Made available in DSpace on 2014-06-11T19:23:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-12Bitstream added on 2014-06-13T20:29:49Z : No. of bitstreams: 1 curtis_kmc_me_araiq.pdf: 767278 bytes, checksum: b9eefeb12752b6e4c943bbfc29b1a46c (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A síndrome do cromossomo X frágil (SXF) é a forma mais comum de deficiência mental herdada. A doença ocorre pela expansão das repetições de trinucleotídeos na região 5’ não traduzida do gene FMR1 no cromossomo X. Dependendo do número de repetições CGG originam-se 4 tipos de alelos: normal (NL), pré-mutado (PM), gray zone (GZ) e mutação completa (FM). A instabilidade e expansão das repetições, aliado à metilação do DNA, causam a diminuição ou ausência na produção da proteína FMRP, a qual é essencial para a função cerebral. O diagnóstico da SXF tem sido realizado principalmente por análise molecular Southern blot. Porém, este método é trabalhoso, demorado e de custo elevado. Recentemente foi desenvolvido um novo método molecular para triagem da SXF por PCR, que segundo os autores, é rápido, de baixo custo, e eficiente na detecção das repetições CGG em homens e mulheres. No entanto, notou-se a ausência de informações importantes para reprodução do método. Os objetivos deste estudo foram: (i) padronizar a técnica de PCR proposta por Tassone et al., (2008), adaptando-a, devido a carência de informações metodológicas; (ii) comprovar a exatidão (acurácia), sensibilidade e especificidade do método, comparando-a ao Southern blot; (iii) avaliar a aplicação da técnica utilizando DNA extraído de diferentes materiais biológicos/métodos de extração; (iv) estimar o custo e o tempo de execução do método no mercado nacional. Os materiais biológicos utilizados foram: sangue coletado por sistema à vácuo e células da mucosa oral, que foram extraídos por solventes orgânicos e sangue coletado em cartões FTA, purificado pelo kit Whatman. Obtevese sucesso na reprodução do método da PCR em 75 indivíduos utilizando a enzima Expand Long Template PCR System (Roche Diagnostics). A exatidão (acurácia), sensibilidade e especificidade foram... / Fragile X Syndrome (FXS) is the most common form of inherited mental retardation. The disease occurs by the expansion of triplet nucleotide repeats in the 5' untranslated of the FMR1 gene on chromosome X. Depending on the number of CGG repeats four types of alleles originate from it: normal (NL), pre-mutated (PM), gray zone (GZ) and full mutation (FM). The instability and expansion of these repetitions, together with the methylation of DNA, cause a decrease or absence in the production of the protein FMRP, which is essential for the brain function. The diagnosis of FXS has been done mainly by molecular analysis Southern blot. However, this method is laborious, time consuming and expensive. Recently we have developed a new molecular method for FXS screening by PCR, which according to the authors, is rapid, inexpensive, and efficient in the detection of CGG repeats in male and female. However, we noted the absence of important information for breeding method. The objectives of this study were: (i) to standardize the PCR technique proposed by Tassone et al. (2008), adapting it, due to the lack of methodological information, (ii) verify the accuracy, sensitivity and specificity of the method, comparing it to the Southern blot, and (iii) to evaluate the technique using DNA extracted from different biological materials / extraction methods, and (iv) estimate the cost and time of the method execution in the domestic market. The biological materials used were: blood collected by vacuum system and oral mucosal cells, which were extracted by organic solvents and blood collected on FTA cards, purified by Whatman kit. Success was achieved in the reproduction of the PCR method in 75 individuals using the enzyme Expand Long Template PCR System (Roche Diagnostics). The accuracy, sensitivity and specificity were 100% when analyzing the total sample, indicating that the technique can detect the presence... (Complete abstract click electronic access below)

