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Der Schlaganfall stellt weltweit eine der häufigsten Todesursachen dar und führt häufig zu einer lebenslangen Behinderung und Hilfsbedürftigkeit im Erwachsenenalter, was die hohe sozioökonomische Bedeutung des Schlaganfalls bedingt. Jenseits rekanalisierender Maßnahmen sind jegliche Ansätze mit dem Ziel, in der Akutphase des Schlaganfalls medikamentös zu intervenieren gescheitert. Daher konzentriert sich die Schlaganfallforschung aktuell vor allem auf die subakute- sowie chronische Phase nach Schlaganfall (Endres et al., 2008). Insbesondere die Schädigung des Gefäßsystems spielt nach Schlaganfall eine entscheidende Rolle. Neuroprotektion und Regeneration durch Wiederherstellung und Stabilisierung der vaskulären Integrität stellen dabei sicherlich einen der vielversprechendsten Ansätze dar. Der vaskuläre endotheliale Wachstumsfaktor (VEGF) ist bekanntermaßen ein hochpotenter proangiogener und neuroprotektiver Faktor. Jedoch wird beim Schlaganfall der Einfluss von VEGF aufgrund der VEGF vermittelten Störung der Blut-Hirn-Schranke, dadurch gesteigerter Ödementwicklung, erhöhter Inflammation und letztlich schlechterer Prognose sehr ambivalent diskutiert. Der Zeitpunkt sowie die Route der VEGF-Applikation scheint hier unter anderem eine entscheidende Rolle zu spielen (Hermann et al., 2009, Geiseler et al., 2018). Aus klinischer Sicht ist VEGF v.a. als Therapieansatz nach Schlaganfall hochinteressant. Ziel der Studie war daher, die Wirkung von VEGF auf das Schlaganfallvolumen und die Ödementwicklung sowie den Effekt auf das vaskuläre Umgestaltung nach Schlaganfall zu untersuchen. Methodisch wurde ein etabliertes Modell zur Okklusion der distalen Arteria cerebri media (dMCAO) benutzt. Ein einzigartiges bi-transgenes Mausmodell ermöglichte die konditionale und hirnspezifische VEGF-Expression, sog. GOF- Tiere sowie eine Inhibition von VEGF durch Expression eines Decoy-Rezeptors, sog. LOF- Tiere (Licht et al., 2011). VEGF wurde in der subakuten Phase nach Schlaganfall induziert bzw. geblockt. Das Schlaganfallvolumen wurde mittels MRT zum Zeitpunkt 24 h und 96 h bestimmt, die immunhistochemische Auswertung erfolgte zum Zeitpunkt 14 und 28 Tage nach Schlaganfall. Die Auswertung der MRT Untersuchungen zeigte weder Unterschiede in der Infarktgröße noch der Ödementwicklung zwischen den GOF- und LOF-Tieren. Die immunhistochemischen Auswertungen mit Ki67/CD31 zeigten dahingegen in den GOF-Tieren in allen Hirnarealen und unabhängig von der Hemisphäre einen signifikanten Anstieg an proliferierenden Endothelzellen und somit eine signifikant erhöhte Dichte an Blutgefäßen. In LOF-Tieren blieb das Gefäßnetz trotz VEGF- Blockade erhalten und so fanden sich zu den Kontrolltieren keine signifikanten Unterschiede in der Gefäßdichte. Die Auswertung von CD31- sowie Desmin-positiven Zellen zeigte sowohl kortikal, als auch striatal eine hohe Perizyten-Endothelzell-Interaktion und somit Dichtigkeit der Gefäße. Unterschiede zwischen den Zeitpunkten 14 und 28 Tage nach Schlaganfall zeigten sich nicht. Zusammenfassend zeigen die Ergebnisse dieser Studie, dass die VEGF Überexpression in der subakuten Phase nach Schlaganfall weder die Läsionsgröße noch Ödementwicklung negativ beeinflusst. Mit dem hier vorgestellten multimodalen Ansatz konnte gezeigt werden, dass VEGF zu einem robusten proangiogenen Effekt führt, einhergehend mit einer hohen Perizyten-Endothelzell-Interaktion. Die Gabe von VEGF in der subakuten Phase nach Schlaganfall kann daher als ein vielversprechender therapeutischer Ansatz angesehen werden., Stroke is among the most common causes of death and morbidity worldwide. As stroke very often leads to lifelong disability, its socioeconomic burden is highly relevant. So far, beyond actions for recanalization, any type of approach going on for drug intervention in the acute phase of stroke failed. Therefore, stroke research is currently concentrating on the subacute and chronic phase after stroke (Endres et al., 2008). The damage and impairment of the vascular system plays an important role after stroke. Thus, neuroprotection and regeneration via restoration and stabilisation of the vascular integrity are certainly among the most promising approaches. The vascular endothelial growth factor (VEGF) is a well known highly potent proangiogenic and neuroprotective factor. However, VEGF may also cause disturbances of the blood brain barrier, increased edema development, aggravate inflammation and finally may be associated with a poorer outcome in ischemic stroke. This causes conflicting discussions about the effects of VEGF. The time point as well as the route of VEGF application seem to play an important role for VEGF mediated effects (Hermann et al., 2009, Geiseler et al., 2018). From a clinical point of view, VEGF is highly interesting as a therapeutic approach especially after stroke. The aim of the study was to investigate the effect of VEGF on stroke volume, edema development as well as the effect on vascular remodelling in the subacute phase after stroke. A unique bi-transgenic mouse model enabled the conditional and brain specific VEGF expression in so called GOF animals while the inhibition of VEGF through the expression of a decoy receptor in so called LOF animals (Licht et al., 2011). Distal middle cerebral artery occlusion (dMCAO) was induced using a well established murine model. VEGF was induced or blocked in the subacute phase after stroke. The stroke volume was assessed by magnetic resonance imaging (MRI) 24 and 96 hours after ischemia. Immunohistochemical analysis was performed 14 and 28 days after stroke. The analysis of the MRI studies showed neither a difference between the infarct size nor the evolution of edema between GOF and LOF animals. The immunohistochemical analysis with Ki67/CD31 in GOF animals revealed a significant increase in proliferating endothelial cells in all brain areas and a significant higher number of vessels. Despite the VEGF-blockage in LOF animals the vascular network maintained unchanged comparing the number of vessels from LOF and control animals. Analysis of CD31 and desmin positive cells showed very high pericyte and endothelial cell interaction and thus no leaky vessels. This was observed in the cortex as well as in the striatum. There were no differences between the time points 14 and 28 days after stroke. In summary the results of this study show that VEGF overexpression in the subacute phase after stroke does neither influence the size of the ischemic lesion nor the evolution of edema in a negative way. With the presented multimodal approach it was possible to clearly show that VEGF induces a robust proangiogenic effect. The administration of VEGF in the subacute phase after stroke is a promising therapeutical approach.

Morphogènes et morphogenèse Le cours de la chaire des Processus morphogénétiques portait cette année sur la question des morphogènes. La première définition du terme de morphogène nous vient d’un article de 1952 d’Alan Turing « Les bases moléculaires de la morphogenèse ». Pour Turing, il s’agit d’évocateurs de formes, en référence aux évocateurs de Waddington. Il s’agit donc là d’une définition assez vague, très éloignée des critères un peu contraignants qui président aujourd’hui au classemen...

