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Forebrain development is controlled by a set of transcription factors which are expressed in dynamic spatiotemporal patterns in the embryonic forebrain and are known to regulate complex gene networks. Pax6 is a transcription factor that regulates corticogenesis and mutations affecting Pax6 protein levels cause neurodevelopmental defects in the eyes and forebrain in both humans and mice. In previous studies, it was shown that the graded expression pattern of Pax6 protein, which is high rostro-laterally to low caudo-medially in the cerebral cortex, is critical for its control of cell cycle progression and proliferation of cortical progenitors. However the underlying mechanisms are still unclear. Based on a microarray analysis carried out in our laboratory, a number of cell cycle-related candidate genes that may be affected by Pax6 have been identified. One such gene, Cell division cycle associated 7 (Cdca7) is expressed in a counter-gradient against that of Pax6. In my current study, I found that Cdca7 mRNA expression in the telencephalon is upregulated in Pax6 null (Small eye) mutants and downregulated in mice that overexpress PAX6 (PAX77) across developing time points from E12.5 to E15.5. There are several potential Pax6 binding motifs located in the genomic locus upstream of Cdca7. However, by chromatin immunoprecipitation, it is showed that none of the predicted binding sites are physically bound by Pax6. Promoter luciferase assays using fragments combining five suspected binding motifs show that Pax6 is functionally critical. Cdca7 is also identified as a Myc and E2F1 direct target and is upregulated in some tumours but its biological role is not fully understood. Current work using in utero electroporation to overexpress Cdca7 around the lateral telencephalon, where Cdca7 expression levels are normally low, tested the effects on the proliferation and differentiation of cortical progenitor cells in this region. In E12.5 mice embryos, overexpression of Cdca7 protein causes fewer intermediate progenitor cells and post-mitotic neurons to be produced but these effects were not found in E14.5 embryos. This result implies that Cdca7 may affect cell fate decision during cortical development.

