Les protéines sont au coeur de la vie. Ce sont d'incroyables nanomachines moléculaires spécialisées et améliorées par des millions d'années d'évolution pour des fonctions bien définies dans la cellule. La structure des protéines, c'est-à-dire l'arrangement tridimensionnel de leurs atomes, est intimement liée à leurs fonctions. L'absence apparente de structure pour certaines protéines est aussi de plus en plus reconnue comme étant tout aussi cruciale. Les protéines amyloïdes en sont un exemple marquant : elles adoptent un ensemble de structures variées difficilement observables expérimentalement qui sont associées à des maladies neurodégénératives. Cette thèse, dans un premier temps, porte sur l'étude structurelle des protéines amyloïdes bêta-amyloïde (Alzheimer) et huntingtine (Huntington) lors de leur processus de repliement et d'auto-assemblage. Les résultats obtenus permettent de décrire avec une résolution atomique les interactions des ensembles structurels de ces deux protéines. Concernant la protéine bêta-amyloïde (AB), nos résultats identifient des différences structurelles significatives entre trois de ses formes physiologiques durant ses premières étapes d'auto-assemblage en environnement aqueux. Nous avons ensuite comparé ces résultats avec ceux obtenus au cours des dernières années par d'autres groupes de recherche avec des protocoles expérimentaux et de simulations variés. Des tendances claires émergent de notre comparaison quant à l'influence de la forme physiologique de AB sur son ensemble structurel durant ses premières étapes d'auto-assemblage. L'identification des propriétés structurelles différentes rationalise l'origine de leurs propriétés d'agrégation distinctes. Par ailleurs, l'identification des propriétés structurelles communes offrent des cibles potentielles pour des agents thérapeutiques empêchant la formation des oligomères responsables de la neurotoxicité. Concernant la protéine huntingtine, nous avons élucidé l'ensemble structurel de sa région fonctionnelle située à son N-terminal en environnement aqueux et membranaire. En accord avec les données expérimentales disponibles, nos résultats sur son repliement en environnement aqueux révèlent les interactions dominantes ainsi que l'influence sur celles-ci des régions adjacentes à la région fonctionnelle. Nous avons aussi caractérisé la stabilité et la croissance de structures nanotubulaires qui sont des candidats potentiels aux chemins d'auto-assemblage de la région amyloïde de huntingtine. Par ailleurs, nous avons également élaboré, avec un groupe d'expérimentateurs, un modèle détaillé illustrant les principales interactions responsables du rôle d'ancre membranaire de la région N-terminal, qui sert à contrôler la localisation de huntingtine dans la cellule. Dans un deuxième temps, cette thèse porte sur le raffinement d'un modèle gros-grain (sOPEP) et sur le développement d'un nouveau modèle tout-atome (aaOPEP) qui sont tous deux basés sur le champ de force gros-grain OPEP, couramment utilisé pour l'étude du repliement des protéines et de l'agrégation des protéines amyloïdes. L'optimisation de ces modèles a été effectuée dans le but d'améliorer les prédictions de novo de la structure de peptides par la méthode PEP-FOLD. Par ailleurs, les modèles OPEP, sOPEP et aaOPEP ont été inclus dans un nouveau code de dynamique moléculaire très flexible afin de grandement simplifier leurs développements futurs. / Proteins are at the center of life. They are formidable molecular nanomachines specialized and optimized during million years of evolution for well-defined functions in the cell. The structure of proteins, meaning the tridimensional setting of their atoms, is closely related to their function. Absence of structure for a subset of proteins is also recognized to be as crucial. Amyloid proteins is a striking example : they fold into an ensemble of various structures hardly observable experimentally that are associated with neurodegenerative diseases. This thesis, firstly, is on the study of the structural ensemble of the amyloid proteins amyloid-beta (Alzheimer) and huntingtin (Huntington) during their folding and aggregation. Our results describe in details, with an atomic resolution, the characteristic interactions present in the structural ensemble of these two proteins. Concerning the amyloid-beta protein (AB), our results show the structural differences between three of its physiological forms during its first aggregation steps in an aqueous environment. We have then compared these results with those obtained during the past few years by several other research groups using various experimental and simulation protocols. Clear trends come out of this comparison regarding the influence of AB physiological form on its structural ensemble during its