Huntington Disease (HD) is a neurodegenerative disorder caused by expanded CAG repeats in the exon1 of huntingtin gene (HTT). The mutant HTT affects the transcriptional profile of neurons by disrupting the activities of transcriptional machinery and alters expression of many genes. In this study, we identified dysregulated non-coding RNAs (ncRNAs) in medium spiny neurons of 4-week-old HD model mouse. Also, we observed the intracellular localizations of Abhd11os and Neat1 ncRNAs by ViewRNA in situ hybridization, which could provide more precise detection, suggesting that it is a useful method to investigate the expression changes of genes with low expression levels. / 博士(理学) / Doctor of Philosophy in Science / 同志社大学 / Doshisha University

Huntington disease (HD) is a fatal neurodegenerative disease caused by CAG tract expansion in the huntingtin (HTT) gene, which results in production of mutant huntingtin (mtHTT) protein. Although mtHTT is expressed throughout life, onset of HD symptoms typically begins in mid-life, around 35 to 50 years of age. Characteristic HD symptoms include motor, cognitive, and psychiatric abnormalities. The emergence of symptoms in adulthood suggests that aging may play a role in HD pathogenesis. Furthermore, markers of accelerated aging can be observed in HD patients, including telomere attrition, epigenetic alterations, and mitochondrial dysfunction. Our lab has previously observed that induction of age-like changes by treatment with progerin, the mutant protein that causes Hutchinson-Gilford Progeria Syndrome, enhances HD phenotypes and contributes to pathogenesis in HD neurons. Taken together, these findings suggest a link between aging and HD, with implications for potential therapeutic benefits from anti-aging treatment. Our lab has conducted a young blood anti-aging trial in which aged HD and wild-type (WT) mice were injected with plasma from young WT or HD mice. Previous work in our lab confirmed that cortical aging markers decline with age at the protein level and are differentially affected by young blood treatment. In this study, we observed a significant effect of age on striatal expression of aging markers, Grin1 and Lmnb1. Varying effects of young blood anti-aging treatment were observed on the genes of interest.

Huntington Disease (HD) is a fatal neurodegenerative disorder that is characterized by motor, cognitive, and psychiatric symptoms. Although HD onset is determined by motor symptoms, psychiatric symptoms, like depression and aggression, can develop earlier, have a larger impact on quality of life, and are understudied due to stigma. Our lab has observed hyper aggression in our humanized HD mouse model (Hu97/18) compared to our knock-in HD mouse model (Q175FDN). We characterized these differences and found that the Hu97/18 mice overreact in neutral situations, behaving as if they are in threatening situations. We are now using this novel model of HD-related aggression to study its neurological basis. Increased reactive aggression has been linked to stress levels and the prefrontal cortex (PFC) due to its role in emotional regulation. This study seeks to determine if HD-related aggression is associated with increased stress levels and changes in the PFC. Our cortisol study shows that the Hu97/18 mice display significantly higher cortisol levels than baseline, suggesting a link between systemic stress and heightened aggression. Additionally, quantified PFC volumes show a moderate relationship between PFC volume and aggression in wild-type (WT) mice that is lost in the Hu97/18 mice. This data will help elucidate factors that modulate aggression in HD and may identify therapies with high potential to alleviate this devastating symptom in patients.