Orientadores: Iscia Teresinha Lopes Cendes, Fábio Rossi Torres / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T06:33:25Z (GMT). No. of bitstreams: 1 Souza_DanielaAguiar_D.pdf: 4993968 bytes, checksum: b0d9bcf5acf25db217c9dedcd7aa4bbc (MD5) Previous issue date: 2013 / Resumo: As malformações do desenvolvimento cortical (MDC) são distúrbios resultantes de defeitos na embriogênese do córtex cerebral e estão entre as principais causas conhecidas de atraso do desenvolvimento e epilepsia. Dentre os principais tipos de MDC podemos citar a heterotopia nodular periventricular (HNP), o espectro lissencefalia/heterotopia subcortical em banda (LIS/HSB) e a esquizencefalia. Alterações moleculares em genes que atuam em mecanismos que vão desde o controle da divisão celular até a migração neuronal foram identificadas como responsáveis pela etiologia de vários tipos de MDC. Tais descobertas, além de auxiliar na compreensão dos mecanismos envolvidos no desenvolvimento do córtex cerebral e doenças relacionadas, fornecem informações importantes para um melhor diagnóstico e tratamento dos pacientes. Neste contexto, o principal objetivo deste trabalho foi analisar do ponto de vista molecular um grande grupo de pacientes com MDC. A casuística foi composta por um grupo de 107 pacientes: 27 possuem HNP, 33 são afetados por LIS/HSB e 47 possuem esquizencefalia. Em um primeiro momento, foi realizada triagem de mutações de ponto em genes candidatos (FLNA, DCX, LIS1, EMX2, TUBA1A, TUBB2B e TUBA8) por sequenciamento (Sanger). Posteriormente, a técnica de MLPA (Multiplex Ligation-dependent Probe Amplification) foi utilizada para detectar variações estruturais em regiões candidatas, incluindo os genes FLNA, DCX e LIS1. Finalmente, a técnica de SNP-Array foi utilizada para análise das variações no número de cópias alélicas em regiões genômicas de alguns pacientes (copy number variation - CNV). Todas as mutações de ponto encontradas nas sequencias de DNA foram verificadas em um grupo de 200 indivíduos controles. Através do sequenciamento, foram identificadas três alterações patogênicas localizadas nos genes FLNA e DCX, através do MLPA foram encontradas seis alterações estruturais patogênicas nos genes FLNA, DCX e LIS1. Os ensaios de SNP-Array permitiram a identificação de vários CNVs com potencial deletério, incluindo 12 pacientes com 17 novas CNVs ainda não descritas na literatura. Tais CNVs envolvem vários potenciais genes candidatos para as MDC, incluindo os genes TSNARE, DAAM1, ARX, PLXNA1 e HAUS7. Concluindo, nossos resultados mostram uma baixa frequência de mutações em genes candidatos previamente relacionados às MDC, somente 2.8% (n=3/107) dos pacientes possuem variantes deletérias nas sequencias dos genes analisados e 18% (n=6/33 testados) dos pacientes analisados possuem alterações estruturais em regiões candidatas. Porém, através da abordagem genômica para identificação de variações estruturais, foram identificadas vá-rias regiões/genes candidatos potencialmente envolvidos com a etiologia das MDC (12/40= 30%). Nossos dados mostram que as MDC apresentam grande heterogeneidade genética, porém uma proporção significativa dos pacientes possuem variações estruturais que podem ser identificadas pela técnica de SNP-Array / Abstract: Malformations of cortical development (MCD) are disorders resulting from defects in the embryogenesis of the cerebral cortex and are one of the most important causes of developmental delay and epilepsy. The main types of MCDs are periventricular nodular heterotopia (PNH), lissencephaly/subcortical band heterotopia spectrum (LIS/SBH) and schizen-cephaly. Mutations in genes acting in mechanisms ranging from control of cell division to neuronal migration were identified as responsible for several types of MCDs. These advances not only improved our understanding about the mechanisms involved in the development of the cerebral cortex but have also provided relevant information that can be used for better diagnosis and management of patients. In this scenario, the main objective of this study was to access and perform a comprehensive molecular genetics study in a large cohort of patients with MCD. We have studied a total of 107 patients with MCDs divided in the following groups: 27 with PNH, 33 with LIS/SBH and 47 with schizencephaly. We first searched for sequence variations in candidate genes (FNLA, DCX, LIS1, EMX2, TU-BA1A, TUBB2B and TUBA8) using the Sanger sequencing method. Subsequently, we checked for structural variants in FLNA, DCX and LIS1 using Multiplex Ligation-dependent Probe Amplification (MLPA) technique. Finally, we took a genomic approach by using single nucleotide polymorphism (SNP)-array technology to studied copy number variations (CNV) in some patients. All sequence variants found were subsequently verified in a group of 200 control individuals. Our results showed 3 pathogenic sequence variants in FNLA and DCX, as well as 6 pathogenic structural variants detected by MLPA in FLNA, LIS1 and DCX. SNP-array technique detected 17 genomic regions, in 12 patients, containing new CNVs. These CNVs involve a number of potential new candidate genes for MCDs, including: TSNARE, DAAM1, ARX, PLXNA1 and HAUS7. In conclusion, our results show a low frequency of mutations in candidate genes previously associated with MCDs, only 2.8% (n=3/107) of our patients have deleterious sequence variants and 18% (n=6/33) have abnormal structural variants in candidate regions. However, by using a genomic approach to look for CNVs we were able to identify several candidate regions/genes that have the potential to be involved in MCDs (12/40= 30%). Our data show that MCDs are genetic heterogeneous; however, it seems that a significant proportion of patients have structural abnormalities that can be identified by SNP-array technology / Doutorado / Fisiopatologia Médica / Doutora em Ciências

The nucleosome remodeling and deacetylase (NuRD) complex makes use of Chromodomain Helicase DNA binding protein 3 (CHD3) to remodel chromatin. Of the three CHD proteins NuRD may incorporate (CHD3/4/5); the function of CHD3 within the brain is the only one to have not been studied through gene knockout. Chd3 mutations in humans cause a neurodevelopmental disorder (SNIBCPS) characterized by intellect and speech deficits, but limited work has been done to describe a phenotype in mice. We have generated mice with forebrain specific deletion of Chd3 to characterize its impact on embryonic and postnatal cortical development. To analyze the consequence of Chd3 ablation on cortical lamination, layer-specific staining was performed and showed a decreased number of cells in layers II-IV. Neuronal birthdating has demonstrated that this is due to a defect in migration, causing the cells to be retained in the lower layers. Behaviour testing has also indicated defect in fear learning and memory in heterozygote males. These initial indications of defects in cortical development and behavioural deficits in the Chd3 mutant mice suggest that the animals are a good first model of the SNIBCP syndrome.