Orientadores: José Antônio Rocha Gontijo, Patrícia Aline Boer Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: Há diversas razões pelas quais têm se buscado o melhor entendimento de alterações causadas pela programação fetal. Entre estas emerge o fato de que estas alterações têm apresentado repercussões evidentes sobre a saúde de populações. Além disso, alterações durante o desenvolvimento embrionário e fetal, podem estar entre as principais causas de doenças metabólicas e endócrinas tais como obesidade, hipertensão arterial, resistência periférica a insulina e doenças cardiovasculares. Isto se deve a manifestação de alterações no desenvolvimento ontogenético, vinculadas à manifestação programada do desenvolvimento morfológico e funcional de órgãos e sistemas. Considerando o fato de que o desenvolvimento estrutural do cérebro se inicia nos primeiros dias do período embrionário e se estende durante os anos iniciais de vida extrauterina, alterações durante períodos críticos do desenvolvimento pré- e pós-natal podem ser altamente prejudiciais para o desenvolvimento desta estrutura. O hipocampo é uma estrutura alvo para diversas alterações provenientes do ambiente materno. Estudos têm mostrado que alterações durante o período pré-natal tiveram influência sobre a neurogênese no hipocampo imaturo, bem como sobre a remodelação de dendritos da região CA3, com possíveis alterações neurocognitivas. Adicionalmente, diferentes sistemas de neurotransmissores, bem como alterações epigenéticas de moduladores do desenvolvimento neural, podem estar implicadas na causa ou consequência desta programação. Para tanto, é importante conhecer quais períodos exatos nos quais o cérebro é susceptível a estas influencias e qual a repercussão destes fatores sobre a estrutura hipocampal. Assim, este trabalho teve como objetivo, avaliar os efeitos da restrição proteica materna sobre o hipocampo da prole de ratos machos com 14 e 40 dias pós-natal. Ratas Wistar foram submetidas a dieta hipoproteica durante toda a gestação. A prole de machos com 14 e 40 dias de vida foram estudadas analisando-se as expressões de genes nos subcampos hipocampais (CA1, CA3 e GD) associando possíveis alterações nestas áreas á respostas encontradas nos modelos de restrição proteica gestacional comparadas àquelas observadas em um grupo controle de mesma idade. Nossos resultados mostraram que animais com 14 dias de vida, submetidos à restrição proteica gestacional mostraram uma diminuição significativa na expressão de DCX no GD e em CA3, 5HT1A no GD e em CA1 e, aumento significativo de MR em CA3; além disso, diminuição destes receptores no GD, associado a uma elevação de CDKN1C em CA1, bem como em AT4 em CA1 e AT1 em CA3. Já com 40 dias observamos elevação significativa da DCX no GD associada a diminuição de SOX nesta mesma região. Tais resultados sugerem que o desenvolvimento de circuitos neuronais no hipocampo está associado com a expressão distinta de genes nas diferentes regiões anatomicas do hipocampo. Também, que a sequencia de eventos incluindo proliferação cellular, migração e diferenciação estão associadas a esta expressão gênica distinta. Além disso, os danos morfológicos e funcionais hipocampais precoces, provenientes da restrição proteica gestacional, podem estar relacionados a danos estruturais temporários. Estes, entretanto, parecem ser revertidos por modificaçoes na expressão gênica que modulam a produção de proteínas e neurotransmissores envolvidos na recuperação cellular destas áreas cognitivas do hipocampo Abstract: There are several reasons why we have sought the best understanding of the changes caused by fetal programming. Among them are the fact that these changes have had evident repercussions on the health of populations. In addition, changes during embryonic and fetal development may be among the leading causes of metabolic and endocrine diseases such as obesity, hypertension, insulin resistance and cardiovascular disease. This is due to changes in ontogeny development, linked to the scheduled manifestation of changes in the morphological and functional development of organs and systems. Considering the fact that the structural development of the brain begins in the early days of the embryonic period and extends into the first years of life, alterations during critical periods of pre and postnatal development can be highly detrimental to the development of this structure. The hippocampus is a target structure for several changes from the maternal environment. Studies have shown that alterations during the prenatal period had influence on neurogenesis in the immature hippocampus, as well as remodeling of the dendrites of the CA3 region, with possible cognitive alterations. In addition, there are several neurotransmitter systems, as well as epigenetic alterations of neural development modulators, which may be implicated in the cause or consequence of this programming. Therefore, it is important to know in what exact periods the brain is more susceptible to these influences and what their repercussion on the hippocampal structure. Thus, this thesis aimed to evaluate the effects of maternal protein restriction on the hippocampus of offspring of male rats at 14 and 40 days postnatal. Female Wistar rats were submitted to a hypoprotein diet throughout pregnancy. In the offspring of males with 14 and 40 days of age, the expression of genes in the hippocampal subfields (CA1, CA3 and GD) that could be modulating the responses found in the gestational protein restriction models were analyzed. Our results showed that animals with 14 days of life undergoing gestational protein restriction had a significant decrease in the expression of DCX in GD and in CA3, of 5HT1A in GD and in CA1. Additionally, the study showed a significant increase of MR in CA3, but with a decrease in GD, Increase in CDKN1C in CA1, as well as in AT4 in CA1 and AT1 in CA3. At 40 days, we observed a significant increase of DCX in GD and a decrease of SOX in this same region. These results suggest that the development of neuronal circuits in the hippocampus is associated with distinct gene expression in the different anatomical regions of the hippocampus. Also, the sequence of events including cell proliferation, migration and differentiation are associated with this distinct gene expression. In addition, early morphological and functional hippocampal damage from gestational protein restriction may be related to temporary structural damage. These, however, appear to be reversed by modifications in gene expression that modulate the production of proteins and neurotransmitters involved in the cellular recovery of these cognitive areas of the hippocampus Doutorado Fisiopatologia Médica Doutora em Ciências FAPESP 2013/20539-1 CAPES

Orientador: Lício Augusto Velloso Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: Em modelos experimentais de obesidade induzida por dieta rica em gordura há desenvolvimento de resistência hipotalâmica à leptina decorrente da ativação de uma resposta inflamatória específica no hipotálamo. Estudos recentes revelaram que além da indução da inflamação, a dieta rica em gordura ativa também vias de sinalização apoptóticas que culminam com a morte de neurônios em regiões hipotalâmicas envolvidas com o controle da fome e do gasto energético. Acredita-se que a perda de sub-populações específicas de neurônios no hipotálamo acarrete em dano dos mecanismos de controle do balanço entre consumo calórico e gasto energético, resultando no desenvolvimento ou perpetuação da obesidade. A composição de ácidos graxos da dieta tem papel central na resposta inflamatória hipotalâmica, sendo os ácidos graxos saturados de cadeia longa os mais lesivos. Por outro lado, em outras regiões do cérebro, alguns ácidos graxos insaturados podem desempenhar papel anti-inflamatório e induzir reparo funcional através da neurogênese. Neste trabalho, nós avaliamos os efeitos dos ácidos graxos poliinsaturados ?-linolênico (ALA C18:3-n3), e docosaexaenoico (DHA 22:6-n3) sobre a indução de neurogênese no hipotálamo de animais com obesidade induzida por dieta rica em gordura (HFD). Nossos resultados mostram que ácidos graxos ômega-3 administrados tanto na dieta ou diretamente no cérebro, são capazes de modular a neurogênese no hipotálamo de camundongos obesos, favorecendo a proliferação de neurônios anorexigênicos POMC, mas sem redução da ingestão alimentar. Embora esses animais não comam menos, eles se movimentam mais e exibem um discreto aumento do gasto energético. A suplementação com ômega-3 em HFD reduz a inflamação e a apoptose no hipotálamo, além de melhorar o metabolismo glicêmico e proteger os animais do ganho de peso. Aparentemente, os neurônios recém-formados surgem a partir da proliferação de células com características de células progenitoras, presentes na parede do terceiro ventrículo. Nesse processo, o receptor de ácidos graxos livres, GPR40, e o fator neurotrófico derivado do cérebro, BDNF, desempenham papel fundamental, mas a participação dessas proteínas em tais eventos parece ser dependente do estado nutricional e metabólico do animal Abstract: In experimental models of high-fat diet (HFD)-induced obesity there is a development of hypothalamic leptin resistance due to the activation of a specific inflammatory responsein the hypothalamus. Recent studies have revealed that besides the induction of inflammation, a fat-rich diet can also activate apoptotic signaling pathways that culminate in neuronal death in hypothalamic areas responsible for the control of feeding and energy expenditure. It is believed that the loss of specific hypothalamic neuronal subpopulations deteriorates mechanisms involved in the control of caloric intake and energy expenditure, resulting in the development and perpetuation of obesity. The fatty acid composition of diet, particularly the saturated fatty acid content, can modulate immune activity in the hypothalamus, inducing an inflammatory response. However, in other regions of the brain, some unsaturated fatty acids can acts as anti-inflammatory agents and induce functional repair through neurogenesis. In this study we evaluated the effects of polyunsaturated fatty acids ?-linolenic (ALA C18:3-n3) and docosahexaenoic (DHA 22:6-n3) on the induction of hypothalamic neurogenesis in HFD-induced obese animals. Our results show that omega-3 fatty acids administered either in the diet or directly into the brain are able to modulate neurogenesis in the hypothalamus of obese mice, leading to the proliferation of anorexigenic POMC neurons. This effect is not accompanied by changes in food intake, but increase unstimulated physical activity resulting in a discrete increase in energy expenditure. In addition, omega-3 supplemented HFD reduces inflammation and apoptosis in the hypothalamus, improve glucose metabolism and protect the mice against body mass gain. Apparently, these newly formed neurons arise from progenitor cell proliferation present in the wall of the third ventricle. In this process, free fatty acid receptor, GPR40, and brain-derived neurotrophic factor, BDNF, play a key role; however, the involvement of these proteins in such events appears to be dependent on the nutritional and metabolic status of the animal Doutorado Biologia Estrutural, Celular, Molecular e do Desenvolvimento Doutor em Ciências