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Les maladies à expansion de triplets nucléotidiques situés dans la région codante, telles que la maladie de Huntington, sont des maladies où les gènes en questions possèdent un nombre de répétitions trinucléotidiques anormalement élevé et inversement corrélé avec l'âge d‟apparition des symptômes. Plusieurs de ces maladies démontrent une anticipation paternelle, où un ajout de répétitions trinucléotidiques a lieu pendant la spermiogenèse, mais les étapes et les mécanismes impliqués sont encore mal compris. Or, la spermiogenèse est caractérisée par un remodelage drastique de la chromatine, où les histones sont ultimement remplacées par les protamines afin de compacter et protéger davantage le matériel génétique. Cette transition implique aussi un changement topologique majeur qui mène à une accumulation de superenroulement négatif qui est éliminé par la topoisomérase 2[beta]. Pour identifier les étapes précises où l'extension trinucléotidique a lieu, j'ai développé une stratégie de séparation des spermatides en utilisant la cytométrie en flux, ce qui m'a permis d'obtenir quatre populations, soit les spermatides aux étapes 1 à 9, 10 à 12, 13-14 et 15-16. J'ai appliqué cette stratégie sur un modèle de souris transgéniques pour la maladie de Huntington, ce qui a permis de démontrer par PCR que l'extension trinucléotidique des répétitions CAG a lieu à la fin du remodelage de la chromatine, soit à l'étape 14. Afin d‟élucider le mécanisme d‟extension trinucléotidique, j'ai utilisé une stratégie in vitro, basée sur l'incubation d‟extraits nucléaires actifs de spermatides avec un plasmide contenant des répétitions CAG. Cette stratégie a démontré que le superenroulement négatif libre, tel que retrouvé pendant le remodelage de la chromatine, est capable d'induire des structures secondaires dans les répétitions CAG, ce qui entraîne une cascade d‟événements menant à l'extension trinucléotidique. J'ai validé ce processus en inhibant aussi les topoisomérases de type 2 qui sont responsables d'éliminer le superenroulement. Finalement, j‟ai démontré que la protamination de l‟ADN, telle qu'observée dans les spermatides, accentue l'accumulation de stress torsionnel aux répétitions CAG, ce qui favorise leur extension. Mes travaux sur le stress torsionnel lors de la protamination suggèrent une nouvelle source potentielle d'instabilité trinucléotidique, nécessitant une caractérisation additionnelle. Cette source d'instabilité, qui est spécifique au mâle, jouerait un rôle majeur dans l'anticipation paternelle des maladies trinucléotiditiques. / Abstract : Trinucleotidic diseases, such as the Huntington disease, are genetic diseases characterized by abnormally long trinucleotidic repeats within a specific gene, which are inversely correlated with the age of onset of symptoms when within exons. Many trinucleotidic diseases display paternal anticipation, where trinucleotidic repeats are added during spermiogenesis, without any details on the mechanism or the steps involved. Interestingly, spermiogenesis is characterized by a drastic chromatin remodeling, where histones are ultimately replaced by protamines in order to achieve greater compaction and protection of DNA. This transition also involves major topological changes, where accumulation of negative supercoils are eliminated by the topoisomerase 2[beta]. In order to identify the specific steps where trinucleotidic extension occurs, I have developed a strategy to separate spermatids from mice, using flow cytrometry. This allowed me to purify four distinct spermatids population, consisting of steps 1-9, 10-12, 13-14 and 15-16 spermatids. The sorting strategy was used on a transgenic mouse model of the Huntington disease, which allowed me to determine, using PCR, that CAG extension occurs at the end of chromatin remodeling, more specifically at step 14. The mechanism of extension was investigated using an in vitro approach, based on the incubation of active nuclear extracts from spermatids with a plasmid containing CAG repeats. Using this strategy, I showed that free negative supercoils, as observed during chromatin remodeling, may lead to secondary structures, and more specifically hairpins in trinucleotidic repeats, which ultimately result in trinucleotidic extension. This hypothesis was validated by inhibiting enzymes such as type 2 topoisomerases, since they are responsible for negative supercoils removal. Moreover, I showed that DNA protamination, as observed in spermatids, may increase torsional stress at CAG repeats and leads to expansion. In conclusion, this work suggest that torsional stress induced by protamination of DNA could be a new potential source of trinucleotidic instability. Moreover, this male specific source of trinucleotidic instability could play a major role in paternal anticipation of trinucleotidic diseases.

