La combinaison de biomatériaux et cellules souches, a pour but de protéger des cellules endommagées et de ralentir la progression des maladies neurodégénératives, comme la maladie de Huntington (MH). Les cellules souches mésenchymateuses et particulièrement une sous-population, les cellules MIAMI, ont déjà démontré leur efficacité dans la maladie de Parkinson. Il est cependant essentiel d’améliorer leur différenciation neuronale, leur survie et évaluer leur sécrétome. L’objectif principal de ce travail fut de proposer une stratégie innovante de médecine régénératrice pour la MH associant cellules souches, nano et micro médecines. Pour l’évaluer, un nouveau modèle animale ex vivo de la MH a été mis en place. Nous avons ensuite développé et optimisé deux nano-vecteurs, des nanocapsules lipidiques et des nanoparticules solides de SPAN, et les avons associés à un inhibiteur de REST qui est un facteur de transcription qui empêche la différenciation neuronale. La transfection de ce siREST a montré une amélioration du phénotype neuronal. Ces cellules ainsi modifiées furent ensuite induites vers un phénotype GABAergic grâce à des facteurs de croissance. Puis elles ont été associées à un support 3D, les microcarriers pharmacologiquement actif (MPA) permettant une meilleure intégration des cellules après greffe. Les MPA sont des microsphères ayant une surface biomimétique de laminine et libérant de façon contrôlée un facteur trophique le « brain derived neurotrophic factor » (inducteur d’un phénotype neuronal et neuro-protecteur). Des résultats prometteurs ont été obtenus, encourageant à continuer l’évaluation de cette stratégie in vivo dans des modèles génétiques de la MH. / The combination of biomaterials and stem cells aims to protect damaged cells and slow the progression of neurodegenerative diseases such as Huntington's disease(HD). Mesenchymal stem cells, particularly a subpopulation known as MIAMI cells, have already demonstrated their effectiveness in Parkinson's disease. However, it is essential to improve their neuronal differentiation, survival, and to assess their secretome. The main objective of this work was to propose an innovative regenerative medicine strategy for HD by combining stemcells, micro and nano medicines. To perform this assessment, a new ex vivo animal model of HD has been set up. We then developed and optimized two nanovectors,lipid nanocapsules and solid SPAN nanoparticles,carrying an inhibitor of REST a transcription factor, which prevents neuronal differentiation. The transfection of this siREST showed an improvement in the neuronal phenotype. These modified cells were then induced into a GABAergic phenotype through growth factors. They were then associated with a 3D support, the pharmacologically active microcarriers (PAM) allowing a high rate of engraftment. The PAM are microspheres which have a biomimetic surface of laminin and release a trophic factor BDNF, brain derived neurotrophic factor (inducer of a neural phenotype and neuroprotective) in a controlled manner. Promising results were obtained, further encouraging continuing the evaluation of this strategy in vivo in genetic models of HD.

Huntington's disease (HD) is an autosomal dominant inherited neuro-psychiatric disease with usual onset in the middle age. The mutation, located on the short shoulder of chromosome 4, is an expansion of a nucleotide triplet, containing cytosine, adenine, guanine (CAG), with critical limit of 40+ repetitions. The principal symptoms include motor symptoms (chorea, dystonia, disorders of voluntary movements), progressive cognitive deterioration and neuropsychiatric symptoms (behaviour disorders, affective symptoms and so on). The clinical diagnosis is confirmed by a genetic test, which may also be carried out presymptomatically in offsprings of the diseased person. The objective of the 1st study consisted in the characterization of differences in psychiatric examination and neuropsychological testing among the people at risk (PAR), in whom it was recommended to delay the test, and people at risk, who were recommended to continue in the so-called predictive protocol. The total of 52 people have been examined (32 females, 20 males). In addition to the common psychiatric examination we have also administered the Eysenck Personality Questionnaire (EPQ-A), self-rating scale of general psychopathology (SCL- 90), three short cognitive tests - Trail making test, test of Verbal fluency and...

This diploma thesis explores problematic areas of patients with Huntington's disease in their performance during activities of daily living (ADLs) from the perspective of patients and their caregivers. The aim of the research was also to assess a possible correlation between cognitive impairment and the patient's performance in ADL. Twenty-five patients with their caregivers met the selection criteria for the research. There were used standardized assessment methods available in Czech: the Montreal Cognitive Assessment (MoCA), the Canadian Occupational Performance Measure (COPM) and the questionnaire for caregivers called Bristol Activities of Daily Living Scale (BADLS-CZ). The statistical analyses consisted of methods of the nonparametric statistics, qualitative analysis was processed by data categorizing. Caregivers reported more problematic areas in ADLs which was significantly confirmed in the statistical hypothesis testing (p <0,05). A significant correlation was seen between the results of the questionnaire and the results of the MoCA assessment (rSp = -0,620; p <0,05). For various reasons, patients with Huntington's disease did not mention as many problematic areas in performing ADL as their caregivers. Therefore, it is appropriate in clinical practice to supplement the assessment of the patient's...

