The TAR DNA-binding protein (TDP-43) is a heterogeneous nuclear ribonucleprotein that is involved in multiple stages of RNA processing. Mutations in the TDP-43 gene and mislocalization of TDP-43 protein have been implicated in a growing number of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Here, we show that TDP-43 negatively regulates innate immune gene expression in response to RNA virus sensing. Perturbation of TDP-43 protein expression leads to an increase in antiviral gene expression in a variety of human and mouse cells. Crosslinked RNA immunoprecipitation (CLIP) experiments revealed that TDP-43 binds to type I interferon (IFN) and interferon stimulated gene (ISGs) transcripts. Using massively parallel 3’ UTR reporter assays coupled with high throughput sequencing (MPRA-seq), we identified polyadenylation signal sequences in the 3' UTRs of innate immune genes to be specifically regulated by TDP-43. Surprisingly, IFN and ISG mRNA decay rates are faster in TDP-43-perturbed cells. Using a metabolic labeling approach to measure nascent transcript generation, we found that perturbation of TDP-43 expression leads to an increase in antiviral gene transcription rates. Additionally, RNA polymerase II (pol II) chromatin immunoprecipitation (ChIP) confirmed that there is greater pol II occupancy on innate immune genes when TDP-43 is depleted. Although TDP-43 perturbation has no effect on an isolated \(IFN\beta\) promoter reporter, we found that TDP-43 inhibits \(IFN\beta\) promoter activity when the \(IFN\beta\) 3' UTR sequence is inserted downstream of the \(IFN\beta\) promoter element, suggesting a novel mechanism of 3' UTR-mediated promoter cis-regulation.

Transactive response DNA-binding protein of 43 kDa (TDP-43) is the major protein found within pathological inclusions in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD) (Neumann et al., 2006). TDP-43 is a ubiquitously expressed protein mainly involved in RNA metabolism. It is a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family and in its normal state is predominantly found in the nucleus. In its pathological state TDP-43 is cleaved, phosphorylated, ubiquitinated, and located in cytoplasmic or nuclear inclusions. Along with ALS and FTLD, TDP-43 is also observed in many other neurodegenerative diseases. Pathological TDP-43 inclusions have been previously reported in cases of Chronic Traumatic Encephalopathy (CTE) (King et al., 2010)(McKee et al., 2010)(Saing et al., 2012)(Hazrati et al., 2013), however no previous study has reported on the incidence and extent of TDP-43 cellular inclusions in a large cohort of autopsy cases diagnosed with CTE. This study finds that TDP-43 inclusions are a frequent feature of CTE, as TDP-43 inclusions are identified in 43% (20/47) of subjects in a CTE+, FTLD-, low-likelihood-of-AD cohort. Furthermore, this study finds that in CTE there is no consistent initial focus of TDP-43 pathology which spreads to neighboring regions as the disease progresses. Despite the lack of a clear progression of TDP-43 pathology, a TDP Staging Scheme for CTE which accurately reflects the extent and severity of TDP-43 pathology in not only the study cohort, but likely in all subjects without FTLD, was established. Four stages were identified: TDP Stage 0 showed no TDP-43 inclusions in the substantia nigra, dorsolateral frontal cortex, or dentate gyrus; TDP Stage 1 showed inclusions in either the substantia nigra or the dorsolateral frontal cortex; TDP Stage 2 showed inclusions either in the dentate gyrus or in both the substantia nigra and the dorsolateral frontal cortex; and TDP Stage 3 showed inclusions in the substantia nigra, dorsolateral frontal cortex, and dentate gyrus. Finally, a correlation was found between the presence of TDP-43 inclusions and the levels of activated microglia in the dorsolateral frontal cortex of CTE+ subjects. This finding aligns with the theory that the pathological changes of TDP-43 found in CTE are driven by the pro-inflammatory cytokines released by chronically activated microglia.

