The Notch receptor is a key signaling protein that also acts as a transcriptional co-activator in numerous cell fate decisions in animals, including Drosophila melanogaster. Like many other transcriptional activators interacting on the DNA, the Notch protein carries a polyglutamine tract encoded by the opa repeats of the Notch gene. Here, I show that considerable variation exists within this tract across populations from the United States and Malawi. This variation is distributed asymmetrically across the range of possible alleles, with a peak in each population at opa31 (typically encoding Q₁₃HQ₁₇) and/or opa32 (typically encoding Q₁₃HQ₁₈) and a tendency towards a large tail of longer alleles and few shorter alleles. Variation in this pattern between populations may be a result of certain tracts being less harmful in certain backgrounds, or it may be due to the ancestry of these populations. This variation has real effects, such that lines bearing alleles longer or shorter than the common 31- and 32-codon alleles exhibit abnormal phenotypes, gene expression disruption, and decreased viability, effects that persist even when the Notch gene is outcrossed or recombined into other backgrounds. I also describe lines bearing CRISPR-Cas9-edited opa repeats and highlight a potential interaction between Notch and the transcriptional activator Dorsal. Dorsal also exhibits variation in the length of its polyglutamine tract, and short tracts at Dorsal most frequently appear alongside short tracts at Notch, while long tracts of each also frequently co-occur.

Polyglutamine expansion disorders are progressive neurodegenerative diseasesthat are caused by the pathological expansion of polyglutamine repeats. Huntington'sdisease (HD) is a polyglutamine disorder caused by the expansion of an existingpolyglutamine tract in a novel protein, Huntingtin (Htt). Oxidative stress has beenimplicated in the neural dysfunction observed in multiple neurodegenerative conditionsincluding HD. The tumor suppressor p53 is a multifunctional protein that has roles inthe cell cycle, apoptosis and neurodevelopment. The role of p53 in HD-associatedneurodegeneration has been studied but not fully elucidated, nor has the role of p53 inoxidative stress toxicity been fully elucidated.Here I present work that demonstrates polyglutamine expansion inducedalterations to p53 stability, localization, and activity. The transcriptional activity of p53was found to have a role in oxidative stress mediated as well as polyglutaminemediated neurotoxicity in vitro. The expression of p53 was also altered in vivo in amouse model of HD as well as in HD brain.Taken together, these data demonstrate a role for p53 in polyglutamine and oxidativestress toxicity.

Polyglutamine (polyQ) diseases are progressive fatal neurodegenerative movement disorders. Although many cellular processes are perturbed in polyQ disease, recent studies highlight the importance of protein misfolding as a central event in polyQ toxicity. Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is a particularly interesting polyQ disease because of the special qualities of the disease protein ataxin-3, which normally participates in cellular protein quality control. Here I use multiple mouse models of disease to explore toxic protein species and the role of protein homeostasis in SCA3 pathogenesis. In Chapter 1, I review the key features of polyQ disease, and outline the background and rationale behind our strategy for identifying toxic protein species in SCA3. In Chapter 2, I examine the role of the protein quality control ubiquitin ligase, CHIP (C-terminus of Hsp70 interacting protein), in regulating the toxicity of expanded ataxin-3 in vivo. Genetic reduction or removal of CHIP increases formation of detergent-resistant ataxin-3 microaggregates specifically in the brain. Concomitant with this, reduction or removal of CHIP exacerbates the phenotype of SCA3 mice, revealing a correlation between high levels of microaggregates and phenotypic severity. Additional cell-based studies confirm that CHIP may not directly mediate ataxin-3 degradation, suggesting that CHIP reduces expanded ataxin-3 toxicity in the brain primarily by enhancing ataxin-3 solubility. In Chapter 3, I use various biochemical techniques to reveal the presence of brain-specific ataxin-3 microaggregates in two genetically distinct mouse models of SCA3. Selective neuropathological evaluation of SCA3 mice reveals that major neuronal loss and reactive glial proliferation are not shared features of phenotypically-manifesting SCA3 mice. Additional studies fail to provide evidence for loss-of-function of endogenous ataxin-3 in SCA3 mice. Our results suggest that neuronal dysfunction in SCA3 is mediated through a toxic gain-of-function mechanism by ataxin-3 microaggregates in the CNS. In Chapter 4, I discuss important areas for future research in polyQ disease. I describe studies that would help elucidate the structural nature of toxic soluble microaggregates, and their effects on other cellular proteins. I also consider how the results described in this thesis inform potential treatment strategies.