La mutation à l’origine de la maladie de Huntington (MH) correspond à une expansion anormale de glutamines sur la protéine huntingtine (HTT). La MH est caractérisée par des symptômes moteurs et cognitifs mais également des troubles psychiatriques tels que l’anxiété et la dépression.Au cours de ma thèse, j’ai montré que la HTT module le statut anxio-dépressif de la souris via ses phosphorylations aux sérines 1181/1201. En effet, l’ablation des phosphorylations sur la HTT endogène améliore significativement le phénotype anxio-dépressif de la souris. Chez la souris, cette modulation dépend d’une augmentation de la maturation et de la survie des nouveaux neurones dans l’hippocampe. En effet, l’irradiation focale de l’hippocampe, dans un contexte où les phosphorylations sont absentes, supprime la neurogenèse et la réduction du statut anxio-dépressif observée en l’absence de phosphorylations. Au niveau moléculaire, la HTT non phosphorylée accroît l’association des moteurs moléculaires et des vésicules de BDNF sur les microtubules, ce qui augmente les dynamiques et la libération du BDNF. Ceci active la voie de signalisation MAPK/CREB dans l’hippocampe, cette voie pouvant ainsi stimuler la neurogenèse.J’ai ensuite étudié le rôle de ces phosphorylations dans un contexte MH et j’ai démontré l’effet anxiolytique/antidépresseur de l’absence de ces phosphorylations.J’ai également montré le rôle de ces phosphorylations de la HTT au cours du développement du cortex embryonnaire.Les résultats obtenus au cours de ma thèse suggèrent que les mécanismes fondamentaux de neurogenèse sont régulés par la HTT et ses phosphorylations. De plus, ils identifient une nouvelle voie de modulation de l’anxiété/dépression faisant intervenir la HTT. / Huntington disease (HD) is a fatal neurodegenerative disorder associated with early psychiatric symptoms including anxiety and depression.During my thesis, I have demonstrated that huntingtin, the protein mutated in HD, modulates anxiety/depression-related behaviors through its phosphorylations at serines 1181 and 1201. Indeed, genetic phospho-ablation at serines 1181 and 1201 in mouse reduces basal levels of anxiety/depression-like behaviors in mouse. Suppression of neurogenesis by focal hippocampal irradiation abolishes this reduction of basal levels of anxiety/depression on some behavioral test demonstrating that neurogenesis is involved in this process. Ablation of HTT phosphorylations may stimulate neurogenesis through BDNF transport, release and signaling.I have also shown that ablation of phosphorylations on HTT is sufficient to ameliorate the anxiety/depression-like behavior of a mouse model of HD, which develops a behavior indicative of depression–like state.I have finally explored the role of HTT phosphorylation at serines 1181 and 1201 during brain development. During early steps of cortical neurogenesis, I have shown that ablation of HTT phosphorylations affects the mitosis of cortical progenitors, the fate of newly generated cells and the migration of new neurons.The results obtained during my thesis support the notion that HTT regulates key molecular mechanisms during neurogenesis both in adult and embryo. It also supports the notion that huntingtin participates to anxiety and depression-like behavior with potential consequences for the etiology of mood disorders and anxiety/depression in HD.

