How do circuits in the mammalian cerebral cortex encode properties of the sensory environment in a way that can drive adaptive behavior? This question is fundamental to neuroscience, but it has been very difficult to approach directly. Various computational and theoretical models can explain a wide range of phenomena observed in the primary visual cortex (V1), including the anatomical organization of its circuits, the development of functional properties like orientation tuning, and behavioral effects like surround modulation. However, so far no model has been able to bridge these levels of description to explain how the machinery that develops directly affects behavior. Bridging these levels is important, because phenomena at any one specific level can have many possible explanations, but there are far fewer possibilities to consider once all of the available evidence is taken into account. In this thesis we integrate the information gleaned about cortical development, circuit and cell-type specific interactions, and anatomical, behavioral and electrophysiological measurements, to develop a computational model of V1 that is constrained enough to make predictions across multiple levels of description. Through a series of models incorporating increasing levels of biophysical detail and becoming increasingly better constrained, we are able to make detailed predictions for the types of mechanistic interactions required for robust development of cortical maps that have a realistic anatomical organization, and thereby gain insight into the computations performed by the primary visual cortex. The initial models focus on how existing anatomical and electrophysiological knowledge can be integrated into previously abstract models to give a well-grounded and highly constrained account of the emergence of pattern-specific tuning in the primary visual cortex. More detailed models then address the interactions between specific excitatory and inhibitory cell classes in V1, and what role each cell type may play during development and function. Finally, we demonstrate how these cell classes come together to form a circuit that gives rise not only to robust development but also the development of realistic lateral connectivity patterns. Crucially, these patterns reflect the statistics of the visual environment to which the model was exposed during development. This property allows us to explore how the model is able to capture higher-order information about the environment and use that information to optimize neural coding and aid the processing of complex visual tasks. Using this model we can make a number of very specific predictions about the mechanistic workings of the brain. Specifically, the model predicts a crucial role of parvalbumin-expressing interneurons in robust development and divisive normalization, while it implicates somatostatin immunoreactive neurons in mediating longer range and feature-selective suppression. The model also makes predictions about the role of these cell classes in efficient neural coding and under what conditions the model fails to organize. In particular, we show that a tight coupling of activity between the principal excitatory population and the parvalbumin population is central to robust and stable responses and organization, which may have implications for a variety of diseases where parvalbumin interneuron function is impaired, such as schizophrenia and autism. Further the model explains the switch from facilitatory to suppressive surround modulation effects as a simple by-product of the facilitating response function of long-range excitatory connections targeting a specialized class of inhibitory interneurons. Finally, the model allows us to make predictions about the statistics that are encoded in the extensive network of long-range intra-areal connectivity in V1, suggesting that even V1 can capture high-level statistical dependencies in the visual environment. The final model represents a comprehensive and well constrained model of the primary visual cortex, which for the first time can relate the physiological properties of individual cell classes to their role in development, learning and function. While the model is specifically tuned for V1, all mechanisms introduced are completely general, and can be used as a general cortical model, useful for studying phenomena across the visual cortex and even the cortex as a whole. This work is also highly relevant for clinical neuroscience, as the cell types studied here have been implicated in neurological disorders as wide ranging as autism, schizophrenia and Parkinson’s disease.

