Alterations in cell cycle progression caused by S375G and S375E phosphomutants of TDP-43

An emerging area of TDP-43 is represented by the study of post-translational modifications and the way they are connected to disease-associated mutations. Recently, we described a novel mutation in TDP-43 in an early-onset ALS case that was affecting a potential phosphorylation site in position 375 (S375G). A preliminary characterization showed that both the S375G variant and its phosphomimic mutant, S375E, displayed altered nuclear-cytoplasmic distribution and cellular toxicity. To better investigate these effects, we established cell lines expressing inducible TDP-43 WT, S375G, and S375E variants. No significant changes were reported in splicing of known regulated targets, TDP-43 autoregulation, or aggregation. However, cell-cycle analysis of the stable clones showed that the number of cells in the G2 phase decreased in the two phospho-mutants (S375G and S375E), as compared to WT. Furthermore, experiments showed that Apoptosis-inducing factor 1 (AIF1) appeared to be released from the mitochondria. The involvement of apoptosis and alterations in cell-cycle induced by these variants were further validated using RNAseq analysis. Taken together, these data strongly support the growing evidence that alterations of TDP-43 post-translational modifications can play a potentially important role in disease pathogenesis. Overall design: Generation of Tet-inducible Hek293 Flp-In T-REx monoclones expressing Wild-Type, S375G, and S375E variants of human TDP-43.