first aggregation steps. Their distinct aggregation pathways are rationalized by the identified differences. For their part, the identified similarities offer targets for therapeutical compounds disrupting the aggregation of the neurotoxic oligomers. Concerning the huntingtin protein, we identify the structural ensemble of its functional region at its N-terminal in an aqueous environment and in a phospholipid membrane. In agreement with the available experimental results on the global structure of this region in aqueous solution, our results reveal the dominant interactions, at an atomic precision, in its structural ensemble as well as the influence of its neighboring regions. We have also characterized the stability and the growth of nanotube-like structures that could occur during the aggregation of the amyloid region of huntingtin. Moreover, we have developed, in collaboration with a group of experimentalists, a precise model describing the main membrane interactions of huntingtin N-terminal, which serves as a membrane anchor that controls the localization of huntingtin in the cell. Secondly, this thesis is on the refinement of a coarse-grained model (sOPEP) and on the development of a new all-atom model (aaOPEP) that are both based on the coarse-grained OPEP force field, commonly used to study protein folding and amyloid protein aggregation. The goal behind the optimization of these models is to improve the de novo structure prediction of the PEP-FOLD method. These three models -- OPEP, sOPEP and aaOPEP -- are now also implemented in a new molecular dynamics software that we have developed specifically to greatly ease their future developments.

La maladie de Huntington (MH) est une maladie neurodégénérative héréditaire autosomique dominante. Elle résulte d’une expansion anormale de glutamines (polyQ) dans la partie N-terminale de la protéine huntingtine (HTT ; codé par HTT). La MH est caractérisée par la dysfonction et la mort de cellules neuronales dans le cerveau, entraînant l’apparition de symptômes cognitifs, psychiatriques et moteurs, dévastateurs chez les patients. De nombreuses études sur des modèles animaux et cellulaires montrent que l’expansion polyQ dans la protéine mutante conduit à un gain de nouvelles fonctions toxiques, ainsi qu’à la perte de fonctions neuroprotectives de la protéine sauvage. Pendant ma thèse, je me suis intéressée à la description et à la validation fonctionnelle d’un nouvel outil pour étudier la HTT : pARIS-htt. pARIS-htt est un gène synthétique construit pour faciliter le clonage et le marquage de la protéine HTT totale. En utilisant différentes approches cellulaires, nous avons montré que pARIS-htt peut remplacer le rôle de la HTT endogène dans le transport de vésicules du Golgi ainsi que du brain derived neurotrophic factor (BDNF). La version mutante de pARIS-htt ne peut pas restaurer cette fonction. Parallèlement, nous avons généré deux variants de pARIS-htt avec soit une délétion dans la région d’interaction de la HTT avec la dynéine, moteur moléculaire se dirigeant vers l'extrémité négative des microtubules, soit avec la huntingtin associated protein 1 (HAP1), l’un de ses interacteurs. Dans les expériences de remplacement du gène, aucun des deux mutants n’a restauré le transport vésiculaire.Un autre aspect de ma thèse a été d’étudier le rôle de la HTT au cours de la mitose. Nous avons mis en évidence l’importance de la HTT dans le contrôle de l’orientation du fuseau. Cette fonction est perdue lorque la HTT est mutée, mais restaurée lorsque celle-ci est phosphorylée par Akt à la sérine 421. Le contrôle de l’orientation du fuseau est particulièrement important durant la neurogénèse puisque cette orientation ainsi que le mode de division sont impliqués dans la détermination des devenirs cellulaires. Cette fonction de la HTT est conservée chez la D. melanogaster.Cette étude a donc permis de mieux comprendre les fonctions de la HTT, et de proposer de nouvelles cibles thérapeutiques pour traiter la MH. / Huntington disease (HD) is an autosomal-dominant neurodegenerative disorder caused by the pathogenic expansion of the poly-glutamine (polyQ) N-terminal stretch in the huntingtin protein (HTT; encoded by HTT). HD is characterized by the dysfunction and death of neurons in the brain, leading to devastating cognitive, psychiatric, and motor symptoms in patients. Studies in multiple cell and animal model systems support the notion that polyQ expansion in mutant HTT leads to the gain of new toxic functions and loss of the neuroprotective functions of the wild-type HTT. During my thesis, I focused on the description and functional validation of a new tool to study HTT: pARIS-htt. pARIS-htt is a synthetic gene built to facilitate cloning and tagging of full-length HTT. Using different cellular