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease with no cure. TAR-DNA binding protein 43 (TDP-43) is the major component of the cytoplasmic inclusions characteristic of ALS. Transgenic Drosophila lines expressing wild-type, mutant and splice variants of human TDP-43 were generated. I find that ubiquitous expression of all TDP-43 transgenes, except for TDP-43∆C-term, is sufficient to cause lethality. I also show that eye-specific expression of a TDP-43∆N-term splice variant, which localizes diffusely to the cytosol, results in increased cell toxicity suggesting an association between cytosolic localization and toxicity. Consistent with this model, I find that the TDP-43∆N-term splice variant is capable of recruiting full length TDP-43 into the cytoplasm, and I suggest this may represent an initiating event in TDP-43-linked ALS. Altogether, my results seem to indicate that exclusion of TDP-43 from the nucleus rather than its presence in aggregates is linked to increased cytotoxicity and lethality in ALS.

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease with no cure. TAR-DNA binding protein 43 (TDP-43) is the major component of the cytoplasmic inclusions characteristic of ALS. Transgenic Drosophila lines expressing wild-type, mutant and splice variants of human TDP-43 were generated. I find that ubiquitous expression of all TDP-43 transgenes, except for TDP-43∆C-term, is sufficient to cause lethality. I also show that eye-specific expression of a TDP-43∆N-term splice variant, which localizes diffusely to the cytosol, results in increased cell toxicity suggesting an association between cytosolic localization and toxicity. Consistent with this model, I find that the TDP-43∆N-term splice variant is capable of recruiting full length TDP-43 into the cytoplasm, and I suggest this may represent an initiating event in TDP-43-linked ALS. Altogether, my results seem to indicate that exclusion of TDP-43 from the nucleus rather than its presence in aggregates is linked to increased cytotoxicity and lethality in ALS.

Yes / Motor neuron disease (MND) is a progressive neurodegenerative disease with no effective treatment. One of the principal pathological hallmarks is the deposition of TAR DNA binding protein 43 (TDP-43) in cytoplasmic inclusions. TDP-43 aggregation occurs in both familial and sporadic MND; however, the mechanism of endogenous TDP-43 aggregation in disease is incompletely understood. This study focused on the induction of cytoplasmic accumulation of endogenous TDP-43 in the motor neuronal cell line NSC-34. The endoplasmic reticulum (ER) stressor tunicamycin induced casein kinase 1 (CK1)-dependent cytoplasmic accumulation of endogenous TDP-43 in differentiated NSC-34 cells, as seen by immunocytochemistry. Immunoblotting showed that induction of ER stress had no effect on abundance of TDP-43 or phosphorylated TDP-43 in the NP-40/RIPA soluble fraction. However, there were significant increases in abundance of TDP-43 and phosphorylated TDP-43 in the NP-40/RIPA-insoluble, urea-soluble fraction, including high molecular weight species. In all cases, these increases were lowered by CK1 inhibition. Thus ER stress signalling, as induced by tunicamycin, causes CK1-dependent phosphorylation of TDP-43 and its consequent cytosolic accumulation. / Funded by a biomedical research grant from the Motor Neurone Disease Association (ref Rattray/Apr15/837-791). The Bioimaging Facility microscopes used in this study were purchased with grants from BBSRC, Wellcome Trust and the University of Manchester Strategic Fund.