Orientadores: Fabio Rogério, Carlos Amilcar Parada Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: A injeção de ácido quinolínico (AQ), um agonista glutamatérgico do receptor N-metil-D-aspartato, no estriado de roedores induz morte seletiva de neurônios espinhosos médios, gliose reativa e neurogênese na zona subventricular, acompanhada da migração dos neurônios recém-gerados para o estriado lesado. Tais achados são também descritos na doença de Huntington (DH). Há indícios de que a via de sinalização JAK/STAT esteja envolvida no mecanismo de ação do AQ, bem como na patogênese da DH. A interação das citocinas da família da IL-6 com seus receptores desencadeia a ativação de enzimas da família das Janus-Quinases (JAKs), que por sua vez permitem o recrutamento e a ativação de fatores de transcrição da família das proteínas transdutoras de sinais e ativadoras da transcrição (STATs). Embora as principais características da DH sejam a presença da coréia e déficits na execução de movimentos voluntários, poucos testes são realizados abordando o comportamento locomotor dos animais no modelo de lesão por AQ. Neste trabalho, estudamos o efeito do AG490, um inibidor da JAK2, na gliose, perda neuronal e neurogênese no estriado de camundongos adultos C57BL/6J após a administração estereotáxica unilateral de AQ (30nmol). Imediatamente após a lesão, os animais receberam uma injeção subcutânea de AG490 (10mg/kg) ou veículo (PBS+DMSO), e injeções diárias por 6 dias adicionais. Além disso, investigamos o possível efeito da lesão por AQ na atividade física voluntária diária (AFVD) em rodas de atividade. A distância percorrida pelos camundongos foi monitorada por 28 dias após a injeção unilateral de QA (30nmol) ou PBS no estriado. Cortes coronais do cérebro (40?m) obtidos em criostato foram utilizados para quantificação de neurônios por estereologia e para a análise de expressão protéica, através de imunoistoquímica e Western Blotting para GFAP e doublecortina, marcadores de gliose e neuroblastos, respectivamente. A área total de células doublecortina-positivas (ACDP) e o número de neurônios (NN) no lado lesado (L) e contralateral à lesão (CL) foram avaliados. O Índice de Neurogênese (IN=ACDP(L)/ACDP(CL)) e o Índice de Sobrevivência Neuronal (ISN=NN(L)/NN(CL)) foram calculados. Após a administração de AQ, o estriado ipsilateral apresentou intensa gliose e células doublecortina positivas com características de células migratórias. O Western Blotting para GFAP mostrou uma redução ipsilateral de 19% nos animais tratados com AG490, em comparação aos animais do grupo tratado apenas com veículo (0.82±0.05; 1.010±0.06, n=9, p0.05). Portanto, nossos resultados suportam um papel para a JAK2 na morte neuronal, gliose, e neurogênese estriatais após lesão com AQ. O tratamento com o inibidor AG490 causou neuroproteção e diminuição da gliose, sugerindo que a reação astrocitária pode prejudicar a sobrevivência neuronal neste modelo experimental Abstract: Injection of quinolinic acid (QA), a N-methyl-D-aspartate receptor agonist, in murine striatum induces death of medium spiny neurons, gliosis and neurogenesis in the subventricular zone with migration of newly synthesized neurons to damaged striatum. Such findings are also described in Huntington's disease (HD). The Janus-kinase (JAK) pathway would take part in QA mechanism of action and HD pathogenesis as well. The interaction of interleukin-6 family of cytokines with its receptor triggers the activation of enzymes of the family of JAKs, which in turn allow the recruitment and activation of transcription factors, known as signal transducers and activators of transcription (STATs). Although the main features of HD are the presence of chorea and deficits in performing voluntary movements, few tests are realized regarding locomotor behavioral on QA model. We studied the effect of AG490, an inhibitor of JAK isoform 2 (JAK2), on gliosis, neuronal loss and neurogenesis in the striatum of adult C57BL/6J mice after unilateral estereotaxic administration of QA (30 nmol). Immediately after injury, animals received a subcutaneous injection of AG490 (10 mg/kg) or vehicle (PBS + DMSO), and then once daily injections for 6 days. Furthermore, in a parallel experiment, we investigated the possible effect of the lesion by AQ on the voluntary daily physical activity (VDPA) in running wheels. The distance traveled by mice was monitored daily for 28 days after unilateral injection of QA (30 nmol) or PBS into the striatum. Frozen brain sections (40?m) were used for neuronal stereological quantification and immunohistochemical and Western Blotting analyses for GFAP and doublecortin, markers of gliosis and neuroblasts, respectively. The total area of doublecortin-positive cells (ADPC) and the number of neurons (NN) in the lesioned (L) and contralateral (CL) sides were evaluated. Neurogenesis index (NI = ADPC in L/ ADPC in CL) and neuronal survival ratio (NSR = NN in L/ NN in CL) were calculated. After QA administration, ipsilateral striatum showed intense gliosis and doublecortin-positive cells with few processes and ovoid bodies, morphological features corroborating a migratory activity. Western Blotting for GFAP showed an ipsilateral decrease of 19% in AG490- vs vehicle-treated animals (0.82 ± 0.05 vs 1.010 ± 0.06; n=9, p 0.05). In conclusion, our results support a role for JAK2 in striatal neuronal death, gliosis and neurogenesis determined by QA. AG490 caused neuroprotection and reduced gliosis suggesting that astrocytic reaction may impair neuronal survival in the present experimental model Mestrado Fisiologia Mestre em Biologia Funcional e Molecular

