<p dir="ltr">Down syndrome (DS) arises from the triplication of human chromosome 21 (Hsa21), leading to a spectrum of phenotypes characterized by neurodevelopmental and cognitive abnormalities. The Ts65Dn mouse model emulates DS by harboring three copies of genes found on Hsa21 resulting in trisomy 21 (Ts21)- like traits, including disruptions in neuronal pathways, delays in sensorimotor and behavior milestones, and deficits in learning and memory tasks. There is no cure for DS and available therapies primarily address symptoms stemming from Ts21-associated phenotypes. <i>DYRK1A</i>, a gene triplicated in Ts21, has a pivotal role in pathways of neurodevelopment and has been a focus of inhibition treatment research aimed at preempting abnormal brain phenotypes. This study aimed to find a point of substantial <i>Dyrk1a </i>expression dysregulation during a period of critical neonatal neurodevelopment and employ targeted pharmacological and genetic knockdown methods to alleviate the presence or severity of characteristically abnormal brain and behavior phenotypes. The hypothesis of this study was that administering a targeted intervention prior to a point of known overexpression in trisomic pups would ameliorate molecular, sensorimotor, and neurobehavioral deficits, redirecting growth trajectories of Ts65Dn neonatal pups towards more neurotypical outcomes. To test this hypothesis, the spatiotemporal pattern of DYRK1A expression was quantified during the first three weeks of neonatal development across the hippocampus, cerebral cortex, and cerebellum of the Ts65Dn mouse model and found to fluctuate according to the genotype, age, sex, and brain region of the subject. <i>Dyrk1a </i>protein and mRNA expression levels were delineated in trisomic animals by age, exploring the correlation between expression and age, sex, genotype, and brain region. Next a constitutive <i>Dyrk1a </i>knockdown model was integrated with the Ts65Dn model to investigate the impact of gene copy number reduction on protein and mRNA expression levels during phases of known DYRK1A dysregulation. On postnatal day 6, protein expression was rescued in all three brain regions of male animals but was rescued only in the cerebellum of females. There were no significant differences in mRNA transcript levels in either sex at this age. Finally, genetic elements were introduced into the Ts65Dn model to facilitate a spatiotemporally controlled functional reduction of <i>Dyrk1a</i> and discern how the timing of gene copy number reduction affects molecular and neurobehavioral development in a trisomic system. Results from these studies suggest that only functionally reducing <i>Dyrk1a </i>gene copy number on the day of birth is not sufficient to rescue the majority of deficits and delays present in the Ts65Dn mouse model of DS. These findings significantly enhance the understanding of trisomic <i>Dyrk1a </i>expression dynamics during neonatal development and shed light on tailored therapeutic approaches to modulate intrinsic DS characteristics based on age, sex, and phenotypic considerations.</p>

DYRK1A is a protein kinase encoded by a gene implicated in Down syndrome pathogenesis. Loss of DYRK1A could promote oncogenic transformation. However, the regulation and substrates of DYRK1A are not fully understood. MudPIT proteomic analysis revealed novel DYRK1A interacting proteins with poorly characterized or even unknown functions. Therefore, the aim of this thesis was to understand the function of DYRK1A through the characterization of its interacting proteins. To achieve this aim, we established stable cell lines expressing these proteins and confirmed the interactions between DYRK1A and seven candidate binding partners. Furthermore, we found that all novel DYRK1A-interacting proteins also bind DCAF7, a previously reported DYRK1A-binding scaffold protein that binds to the N-terminus of DYRK1A. Using cyto-nuclear fractionation and immunostaining we found that DYRK1A-interacting proteins were present in different cellular compartments, suggesting that DYRK1A could play distinct roles in the cell depending on its localization. DYRK1A has been shown to regulate cell proliferation and actin cytoskeleton therefore we used cell proliferation assays and actin staining to determine the role of DYRK1A-interacting proteins in these processes. Here we report functional characterization of the interacting partners of DYRK1A and present cell-based models that will help to understand the function and regulation of this important protein kinase.

