Dissertations / Theses on the topic 'Fused in Sarcoma'

Neurodegenerative disease patients suffer from cognitive decline and/or motor dysfunctions, depending on the different regions affected by the neuron loss. With aging being the major risk factor and a society with increased life expectancy, there is an urgent need to develop new effective treatments to alleviate the situation faced by patients, their families and society. Although neurodegenerative diseases including Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD) lead to different clinical symptoms, they share common pathomechanisms, such as protein aggregation and altered RNA metabolism. A subset of ALS and FTD cases, for instance, is pathologically characterized by neuronal cytoplasmic inclusions containing aggregated Fused in Sarcoma (FUS) protein. There is also a genetic link, since FUS mutations cause ALS with FUS pathology. FUS is a DNA/RNA-binding protein known to regulate different steps of RNA metabolism, however, its exact function and target genes in neurons were unknown. In this study, I evaluated the neuronal role of FUS in alternative splicing using a candidate approach focusing on the microtubule-associated protein TAU. TAU is one of the most widely studied proteins in neurodegeneration research due to its aggregation in different tauopathies, most notably AD. Mutations in the TAU gene MAPT, that affect alternative splicing of exon 10, are known to cause another subtype of FTD. Here, I demonstrate that FUS depleted rat neurons, although having normal viability, show aberrant alternative splicing of TAU, with increased inclusion of exon 3 and exon 10, resulting in higher expression of the 2N and 4R TAU isoforms. Importantly, reintroduction of human FUS rescues aberrant splicing of TAU in FUS depleted neurons. Accordingly, overexpression of FUS decreases expression of 2N and 4R TAU isoforms. In mouse brain lysates, I detected direct FUS binding to TAU pre-mRNA, with strong binding around the regulated exon 10, often at AUU-rich RNA stretches. Since TAU splicing is regulated differently in humans and rodents, I also confirmed the role of human FUS in TAU exon 10 splicing using a TAU minigene and a human neuronal cell line. In addition, I analyzed the morphology and development of axons to evaluate the functional consequences of FUS depletion in neurons. Although FUS depleted neurons develop neurites normally, their axons are significantly shorter than in the control cells. Similar to observations in TAU/MAP1B knockout neurons, axons of FUS depleted neurons develop significantly larger growth cones with abnormal cytoskeletal organization. The development of growth cones in vivo is an essential step in axonal maintenance and repair. Altogether, this study identified TAU as the first physiological splice target of FUS in neurons. The newly discovered role of FUS in regulating the axonal cytoskeleton indicates that aberrant axonal function could contribute to the neuron loss seen in ALS/FTD cases with FUS aggregates.
Patienten mit neurodegenerativen Erkrankungen können an kognitivem Abbau und/oder motorische Störungen leiden, je nachdem welche Gehirnregion von dem Verlust von Neuronen betroffen ist. Da sich das Risiko einer neurodegenerativen Erkrankung mit zunehmendem Alter drastisch erhöht und wir eine Gesellschaft mit steigender Lebenserwartung haben, ist es dringend notwending, neue wirksame Behandlungsmethoden zu entwickeln, um die Situation, mit der sich Patienten, ihre Familien und die Gesellschaft konfrontiert sehen, zu erleichtern. Obwohl sich verschiedene neurodegenerative Erkrankungen wie die Alzheimer-Erkrankung (AD), Amyotrophe Lateralsklerose (ALS) oder Frontotemporale Demenz (FTD) klinisch unterscheiden, gibt es gemeinsame Pathomechanismen, wie Proteinaggregation und Störungen im RNA-Metabolismus. Bei einem Teil der ALS und FTD Patienten beobachtet man Ablagerungen aus aggregiertem Fused in Sarcoma (FUS) Protein. Des Weiteren verursachen FUS Mutationen ALS mit FUS neuronalen Aggregaten. FUS ist ein DNA/RNA-bindendes Protein, das verschiedene Schritte des RNA-Metabolismus reguliert. Die genaue Funktion von FUS und seine Zielgene in Neuronen waren jedoch bisher unbekannt. In dieser Studie habe ich die Funktion von FUS auf neuronales alternatives Spleißen mit einem Kandidaten-Ansatz untersucht, und mich insbesondere auf das Mikrotubuli-bindende Protein