Orientadores: Iscia Teresinha Lopes Cendes, André Schwambach Vieira e Fabio Rossi Torres Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: As displasias corticais focais (DCFs) são uma importante causa de epilepsia refratária às drogas antiepilépticas e nossos objetivos neste trabalho foram ajudar a elucidar os mecanismos moleculares envolvidos na DCF tipo II e, identificar e validar biomarcadores não invasivos para serem utilizados no diagnóstico desta malformação cortical, usando para essas duas abordagens os microRNAs. Foi utilizado o tecido cerebral de pacientes com DCF tipo II submetidos à cirurgia para controles das crises epilépticas e tecido cerebral de indivíduos controles; além de plasma sanguíneo desses mesmos pacientes e de pacientes com epilepsia do lobo temporal mesial, bem como de indivíduos normais. Foram utilizadas as seguintes técnicas neste trabalho: microarranjos de microRNAs, PCR quantitativa, hibridização in situ, ensaios de gene repórter e sequenciamento de nova geração. Identificamos três microRNAs, hsa-let-7f, hsa-miR-31 e hsa-miR34a, com a expressão diminuída nos tecidos do pacientes. Em contrapartida, observamos um aumento da expressão de um fator de transcrição envolvido na diferenciação neuroglial, NEUROG2. Também encontramos o gene RND2, um alvo de NEUROG2, com a expressão aumentada, e sabe-se que tem um papel fundamental na migração radial dos neurônios corticais. Experimentos in vitro mostraram que NEUROG2 é regulado na região 5¿ não traduzida por hsa-miR-34a. Além disso, foi observada uma forte expressão nuclear de NEUROG2 em células aberrantes presentes neste subtipo da DCF. Juntos, esses achados indicam que provavelmente há uma falha na regulação pós-transcricional da expressão de NEUROG2 e que isso tem uma repercussão em cadeia, levando a falhas tanto no processo de diferenciação neuroglial como de migração neuronal na DCF tipo II, o que pode explicar a presença de células aberrantes e a epileptogênese nesta malformação cortical. Outro achado importante, em nosso estudo, consistiu da identificação do hsa-miR-134 como potencial biomarcador não invasivo para ajudar no diagnóstico de pacientes com epilepsia do lobo temporal mesial. Este microRNA circulante no plasma sanguíneo é capaz de discriminar o grupo de pacientes do grupo de controles com uma área sob a curva (AUC) de 0.75, independentemente da sua resposta ao tratamento medicamentoso ou à presença de sinais de esclerose hipocampal nos achados de ressonância magnética. Com isso, por meio dos microRNAs, foi possível sugerir um mecanismo molecular envolvido na DCF tipo II, bem como identificar um microRNA circulante com potencial uso no diagnóstico de epilepsia do lobo temporal Abstract: Focal cortical dysplasias (FCDs) are an important cause of drug-resistant epilepsy and our goals were to help elucidate the molecular mechanisms involved in FCD type II and to identify and validate reliable non-invasive biomarkers to be used in diagnosis of epilepsy related with this cortical malformation, using for these two approaches microRNAs as a molecular tool. We used brain tissue from patients with FCD type II and normal controls, besides blood plasma was used from patients with FCD type II, patients with mesial temporal lobe epilepsy (MTLE), as well as controls individuals. Microarrays of microRNAs, quantitative PCR, in situ hybridization, luciferase reporter assays and exome sequencing was performed to validate and further explore our data. We identified hsa-let-7f, hsa-miR-31 and hsa-miR34a were downregulated in tissue from FCD type II, whereas a transcription factor involved in neuronal and glial fate specification, NEUROG2, was found upregulated. We also found RND2 gene, a NEUROG2-target, upregulated, which has a key role in radial migration of cortical neurons. In vitro experiments showed that hsa-miR-34a downregulates NEUROG2 by binding to its 5'-untranslated region. Moreover, we observed strong nuclear expression of NEUROG2 in aberrant cells present in FCD type II, balloon cells and dysmorphic neurons. Together, these findings indicate that a faulty coupling in neuronal differentiation and migration mechanisms may explain the presence of aberrant cells and intrinsic epileptogenesis in this cortical malformation. Another important finding, in our study, was the identification of the downregulation of hsa-miR-134 as a potential non-invasive biomarker to support the diagnosis of patients with MTLE. This circulating microRNA could be used to discriminate MTLE patients with an area under the curve (AUC) of 0.75, regardless of their response to pharmacotherapy or the presence of MRI signs of hippocampal sclerosis. Together, these findings allowed us to suggest a potential molecular mechanism involved in FCD type II, due to microRNAs, as well to identify a circulating microRNA as biomarker of epilepsy Doutorado Fisiopatologia Médica Doutora em Ciências CAPES

Genetic studies have long suggested the important role of the DRD2 gene in psychiatric disorders and behavior. Further research has shown a conjoined effect of genes in the Chr11q22-23 region, which includes the NCAM1, TTC12, ANKK1 and DRD2 genes, or NTAD cluster. Despite a growing need to unravel the role of this cluster, few studies have taken into account interspecies and evolutionary approaches. This study shows that behaviorally relevant SNPs from the NTAD cluster, such as rs1800497 (Taq1A) and rs6277, are ancient polymorphisms that date back to the common ancestor between modern humans and Neanderthals/Denisovans. Conserved synteny and neighborhood indicate the NTAD cluster seems to have been established at least 400 million years ago, when the first Sarcopterygians emerged. The NTAD genes are apparently co-regulated and this could be attributed to adaptive functional properties, including those that emerged when the central nervous system became more complex. Finally, our findings indicate that NTAD genes, which are related to neurogenesis and dopaminergic neurotransmission, should be approached as a unit in behavioral and psychiatric genetic studies.

Differents elements exterieurs au systeme nerveux central (SNC) peuvent influencer son fonctionnement et sa reponse a des agressions. D’une part, l’alimentation, selon sa composition, peut augmenter ou diminuer les risques de neurodegenerescence. D’autre part, des etudes epidemiologiques et des experiences sur des modeles animaux montrent que le surpoids et l’obesite augmentent le risque de developper des maladies neurodegeneratives (Alzheimer, Parkinson…) ainsi que la susceptibilite a des neurodegenerescences aigues (traumatisme, ischemie, chimique…). La neurogenese constitutive ou induite dans le SNC adulte represente un espoir therapeutique permettant de compenser les pertes liees aux phenomenes neurodegeneratifs. Cependant, meme s’il a ete montre que l’alimentation peut affecter la production de nouveaux neurones, l’influence de l’exces de tissu adipeux sur la neurogenese n’est pas encore determinee.

Orientador: Marcio Alberto Torsoni Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: Estudos mostram que o consumo materno de dieta hiperlipídica (HFD) é capaz de prejudicar a formação de circuitos neuronais hipotalâmicos em proles de camundongos, contribuindo para um fenótipo hiperfágico que pode estar relacionado com obesidade na vida adulta. Este distúrbio parece ser mediado pela ativação da via de sinalização Notch1 levando à repressão de fatores proneurais como Mash1 e Ngn2/3 que são importantes na neurogênese hipotalâmica e diferenciação neuronal. O objetivo do estudo foi avaliar os efeitos do consumo materno de dieta hiperlipídica durante a gestação e lactação sobre a via Notch1 no hipotálamo e no núcleo arqueado hipotalâmico (ARC) da prole de camundongos. Para isso utilizou-se proles de camundongos Swiss (machos) de mães alimentadas com HFD e com ração comercial durante a gestação e lactação. Foram avaliados neonatos (P0), proles com 28 dias de vida (P28) e proles adultas (P82). Determinou-se peso corporal e adiposidade das proles. Investigou-se os níveis de mRNA dos genes que codificam proteínas Notch1, Hes5 e Mash1, citocinas pró-inflamatórias Il-1b e TNF-a e neuropeptídios AgRP e POMC. Fez-se análise do conteúdo das proteínas da via Notch1 e das proteínas inflamatórias JNK e NF-kB. Avaliou-se a proliferação celular por marcação com BrDU e a presença de Mash1 e neurônios recém-formados, por imunohistoquímica. Além de avaliar a população de neurônios POMC e NPY no ARC da prole de camundongos, por imunofluorescência. Observou-se maior peso corporal e adiposidade nas proles de mães alimentadas com HFD. Notou-se também aumento nos níveis hipotalâmicos de mRNA de Il-1?. Acompanhado de aumento nos níveis de mRNA de Hes5 e redução nos níveis de mRNA de Ascl1 (Mash1) no hipotálamo e ARC das proles de mães HFD. Além disso, os dados demonstraram maior número de células marcadas com BrDU seguido de aumento na população de neurônios NPY ao passo que a população de neurônios POMC apresentou-se diminuída no ARC das proles de mães HFD. Os resultados apontam alterações na neurogênese hipotalâmica e diferenciação neuronal, levando a uma redução de neurônios POMC que parece ser mediada pela supressão de Mash1, acompanhada de aumento compensatório de neurônios NPY. Estas alterações contribuem para hiperfagia e aumento do peso corporal, podendo compor um dos mecanismos responsáveis pela obesidade na vida adulta Abstract: Studies show that maternal high-fat diet (HFD) consumption can be able to impair the formation of hypothalamic neuronal networks in mice offspring contributing to a phenotype of hyperphagia that can be related to obesity in adulthood. This disturbance seems to be mediated by Notch1 signaling pathway activation leading to the repression of proneural factors as Mash1 and Ngn2/3 which are important in hypothalamic neurogenesis and neuronal differentiation. The aim of the study was to evaluate the maternal HFD consumption effects during gestation and lactation on the Notch1 signaling in the hypothalamus and hypothalamic arcuate nucleus (ARC) of mice offspring. To do this, we used mice offspring Swiss (males) from dams fed with HFD and standard chow (SC) during pregnancy and lactation. It was evaluated neonates (P0), young and adult offspring (P28 and P82, respectively). We determined body weight and adiposity of mice offspring, the mRNA levels of genes that code Notch1, Hes5 and Mash1 proteins; pro-inflammatory cytokines as Il-1b and TNF-a, and neuropeptides as AgRP and POMC. We evaluated the content of Notch1 pathway proteins and inflammatory proteins as JNK and NF-kB. To measure the cell proliferation, we used BrDU labeling. We analyzed presence of Mash1 and newborn neurons by immunohistochemistry. Besides, we measured the population of POMC and NPY neurons in the ARC of mice offspring by immunofluorescence. We observed an increased body weight and adiposity in mice offspring from dams fed with HFD. We noticed an increase in the mRNA levels of Il-1? accompanied by higher mRNA levels of Hes5 and lower mRNA levels of Ascl1 (Mash1) in the hypothalamus and ARC of mice offspring from HFD dams. In addition, data showed higher number of BrDU-labeled cells followed by increase in the NPY neurons population and reduction in the POMC neurons population in the ARC of these mice offspring. The results indicate alterations in the hypothalamic neurogenesis and neuronal differentiation leading to a reduction of POMC neurons that seems to be mediated by Mash1 suppression accompanied by compensatory increase of NPY neurons. These alterations contribute to hyperphagia and increase of body weight that could be one of the responsible mechanisms for obesity in adulthood Doutorado Fisiologia Doutora em Biologia Funcional e Molecular CAPES 94328641115 FAPESP FAEPEX CNPQ

