La maladie de Huntington (MH) est une maladie autosomale dominante causée par une expansion de la répétition CAG codant pour une expansion de la polyglutamine dans le premier exon du gène Huntingtine (HTT). Ce gène code pour une protéine ubiquitaire dont la mutation entraine de graves symptômes moteurs, psychiatriques et cognitifs, dus à la dégénérescence spécifique des neurones GABAergique épineux moyens du striatum. Nous proposons d'utiliser le trans-épissage pour développer un vecteur de thérapie génique qui réduira significativement voir éliminera l'expression de la protéine mutée tout en restaurant un niveau physiologique de HTT normale dans les cellules affectées par la mutation du gène Huntingtine. Cette technologie est basée sur le remplacement de l'exon muté par un exon sans mutation pendant l'étape de maturation de l'ARNm. Du fait du caractère dominant de la mutation,l'efficacité thérapeutique nécessitera une réaction de trans-épissage très efficace capable de convertir une portion significative de pre-ARNm HTT mutés en en ARNm HTT normaux. Nous avons donc développé un système rapporteur fluorescent permettant la détection des évènements de trans-épissage afin d’identifier les séquences les plus performantes parmi une centaine de molécules candidates. Nous avons validé notre stratégie de criblage basée sur la fluorescence et réalisé le criblage sur plusieurs introns HTT (3, 9 et 20) qui ont démontré des zones favorables au trans-épissage. Une méthode de quantification directe et absolue du taux de trans-épissage a également été validée pour déterminer très précisément le taux de correction. L’ensemble de ce travail a permis de contribuer à la mise en évidence de la faisabilité du trans-épissage dans le contexte de la MH. / Huntington’s disease (HD) is an autosomal dominant genetic disorder caused by the expansion of a CAG repeat encoding a polyglutamine tract in the first exon of the Huntingtin gene (HTT). This gene encode a ubiquitous protein in which mutation lead to severe motor, psychiatric and cognitive deficits and causes degeneration of specific neuronal populations, in particular the GABAergic medium spiny neurons of the striatum. We propose to use trans-splicing to develop a gene therapy vector that will significantly reduce or eliminate the expression of the mutant protein while restoring a physiological level of normal HTT in cells affected by the HD mutation. This technology is based on replacement of the mutated exon by a normal version during the mRNA maturation process. HTT mutation being dominant, therapeutic benefits necessitates a highly efficient trans-splicing reaction that would convert a significant proportion of mutant-HTT pre-mRNA into normal HTT mRNA. For this purpose, we developed a fluorescent reporter system enabling the detection of trans-splicing events in high content screening in order to identify the most potent trans-splicing sequences among hundreds of molecules. We validated our fluorescent screening strategy and implement trans-splicing screening on 3 HTT introns (3, 9 and 20), in which we demonstrated the presence of hotspot promoting trans-splicing reactions. A direct and absolute quantification method was also validated to accurately assess the correction rate. Overall, this work generated additional evidences of trans-splicing feasibility in HD.

Huntington's disease (HD) is a fatal neurodegenerative disorder characterized by progressive loss of striatal and cortical neurons. HD is caused by an abnormal polyglutamine (polyQ) expansion in Huntingtin protein (HTT). HTT controls vesicular trafficking along axons in neurons through interaction with components of the molecular motor machinery. Arginine methylation is one of the most abundant post-translational modifications (PTMs) and is catalyzed by protein arginine methyltransferases (PRMTs). Recent evidence supports a key role for arginine methylation in neurodegeneration and particularly in polyglutamine diseases. However, whether HTT is methylated at arginine residues has not been investigated yet and the role of arginine methylation in HD pathogenesis remains to be fully elucidated. In this thesis, I show that vesicle-associated HTT is methylated in vivo at two evolutionarily conserved arginine residues, namely R101 and R118. Methylation of HTT at R118 is catalyzed by Protein Arginine Methyltransferase 6 (PRMT6), which localizes on vesicles together with HTT, whereas further analyses are required to identify the enzyme(s) responsible for R101 methylation. Interestingly, loss of PRMT6-mediated R118 methylation reduces the association of HTT with vesicles, impairs anterograde axonal transport and exacerbates polyQ-expanded HTT toxicity. Conversely, PRMT6 overexpression improves the global efficiency of anterograde axonal transport and rescues cell death in neurons expressing polyQ-expanded HTT. These findings establish a crucial role of arginine methylation as a modulator of both normal HTT function and polyQ-expanded HTT toxicity and identify PRMT6 as a novel modifier of HD pathogenesis. Importantly, defects in HTT methylation may contribute to neurodegeneration in HD and promoting arginine methylation of HTT might represent a new therapeutic strategy for HD.