Orientador: Fernando Cendes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-17T18:37:25Z (GMT). No. of bitstreams: 1 Fonseca_VivianedeCarvalho_M.pdf: 1655188 bytes, checksum: bcdca0376058479aae51c23d91464f89 (MD5) Previous issue date: 2011 / Resumo: A epilepsia parcial secundária a displasia cortical focal (DCF), comumente se origina nos primeiros anos de vida, entretanto, alguns casos podem apresentar início após os 40 anos. Atualmente, a DCF é identificada em 20 - 25% de pacientes com epilepsia parcial extratemporal e aproximadamente 76% dos pacientes supostamente apresentam epilepsia refratária à medicação. Trata-se de uma malformação do desenvolvimento cortical (MDC), identificada por uma diferenciação anormal do córtex e neurônios displásicos na substância branca (SB). O exame de imagem por tensor de difusão (DTI) tem a capacidade de descrever a integridade da SB, através da quantificação da difusão e orientação das moléculas de água nos tecidos de forma não invasiva, podendo detectar anormalidades no tecido cerebral em estágios precoces em relação ao exame convencional de imagem por RM ponderado em T1 ou T2. Com o objetivo de detectar alterações microestruturais no tecido cerebral, utilizamos o exame de DTI para investigar a SB desses pacientes. Para isso, utilizamos uma medida de direcionamento da difusão, conhecida como anisotropia fracional (AF), que representa a orientação do eixo das estruturas dos feixes de fibras ao longo do qual as moléculas de água se movem de modo preferencial, indicando mudanças da microestrutura tissular. Foram analisados 53 sujeitos, sendo 22 pacientes e 31 indivíduos saudáveis. Todos os pacientes tinham diagnóstico clínico e eletroencefalográfico de epilepsia extratemporal (lobo frontal) secundária a provável DCF. Processamos o DTI com os programas: MRIcroN, FSL e TBSS (Tract-based Spatial Statistics). A comparação entre o grupo de pacientes e grupo controle foi realizada usando two-sample teste-t, com nível de significância de p <0,05. Identificamos áreas com redução da FA, nos lobos frontal, parietal, temporal e occipital, sendo elas: fórceps menor à direita (p=0,032), fórceps menor à esquerda (p=0,042), giro do cíngulo à esquerda (p=0,048), trato córtico-espinhal direito e esquerdo (p=0,022), fascículo fronto-occipital inferior direito (p=0,022), fascículo longitudinal superior e inferior esquerdo (p=0,034), radiação talâmica anterior à direita (p=0,034) e fascículo uncinado à esquerda (p=0,042). Nossos resultados mostraram um padrão extenso de anormalidades estruturais em regiões da SB que se estendem além do foco epileptogênico (lobo frontal), provavelmente decorrente da cronicidade da epilepsia. É possível que essas alterações sejam secundárias às descargas epilépticas muito freqüentes, associadas à generalização e bissincronia secundária / Abstract: Epilepsy secondary to FCD usually begins early in life, however, some cases may have onset after 40 years. Currently FCD has been identified in 20-25% of patients with extratemporal epilepsy and approximately 76% of patients with epilepsy refractory to antiepileptic drug treatment. FCD is a malformation of cortical development (MCD), identified by an abnormal differentiation of cortex and dysplastic neurons on the white matter. Diffusion tensor imaging (DTI) has a powerful ability to describe white matter integrity, through the quantification of the spread and direction of water molecules in tissues noninvasively, which can detect the abnormalities of the brain tissue in an earlier stage than conventional T2- or T1-weighted MRI. Aiming to detect microstructural changes in brain tissue, we used DTI to investigate the WM these patients. For this, we used a measure of diffusion direction, known as fractional anisotropy (FA), which represents the axis orientation of the structures of the fiber bundles along which the water molecules move preferentially, indicating changes in tissue microstructure. We analyzed 53 subjects, 22 patients and 31 healthy individuals. All the patients had clinical and EEG diagnosis of extratemporal epilepsy (frontal lobe), probably secondary to FCD. To process the DTI we used the following softwares: MRIcroN, FSL, TBSS. The comparison between the patients group and control group was performed using two-sample t-test, and the level of significance was set at <0.05. FA reduction in patients were identified in the frontal, parietal, temporal and occipital lobes, which were: right forceps minor (p =0.032), left forceps minor (p = 0.042), left cingulum (p = 0.048), right and left corticospinal tracts (p = 0.022), inferior right fronto-occipital fasciculus (p = 0.022),right and left superior longitudinal fasciculus (p = 0.034), right