<p>The relationship between gene copy number and protein expression levels has not thoroughly been examined in humans or mouse models of Down syndrome (DS) in relationship to developmental changes in the trisomic brain. Found on human chromosome 21 (Hsa21) and triplicated in DS, Dual-specificity tyrosine-phosphorylated regulated kinase 1A (<i>DYRK1A)</i> has been linked in DS to neurological deficits by restricting cell growth and proliferation. Little information exists regarding DYRK1A during perinatal development and how its expression may lead to cognitive deficits, and none exists that explores the gene-to-protein relationship during these critical time periods. This study aims to 1) Quantify variable DYRK1A expression across development as a function of age, sex, and brain region in trisomic Ts65Dn mice compared to euploid counterparts and 2) establish that the spatiotemporal pattern of developmental DYRK1A in the brain is not influenced solely by gene copy number, and that reduction of <i>Dyrk1a</i> in euploid and trisomic mice does not result in a corresponding global reduction of DYRK1A expression. DYRK1A was quantified in three areas of the postnatal brain at seven ages using the Ts65Dn mouse, the most studied model of DS, and found that trisomic expression is significantly increased on postnatal day ([P]6), declining by the third week to near euploid levels. We also uncovered a sexual dimorphic expression of DYRK1A when comparing animals of different sexes within the same genotype. Data from <i>Dyrk1a</i> knockdown mice indicated that reducing only <i>Dyrk1a</i> in euploid and in otherwise trisomic animals yields highly variable levels of DYRK1A, dependent on sex and tissue type, supporting the non-intuitive relationship between gene dosage and protein expression. These data emphasize the need to understand the age-dependent regulation of antecedent conditions that are causing changes in <i>Dyrk1a</i> expression in the brain.</p>

Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) is a complex genetic disorder caused by the triplication of human chromosome 21 (Hsa21). The presence of an extra copy of an entire chromosome greatly disrupts the copy number and expression of over 350 protein coding genes. This gene dosage imbalance has far-reaching effects on normal development and aging, leading to cognitive and skeletal defects that emerge earlier in life than the general population. The present study begins by characterizing skeletal development in young male Ts65Dn mice to test the hypothesis that skeletal defects in male Ts65Dn mice are developmental in nature.Femurs from young mice ranging from postnatal day 12- to 42-days of age (P12-42) were measured and analyzed by microcomputed tomography (μCT). Cortical defects were present generally throughout development, but trabecular defects emerged at P30 and persisted until P42. The gene Dual-specificity tyrosine-regulated kinase 1a (Dyrk1a) is triplicated in both DS and in Ts65Dn mice and has been implicated as a putative cause of both cognitive and skeletal defects. To test the hypothesis that trisomic Dyrk1a is related to the emergence of trabecular defects at P30, expression of Dyrk1a in the femurs of male Ts65Dn mice was quantified by qPCR. Expression was shown to fluctuate throughout development and overexpression generally aligned with the emergence of trabecular defects at P30. The growth rate in trabecular measures between male Ts65Dn and euploid littermates was similar between P30 and P42, suggesting a closer look into cellular mechanisms at P42. Assessment of proliferation of BMSCs, differentiation and activity of osteoblasts showed no significant differences between Ts65Dn and euploid cellular activity, suggesting that the cellular microenvironment has a greater influence on cellular activity than genetic background. These data led to the hypothesis that reduction of Dyrk1a gene expression and pharmacological inhibition of DYRK1A could be executed during a critical period to prevent the emergence of trabecular defects at P30. To tests this hypothesis, doxycycline-induced cre-lox recombination to reduce Dyrk1a gene copy number or the DYRK1A inhibitor CX-4945 began at P21. The results of both genetic and pharmacological interventions suggest that trisomic Dyrk1a does not influence the emergence of trabecular defects up to P30. Instead, data suggest that the critical window for the rescue of trabecular defects lies between P30 and P42.

No description available.