approaches, we showed that pARIS-htt can replace endogenous HTT in the transport of Golgi and brain derived neurotrophic factor (BDNF) containing vesicles. pARIS-htt mutant version could not restore vesicular transport when endogenous HTT was knocked-down. Moreover, we generated pARIS-htt deletion mutants for HTT interaction domain with dynein, a minus-end directed motor protein, and huntingtin associated protein 1 (HAP1), a HTT interactor. Both deletion mutants failed to restore vesicular transport in gene replacement assays. Another aspect of my thesis was the study of HTT during mitosis. We showed that HTT monitors spindle orientation though its interaction with diverse proteins involved in cell division. This function is lost when HTT is mutated and can be reverted by Akt phosphorylation at serine 421. The control of spindle orientation is particularly important during neurogenesis since spindle orientation and the mode of division of apical progenitors are implicated in the determination of cell fate. This function of HTT is conserved in D. melanogaster. This study contributes to the understanding of HTT functions and suggests new therapeutical approaches to treat HD.

Huntington’s disease (HD) is a neurodegenerative disorder caused by the inheritance of one mutant copy of the huntingtin gene. Mutant huntingtin protein (mHtt) contains an expanded polyglutamine repeat region near the N-terminus. Cleavage of mHtt releases an N-terminal fragment (N-mHtt) which translocates, and accumulates in the nucleus. Nuclear accumulation of N-mHtt has been directly associated with cellular toxicity. Decreased transcription is among the earliest detected changes that occur in the brains of HD patients and is consistently observed in all animal and cellular models of HD. Transcriptional dysregulation may trigger many of the perturbations that occur later in disease progression and an understanding of the effects of mHtt may lead to strategies to slow the progression of the disease. Current models of N-mHtt-mediated transcriptional dysregulation suggest that abnormal interactions between N-mHtt and transcription factors impair the ability of these transcription factors to associate at N-mHtt-affected promoters and properly regulate gene expression. We tested various aspects of these models using two N-mHtt-affected promoters in in vitro transcription assays and in two cell models of HD using techniques including overexpression of known N-mHtt-interacting transcription factors, chromatin immunoprecipitation, promoter deletion and mutation analyses and in vitro promoter binding assays. Based on our results and those in the literature, we proposed a new model of N-mHtt-mediated transcriptional dysregulation centered on the presence of N-mHtt at affected promoters. We concluded that simultaneous interaction of N-mHtt with multiple binding partners within the transcriptional machinery would explain the gene-specificity of N-mHtt-mediated transcriptional dysregulation, as well as the observation that some genes are affected early in disease progression while others are affected later. Our model explains why alleviating N-mHtt-mediated transcriptional dysregulation through overexpression of N-mHtt-interacting proteins has proven to be difficult and suggests that the most realistic strategy for restoring gene expression across the spectrum of N-mHtt affected genes is by reducing the amount of soluble nuclear N-mHtt.

No description available.

The causative role of the huntingtin (HTT) gene in Huntington's disease (HD) has been identified more than 25 years ago. The extension of CAG repeat stretch over 39 repeats in exon 1 of one HTT allele results in full penetrance of this neurodegenerative disorder. While the identification of the causative mutation raised hopes that development of the therapeutic compound will be easily achievable, the patients and their families are still waiting for treatment until now. The main reason for that might be the complex cellular function HTT that makes the determination of the pathologic mechanism difficult and the development of treatments even more challenging. Although a lot of different animal models have been generated until now, establishing a suitable model has still not been achieved yet. Due to its anatomy, physiology, and genetics, the minipig seems to be a suitable candidate for neurodegenerative disease models. Indeed, the existing Transgenic (Tg) Libechov minipig model manifests signs typical for HD in patients, but on the other hand significant inconsistencies have also been observed. The finding of malformation that partially shows the situation in human patients is true for both, the male reproductive tract as well as for the brain. The reason for this might be the fact the genetic...