<p>Trafficking of the glutamatergic AMPA receptors (AMPARs) has been implicated in synaptic plasticity regulation, including long-term potentiation, long-term depression, and synaptic scaling. Two proteins, GRIP for stabilization at the synapse and PICK for internalization, are involved in trafficking GluR2-containing AMPARs in and out of the synapse. In this thesis, I addressed the changes in the mechanisms of AMPAR trafficking by characterizing the developmental trajectories of GluR2, the phosphorylated form pGluR2, GRIP, and PICK and comparing expression in visual vs. frontal cortex. I found significant differences between cortical areas in the developmental trajectories of GluR2 and pGluR2. In visual cortex, expression levels exhibited smooth developmental increases. In frontal cortex, GluR2 and pGluR2 rose to an exuberant expression between P18 and P35. Developmental trajectories for GRIP and PICK showed smooth increases that were consistent across cortical areas. Furthermore, looking at the correlation between the surface components (GluR2 and GRIP) and internalized components (pGluR2 and PICK), I found that the development of AMPAR trafficking components is tightly regulated across the cortex.</p> <p>In this thesis, I also looked at AMPAR expression in adult cortex. Fluoxetine has previously been reported to induce a juvenile like state of plasticity in visual cortex and this plasticity was assessed through monocular deprivation. My results indicated that fluoxetine administration was not associated with significant changes in AMPAR expression levels. However, monocular deprivation induced significant upregulation in expression levels of all four proteins. These results imply the presence of AMPAR-mediated plasticity in the adult brain.</p> / Master of Science (MSc)

Des mutations du gène ARX (aristaless-related homeobox gene) ont été identifiées dans un large spectre de désordres neurologiques précoces, incluant ou non des malformations cérébrales, le plus souvent associés à des épilepsies. Il est proposé que le gène ARX, codant pour un facteur de transcription, joue un rôle primordial au cours du développement cérébral, notamment sur la migration des neurones GABAergiques, mais son implication au cours de la mise en place du système nerveux central reste cependant encore mal connue. L’objectif de ce travail a été d’étudier le rôle du gène ARX et les conséquences de ses mutations sur le développement cérébral dans le but de mieux comprendre ces pathologies. Dans un premier temps, nous avons étudié l’effet d’une mutation particulière du gène, la mutation ARX(CGC)7, une expansion polyalanine retrouvée principalement dans des pathologies sans malformation cérébrale mais avec des épilepsies, tels que les syndromes de West ou d’Ohtahara. Des analyses réalisées sur une lignée de souris knock-in pour cette mutation (GCG)7 et sur des rats après électroporation in utero ont montré que la migration neuronale des neurones glutamatergiques et GABAergiques ainsi que la maturation des neurones GABAergiques ne sont pas altérées par cette mutation. De façon intéressante, nos données suggèrent que les épilepsies observées chez les souris knock-in résulteraient plutôt d’une réorganisation du réseau glutamatergique. Etant donné que le gène ARX n’est pas exprimé dans les neurones glutamatergiques, l’ensemble de ce travail suggère donc que les épilepsies chez les souris knock-in pour la mutation (GCG)7 sont la conséquence d’une altération développementale secondaire à la mutation initiale du gène, et ceci aurait d’importantes répercussions thérapeutiques qui requièrent d’avantages d’études. Des expériences nous ont ensuite permis d’étudier l’effet de plusieurs mutations du gène ARX sur la morphologie des interneurones in vitro. Celles-ci ont montré que les mutations d’ARX n’engendrent pas une localisation subcellulaire anormale de la protéine dans les interneurones en culture. De façon intéressante, ces expériences suggèrent que la morphologie des interneurones est altérée seulement par certaines mutations, notamment les mutations P353R et Dup24. Ces données soulignent ainsi l’importance d’étudier de façon spécifique chaque mutation du gène pour expliquer les mécanismes engendrant l’hétérogénéité phénotypique liée aux mutations d’ARX. L’ensemble de ces travaux contribuent à une meilleure compréhension du rôle du gène ARX dans le développement cortical et à une meilleure caractérisation des mécanismes physiopathologiques des désordres neurologiques précoces liés aux mutations de ce gène. / Several mutations in ARX gene (aristaless-related homeobox gene) have been found in a large spectrum of infantile neurological disorders, with or without cerebral malformation, but frequently linked to epilepsy. It has been proposed that ARX, coding for a transcription factor, plays a crucial role in brain development, especially in migrating interneurons, but its involvement in nervous system development still remains to be clarified. The aim of this work has been to study the role of ARX gene and the consequences of ARX mutations on cerebral development in order to better understand these pathologies.We have first investigated the effects of an ARX polyalanine expansion, the mutation (GCG)7, which was found in pathologies without brain malformation but associated to epilepsy, such as West and Ohtahara syndromes. Analysis performed on knock-in mice for this mutation and in utero electroporated rat brains have shown that this mutation doesn’t alter neither glutamatergic and GABAergic neuronal migration, nor GABAergic neuron maturation. Interestingly, our data suggest that epilepsy observed in knock-in mice would result rather from a reorganization of glutamatergic networks. Since ARX gene is not expressed in excitatory neurons, our work suggests that epilepsy observed in knock-in mice is the consequence of developmental alterations secondary to the initial mutation, and this would have crucial therapeutic implications that require additional investigations. In vitro experiments have then allowed us to study the effect of several ARX mutations on interneurons morphology. These experiments have shown no abnormal subcellular localization of ARX protein following transfection of these different mutations in cultured interneurons. Interestingly, our data show that interneuron morphology is altered only by some mutations, particularly the P353R and the Dup24 ARX mutations. Our data underline the importance to study specifically each mutation in order to explain mechanisms generating phenotypic heterogeneity linked to ARX mutations.Taken together, this study contributes to a better understanding of ARX involvement in cerebral development and to a better characterization of pathophysiological mechanisms linked to ARX mutations.

