In 2006, human Trans-Active Regulator DNA Binding Protein (TDP-43) was identified as the major ubiquitinated component of inclusion bodies in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD), two important neurodegenerative diseases in the human population. In ALS/FTLD, TDP-43 that is normally present in the nucleus, is found aggregated in the cytoplasm where it is also abnormally phosphorylated, poly-ubiquitinated, and cleaved to release toxic C-terminal fragments. Recently, evidences of TDP-43 involvement were also reported by our laboratory in Niemann-Pick type C (NPC) disease, a Lysosomal Storage Disorder (LDS) with visceral and neurological symptoms. In particular, in this disease, the protein TDP-43 does not aggregate in the cytoplasm like in the motor neurons of ALS patients. However, it is found mislocalized and abnormally phosphorylated in the cytoplasm of three different models: mouse NPC1-/- brain; human NPC cellular model (multipotent stem cells derived from skin biopsies, reprogrammed to neuronal cells); and in patients’ brain Purkinje cells.
Keeping these two observations in mind, the general aim of my work for this thesis has been to specifically study one of the major TDP-43 post-translational modifications (PTMs), phosphorylation, in two different diseases models: disease-associated TDP-43 mutations in ALS patients and aberrant phosphorylation of TDP-43 in NPC disease.
Regarding ALS, together with a group in Indiana/Kansas University I described a novel mutation in TDP-43 affecting a Serine residue changing to a Glycine in position 375 (S375G). The reason why this mutation was interesting is because it was discovered in an early-onset ALS case. The results of my study showed that the TDP-43 carrying this S375G variant localized more in the nucleus with respect to the wild-type (WT) form. This nuclear localization leads to a stronger cytotoxicity probably due to the lack of the phosphorylation site, that was suggested to strongly destabilize an amyloid-like structure in its C-terminal tail that promoted TDP-43 multimerization. In order to study in depth, the physiological/pathological behavior of this Serine residue, I created a cell line expressing constitutively the WT, S375G, and S375E (phosphomimic) TDP-43 forms. No significant changes were reported in splicing activity, autoregulation, or aggregation, but a cell-cycle analysis of the stable clones showed that the number of cells in the G2 phase decreased in the two phospho-mutants compared to WT. The exact reason for this alteration is still not known. However, preliminary experiments on the mitochondria apoptotic signal that I performed showed that Apoptosis-Inducing Factor 1 (AIF1) seemed to be released from the mitochondria.
Regarding Niemann Pick C, in order to better understand the molecular mechanisms that lead to TDP-43 phosphorylation in NPC, I performed an RNA sequencing analysis using a human NPC cellular model and compared the detected list of gene expression changes with a list of changes that we previously described for neuronal SHSy5Y cells depleted of TDP-43. As described in depth in the thesis, approximately 800 genes were found differentially regulated between NPC patients and healthy controls, involving neuronal, inflammatory, and lipid metabolism pathway. Among these 800 genes, 64 were found to be commonly misregulated in the RNA sequencing performed on SHSy5Y cells upon TDP-43 silencing. Based on these preliminary results, I identified two particular targets of TDP-43 that were previously unknown: Inositol 1,4,5-Trisphosphate Receptor Type 1 (ITPR1), and the Ependymin Related 1 (EPDR1). Interestingly, the depletion (down regulation) of ITPR1 gene induced changes in TDP-43 protein cellular localization, thus suggesting a direct link
between alterations in this gene and aberrant TDP-43 regulation.
Taken together, the data contained in my thesis strongly support growing evidence that alterations of TDP-43 post-translational modifications, either due to disease-associated mutations or genes that control its cellular localization, can play a potentially important role in disease pathogenesis