La maladie de Huntington (MH) est une maladie neurodégénérative causée par une extension de répétitions du codon CAG dans le gène de la Huntingtine (Htt). Cette maladie est caractérisée par la mort des neurones striataux et la présence d’agrégats de Htt mutée (mHtt). De plus, au cours de la MH, les astrocytes, qui sont essentiels au bon fonctionnement neuronal, changent d’état et deviennent réactifs. La réactivité astrocytaire est caractérisée par des changements morphologiques et transcriptomiques mais l’impact fonctionnel de cette réactivité reste peu compris.Afin d’étudier le rôle des astrocytes réactifs dans la MH, nous avons utilisé des vecteurs viraux récemment développés par notre équipe, qui induisent ou bloquent la réactivité astrocytaire in vivo en ciblant la voie JAK2-STAT3. Nous avons montré que les astrocytes réactifs diminuent le nombre et la taille des agrégats de mHtt majoritairement présents dans les neurones. Ceci est associé à l’amélioration de plusieurs altérations neuronales observées dans ces modèles. Une analyse transcriptomique réalisée sur des astrocytes réactifs révèle des changements majeurs d’expression de gènes liés aux systèmes de protéostasie. De plus, l’activité du lysosome et du protéasome est augmentée dans les astrocytes réactifs de souris modèles de la MH. Nous montrons également que les astrocytes réactifs éliminent plus efficacement leurs propres agrégats de mHtt, suggérant qu’au cours de la MH, ces cellules pourraient dégrader plus efficacement la mHtt provenant des neurones. De plus, certaines protéines chaperonnes sont induites dans les astrocytes réactifs. En particulier, la co-chaperonne DNAJB1/Hsp40 est surexprimée dans les astrocytes réactifs et est retrouvée dans les exosomes isolés à partir de striata de souris MH. Des expériences de gain et perte de fonction suggèrent que cette chaperonne est impliquée dans les effets bénéfiques des astrocytes réactifs sur l’agrégation de la mHtt et l’état des neurones. Les astrocytes réactifs pourraient donc libérer des protéines anti-agrégantes qui favorise l’élimination de la mHtt dans les neurones.Notre étude montre que les astrocytes peuvent, en devenant réactifs au cours de la MH, acquérir des propriétés bénéfiques pour les neurones et favoriser, via un dialogue complexe avec les neurones, l’élimination des agrégats de mHtt. / Huntington’s disease (HD) is a hereditary neurodegenerative disease caused by an expansion of CAG codons in the Huntingtin gene. It is characterized by the death of striatal neurons and the presence of mutant Huntingtin (mHtt) aggregates. In pathological conditions, as in HD, astrocytes change and become reactive. Astrocyte reactivity is characterized by morphological and significant transcriptomic changes. Astrocytes are essential for the proper functioning of neurons but the functional changes associated with reactivity are still unclear.To better understand the roles played by reactive astrocytes in HD, we took advantage of our recently developed viral vectors that infect selectively astrocytes in vivo and either block or induce reactivity, through manipulation of the JAK2-STAT3 pathway. We used these vectors in two complementary mouse models of HD and found that reactive astrocytes decrease the number and the size of mHtt aggregates that mainly form in neurons. Reduced mHtt aggregation was associated with improvement of neuronal alterations observed in our mouse models of HD. A genome-wide transcriptomic analysis was performed on acutely sorted reactive astrocytes and revealed an enrichment in genes linked to proteolysis. Lysosomal and proteosomal activities were also increased in reactive astrocytes in HD mice. Moreover, we show that reactive astrocytes degrade more efficiently their own mHtt aggregates, suggesting that these cells could siphon mHtt away from neurons. Alternatively, several chaperones were induced in reactive astrocytes. In particular, the co-chaperone DNAJB1/Hsp40 was upregulated in reactive astrocytes and was present in exosomal fraction from HD mouse striatum. Loss and gain of function experiments suggest that this chaperone is involved in the beneficial effects of reactive astrocytes on mHtt aggregation and neuronal status. Therefore, reactive astrocytes could release anti-aggregation proteins that could promote mHtt clearance in neurons.Overall, our data show that astrocytes, by becoming reactive in HD, develop a protective response that involves complex bidirectional signaling with neurons to reduce mHtt aggregation.

