<p>Huntington’s disease (HD) is a progressive, neurodegenerative disorder that leads to the selective loss of neurons in the striatum and the cerebral cortex. HD is caused by a CAG trinucleotide repeat expansion beyond the normal length in the <em>IT15 </em>(<em>Htt</em>) gene. The CAG stretch codes for an elongated polyglutamine tract within the amino‐terminus of the huntingtin protein. Polyglutamine tracts with lengths exceeding 37 repeats cause HD whereas repeat lengths below do not. This phenomenon has plagued the HD community since the discovery of the gene in 1993. In this thesis, we sought to elucidate the molecular mechanism by which huntingtin becomes toxic at polyglutamine lengths above 37. Using Förster resonance energy transfer (FRET) techniques, we describe an intramolecular proximity between the first 17 residues (N17) and the proline-rich regions, which flank the polyglutamine tract of huntingtin. We report that we can precisely measure differences between the conformations adopted by the huntingtin protein with polyglutamine tracts below and above the pathogenic repeat threshold of 37 repeats. Our data supports the hypothesis that polyglutamine tracts below the pathogenic threshold can act as a flexible hinge allowing the N17 domain to freely fold back upon huntingtin and come into close 3D proximity with the polyproline region. This flexibility is lost in polyglutamine tracts with >37 repeats resulting in a diminished spatial proximity between N17 and the polyproline domain.</p> / Doctor of Philosophy (PhD)

The purpose of this thesis was to develop methodology by which treatments for the cognitive impairments in Huntington's disease (HD) could be tested. As such, the thesis focused mainly on evaluating rats with quinolinic acid (QA) lesions of the striatum, as this manipulation mimics some aspects of the neural damage in Huntington's disease, to try to identify cognitive deficits of HD resulting from cell loss in the striatum. In the first part (Chapters 3-5), the role of the striatum in implicit memory was investigated. Chapter 3 compared the performance of rats and humans on a reaction time task that evaluated implicit memory by presenting visual stimuli with differing probabilities which change over time. Although rats made higher percentage of incorrect responses and late errors, both groups showed a similar pattern of reaction times. Chapter 4 investigated whether implicit memory (the computation of probabilities to predict the location of a stimulus) was affected by selective blockade of dopaminergic transmission at the D1 or D2 receptors by SCH-23390 and raclopride, respectively. Reaction times were slower with SCH-23390 and raclopride, but only SCH-23390 reduced errors to the least probable target location. Chapter 5 used the same task to evaluate implicit memory in rats with QA lesions of the dorsomedial striatum (DMS). Implicit memory was not affected by lesions of the DMS, which suggested that once a task that requires implicit memory has been learned, the DMS was not involved in sustaining the performance of the task. The second part of this thesis (Chapter 6), explored the contribution of the DMS in habit formation. DMS lesioned rats did not show habitual responding, and were not impaired in learning a new goal-directed behaviour. The third part (Chapters 7 and 8), investigated the role of the dorsal striatum in reversal learning, attentional set-formation, and set-shifting. Dorsal striatum lesioned rats were not impaired in reversal learning, but had a diminished shift-cost, which suggested that dorsal striatum lesions disrupted the formation of attentional sets. These results showed that although QA lesions of the dorsal striatum mimic some aspects of the neural damage in HD, they did not result in the same cognitive deficits observed in patients with HD, at least using the tasks presented in this thesis. However, other animal models of HD could be evaluated using the different tasks presented in this thesis to continue the search of a reliable animal model of HD in which treatments for the disease could be evaluated.

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.

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.

Stable porcine ES cell lines have not been succesfully established yet. Ability to selfrenew or to differentiate has been limited in different porcine ES-like cell lines so far. PiPSCs represent an alternative to pESCs. PiPSCs can be generated by reprogramming of somatic cells by introduction of several transcription factors on viral vectors and were established by several groups. However, the majority of piPS cell lines depend on transgene expression because of incomplete reprogramming and weak activation of endogenous pluripotency genes. Transgene expression can infuence differentiation potential of piPSCs. Therefore, we have used integrative and reexcisable PiggyBac transposons to generate viral free piPSCs. At the same time, small molecules (low-molecular inhibitors) with potential to increase reprogramming efficiency and to activate endogenous pluripotency genes were used in the reprogramming media. This strategy has a potential for generation of naive piPSCs. Successful excision of transgenes would generate transgene-free piPSCs with uncompromised differentiation potential. Pig (Sus Scrofa) is at the same time an important animal model in preclinical stage research of the diseases. Somatic cells used for generation of piPSCs were isolated from pigs carrying mutated huntingtin. Integration of the...

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

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.

