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.

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.

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-43S375) located in the low-complexity domain, and used it to investigate the presence of S375 phosphorylation in a series (n = 44) of FTLD-TDP and ALS-TDP cases. Immunoblot analysis demonstrated phosphorylation of S375 to be a consistent feature of pathological TDP-43 species, including full-length and C-terminal fragments, in all FTLD-TDP subtypes examined (A-C) and in ALS-TDP. Of particular interest, however, detailed immunohistochemical analysis showed striking differences in the immunoreactivity profile of inclusions with the pTDP-43S375 antiserum among pathological subtypes. TDP-43 pathology of ALS-TDP, FTLD-TDP type B (including cases with the C9orf72 mutation), and FTLD-TDP type C all showed strong pTDP-43S375 immunoreactivity that was similar in amount and morphology to that seen with an antibody against TDP-43 phosphorylated at S409/410 used as the gold standard. In stark contrast, TDP-43 pathology in sporadic and genetic forms of FTLD-TDP type A (including cases with GRN and C9orf72 mutations) was found to be almost completely negative by pTDP-43S375 immunohistochemistry. These data suggest a subtype-specific, conformation-dependent binding of pTDP-43S375 antiserum to TDP-43 aggregates, consistent with the idea of distinct structural TDP-43 conformers (i.e., TDP-43 strains) as the molecular basis for the phenotypic diversity in TDP-43 proteinopathies.

Pre-mRNA splicing defects and RNA binding protein involvement in Niemann Pick type C disease.

Niemann-Pick type C (NPC) is an autosomal recessive lysosomal storage disorder due to mutations in NPC1 (95 % cases) or NPC2 genes, encoding NPC1 and NPC2 proteins, respectively. Both NPC1 and NPC2 proteins are involved in transport of intracellular cholesterol and their alteration leads to the accumulation of unesterified cholesterol and other lipids within the lysosomes. The disease is characterized by visceral, neurological and psychiatric symptoms. However, the pathogenic mechanisms that lead to the fatal neurodegeneration are still unclear. To date, several mutations leading to the generation of aberrant splicing variants or mRNA degradation in NPC1 and NPC2 genes have been reported. In addition, different lines of experimental evidence have highlighted the possible role of RNA-binding proteins and RNA-metabolism, in the onset and progression of many neurodegenerative disorders, that could explain NPC neurological features and in general, the disease pathogenesis. In this review, we will provide an overview of the impact of mRNA processing and metabolism on NPC disease pathology.

Dysregulation of TDP-43 intracellular localization and early onset ALS are associated with a TARDBP S375G variant.

We investigated the Central Nervous System (CNS) and skeletal muscle tissue from A woman was 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 Deoxyribonucleic acid (DNA) revealed a AGT>GGT change resulting in an 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.