Huntington's disease (HD) is an inherited autosomal dominant neurodegenerative disorder caused by an unstable expansion of the CAG repeat sequence within the huntingtin gene. Huntingtin associates with ubiquitin-proteasome system that ensures degradation of particular proteins including β-catenin which is an important molecule whose equilibrated degradation is necessary for the proper functioning of the Wnt signaling pathway. The binding of β-catenin to the destruction complex is altered in HD, leading to the toxic stabilization of β-catenin. The main goal of my thesis was to determine whether the accumulation of β-catenin due to the presence of mutant huntingtin is also characteristic of Liběchov minipigs, a large animal model of Huntington's disease stably expressing N-truncated human mutant huntingtin. Using immunoblot and specific antibodies, we have revealed age-dependent accumulation of mutant huntingtin in transgenic minipigs. Unlike endogenous huntingtin, no decrease of the level of mutant huntingtin was observed in the striatum of transgenic animals. Surprisingly, this was followed by a decrease of phosphorylated β-catenin. Nevertheless, our results demostrate the accumulation of β-catenin in mesenchymal stem cells isolated from the oldest boars during ontogenesis. Furthermore, we have revealed a...

Huntington's disease (HD) is an autosomal dominant hereditary neurodegenerative disease characterized by motor, cognitive and behavioral disorders. HD is caused by expansion of CAG triplet (cytosine-adenosine-guanine) located in a gene on the short arm of the fourth chromosome. This expansion encodes an aberrant polyglutamine chain in the protein huntingtin. Physiological and mutated huntingtin (in case of HD) are expressed in almost all tissues and influences many cellular functions. The prevalence of HD in population is about 1 per 10.000. The disease is currently incurable and its mechanisms are not sufficiently understood. Besides affecting the central nervous system HD also affects peripheral tissues, including skeletal muscles. HD disrupts mitochondrial function and damages oxidative phosphorylation system, which has the task of producing energy in the form of ATP in cells. Research of transgenic minipig model for HD could help elucidate the mechanisms of disease's pathogenesis and potential therapeutic strategy. In this diploma thesis, immunodetection with help of specific antibodies to detect changes in amount of 14 selected mitochondrial proteins in skeletal muscle tissue of three age groups of transgenic HD minipigs - 24, 36 and 48 months old was used. Gradual progression in reduced...

The causative role of the huntingtin (HTT) gene in Huntington's disease (HD) has been identified more than 25 years ago. The extension of CAG repeat stretch over 39 repeats in exon 1 of one HTT allele results in full penetrance of this neurodegenerative disorder. While the identification of the causative mutation raised hopes that development of the therapeutic compound will be easily achievable, the patients and their families are still waiting for treatment until now. The main reason for that might be the complex cellular function HTT that makes the determination of the pathologic mechanism difficult and the development of treatments even more challenging. Although a lot of different animal models have been generated until now, establishing a suitable model has still not been achieved yet. Due to its anatomy, physiology, and genetics, the minipig seems to be a suitable candidate for neurodegenerative disease models. Indeed, the existing Transgenic (Tg) Libechov minipig model manifests signs typical for HD in patients, but on the other hand significant inconsistencies have also been observed. The finding of malformation that partially shows the situation in human patients is true for both, the male reproductive tract as well as for the brain. The reason for this might be the fact the genetic...