TAU fokussiert. Tau ist eines der bekanntesten Proteine in der Demenzforschung, da TAU Aggregate in verschiedenen sogenannten Tauopathien, insbesondere AD, gefunden wurden. Mutationen im TAU Gen MAPT, die das alternative Spleißen von TAU Exon 10 beeinflussen, können einen anderen Subtyp der FTD verursachen. Diese Studie zeigt, dass die Herunterregulierung (Gen-Knockdown) von FUS in murinen Neuronen das Überleben der Neuronen nicht beeinträchtigt, aber zu verändertem alternativen Spleißen von TAU mit einem erhöhten Einschluss von Exon 3 und Exon 10 führt und somit eine höhere Expression von den 2N und 4R TAU Isoformen verursacht. Eine wichtige Beobachtung dieser Studie war auch, dass die Expression von humanem FUS in FUS knockdown Neuronen aberrantes TAU Spleißen korrigieren kann. Dementsprechend führte auch die alleinige Überexpression von FUS zu einer verminderten Expression von 2N und 4R TAU. In Lysaten von Mausgehirnen konnte ich eine direkte Interaktion zwischen FUS und TAU RNA nachweisen, und zwar mit bevorzugter FUS Bindung nahe am regulierten TAU Exon 10 und oft an AUU-reichen RNA-Abschnitten. Da das Spleißen von TAU in Menschen und Nagetieren unterschiedlich reguliert wird, bestätigte ich mit sowohl einer menschlichen neuronalen Zelllinie als auch einem TAU-Minigen Konstrukt die Rolle von humanem FUS in TAU Exon 10 Spleißen. Um die funktionalen Konsequenzen von FUS knockdown in Neuronen zu bewerten, analysierte ich die Morphologie und Entwicklung der Axone. Obwohl Neuronen mit FUS knockdown normalen Neuriten bilden, sind ihre Axone deutlich kürzer als die der Kontroll-Neuronen. Wie auch schon in TAU/MAP1B knockout Neuronen beobachtet wurde, entwickeln FUS knockdown Neuronen Axone mit einem deutlich größeren Wachstumskegel und abnormer Zytoskelett-Organisation. Die dynamische Bildung axonaler Wachstumskegel ist ein wesentlicher Schritt in der axonalen Aufrechterhaltung und Reparatur in vivo. Insgesamt konnte diese Studie TAU als erstes physiologisches splice Zielgen von FUS in Neuronen identifizieren. Die neu entdeckte Funktion von FUS bei der Regulation des axonalen Zytoskelettes spricht für eine mögliche Rolle der veränderten axonalen Funktion beim Verlust von Neuronen in ALS/FTD Fällen mit FUS Aggregaten.

Amyotrophic lateral sclerosis (ALS) is an aggressive neurodegenerative disease characterised by the loss of upper and lower motor neurons, resulting in progressive paralysis, muscular atrophy and eventual death, on average, within 2-5 years post diagnosis. In ∼5% of patients with familial ALS (fALS), causative mutations occur within the gene encoding the RNA-binding protein, Fused in Sarcoma (FUS). Normally, FUS is predominantly localised to the nucleus and has several known roles in transcription, splicing and mRNA transport. Yet, in ALS patients with mutant forms of FUS, the protein becomes dramatically mislocalised to the cytoplasm and abnormal proteinaceous inclusions of FUS in the cytoplasm are observed post-mortem. Several questions remain: How do large pathological inclusions of FUS form? Is pathology induced via a gain or loss of protein function? Can aggregation in the cytoplasm of this normally nuclear protein be sufficient to produce toxicity? This thesis provides detailed characterisation of a novel pathway through which FUS may aggregate following its mislocalisation to the cytoplasm. This pathway is distinct from recruitment into stressinduced stress granules and can lead to the formation of large RNA-based FUS aggregates in a concentration-dependent manner. It was demonstrated that reduced protein-RNA interaction through transcriptional inhibition resulted in the dissolution and reassembly of these FUS aggregates into higher order RNA-free structures, reminiscent of inclusions seen in ALS-FUS patients. We also show in vivo that an initial insult of FUS aggregation in the cytoplasm is sufficient to elicit ALS-like pathology. In addition, how loss of FUS from the nucleus could affect the nuclear architecture was investigated, highlighting an important role for FUS in the maintenance of a protective subnuclear body, the paraspeckle, the disruption of which may contribute to the pathogenesis of FUSopathies. As such, this thesis identifies several novel mechanisms involved in the development and progression of FUSopathy, which may be useful for future therapeutic strategies targeting ALS caused by FUS mutation.