Le développement d'outils de phénotypage comportemental sophistiqués est indispensable pour comprendre le fonctionnement cognitif. A partir d'une analyse élaborée de tests comportementaux classiques, mes résultats suggèrent que l'hypersensibilité sensorielle associée à un canal potassique spécifique (BkCa) participe aux divers troubles comportementaux du syndrome de l'X-Fragile et du spectre autistique. Grâce à un dispositif expérimental nouveau et original, comprenant des capteurs de pression hyper-sensibles à même de détecter les moindres mouvement d'un rat ou d'une souris avec une sensibilité et une précision temporelle exceptionnelles, j'ai pu identifier des composantes comportementales normales et pathologiques inédites, telles que des tremblements ou la dynamique des forces mises en jeu dans divers mouvements, qui modifieront certainement nos capacités d'investigation des mécanismes impliqués dans la douleur, la peur ou la locomotion, dans les conditions normales et pathologiques. / Modern neuroscience highlights the need for designing sophisticated behavioral readout of internal cognitive states. From a thorough analysis of classical behavioural tests, my results support the hypothesis that sensory hypersensitivity might be the cause of other behavioural deficits, and confirm the potassium channel BKCa as a potentially relevant molecular target for the development of drug edication against Fragile X Syndrome / Autism Spectrum Disorders. I have also used an innovative device, based on pressure sensors that can non-invasively detect the slightest animal movement with unprecedented sensitivity and time resolution, during spontaneous behaviour. Analysing this signal with sophisticated computational tools, I could demonstrate the outstanding potential of this methodology for behavioural phenotyping in general, and more specifically for the investigation of pain, fear or locomotion in normal mice and models of neurodevelopmental and neurodegenerative disorders.

Mise en contexte : Le syndrome du X fragile (SXF) résulte de la perte d’expression de la protéine FMRP. L’absence de FMRP est responsable d’une série de perturbations signalétiques, notamment une hyperactivation de la voie MAPK/ERK. La lovastatine, un médicament hypocholestérolémiant, possède comme effet pléiotrope la capacité d’inhiber la voie MAPK/ERK et a permis de corriger certains phénotypes pathologiques clés du modèle murin du SXF, mettant en lumière son potentiel thérapeutique chez l’humain. Ainsi, nous avons réalisé en 2013 une étude ouverte visant à étudier l’effet d’un traitement de 12 semaines à la lovastatine sur les troubles cognitifs et comportementaux des enfants et des adultes avec SXF. La plupart des individus ont présenté des améliorations cognitives et comportementales, telles qu’évaluées par les échelles cliniques Vineland-II Adaptive Behavior Scale (VABS-II) et Aberrant Behavior Checklist-Community (ABC-C), respectivement. Ces échelles remplies par les tuteurs et les soignants sont toutefois évaluateur-dépendantes et sujettes à l’effet expérimentateur. Ces variables parasites, qui s’ajoutent à l’effet placebo inhérent à la conception ouverte de l’essai thérapeutique, peuvent ainsi avoir faussé l’évaluation de la réponse au traitement. Nous avons donc étudié si les cascades signalétiques des plaquettes sanguines peuvent être utilisées comme biomarqueurs objectifs pour surveiller la réponse au traitement. Méthode : Des échantillons sanguins des 15 individus SXF ayant participé à l’essai clinique ont été recueillis au début et à la fin de l’étude afin d’évaluer par Western Blot l’effet in vivo de la lovastatine sur l’activité de ERK dans les plaquettes sanguines, et ainsi de pouvoir corréler les réponses biologiques et cliniques. L’état de phosphorylation de ERK a également été étudié dans les plaquettes d’une cohorte contrôle. Résultats : Nos résultats démontrent une augmentation significative de près du double de la phosphorylation basale de ERK dans les plaquettes sanguines des individus avec SXF en comparaison avec les sujets contrôles (p=0,002). De plus, nous avons observé une normalisation de la phosphorylation de ERK chez 13 des 15 individus SXF après le traitement de 12 semaines à la lovastatine (p=0,007). Notre étude fournit ainsi les premières évidences d’un effet bénéfique de la lovastatine dans le SXF chez l’humain. Nous avons également démontré que les changements de la phosphorylation de ERK étaient partiellement corrélés à la réponse clinique, et ce, pour le score total et les scores des sous-domaines ‘socialisation’ et ‘compétences de la vie quotidienne’ de l’échelle VABS-II (p=0,003). Conclusion : De façon générale, ces résultats suggèrent que les cascades signalétiques plaquettaires peuvent être utilisées comme biomarqueurs pour évaluer de façon objective la réponse au traitement lors de futurs essais thérapeutiques. / Abstract: Background: Fragile X syndrome (FXS) results from loss of FMRP expression, which causes several signaling dysregulations, including the hyperactivation of the Mitogen-activated protein kinase (MAPK)/Extracellular signal-regulated kinase (ERK) pathway. Lovastatin, a drug used for the treatment of hypercholesterolemia, pleiotropically inhibits the MAPK/ERK cascade and has successfully corrected key pathological phenotypes in the FXS mouse model, underscoring its ‘disease-modifying’ potential. Thereby, we conducted in 2013 the first open-label clinical trial investigating the effect of a 12-week lovastatin regimen on cognitive and behavioral disabilities in FXS. Most individuals presented subtle positive cognitive changes as assessed by the Vineland-II Adaptive Behavior Scale (VABS-II) as well as behavior improvements using the widely used scale Aberrant Behavior Checklist-Community (ABC-C). The latter two scales are filled up by caregivers making them rater-dependent and prone to observer-expectancy effect. This might result in a placebo effect which is inherent to the open-label design of the trial. We therefore investigated whether blood platelets’ signaling cascades may be used as objective biomarkers to monitor treatment response. Methods: Blood samples were gathered from 15 FXS individuals during the trial in order to evaluate by quantitative Western Blotting the in vivo effect of lovastatin on ERK activity in blood platelets, and to correlate clinical and biological responses. The basal phosphorylation status of ERK was also assessed in platelets from a control cohort. Results: Our results showed a more than two-fold significant increase in FXS blood platelet basal ERK phosphorylation as compared to controls (p=0.002). Of note, we found that this hyperphosphorylation was normalized following the 12-week lovastatin trial (p=0.007) in 13 of the 15 FXS individuals enrolled in the trial. This represents the first evidence for a beneficial effect of lovastatin in human FXS. The extent of changes in ERK phosphorylation was also found to partly correlate with the clinical response scales’ scores, especially for the VABS-II. Indeed, the composite total score and the ‘daily living skills’ as well as the ‘socialization’ subscales scores of the VABS-II were correlated with the biological response (p=0.03). In comparison, no correlation was observed with the ABC-C scale. Conclusion: Broadly, these results suggest that platelets’ signaling cascades could be used as biomarkers to objectively assess treatment response during future clinical trials.