As doenças neurodegenerativas (DN) estão entre as principais causas de mortalidade e morbidade nos países ocidentais. Não há ainda um tratamento definitivo para estas neuropatias, mas estudos têm indicado mecanismos comuns de toxicidade que incluem disfunção mitocondrial, estresse oxidativo, apoptose e neuroinflamação. Adicionalmente, o efeito benéfico da neuroplasticidade induzida por fatores neurotróficos no retardamento ou inibição do processo neurodegenerativo também tem sido sugerido por muitos estudos. O beta-cariofileno é um sesquiterpeno bi-cíclico encontrado no óleo essencial de algumas plantas, e que possui efeito anti-inflamatório e antioxidante. Assim, este composto possui características e é capaz de induzir efeitos que o tornam um potencial candidato ao tratamento/prevenção dos processos envolvidos na neurodegeneração. Apesar disso, pouco se sabe sobre os efeitos e os mecanismos de ação do beta-cariofileno no processo de degeneração neuronal. Então, neste estudo, avaliou-se o efeito do beta-cariofileno em modelos celulares (PC 12) de neurotoxicidade que mimetizam in vitro os mecanismos moleculares envolvidos nas doenças de Parkinson, Huntington e Alzheimer, os quais, para efeitos práticos, denominaremos de \"modelos celulares de Parkinson, Huntington e Alzheimer\". Estes modelos são induzidos experimentalmente pela neurotoxina dopaminérgica iodeto de 1-metil 4-fenil piridina (MPP+), pela neurotoxina mitocondrial ácido 3-nitropropiônico (3NP) e pelo peptídeo neurotóxico B-amiloide (AB42), respectivamente. O beta-cariofileno apresentou efeitos benéficos nestes três modelos de neurotoxicidade, e adicionalmente induziu neuritogênese e a expressão de proteínas neurotípicas no modelo neuronal. Este é o primeiro estudo a demonstrar tais efeitos do beta-cariofileno. / Neurodegenerative diseases (ND) are among the leading causes of mortality and morbidity in Western countries. There is not a definitive treatment for these neuropathies, but studies have indicated common mechanisms of toxicity that include mitochondrial dysfunction, oxidative stress, neuroinflammation and apoptosis. Additionally, the beneficial effect of the neuroplasticity induced by neurotrophic factors on the retardation or inhibition of neurodegeneration has also been suggested by several studies. Beta-caryophyllene is a bicyclic sesquiterpene found in essential oils of some plants, and possesses anti-inflammatory and antioxidant effects. Thus, this compound has characteristics and is capable of inducing effects that make it a potential candidate for treatment / prevention of the processes involved in neurodegeneration. Despite this, little is known about the effects and mechanisms of action of beta-caryophyllene in the neuronal degeneration process. Then, this study evaluated the effect of beta-caryophyllene in cellular models of neurotoxicity (PC 12) that mimic in vitro the molecular mechanisms involved in Parkinson\'s, Huntington\'s and Alzheimer\'s diseases, which, for practical purposes, we will denominate \"Cellular models of Parkinson\'s, Huntington\'s and Alzheimer\'s diseases.\" These models are experimentally induced by the dopaminergic neurotoxin 1-methyl iodide, 4-phenyl pyridine (MPP+), by the mitochondrial neurotoxin 3-nitropropionic acid (3NP) and the neurotoxic peptide B-amyloid (AB42), respectively. Beta-caryophyllene showed beneficial effects on these three models of neurotoxicity, and additionally induced neuritogenesis and the expression of neurotypic proteins in the neuronal model. This is the first study to demonstrate such effects of beta-caryophyllene.

