The purpose of this study was to examine the expression of GABAergic proteins in Fmr1 knockout mice during brain maturation and to assess behavioural changes potentially linked to perturbations in the GABAergic system. Quantitative western blotting of the forebrain revealed that compared to wild-type mice, the GABAA receptor α1, β2, and δ subunits, and the GABA catabolic enzymes GABA transaminase and SSADH were down-regulated during postnatal development, while GAD65 was up-regulated in the adult knockout mouse forebrain. In tests of locomotor activity, the suppressive effect on motor activity of the GABAA β2/3 subunit-selective drug loreclezole was impaired in the mutant mice. In addition, sleep time induced by the GABAA β2/3-selective anaesthetic drug etomidate was decreased in the knockout mice. Our results indicate that disruptions in the GABAergic system in the developing brain may result in behavioural consequences in adults with fragile X syndrome.

The purpose of this study was to examine the expression of GABAergic proteins in Fmr1 knockout mice during brain maturation and to assess behavioural changes potentially linked to perturbations in the GABAergic system. Quantitative western blotting of the forebrain revealed that compared to wild-type mice, the GABAA receptor α1, β2, and δ subunits, and the GABA catabolic enzymes GABA transaminase and SSADH were down-regulated during postnatal development, while GAD65 was up-regulated in the adult knockout mouse forebrain. In tests of locomotor activity, the suppressive effect on motor activity of the GABAA β2/3 subunit-selective drug loreclezole was impaired in the mutant mice. In addition, sleep time induced by the GABAA β2/3-selective anaesthetic drug etomidate was decreased in the knockout mice. Our results indicate that disruptions in the GABAergic system in the developing brain may result in behavioural consequences in adults with fragile X syndrome.

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.

L’autisme est un trouble envahissant du développement défini uniquement sur des critères comportementaux et l’âge d’apparition. Le X fragile est une pathologie d’origine monogénique dont 15-25% des patients présente le diagnostique complet de l’autisme et dont de nombreux symptômes chevauchent avec l’autisme. Une souris Fmr1 KO a été créée et validée comme modèle pour le X fragile. A l’instar de la variabilité des phénotypes du X fragile chez l’humain, les données préliminaires montrent que la mutation nulle Fmr1 chez la souris interagit avec l'arrière fond génétique. Les travaux présentés visaient à déterminer les caractéristiques autistiques exprimées par les souris Fmr1 KO, ainsi que l’interaction de la mutation nulle avec le fond génétique (souches C57BL/6J, FVB.129P2tm1Cgr /J et leurs hybrides). Les résultats de ces travaux montrent notamment que les souris Fmr1 KO présentent un évitement initial d’approche sociale, des altérations principalement qualitatives des vocalisations, de l’hyperactivité et une augmentation de l’activité diurne. La mutation interagit avec le fond génétique et les résultats actuels indiquent que les KO de fond FVB.129P2tm1Cgr /J ont le phénotype le plus marqué. / Autism is a pervasive developmental disorder defined by behavioural criteria and age of onset. Fragile X is a disorder due to the silencing of the Fmr1 gene. About 15-25% of Fragile X patients are diagnosed as autistic and many symptoms overlap between the two disorders. A mouse Fmr1 KO was created and validated as a model for Fragile X Syndrome. Preliminary data also show that the null mutation interacts with the genetic background. The work presented in this thesis aimed to determine the autistic features expressed in Fmr1 KO mice, as well as the influence of the genetic background (C57BL/6J and FVB.129P2tm1Cgr/J strains, and their reciprocal hybrids) on the expression of the Fmr1 mutation. Our results show an initial inhibition of social approach in Fmr1 KO mice and a qualitative alteration of ultrasonic vocalizations in isolated pups, as well as an increase in activity, especially during the diurnal period. The Fmr1 mutation interacts with the genetic background and the results indicate that KO on the FVB.129P2tm1Cgr/J background show the most marked phenotype.