This dissertation describes separate but related studies that explore visual spatial learning and memory in Fragile X Syndrome. Across all studies, either the performance of individuals affected by FXS and/or fmr1 KO mice was compared to comparison controls on seven H-W mazes of increasing difficulty levels. Study one employed the traditional configuration of the H-W mazes to evaluate performance variables that include latency to complete the maze and number of the errors. The results of study 1 revealed significant differences in performance for both FXS groups as compared to mental age-matched comparison individuals and wild type mice, respectively. In contrast to the FXS group, performance of the comparison group improved as indicated by significantly fewer errors across trials. A similar pattern of results was observed when latency across trials was analyzed. Taken together, the results of study one support the hypothesis that a selective deficit in spatial learning and memory characteristic of the FXS phenotype can be observed in the murine model of FXS, if equivalent tasks are employed in testing humans and mice. Study two expanded on these findings by adding landmarks to the maze environment to evaluate how these may impact spatial learning and memory in fmr1 KO mice. Contrary to our hypotheses, landmarks significantly impaired wild type control performance. In addition, results revealed that the performance of the fmr1 KO mice generally did not differ between landmark and non-landmark tasks, indicating that the presence of landmarks neither enhanced nor hindered mouse performance. Lastly, study three entailed a more in-depth behavior analysis of maze navigation performance for FXS individuals from study 1. Consistent with the hypotheses and findings from study 1, results revealed significant differences in performance variables between individuals, with FXS participants generally performing worse than the comparison group participants. Taken together, the results of study 3 generally supported the hypothesis that there was greater impairment in performance for individuals affected by FXS as compared to controls. This impairment was evident in the pattern of pathways taken to solve H-W mazes, consistent with the notion that affected individuals employed different behavioral strategies.

Healthy brain development requires coordinated synapse growth and synapse elimination, with disruptions to these processes often resulting in neurodevelopmental disorder. While glia, the non-neuronal cells of the brain, are increasingly recognized as important regulators of both of these processes, the extent of this regulation and, in the case of disorder, dysregulation is still unknown. In this dissertation, I made classic use of the mouse visual system to outline the contours of glial regulation of synapse development in both synapse growth and synapse elimination. First, I examined astrocytes and microglia in the context of normal brain development, characterizing their spatiotemporal expression patterns in and around the mouse optic tract throughout late embryonic and early postnatal development, as RGC axons are growing into their synaptic target, the dLGN (Chapter 2). Next, I examined astrocyte and microglia in the context of disorder. Here, I found that synapses are reduced in size and eye-specific RGC synapse segregation is enhanced in a mouse model of Fragile X Syndrome, the most common single-gene cause of autism and intellectual disability, (Fmr1 KO mouse) during brain development. I identified glial phagocytic genes as disrupted within the developing Fmr1 KO dLGN and demonstrated that both microglial and astrocytic engulfment of synapses were aberrantly increased during this period of enhanced segregation, providing evidence that over-active glial engulfment may drive aberrant synapse refinement during development in a model of Fragile X Syndrome (Chapter 3).