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<p>Huntington’s disease (HD) is a devastating autosomal dominant neurodegenerative disorder for which there are no disease modifying treatments. Owing to this are the multiple biological functions of the huntingtin protein and the lack of understanding of the exact pathways being affected in HD. It is clear that the huntingtin protein normally provides anti-apoptotic support and that there are underlying energetic problems and cell stress defects associated with disease. Work from our group has shown that huntingtin acts as a stress sensor and translocates from the endoplasmic reticulum to the nucleus upon cell stress. We therefore hypothesized that huntingtin has a nuclear function in the cell stress response; which would tie together what is currently known about huntingtin, its pro-apoptotic function and the energetic defects of neurodegeneration. In this thesis we describe huntingtin as having a role in the nuclear cofilin-actin rod stress response. Cofilin is an actin binding protein normally involved in actin treadmilling. During stress, cofilin saturates F-actin leading to rod formation which functions to alleviate ATP. We show that this response is impaired in the presence of mutant huntingtin and that the aberrations in this response can be mediated through the enzyme tissue transglutaminase. Little is known about the physiological role and requirement of the cofilin-actin rod response. Therefore we created a system to test if rod formation was required in cells during stress, which indicates if and how targeting this pathway will be possible. We additionally looked at targeting the nuclear import and export properties of the cofilin protein, which directly affect rod formation and may be targetable in cofilin modifying drug discovery efforts. Overall, this work has described a specific and relevant pathway affected by mutant huntingtin and started the process of assessing this pathway as a therapeutic avenue for Huntington’s disease.</p> / Doctor of Philosophy (PhD)

<p>Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein. Recent research demonstrates that post-translational modifications of huntingtin could be an important determinant of mutant huntingtin’s toxicity in HD. In particular, phosphorylation at residues serine 13 and 16 within the first 17 amino acids of huntingtin (N17), have been shown to be critical modulators of mutant huntingtin’s toxicity and localization, and can be triggered by stress. This project aims to study how phosphorylation within N17 alters the interactome at this site and what physiological stress results in the nuclear localization of N17 and huntingtin. The initial search to identify potential interactors was conducted through an affinity chromatography assay using a wild type striatal cell line derived from knock in mouse model of HD. Fluorescent lifetime imaging microscopy (FLIM) to determine Fӧrester resonance energy transfer (FRET), co-immunoprecipitation and co-immunofluorescence assays were then used to validate real interactors of N17. Analysis from this project has validated two previously unidentified interactors of N17; SET, a small nucleo-oncoprotein, and vimentin, a type 3 intermediate filament. Both interactors have suggested two potentially novel roles for N17 within huntingtin, in cell cycle regulation and intermediate filament dynamics. Finally, smart screening techniques using stress-inducing compounds reveal that the sensitivity of N17 to stress and its subsequent nuclear localization can be attributed in part to activation of oxidative stress pathways. Data shown here can be expanded upon to elucidate how huntingtin function and response to cell stress are regulated and mediated via N17 and potentially how this mechanism is disrupted within HD.</p> / Master of Science (MSc)

In healthy cells, huntingtin is primarily found in the cytoplasm; however, upon cellular stress, huntingtin is phosphorylated (phospho-huntingtin) at serines 13 and 16 of the amino-terminal N17 domain and shuttled into the nucleus. Such dynamism in nucleocytoplasmic translocation and post-translational modification suggests an important role for huntingtin in Huntington’s disease (HD) pathogenesis as these phenotypes propose possible mechanisms for disease progression. Huntingtin nuclear import is also facilitated by its proline-tyrosine nuclear localization signal (PY-NLS), which harbours a highly conserved intervening sequence specific to the huntingtin gene. This encouraged a proteome investigation to identify potential protein partners of the PY- NLS. Results of this study revealed that high mobility group box 1 (HMGB1), a cofactor of base excision repair, uniquely bound to the wild-type PY-NLS, but not the PY-NLS KK177/178AA mutant. Immunofluorescence microscopy in human telomerase reverse transcriptase (hTERT) immortalized fibroblast cells using HMGB1- and phospho- huntingtin-specific antibodies revealed a promising association between the two, as changes in nuclear levels of HMGB1 positively correlated with nuclear levels of phospho- huntingtin. This relationship was further confirmed by co-immunoprecipitation of HMGB1 by the PY-NLS and N17 domain. Also, when exogenous oxidative stress was introduced, increased interaction between HMGB1 and huntingtin was observed. This suggests that HMGB1 facilitates the nuclear import of huntingtin in a ROS-dependent manner, and thus, presents a novel avenue to a potential therapeutic target in HD pathogenesis. / Thesis / Master of Science (MSc)