The fused in sarcoma (FUS) protein has been shown to be a significant disease protein in a subgroup of patients with frontotemporal lobar degeneration (FTLD). Nevertheless, the mechanism underlying FUS associated FTLD is only poorly understood. Recent research has identified a large hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9orf72), reinforcing the association between C9orf72 and FTLD. Moreover, an unusual histopathological change has been observed within the granule cell layer of the cerebellum in chromosome 9p-linked frontotemporal dementia with motor neuron disease. Whether this type of cerebellar pathology is a pathological marker for chromosome 9p-linked families remains unknown. The purpose of this study was to genetically, neuropathologically and biochemically characterize FUS and C9orf72 in FTLD, and also to investigate the association between the cerebellar pathology and chromosome 9p-linked families. The genetic sequencing study searching for potential genetic factors of FUS in FTLD failed to detect any pathogenic mutations or variations. Immunohistochemical study for FUS pathology in FTLD provided strong evidence for FUS being the specific pathological protein in all forms of FTLD-FUS. Immunoblotting for FUS in FTLD detected one novel disease-associated FUS aggregate (~37 kDa) in the urea fraction of atypical FTLD with ubiquitinated inclusions (aFTLD-U) frontal cortical samples, suggesting this unique protein product might be more associated with disease than the full-length protein itself. Immunohistochemical study of C9orf72 in FTLD detected a 'synaptic' staining in CA sectors, as the most prominent histological feature identified. Immunoblotting for C9orf72 protein demonstrated no distinctive bands among different diagnostic groups, in frontal and cerebellar cortical regions. The present study also confirmed the presence of cerebellar p62 neuronal cytoplasmic inclusions (NCI) in a proportion of FTLD-TDP cases. Although most of these cases showed an autosomally dominant pattern of inheritance, not all of them shared a common C9orf72 haplotype, or mutation in C9orf72.Much work is still needed to investigate the underlying pathogenesis of FTLD-FUS. Attention should still be given to identifying possible genetic risk factors in FUS using a large series of FTLD samples and searching for other possible proteins within the FUS immunoreactive neuronal inclusions. Moreover, the target protein within the cerebellar p62 NCI remains unknown, but it is clear that it is not C9orf72 protein.

La present tesi té com a objectiu estudiar des de diferents punts de vista la degeneració lobular frontotemporal (FTLD), una malaltia neurodegenerativa que es caracteritza per la pèrdua neuronal focal en els lòbuls frontals i temporals. Aquesta malaltia és molt heterogènia des del punt de vista clínic, neuropatològic i genètic, com també en els mecanismes patogènics. Aquesta heterogeneïtat fa que sovint el diagnòstic no sigui fàcil i que la seva classificació nosològica i la recerca en els seus mecanismes sigui especialment complexa. En aquesta tesi s’aborda aquesta heterogeneïtat estudiant diferents aspectes de la FTLD. Primer, s’estudia la FTLD des de un punt de vista clínic: s’avaluen els nous criteris de la variant conductual d’aquesta malaltia (bvFTD) en la cohort de pacients seguits a la Unitat de Memòria de l'hospital de la Santa Creu i Sant Pau. Des del punt de vista genètic, es caracteritzen aspectes clínics i radiològics específics dels casos de FTLD que presenten una expansió de la repetició d’hexanucleòtids en el gen C9orf72. Seguidament, des del punt de vista dels biomarcadors, es mesura en sang i líquid cefaloraquidi la proteïna TDP-43 i la seva forma fosforilada (pTDP-43). En l’últim capítol, s’aprofundeix en els mecanismes moleculars de la patogènia de la FTLD amb dipòsits de proteïnes FUS i FET (FTLD-FET), s’estudien la metilació en les arginines presents en aquests proteïnes i com aquest canvi postraduccional regula el transport citoplasma-nucli de la proteïna FUS. Finalment, es realitza un estudi neuropatològic a on es comparen les diferències en el patró de metilació de la proteïna FUS entre la FTLD-FET i la esclerosi lateral amiotròfica amb mutacions en el gen de FUS (ALS-FUS). Aquest estudi afegeix una nova diferència entre la FTLD-FET i la ALS-FUS, tot i que ambdues comparteixen els dipòsits de la proteïna FUS.
