The expansion of gene-specific trinucleotide repeats is responsible for a growing list of human disorders, including myotonic dystrophy type 1 (DM1). Repeat instability for most of these disorders, including DM1, is characterized by complex patterns of inherited and ongoing tissue-specific instability and pathogenesis. While the mechanistic basis behind the unique locus-specific instability of trinucleotide repeats is currently unknown, DNA metabolic processes are likely to play a role. My thesis involves investigating the contribution of DNA replication to the trinucleotide instability of myotonic dystrophy type 1. Herein I have designed an in vivo primate model system, based on the SV40 replication system, to assess the contribution of DNA replication to DM1 repeat instability. This system allows the assessment, under controlled conditions, and manipulation of variables that may affect replication-associated repeat instability, under a primate cellular system. Using the SV40 model system, I not only confirmed previous observations that repeat length and replication direction affect repeat instability, but also for the first time determined that the location of the replication origin relative to the repeat tract plays an important role in repeat instability. This novel observation allowed for the development of a fork-shift model of repeat instability, in which cis-elements adjacent to the repeat tract affect replication, in turn altering the propensity for repeat instability. To further my study of DNA replication in DM1 repeat instability, I have mapped the origin of replication adjacent to the DM1 locus in human patient cells and the tissues of DM1 transgenic mice actively undergoing repeat instability. The position of the replication origins adjacent to the repeat tract at the DM1 locus places several known cis-elements, including CTCF binding sites, in a position to alter replication as predicted by the fork-shift model. My analysis of the CTCF sites showed them capable of altering replication and repeat instability at the DM1 locus. Taken together these results suggest that the placement of replication origins, repeat tracts and cis-elements, may mark trinucleotide repeat tracts, such as the DM1, for locus-, tissue- and development-specific replication-associated repeat instability.

No description available.

Neurodegenerative disorders (NDD) are an ever-increasing burden on healthcare; consequently, elucidating the mechanisms underlying neurodegeneration (ND) is critical for the development of effective treatments for these diseases. In order to unravel the molecular pathways underlying movement disorders and identify new genes involved in ND, two ataxic mouse mutants characterised by cell death in the cerebellum were studied in detail using a combination of in vitro and in vivo techniques. The robotic mouse demonstrated the key role of a transcription factor, Af4, in Purkinje cell (PC) survival and how only small changes in the levels of a single transcriptional cofactor could deleteriously affect normal cerebellum function. Expression array studies of the robotic PCs revealed the first confirmed targets of Af4-mediated transcription, including insulin-like growth factor 1 (Igf-1). It was demonstrated that Igf-1 is critical for PC survival, highlighting the role of the IGF-1 signalling pathway as a potential therapeutic target for the treatment of cerebellar ataxia in humans. Detailed analysis of the bella mutant demonstrated that ataxia and apoptotic cerebellar degeneration is caused by loss of the oxidative resistance 1 (Oxr1) gene. In vitro modelling experiments went on to show that the levels of this previously uncharacterised gene are critical for controlling the sensitivity of neuronal cells to oxidative stress (OS). Moreover, this study showed that Oxr1 was up-regulated both in human and pre-symptomatic mouse models of amyotrophic lateral sclerosis (ALS), demonstrating that Oxr1 was an early marker of ROS defence, prior to pathology, and potentially a novel neuroprotective factor in NDD. Preliminary interaction studies show that Oxr1 is likely to be a multi-functional protein that forms complexes with proteins known to be mutated in NDD. Thus, the study of both the robotic and the bella mouse has demonstrated the value of the phenotype-driven approach to investigate novel neurodegenerative pathways.

No description available.