A doença de Huntington (DH) é um distúrbio neurodegenerativo hereditário e autossômico dominante e tem como características alterações motoras e mentais progressivas. Recentemente, além das alterações verificadas no sistema nervoso central, também têm sido descritas alterações em órgãos periféricos, tais como osteoporose, atrofia muscular, problemas intestinais, alterações cardíacas e, sobretudo, alterações no sistema nervoso autônomo. São evidentes as alterações autonômicas do coração nos portadores da DH, as quais, são, sobretudo, um risco potencial, tornando os pacientes suscetíveis a problemas cardiovasculares. No entanto, os mecanismos pelos quais a doença afeta os componentes autonômicos do coração não são totalmente conhecidos, por isso a importância de se estudar os componentes da inervação cardíaca, sobretudo o gânglio estrelado (GE). A DH pode ser induzida através do ácido 3-nitropropiônico (3-NP), pois essa substância produz efeitos neurotóxicos inibindo a succinato desidrogenase. Esta pesquisa objetiva analisar, por meio da indução através do 3-NP, os efeitos da DH no GE, identificando possíveis alterações morfoquantitativas dos neurônios ganglionares, com uso de técnicas baseadas em delineamento estereológico 3D e de bioimagem associadas à teste comportamental e perfil hemodinâmico, a fim de contribuir para o entendimento de como a doença age na inervação do coração. Para isso foram utilizados 14 camundongos C57BL-6 machos que foram alocados em dois grupos: Grupo Controle com 7 animais induzidos com solução salina (0,9%); Grupo 3NP com 7 animais induzidos com doses subagudas de 60 mg.kg-1dia-1 de 3-NP. Foram realizados o teste comportamental, a avaliação cardíaca e a análise estereológica. Os principais achados dessa pesquisa foram: (I) diminuição da atividade exploratória dos animais; (II) prejuízo da função sistólica; (III) aumento de 76% no volume ganglionar; (IV) aumento de 70% no volume médio dos neurônios, concluindo-se que o 3-NP produz efeitos na função cardíaca, ocasionando hipertrofia do gânglio / Huntington\'s disease (HD) is a hereditary and autosomal dominant neurodegenerative disorder and is characterized by progressive motor and mental changes. Recently, in addition to changes in the central nervous system, alterations in peripheral organs such as osteoporosis, muscular atrophy, intestinal problems, cardiac alterations and, above all, changes in the autonomic nervous system have also been described. Autonomic heart alterations in DH patients are evident, which are a potential risk, making patients susceptible to cardiovascular problems. However, the mechanisms by which the disease affects the autonomic components of the heart are not fully understood, therefore, the importance of studying the components of cardiac innervation, especially the stellate ganglion (SG). HD can be induced through 3-nitropropionic acid (3-NP), as this substance produces neurotoxic effects inhibiting succinate dehydrogenase. The aim of this research was to analyze the effects of HD on the SG by means of 3-NP induction, identifying possible morpho-quantitative changes in ganglion neurons, using techniques based on 3D stereological and bioimaging techniques associated with behavioral and hemodynamic profile, In order to contribute to the understanding of how the disease acts in the heart innervation. For this, 14 male C57BL-6 mice were used, which were allocated in two groups: Control Group with 7 animals induced with saline solution (0.9%); Group 3NP with 7 animals induced with subacute doses of 60 mg.kg-1day-1 of 3-NP. Behavioral test, cardiac evaluation and stereological analysis were performed. The main findings of this research were: (I) decrease in the exploratory activity of the animals; (II) impairment of systolic function; (III) 76% increase in ganglion volume; (IV) increase of 70% in the mean volume of the neurons, concluding that 3-NP produces effects on cardiac function, causing hypertrophy of the ganglion