anterior thalamic radiation (p = 0.034) and the left uncinate fasciculus (p = 0,042). Our results showed a widespread pattern of WM micro structural abnormalities extending beyond the ictal onset zone (frontal lobe), probably due to the epilepsy chronicity. It is possible that this damage is secondary to persistent epileptic discharges with frequent generalization and secondary bilateral synchrony / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Orientadores: IsciaTeresinha Lopes Cendes, Fábio Rossi Torres / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T05:32:00Z (GMT). No. of bitstreams: 1 Avansini_SimoniHelena_M.pdf: 4063977 bytes, checksum: fd585dcd7befa8b76aa15db5e445d1d2 (MD5) Previous issue date: 2012 / Resumo: A displasia cortical focal (DCF) é uma malformação do córtex cerebral humano que ocorre na fase de proliferação e diferenciação neuronal e está frequentemente associada com a refratariedade das crises epilépticas. É designada como um espectro de anormalidades da estrutura laminar do córtex, associada com características citopatológicas que incluem neurônios gigantes, dismórficos e células em balão e, sua etiologia é pouco conhecida. Os microRNAs (miRNAs) são uma classe de RNAs de fita simples não codificadores de proteínas que regulam a expressão gênica pós-transcricional. Há evidências que indicam que eles estão envolvidos em importantes processos do sistema nervoso e que os mesmos podem ter um papel nas DCF. A elucidação das vias moleculares desta malformação pode permitir uma melhor compreensão dos mecanismos subjacentes e levar a novas estratégias de tratamento, melhora na conduta clínica e identificação de novos alvos terapêuticos, bem como a descoberta de biomarcadores que possam ser associados ao diagnóstico, prognóstico e resposta ao tratamento. Com isso, o objetivo deste trabalho foi investigar o padrão de expressão dos miRNAs em tecidos com DCF obtidos através de cirurgia para o controle de crises refratárias, verificar os prováveis genes alvos para esses miRNAs diferencialmente expressos e comparar a assinatura molecular baseada nos miRNAs em tipos distintos de DCF do tipo 2. Foi utilizado para isso o RNA total de 17 pacientes com DCF e de 20 controles oriundos de autópsia. Os experimentos de microarranjos de miRNAs revelaram 39 miRNAs diferencialmente hipoexpressos e um miRNA hiperexpresso. Utilizouse a técnica de qPCR para validação desses miRNAs e foi possível identificar uma diferença na expressão de três miRNAs: hsa-miR-31, hsa-miR-34a e hsa-let-7f em pacientes com DCF tipo 2 em relação ao grupo controle. Além disso, o hsa-miR-31 foi identificado como um possível biomarcador para o subtipo 2b de DCF. Na busca por genes alvos foi encontrado hiperexpresso o NEUROG2. Também foi verificada a desregulação do gene DICER1, encontrado hipoexpresso, o que justifica a predominância de miRNAs com expressão diminuída encontrados. E por fim, observou-se que o padrão diferencial de expressão dos três miRNAs e os dois genes identificados em nosso estudo fornecem subsídios importantes para esclarecer os mecanismos moleculares envolvidos na falha da diferenciação neuroglial em DCF tipo 2 / Abstract: Focal cortical dysplasia (FCD) is a malformation of human cerebral cortex that occurs during proliferation and neuronal differentiation frequently associated with drug-resistant epilepsy. It is designated as a spectrum of abnormalities of laminar structure of the cortex, associated with cellular abnormalities that consist of giant and dysmorphic neurons and balloon cells; however, its etiology is poorly understood. MicroRNAs (miRNAs) are small noncoding RNAs which regulate post-transcriptional gene expression. There is evidence that they are involved in important processes in the nervous system and that they may play a role in FCD. The elucidation of the molecular pathways involved in FCD may allow a better understanding of the underlying mechanisms and may lead to new treatment strategies, improvement in clinical management and identification of new therapeutic targets, as well as the discovery of biomarkers that may be associated with the diagnosis, prognosis and response to treatment. Thus, the aim of this study was to investigate miRNAs expression pattern in tissue with FCD obtained at surgery for control of refractory seizures. In addition, we aimed to identify target genes for these miRNAs differentially expressed, as well as to compare the molecular signature based on miRNAs in different types of FCD. We used total RNA isolated from brain tissue obtained after surgery for the treatment of medically refractory seizures from 17 patients with DCF and 20 controls from autopsy. Microarray analysis revealed 39 miRNAs