The aim of the present thesis is to study frontotemporal lobar degeneration (FTLD), a neurodegenerative disease characterised by a focal neural loss in the frontal and the temporal lobes, from different perspectives. FTLD is a very heterogeneous disease either from the clinical or the neuropathological, genetic and pathogenic perspectives. This heterogeneity makes its diagnosis challenging and its nosologic classification and pathogenesis research especially puzzling. In the present thesis, this heterogeneity is addressed through studying FTLD from different angles. First, FTLD is investigated from a clinical viewpoint: the most recent clinical criteria about the behavioural variant of FTLD (bvFTD) are evaluated through the cohort of patients followed in the Memory Unit of Hospital de la Santa Creu i Sant Pau. From a genetic perspective, specific clinical and radiological features of the FTLD patients carrying a hexanucleotide repeat expansion in the C9orf72 gene are defined. Next, and from a biomarkers angle, TDP-43 protein and its phosphorylated form (pTDP-43) are measured in blood and in cerebrospinal fluid (CSF). In the last chapter, the molecular pathogenic mechanisms of FTLD with FUS- and FET-positive inclusions (FTLD-FET) are scrutinized, in order to study the methylation pattern of the arginines present in FUS protein and how this posttranslational modification regulates the cytoplasmic-nuclear transport. Finally, a neuropathological study is performed by means of the comparison of the differences in FUS methylation pattern between FTLD-FET and amyotrophic lateral sclerosis with FUS mutations (ALS-FUS). This study provides further evidence of the differences between FTLD-FET and ALS-FUS, despite the fact that both diseases share the deposition of the protein FUS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron death and subsequent muscle atrophy. Approximately 15% of ALS cases are inheritable, and mutations in the Fused in Sarcoma (FUS) gene contribute to approximately 5% of these cases, as well as about 2% of sporadic cases. FUS performs a diverse set of cellular functions, including being a major regulator of RNA metabolism. FUS undergoes liquid- liquid phase transition in vitro, allowing for its participation in stress granules and RNA transport granules. Phase transition also contributes to the formation of cytoplasmic inclusions found in the cell bodies of FUS ALS patients motor neurons. The nature of these inclusions has remained elusive, as the proteins localized to them have not been identified. Additionally, the functional consequence of the accumulation of cytoplasmic FUS inclusions has not been established, nor is it understood how they contribute to selective motor neuron death. We carried out two related, but independent studies to characterize the proteins that may be included in FUS-positive inclusions. In this first study, we utilized immunoprecipitation of wild-type and mutant FUS in the presence and absence of RNase, followed by LC MS/MS. The identified proteins represent those that directly or indirectly interact with FUS, with relatively high affinity that can be pulled down with immunoprecipitation. A wide variety of interacting proteins were identified and they are involved in a multitude of pathways including: chromosomal organization, transcription, RNA splicing, RNA transport, localized translation, and stress response. Their interaction with FUS varied greatly in their requirements for RNA. Most notably, FUS interacted with hnRNPA1 and Matrin-3, proteins also known to cause familial ALS. Immunofluorescent staining of proteins interacting with mutant FUS were localized to cytoplasmic inclusions. We concluded that mis-localization of these proteins potentially lead to their dysregulation or loss of function, thus contributing to FUS pathogenesis. In the second study, we developed a protocol to isolate dynamic FUS inclusions and employed LC MS/MS to identify all proteins associated with FUS inclusions. We identified a cohort of proteins involved in translation, splicing, and RNA export to be associated with the FUS inclusions. Further pathway and disease association analysis suggested that proteins associated with translation and RNA quality control pathways may be the most significant. Protein translation assays using both N2A and ALS patient fibroblasts demonstrated suppression of protein biosynthesis in mutant FUS expressing cells. However, translation initiation was not impaired. To understand how protein synthesis is suppressed by mutant FUS mediated defects in RNA metabolism, we examined changes in a well conserved RNA turnover pathway namely: nonsense mediated decay (NMD). We found that NMD is hyperactivated in cells expressing mutant FUS, likely due to chronic suppression of protein translation shifting the pathways autoregulatory circuit to allow for hyperactivation. We concluded that mutant FUS suppresses protein biosynthesis and disrupts NMD regulation. These defects together likely contribute to motor neuron death.

