<p>The primary cilium is a singular signaling organelle found on most mammalian cell types. Dysfunction of the primary cilium or associated structures form a group of genetic disorders called ciliopathies. Recently, Huntington’s disease (HD), a monogenetic neurodegenerative disorder, was classified, at least in part, as a ciliopathy. How the primary cilium contributes to the pathogenesis of HD is the focus of this work. We demonstrate that huntingtin localization to the basal body or primary cilium is dependent on the phosphorylation status of serine residues 13 and 16. Furthermore, we demonstrate that, compared to controls, HD cell models have an increased number of cells with a primary cilium and that these cells have higher presence of huntingtin within the ciliary compartment. The primary cilia that form in HD cell lines demonstrate abnormal dynamics and morphology with bulging tips, characteristic of defective retrograde trafficking. We also demonstrate that alpha tubulin acetyltransferase 1 (αTAT1) expression and localization is increased in the primary cilium of HD cell lines. Subsequently, the primary cilium of HD cell lines are highly acetylated when compared to controls. These data support that primary cilia structure, ciliogenesis and ciliome are altered in HD.</p> / Master of Science (MSc)

Huntington’s disease (HD) is a late-onset neurodegenerative disorder caused by the expansion of the CAG repeat in the HTT allele. This forms an expanded huntingtin protein which disrupts various cellular processes including DNA damage repair pathways. Many models have been developed to study HD. This includes the TruHD cells which are immortal patient-derived fibroblasts that retain characteristics of their original primary culture. However, the current TruHD cell library is limited. In this project, we expanded the library of TruHD cells, characterized their basal growth rate and huntingtin levels, identified a standard set of cells for future experiments, and amended the TruHD name to better guide future investigations. Furthermore, we developed and optimized qPCR as a tool for the TruHD cells. N6-furfuryladenine was also assessed and determined to not modulate HTT mRNA levels and not induce apoptosis in control cells. / Thesis / Master of Science (MSc)

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

HTT ist ein Protein, das durch seine Verbindung mit mehreren Interaktionspartnern an einer Vielzahl von zellulären Prozessen beteiligt ist. Darüber hinaus verursacht eine Mutation im HTT-Gen eine Krankheit, die als Huntington-Krankheit (HD) bezeichnet wird. Aufgrund der gerüstbildenden Eigenschaften von HTT wurde eine Vielzahl von Studien durchgeführt, um potenzielle therapeutische Ziele zu identifizieren. Die auf dem Resonanzenergietransfer (RET) basierenden Ansätze sind jedoch im Bereich der Huntington-Krankheit noch nicht vollständig genutzt worden. Daher habe ich versucht, solche Ansätze in Interaktionsstudien mit dem HTT-Exon 1 (HTTexon1) und dem Protein in voller Länge zu bewerten. Ich habe eine Benchmarking-Studie mit einem zuvor beschriebenen Huntingtin-interagierenden Protein (HIP) und HTTex1 (Wildtyp und mutiert) unter Verwendung eines BRET-Ansatzes durchgeführt. Meine Studien bestätigten die binäre Interaktion zwischen HTTex1 und sieben Proteinen. Ich habe auch drei Interaktionen mit der mutierten Version von HTTex1 bestätigt. Zusätzlich bewertete ich die Interaktionen durch FRET-Messungen mit Hilfe der Durchflusszytometrie. Der zweite Teil dieser Arbeit zielte darauf ab, ein Hochdurchsatz-Screening für den Nachweis von Protein-Protein-Interaktionen (PPIs) mit HTT in voller Länge (FL) unter Verwendung von Biolumineszenz-Resonanz-Energie-Transfer zu etablieren. Auf diese Weise konnte ich die Wechselwirkung zwischen FL HTT und einer Bibliothek von 580 Proteinkinasen bewerten. Schließlich analysierte ich die Spezifität der entdeckten Wechselwirkungen, indem ich die unspezifische Bindung durch Donor-Sättigungstests bewertete. Zusammenfassend belegen meine Ergebnisse die potenzielle Verwendung von Resonanzenergietransferansätzen zur Validierung von HTT-Wechselwirkungen. Außerdem wird ein neues Screening-Tool vorgestellt, das dazu beitragen soll, HTT-Interaktoren zu identifizieren und zu verifizieren. / HTT is a protein involved in a plethora of cellular processes through its association with several interaction partners. Furthermore, a mutation in the HTT gene, causes a disease denominated Huntington’s disease (HD). Due to the scaffolding properties of HTT, a large variety of studies have been performed to identify potential therapeutical targets. However, resonance energy transfer-based (RET) approaches have not been fully exploited in the HD field. Therefore, I aimed to evaluate such approaches in interaction studies using the HTT exon 1 (HTTexon1) as well as the full-length protein. I performed a benchmarking study with a previously described huntingtin interacting protein (HIP) and HTTex1 (wild type and mutated) using a BRET approach. My studies confirmed the binary interaction between HTTex1 and seven proteins. I also confirmed three interactions with the mutated version of HTTex1. Additionally, I also evaluated the interactions by measuring FRET using flow cytometry. The second part of this work aimed to stablish a high-throughput screening for the detection of protein-protein interactions (PPIs) with full-length (FL) HTT using bioluminescence resonance energy transfer. With this, I was able to evaluate the interaction between FL HTT and a library composed by 580 protein kinases. Finally, I analysed the specificity of the detected interactions by assessing unspecific binding through donor saturation assays. In summary my results provide evidence of the potential use of resonance energy transfer approaches to validate HTT interactions. Additionally, a new screening tool is presented to contribute to identify and verify HTT interactors.