Emerging evidence suggests that opioid drugs can exacerbate neuroAIDS. Microglia are the principal neuroimmune effectors thought to be responsible for neuron damage in HIV-infected individuals, and evidence suggests that drugs acting via opioid receptors in microglia aggravate the neuropathophysiological effects of HIV. The P2X family of ATP activated ligand-gated ion channels regulates key aspects of microglial function. In addition, opioid-dependent microglial activation has been reported to be mediated through P2X4 signaling, prompting us to investigate P2X receptors contribution to the neurotoxic effects of HIV and morphine. In vitro experiments showed treatment with TNP-ATP prevented the neurotoxic effects of morphine and/or HIV Tat, or ATP alone in a concentration dependent manner. This evidence suggests P2X receptors mediate the neurotoxic effects of these insults in striatal neurons. P2X1, P2X3, and P2X7 selective receptor antagonists did not prevent Tat- and/or morphine-induced neurotoxicity, implying cellular pathways activated may not involve these subtypes. Cells from P2X4KO mice show that activation of the P2X4 receptor on glia are necessary to cause Tat and/or morphine toxicity. However, data implied that baseline neuronal function may be altered due to lack of P2X4 receptor expression, and also gave evidence for altered Tat and morphine cellular signaling when the two are given in combination versus alone. Surgeries were performed on P2X4 KO and WT mice, which received intrastriatal Tat injections and morphine and/or naltrexone pellets. WT mice showed significant increases in inflammatory markers when treated with Tat and/or morphine. Increases in inflammatory markers were not seen in P2X4 KO mice, implying P2X4 receptors play a role in neuroinflammation resulting from Tat and/or morphine. Finally, human tissue samples from the National NeuroAIDS Tissue Consortium were analyzed. Changes in P2X5 and P2X7 mRNA were found in microarray data, but only changes in P2X7 mRNA levels were confirmed by RT-PCR. No changes in P2X4 mRNA levels were detected. Our experiments indicate the P2X receptor family contributes to Tat- and morphine- related neuronal injury, and reveal that members of the P2X receptor family, especially P2X4, may be novel therapeutic targets for restricting the synaptodendritic injury and neurodegeneration that accompany neuroAIDS and opiate abuse.

L’incidence constante des maladies liées à l’âge reflète un réel enjeu dans nos sociétés actuelles, principalement lorsqu’il est question des cas de cancers, d’accidents cérébraux et de maladies neurodégénératives. Ces désordres sont liés à l’augmentation de l’espérance de vie et à un vieillissement de la population. Les coûts, estimés en milliards de dollars, représentent des sommes de plus en plus importantes. Bien que les efforts déployés soient importants, aucun traitement n’a encore été trouvé. Les maladies neurodégénératives, telles que la maladie d’Alzheimer, de Parkinson, d’Huntington ou la sclérose latérale amyotrophique (SLA), caractérisées par la dégénérescence d’un type neuronal spécifique à chaque pathologie, représentent un défi important. Les mécanismes de déclenchement de la pathologie sont encore nébuleux, de plus il est maintenant clair que certains de ces désordres impliquent de nombreux gènes impliqués dans diverses voies de signalisation induisant le dysfonctionnement de processus biologiques importants, tel que le métabolisme. Dans nos sociétés occidentales, une problématique, directement lié à notre style de vie s’ajoute. L’augmentation des quantités de sucre et de gras dans nos diètes a amené à un accroissement des cas de diabètes de type II, d’obésité et de maladies coronariennes. Néanmoins, le métabolisme du glucose, principale source énergétique du cerveau, est primordial à la survie de n’importe quel organisme. Lors de ces travaux, deux études effectuées à l’aide de l’organisme Caenorhabditis elegans ont porté sur un rôle protecteur du glucose dans un contexte de vieillissement pathologique et dans des conditions de stress cellulaire. Le vieillissement semble accéléré dans un environnement enrichi en glucose. Cependant, les sujets traités ont démontré une résistance importante à différents stress et aussi à la présence de protéines toxiques impliquées dans la SLA et la maladie de Huntington. Dans un deuxième temps, nous avons démontré que ces effets peuvent aussi être transmis à la génération suivante. Un environnement enrichi en glucose a pour bénéfice de permettre une meilleure résistance de la progéniture, sans pour autant transmettre les effets néfastes dû au vieillissement accéléré. / The constant increase of the cases of age-related diseases, including cancers, cerebral accidents and neurodegenerative diseases raises a real problem in our current societies. These disorders are very strongly linked to the increase of life expectancy and to the ageing population. The costs, estimated in billion dollars, requiring vast medical resources and very few treatments exist today. Neuronal diseases, such as the Alzheimer's, Parkinson’s, Huntington’s disease and amyotrophic lateral sclerosis (ALS) are characterized by the degeneration of various types of neurons. This represents an important challenge because besides the lack of understanding the underlying mechanisms related to their pathology, it is now clear that some of these disorders involve several genes and lead to the dysfunction of fundmental biological processes such as metabolism. In western societies lifestyle and dietary practices may contribute to disease. The increased quantities of sugar and fat in western diets are thought to contribute to the rise of metabolic disorders, including Type II diabetes, obesity and coronary diseases. Nevertheless, it is important to understand that the metabolism of glucose, the brain’s main energy source, is essential for survival. In this thesis, two studies using the model organism Caenorhabditis elegans investigated a potential protective role of the glucose in a context of pathological ageing and in conditions of cellular stress. Although ageing seems accelerated in a glucose enriched environment, the test subjects demonstrated an improved resistance to numerous stresses including against toxic proteins involved in the ALS and Huntington's disease. Secondly, it appeared that these effects can be heritably transmitted to successive generations of animals. Thus, a glucose enriched environment allows for increased stress resistance in the offspring, without transmitting the negative effects of accelerated ageing.