During stress, eukaryotes regulate protein synthesis in part through formation of cytoplasmic, non-membrane-bound complexes called stress granules (SGs). SGs transiently store signaling proteins and stalled translational complexes in response to stress stimuli (e.g. oxidative insult, DNA damage, temperature shifts and ER dysfunction). The functional outcome of SGs is proper translational regulation and signaling, allowing cells to overcome stress. The fatal motor neuron disease Amyotrophic Lateral Sclerosis (ALS) develops in an age-related manner and is marked by progressive neuronal death, with cytoplasmic protein aggregation, excitotoxicity and increased oxidative stress as major hallmarks. Fused in Sarcoma/Translocated in Liposarcoma (FUS) is an RNA-binding protein mutated in ALS with roles in RNA and DNA processing. Most ALS-associated FUS mutations cause FUS to aberrantly localize in the cytoplasm due to a disruption in the nuclear localization sequence. Intriguingly, pathological inclusions in human FUSALS cases contain aggregated FUS as well as several SG-associated proteins. Further, cytoplasmic mutant FUS incorporates into SGs, which increases SG volume and number, delays SG assembly, accelerates SG disassembly, and alters SG dynamics. I posit that mutant FUS association with stress granules is a toxic gain-of-function in ALS that alters the function of SGs by interaction with SG components. Here, I show that mutant FUS incorporates in to SGs via its Cterminal RGG motifs, the methylation of which is not required for this localization. Further, I identify protein interactions specific to full-length mutant FUS under stress conditions that are potentially capable of interacting with FUS in SGs. Finally, I demonstrate a potential change in the protein composition of SGs upon incorporation of mutant FUS. These findings advance the field of ALS and SG biology, thereby providing groundwork for future investigation.

During stress, eukaryotes regulate protein synthesis in part through formation of cytoplasmic, non-membrane-bound complexes called stress granules (SGs). SGs transiently store signaling proteins and stalled translational complexes in response to stress stimuli (e.g. oxidative insult, DNA damage, temperature shifts and ER dysfunction). The functional outcome of SGs is proper translational regulation and signaling, allowing cells to overcome stress. The fatal motor neuron disease Amyotrophic Lateral Sclerosis (ALS) develops in an age-related manner and is marked by progressive neuronal death, with cytoplasmic protein aggregation, excitotoxicity and increased oxidative stress as major hallmarks. Fused in Sarcoma/Translocated in Liposarcoma (FUS) is an RNA-binding protein mutated in ALS with roles in RNA and DNA processing. Most ALS-associated FUS mutations cause FUS to aberrantly localize in the cytoplasm due to a disruption in the nuclear localization sequence. Intriguingly, pathological inclusions in human FUSALS cases contain aggregated FUS as well as several SG-associated proteins. Further, cytoplasmic mutant FUS incorporates into SGs, which increases SG volume and number, delays SG assembly, accelerates SG disassembly, and alters SG dynamics. I posit that mutant FUS association with stress granules is a toxic gain-of-function in ALS that alters the function of SGs by interaction with SG components. Here, I show that mutant FUS incorporates in to SGs via its Cterminal RGG motifs, the methylation of which is not required for this localization. Further, I identify protein interactions specific to full-length mutant FUS under stress conditions that are potentially capable of interacting with FUS in SGs. Finally, I demonstrate a potential change in the protein composition of SGs upon incorporation of mutant FUS. These findings advance the field of ALS and SG biology, thereby providing groundwork for future investigation.