Les maladies par expansion de polyglutamines sont des maladies neurodégénératives dues à l’expansion du trinucléotide cytosine-adénine-guanine (CAG) dans les gènes correspondants codant pour une expansion d’homopolymère de glutamine dans les protéines mutées. Ce projet concerne les formes les plus courantes qui sont la maladie de Huntington (MH) et les ataxies spinocérébelleuses (SCA) types 1, 2, 3 et 7. Ce sont des maladies autosomiques dominantes, responsables de troubles graves de la motricité partageant des voies physiopathologiques communes, avec un effet notable sur la dysfonction métabolique. La disponibilité des tests génétiques et le fait que la plupart du temps la maladie débute à l’âge adulte offre la possibilité d’une intervention thérapeutique avant l’apparition de symptômes. Toutefois, les échelles cliniques ne sont pas assez sensibles et ne peuvent effectivement être utilisés pour évaluer les personnes au stade présymptomatique de la maladie. Les techniques d’imagerie par résonance magnétique (IRM) et de spectroscopie (SRM) sont des approches non invasives qui permettent de recueillir des informations pertinentes et sensibles. Ainsi, dans ce travail, nous présentons une combinaison de différentes techniques d’IRM et SRM afin d’identifier de robustes biomarqueurs de la MH et des SCA. Nous présentons aussi des approches thérapeutiques prometteuses dans la MH. De la même manière, nous voulons démontrer que des biomarqueurs d’imagerie sont plus sensibles que des échelles cliniques. Pour conclure, nous combinons des données multimodales – volumétrie, SRM, métabolomique et lipidomique – à partir de SCA dans un modèle qui explique mieux la pathologie. / Mutations in different gene loci that lead to the encoding of the unstable and expanded glutamine-encoding cytosine-adenine-guanine (CAG) repeats results in the group of diseases known as the polyglutamine diseases. This project focuses on the most common forms which are Huntington disease (HD) and spinocerebellar ataxia (SCA) types 1, 2, 3 and 7. These are autosomal dominant diseases responsible for severe movement disorders and are thought to share common pathophysiological pathways with a major emphasis on metabolic dysfunction. The availability of genetic testing and their predominantly adult onset opens a window for therapeutic intervention before symptoms onset. However, current clinical scales are not sensitive and cannot effectively be used to evaluate individuals at the presymptomatic stage of the diseases. This prompts the need for biomarkers that are sensitive to macroscopic and microscopic changes that may occur prior to disease onset. Magnetic resonance imaging (MRI) and spectroscopy (MRS) techniques present non-invasive approaches to extract pertinent information that otherwise would not be possible with clinical scales. In this work therefore, we present a combination of different MRI and MRS techniques to identify robust biomarkers in HD and SCA. We also present therapeutic approaches that hold promise in HD. Likewise, we show that imaging biomarkers have higher effect sizes than clinical scales. Finally, we combine multimodal data – volumetry, MRS, metabolomics and lipidomic – from SCA into a model that best explains the pathology.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Gabriela Kocurová Supervisor: Prof. MUDr. Jaroslav Dršata, CSc. Title of diploma thesis: Investigation of proteolytic enzymes expression in different tissues at the transgenic animal model of Huntington's disease by means of biochemical and immunohistochemical methods Background: Huntington's disease (HD) is a neurodegenerative disorder that is caused by an expansion of a polyglutamine (polyQ) domain in the huntingtin (Htt) protein. Because it is known that mutant Htt and especially its small proteolytic fragments are toxic to neurons (particularly those in the striatum and cortex), it has been suggested that proteolysis of mutant huntingtin (mHtt) might play an important role in HD pathogenesis. Therefore, the aim of the present study was to examine the expression of endogenous and mtHtt and possible participation of the proteolytic enzymes from the group of caspases, matrix metalloproteinases (MMPs), kallikreins (KLKs) and calpains in HD pathology of brain tissue. Methods: In this study we used WT and TgHD minipigs for N-terminal part of the human mtHtt (548aaHTT-145Q, both F2 generation, age 36 months; F3 generation, age 48 months in additional experiment), R6/2 mice were used as...