differentially downregulated and only one miRNA overexpressed. Decreased expression of three miRNAs was confirmed by qPCR when patients with type 2 FCD were compared with controls: hsa-miR-31, hsa-miR34a and hsa-let-7f. In addition, we found that hsa-miR-31 could be a potential biomarker for type 2b FCD. In the search for target genes, NEUROG2 was found upregulated and DICER1 was found underexpressed, which explains the predominance of miRNAs with decreased expression. Finally, we observed that the differential pattern of expression of three miRNAs, and the two genes identified in our study provide important information which may help to clarify the molecular mechanisms involved in the failure of neuroglial differentiation in type 2 FCD / Mestrado / Neurociencias / Mestra em Fisiopatologia Médica

Orientador: Iscia Teresinha Lopes Cendes, Fábio Rossi Torres / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T06:16:28Z (GMT). No. of bitstreams: 1 Tsuneda_SimoneSayuri_D.pdf: 5548129 bytes, checksum: b0213ba3907f298ca16dddb2df837b62 (MD5) Previous issue date: 2012 / Resumo: A polimicrogiria (PMG) é uma malformação do córtex cerebral causada por falhas no seu desenvolvimento, caracterizando-se por um número excessivo de pequenos giros e laminação anormal, dando à superfície cortical uma aparência irregular e grosseira. A gravidade de suas manifestações clínicas se relaciona diretamente com a extensão da malformação e das regiões cerebrais afetadas, sendo que a presença de lesões bilaterais ou unilaterais extensas indica um pior prognóstico. Uma das síndromes de polimicrogiria mais frequentes e, consequentemente, mais bem descritas clinicamente, é a polimicrogiria perisylviana bilateral (PPB). Essa forma de polimicrogiria atinge a região que tange a fenda Sylviana, podendo apresentar-se tanto unilateralmente quanto em ambos os hemisférios. O padrão de herança da PPB foi descrito inicialmente como ligada ao cromossomo X por Borgatti et al. em 1999. Já em 2000, Guerreiro et al. confirmaram o padrão de herança consistente com herança ligada ao cromossomo X, mas ainda nenhum gene havia sido identificado como responsável pelo distúrbio. Nosso grupo recentemente mapeou uma nova região candidata para a PPB em Xq27.1-q27.3, e esta tese se propôs a avaliar essa região através da técnica de sequenciamento em larga escala aliada à tecnologia de captura para o cromossomo X. Os resultados apontaram como potenciais patogênicos os genes MAGEC1, UBE2NL, além da região do gene SPANXC, todos localizados na região candidata, mas uma avaliação mais detalhada levantou a hipótese de uma relação complexa entre as alterações encontradas no gene MAGEC1 e o quadro clínico dos pacientes. Além da análise da região Xq27.1-q27.3, considerando o grande número de genes de microtúbulo que tem sido relacionado a malformações do córtex cerebral, esse trabalho também avaliou pacientes esporádicos e famílias com histórico de PPB realizando triagem de mutações nas regiões codificantes dos genes AFF2, SLITRK2 e SLITRK4, localizados na região candidata, nos genes de microtúbulo TUBA1A, TUBB2B e TUBA8, além dos genes SRPX2 e WDR62, presentes em trabalhos na literatura de malformações corticais. A triagem foi realizada utilizando as técnicas de DHPLC e de sequenciamento utilizando a técnica de Sanger por eletroforese capilar. Foi encontrada uma alteração potencialmente patogênica no gene AFF2. As alterações identificadas neste estudo que resultam em troca de aminoácidos foram avaliadas utilizando as ferramentas in silico MutPred, SNPs&GO, Polyphen 2, Panther e SIFT, de forma a fornecer mais informações a respeito de seu potencial patogênico. Além disso, as variantes inéditas identificadas nesse trabalho foram estudadas em uma amostra de indivíduos normais (grupo controle). Com esses dados foi possível sugerir que algumas dessas variantes encontradas possuem potencial patogênico que deve ser futuramente investigado através de estudos funcionais / Abstract: Polimicrogyria (PMG) is a cortical malformation caused by failures during the brain cortex development process and is characterized by an excessive number of small gyri, resulting in an irregular cortical surface. The severity of its clinical manifestations is directly related to the extension of the tissue abnormalities. Bilateral Perisylvian Polimicrogyria (BPP) is the most comum and, consequently, a very well described syndrome that affects the cortex surrounding the Sylvian fissures in both hemispheres. The genetic pattern for BPP was initially described by Borgatti et al. as an X-linked pattern, confirmed by Guerreiro et al. in 2000, but with no specific gene identified. We have recently described a candidate site for BPP