Temporal lobe epilepsy (TLE), the most frequent type of focal epilepsy in adults, is characterized by resistance to pharmacological treatment, progressive hipocampal neuronal loss and impairment of cognitive and neuropsychological performances. Studies with human specimens and experimental animal models have demonstrated exuberant and dynamic neuronal plasticity phenomena in the hipocampal formation associated with TLE. The study of these phenomena is fundamental for the definition of the genesis and progression mechanisms of epilepsy and for the development of better therapeutic strategies than those current available. In this article, we review neuronal and glial plastic alterations as neuronal death and birth, dendritic spine formation, axonal sprouting, gliosis and gap junction establishment and their role in the pathophysiology of TLE.

Orientador: Fabio Papes Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: Em mamíferos, os neurônios sensoriais do Sistema Olfatório (OSNs) se encontram no interior da cavidade nasal, mas estão diretamente expostos ao ambiente externo. Por um lado, tal localização permite a esses neurônios o acesso imediato aos estímulos químicos ambientais, tomando vantagem do fluxo respiratório. Por outro lado, esses neurônios estão constantemente sujeitos a injúrias por agentes nocivos, como toxinas e patógenos, capazes de destruir essas células sensoriais. Sua perda constante, contudo, é contrabalanceada pela geração de novos OSNs durante toda a vida do indivíduo, fato que torna o Sistema Olfatório um dos poucos locais do organismo com neurogênese contínua na idade adulta. A regeneração dos OSNs tem atraído a atenção da comunidade científica tanto pelo seu potencial uso como modelo de estudo do Sistema Nervoso quanto pela sua potencial aplicação para o tratamento de doenças neurodegenerativas. Nesse sentido, muito conhecimento já foi produzido sobre a dinâmica de fatores de transcrição que acompanha a diferenciação dos progenitores neuronais olfatórios em OSNs. Porém, uma grande lacuna no conhecimento diz respeito a outros elementos capazes de coordenar esse processo, como os fatores moduladores da cromatina. Diante desse cenário, escolhemos como objeto de estudo as proteínas do Grupo Polycomb (PcG), que constituem uma maquinaria de controle transcricional relacionada a modificações na organização da cromatina. Neste trabalho, genes PcG selecionados foram caracterizados molecular e funcionalmente no epitélio olfatório principal de camundongos (MOE). Através de ensaios de hibridação in situ, cinco dos seis genes avaliados apresentaram expressão ubíqua por todo o epitélio (Cbx2, Cbx4, Phc2, Ezh1, Bcl6), enquanto um (Ezh2) mostrou-se expresso somente nos estratos basais do MOE. Em ensaios de colocalização, provamos que Ezh2 é expresso exclusivamente nos progenitores olfatórios, onde o processo de diferenciação se inicia, e em parte dos OSNs recém-diferenciados, ainda não funcionais. Esta foi a primeira vez que a expressão de um gene PcG foi analisada detalhadamente no Sistema Olfatório. O interessante perfil de expressão de Ezh2 foi sugestivo de um possível papel funcional relacionado à diferenciação dos progenitores olfatórios. Para investigar essa hipótese, utilizamos como ferramenta experimental a habilidade do MOE em se regenerar após a indução de injúrias específicas. Para isso, o MOE de camundongos foi lesionado quimicamente com o composto diclobenil, que leva à perda abrupta de OSNs, estimulando a proliferação e a diferenciação dos progenitores olfatórios para repovoar as regiões lesionadas. Os animais assim tratados receberam, por via intranasal, o fármaco GSK126, uma molécula inibidora específica da atividade da proteína EZH2. Acompanhando a regeneração subsequente do MOE, observamos que a inibição da atividade de EZH2 levou ao incremento de OSNs no epitélio, favorecendo a sua regeneração. Interessantemente, esse incremento também foi observado em MOEs não lesionados, mostrando que o efeito de GSK126 não é dependente da indução de injúrias prévias. Através dessa investigação molecular e funcional, buscamos contribuir para o melhor entendimento da diferenciação neuronal do MOE, e apontamos as proteína PcG como elementos importantes para esse processo Abstract: In mammals, the olfactory sensory neurons (OSNs) are located inside the nasal cavity, but they are directly exposed to the external environment. Taking advantage of the respiratory flux, this location favors the access to the chemical stimuli presented by the environment. On the other hand, it leads OSNs to be continually damaged by pathogens and toxic substances carried by the inhaled air. However, the persistence of neuronal progenitors in the olfactory epithelium makes the constant reposition of the OSNs possible. This unique ability of regeneration makes the Olfactory System one of the few sites of neurogenesis throughout the adult life. Olfactory regeneration has attracted the attention scientific community because of its potential as a model of study of the Nervous System and application in the treatment of neurodegenerative diseases. A great amount of knowledge has been accumulated about the transcription factor dynamics that follows the differentiation of neuronal progenitors into OSNs. However, there is a great gap about other elements that could coordinate this process, such as chromatin modulator factors. In this scenario, we decided to study the Polycomb Group (PcG) proteins, a transcription control machinery involved in chromatin structure organization. In the present study, selected PcG genes were molecular and functionally analyzed in the mouse main olfactory epithelium (MOE). Using in situ hybridization assays, we characterized the expression of six PcG genes. Five of them were shown to be expressed throughout the MOE (Cbx2, Cbx4, Phc2, Ezh1, Bcl6), while one (Ezh2) was found only in the basal layers of this epithelium. Using colocalization strategies, we proved that Ezh2 gene is expressed exclusively in the olfactory progenitor cells, where the differentiation process begins, and in part of the newly differentiated OSNs that are still not functional. It was the first time that a PcG gene expression profile was finely analyzed in the Olfactory System. This interesting expression profile presented by Ezh2 suggested a possible involvement with the MOE neuronal progenitor differentiation. For this functional investigation, we used MOE¿s neuronal regeneration after specific injuries as an experimental tool. For this purpose, the MOE was chemically damaged by the compound dichlobenil, which causes a great loss of OSNs, stimulating proliferation and differentiation of neuronal progenitor cells, leading to the repopulation of the damaged tissue. Next, mice received by intranasal route the pharmacological inhibitor GSK126, which blocks EZH2 protein activity. The observation of the MOE regeneration that followed showed us that GSK126 application resulted in an increased number of OSNs, improving MOE regeneration. Interestingly, this increase was also found in intact MOEs, pointing that GSK126¿s effects do not depend on previous olfactory injuries. By this molecular and functional investigation, we aimed at a better understanding of olfactory neuronal differentiation, and we targeted the PcG proteins as relevant elements to this process Mestrado Genética Animal e Evolução Mestre em Genética e Biologia Molecular