Chorea Huntington (HD) ist eine neurodegenerative Erkrankung, die durch Ablagerungen von N-terminal Polyglutamin-reichen Huntingtin (Htt) -Fragmenten in den betroffenen Neuronen charakterisiert ist. Das mutierte Htt (mHtt) Protein wird ubiquitär exprimiert. Das zellspezifische Absterben von „medium-sized spiny neurons“ (MSN) wird jedoch im Striatum von HD Patienten verursacht (Albin, 1995). Es wird angenommen, dass Striatum-spezifische Proteine, die mit Htt interagieren, eine wichtige Rolle in der Pathogenese von HD spielen (Ross, 1995). Protein-Protein-Interaktionsstudien haben gezeigt, dass einige der Htt-Interaktionspartner mit unlöslichen Htt-Ablagerungen in den Gehirnen von HD-Patienten kolokalisieren und die Bildung von Protein-Aggregaten beeinflussen (Goehler, 2004). Kürzlich wurde durch die Integration von Genexpressions- und Interaktionsdaten ein Striatum-spezifisches Protein-Interaktionsnetzwerk erstellt (Chaurasia, unveröffentlichte Daten). Eines der identifizierten Proteine ist CRMP1 (collapsin response mediator protein 1), das spezifisch in Neuronen exprimiert wird und möglicherweise eine wichtige Rolle bei der Pathogenese von HD spielt. Experimentelle Untersuchungen mithilfe eines Filter-Retardationsassays zeigten, dass CRMP1 die Anordnung von Htt zu fibrillären, SDS-unlöslichen Aggregaten verringert. Durch Rasterkraftmikroskopie wurde der direkte Effekt von CRMP1 auf den Aggregationsprozess von Htt bestätigt. Ko-Immunopräzipitationsstudien zeigten, dass CRMP1 und Htt in Säugerzellen unter physiologischen Bedingungen miteinander interagieren. Es wurde nachgewiesen, dass CRMP1 die Polyglutamin-abhängige Aggregation und Toxizität von Htt in Zell- und Drosophila-Modellen von HD moduliert. Außerdem konnte CRMP1 in neuronalen Ablagerungen in R6/2 Mäusegehirnen und dessen selektive Spaltung durch Calpaine gezeigt werden. Diese Ergebnisse deuten darauf hin, dass die Lokalisation und Funktion von CRMP1 bei der Krankheitsentstehung verändert werden. / Huntington’s disease (HD) is a neurodegenerative disorder characterized by the accumulation of N-terminal polyglutamine (polyQ)-containing huntingtin (Htt) fragments in affected neurons. The mutant Htt (mHtt) protein is ubiquitously expressed but causes specific dysfunction and death of striatal medium-sized spiny neurons (MSNs) (Albin, 1995). It is assumed that striatum specific proteins interacting with Htt might play an important role in HD pathogenesis (Ross, 1995). Previous protein-protein interaction (PPI) studies demonstrated that many Htt-interacting proteins colocalize with insoluble Htt inclusions in HD brains and modulate the mHtt phenotype (Goehler 2004). A striatum-specific, dysregulated PPI network has been created recently by integrating PPI networks with information from gene expression profiling data (Chaurasia, unpublished data). One of the identified dysregulated proteins potentially involved in HD pathogenesis was the neuron-specific collapsin response-mediator protein 1 (CRMP1). Here, I show that CRMP1 reduces the self-assembly of SDS-insoluble mHtt protein aggregates in vitro, indicating a direct role of CRMP1 on the mHtt aggregation process. Coimmunoprecipitation studies showed that CRMP1 and Htt associate in mammalian cells under physiological conditions. In addition, CRMP1 localizes to abnormal neuronal inclusions and efficiently modulates polyQ-mediated Htt aggregation and toxicity in cell and Drosophila models of HD. This suggests that dysfunction of the protein is crucial for disease pathogenesis. Finally, I observed that CRMP1 localizes to neuronal inclusions and is selectively cleaved by calpains in R6/2 mouse brains, indicating that its distribution and function are altered in pathogenesis. In conclusion, this study presents new findings on the function of CRMP1 and its role in the pathogenesis of HD. The protein interacts with Htt and modulates its aggregation and toxicity, in this way influencing the molecular course of the disease.