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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 (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.

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.

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.