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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-43 S375 ) 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-43 S375 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-43 S375 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-43 S375 immunohistochemistry. These data suggest a subtype-specific, conformation-dependent binding of pTDP-43 S375 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.

Due to a mistake with incorrect assignment of the originally planned amino acid residues 368-379 to the protein sequence of TDP-43 for antibody production by the contracted company, a peptide corresponding to amino acids 362-373 with a phosphorylated serin at 369 instead of a peptide corresponding to amino acids 368-379 with a phosphorylated serin at 375 was synthesized and used for antibody generation and purification.

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.

Aggregation and nuclear depletion of the RNA binding protein TDP-43 are the crucial pathological features of amyotrophic lateral sclerosis (ALS) and inclusion body myositis (IBM), two degenerative diseases of the CNS and muscle. The loss of TDP-43 nuclear function results in the aberrant inclusion of cryptic exons in mRNA transcripts, leading to the expression of proteins. Clonally expanded and highly differentiated CD8 T cells have been observed in individuals with TDP-43 proteinopathies and therapeutics modulating the T cell response have recently been found to extend survival. However, the target antigens mediating T cell activation have remained elusive. Here, we investigate whether the proteins induced by aberrant cryptic splicing due to TDP-43 nuclear loss can act as neo-antigens. We detect the HDGFL2 cryptic peptide and multiple other TDP-43 cryptic exons in IBM skeletal muscle, where their presence correlates with enrichment of T cells and class I antigen presentation pathways. Furthermore, we identify epitopes deriving from HDGFL2 and IGLON5 cryptic peptides which are recognized by clonally expanded and functionally differentiated populations of CD8 T cells in ALS and IBM Patients. Finally, we demonstrate that T cells engineered to express the identified TCRs can bind and activate in response to the cryptic peptide derived epitopes (cryptic epitopes) and are able to kill TDP-43 deficient astrocytes. This work identifies for the first time specific T cell antigens in ALS and IBM, directly linking adaptive immune response to TDP-43 pathology.

Rho guanine nucleotide exchange factor (RGNEF) is a guanine nucleotide exchange factor (GEF) mainly involved in regulating the activity of Rho-family GTPases. Previous work has shown that RGNEF inclusions in the spinal motor neurons of ALS patients co-localise with TDP-43, the major RNA binding protein aggregating in the brain and spinal cord of ALS patients. To further characterise their relationship, we have compared the transcriptomic profiles of neuronal-like cells depleted of TDP-43 and RGNEF and show that these two factors predominantly act in an antagonistic manner when regulating the expression of axon guidance genes. From a mechanistic point of view, our experiments show that the effect of these factors on the processivity of long introns can explain their mode of action. Our findings highlight that neurodegenerative processes at the RNA level can often represent the result of combinatorial interactions between different RNA binding factors, leading to a better understanding of pathogenic mechanisms occurring in patients where more than one specific protein may be aggregating in their neurons.

Bringing together international academics and professionals who are actively researching and working in the field, this pioneering scholarly volume covers the issues faced by individuals with Autism Spectrum Disorder(ASD) in mid and later life. Including a range of personal, academic and clinical perspectives, the book considers historical and contemporary perspectives on autism, including diagnosis, developmental outcomes and life course issues. Attention is given to medical, care and psychological issues that arise as people with ASD age, such as declining cognitive function and speech and communication issues. Family, community support, housing, advocacy, and socio-cultural considerations for older adults with ASD are also given careful consideration, and there are chapters on relationship and sexuality issues and on environmental design.