Die Spinozerebelläre Ataxie Typ 3 (SCA3) oder Machado-Joseph-Krankheit (MJD) gehört zur Gruppe der neurodegenerativen Polyglutaminerkrankungen (PolyQ-Erkrankungen) und ist die häufigste autosomal-dominante zerebelläre Ataxie weltweit. Ein in der Länge hochvariabler Polyglutaminabschnitt ist vermutlich die Ursache für die Toxizität der ansonsten nicht verwandten Proteine, welche die PolyQ-Erkrankungen verursachen. Abgesehen von dem verlängerten Polyglutaminbereich scheinen die physiologische Funktion und der zelluläre Kontext dieser Proteine und ihrer Interaktionspartner entscheidend für die spezifische Pathogenese und den Krankheitsverlauf zu sein. Diese Arbeit soll dazu beitragen, genetische Interaktoren zu identifizieren, welche die PolyQ-Toxizität verstärken oder vermindern, um somit die molekularen Krankheitsmechanismen zu entschlüsseln, die durch die Trinukleotid-Wiederholungen ausgelöst werden.  Dafür wurde ein humanes, von Ataxin-3 abgeleitetes Transgen in den Facettenaugen von Drosophila exprimiert.  Die daraus resultierende Degeneration der Photorezeptoren induziert einen Raue-Augen-Phänotyp (Rough Eye Phenotype, REP) in adulten Fliegen. Um genetische Modifikatoren des REP zu identifizieren, wurde die Expression bestimmter Gene (Fliegengene mit einem humanen Ortholog, insgesamt ca. 7.500) augenspezifisch per RNAi vermindert. Mögliche Veränderungen im beobachteten REP sind dann höchstwahrscheinlich auf den RNAi-vermittelten Knockdown der Genexpression zurückzuführen. Damit wären die stummgeschalteten Kandidatengene zur Modifizierung der PolyQ-induzierten Neurotoxizität fähig.  Die auf diese Weise identifizierten Genprodukte sind in verschiedene biologische Prozesse involviert und stehen stellvertretend für unterschiedlichste molekulare Funktionen. Für eine Auswahl von Kandidatengenen wurden zusätzliche Untersuchungen angestellt, um die Art und das Ausmaß der Interaktionen zu bestimmen. Dabei wurden neue Modifikatorengene analysiert, welche z. B. in die Methylierung von tRNA oder den Sphingolipid-Metabolismus involviert sind. Diese Ergebnisse können neue Erkenntnisse bei der Aufklärung der Pathogenese der MJD und anderer PolyQ-Erkrankungen hervorbringen und gleichzeitig zum Verständnis der Rolle von Ataxin-3 und seinen Modulatorproteinen beitragen.

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.

Heat shock proteins function as molecular chaperones which help protein folding and prevent protein aggregation. My study shows that celastrol, a pharmacological compound capable of up-regulating the levels of heat shock proteins, inhibits cell death and protein aggregation caused by expanded polyglutamine containing protein, and the protective effects of celastrol are dependent on heat shock factor 1. These results suggest the potential of celastrol as a therapeutic agent in the treatment of polyglutamine diseases. Sumoylation is a protein modification which plays diverse roles in regulating the target proteins. My study shows that lamin A is a target of protein sumoylation, and two lamin A mutants associated with familial dilated cardiomyopathy, E203G and E203K, exhibit decreased sumoylation. My results also indicate that sumoylation is important for the normal localization of lamin A, and support a role for altered sumoylation in the underlying molecular mechanism of cardiomyopathies associated with the E203G/E203K lamin A mutations. In the third project, my results show that amyloid precursor protein is another target of SUMO modification, and sumoylation of amyloid precursor protein reduces the levels of amyloid β aggregates, which are the primary causative factor for Alzheimer’s disease. My results provide a new mechanism for the generation of amyloid β, and indicate the potential of up-regulating activity of the cellular sumoylation machinery as an approach against Alzheimer’s disease. My results also provide the first demonstration that SUMO E2 enzyme exists in the lumen of the endoplasmic reticulum, extending the sub-cellular reach of sumoylation to include the regulation of proteins in secretory pathways.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