The eukaryotic stress response involves translational suppression of non-housekeeping proteins, and the sequestration of unnecessary mRNA transcripts into stress granules (SGs). This process is dependent on mRNA binding proteins (RBPs), such as T- cell intracellular antigen (TIA-1). RBPs interact with unnecessary mRNA transcripts through prion and poly-glutamine like domains, and their aggregation mirrors proteins linked to neurodegenerative diseases. Recent advances in molecular genetics emphasize the importance of SG biology in disease by associating multiple RBPs linked to SGs with neurodegenerative disease. The major difference between SG proteins and aggregation prone proteins in neurodegeneration is that aggregation of SGs is transient and rapidly reverses when the stress is removed. In contrast, aggregates associated with disease are stable and accumulate over time. This study identifies overabundant SGs as a novel pathology in Alzheimer's disease and related tauopathies. The data suggest that TIA-1 is intimately linked to tau pathogenesis, acting as a modifier of tau aggregation and associated toxicity. TIA-1 is present in a protein complex with tau protein including hyper-phosphorylated and misfolded tau. The expression of WT or P301L mutant tau increases the formation and size of TIA-1 positive SGs, and the localization and dynamics of these SGs are altered. Conversely, the expression of TIA-1 increases the formation and stabilization of phospho- and misfolded tau inclusions, as well as visible alterations in microtubule morphology, perhaps reflecting a loss of tau function. The data further show that co-expression of TIA-1 and tau leads to dendrite shortening, increases in caspase cleavage, and apoptosis in primary neurons, suggesting that an interaction between TIA-1 and tau results in neurotoxicity. This toxicity is SG-dependent and is rescued by microtubule stabilizing drugs. The results of this thesis research suggest that the aggregation of tau may proceed through the SG pathway, with SG formation accelerating the pathophysiology of tau aggregation. These studies propose that these tau aggregates serve as a nidus for further accelerated aggregation of SGs, leading to formation of long-lived pathological SG.

A doença de Alzheimer (DA) é caracterizada por déficit cognitivo, associada com prejuízos no metabolismo energético e na via de sinalização da insulina encefálicos. A injeção intracerebroventricular de baixas doses de estreptozotocina (STZ) tem sido utilizada como um modelo experimental da DA em ratos. Nesse sentido, tem sido demonstrada a participação do sistema canabinoide em processos neurodegenerativos e seus efeitos neuroprotetores e anti-inflamatórios. O objetivo deste trabalho foi caracterizar as alterações comportamentais e moleculares em modelos experimentais (in vivo e in vitro) expostos à STZ e avaliar a participação do sistema canabinoide. A STZ produziu prejuízo cognitivo, morte celular por apoptose, deficiência na resposta à insulina e alterações na via IR/PI3K, semelhantes às encontradas na DA. O agonista canabinoide ACEA foi capaz de reverter o prejuízo cognitivo, modificar as alterações proteicas da via IR/PI3K, e regular positivamente a via anti-apoptótica, gerando uma neuroproteção. / Alzheimer\'s disease (AD) is characterized by cognitive deficit associated with energy metabolism impairment and changes in insulin signaling. In this context, low doses of intracerebroventricular streptozotocin (STZ) injection has been used as an experimental model of AD in rats. Several studies have demonstrated the participation of the cannabinoid system in neurodegenerative processes and its neuroprotective and anti-inflammatory properties. Thus, the aim of this work was to characterize the molecular and behavior alterations in experimental models (in vitro and in vivo) produced by STZ exposure and evaluate the cannabinoid system participation in these models. STZ was able to induce cognitive impairment, apoptosis cell death, impaired insulin response and alterations in the IR/PI3K signaling pathway, similar to those found in AD. CB1 agonist, ACEA reversed cognitive impairment and modified some protein changes in IR/PI3K pathway caused by STZ, and positively regulate the anti-apoptotic pathway, generating neuroprotection.