Les travaux de la thèse se sont articulés autour de la synthèse totale d’un composé naturel isolé à partir de la plante Scorzoneraradiata, la RCZ. Lors d’un criblage sur un panel de kinases, dont la kinase DYRK1A, les résultats ont montré la très grande sélectivité de ce composé avec une action d’inhibition sur DYRK1A (IC50 = 220 nM). De nombreuses études disponibles dans la littérature ont établi le lien entre la dérégulation de la protéine DYRK1A avec le syndrome de Down (trisomie 21) et certaines maladies neurodégénératives tel que la maladie d’Alzheimer. En vue de l’obtention de ce composé prometteur présentant un squelette dihydrostilbène porteur d’une fonction cétone et d’un groupement osidique. Trois voies d’approche ont été successivement mises en œuvre. Le groupement cétone est construit par le biais d’une réaction de Sonogashira suivit par une hydratation en présence d’un sel mercurique. La réaction de Wittig permet l’assemblage du squelette stilbène. Lors de la première approche, un isomère de position du composé final avec un pouvoir inhibiteur divisé par dix par rapport à RCZ a été obtenu. L’utilisation de différents groupements protecteurs a permis l’obtention du composé final RCZ en seulement 15 étapes. Ce travail ouvre de nouvelles perspectives dans le développement d’analogues par des pharmacomodulations en se basant sur les informations fournies par le co-cristal de RCZ avec la protéine DYRK2. / This thesis focuses on the synthesis of RCZ, a natural compound isolated from the plant Scorzonera radiata. During a high-throughput screening, this compound has demonstrated, on a large panel of kinases, a high and unusual degree of selectivity against the kinase DYRK1A. On a structural point of view, the compound is a glycosylated dihydrostlibene, which also bears two phenols and an acetyl group. Recently, a link between the deregulation of the kinase DYRK1A with Down syndrom and some neurodegenerative diseases such as Alzheimer disease has been established. In order to perform the synthesis of this compound, three approaches were undertaken. In these approaches, the acetyl group was built through a Sonogashira coupling followed by a mercury salt catalyzed hydration of the acetylenic group. Also, the stilbene scaffold was always obtained by Wittig condensdation. In the first approach, an isomer was obtained with approximately a ten times less potent inhibitory activity against DYRK1A than RCZ. By the use of different protective groups the final compound RCZ was successfully obtained. In conclusion, a total and efficient synthesis of RCZ has been constructed in 15 steps. This work opens future perspective in the design of new inhibitors based on the determination of the crystal structure of the RCZ-DYRK2 complex.

La trisomie 21 (T21) résulte de la présence d’une extra-copie du chromosome 21 (Chr21). Parmi les gènes candidats impliqués dans les déficits cognitifs liés à la T21, DYRK1A est un des plus pertinents. Des études ont montré une corrélation entre l’augmentation de son activité kinase avec les déficits mnésiques observés dans les modèles murins de T21. Afin de comprendre les mécanismes sous-jacents aux altérations cognitives causées par son surdosage, nous avons utilisé des modèles murins sur-exprimant DYRK1A seule ou en plus d’autres gènes orthologues au Chr21, ainsi que des inhibiteurs spécifiques de DYRK1A (Leucettines) synthétisés par ManRos Therapeutics. Ici sont présentées les conséquences d’un traitement chronique avec ces Leucettines sur la cognition de ces animaux. Des analyses du phosphoprotéome de ces souris traitées ou non avec une des Leucettines, la L41, a mis en lumière des cibles et mécanismes biologiques impliqués dans les perturbations mnésiques de ces animaux. Enfin, ce projet a surtout permis de mettre en évidence un nouvel inhibiteur plus sélectif pour DYRK1A comme candidat-médicament pour l’amélioration des fonctions cognitives des porteurs de la T21. / Down Syndrome (DS), is due to the presence of an extra copy of chromosome 21 (Ch21). Among the candidates implicated in DS intellectual disabilities, DYRK1A is one of the most relevant. Several studies have shown a correlation between an increase of its kinase activity and the intellectual defects observed in DS models. In order to understand the mechanisms underlying the impact of DYRK1A overdosage on cognitive alterations, we used several trisomic mouse models expressing DYRK1A alone or with additional Hsa21 orthologous genes and specific DYRK1A inhibitors (Leucettines) from ManRos Therapeutics. We will present here the consequence of the Leucettines treatment following repetitive administration to several DS mouse models on the behavior and cognition. Further analysis of the phosphoproteome of DS mouse models treated or not with one of the Leucettine, the L41, unravels a few targets and pathways which are involved in the cognitive alterations observed in these trisomic mice. These results support the potential of more selective DYRK1A inhibitor as a therapeutic approach to improve cognitive functions in DS patients.