Brain injury is a serious clinical problem. The success of recovery from brain injury involves functional compensation in the affected brain area. We are interested in general mechanisms that underlie compensatory plasticity after brain damage, particularly when multiple brain areas or multiple modalities are included. In this thesis, I studied the function of auditory cortex after recovery from neonatal midbrain damage as a model system that resembles patients with brain damage or sensory dysfunction. I addressed maladaptive changes of auditory cortex after invasion by ectopic visual inputs. I found that auditory cortex contained auditory, visual, and multisensory neurons after it recovered from neonatal midbrain damage (Mao et al. 2011). The distribution of these different neuronal responses did not show any clustering or segregation. As might be predicted from the fact that auditory neurons and visual neurons were intermingled throughout the entire auditory cortex, I found that residual auditory tuning and tonotopy in the rewired auditory cortex were compromised. Auditory tuning curves were broader and tonotopic maps were disrupted in the experimental animals. Because lateral inhibition is proposed to contribute to refinement of sensory maps and tuning of receptive fields, I tested whether loss of inhibition is responsible for the compromised auditory function in my experimental animals. I found an increase rather than a decrease of inhibition in the rewired auditory cortex, suggesting that broader tuning curves in the experimental animals are not caused by loss of lateral inhibition. These results suggest that compensatory plasticity can be maladaptive and thus impair the recovery of the original sensory cortical function. The reorganization of brain areas after recovery from brain damage may require stronger inhibition in order to process multiple sensory modalities simultaneously. These findings provide insight into compensatory plasticity after sensory dysfunction and brain damage and new information about the role of inhibition in cross-modal plasticity. This study can guide further research on design of therapeutic strategies to encourage adaptive changes and discourage maladaptive changes after brain damage, sensory/motor dysfunction, and deafferentation.

Human genetics has identified essential roles for many centriole- and cilia-related proteins during human development. Mutations in centrosome-associated genes commonly cause microcephaly, or "small brain," and mutations in cilia-associated genes cause a diverse spectrum of diseases termed "ciliopathies." However, the functional relationships between these two crucial organelles are less well studied. The activities of centrosome-related proteins during mitosis and cytoskeletal remodeling are well-characterized, but their in vivo functions are incompletely understood. Here, we identify novel human mutations in a centrosomal gene which encodes a regulatory subunit of a microtubule interacting protein, and uncover unexpected pathways during vertebrate development. Human mutations cause severe microlissencephaly, reflecting defects in cerebral cortical neurogenesis, and loss of function in mice and zebrafish confirm essential roles in embryonic development, neurogenesis, and cell survival. Surprisingly, null mutant embryos display hallmarks of aberrant Sonic hedgehog signaling, including holoprosencephaly. Deficient induced pluripotent stem cells and lymphoblasts show defective proliferation and spindle structure, while deficient fibroblasts also demonstrate a remarkable excess of centrioles, including excessive maternal centrioles, with supernumerary cilia but deficient Hedgehog signaling. Our results reveal novel roles for this protein in regulating overall centriole number, mother centriole and cilia number, and as an essential gene for normal Hedgehog signaling during neocortical development.

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