A doença de Alzheimer (DA) é a principal causa de demência na população idosa mundial. O tratamento de primeira escolha em pacientes com DA de grau leve a moderado é com anticolinesterásicos, que promovem modesta melhora cognitiva. Trabalhos mostram que o lítio também possui efeito benéfico para o tratamento da DA e que é ainda mais eficaz se utilizado como prevenção dos sintomas de demência, mesmo em doses mais baixas que as terapêuticas. O objetivo desse trabalho foi avaliar o efeito de uma microdose de lítio em associação com um anticolinesterásico, para a memória e neuroproteção em modelos in vitro e in vivo de neurodegeneração induzida com o peptídeo <font face = \"symbol\">bA1-40. Em culturas organotípicas de hipocampo de camundongos c57Bl/6 de 12 meses de idade, foi observado aumento significativo da morte neuronal após incubação de <font face = \"symbol\">bA (10 <font face = \"symbol\">mg/mL). O tratamento com 10 <font face = \"symbol\">mM de lítio protegeu as fatias de hipocampo da morte celular, independente do tratamento com rivastigmina. O tratamento das fatias com lítio não alterou a atividade da acetilcolinesterase, diferente do observado com rivastigmina, porém, houve aumento não significativo da densidade de BDNF, em relação às fatias não tratadas. Para os estudos in vivo, camundongos C57Bl/6 machos de 10 meses de idade foram submetidos a três sessões de aquisição de memória em esquiva ativa (EA) e, em seguida, à cirurgia estereotáxica para implante de minibomba osmótica contendo veículo ou <font face = \"symbol\">bA (0,46 nmol). No dia seguinte, iniciou-se o tratamento com rivastigmina (Riv; 0,3 mg/kg/dia). Formaram-se 5 grupos: veículo, <font face = \"symbol\">bA, Li, Riv e Li+Riv. Os grupos tratados com lítio receberam o fármaco (0,3 mg/kg/dia) desde os 2 meses de idade e o tratamento foi mantido até o final dos experimentos. Todos os grupos tratados com Li e/ou Riv foram infundidos com <font face = \"symbol\">bA. O grupo veículo foi tratado com água. Os animais passaram por dois testes em EA, aos 7 e 35 dias após a cirurgia; a partir do 28º dia, foram realizadas avaliações de atividade motora e de memória de curta duração, utilizando-se caixa de atividade motora e teste de reconhecimento de objeto novo, respectivamente. Observou-se que o grupo <font face = \"symbol\">bA apresentou perda de memória em EA e no teste de reconhecimento de objeto novo, quando comparado ao grupo veículo. Nenhum dos tratamentos, no entanto, foi eficaz em prevenir esse prejuízo ou promover melhora no desempenho dos animais nos testes de memória. Não houve formação de placas amiloides em nenhum dos grupos, avaliada por meio da coloração com tioflavina S, sugerindo que o prejuízo cognitivo observado foi devido à forma solúvel do peptídeo. O peptídeo <font face = \"symbol\">bA induziu o processo autofágico no grupo <font face = \"symbol\">bA, verificado pelo aumento da formação de autofagossomos (avaliados por LC3-II) e níveis inalterados de p62 em relação ao veículo. Os tratamentos promoveram uma diminuição não significativa de autofagia, em relação ao grupo <font face = \"symbol\">bA. Dados os resultados in vivo e in vitro, concluiu-se que ambos os fármacos, em monoterapia, promovem efeitos benéficos, porém, quando associados, os efeitos são perdidos. / Alzheimer\'s disease (AD) is the leading cause of dementia in the elderly. The first- choice treatment for patients with mild to moderate AD is a cholinesterase inhibitor, which leads to a modest cognitive improvement. Previous studies have shown that lithium also has a beneficial effect for the treatment of AD, but more effective if used to prevent the symptoms of dementia, even at lower doses than the therapeutic ones. The objective of this work was to evaluate the effect of a lithium microdose in association with a cholinesterase inhibitor on memory and neuroprotection in both in vivo and in vitro models of neurodegeneration induced by <font face = \"symbol\">bA1-40 peptide. By using organotypic culture of hippocampus of 12-month-old c57Bl/6 mice, it was verified a significant increase in neuronal death after incubation with <font face = \"symbol\">bA (10 <font face = \"symbol\">mg/ml). Treatment with 10 <font face = \"symbol\">bM lithium protected the hippocampal slices from cell death, independently of rivastigmine. Treatment with lithium in slices did not alter acetylcholinesterase activity, different from that observed with rivastigmine, however, there was a non-significant increase in BDNF density, in relation to untreated slices. For the in vivo evaluation, 10- month-old male C57Bl/6 mice underwent three sessions of active avoidance memory acquisition (AA) and then, stereotactic surgery for the implant of an osmotic minipump containing vehicle or <font face = \"symbol\">bA (0.46 nmol). At the next day, the treatment with rivastigmine (Riv, 0.3 mg/kg/day) was started. Five groups were formed: vehicle, <font face = \"symbol\">bA, Li, Riv and Li+iv. The lithium-treated groups received the drug (0.3 mg/kg/day) from 2 months of age and the treatment was kept until the end of the experiments. All drug-treated groups were infused with <font face = \"symbol\">bA. Vehicle group was treated with water. The animals underwent two tests in AA: at 7 and 35 days after surgery; from the 28th day, motor activity and short-term memory evaluations were performed, using a motor activity cage and the novel object recognition test, respectively. <font face = \"symbol\">bA group presented memory loss in AA and in the novel object recognition test when compared to vehicle group. None of the treatments, however, was effective in preventing this injury or promoting improvement in the performance of the animals in memory tests. There was no amyloid plaque formation in any of the groups, which was evaluated by thioflavin S staining, suggesting that the observed cognitive impairment was due to the soluble forms of the peptide. <font face = \"symbol\">bA peptide induced autophagic process in <font face = \"symbol\">bA group, as evidenced by an increase in formation of autophagosomes (assessed by LC3-II) and unaltered levels of p62 when compared to vehicle group. The treatment with Li and Riv promoted a non-significant decrease of autophagy, in relation to <font face = \"symbol\">bA group. Together, the results suggest that the drugs promote beneficial effects when used in monotherapy, however, when associated, the effects are no longer observed.