Orientador: Jose Antonio Rocha Gontijo Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: O estresse gestacional afeta diversas regiões neurais incluindo hipocampo, amígdala, corpo caloso, neocortéx, cerebelo e hipotálamo e frequentemente resulta em redução no volume dos tecidos que compõem estas estruturas. A formação hipocampal tem sido alvo de diversos estudos devido a sua importância na plasticidade neural, na neurogênese e na regulação de processos cognitivos. Dessa forma, este estudo buscou avaliar os efeitos da restrição proteica, durante a gestação e amamentação, sobre a estrutura do hipocampo e o comportamento relacionado à memória e emoções (ansiedade/medo) bem como sobre a composição celular desta estrutura cerebral e, a influência sobre estes parâmetros morfológicos e comportamentais, da exposição da prole de ratos machos ao ambiente enriquecido. Os achados deste estudo representam o impacto pré e perinatal da desnutrição proteica correspondente à situação de estresse nutricional, no hipocampo que está envolvido no comportamento emocional bem como na memória e no aprendizado. O estudo revelou dissociação entre a resposta do teste comportamental e alterações no número de neurônios hipocampais, como consequência da programação fetal. A ausência de alterações basais no desempenho destes testes, ocorreram a despeito de redução no número de neurônios no giro denteado do hipocampo. Vários autores têm sugerido que a atrofia observada no hipocampo pode ser uma resposta compensatória para proteger o hipocampo de danos adicionais. Nós demonstramos, pela primeira vez, que a exposição materna a restrição proteica durante o desenvolvimento neural da prole causa importantes mudanças morfológicas no hipocampo podendo tornar estes animais vulneráveis a distúrbios neurais na idade adulta. O presente estudo pelo menos sob aspecto morfológico ponderal confirma a teoria do "cérebro egoísta", um paradigma recente que postula que, para manter estável seu próprio fornecimento de energia, o cérebro modula o metabolismo da energia na periferia regulando tanto a alocação quanto a ingestão de nutrientes. Neste trabalho, a ausência de alterações ponderais encefálicas não está associada às intensas modificações na composição citológica, particularmente hipocampal, nos diferentes grupos experimentais. Embora pareça que as alterações nutricionais promovam alterações irreversíveis ponderais na massa corporal, mas não no encéfalo e algumas de suas estruturas fundamentais, a composição e estrutura neuronal e sua recuperação a partir de células primordiais, são profundamente modificadas pela restrição dietética materna e, surpreendentemente, pela exposição ao ambiente enriquecido. Assim, podemos afirmar que a teoria do cérebro egoísta explica a manutenção da massa encefálica entretanto, a proporção dos diferentes tipos celulares é profundamente alterada o que pode expandir nosso entendimento sobre a adaptação ao estresse e a neuro-regeneração em estados neuro-comportamentais tidos como anormais. Além disso, devemos ressaltar que, embora tenhamos observado redução significativa no número de neurônios após o período de amamentação, demonstramos pela primeira vez que este parâmetro é revertido pelo estimulo em ambiente enriquecido Abstract: he stress affects neural regions including gestational hippocampus, amygdala, corpus callosum, neocortex, cerebellum and hypothalamus and often results in a reduction in the volume of the tissues that make up these structures. The hippocampal gyrus training has been the subject of several studies due to its importance in neural plasticity in neurogenesis and regulation of cognitive processes. Thus, this study sought to assess the effects of protein restriction, during pregnancy and breastfeeding, on the structure of hippocampus, their duties on the memory and emotions (anxiety/fear) as well as on the cellular composition of this brain structure and influence over these morphological and behavioral parameters, the exposure of the offspring of male rats to the enriched environment. The findings of this study represent the pre and perinatal impact of malnutrition protein corresponding to situation of nutritional stress in the hippocampus, which is involved in emotional behavior as well as in memory and learning. The study revealed decoupling the behavioral test response and changes in the number of hipocampais neurons, as a consequence of fetal programming. The absence of basal changes in performance of these tests, occurred in spite of reduction in the number of neurons in the dentate gyrus of the hippocampus. Several authors have suggested that the observed atrophy in the hippocampus may be a compensatory response to protect the hippocampus of additional damage. We have demonstrated, for the first time, that maternal exposure to protein restriction during neural development of offspring cause important morphological changes in hippocampus may make these animals vulnerable to neural disorders in adulthood. The present study at least under morphological aspect by confirming the "selfish brain" theory, a recent paradigm that posits that in order to keep stable its own energy supply, the brain modulates the energy metabolism in the periphery by regulating both the allocation as the intake of nutrients. In this work, the unmodified brain mass, do not match with the intensity of cytological composition changes, particularly of the hippocampal nucleus, in different experimental groups. Although it seems that the nutritional changes promote irreversible changes in body mass, but not in the brain and some of its fundamental structures, composition and neuronal structure and its recovery from primordial cells, are deeply modified by the maternal dietary restriction and, surprisingly, by exposure to oxygen-enriched environment. Thus, we can affirm that the selfish brain theory explains the maintenance of brain matter however, the proportion of the different cell types is profoundly changed what can expand our understanding of the adaptation to stress and neuro-regeneration in neuro-behavioral States regarded as abnormal. Moreover, we must emphasize that, while we have observed a significant reduction in the number of neurons after the period of breastfeeding, we demonstrate for the first time this parameter is reversed by stimulus in enriched Mestrado Fisiopatologia Médica Mestre em Ciências CAPES

In stroke patients with severe persistent neurological deficits, alternative therapeutic modalities are limited. Stem cell therapy might be an opportunity when the safety profile of this approach will be achieved. This review will give possible mechanisms of restoration of function in animals and a statement of clinical trials in humans. The sources of neural stem cells for therapeutic use will be detailed. Potentials mechanisms of transplanted cell-mediated recovery are described with a particular emphasis on ipsilesional post-stroke plasticity. The optimal conditions for cell transplant therapy after stroke are evoked but not yet clearly defined. Finally, since multimodality imaging will be crucial in the post-transplantation patient assessment, the final part describes recent advances in the in vivo monitoring of repair progress.