at the Xq27.1-27.3 region and, in this project, we proposed to evaluate this site through next generation sequencing technology combined with capture technology. Our results suggest that MAGEC1 and UBE2NL genes, or the SPANXC gene area might be related to the pathogeny in this case, however a further analysis brought up the hypothesis of a complex relation between the MAGEC1 mutations and the clinical manifestations in each different patient. Considering recurrent description of relations between microtubule genes and cortex malformations, we also performed the evaluation of exon regions of eight selected genes from sporadic patients and BPP families through DHPLC and sequencing. The analysis focused on AFF2, SLITRK2 and SLITRK4 genes, located at the identified site, microtubule genes TUBA1A, TUBB2B and TUBA8, and SRPX2 and WDR62 genes, also related to cortical malformations. As a result from this screening, we identified a potentially pathogenic mutation in gene AFF2. All non-synonymous SNPs were evaluated using the in silico tools MutPred, SNPs&GO, Polyphen 2, Panther and SIFT, providing further insights for their analysis. A control group of individuals was analyzed for the presence of the non-described SNPs. These data suggest a pathogenic potential for these genetic alterations that must be investigated through function studies / Doutorado / Fisiopatologia Médica / Doutora em Ciências

Le développement du cortex cérébral se déroule selon des étapes bien définies qui sont essentielles à la formation d’un cerveau fonctionnel. La perturbation de l’une ou plusieurs de ces étapes peut conduire à des malformations neuro-développementales, responsables de différents troubles cognitifs, d’épilepsies ou encore de déficience intellectuelle. De nombreuses mutations dans des gènes codant pour les tubulines ou bien les kinésines, sont retrouvées chez des individus présentant diverses anomalies neuro-développementales. Bien que les kinésines soient impliquées dans le développement cortical, les mécanismes fonctionnels par lesquels elles conduisent aux malformations demeurent encore méconnus. Mon travail de thèse identifie la kinésine Kif21b, jusqu’alors peu connue, comme étant essentielle au développement cortical. Nous montrons que Kif21b régule la migration neuronale dans le cortex et identifions quatre variants chez des individus présentant des malformations neuro-développementales. Nous montrons que l’expression ectopique des variants chez la souris et le poisson zèbre récapitulent les phénotypes observés chez ces patients. / The development of the cerebral cortex is a highly regulated process that is crucial for the establishment of functional cortical networks. Disruption of one or several of these steps can lead severe neurodevelopmental disorders that are associated with intellectual disabilities, epilepsies and cognitive impairment. Over the past few years, several genetic mutations in genes encoding either tubulin or microtubule-associated motors such as kinesins, have been found in individuals with neurodevelopmental disorders. Although kinesins have been found to be essential for a proper cortical development, the exact functions of kinesins in these processes are still poorly understood. My work clearly identified Kif21b, a poorly-known kinesin, as a novel key regulator of cortical development both in mouse and human. We show that Kif21b regulates both radial and tangential migration of cortical neurons, and identify four KIF21B variants in individuals presenting neurodevelopmental disorders. We show that ectopic expression of variants recapitulate phenotypes both in mice and zebrafish.

Les malformations du développement cortical (MDC) résultent d’altérations au niveau de différentes étapes de la corticogénèse telles que la prolifération, la migration et la différenciation neuronale et sont généralement associées à des épilepsies pharmaco-résistantes et à des déficiences intellectuelles sévères. Les causes génétiques des MDC restent encore inconnues dans de nombreux cas, nous avons donc réalisé le séquençage de l’exome entier de nombreux patients présentant des MDC et les analyses ont permis de mettre en évidence l’implication des gènes KIF2A et NEDD4L dans les MDC. Dans le cadre de ma thèse, nous proposons de focaliser sur les conséquences cellulaires et neurodéveloppementales résultant des mutations dans les gènes KIF2A et NEDD4L retrouvées chez les patients atteints de MDC. KIF2A code pour une kinésine-13 qui a pour fonction de réguler la dynamique des microtubules (MT) via son activité MT dépolymérase ATP-dépendante aux niveaux des extrémités des MT. L’approche basée sur la technique d’électroporation in utero nous a permis de mettre