O sistema colinérgico está sabidamente envolvido com processos cognitivos. Em trabalho recente mostramos que a infusão do peptídeo A promoveu neurodegeneração e redução da memória de ratos. O treino semanal dos animais em equipamento de esquiva ativa recuperou o desempenho na tarefa e aumentou a densidade de receptores nicotínicos &alpha;7 em áreas relacionadas à memória. No presente trabalho, o antagonismo de &alpha;7 com metilicaconitina (MLA), em camundongos, promoveu perda cognitiva, porém a recuperação com o treino foi parcial. A infusão conjunta de &beta;A e MLA causou perda da memória, mas essa não foi revertida com o treino semanal. Os animais com MLA apresentaram aumento da atividade da acetilcolinesterase (AChE) e aumento de BDNF, que poderia ser relacionado à resiliência a injúrias. Porém, animais com A e MLA apresentaram aumento da atividade da AChE e redução de BDNF, sugerindo perda dos mecanismos de neuroproteção deflagrados por &alpha;7. Com isso, sugere-se que &alpha;7 tenha um papel determinante na recuperação da memória e resiliência tecidual, frente à neurodegeneração. / Cholinergic system plays an important role in cognitive processes. In a recent work we showed that infusion of A&beta; promoted neurodegeneration and reduction of memory of rats. Week training of animals in active avoidance shuttle box recovered their performance and increased the density of &alpha;7 nicotinic receptors in brain areas related to memory. In the present work, infusion of the &alpha;7 antagonist methyllycaconitine (MLA), in mice, caused cognitive impairment, but memory recover with week training was partial. Infusion of &beta;A together with MLA promoted memory loss, but this was not recovered with the week training. MLA infused mice presented increase in acetylcholinesterase (AChE) activity and increase in BDNF, which could be related to resilience to tissue injuries. However, animals infused with &beta;A and MLA showed increase in AChE activity and reduction of BDNF, suggesting loss of neuroprotection mechanisms triggered by &alpha;7. It is suggested that &alpha;7 has a determinant role in memory recover and brain resilience, in neurodegenerative processes.