Lors du screening de composés du ‘human chemical exposome’, les herbicides triazines et les insecticides pyrazoles, notamment le fipronil, ont révélé leur capacité à induire la production de peptides amyloïde β -42 et -43. Ayant tendance à s’agréger en oligomères puis en plaques, ils sont une caractéristique de la maladie d’Alzheimer (MA). Cette découverte informe sur le danger potentiel de ces produits et apporte des outils pour décrypter les mécanismes menant à l’altération du rapport des différentes formes d’amyloïdes. Par ailleurs, la dérégulation de DYRK1A, impliquée dans la trisomie 21, affecte aussi ces peptides amyloïdes, ainsi que la protéine Tau qui est alors hyperphosphorylée et a tendance à s’agréger en enchevêtrements neurofibillaires, une autre caractéristique de la MA. Ce même effet sur Tau est observé avec CDK5 dans la MA, les AVC et les traumatismes crâniens. Le développement d’inhibiteurs pharmacologiques spécifiques de ces deux kinases est donc un enjeu pour ManRos Therapeutics. / In the screening of ‘human chemical exposome’ compounds, triazine herbicides and pyrazole insecticides, especially fipronil, have shown their ability to induce β-amyloid -42 and -43 peptide production. As they tend to aggregate into oligomers then into plaques, they are a characteristic of Alzheimer's disease (AD). This discovery informs about the potential danger of these products and provides tools to decipher the mechanisms leading to the alteration of the ratio of the different forms of amyloid.Moreover, dysregulation of DYRK1A, involved in trisomy 21, also affects these amyloid peptides, as well as the Tau protein which is then hyperphosphorylated and tends to aggregate into neurofibillar tangles, another characteristic of AD. This same effect on Tau is observed with CDK5 in AD, stroke and brain injury. The development of specific pharmacological inhibitors of these two kinases is therefore an issue for ManRos Therapeutics.

La maladie d’Alzheimer (MA) est actuellement conceptualisée comme un continuum : la démence représentant la conséquence clinique d’une longue période où s’accumule des éléments pathologiques dans le cerveau d’individus indemnes de symptômes comportementaux. Les futures thérapies devront idéalement débuter avant l’apparition des symptômes mais le manque actuel d’outils reproduisant avec pertinence cette phase préclinique empêche leurs développements. Dans ce travail de thèse, nous avons d’abord levé ce verrou technologique. Un nouveau modèle a été créé dans lequel la production de peptides Aβs solubles en quantité faible, mais néanmoins suffisante pour induire une hyper-phosphorylation des protéines Tau, perturbe la fonction cognitive bien avant l’apparition d’éléments caractéristiques de la MA (plaques amyloïdes, dégénérescences neurofibrillaires, inflammation). Cette avancée technologique, nous a ensuite permis d’évaluer l’implication de la protéine kinase DYRK1A et le potentiel thérapeutique de molécules modulant ses fonctions dans l’ensemble de la pathologie Alzheimer. Nos résultats démontrent que l’inhibition de son activité kinase réduit l’hyper-phosphorylation des protéines Tau et améliore la fonction mnésique chez notre animal modélisant la phase préclinique de la MA. A contrario, nous démontrons que DYRK1A est sensible à un clivage protéolytique dans le cerveau de patients atteints de démence Alzheimer et acquiert de nouvelles fonctions biologiques. Dans ce contexte, la prévention de ce clivage réduit l’inflammation et restaure les déficits cognitifs chez la souris modélisant la phase clinique de la MA. En ciblant différentes phases de la MA, ces données ouvrent donc la voie à la médecine personnalisée et à des stratégies de traitement plus ciblées. / Current view conceptualizes Alzheimer’s disease (AD) as a continuum, with dementia representing the clinical outcome of a long period of cumulative pathological events in the brain of individual with free cognitive symptoms. New therapies for AD should ideally be started before the onset of symptoms but the lack of suitable tools mimicking preclinical stage of AD limits their future evaluations. In this work, we break this technological limitation. A new animal model have been developed in which a small amount of soluble Aβs forms able to induce hyper-phosphorylation of Tau is sufficient to disturb cognitive function long before classical lesions occur (amyloid plaques, neurofibrillary tangles and inflammation). This technological breakthrough allows us to evaluate involvement of the protein kinase DYRK1A and therapeutic potential of molecules modifying its functions in different stages of AD. Our results demonstrate that inhibition of its kinase activity reduces hyper-phosphorylation of Tau proteins and alleviates memory function in our preclinical AD-like animal model. In contrast, we provide evidences that DYRK1A undergoes a cleavage in brain of patient with clinical AD and gains new biological functions. Prevention of this proteolysis reduces inflammation and restores cognitive impairments in a clinical AD-like mice model. By targeting distinct phases of the disease, these data open avenue for personalized medicine and more-targeted treatment strategies.