Le but des recherches du laboratoire de génétique du développement est de mieux comprendre les mécanismes moléculaires qui contrôlent le développement neural des vertébrés. C’est la raison pour la quelle, j’ai identifié deux EST (BC071005 et BC077938) spécifiques de l’expression génique chez le Xenopus laevis. Sur base de la littérature, ces deux gènes présentent des profils d’expression intéressants, caractéristiques des gènes impliqués dans la neurogenèse. Le premier clone d’ADNc code pour l’homologue du facteur de transcription Homez, contenant trois homéodomaines et deux motifs leucine zipper et dont la fonction est inconnue. Mes résultats ont montré que chez l’embryon de xénope au stade neurula, Homez est exprimé préférentiellement dans la plaque neurale, l’expression la plus forte étant détectée dans les domaines où les neurones primaires apparaissent. Plus tard, Homez est détecté dans le tube neural dans des cellules neurales postmitotiques en cours de différenciation. En accord avec ce profil d’expression, j’ai observé que Homez est régulé positivement par l’atténuation des signaux BMPs et par le facteur proneural Ngnr1 et négativement par la voie Notch. Bien que le facteur Homez ne soit pas suffisant pour induire une expression ectopique de marqueurs neuronaux dans l’embryon de xénope, j’ai pu montrer, en utilisant une approche de morpholino antisens, que celui-ci est requis en aval du facteur Ngnr1 pour la différenciation des précurseurs neuraux en neurones primaires. Le deuxième clone code pour l’homologue du facteur CBFβ qui s’associe avec une famille de protéines CBFα1-3/Aml1-3/Runx1-3 pour former un complexe hétérodimérique liant l’ADN. Alors que chez la souris, les facteurs Runx1 et Runx3 jouent un rôle important dans la neurogenèse dans les ganglions spinaux et que chez le xénope, Runx1 est requis pour la formation des neurones Rohon-Beard, le rôle de CBFβ au cours du développement du système nerveux est actuellement mal connu. Mes résultats ont montré que chez l’embryon de xénope au stade neurula, CBFβ est coexprimé avec les facteurs Runx1-3 en bordure de la plaque neurale, mais de manière plus étendue et plus précoce. Comme attendu pour un marqueur de la bordure de la plaque neurale, j’ai observé que l’expression de CBFβ est régulée par les signaux BMP, Wnt, FGF et Notch. De manière intéressante, son expression est induite par les facteurs proneuraux alors que celle de Runx1 est inhibée. Des expériences de perte de fonction à l’aide de morpholinos antisens bloquant la traduction de CBFβ ont été réalisées. Ces expériences suggèrent que le facteur CBFβ est nécessaire à la mise en place de la CN et à la différenciation des neurones de Rohon-Beard., Doctorat en Sciences, info:eu-repo/semantics/published

International audience; Increased consumption of high-fat diet (HFD) leads to obesity and adverse neurocognitive outcomes. Childhood and adolescence are important periods of brain maturation shaping cognitive function. These periods could consequently be particularly sensitive to the detrimental effects of HFD intake. In mice, juvenile and adulthood consumption of HFD induce similar morphometric and metabolic changes. However, only juvenile exposure to HFD abolishes relational memory flexibility, assessed after initial radial-maze concurrent spatial discrimination learning, and decreases neurogenesis. Our results identify a critical period of development covering adolescence with higher sensitivity to HFD-induced hippocampal dysfunction at both behavioral and cellular levels.

vorgelegt von: Stephan Wagner Zusammenfassung in deutscher und englischer Sprache Molecular Biotechnology Masterarbeit FH Campus Wien 2011

A dysfunction of retinoid hippocampal signaling pathway has been involved in the appearance of affective and cognitive disorders. However, the underlying neurobiological mechanisms remain unknown. Hippocampal granule neurons are generated throughout life and are involved in emotion and memory. Here, we investigated the effects of vitamin A deficiency (VAD) on neurogenesis and memory and the ability of retinoic acid (RA) treatment to prevent VAD-induced impairments. Adult retinoid-deficient rats were generated by a vitamin A-free diet from weaning in order to allow a normal development. The effects of VAD and/or RA administration were examined on hippocampal neurogenesis, retinoid target genes such as neurotrophin receptors and spatial reference memory measured in the water maze. Long-term VAD decreased neurogenesis and led to memory deficits. More importantly, these effects were reversed by 4 weeks of RA treatment. These beneficial effects may be in part related to an up-regulation of retinoid-mediated molecular events, such as the expression of the neurotrophin receptor TrkA. We have demonstrated for the first time that the effect of vitamin A deficient diet on the level of hippoccampal neurogenesis is reversible and that RA treatment is important for the maintenance of the hippocampal plasticity and function.

It is well established that persistent viral infection may impair cellular function of specialized cells without overt damage. This concept, when applied to neurotropic viruses, may help to understand certain neurologic and neuropsychiatric diseases. Borna disease virus (BDV) is an excellent example of a persistent virus that targets the brain, impairs neural functions without cell lysis, and ultimately results in neurobehavioral disturbances. Recently, we have shown that BDV infects human neural progenitor cells (hNPCs) and impairs neurogenesis, revealing a new mechanism by which BDV may interfere with brain function. Here, we sought to identify the viral proteins and molecular pathways that are involved. Using lentiviral vectors for expression of the bdv-p and bdv-x viral genes, we demonstrate that the phosphoprotein P, but not the X protein, diminishes human neurogenesis and, more particularly, GABAergic neurogenesis. We further reveal a decrease in pro-neuronal factors known to be involved in neuronal differentiation (ApoE, Noggin, TH and Scg10/Stathmin2), demonstrating that cellular dysfunction is associated with impairment of specific components of the molecular program that controls neurogenesis. Our findings thus provide the first evidence that a viral protein impairs GABAergic human neurogenesis, a process that is dysregulated in several neuropsychiatric disorders. They improve our understanding of the mechanisms by which a persistent virus may interfere with brain development and function in the adult., Author Summary When a virus enters the brain, it most often induces inflammation, fever, and brain injury, all signs that are indicative of acute encephalitis. Under certain conditions, however, some neurotropic viruses may cause disease in a subtler manner. The Borna disease virus (BDV) is an excellent example of this second class of viruses, as it impairs neural function without cell lysis and induces neurobehavioral disturbances. Recently, we have shown that BDV infects human neural progenitor cells (hNPCs) and impairs neurogenesis, revealing a new mechanism by which BDV may interfere with brain function. In the present study, we identify that a singled-out BDV protein called P causes similar impairment of human neurogenesis, and further show that it leads to diminution in the genesis of a particular neuronal subtype, the GABAergic neurons. We have also found that the expression of several genes involved in the generation and the maturation of neurons is dysregulated by this viral protein, which strongly suggests their implication in P-induced impairment of GABAergic neurogenesis. This study is the first to demonstrate that a viral protein interferes with human GABAergic neurogenesis, a process that is frequently impaired in neuropsychiatric disorders. It may thus contribute to elucidating the molecular bases of psychiatric disorders.

Au cours de la différenciation neuronale, les gènes proneuraux induisent l'expression de nombreux gènes appartenant à différentes familles. Deux de ces familles constituent l'intérêt de cette étude à savoir les facteurs de transcription à doigts à zinc Myt/NZF et IA1/INSM1. Chez le xénope, il a été démontré que XMyT1 coopère avec les facteurs bHLH afin d'induire la neurogenèse de manière insensible à la voie de signalisation Delta/Notch (Bellefroid et al. 1996). Son mode d'action n'est pas connu et nécessite d'être approfondi afin de mieux comprendre son rôle au cours de la neurogenèse. Lors d'expériences utilisant un gène rapporteur, la protéine XMyT1 a été décrite comme activateur de la transcription tandis que la protéine orthologue NZF3 chez le rat se comporte comme répresseur de la transcription (Yee et al. 1998). Récemment, il a été rapporté que les protéines NZF1 et NZF2 chez la souris interagissent avec le corépresseur Sin3 (Romm et al. 2005). Néanmoins aucunes données n'existent concernant l'interaction entre Myt/NZF et d'autres co-facteurs. Suite à la recherche de partenaires protéiques en utilisant la technique du double hybride en levures, nous avons identifié le corépresseur XCtBP. Nous avons confirmé in vivo et in vitro que XMyT1 interagit avec XCtBP. Cette interaction est propre à la région centrale de XMyT1 et trois motifs de recrutement de CtBP ont été identifiés dans celle-ci à savoir PGDLS, PENLS et TLDLS. Le motif de liaison TLDLS constitue un site dégénéré non décrit dans la littérature. Ce motif est conservé évolutivement parmi les trois membres NZF1, NZF2 et NZF3. Aussi, le motif TLDLS présente le plus d'affinité pour XCtBP. Les parties N-terminale, C-terminale et centrale de XMyT1 se comportent comme répresseur de la transcription et la région centrale continue de réprimer la transcription même lorsqu'elle n'est plus capable de recruter XCtBP. Cette activité est antagoniste à celle exercée par la protéine sauvage XMyT1. En effet, dans des expériences de gain de fonction, nous avons montré que XMyT1 induit l'expression des gènes XIA1, XPak3 et elrC. Nous avons montré que leur expression est induite de manière ectopique lorsque XMyT1 et XAsh3 sont surexprimés en même temps. Nous avons montré en embryons de xénope que XIA1 est exprimé dans les neurones primaires à partir du stade neurula précoce et qu'il a un profil d'expression similaire à XMyT1. Nos résultats ont permis de mettre en lumière une fonction biologique de l'interaction entre XMyT1 et XCtBP. Le recrutement de XCtBP réduit l'activation de l'expression de XIA exercée par la protéine XMyT1 seule ou combinée à XAsh3 et ce aux stades précoces de la différenciation neuronale. Ainsi, XCtBP jouerait un rôle dans la régulation transcriptionnelle de XMyT1 durant la neurogenèse. Cette étude suggère que XMyT1 pourrait également interagir avec des co-activateurs pour contrebalancer l'effet des co-répresseurs étant donné que la protéine sauvage a été décrite, à ce jour, aussi bien dans des expériences in vivo qu'in vitro, comme activatrice de la transcription., Doctorat en Sciences, info:eu-repo/semantics/published