Mutations in TSC2 cause the disorder tuberous sclerosis (TSC), which has a high incidence of autism and intellectual disability. TSC2 regulates mRNA translation required for group 1 metabotropic glutamate receptor-dependent synaptic long-term depression (mGluR-LTD), but the identity of mRNAs responsive to mGluR-LTD signaling in the normal and TSC brain is largely unknown. We generated Tsc2+/- mice to model TSC autism and performed ribosome profiling to identify differentially expressed genes following mGluR-LTD in the normal and Tsc2+/- hippocampus. Ribosome profiling reveals that in Tsc2+/-mice, RNA-binding targets of Fragile X Mental Retardation Protein (FMRP) are increased. In wild-type hippocampus, induction of mGluR-LTD caused rapid changes in the steady state levels of hundreds of mRNAs, many of which are FMRP targets. Moreover, mGluR-LTD signaling failed to promote phosphorylation of eukaryotic elongation factor 2 (eEF2) in Tsc2+/- mice, and chemically mimicking phospho-eEF2 with low cycloheximide enhances mGluR-LTD in the Tsc2+/- brain. These results suggest a molecular basis for bidirectional regulation of synaptic plasticity by TSC2 and FMRP. Furthermore, deficient mGluR-regulated translation elongation contributes to impaired synaptic plasticity in Tsc2+/- mice.

Le but de ce travail est l'étude de la connectivité anatomique et fonctionnelle desréseaux neuronaux et le développement des nouveaux outils à cet effet. Car le dernieraspect est une préoccupation majeure de la neuroscience actuelle, nous avonsdeveloppé d'abord un nouveau traceur virale permettant la reconstruction neuronale.Nous avons ensuite appliqué cet et d'autres techniques pour sonder les défauts deconnectivité neuronale dans le syndrome de l'X fragile.Dans la première partie, nous avons discuté les avantages et inconvénients d'unetechnique émergente en utilisant un nouveau type de vecteur viral qui permet uneunique application pour l’étude du cerveau.Dans la deuxième partie, nous avons développé, au départ de ce vecteur viral, unenouvelle variante de faciliter le traçage et reconstruction des caractéristiquesmorphologiques de neurones. Nous avons montré la force de cette varianteantérograde du virus de la rage recombinant glycoprotéine supprimé pour lareconstruction de calcul de toutes les caractéristiques morphologiques clés deneurones: les dendrites, épines, les axones longs envergure dans tous les terminaux ducerveau et les boutons.Dans la troisième partie, nous avons examiné les modifications dans la connectivitédes structures cérébrales dans le syndrome du X fragile (FXS). FXS est le retardmental héréditaire la plus fréquente et la forme génétique la plus fréquente del'autisme, ce qui conduit à l'apprentissage et de la mémoire des déficits, lescomportements répétitifs, des convulsions et une hypersensibilité à des stimulisensoriels (visuels). Une des hypothèses éminents dans le domaine de l'autismesuppose une phénotype de hyper-connectivité locale mais de hypo-connectivité pourles connexions longue portée. Pour tester cette hypothèse dans un modèle de sourisFXS nous avons utilisé l'imagerie par résonance magnétique, pour balayer la totalitédu cerveau et de mesurer la connectivité anatomique et fonctionnel. Cela nous apermis d'identifier des altérations de connectivité dans plusieurs domains. Après nous8avons utilisé des traceurs viraux pour explorer un de ceux domains plus detaillé. Enutilisant le virus de la rage rétrograde à quantifier le nombre de neurones projetantvers ces zones, nous avons confirmé une connectivité d'entrée modifié pour le cortexvisuel primaire, ce qui pourrait contribuer au traitement visuel altéré de l'information.Nous avons découvert une connectivité réduite à longue portée globale anatomique etfonctionnelle entre plusieurs régions du cerveau, l'identification FXS comme unepathologie de la connectivité neuronale, ce qui pourrait expliquer les difficultés deplusieurs stratégies de sauvetage visant des cibles moléculaires sont actuellementconfrontés. / The goal of this work was the investigation of the anatomical and functionalconnectivity of neuronal networks and the development of novel tools for thispurpose. Since the latter aspect is a major focus of current neuroscience, we firstsought a novel viral tracer enabling sparse neuronal reconstruction and neuronclassification. We then applied this and other techniques to probe neuronalconnectivity defects in Fragile X Syndrome.In the first part we discussed the merits and drawbacks of a emergingtechnique using a new type of viral vector that allows in a unique manner mapping ofthe input of a given brain area.In the second part we developed, departing from this viral vector, a newvariant to facilitate the tracing and reconstructing of morphologic features of neurons.We showed the strength of this anterograde variant of the recombinant glycoproteindeletedrabies virus for computational reconstruction of all key morphologicalfeatures of neurons: dendrites, spines, long-ranging axons throughout the brain andbouton terminals.In the third part we examined alterations in the wiring of brain structures inthe Fragile X Syndrome (FXS). FXS is the most common inherited mental retardationand most frequent genetic form of autism, leading to learning and memory deficits,repetitive behavior, seizures and hypersensitivity to sensory (e.g. visual) stimuli. Oneof the eminent hypotheses in the autism field assumes a local hyper- connectivityphenotype but hypo-connectivity for long-ranging connections. To test this hypothesisin a FXS mouse model we used magnetic resonance imaging, to scan the entire brainand measure the anatomical and functional connectivity. This allowed us to identifyconnectivity alterations in several areas that we further explored using viral tracers.Using retrograde rabies virus to count the number of neurons projecting to such areaswe confirmed an altered input connectivity to the primary visual cortex, which couldcontribute to the altered visual information processing. We discovered an overallreduced anatomical and functional long-range connectivity between several brainareas, identifying FXS as pathology of neuronal connectivity, which might explain thedifficulties several rescue strategies aiming at molecular targets are currently facing.