La maladie de Huntington (MH) est une maladie neurodégénérative héréditaire autosomique dominante. Elle est due à l’expansion anormale de polyglutamine dans la partie N-terminal de la protéine huntingtine (HTT). Une des étapes clés de la pathologie est le clivage de la HTT pleine longueur en fragments N-terminaux plus petits, contenant l’expansion de polyglutamine, et qui sont toxiques pour les neurones. En effet, les clivages de la HTT mutée génère des fragments N-terminaux (N-ter) de tailles comprises entre les acides aminés 1-105 et 1-586 observés dans des extraits de cerveaux de patients MH post-mortem et dont l’implication dans la mort neuronal est bien caractérisée. Mes travaux de thèse ont visé à modéliser le clivage de la HTT et à évaluer les conséquences sur la survie neuronale.Au cours de ma thèse, j’ai développé un outil permettant de contrôler le clivage de la HTT dans le temps et à des sites spécifiques. J’ai étudié le clivage de la HTT à deux sites stratégiques : les positions clivées par la caspase-6 et par la bléomycine hydrolase/cathepsine Z. A l’aide de cet outil, j’ai montré que le clivage de la HTT confère une toxicité cellulaire qui dépend du profil du clivage. Plus précisément, J’ai décrit une interaction intramoléculaire au sein des domaines de la HTT. Mes résultats indiquent que cette interaction protège les cellules de la toxicité induite par le clivage de la HTT mutée. En effet, les clivages successifs de la HTT annulent cette interaction, ce qui induit la libération des fragments N-ter mutants et provoque la mort cellulaire à l’issue de leur translocation nucléaire. Pour conclure, au cours de ma thèse, j’ai montré que la protéolyse successive de la HTT induit des processus cytotoxiques différents. / Huntington’s disease (HD) is an autosomal dominant inherited neurodegenerative disorder caused by an abnormal polyglutamine (polyQ) expansion in the N-terminus of the protein huntingtin (HTT). A crucial step in HD pathogenesis is the cleavage of full-length HTT into smaller N-terminal (N-ter) fragments that contain the polyQ stretch and that are toxic to neurons. HTT cleavage generates short N-ter fragments whose amino-acid positions range from 1-105 to 1-586. These fragments are observed in HD post mortem brain samples and their participation in neuronal death in HD is well characterized. During my PhD research, I investigated the consequences of full-length mutant HTT proteolysis by developing a time and site-specific controlled system for HTT proteolysis. I have assessed HTT cleavage on two sites caspase-6 and cathepsin Z. My results show that HTT cleavage induces neurotoxicity in vitro as well as in vivo, toxicity which depends on HTT proteolysis pattern. Briefly, we described an intramolecular interaction within the HTT domains which is impaired upon successive proteolysis of HTT. We found that HTT intramolecular interaction buffer mutant N-ter HTT-induced toxicity. Moreover, specific cleavages of the mutant HTT generated toxic N-ter fragments as they translocate into the nucleus. To conclude, my PhD work has shown that additional cleavage of mutant HTT induces cytotoxicity by different mechanisms.

