compare with unsilenced controls. Following stress stimuli, we observed, using MTT assay, a significant higher cytotoxicity in fibroblasts from patients carriying TARDBPA382T mutation compare with healthy controls. Expression of G3BP, a core stress granule component, was significantly lower after sodium arsenite treatment in fibroblasts from patients carriying TARDBPA382T mutation compare with healthy controls. We can conclude that TARDBPA382T mutation caused a reduction in the ability of human fibroblasts to respond to stress through loss of TDP-43 function in stress granule nucleation. The pathogenetic action revealed in our study model does not seem to be mediated by changes in the localization of the TDP-43 protein, but we found that this protein contributes to stress granule formation through a regulatory effect on the G3BP core protein. These data demonstrate that TDP-43 may modulate stress granule formation contributing to the cellular response to acute stress and suggest that TARDBPA382T mutation may compromise the cellular stress response, contributing to neuronal vulnerability in ALS.