La huntingtine (HTT) est une protéine d’échafaudage participant à des fonctions indispensables au bon fonctionnement cellulaire. Elle est codée par le gène HTT qui présente une répétition polymorphique de triplet CAG. Une répétition excédant 35 CAG dans la HTT est à l’origine de la maladie de Huntington, une maladie neurodégénérative héréditaire sévère. Ainsi, bien que d’expression ubiquitaire, la HTT est principalement étudiée dans le système nerveux. Par exemple, ses implications dans le tissu mammaire, en condition normale et pathologique, sont inconnues. Nous avons observé que la forme mutante de la HTT accélère le développement de cancer du sein et en accentue la sévérité et que la forme sauvage est impliquée dans le développement normal de la glande mammaire. Mon projet principal de thèse était de caractériser le rôle de la HTT, de sa phosphorylation à la sérine 421 (S421-P-HTT) ainsi que du polymorphisme des répétitions CAG dans les cancers du sein.En utilisant des modèles cellulaires et murins et par des études d’expression chez des patientes atteintes d’un cancer du sein, j’ai observé que l’expression de la HTT et de la S421-P-HTT corrèlent avec le stade de différenciation tumorale. Au niveau moléculaire, la HTT régule, par sa phosphorylation à la S421, l’expression et la localisation d’une des protéines des jonctions serrées, ZO1 et module ainsi l’adhésion intercellulaire. ZO1 colocalise avec la S421-P-HTT aux jonctions intercellulaires et forme un complexe avec la HTT. La perte d’expression de HTT est pro-Métastatique chez la souris et est moindre dans les cancers du sein métastatiques. De plus, les niveaux d’expression de HTT et de ZO1 sont diminués en parallèle dans les carcinomes humains de bas grades.J’ai également montré que le polymorphisme CAG présent dans la HTT sauvage joue un « double emploi » : tandis que de longues répétitions protègent de l’apparition de cancers, elles accentuent sa sévérité lorsque la maladie se développe. Dans le sous-Type HER2 spécifiquement, la longueur de la répétition CAG est un facteur pronostic indépendant du développement de métastases.Ainsi, ces travaux ont permis de mettre en évidence un rôle clé pour la HTT au cours de la progression tumorale mammaire, et devraient conduire à une meilleure compréhension des mécanismes moléculaires impliqués dans le développement de métastases dans le cancer du sein. / Huntingtin (HTT) is a scaffold protein involved in numerous cellular mechanisms essentials for appropriate physiological functions. HTT is encoded by HTT gene which carries a polymorphic repetition of CAG triplet. When the CAG repetition exceeds 35, it leads to Huntington’s disease, a hereditary severe neurodegenerative disorder. While HTT expression is ubiquitous, it is mainly studied in nervous system. For example, HTT roles in breast physiology and cancer are unknown. We demonstrated that mutant HTT accelerates breast tumor and metastasis development and that wild-Type HTT is involved in normal mammary gland development. My main project was to characterize the roles of HTT and of its phosphorylation at S421 (S421-P-HTT) and that of the polymorphic CAG length in mammary carcinomas.First, leaning on cellular and murine models as well as on expression studies in breast cancer patients, I observed that HTT and S421-P-HTT expression correlates with tumoral differentiation stage. At the molecular level, HTT regulates through its phosphorylation at S421, the expression and localization of ZO1, a marker of intercellular junction and therefore modulates intercellular adhesion. ZO1 colocalizes with S421-P-HTT specifically at tight junctions and forms a complex with HTT. Loss of HTT is itself pro-Metastatic in mice and is decreased in metastatic human breast cancer. Moreover, HTT and ZO1 are concomitantly downregulated in low-Grade human carcinomas.On the other hand, the polymorphism of CAG repetitions in HTT has a dual-Purpose: while long repetitions protect against cancer development, it increases its severity once cancer is developed. In HER2 subtype specifically, HTT appears as an independent prognostic factor of metastasis development.Thus, these studies point out a key function of HTT outside the brain during mammary carcinoma progression and should lead to a better understanding of molecular mechanisms involved in metastasis development.