Background: Prevalence of metabolic disturbances is higher among individuals with neurodevelopmental disorders (NDDs), yet this association has been poorly studied. Investigation into human disease remains challenging, as a complete pathophysiological understanding relies on accurate modeling and highly controlled variables. As such, genetically engineered mouse models are increasingly used to gain insight into the biology of human NDDs, but preclinical research focus has been mainly on behavioral and neurophysiological abnormalities. Mouse models engineered to embody human-equivalent genetic variations can display discrepancies to human phenotypes, therefore a thorough characterization of mouse phenotypes must be conducted in order to evaluate how accurately a mouse model embodies a human phenotype. Also, mouse models can help discover unsuspected abnormalities that can be further validated in humans. Objective: In this study, we sought to investigate the metabolic alterations derived from NDD-associated genetic polymorphisms in previously-validated genetic mouse models. Due to the similarities in NDD-associated phenotypic expression, we hypothesized that our NDDs of interest would express similar metabolic signatures. Further, we anticipated that we might uncover unknown metabolic anomalies, and that sex may alter these differences. Methods: We used the Comprehensive Lab Animal Monitoring System coupled to EchoMRI, as well as quantification of key plasma metabolites by liquid chromatography-mass spectrometry to characterize and compare basal metabolism in three mouse models of NDDs, namely Down syndrome (Dp(16)Yey/+ mice), 16p11.2 deletion syndrome (16p11.2df/+ mice) and Fragile X syndrome (Fmr1-/- KO mice) and their wild-type (WT) counterparts. Results: Our study reveals that each mouse model expresses a unique metabolic signature that is sex-specific, independent of the amount of food consumed and minimally influenced by physical activity. We found striking differences in body composition, respiratory exchange ratio, caloric expenditure and concentrations of circulating plasma metabolites related to mitochondrial function. Conclusion: Providing novel insight into NDD-associated metabolic alterations provides a basis for future studies aimed at understanding physiological mechanisms and provides a point of reference for research aimed at detecting changes in response to intervention.

Rare conditions can be catastrophic for families and the implications for public health can be substantial. Our study compared basic surveillance through active medical record review with a linked administrative data file to assess the number of cases of two rare conditions, fragile X syndrome (FXS) and muscular dystrophy (MD) in a population. Methods: Two methods of data collection were used to collect information from five counties comprising two standard metropolitan statistical areas of South Carolina. The passive system relied mostly on health claims data using ICD-9 CM diagnostic codes. The active system relied on a nurse abstracting records from a list of all licensed physicians with specialties in neurology, orthopedics, and genetics. Results: There were 141 FXS cases and 348 MD cases that met the case definitions using active surveillance. Additional cases were found for both conditions but they were determined to not be true cases. After linking the actively collected MD and FXS cases to passive datasets, we found that the estimated total numbers of cases were similar to using capture-recapture analysis; the positive predictive values for cases identified in the passive system were 56.6% for MD and 75.7% for FXS. Conclusions: Applying capture-recapture methods to passively collected surveillance data for rare health conditions produced an estimate of the number of true cases that was similar to that obtained through active data collection.

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