en évidence le rôle crucial joué par KIF2A dans la régulation de la neurogénèse, la migration neuronale et le positionnement des neurones dans le cortex. En particulier, nos données révèlent que l’expression des mutants KIF2A responsables de MDC entraîne une augmentation du nombre de cellules à l’état de progéniteurs qui est conséquente à un allongement du temps passé dans le cycle cellulaire. Nos premières données cellulaires et au cours du développement montrent que l’expression des mutants KIF2A induit des altérations dans l’intégrité du fuseau mitotique, dans la progression mitotique et également une localisation anormale de KIF2A au niveau du cil primaire. NEDD4L code pour une E3 ubiquitine ligase qui joue un rôle dans l’ubiquitination de nombreux substrats permettant la régulation de leur dégradation et de leur localisation subcellulaire. Dans un premier temps, nos données cellulaires ont montré que les mutants associées à des MDC ont une sensibilité accrue pour la dégradation par le protéasome. De plus, l’approche d’électroporation in utero a permis de montrer que l’expression des mutants NEDD4L ainsi qu’un excès de NEDD4L WT dérégulent la neurogenèse, le positionnement des neurones et le processus de translocation terminal. Des études complémentaires, incluant le traitement à la rapamycine, ont révélé qu’un excès de NEDD4L WT mène à la dérégulation des voies de signalisations mTORC1 et Dab1 tandis que l’expression des mutants est associée à une dérégulation des voies mTORC1 et Akt. L’ensemble de ces résultats renforce donc dans un premier temps l’importance des protéines liées aux MT dans le développement cortical en décrivant le rôle crucial de la kinésine KIF2A dans des mécanismes tels que la dynamique de migration neuronale et dans la régulation du cycle cellulaire des progéniteurs neuronaux. D’autre part, nous fournissons également de nouvelles données permettant de mieux comprendre le rôle critique de NEDD4L dans la régulation des voies mTOR et de leurs contributions dans le développement cortical. / Malformations of cortical development (MCD) result from alterations in different stages of corticogenesis such as proliferation, migration and neuronal differentiation, and are generally associated with drug-resistant epilepsy and severe intellectual disabilities. The genetics causes of MCD remain largely unknown, we have thus performed the whole-exome sequencing of many patients with MCD and reported the identification of multiple pathogenic missense mutations in KIF2A and NEDD4L genes. Within the frame of my thesis project, we propose to focus on the cellular and neurodevelopmental consequences resulting from KIF2A and NEDD4L mutations shown to be involved in MCD. KIF2A is a member of the kinesin-13 family, which rather than regulating cargos transport along microtubules (MT), regulates MT dynamics by depolymerizing MTs. The in utero electroporation approach allowed us to highlight the crucial role of KIF2A in the regulation neurogenesis, neuronal migration and the neuronal positioning in the cortex. Particularly, our data show that the expression of the KIF2A mutants involved in MDC lead to an increase in the number of cells in proliferative state which is a consequence of a prolonged time spent in the cell cycle. Our first cellular data and during development show that the expression of pathogenic KIF2A mutations induce alterations in the mitotic spindle integrity, in the mitotic progression and also an abnormal localization of KIF2A in the primary cilium. NEDD4L encodes a member of the NEDD4 family of HECT-type E3 ubiquitin ligases known to regulate the turnover and function of a number of proteins involved in fundamental cellular pathways and processes. Firstly, cellular and expression data showed sensitivity of MCD-associated mutants to proteasome degradation. Moreover, the in utero electroporation approach showed that PNH-related mutants and excess wild-type NEDD4L affect neurogenesis, neuronal positioning and terminal translocation. Further investigations, including rapamycin-based experiments, found differential deregulation of pathways involved. Excess wild-type NEDD4L leads to disruption of Dab1 and mTORC1 pathways, while MCD-related mutations are associated with deregulation of mTORC1 and AKT activities. Altogether, these results reinforce the importance of MT-related proteins in cortical development describing the crucial role of KIF2A kinesin in mechanisms such as neuronal migration dynamics and neuronal progenitor’s cell cycle regulation. On the other hand, we also provide new data to better understand the critical role of NEDD4L in the regulation of mTOR pathways and their contributions in cortical development.

