TDP-43 Aberrant Phosphorylation in Amyotrophic Lateral Sclerosis (ALS) and in Niemann-Pick type C (NPC) Disease

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

Unraveling the toxic effects mediated by the neurodegenerative disease-associated S375G mutation of TDP-43 and its S375E phosphomimetic variant

TAR DNA-binding protein 43 (TDP-43) is a nucleic acid-binding protein found in the nucleus that accumulates in the cytoplasm under pathological conditions, leading to proteinopathies, such as frontotemporal dementia and ALS. An emerging area of TDP-43 research is represented by the study of its post-translational modifications, the way they are connected to disease-associated mutations, and what this means for pathological processes. 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 mutation and its phosphomimetic variant, S375E, displayed altered nuclear-cytoplasmic distribution and cellular toxicity. To better investigate these effects, here we established cell lines expressing inducible WT, S375G, and S375E TDP-43 variants. Interestingly, we found that these mutants do not seem to affect well-studied aspects of TDP-43, such as RNA splicing or autoregulation, or protein conformation, dynamics, or aggregation, although they do display dysmorphic nuclear shape and cell cycle alterations. In addition, RNA-Seq analysis of these cell lines showed that although the disease-associated S375G mutation and its phosphomimetic S375E variant regulate distinct sets of genes, they have a common target in mitochondrial apoptotic genes. Taken together, our data strongly support the growing evidence that alterations in TDP-43 post-translational modifications can play a potentially important role in disease pathogenesis and provide a further link between TDP-43 pathology and mitochondrial health

Dysregulation of TDP-43 Intracellular Localization and Early-Onset ALS are Associated with a TARDBP S375G Variant

11siWe investigated the CNS and skeletal muscle tissue from a woman clinically diagnosed with amyotrophic lateral sclerosis (ALS) at the age of 22. Neuropathologic evaluation showed upper and lower motor neuron loss, corticospinal tract degeneration, and skeletal muscle denervation.Analysis of the patient's DNA revealed a AGT>GGT change resulting in a S375G substitution in the C-terminal region of TDP-43. This variant was previously reported as being benign. Considering the early onset and severity of the disease in this patient, we tested the effects of this genetic variant on TDP-43 localization, pre-mRNA splicing activity, and toxicity, in parallel with the effects on known neighboring disease-associated mutations. In cell lines, expressed in culture, S375G TDP-43 appeared to be more significantly localized in the nucleus and to exert higher toxicity than wild-type TDP-43. Strikingly, a phosphomimic mutant at the same residue (S375E) showed a strong tendency to accumulate in the cytoplasm, especially under stress conditions, and molecular dynamics simulations suggest that phosphorylation of this residue can disrupt TDP-43 intermolecular interactions. The results of the current study highlight the importance of phosphorylation and regulation of TDP-43 nuclear-cytoplasmic shuttling/redistribution, in relation to the pathogenetic mechanisms involved in different forms of ALS. This article is protected by copyright. All rights reserved.partially_openopenNewell, Kathy; Paron, Francesca; Mompean, Miguel; Murrell, Jill; Salis, Elisa; Stuani, Cristiana; Pattee, Gary; Romano, Maurizio; Laurents, Douglas; Ghetti, Bernardino; Buratti, EmanueleNewell, Kathy; Paron, Francesca; Mompean, Miguel; Murrell, Jill; Salis, Elisa; Stuani, Cristiana; Pattee, Gary; Romano, Maurizio; Laurents, Douglas; Ghetti, Bernardino; Buratti, Emanuel

Somatic TARDBP variants as a cause of semantic dementia

The aetiology of late-onset neurodegenerative diseases is largely unknown. Here we investigated whether de novo somatic variants for semantic dementia can be detected, thereby arguing for a more general role of somatic variants in neurodegenerative disease. Semantic dementia is characterized by a non-familial occurrence, early onset (<65 years), focal temporal atrophy and TDP-43 pathology. To test whether somatic variants in neural progenitor cells during brain development might lead to semantic dementia, we compared deep exome sequencing data of DNA derived from brain and blood of 16 semantic dementia cases. Somatic variants observed in brain tissue and absent in blood were validated using amplicon sequencing and digital PCR. We identified two variants in exon one of the TARDBP gene (L41F and R42H) at low level (1-3%) in cortical regions and in dentate gyrus in two semantic dementia brains, respectively. The pathogenicity of both variants is supported by demonstrating impaired splicing regulation of TDP-43 and by altered subcellular localization of the mutant TDP-43 protein. These findings indicate that somatic variants may cause semantic dementia as a non-hereditary neurodegenerative disease, which might be exemplary for other late-onset neurodegenerative disorders

Somatic TARDBP variants as a cause of semantic dementia

The aetiology of late-onset neurodegenerative diseases is largely unknown. Here we investigated whether de novo somatic variants for semantic dementia can be detected, thereby arguing for a more general role of somatic variants i

Antibody against TDP-43 phosphorylated at serine 375 suggests conformational differences of TDP-43 aggregates among FTLD–TDP subtypes

Aggregation of hyperphosphorylated TDP-43 is the hallmark pathological feature of the most common molecular form of frontotemporal lobar degeneration (FTLD-TDP) and in the vast majority of cases with amyotrophic lateral sclerosis (ALS-TDP). However, most of the specific phosphorylation sites remain to be determined, and their relevance regarding pathogenicity and clinical and pathological phenotypic diversity in FTLD-TDP and ALS-TDP remains to be identified. Here, we generated a novel antibody raised against TDP-43 phosphorylated at serine 375 (pTDP-4