Le lymphome à cellules du manteau (LCM) est une hémopathie maligne B mature, appartenant à la famille des lymphomes non hodgkiniens. Le LCM est caractérisé par la translocation t(11;14)(q13;q32) qui provoque une expression aberrante de cycline D1. C’est une pathologie rare mais à haut risque de rechute, et qui reste le plus souvent incurable suite à l’apparition de clones chimiorésistants. L’acquisition de résistance est intimement liée aux interactions entre les cellules tumorales et leur microenvironnement. Afin de mimer de la manière la plus pertinente possible ces interactions, nous avons mis en place un modèle murin de xénogreffe en utilisant les lignées cellulaires de LCM JeKo1, REC1, Z138 et Granta-519 que nous avons modifiées afin qu’elles expriment un fluorophore (GFP ou m-cherry) et/ou le gène codant pour la luciférase. Après injection aux souris du substrat de la luciférase, la luciférine, nous sommes en mesure de suivre au cours du temps la progression tumorale. Nous pouvons également évaluer le degré d’infiltration tumorale dans la moelle osseuse, la rate, le cerveau et le sang après euthanasie des animaux, par des techniques de cytométrie en flux et d’immunocytochimie. Ce modèle nous a permis de montrer l’intérêt thérapeutique d’un inhibiteur de l’exportine 1 (XPO1) : le KPT 330 (ou selinexor) qui est capable de contenir cycline D1 uniquement au niveau nucléaire. Nous avons montré que la localisation subcellulaire de cycline D1, est retrouvée majoritairement cytoplasmique dans certaines lignées cellulaires de LCM (2/7) et chez un certain nombre de patients (6/42, 14%), et est associée à un fort potentiel d’invasion, de migration et à un phénotype agressif. Par ailleurs, grâce à ce modèle, nous avons pu objectiver le manque d’efficacité in vivo d’agonistes aux récepteurs aux œstrogènes de type β (ER β). Ces récepteurs, présents sur les lymphocytes B étaient supposés inhiber la prolifération cellulaire et provoquer la mort des cellules par apoptose. L’utilisation de deux agonistes des ER β, le diarylpropionitrile (DPN) et l’ERB-041 a montré une absence d’effet de ces molécules, lorsque les cellules tumorales sont au contact de leur microenvironnement. D’autre part, afin de mieux comprendre les mécanismes de résistance aux chimiothérapies, nous avons étudié la résistance de la lignée cellulaire REC-1 traitée par des agents génotoxiques. Nous avons montré que cette lignée présentait une anomalie de dégradation de cycline D1 associée à une activité diminuée du protéasome 26S. Enfin, nous avons montré dans des travaux préliminaires que la protéine fused in sarcoma (FUS) pourrait, lorsqu’elle est associée à cycline D1, être capable de réguler les voies de réparation des dommages à l’ADN. Des anomalies de ces voies induisent une grande instabilité génétique responsable de l’échappement des tumeurs aux traitements, le ciblage de FUS pourrait par conséquent présenter un intérêt thérapeutique.Pris dans leur ensemble, ces résultats permettent de renforcer ou d’infirmer l’intérêt de certaines cibles thérapeutiques dans l’espoir de pouvoir continuer à améliorer la prise en charge des patients. Ils fournissent également un outil pour l’évaluation de nouvelles molécules dans un modèle murin prenant en compte les interactions entre la cellule tumorale et son microenvironnement
Mantle cell lymphoma (MCL) is a mature malignant hemopathy, belonging to the non-Hodgkin's lymphoma family. The MCL is characterized by the translocation t(11;14)(q13;q32) which causes an aberrant expression of cyclin D1. It is a rare disease but at high risk of relapse, and it is most often incurable due to the appearance of chemoresistant clones. The acquisition of resistance is intimately linked to the interactions between the tumor cells and their microenvironment. In order to mimic, in the most relevant way, these interactions, we have implemented a mouse xenograft model using the MCL cell lines JeKo1, REC1, Z138 and Granta-519 which we have modified so that they express a fluorophore (GFP or m-cherry) and / or the gene encoding the luciferase. After injection to the mice of the luciferase substrate, luciferin, we are able to follow over time the tumor progression. We can also assess the degree of tumor infiltration in bone marrow, spleen, brain and blood after euthanasia of animals, by flow cytometry and immunocytochemistry. This model allowed us to show the therapeutic interest of an inhibitor of exportin 1 (XPO1): the KPT 330 (or selinexor) which is able to contain cyclin D1 only on the nuclear level. We have shown that the subcellular localization of cyclin D1 is mainly cytoplasmic in some LCM (2/7) cell lines and in a number of patients (6/42, 14%), and is associated with a high potential Invasion, migration and an aggressive phenotype. Moreover, thanks to this model, we have been able to objectify the in vivo lack of efficacy of agonists to β-type estrogen receptors (ER β). These receptors, present on B lymphocytes, were thought to inhibit cell proliferation and cause cell death by apoptosis. The use of two ER β agonists, diarylpropionitrile (DPN) and ERB-041 showed an absence of effect of these molecules, when the tumor cells are in contact with their microenvironment. On the other hand, in order to better understand the mechanisms of resistance to chemotherapies, we studied the resistance of the REC-1 cell line treated with genotoxic agents. We have shown that this line has an abnormality of cyclin D1 degradation associated with decreased activity of the 26S proteasome. Finally, we have shown in preliminary work that the fused in sarcoma protein (FUS) could, when associated with cyclin D1, be able to regulate the repair pathways of DNA damage. Abnormalities of these pathways induce a great genetic instability responsible for the escape of tumors to treatments, the targeting of FUS could therefore be of therapeutic interest.Taken as a whole, these results reinforce or invalidate the interest of certain therapeutic targets in the hope of continuing to improve the management of patients. They also provide a tool for evaluating new molecules in a murine model that takes into account the interactions between the tumor cell and its microenvironment

Background: The submitted cumulative dissertation is based on two intertwined main studies with biomolecular foundation and clinical perspective on FUS-ALS complemented by two follow-up projects. This subtype of Amyotrophic lateral sclerosis is caused by heterozygous mutations mainly in the NLS of the FUS gene, which interferes with the proper nuclear import of the gene product. To date, there is no sufficient therapy available for this devastating neurodegenerative disease due to an incomplete pathophysiological understanding. Furthermore, not much is known about the specific clinical phenotype of FUS-ALS patients, including the influence of distinct FUS mutations due to the rarity of the disease. FUS is a DNA/RNA-binding protein that is mainly located in the nucleus and has essential functions in splicing, mRNA transport, transcription, and DNA damage repair. Hypothesis:1. It was hypothesized that the human-induced pluripotent stem-cell technique enables to create a sufficient motor neuron in-vitro cell model, which should provide new insights into unknown pathophysiological processes compared to previous cell models of FUS-ALS due to its patient-specific and human character. Thus, screening for potential therapeutic substances should be feasible using this model system. 2. Judging from the previously demonstrated, essential function of FUS in the DNA damage repair, FUS mutations are expected to increase the risk of malignant diseases in affected patients. Moreover, specific correlations between the nature of the mutation and the clinical, neurological phenotype appear plausible.Material & methods: First, an in-vitro cell culture model of FUS-ALS was established. For this purpose, a patient-specific, induced pluripotent stem cell-derived sMN culture was generated, which contained spinal motor neurons with mutations in the gene FUS or WT control cells. The Microfluidic Chamber system was used for the selective analysis of axons, which enabled the live-cell imaging of lysosomes and mitochondria using TIRF microscopy. For the analysis of DNA damage and its repair, gamma-H2A.X immunofluorescence staining was used on the one hand and live-cell laser ablation microscopy on the other, which allowed the precise induction of DNA damage and the monitoring of the repair response. For this purpose, isogenic FUS-GFP cell lines generated via CRISPR-Cas9n were used. A multicentre, retrospective cross-sectional study was conducted to determine genotype-phenotype correlations and the prevalence of malignant neoplasms in FUS-ALS. Previously published FUS-ALS cases have been added to perform a meta-analysis of clinical features.Results: Primarily, correct neuronal differentiation was observed prior to neurodegenerative phenotypes, perfectly mimicking a neurodegenerative disease in the dish. The typical cellular pathology of cytoplasmatic FUS deposition could be reproduced, making it a suitable model for more in-depth pathophysiological studies. Furthermore, the use of Microfluidic Chambers enabled the guided cultivation of neurons with somato-axonal direction of neurite outgrow along tiny microchannels in silico, resulting