De plus en plus de preuves montrent que le transport axonal rapide (FAT) joue un rôle crucial au cours des maladies neurodégénératives (NDs). La maladie de Huntington est une maladie neurodégénérative causée par une expansion anormale de polyglutamines dans la partie Nterminale de la protéine huntingtine (HTT) : une grande protéine d’échafaudage impliquée dans la régulation du transport. La présence de HTT mutante comme l’absence de la HTT induisent des défauts de transport chez les mammifères. Chez la Drosophile, la HTT mutante reproduit le phénotype observée chez les mammifères, cependant la fonction conservée de la HTT chez la Drosophile melanogaster (DmHTT) n’est pas encore clairement établie. Ici nous mettons en évidence que DmHTT s’associe aux vésicules, aux microtubules et intéragit avec la proteine dynéine. Dans les neurones corticaux de rat, DmHTT remplace partiellement la HTT de mammifère dans le transport axonal rapide, et les drosophiles invalidées pour la HTT montrent des défauts de transport axonal in vivo. Ces résultats suggèrent que la fonction de la HTT est conservée dans le modèle Drosophile.Le FAT est un processus qui requiert un apport constant d’énergie. Les mitochondries sont les principales sources de production d’ATP de la cellule. Cependant nous avons démontré que le FAT ne dépend non pas de cette source d’énergie là, contrairement à ce que l’on pensait, mais de l’ATP glycolytique produit par les vésicules. La dérégulation de GAPDH ou de PK, les deux enzymes glycolytiques productrices d’ATP, ralentit le transport vésiculaire. Néanmoins, l’invalidation de GAPDH n’affecte pas le transport mitochondrial. En outre, toutes les enzymes glycolytiques sont associées à des vésicules dynamiques et sont capables de produire leur propre ATP. Enfin nous montrons que l’ATP produit est suffisant pour assurer leur propre transport, prouvant l’autonomie énergétique des vésicules pour le transport. / Growing evidence support the idea that impairments in Fast Axonal Transport (FAT) play a crucial role in Neurodegenerative Diseases (NDs). Huntington’s Disease is neurodegenerative disorder caused by an abnormal polyglutamine expansion in the N-Terminal part of huntingtin (HTT), a large scaffold protein implicated in transport regulation. Both the presence of the mutated HTT as the loss of HTT leads to transport defects in mammals. In the fruit fly overexpression of the mutant HTT recapitulates the phenotype observed in mammals. However, it is still unclear whether HTT’s function is conserved in D. melanogaster. Here, we show that D. melanogaster HTT (DmHTT) associates with vesicles, microtubules, and interacts with dynein. In rat cortical neurons, DmHTT partially replaces mammalian HTT in fast axonal transport, and DmHTT KO flies show axonal transport defects in vivo. These results suggest that HTT function in transport is conserved in D. melanogaster.FAT is a process that requires a constant supply of energy. Mitochondria are the main producers of ATP in the cell. However, we have demonstrated that FAT does not depend on this source of energy, as previously thought, but it depends on glycolytic ATP produced on vesicles. Perturbing GAPDH or PK, the two ATP generating glycolytic enzymes, slows down vesicular transport. However, knocking down GAPDH does not affect mitochondrial transport. Furthermore, all of the glycolytic enzymes are associated with dynamic vesicles, and are capable of producing their own ATP. Finally, we show that this ATP production is sufficient to sustain their own transport, demonstrating the energetical autonomy of vesicles for transport.

Huntington's disease (HD) belongs to neurodegenerative disorders. It is a monogenic disease caused by trinucleotic CAG expansion in exon 1 of gene coding protein huntingtin. Even though the cause of HD is known since 1993, the pathophysiology and cure for HD reminds to be found. The animal models are being used for better understanding of HD. The most common animal models for HD are rodents, especially mice but it was also important to create large animal models, which will be more like human. Therefore, TgHD minipig was created in Academic of Science in Liběchov in 2009. This model was created by microinjection of lentiviral vector carrying N-terminal part of human HTT with 124 repetitive CAG in exon 1. This model is viable and in every generation, is part of the offspring transgenic. In this thesis, I specialized to biochemical and behavioral changes of this model. I compared transgenic and wild type siblings. I found that biochemical changes are manifested mostly by increased level of mtHtt fragments in testes and brain. In behavioral part of this thesis I established new methods for testing behavioral changes in this model. The introduced methods showed some changes between wild type and transgenic animals at the tested ages but these changes were not significant due to the low number of...

Huntington's disease (HD) is inherited fatal disorder caused by CAG triplet expansions in the huntingtin gene resulting in the expression of mutated huntingtin protein (mtHtt). The main symptoms of HD are neurodegeneration, osteoporosis, testicular degeneration, loss of muscle tissue and heart muscle malfunction, weight loss, metabolic changes, and sleeping disturbances. Since huntingtin protein (Htt) has a role in several biological processes, many molecular mechanisms, like oxidative stress, mitochondrial dysfunction, DNA-damage, and others, are affected by mtHtt. However, its exact pathogenic mechanisms in HD are still not well understood. Transgenic minipig model of HD (TgHD) serves an opportunity to isolate unlimited number of primary cells and unlike primary cells obtained from HD patients, often in the late stages of the disease, the TgHD minipig model allows to monitor molecular changes occurring gradually with age and progression of the disease. Thus, TgHD minipig model and primary cells isolated from it play an important role in investigating and understanding the underlying mechanistic cause of HD. We focused on molecular and cellular changes in primary cells isolated from TgHD minipigs and their wild type (WT) controls at different ages (24, 36, and 48 months). In mesenchymal stem cells...