La maladie de Huntington (MH) est une maladie neurodégénérative autosomale dominante causée par une expansion anormale de CAG dans le gène codant la huntingtine (HTT) qui se traduit dans la protéine HTT par une répétition de polyglutamine, entrainant la mort neuronale. Néanmoins, la MH entraine aussi le développement de symptômes périphériques comme la HTT est une protéine exprimée de façon ubiquitaire. Notamment, la MH a été associé à une plus faible incidence des cancers, mais les mécanismes sous-jacents ne sont pas décrits. Nous avons étudié le rôle de HTT mutée et sauvage dans le cancer du sein, où la protéine est fortement exprimée. Des modèles murins de cancer du sein (MMTV-PyVT et MMTV-ErbB2) exprimant la HTT mutée (souris knock-in transportant 111 GAC) développent des tumeurs mammaires agressives par rapport aux souris exprimant la HTT sauvage. La transition épithéliale-mésenchymateuse est accélérée avec une augmentation de la motilité cellulaire ainsi que de la formation de métastases. Ces tumeurs accumulent le récepteur tyrosine-kinase HER2 à la membrane, en raison d'un défaut d'endocytose dynamine-dépendante en présence de la HTT mutée. La signalisation accrue de HER2 est responsable de l'agressivité des tumeurs exprimant la HTT mutée, comme en témoigne le traitement trastuzumab, un anticorps dirigé contre HER2 qui restaure la motilité et l'invasion des cellules tumorales porteuses de la mutation responsable de la MH. La HTT sauvage a elle-même un rôle protecteur dans le cancer, retardant l’apparition des métastases en raison d'un potentiel rôle dans l’adhésion intercellulaire. Ainsi, notre travail met en évidence des rôles clés de la HTT mutée et sauvage au cours de la progression du cancer du sein. / Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by an abnormal CAG expansion in the huntingtin (HTT) gene. The corresponding polyglutamine expansion in the HTT protein causes specific neuronal death, but the consequences of HTT mutation in other tissues are less well understood. Nevertheless, HD mutation causes peripheral symptoms as HTT is an ubiquitous protein. HD was associated to lower cancer incidence, however, the mechanisms behind this effect were not described. Here we have studied the role of wild-type and mutant HTT in breast cancer, where we found the protein to be highly expressed. We demonstrate that mouse breast cancer models (MMTV-PyVT and MMTV-ErbB2) expressing mutant HTT (knock-in mice carrying 111 CAGs) develop aggressive mammary tumors as compared to control mice. Epithelial-to-mesenchymal transition is enhanced with subsequent increased cell motility and metastasis. These tumors accumulate tyrosine-kinase receptor HER2 at the membrane, due to a dynamin-dependent endocytosis defect in the presence of mutant HTT. HER2 enhanced signaling is responsible for the aggressiveness of the mutant HTT expressing tumors, as demonstrated by Trastuzumab treatment, an antibody against HER2 that restores motility and invasion in tumor cells carrying HD mutation. The wild-type HTT has itself a protective role in cancer, inhibiting metastasis due to a possible role in cellular junction maintenance. Thus, our work unravels a key role of HTT in breast cancer progression, with the mutant HTT triggering the development of aggressive and metastatic tumors.