La maladie de Huntington (MH) est un trouble neurologique transmis selon un mode autosomique dominant qui conduit à l’apparition de symptômes moteurs, psychiatriques et cognitifs chez l’adulte. La MH est caractérisée par une neurodégénérescence massive des neurones striataux et corticaux. La MH est causée par une mutation de la séquence codante de la protéine Huntingtine (HTT) conduisant à la production d’une protéine mutée (mHTT). La mHTT gagne de nouvelles fonctions toxiques mais perd aussi certaines fonctions normales. L’étude de ces deux aspects (à la fois gain et perte de fonction) est donc indispensable à la compréhension du processus pathologique de la MH.La HTT et mHTT participent au développement des structures cérébrales. Notre hypothèse est donc que les défauts développementaux induits par la mHTT contribuent à la progression physiopathologique de la MH. Notre équipe s’intéresse tout particulièrement au développement du cortex cérébral, largement atteint dans la MH. Nos précédentes études ont démontré le rôle de la HTT et l’effet de sa mutation dans la prolifération des précurseurs neuronaux du cortex cérébral. Cependant, les fonctions de la HTT et de la mHTT lors des étapes plus avancées du développement cortical, restent à ce jour inconnues.Mon projet de thèse se décompose en deux axes principaux : (i) l’étude des fonctions de la HTT dans les neurones nouvellement produits dans le cortex en développement, notamment au cours de leur migration et de leur maturation dendritique et (ii), la caractérisation de la neurogenèse corticale dans un modèle génétique de la MH : zQ175. / Huntington disease (HD) is an autosomal dominant inherited neurological disorder conducting to the appearance of motors, psychiatrics and cognitives symptoms during mid-adulthood. HD is characterised by a massive neurodegenerescence of both striatal and cortical neurons. HD is caused by a mutation in coding region of the protein Huntingtin (HTT) leading to the production of a mutated form (mHTT). mHTT gain new toxic function but also loss some of normal function of HTT. Therefore, studying both gain and loss of function is mandatory to better understand the physiopathological progression of HD.HTT and mHTT both contribute to development of cerebral structures. Our hypothesis is that developmental defects induced by mHTT could contribute at least in part to the physiological progression of HD. Our work focuses on cerebral cortex development a structure which is largely impacted in HD. Our previous studies demonstrated roles of HTT and the effect of mHTT in neuronal precursor proliferation during neurogenesis. However, roles and functions of HTT and mHTT during later step of cortical neurogenesis remain elusive.My PhD project has focused on two main aspects: (i) study the function of HTT in newborn post-mitotic neurons in cerebral cortex, notably during their migration and maturation, and (ii), characterising cortical neurogenesis in genetically integrated mouse model of HD: zQ175.

<p>Huntington’s disease (HD) is an adult-onset, neurodegenerative disease caused by an autosomal dominant mutation in the huntingtin (HTT) gene. HD patients suffer from motor, cognitive, and psychiatric symptoms. The pathogenic mutation of HD is expansion of a CAG repeat in the first exon of the HTT that encodes for a polyglutamine (poly-Q) repeat in the huntingtin protein (Htt). HD results in neurodegeneration of the striatum and cortex, which is thought to underlie the development of HD symptoms, but recent evidence has shown that there are alterations to the connectivity of patients’ brains preceding degeneration. This study focuses on how wild type Htt contributes to establishing and maintaining synaptic connectivity and how a loss of normal Htt function may contribute to the synaptic alterations in HD.</p><p>In this study, I examined the role of wild type Htt in synapse formation and maturation synapses in the basal ganglia circuit, and I examined how loss of wild type Htt function may affect the pathogenesis of HD. To do so, I created conditional deletions of Htt in the mouse brain by crossing a floxed allele of Huntingtin to transgenic Cre lines. I conditionally deleted Htt from the cortex and the indirect pathway spiny neurons (iSPNs) of the striatum using Emx1-Cre and Adora2A-Cre, respectively. I also used a knock-in mouse model of HD, the zQ175 mouse, to examine alterations caused by the HD mutation. I used imaging and electrophysiological techniques to determine how loss of Huntingtin affected synapse number, function, and morphology in the cortex, striatum, and basal ganglia. </p><p>In the cortex and striatum, loss of Htt leads to disruptions in synaptic connectivity followed by neuronal stress and death. Htt is critical for moderating the formation of excitatory synapse formation in both the cortex and striatum, and that in the cortex this function is lost in HD. In the striatum, Htt is required for stabilizing striatopallidal synapses, and for proper basal ganglia function. </p><p>In order to explore the molecular mechanisms behind Htt’s control of excitatory synapse formation, I investigated its interaction with α2δ-1. α2δ-1 is a genetic modifier of mutant Htt toxicity that our lab had previously identified as the neuronal receptor of the synaptogenic Thrombospondin family (TSP) of proteins. I used in vitro neuronal cultures and biochemical analysis to determine how Htt interacts with α2δ-1 and how Htt affects TSP/α2δ-1 excitatory synapse formation. I characterized α2δ-1’s biochemical interaction with Htt and discovered that Htt postsynaptically suppresses excitatory synapses.</p><p>Taken together, these results suggest that wild type Htt functions to moderate excitatory activity in the brain. It slows the formation of excitatory connections and stabilized inhibitory ones, which may protect the brain from excitotoxic damage. These results show that Htt plays an important role in maintaining neuronal health and the establishment of synaptic connectivity in cortical and striatal circuits.</p> / Dissertation