Fragile X Syndrome (FXS) is the leading cause of inherited intellectual disability. The disorder is caused by a trinucleotide expansion that silences the Fragile X Mental Retardation 1 (Fmr1) gene resulting in the loss of its protein product, the Fragile X Mental Retardation Protein (FMRP). FXS patients show broad clinical phenotypes including intellectual disability, as well as a number of cognitive and behavioral problems. The lack of FMRP is believed to be the direct cause of the deficits seen in FXS patients. FMRP is an RNA-binding protein that is expressed in the brain and testes. This protein is believed to form a messenger ribonucleoprotein complex with mRNAs in the nucleus and subsequently export them to polyribosomes in the cytoplasm, therefore influencing translation of its bound mRNAs. Importantly, FMRP has long been suspected to be involved in synaptic plasticity due to its ability to bind several mRNAs that encode for proteins important in synaptic plasticity. Such proteins include the GluN1, GluN2A and GluN2B subunits of the N-methyl-D- aspartate receptor (NMDAR). FMRP is expressed in the hippocampus, a region of the brain involved in learning and memory processes. Recently, impaired NMDAR functioning in the dentate gyrus (DG) subregion of the hippocampus has been observed in Fmr1 knockout (-/y) mice. This impairment also resulted in reduction in long-term potentiation (LTP) and long-term depression (LTD) of synaptic efficacy, two biological models of learning and memory. In the present study, I focused on the levels of the NMDAR GluN1, GluN2B and Glu2B subunits in order to determine the synaptic plasticity alterations seen in the DG of Fmr1-/y mice. Using Western blotting, I found that there is a decrease in the GluN1, GluN2A and GluN2B subunits in the DG of young adult Fmr1-/y mice, indicating that these mice have significantly lower amounts of total NMDARs. These results could explain the altered LTP and LTD seen in Fmr1-/y mice at the molecular level and might contribute to the intellectual impairments seen in these KO mice. NMDARs appear to be important in the development and maturation of synapses. The GluN2A and GluN2B subunits are developmentally regulated, where GluN2B is predominantly expressed early in development and GluN2A in the adult brain. A dysregulation of GluN2A and GluN2B subunits has been proposed to affect the maturation and formation of synapses. Intriguingly, FMRP is also believed to play a functional role in early brain development. Thus, this study also focused on the developmental expression of the GluN1, GluN2A and GluN2B subunits in the DG, Cornu Ammonis, prefrontal cortex and cerebellum of Fmr1-/y mice, all of which are brain regions implicated in FXS. We found that the developmental expression of these subunits is altered in Fmr1-/y mice in specific brain regions. Together, these results demonstrate that the loss of FMRP differentially affects GluN1, GluN2A and GluN2B subunit expression both developmentally and spatially, further implicating NMDARs in the pathophysiology of FXS. / Graduate