Le développement du cortex cérébral résulte de processus de prolifération, neurogenèse, migration et différenciation cellulaire qui sont contrôlés génétiquement. Les malformations corticales qui résultent d'anomalies de ces processus sont associées à l'épilepsie et la déficience intellectuelle. Nous avons étudié la souris mutante HeCo (heterotopic cortex), qui présente une hétérotopie sous-cortical bilatérale (neurones présents dans la substance blanche) et nous avons identifié la présence d'une mutation sur le gène Eml1 (Echinoderm Microtubule-associated protein-Like 1). De plus, des mutations du gène EML1 ont été identifiées chez des patients atteints d'une forme sévère et rare d'hétérotopie. Dans le cerveau embryonnaire des souris HeCo, des progéniteurs ont été identifiés en dehors de la zone de prolifération, ce qui représente une nouvelle cause de cette malformation. Nous avons étudié la fonction d'Eml1 dans les progéniteurs de la glie radiaire, qui sont clés au cours de la corticogenèse. Nous avons montré qu'Eml1 se localise dans le fuseau mitotique où elle est susceptible de réguler la dynamique des microtubules. Nos données suggèrent qu'Eml1 peut jouer un rôle dans la régulation de la longueur du fuseau puisque celle-ci est perturbée dans les cellules de la glie radiaire chez la souris HeCo. Ceci pourrait représenter la cause primaire de leur ectopie. Nous avons analysé le nombre et la taille des cellules en métaphase dans la partie apicale de la zone ventriculaire où ont lieu les mitoses. Nous proposons ici de nouveaux mécanismes qui régissent l'organisation des progéniteurs dans la zone ventriculaire au cours du développement cortical normal et pathologique. / The cerebral cortex develops through genetically regulated processes of cellular proliferation, neurogenesis, migration and differentiation. Cortical malformations represent a spectrum of heterogeneous disorders due to abnormalities in these steps, and associated with epilepsy and intellectual disability. We studied the HeCo (heterotopic cortex) mutant mouse, which exhibits bilateral subcortical band heterotopia (SBH), characterized by many aberrantly positioned neurons in the white matter. We found that Eml1 (Echinoderm Microtubule-associated protein-Like 1) is mutated in these mice. Screening of EML1 in heterotopia patients identified mutations giving rise to a severe and rare form of atypical heterotopia. In HeCo embryonic brains, progenitors were identified outside the normal proliferative ventricular zone (VZ), representing a novel cause of this disorder. We studied Eml1 function in radial glial progenitors (RGCs), which are important during corticogenesis generating other subtypes of progenitors and post-mitotic neurons, and serving as guides for migrating neurons. We showed that Eml1 localizes to the mitotic spindle where it might regulate microtubule dynamics. My data suggest a role in the establishment of the steady state metaphase spindle length. Indeed, HeCo RGCs in the VZ showed a perturbed spindle length during corticogenesis, and this may represent one of the primary mechanisms leading to abnormal progenitor behavior. I also analyzed cell number and metaphase cell size at the apical side of the VZ, where mitosis occurs. I thus propose new mechanisms governing normal and pathological VZ progenitor organization and function during cortical development.

Η δημιουργία του εγκεφαλικού φλοιού στηρίζεται στη διαδοχική εμφάνιση πληθυσμών προγονικών νευρώνων, οι οποίοι δίνουν γένεση σε νευρικά και γλοιακά κύτταρα. Κατά την νευρογένεση όλοι οι νευρώνες του εγκεφαλικού φλοιού προέρχονται από το νευροεπιθήλιο που βρίσκεται δίπλα από τις πλευρικές κοιλίες. Τα νευροεπιθηλιακά κύτταρα αρχικά διαιρούνται με σκοπό την δημιουργία ικανού αριθμού πρόδρομων κυττάρων που θα δώσουν γένεση στον αναπτυσσόμενο φλοιό. Αργότερα, τα κύτταρα αυτά, διαφοροποιούνται προς τις άλλες κατηγορίες πρόδρομων κυττάρων και προς τους διαφοροποιημένους νευρώνες. Η πρωτεΐνη Geminin έχει προταθεί ως ένα μόριο που ρυθμίζει τόσο τον κυτταρικό πολλαπλασιασμό όσο και την κυτταρική διαφοροποίηση. Προκειμένου να διερευνηθεί ο ρόλος της πρωτεΐνης Geminin in vivo στη δημιουργία νευρώνων, πραγματοποιήθηκαν πειράματα υπερέκφρασης της Geminin στον αναπτυσσόμενο εγκεφαλικό φλοιό του μυός κατά την Ε14.5 dpc. Τα αποτελέσματα της παρούσας εργασίας δείχνουν ότι η υπερέκφραση της Geminin οδηγεί στην αύξηση του αριθμού των κυττάρων της ανώτερης στοιβάδας και μείωση του αριθμού των κυττάρων της κατώτερης στοιβάδας. Συνοψίζοντας, τα