La maladie de Huntington (MH) est une maladie neurodégénérative progressive héréditaire. Aucun traitement curatif n’a encore été trouvé. La MH est provoquée par une mutation dans le gène HTT induisant l’augmentation anormale du domaine PolyQ (>36) contenue dans la Huntingtine (Htt), la protéine codée par le gène HTT. Les conséquences et les mécanismes de cette mutation sont maintenant bien décrits et ont permis d’identifier une interaction importante entre la Htt et la voie de signalisation du BDNF. Le BDNF est un facteur neurotrophique qui joue des rôles importants, à travers l’activation de son récepteur TrkB, dans le développement et le maintien des neurones et également dans la plasticité des réseaux synaptiques. La Htt mutante (mHtt) diminue l’expression et le transport du BDNF et de TrkB dans les neurones.P42, une partie de 23 acides aminés de la Htt, est capable de sauver de nombreux phénotypes pathologiques induits dans la MH.L’objectif de ma thèse était de mieux comprendre les mécanismes d’action de P42, dans le but d’optimiser son potentiel thérapeutique. Pour cela, j’ai développé plusieurs expériences sur plusieurs modèles. Dans un article, publié dans HMG, je montre les effets d’un traitement avec P42 sur la voie de signalisation BDNF/TrkB dans un modèle de MH : les souris R6/2. Pour cela, j’ai analysé plusieurs phénotypes pathologiques, comportements et mécanismes cellulaires développés par les souris R6/2 et connus pour être dépendants de la voie BDNF/TrkB. J’ai également mesuré les taux d’ARNm et de protéine, du BDNF et de TrkB, dans le cortex et le striatum. Ce que j’ai trouvé est que P42 agit sur la voie BNDF/TrkB principalement en augmentant l’expression de TrkB dans le striatum.Afin d’observer l’effet de P42 sur le transport vésiculaire, j’ai également utilisé la drosophile comme modèle. Grâce à ce modèle, j’ai pu observer in vivo le transport vésiculaire dans différentes conditions avec ou sans mHtt ou P42.Egalement, dans le but de mieux suivre l’évolution des différents phénotypes pathologiques induits par la MH et l’effet du traitement par P42, j’ai expérimenté le Hamlet test®, un test innovant multi-comportemental.Enfin, j’ai observé sur les souris R6/2, l’effet d’une bithérapie P42 avec P3, un peptide ciblant les effets toxiques induits par les ARN codants pour un PolyQ.Tous ces résultats permettent ou vont permettre de mieux comprendre les mécanismes d’action de P42. / Huntington’s disease (HD) is a rare genetic neurodegenerative disorder. Curative treatments are still actively sought. HD is induced by a mutation in the HTT gene inducing an abnormal expansion of the polyQ domain contained in the Huntingtin protein (Htt). Mechanisms and consequences of this mutation are now well described and allowed to identify an interaction of the Htt with the brain derived neurotrophic factor (BDNF) signaling pathway. BDNF is a neurotrophic factor, which plays important roles, through TrkB, one of its receptor, in neuronal development and plasticity. Mutant Htt (mHtt) down-regulates BDNF and TrkB transcription and transport along the axons.P42, a part of the Htt protein, is a 23aa peptide able to rescue HD pathological phenotypes, such as aggregation, axonal transport and neuronal viability.The aim of my PhD was to better understand the mechanisms of action of P42, in a purpose to optimize its therapeutic potential. To this end I developed different studies using different models.In a paper now accepted for publication in HMG, I first used a P42-based treatment on R6/2 HD mice, to analyze the effect of P42 on the BDNF/TrkB signaling pathway. To this end I analyzed pathologic phenotypes: behaviors or cell mechanisms developing in R6/2 mice and are related to the BDNF/TrkB pathway. I also measured BDNF and TrkB, mRNA or protein levels in both striatum and cortex. What I found is that P42 is acting on BDNF/TrkB pathway mainly by increasing the protein level of TrkB in the striatum.To observe the effect of P42 on vesicular transport, I rather used a Drosophila model, to perform live imaging based studies, in different transgenic conditions: with or without mHTT or P42.Also, in a way to better follow the progression of different pathological phenotypes and the effect of treatments on R6/2 mice, I benefited from a very recent and innovative tool, the HAMLET, which allows a multi-behavioral test.Finally, a bitherapy was used on R6/2 mice combining P42 and P3, a peptide raised against PolyQ mRNA that are also toxic.All those results contribute or will contribute to a better understanding of P42 mechanisms of action.