The model of basal ganglia function proposed by Albin, Young and Penney (1989) describes two anatomically independent motor pathways, the direct and indirect. However, under normal conditions striatal dopamine (DA) is required for the expression of motor behavior, and DAergic control of the two pathways (via D1 and D2 receptors, respectively) is dependent on co-activation. We tested for a possible breakdown of D1/D2 synergism using transgenic R6/1 mice bearing the human huntingtin allele (Htt). Motor stereotypy, observed prior to the onset of HD-related symptoms, was rated on a 5-point scale following activation of: A) D1 receptors alone, B) D2 receptors alone, and C) stimulation of both D1 and D2 receptors. Results revealed that mice with the HD allele, like their WT litter mates, depend on the co-activation of the indirect and direct motor pathways to facilitate deliberate behavior.

Degeneration of the striatum and striatal-frontal circuits are generally thought to cause most of the neuropsychological symptoms experienced in Huntington‘s Disease (HD). Advances in cortical thickness mapping (an automated MRI method for precisely measuring the cortical thickness across the entire cortex) provide a new technique for examining changes in the brain in HD. Recent studies using this technology have reported provocative results. They found significant cortical thinning in participants with early HD (Rosas et al., 2002; Rosas et al., 2008) and even in presymptomatic HD (Rosas et al., 2005). Moreover, cortical thinning was most prominent in posterior regions of the brain, with relative preservation of the anterior frontal regions. The present study replicated Rosas et al.‘s (2005) study but used a larger sample of presymptomatic HD participants (n = 19) and a control group matched for age, gender and education (n = 19). Presymptomatic HD participants were divided into two groups, PreHDclose and PreHDfar, based on their estimated proximity to clinical onset. The distribution of cortical thinning was assessed using an identical MRI method to previous cortical thinning studies with HD participants. Specific neuropsychological tests were used to assess cognitive and mood changes that may be associated with cortical thinning. It was hypothesised that cortical thinning would be more evident in posterior than frontal cortical regions. It was also hypothesised that presymptomatic HD participants would perform more poorly than controls on tests that are subserved primarily by specific posterior cortical regions, but not on tests that are subserved by anterior cortical regions. Lastly, it was predicted that poorer performance in the neuropsychological measures would be associated with greater thinning in cortical regions that are important during performance of these tasks. Consistent with predictions, the presymptomatic HD group showed regionally-specific cortical thinning which was most prominent in the posterior cortices, particularly around the right parieto-temporal-occipital (PTO) junction. Thinning occurred in people up to 15 years before clinical onset, with little to no thinning before that. The presymptomatic HD group, and particularly the PreHDclose participants, performed significantly worse than controls in 2 of the 6 cognitive tests that are subserved primarily by posterior cortical regions (the Judgment of Line iii Orientation test and modified Roadmap Test), but not in tests that are subserved primarily by frontal cortical regions. Correlational analyses showed a number of regionally-specific relationships between thinning and cognitive performance, although the distribution of these relationships did not generally support our region-of-interest predictions. The results contribute to a better characterisation of the cortical and neuropsychological changes that occur early in the development of HD, and provide tentative support for cortical thickness mapping as a valid and sensitive measure for assessing cortical changes in presymptomatic HD.

Huntington’s disease (HD) is a neurodegenerative disorder caused by the inheritance of one mutant copy of the huntingtin gene. Mutant huntingtin protein (mHtt) contains an expanded polyglutamine repeat region near the N-terminus. Cleavage of mHtt releases an N-terminal fragment (N-mHtt) which translocates, and accumulates in the nucleus. Nuclear accumulation of N-mHtt has been directly associated with cellular toxicity. Decreased transcription is among the earliest detected changes that occur in the brains of HD patients and is consistently observed in all animal and cellular models of HD. Transcriptional dysregulation may trigger many of the perturbations that occur later in disease progression and an understanding of the effects of mHtt may lead to strategies to slow the progression of the disease. Current models of N-mHtt-mediated transcriptional dysregulation suggest that abnormal interactions between N-mHtt and transcription factors impair the ability of these transcription factors to associate at N-mHtt-affected promoters and properly regulate gene expression. We tested various aspects of these models using two N-mHtt-affected promoters in in vitro transcription assays and in two cell models of HD using techniques including overexpression of known N-mHtt-interacting transcription factors, chromatin immunoprecipitation, promoter deletion and mutation analyses and in vitro promoter binding assays. Based on our results and those in the literature, we proposed a new model of N-mHtt-mediated transcriptional dysregulation centered on the presence of N-mHtt at affected promoters. We concluded that simultaneous interaction of N-mHtt with multiple binding partners within the transcriptional machinery would explain the gene-specificity of N-mHtt-mediated transcriptional dysregulation, as well as the observation that some genes are affected early in disease progression while others are affected later. Our model explains why alleviating N-mHtt-mediated transcriptional dysregulation through overexpression of N-mHtt-interacting proteins has proven to be difficult and suggests that the most realistic strategy for restoring gene expression across the spectrum of N-mHtt affected genes is by reducing the amount of soluble nuclear N-mHtt.