Les mitochondries sont la principale source d’énergie dans les neurones. Les défauts mitochondriaux participent à l’apparition de maladies neurodégénératives, cependant ils peuvent être contrés par un système de contrôle qualité. Le but de ma thèse a été de déterminer si ce système est dérégulé dans la maladie de Huntington (MH) et la sclérose latérale amyotrophique (SLA) et si sa restauration est neuroprotectrice, en utilisant principalement des modèles drosophile. La MH, caractérisée par une atteinte des neurones du striatum, est due à la protéine Huntingtin mutée (mHtt). Nous avons montré que la mHtt induit une accumulation des mitochondries dans la rétine. Ceci pourrait être dû à un défaut de la mitophagie, un mécanisme qui permet l’élimination des mitochondries défectueuses et qui est orchestré par la protéine PINK1. De manière intéressante, la surexpression de PINK1 corrige le phénotype pathologique des drosophiles exprimant la mHtt. Je me suis aussi intéressé à la SLA, chez laquelle les motoneurones dégénèrent, plus exactement au gène TDP-43 qui est un contributeur majeur à la maladie. Nous avons montré que la surexpression de TDP-43 dans les neurones de drosophiles entraîne une fragmentation des mitochondries liée à une sous-expression du gène mitofusin. Ce dernier contrôle le processus de fusion entre les mitochondries saines et endommagées et donc l’intégrité de cet organite. La surexpression de Mitofusin améliore les défauts locomoteurs et l’activité neuronale altérée chez les drosophiles exprimant TDP-43. Nos résultats montrent l’importance du contrôle qualité mitochondrial dans la pathogenèse de ces maladies, et que de le renforcer pourrait être bénéfique. / Mitochondria are the main energy source in neurons. Mitochondrial defects contribute to the development of neurodegenerative diseases, however they can be countered by a quality control system. The purpose of my thesis has been to determine if this system is dysregulated in Huntington’s disease (HD) and in amyotrophic lateral sclerosis (ALS) and if restoring it can be neuroprotective, by mainly using Drosophila models. HD, which is characterized by loss of striatal neurons, is caused by the mutant Huntingtin protein (mHtt). We showed that mHtt induces the accumulation of mitochondria in the retina. This could be due to a defect in mitophagy, a mechanism which allows the elimination of defective mitochondria and which is orchestrated by the protein PINK1. Interestingly, PINK1 overexpression ameliorates the abnormal phenotype of flies expressing mHtt. I also got interested in ALS, in which motor neurons degenerate, and mainly in the TDP-43 gene which is a major contributor to the disease. We showed that TDP-43 overexpression in Drosophila neurons leads to fragmentation of mitochondria due to decreased expression levels of the mitofusin gene. The latter controls the fusion process between healthy and damaged mitochondria and therefore the organelle integrity. We show that Mitofusin overexpression ameliorates locomotor defects and abnormal neuronal activity in flies expressing TDP-43. Our results show the importance of mitochondrial quality control in the pathogenesis of these diseases, and that reinforcing it can be beneficial.

No description available.