Depuis le début des années 2000, la connaissance précise du kinome a entraîné l’émergence de nouvelles stratégies thérapeutiques ciblant des protéines kinases impliquées dans de nombreuses pathologies en oncologie et dans les maladies du système nerveux central. Afin de cibler les kinases d’intérêts identifiées au sein de ces travaux, nous avons effectué, dans une démarche orientée vers la diversité moléculaire, la synthèse de nouveaux hétérocycliques a fort potentiel de valorisation. Nous nous sommes appuyé sur la création et la fonctionnalisation de bicycles à 5 ou 6chaînons de type [6-5] ou [5-5], ces espèces chimiques représentant la voie d’accès à des inhibiteurs compétitifs du substrat naturel des kinases, l’ATP. Nous avons dans un premier temps travaillé autour des imidazo[1,2-b]pyridazines puis des[1,2,4] triazolo[4,3-b]pyridazines, scaffold plus original, pour concevoir des inhibiteurs plus actifs et spécifiques de la kinase HASPIN, nouvelle cible prometteuse en oncologie.Nous avons ensuite poursuivi les études précédentes du laboratoire sur les imidazo[2,1-b][1,3,4]thiadiazoles. Nous basant sur une méthodologie de synthèse bien développée, nous avons créé une librairie de composés dirigés contre les kinases DYRK1A et CLK1 impliquées dans les processus de neuro dégénération, notamment dans la maladie d’Alzheimer. Ainsi, au travers d’analogues des imidazothiadiazoles originaux, nous avons proposé des méthodologies de synthèses de ces nouveaux hétérocycles permettant des pharmaco modulations aisées.Ces divers projets de chimie médicinale ont pu être entrepris de façon à améliorer les connaissances des relations structure-activité, et concevoir de nouveaux inhibiteurs puissants des kinases HASPIN, DYRK1A etCLK1. / Since the early 2000s, precise knowledge of kinome has induced the emergence of novel therapies targeting kinases involved in several kinds of pathologies in oncology and nervous central systems disorders.In order to target original kinases of interest identified in this work, we have developed diversity-oriented synthesis to create new high-valuable heterocycles. We have focused our efforts on the design and functionalization of [6-5] or [5-5] fused ring bicycles. Those chemical species representing a great pathway tocreate competitive inhibitors of ATP; the natural substrate of kinases.First-of-all, we have worked on imidazo[1,2-b]pyridazines and then on [1,2,4]triazolo[4,3-b]pyridazinesscaffolds, to create more active and selective HASPIN kinase inhibitors, a new hot-target in oncology.Then, we have pursued previous laboratory studies on imidazo[2,1-b][1,3,4]thiadiazoles. Based on a well-built methodology, we have synthesized severals DYRK1A and CLK1 kinases inhibitors involved in neurodegenerative disorders, as Alzheimer’s disease. Thus, through original imidazothiadiazoles analogues,we have proposed synthetic methodologies to design these novel heterocycles allowding esay pharmacomodulations.These medicinal chemistry projects have been undertaken to improved knowledge of structure-activityrelashionship, and providing novel strong inhibitors of HASPIN, DYRK1A or CLK1 kinases.