<p>Huntington's Disease brains display a striking accumulation of huntingtin in the nucleus of striatal and cortical neurons, suggesting that nuclear functions may be key to the onset of cellular pathogenesis. Entry to the nucleus is tightly regulated by a family of import receptors called karyopherins that limit their binding to proteins bearing specific nuclear localization signals (NLS). Although huntingtin is primarily cytoplasmic in healthy neurons, it is also found in the nucleus at low levels and contains a nuclear export signal (NES), suggesting that shuttling to and from the nucleus is part of the protein's normal function. Indeed, recent publications from our lab (Atwal <em>et al.</em> 2007 and 2011, and Munsie <em>et al</em>. 2011) describe huntingtin's ability to enter the nucleus in response to cell stress, and localize to cofilin-actin rods. We have identified an active NLS near the amino terminus of huntingtin between amino acids 174-207, which is recognized by both karyopherin β2 and β1. While functional in ST<em>Hdh </em>and NIH 3T3 cells, the huntingtin NLS lacks activity in HEK 293 and MCF-7 cell lines. This surprising observation suggests that additional levels of regulation exist amongst cell types. We have isolated a shorter sequence within the NLS that localizes to membrane structures throughout the cell (such as the plasma membrane and vesicles). This thesis explores several putative secondary regulatory mechanisms, such as, nuclear export activity, transcriptional dependence, palmitoylation and acetylation. However, the most promising mechanism thus far is masking of the NLS by HAP1 and HMGB1. The functions of these proteins, in vesicular trafficking and autophagy/apoptosis, respectively, may offer further insight into huntingtin’s nuclear function. By understanding the underlying regulatory mechanisms of huntingtin nuclear import, we hope to gain further insight into why huntingtin accumulates in the nucleus of specific neurons in HD, and whether or not this mislocalization directly contributes to disease.</p> / Doctor of Philosophy (PhD)

<p>Huntington’s disease (HD) is a neurodegenerative disorder caused by the expansion of a polyglutamine tract in the huntingtin protein. The huntingtin protein has many roles in vesicular and endocytic trafficking, which can be modified in HD cells. When mutant huntingtin is expressed in HD, protein levels of the huntingtin interacting protein, Huntingtin-associated protein of 40kDa (HAP40) are increased. This increase in HAP40 protein levels causes the formation of a complex between carboxyl terminal huntingtin, HAP40 and active Rab5 on the early endosome. This complex induces a switch from early endosomal movements on microtubules to movements on actin filaments, greatly reducing both the speed and distance of movement. The main objective of this research is to determine where the interaction occurs between huntingtin and HAP40 on the huntingtin protein. Here we show that HAP40 interacts with the amino terminus of huntingtin, specifically the first 17 amino acids (N17). In co-localization studies, we found that HAP40 co-localizes with the carboxyl terminal fragment of huntingtin corresponding to amino acids 2570-2634; however, GFP trap immunoprecipitation analysis revealed no interaction between the carboxyl terminal fragments of huntingtin and HAP40. An interaction was discovered between HAP40 and N17, which was then confirmed by far western blot. These results demonstrate that HAP40 interacts with N17. By identifying the interaction site between HAP40 and huntingtin, modifications can be explored to prevent the interaction of HAP40 with huntingtin. This would restore motility on microtubules reinstating fast, long-range movements of early endosomes.</p> / Master of Science (MSc)