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-25T21:04:26Z No. of bitstreams: 1 Marcela Kelly Astete Gómez Estudo dos alelos da região....pdf: 1187772 bytes, checksum: d35081c0e79dae64122fa5e5a15c60c3 (MD5) / Made available in DSpace on 2012-07-25T21:04:26Z (GMT). No. of bitstreams: 1 Marcela Kelly Astete Gómez Estudo dos alelos da região....pdf: 1187772 bytes, checksum: d35081c0e79dae64122fa5e5a15c60c3 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A Síndrome do X-Frágil (SXF) é a principal causa hereditária de deficiência mental de herança dominante ligada ao cromossomo X. Em 1991, o gene FMR1 (Fragile X Mental Retardation 1) foi descoberto como responsável pela SXF. De acordo com o número de repetições, os alelos se dividem em: (a) alelo normal compreendendo 6 a 55 repetições (b) pré-mutado: 61 a 200 repetições e (c) mutado: com >200 repetições. Os alelos que apresentam 45 a 60 repetições são considerados como zona intermediária ou gray-zone. A base molecular dessa doença é bastante peculiar quando comparada com os padrões típicos observados em outras desordens de etiologia genética. As pré-mutações podem agregar-se de forma silenciosa por muitas gerações de uma família antes de se expandir para a mutação completa, levando aos sinais clínicos da doença. Na Bahia existem poucas pesquisas envolvendo esta doença, consequentemente, é desconhecida sua frequência nessa região. Portanto, este trabalho tem como objetivo detectar a frequência das classes alélicas do gene FMR1 em uma amostra de indivíduos do sexo masculino da população geral de Salvador-BA. Este estudo foi realizado em 511 homens provenientes de outro amplo estudo desenvolvido pelo Instituto de Saúde Coletiva (ISC) da Universidade Federal da Bahia (UFBA) sendo utilizada a técnica da PCR e posterior classificação dos alelos baseando-se na relação entre o número de repetições CGG e o tamanho do fragmento obtido na PCR. Dos 511 homens analisados no presente estudo observou-se predominância de alelos normais pertencentes à classe 2 (11 a 26 repetições CGG) em 73,70% dos indivíduos analisados, seguido da classe 3 (27 a 40 repetições CGG) em 25,10% dos indivíduos. Apenas 1,20% foram incluídos na classe 1 (<10 repetições CGG) e nenhum alelo foi encontrado nas classes 4 (41 a 60 repetições CGG), classe 5 ( >60 repetições CGG) e classe 6 (>200 repetições CGG). Este é o primeiro estudo utilizando a técnica da PCR para detecção dos alelos do gene FMR1 em uma população geral de Salvador-BA, podendo direcionar futuros trabalhos envolvendo o gene FMR1 tanto para o estado da Bahia, quanto para a região Nordeste e também minimizar deficiências existentes em termos de diagnóstico da SXF na cidade de Salvador-BA. / The Fragile X syndrome (FXS) is the leading cause of inherited mental deficiency (MD) of dominant inheritance linked to X chromosome. In 1991, the FMR1 gene (Fragile X Mental Retardation 1) was discovered as responsible for FXS. According to the number of repetitions, the alleles are divided in: (a) normal stable allele comprising 6 to 55 repetitions; (b) premutation: 61 to 200 repetitions and (c) mutant with> 200 repeats. The molecular basis of this disease is quite unusual when compared with the typical patterns seen in other disorders of genetic etiology. The pre-change can add up so silent for many generations of a family before they expand to full mutation, leading to clinical signs of disease. In Bahia there are few studies involving this disease, therefore, its frequency is unknown in this region. So, this study aims to detect the frequency of allelic classes of FMR1 gene in a sample of males from the general population of Salvador-BA city. This study was conducted on 511 samples using the PCR technique and subsequent classification of alleles based on the number of CGG repeats and the size of the fragments in PCR. Of the 511 individuals examined was found to predominate among the normal alleles the class 2 (11 to 26 repetitions) with 73,70% followed alleles analyzed in Class 3 (27 to 40 repetitions) with 25,10%, and Class 1 (<10 repetitions) with only one 1,20% . No allele was found in class 4 (41 to 60 repetitions), which corresponds to gray zone and class 5 (> 60 repetitions) for the premutation and class 6 (>200 repetitions) the corresponds full mutation.This is the first study using the PCR technique for detection of alleles of the FMR1 gene in a general population of Salvador, Bahia, and may direct future studies involving the FMR1 gene for both the state of Bahia, the Northeast and to also minimize deficiencies existing in the diagnosis of FXS in Salvador, Bahia.

<p> Fragile X Syndrome (FXS) is the most common inherited disease of mental impairment, typically caused by a mutation in the Fragile X mental retardation 1 (FMRJ) gene. The clinical features are thought to result from abnormal neurobiology due to a lack of the Fragile X mental retardation protein (FMRP). Previously, it was thought that FMRP was confined exclusively to neurons; however, our laboratory recently discovered that astrocytes also express FMRP. Consequently, it is possible that astrocytes also suffer abnormalities as a result of a lack of FMRP. Astrocytes play integral roles in the development and maintenance of communication in the central nervous system. Therefore, it is now important to determine the contribution of astrocytes to the abnormal neuronal phenotype seen in FXS. In these experiments, neurons and astrocytes were independently isolated from wild type (WT) or FMRJ null mice and grown in a coculture. Neurons were evaluated using immunocytochemistry in combination with computer-aided morphometric and synaptic protein analyses. The findings presented here provide convincing evidence that Fragile X astrocytes contribute to the abnormal neurobiology seen in FXS . Fragile X astrocytes alter the dendrite morphology and excitatory synaptic protein expression of WT neurons in culture; and, importantly, when Fragile X neurons are grown with WT astrocytes these changes are prevented. Interestingly, the Fragile X astrocytes appear to act by causing a delay in development; even WT neurons grown in the presence of Fragile X astrocytes, that displayed an abnormal phenotype at 7 days in culture, exhibited nearly normal dendrite morphology and expression of excitatory synapses at 21 days. Furthermore, the results suggest that the dendritic abnormalities induced by the Fragile X astrocytes specifically target neurons with a spiny stellate morphology. This research establishes a role for astrocytes in the development of the abnormal neurobiology seen in FXS, and as such, the results presented here have significant implications for Fragile X research. The novel prospect that astrocytes are key contributing components in the development of FXS provides an exciting new direction for investigations into the mechanisms underlying FXS, with many unexplored avenues for potential treatment strategies. </p> / Thesis / Doctor of Philosophy (PhD)