La maladie de Huntington est une maladie neurodégénérative héréditaire qui est devenue un modèle d'étude pour comprendre la physiopathologie des maladies du cerveau associées à la production de protéines mal conformées et à la neurodégénérescence. Bien que plusieurs mécanismes aient été mis en avant pour cette maladie, dont plusieurs seraient aussi impliqués dans des pathologies plus fréquentes comme la maladie d’Alzheimer ou la maladie de Parkinson, nous ne savons toujours pas quels sont les mécanismes ou les profils moléculaires qui déterminent fondamentalement la dynamique des processus de dysfonction et de dégénérescence neuronale dans cette maladie. De même, nous ne savons toujours pas comment le cerveau peut résister aussi longtemps à la production de protéines mal conformées, ce qui suggère en fait que ces protéines ne présentent qu’une toxicité modérée ou que le cerveau dispose d'une capacité de compensation et de résilience considérable. L'hypothèse de mon travail de thèse est que l'intégration de données génomiques et transcriptomiques au travers des modèles qui récapitulent différentes phases biologiques de la maladie de Huntington peut permettre de répondre à ces questions. Dans cette optique, l'utilisation des réseaux de gènes et la mise en application de concepts issus de la théorie des graphes sont particulièrement bien adaptés à l'intégration de données hétérogènes, au travers des modèles et au travers des espèces. Les résultats de mon travail suggèrent que l'altération précoce (avant les symptômes, avant la mort cellulaire) et éventuellement dès le développement cérébral) des grandes voies de développement et de maintenance neuronale, puis la persistance voire l'aggravation de ces effets, sont à la base des processus physiopathologiques qui conduisent à la dysfonction puis à la mort neuronale. Ces résultats permettent aussi de prioriser des gènes et de générer des hypothèses fortes sur les cibles thérapeutiques les plus intéressantes à étudier d'un point de vue expérimental. En conclusion, mes recherches ont un impact à la fois fondamental et translationnel sur l'étude de la maladie de Huntington, permettant de dégager des méthodes d'analyse et des hypothèses qui pourraient avoir valeur thérapeutique pour les maladies neurodégénératives en général. / Huntington’s disease is a hereditary neurodegenerative disease that has become a model to understand physiopathological mechanisms associated to misfolded proteins that ocurs in brain diseases. Despite exciting findings that have uncover pathological mechanisms occurring in this disease and that might also be relevant to Alzheimer’s disease and Parkinson’s disease, we still do not know yet which are the mechanisms and molecular profiles that rule the dynamic of neurodegenerative processes in Huntington’s disease. Also, we do not understand clearly how the brain resist over such a long time to misfolded proteins, which suggest that the toxicity of these proteins is mild, and that the brain have exceptional compensation capacities. My work is based on the hypothesis that integration of ‘omics’ data from models that depicts various stages of the disease might be able to give us clues to answer these questions. Within this framework, the use of network biology and graph theory concepts seems particularly well suited to help us integrate heterogeneous data across models and species. So far, the outcome of my work suggest that early, pre-symptomatic alterations of signaling pathways and cellular maintenance processes, and persistency and worthening of these phenomenon are at the basis of physiopathological processes that lead to neuronal dysfunction and death. These results might allow to prioritize targets and formulate new hypotheses that are interesting to further study and test experimentally. To conclude, this work shall have a fundamental and translational impact to the field of Huntington’s disease, by pinpointing methods and hypotheses that could be valuable in a therapeutic perspective.

Mutant mRNA and protein both contribute to the clinical manifestation of many repeat-associated neurodegenerative and neuromuscular disorders. The presence of nuclear RNA clusters is a feature shared amongst these diseases, such as C9ORF72/ALS and myotonic dystrophy 1/2 (DM1/2); however, this pathological hallmark has not been conclusively demonstrated in Huntington’s disease (HD) in vivo. Investigations into HD – caused by a CAG repeat expansion in exon 1 of the huntingtin (HTT) gene – have largely focused on toxic protein gain-of-function as a disease-causing feature, with fewer studies investigating the role of mutant HTT mRNA in pathology or pathogenesis. Here we report that in two HD mouse models, YAC128 and BACHD-97Q-ΔN17, mutant HTT mRNA is preferentially retained in the nucleus in vivo. Furthermore, we observed the early, widespread formation of large mutant HTT mRNA clusters (approximately 0.6 to 5 µm3 in size) present in over 50-75% of striatal and cortical neurons. Affected cells were limited to one cluster at most. Endogenous wild-type mouse Htt or human HTT mRNA containing 31 or fewer repeats did not form clusters. Additionally, the aberrantly spliced N-terminal exon 1-intron 1 RNA fragment, HTT1a, also formed clusters that fully co-localized with the mutant HTT mRNA clusters. These results suggest that multiple repeat-containing transcripts can coalesce to form a single cluster in a given cell. Treating YAC128 mice with antisense oligonucleotides efficiently silenced individual HTT mRNA foci but had limited impact on clusters. Our findings identify mutant HTT mRNA clustering as an early, robust molecular signature of HD, further supporting HD as a repeat expansion disease with suspected mRNA involvement.

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

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

Huntingtons's disease (HD) is devastating neurodegenerative disorder manifesting by motor disturbances, cognitive decline and personal changes. The huge effort to find a cure for HD has brought several promising therapeutic treatments on the scene. Each of the prospective approaches needs to be investigated for safety, tolerability and efficacy. Mouse and rat models were a lot helpful in examination of pathological mechanisms of HD, but they are not sufficient for completion of pre-clinical testing. Therefore, we aimed to generate transgenic HD minipig to overcome the gap between rodents and humans. Minipig transgenic for the first 548 aminoacids of human mutant huntingtin gene (TgHD) under the control of human HD promotor was manipulated by lentiviral transduction of porcine one-cell stage embryos. Currently, six generations of minipigs expressing single copy of N-truncated human mutant huntingtin protein (mtHtt) with a repetition of 124 glutamines are at disposal. The more the model simulates the disease symptoms the better it is for translational research as the efficacy of the cure can be finer evaluated. Hence, the second aim was to demonstrate HD-like phenotype in our model. Testicular degeneration that preceded the clinical symptoms onset was observed as a consequence of expression of mtHtt....