The polyglutamine diseases are a clinically heterogeneous group of inherited neurodegenerative disorders caused by expansion of polyglutamine-encoding (CAG)n trinucleotide repeats within the disease genes. It is increasingly clear that the amino acid sequences flanking the polyglutamine expansion in each disease protein, i.e. the specific protein context, contribute to selective neuronal toxicity by influencing the behavior of the disease protein within selectively vulnerable neuronal populations. In the studies described here, I explore the role that protein context plays in the polyglutamine disease, Spinocerebellar ataxia type 3 (SCA3). Toward this end, I utilize biochemical, cell-based, and animal models to gain a broader understanding of the SCA3 disease protein, ataxin-3, and generate tools for further exploration of the molecular properties of ataxin-3 that modulate its toxicity during disease. In Chapter 1, I provide an overview of the recognized polyglutamine diseases, emphasizing the elements of protein context that are distinct among the polyglutamine disease proteins and may contribute to the neuropathological and clinical heterogeneity within this family of diseases. Alternative splicing of the polyglutamine disease gene products adds an additional level of complexity to the tissue-specific protein context of expanded polyglutamine, yet this phenomenon has been underinvestigated. In Chapter 2, I examine the significance of ataxin-3 splice variation. Several minor 5' variants and both known 3' splice variants of ataxin-3, a deubiquitinating enzyme, are expressed at the mRNA level in brain. At the protein level, however, the C-terminal splice isoform with three ubiquitin interacting motifs (3UIM ataxin-3) is the predominant isoform in brain, independent of age or (CAG)n expansion. Although both C-terminal ataxin-3 splice isoforms display similar in vitro deubiquitinating activity, 2UIM ataxin-3 is more prone to aggregate and is more rapidly degraded by the proteasome. These observations demonstrate how alternative splicing of sequences distinct from the polyglutamine-encoding (CAG)n repeat can alter disease-related components of protein context. Knock-in models of polyglutamine diseases utilize pathogenic (CAG)n expansions within the endogenous genomic, transcript, and protein context to recreate key features of individual polyglutamine diseases. In chapter 3, I describe the creation of the first knock-in mouse model of SCA3. Hemizygous knock-in mice transmit the knock-in allele in Mendelian ratios and broadly express both the expanded Atxn3(Q3KQ82) protein and the wildtype murine Atxn3(Q6) protein. In this chapter, I also compare the gene targeting efficiencies and rates of chromosomal instability of a novel C57BL/6J ES cell line (UMB6JD7) and two well established ES cell lines (W4 and Bruce4.G9). Of these, Bruce4.G9 ES cells proved superior based on lower rates of aneuploidy and the production of germline transmitting chimeras. Finally, in Chapter 4 I discuss questions and concepts raised during the course of these studies, and suggest avenues of future research aimed at broadening our understanding of ataxin-3 physiology and of protein context-dependent elements in polyglutamine disease pathogenesis.

<p>Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease characterized by cerebellar ataxia and visual problems due to a progressive and selective loss of neurons within the cerebellum, brainstem and retina. The disease is caused by the expansion of a CAG repeat in the first coding exon of the SCA7 gene, resulting in an expanded polyglutamine domain in the N-terminal part of ataxin-7, a protein of unknown function.</p><p>To expand our knowledge of the ataxin-7 protein and the mechanism by which mutant ataxin-7 causes disease, we have studied the expression and function of both the normal and the mutated ataxin-7 protein. </p><p>Ataxin-7 expression was examination in brain and non-CNS tissues from SCA7 patients and age-matched controls. Expression was predominantly nuclear in neurons throughout the brain of both healthy and SCA7 individuals. We also observed aggregation of mutant ataxin-7 in the nuclei of neurons. No obvious difference in the expression level of ataxin-7 or the formation of aggregates could be observed between affected and non-affected brain regions in SCA7 patients. Based on these findings, we could conclude that the cell type specific neurodegeneration in SCA7 is not due to differences in expression levels or to the formation of ataxin-7 aggregates.</p><p>To widen our studies on ataxin-7 expression, we isolated and characterized the mouse SCA7 gene homolog. Cloning of the mouse SCA7 gene revealed two SCA7 mRNA isoforms that were highly homologous to their human counterparts. Immunohistochemical analysis also revealed a conserved expression pattern of ataxin-7 in adult mouse brain. In addition, ataxin-7 expression was observed during embryonic development in brain as well as in several non-neuronal tissues such as heart, liver and lung. </p><p>Besides SCA7, eight neurodegenerative disorders are known to be caused by expanded polyglutamine repeats, including SCA 1-3, 6 and 17, DRPLA, SBMA and Huntington’s disease. The polyglutamine disorders have many features in common and a common pathological disease mechanism involving transcriptional dysregulation has been proposed. To investigate the possible involvement of transcriptional dysregulation in SCA7 pathology, we analyzed the effects of both wild-type and expanded ataxin-7 on transcription driven by the co-activator CBP, the Purkinje cell-expressed nuclear receptor RORα1 or a basic TATA promoter. As previously shown for other polyglutamine disease proteins, expansion of the polyglutamine domain in ataxin-7 leads to reduced transcription. Surprisingly, strong repression of CBP-mediated, RORα1-mediated and basal transcription was also observed with wild-type ataxin-7, suggesting that the normal ataxin-7 protein may have a role in transcriptional regulation. </p>