Huntington’s disease (HD) is a fatal neurodegenerative disease caused by a CAG repeat expansion in exon 1 of the huntingtin gene, resulting in an expanded polyglutamine (polyQ) repeat in the huntingtin protein. Patients receive symptomatic treatment for motor, emotional, and cognitive impairments; however, there is no treatment to slow the progression of the disease, with death occurring 15-20 years after diagnosis. Mutant huntingtin protein interferes with multiple cellular processes leading to cellular dysfunction and neuronal loss. Due to the complexity of mutant huntingtin toxicity, many approaches to treating each effect are being investigated. Unfortunately, addressing one cause of toxicity might not result in protection from other toxic insults, necessitating a combination of treatments for HD patients. Ideally, single therapy targeting the mutant mRNA or protein could prevent all downstream toxicities caused by mutant huntingtin. In this work, I used animal models to investigate a potential therapeutic target for decreasing mutant huntingtin protein, and I apply bioluminescent imaging to investigate RNA interference to silence mutant huntingtin target sites. The enzyme puromycin sensitive aminopeptidase (PSA) has the unique property of degrading polyQ peptides and been implicated in the degradation of huntingtin. In this study, we looked for an effect of decreased PSA on the pathology and behavior in a mouse model of Huntington’s disease. To achieve this, we crossed HD mice with mice with one functional PSA allele and one inactivated PSA allele. We found that PSA heterozygous HD mice develop a greater number of pathological inclusion bodies, representing an accumulation of mutant huntingtin in neurons. PSA heterozygous HD mice also exhibit worsened performance on the raised-beam test, a test for balance and coordination indicating that the PSA heterozygosity impairs the function of neurons with mutant huntingtin. In order to test whether increasing PSA expression ameliorates the HD phenotype in mice we created an adeno-associated virus (AAV) expressing the human form of PSA (AAV-hPSA). Unexpectedly, testing of AAV-hPSA in non-HD mice resulted in widespread toxicity at high doses. These findings suggest that overexpression of PSA is toxic to neurons in the conditions tested. In the second part of my dissertation work, I designed a model for following the silencing of huntingtin sequences in the brain. Firefly luciferase is a bioluminescent enzyme that is extensively used as a reporter molecule to follow biological processes in vivo using bioluminescent imaging (BLI). I created an AAV expressing the luciferase gene containing huntingtin sequences in the 3'-untranslated region (AAV-Luc-Htt). After co-injection of AAV-Luc-Htt with RNA-silencing molecules (RNAi) into the brain, we followed luciferase activity. Using this method, we tested cholesterol-conjugated siRNA, un-conjugated siRNA, and hairpin RNA targeting both luciferase and huntingtin sequences. Despite being able to detect silencing on isolated days, we were unable to detect sustained silencing, which had been reported in similar studies in tissues other than the brain. We observed an interesting finding that co-injection of cholesterol-conjugated siRNA with AAV-Luc-Htt increased luminescence, findings that were verified in cell culture to be independent of serotype, siRNA sequence, and cell type. That cc-siRNA affects the expression of AAV-Luc-Htt reveals an interesting interaction possibly resulting in increased delivery of AAV into cells or an increase in luciferase expression within the cell. My work presents a method to follow gene silencing of huntingtin targets in the brain, which needs further optimization in order to detect sustained silencing. Finally, in this dissertation I continue the study of bioluminescent imaging in the brain. We use mice that have been injected in the brain with AAV-Luciferase (AAV-Luc) to screen 34 luciferase substrate solutions to identify the greatest light-emitting substrate in the brain. We identify two substrates, CycLuc1 and iPr-amide as substrates with enhanced light-emitting properties compared with D-luciferin, the standard, commercially available substrate. CycLuc1 and iPr-amide were tested in transgenic mice expressing luciferase in dopaminergic neurons. These novel substrates produced luminescence unlike the standard substrate, D-luciferin which was undetectable. This demonstrates that CycLuc1 and iPr-amide improve the sensitivity of BLI in low expression models. We then used CycLuc1 to test silencing of luciferase in the brain using AAV-shRNA (AAV-shLuc). We were unable to detect silencing in treated mice, despite a 50% reduction of luciferase mRNA. The results from this experiment identify luciferase substrates that can be used to image transgenic mice expressing luciferase in dopaminergic neurons. My work contributes new data on the study of PSA as a modifier of Huntington’s disease in a knock-in mouse model of Huntington’s disease. My work also makes contributions to the field of bioluminescent imaging by identifying and testing luciferase substrates in the brain to detect low level of luciferase expression.