Embryonen von Höheren Krebsen (Malacostraca) zeigen ein stereotypes Zellteilungsmuster im Ektoderm des Rumpfes, im Verlaufe dessen paarige seitliche Reihen von Zellen und eine unpaare mittlere Reihe von Zellen gebildet werden. Das Muster der seitlichen Zellen ist von Dohle (1970, 1976) und Mitarbeitern geklärt worden. Die vorliegende Arbeit untersucht die cell lineage und Zelldifferenzierung der Mittellinienzellen im Thorax. Diese Zellen sind von besonderem Interesse, weil sie bei Insekten bereits intensiv erforscht wurden. (i) Die DiI Markierungen von Mittellinienzellen von Orchestia cavimana zeigen: Die Bildung der Mittellinie beginnt mit einer Zelle, die sich zweimal in Längsrichtung teilt. Die resultierenden vier Zellen werden mit a0, b0, c0 und d0 bezeichtet. Aus den Zellen a0, b0 und c0 gehen Paare von Gliazellen hervor. Die Tochterzellen von a0 und c0 umhüllen die Kommissuren. Die Zelle d0 ist ein medianer Neuroblast, aus dem mehrere Neurone hervorgehen, unter anderem ein unpaares Neuron im medianen Fasertrakt, interneurone und ein Motoneuron. (ii)BrdU Markierungen von Porcellio scaber zeigen: In den Ganglienanlagen liegt in der Mittellinie je eine Zelle, die größer ist als die benachbarten, schneller proliferiert und deshalb vermutlich ein medianer Neuroblast ist. (iii) Die Expression von engrailed setzt bei Orchestia und Porcellio ein in der Zelle a0 und wird in den zwei Tochterzellen fortgesetzt. Für Orchestia wird gezeigt, daß diese Expression zurückgeht und die Tochterzellen der Zelle d0 de novo mit einer Expression von engrailed beginnen.Aus den Ergebnissen kann abgeleitet werden, daß der gemeinsame Vorfahr von Insekten und Höheren Krebsen eine Mittellinie differenziert, die Vorläufer für Glia der Kommissuren und einen medianen Neuroblasten umfaßt und eine Expression von engrailed in den Tochterzellen des Neuroblasten zeigt., Embryos of higher crustaceans (Malacostraca) show a highly stereotypic cell division pattern in the ectoderm of the trunk region while forming paired rows of lateral cells and an unpaired median row of midline cells. By using nuclear dyes, the pattern of the lateral cells has been determined by Dohle (1970, 1976) an co-workers. This study addresses the cell lineage and cell differentiation of the midline cells in the thorax. These kind of cells are of particular interest as they have been investigated extensively in insects. (i) The DiI labelling of midline cells in Orchestia cavimana reveals: Formation of the midline starts with a single midline cell that divides twice in longitudinal direction. The resulting four cells are termed a0, b0, c0, and d0. The cells a0, b0, and c0 give rise to pairs of glial cells. The progeny of a0 and c0 enwrap the commissures. The cell d0 is a median neuroblast that gives rise to several neurons, among them an unpaired neuron in the median fibre tract, interneurons and probably a single motoneuron. (ii) BrdU labelling in Porcellio scaber shows: there is a single larger and faster dividing cell in the midline in each segmental ganglion anlage that is a putative median neuroblast. (iii)The expression of engrailed starts in Orchestia and Porcellio the cell a0 and continues in the two daughter cells during segmentation. In Orchestia it can be shown that this expression ceases and progenies of the cell d0 start de novo with the expression of engrailed. From the results can be concluded that the common ancestor of insects and higher crustaceans differentiated an unpaired midline comprising precursors for glial cells enwrapping the commissures and a single median neuroblast whose derivatives express engrailed.

Neurogenesis is the ability of the adult brain to build new neurons. This process induces structural and functional changes in the brain that can reduce cognitive decline during aging. This neuroplasticity exists throughout life but it gradually decreases with aging. In this study, we characterized the evolution of cognitive functions and neurogenesis during aging in the grey mouse lemur (Microcebus murinus) that shares morphological, behavioural and physiological changes with aged humans. We observed that some aged animals presented a specific deficit in learning and memory whereas others had cognitive performances equivalent or better than young animals. It might be due to the neurogenesis process that would preserve cognitive functions during aging. Indeed, in the subventricular zone, the balance between neurons and glial cells would be in favour of neurogenesis in the dorsal part while oligodendrogenesis would be favoured in the horn. Stimulation of neurogenesis could help replace neurons lost due to injury or aging. Among the possible strategies to stimulate neurogenesis, food and physical activity seem pertinent. During this thesis project, we studied, in particular, the impact of n-3 polyunsaturated fatty acid supplementation and the combination of caloric restriction and physical activity in adulthood. These interventions induced an improvement of cognitive functions associated with an increase in the number of new neurons. These different approaches constitute a promising strategy without drugs against cognitive decline during aging by participating in brain plasticity.; La neurogenèse correspond à la capacité du cerveau à former de nouveaux neurones. Ce mécanisme permet d’induire des changements structurels et fonctionnels dans le cerveau pouvant atténuer le déclin cognitif observé avec l’âge. Cette neuroplasticité persiste à l’âge adulte mais diminue au cours de la vie. Au cours de ce travail, nous avons caractérisé l’évolution des fonctions cognitives et de la neurogenèse avec l’âge chez le Microcèbe (Microcebus murinus) qui présente des changements morphologiques, comportementaux et physiologiques similaires à ceux observés chez l’Homme au cours du vieillissement. Nous avons pu montrer qu’une partie des animaux âgés présentaient une diminution de leurs capacités cognitives tandis que d’autres ont conservé des performances à un niveau équivalent à celui des individus jeunes. Ce maintien des fonctions cognitives avec l’âge pourrait être dû en partie au processus de neurogenèse. En effet, au niveau de la zone sous-ventriculaire, la balance neurone/glie serait en faveur de la neurogenèse dans la partie dorsale tandis que l’oligodendrogenèse serait favorisée dans la corne. La stimulation de la neurogenèse pourrait permettre de remplacer les neurones lésés avec l’âge ou détruits par un traumatisme. Parmi les stratégies possibles pour stimuler ce mécanisme, l’alimentation et l’activité physique apparaissent être des interventions pertinentes. Au cours de ce travail de thèse, nous nous sommes intéressés, en particulier, à l’impact d’une supplémentation en acides gras polyinsaturés n-3 ainsi qu’à la combinaison de la restriction calorique et de l’activité physique à l’âge adulte. Ces interventions ont induit une amélioration des fonctions cognitives associée à une hausse du nombre de nouveaux neurones. Ces différentes approches constituent donc une stratégie non médicamenteuse prometteuse afin de lutter contre le déclin des fonctions cognitives au cours du vieillissement en participant à la plasticité cérébrale.