TDP-43 est une protéine nucléaire de type hnRNP impliquée dans la pathogénèse de plusieurs maladies neurodégénératives telles la sclérose latérale amyotrophique et la dégénérescence lobaire fronto-temporale. Cette protéine hautement conservée est impliquée dans une gamme de processus cellulaires allant de la transcription à la traduction. Dans les cellules neuronales de patients atteints dé maladies neurodégénératives, TDP-43 est délocalisée sous forme d'agrégats cytoplasmiques, suggérant une perte de fonctions nucléaires. Une meilleure compréhension des fonctions nucléaires de TDP-43 pourrait permettre de mieux adresser cette possibilité. L'activité nucléaire de TDP-43 la mieux caractérisée est son rôle dans la régulation de l'épissage alternatif comme répresseur de sites d' épissage 3'. La régulation de sites d'épissage 5' demeure moins bien comprise, bien que TDP-43 puisse agir comme enhancer ou répresseur pour ce site. Dans cette étude, nous avons montré l'impact de la présence de sites de haute affinité à TDP-43 sur l'épissage in vivo. Afin de mieux comprendre les bases moléculaires de cette modulation, nous avons utilisé une approche in vitro à l'aide de transcrits possédant des sites d' épissage 5' et des sites de haute affinité pour TDP-43. Nous avons ainsi pu démontrer que la présence d'un site de liaison pour TDP-43 près d'un site d' épissage 5' peut stimuler la liaison du snRNP U1 à ce site. Nos résultats suggèrent cependant que la présence de plusieurs sites de liaison pour TDP-43 peut possiblement par des interactions entre ces protéines, provoquer un changement structural modulant la sélection des sites sans changer la liaison de U1. Nous avons utilisé lá capacité stimulatrice de TDP-43 pour améliorer l'inclusion de l'exon 7 du gène SMN2. Ainsi, un oligo bifonctionnel capable de recruter TDP-43 positionné dans l'intron à proximité du site d'épissage 5' stimule son utilisation. Notre étude a donc permis de développer des outils qui pourront éventuellement servir à mieux comprendre l'impact de mutations de TDP-43 associées à diverses maladies et possiblement utiliser TDP-43 comme nouvel outil de reprogrammation de l'épissage.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative condition that affects corticospinal and spinal motor neurons and leads to death within 30 months of symptom onset in half of all cases. It remains incurable and treatment is supportive. The genetic and molecular understanding of ALS has gone through a rapid expansion in recent years, notably with the discoveries of TDP-43, a heterogeneous ribonucleoprotein as a major component of neuronal inclusions in ALS, as well as the discovery of the C9orf72 hexanucleotide expansion as the most common genetic cause of this disease. This first part of this thesis addresses the question of which of the various pathological hallmarks of the C9orf72 Hexanucleotide Repeat Expansion (HRE) in autopsy material correlates best with the clinical presentation. The main finding is that TDP-43 distribution, rather than C9orf72 RNA foci or dipeptide aggregation in the brain, corresponds best with the areas relevant to the clinical subtype of ALS-FTD. Subsequently the role of TDP-43 was investigated in induced pluripotent stem cell derived motor neurons, and no evidence of the hallmarks of TDP-43 dysfunction, were seen in this model of the disease. No mislocalisation is found on immunofluorescence, and biochemical analysis shows no differences in insoluble species between the patient and control cell lines. In the final section, RNA sequencing was used to study the transcriptome of a BAC transgenic mouse carrying a human M337V transgene expressed at low levels, to identify early presymptomatic differences in gene expression. Interestingly, no changes were found in genes known to be associated with ALS through mutations, and the constitutive nuclear functions of TDP-43 in the regulation of splicing was maintained, prior to the emergence of a clinical phenotype in the mouse. This favours a gain of function mechanism for TDP-43 mutations in ALS.

Old-age hippocampal sclerosis (HS), characterised by severe neuron loss in hippocampal CA1, is a poorly understood cause of dementia. At present no objective pathological HS criteria exist. In life HS is commonly diagnosed as Alzheimer's disease. HS aetiology is unclear, although it has been associated with both ischaemia and TAR-DNA-binding protein-43 (TDP-43)-related neurodegeneration. Variations in genes GRN, TMEM106B and ABCC9 are proposed as HS risk factors. The aim of this thesis was to investigate epidemiological, clinical, pathological and genetic characteristics of HS in older European populations. 976 brains donated for the Cambridge City over-75s Cohort, the Cognitive Function and Ageing Study and the Finnish Vantaa 85+ study were available for evaluation -including bilateral hippocampi from 302 individuals. A protocol capturing the extent and severity of hippocampal neuron loss was developed, establishing objective HS diagnosis criteria and allowing observation of distinct neuron loss patterns associated with ischaemia and neurodegeneration. 71 HS cases (overall prevalence: 7.3%) were identified. HS was significantly associated with an advanced age at death as well as dementia at the end of life. Neuropsychological and cardiovascular characteristics were similar between HS and AD, except for a longer duration of dementia and more disability in HS. HS was not associated with neurofibrillary tangles, amyloid plaques, or vascular pathologies, but all HS cases evaluated for TDP-43 showed neuronal inclusions in the hippocampal dentate and a high frequency of other glial, neuronal and neurite TDP-43 pathologies. GRN and TMEM106B but not ABCC9 variations were linked to HS. A moderating effect of TDP-43 on this association was detected. HS presented pathologically similarly to frontotemporal dementia cases with TDP-43 (FTLD-TDP) caused by mutations in GRN, but differed from other FTLD-TDP subtypes. Results of this thesis reveal the importance of HS in the oldest old in the population, the key role of TDP-43, as well as providing robust methods to capture HS characteristics for an area that has been under-researched but is clearly vital to understanding dementia in the oldest old.