αποτελέσματά μας προτείνουν ότι η Geminin συμμετέχει στη ρύθμιση του πληθυσμού των φλοιϊκών νευρώνων. / Cortical development is a highly ordered process, involving the timely orchestration of the appearance of different neural progenitor lineages, which succeed one another in order to generate the neurons and glia comprising the cortex.During neurogenesis, the cortical neurons are originated from the neuroepithelium that lies next to the lateral vesicles. At the beginning, neuroepithelial cells divide in order to expand their population and to create the number of progenitor cells that would give rise to the neurons and glia that comprise the cortex. Geminin has been shown to regulate cell proliferation, fate determination and organogenesis, representing a potential link between these processes. In order to investigate the in vivo role of Geminin in the creation of the cortical neurons, we performed overexpression experiments with of Geminin in the developing mouse cortex. Our results indicate that overexpression of Geminin in the developing cerebral cortex increases the number of the upper layer cells and reduces the number of the deep layer cells at E14.5 dpc. Our work suggests that Geminin is a molecule that participates in the regulation of the correct number of cortical progenitors and neurons in the cerebral cortex.

No description available.

La migration radiaire est un des processus clefs de la corticogenèse menant à l’établissement d’un cortex en six couches chez les mammifères. La compréhension de ce mécanisme complexe est nécessaire à une meilleure appréhension du développement cortical. Dans ce travail de thèse, j’ai étudié la migration des neurones pyramidaux du cortex sous deux angles distincts. La 1ère partie se place d’un point de vue développemental en appréciant comment le positionnement laminaire résultant d’une migration normale ou anormale affecte la maturation neuronale. La 2nde partie se concentre sur une pathologie migratoire, l’hétérotopie en bande sous-corticale, et les altérations cognitives parfois associées à cette malformation. Pour ces deux projets, la migration neuronale a été altérée chez le rat par knockdown (KD) in utero de la doublecortine (Dcx), un effecteur majeur de la migration. Les neurones positionnés anormalement présentent une orientation incorrecte, un arbre dendritique moins développé, une spinogenère réduite et une altération morpho-fonctionnelle de la synaptogenèse glutamatergique. De plus, notre étude a mis en évidence l’implication de Dcx dans la dendritogenèse et la régulation fine des synapses glutamatergiques in vivo. Enfin, nous avons utilisé les rats Dcx-KD comme modèle d’hétérotopie en bande afin d’étudier comment un déficit de migration neuronale impacte le fonctionnement du cortex. La caractérisation comportementale, réalisée à l’aide d’une large gamme de tests, n’a pas mis en évidence de déficits majeurs des capacités motrices, somatosensorielles ou cognitives chez ces animaux. / Radial migration is one of the key processes leading to the formation of a six-layered cortex in mammals. Understanding this mechanism is necessary to get a better grasp of cortical development. During my PhD, I studied neuronal migration of pyramidal neurons from two different points of views. The 1st part is related to fundamental biology and assesses how laminar misplacement resulting from migration failure influences neuronal maturation. The 2nd one focuses on pathology by investigating a migration disorder, subcortical band heterotopia, and associated cognitive deficits. For both projects, neuronal migration was impaired in rat through in utero knockdown (KD) of doublecortin (Dcx), a major effector of cortical migration. Misplaced neurons display an abnormal orientation, a simplified dendritic arbor, a decreased spinogenesis and morpho-functional alterations of glutamatergic synaptogenesis. Moreover, our study shows that Dcx plays a role in dendritogenesis, in shaping spine morphology and in fine-tuning glutamatergic synaptogenesis. Finally, we used Dcx-KD rats as an animal model of subcortical band heterotopia to assess how migration failure would impact cortical functions. The behavioral characterization carried out through a wide range of tests did not bring to light any major shortcoming regarding motor, somatosensory or cognitive abilities in these animals. Therefore, although Dcx-KD rats display a SBH and develop spontaneous seizures, it does not seem to recapitulate cognitive deficits found in patients.