in a pure motoneuronal, axonal model. Within the distal axonal compartment of these channels, a loss of motility of both lysosomes and mitochondria was observed in parallel with a loss of the mitochondrial membrane potential, followed by the secondary degeneration of the distal axons of the sMNs with FUS mutation. A pathological increase in nuclear DNA damage has been identified as the cause of the distal-axonal phenotypes. This was due to a reduced nuclear FUS abundance as a result of the FUS-NLS mutation, which impaired proper nuclear import. There was evidence of a vicious cycle in this setting because the loss of the nuclear function of FUS disrupted the proper PAR-dependent DNA damage response, resulting in sustained DNA damage. Moreover, the remaining nuclear FUS was transferred into the cytoplasm upon phosphorylation by DNA-PK in a DNA damage response dependent manner, which is to date a process of unclear biological relevance. However, pharmacological inhibition of either the degradation of the PAR biopolymer or DNA-PK improved the nuclear presence of mutant FUS, restored its function in the DNA damage response, and finally prevented the distal axonal phenotype. Furthermore, the multicentric cohort study included 36 newly diagnosed patients. Only one in 40 patients was diagnosed with a malignant disease. By combining the newly diagnosed patients with previously published cases (186 cases in total), the so far most comprehensive database of FUS-ALS patients has been created. This allowed a thorough genotype-phenotype analysis, which showed a clear correlation between individual FUS mutations and the clinical phenotype. Conclusion: The experimental results indicated a primary nuclear insufficiency of mutated FUS, which is due to an impaired nuclear import and leads to a secondary axonal degeneration and finally to neuronal demise (“Dying-Back”). Potential therapeutic options have been identified, but their applicability and safety must be determined in prospective studies. The hypothesis of a generally increased risk of malignant diseases in the analysed FUS-ALS patient group was rejected. However, the clinical data of the meta-analysis are helpful in the counselling of newly diagnosed FUS-ALS patients, including the decision making of the therapeutic management and clearly add FUS-ALS to the family of diseases characterised by deficient DNA damage repair with purely neurological phenotypes such as AOA1, AOA2, and SCAN1.

Amyotrophic Lateral Sclerosis is a fatal neurodegenerative disease affecting upper and lower motor neurons. Patients display progressive paralysis with death typically resulting from diaphragmatic failure. Considerable evidence points out for a significant, but nevertheless complex genetic contribution for both ALS forms, familial and sporadic. Hence, subsequent work has identified a broad set of mutated genes associated with ALS symptoms, including Fused in Sarcoma (FUS). Not surprisingly, current efforts to develop new treatments for ALS involve the identification of small molecules that counteract the cellular hallmarks of the pathology. Ample evidence suggests that small bioactive molecules such as polyphenols have protective effects in neurodegenerative disorders. In fact, many studies have been supporting the possibility of changing the progression of the neurodegeneration through diet. Our study had a two-fold objective: to characterize the neuronal and kinematic decay of a Drosophila model of ALS; to test the protective effect of a small molecule previously identified for its capability to improve cellular growth of a yeast model overexpressing FUS. Since Drosophila has a relatively complex nervous system and a stereotyped set of motor outputs, it represents a step forward in the validation of this promising small molecule. Consistent with previous transgenic models, the Drosophila model of ALS overexpressing human FUS alleles (wild type and R521C) exhibited locomotor deficits, impairment of the climbing ability, reduced reproduction and shortened life span, with the degree of severity of mutant FUS phenotypes more aggressive than the wild type form. Furthermore, a detailed analysis of the locomotor pattern of the flies modelling ALS, using the custom-made FlyWalker system, revealed that the motor phenotype of these flies is evident after 14 days of FUS wild type expression. In addition, FUS wild type transgenic flies exposed to 10 mM of the small molecule showed a significant increase in the survival rate. Collectively, we conclude that the Drosophila model captures important aspects of human FUS-based ALS, providing a useful tool to test the efficacy of bioactive molecules.