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Les mutations somatiques sur le variant d’histone H3.3 et les régulateurs épigénétiques associés à H3.3 ont été identifiés dans 30 % des glioblastomes pédiatriques. Ces mutations sont caractérisées par des substitutions d'acides aminés à des positions spécifiques dans la région N-terminale de l'histone H3.3 telles que la glycine 34 en valine/arginine (G34V/R), l'alanine 29 en proline (A29P), ou une haplo-insuffisance de la protéine Zinc Finger MYND-Type Containing 11 (ZMYND11). ZMYND11 est un co-répresseur de la transcription qui se lie spécifiquement à H3.3K36me3 pour moduler l'activité de l'ARN polymérase II. De plus, il est intéressant de mentionner que l’interaction entre ZMYND11 et H3.3K36me3 est altérée lorsque le résidu G34 est muté en G34V. Récemment, les mutations germinales H3.3G34V, H3.3A29P et ZMYND11 ont été identifiées chez des patients présentant une déficience neurologique. Nous émettons l'hypothèse que les mutants H3.3G34V et H3.3A29P empêchent ZMYND11 de se lier à H3.3K36me3 et pourrait converger mécaniquement avec la perte de fonction de ZMYND11, ce qui perturberait la neurogenèse. À l'aide de la technologie CRISPR Cas9, nous avons généré des modèles mutants isogéniques à partir de cellules souches pluripotentes (iPSC) pour H3F3B-A29P, H3F3B-G34V et ZMYND11-knock-out (KO). Par la suite, nous avons stimulé la différenciation de ces modèles vers des lignées neuronales afin d’identifier si ces mutations affectent la neurogenèse. Enfin, en utilisant des méthodes de séquençage à haut-débit nous avons analysé le profil épigénomique et transcriptomique pour déterminer comment l’interaction entre ZMYND11 et H3K36me3 est perturbée et à quels degrés ces mutations impactent sur les modifications post-traductionnelles des histones. Ce projet permettra de mieux comprendre les fonctions de ZMYND11 sur le remodelage de la chromatine et sa fonction biologique au cours du développement cérébral., Somatic mutations on the histone 3 variant H3.3 and H3.3-associated chromatin modifiers have been identified in 30% of pediatric high-grade gliomas (pHGG). The mutations are characterized by amino acid substitutions at specific positions within the histone H3.3 tail such as glycine 34 to valine/arginine (G34V/R), alanine 29 to proline (A29P), or haploinsufficiency of the chromatin reader Zinc Finger MYND-Type Containing 11 (ZMYND11). ZMYND11 is a transcriptional co-repressor that specifically reads H3.3K36me3 to modulate RNA polymerase II activity. Notably, binding of ZMYND11 to H3.3K36me3 is altered when G34 residue is mutated to G34V. Recently, germline mutations of H3.3G34V, H3.3A29P, and ZMYND11 have been identified in patients with Intellectual disability. We hypothesize that H3.3 G34V and H3.3A29P mutants impede the binding of ZMYND11 to H3.3K36me3 and may mechanistically converge with ZMYND11 loss-of-function mutation to perturb neurogenesis. Using CRISPR Cas9-mediated gene editing, we will generate isogenic human induced pluripotent stem cell (iPSC) models for H3F3B-A29P, H3F3B-G34V and ZMYND11-KO, and perform in vitro neural differentiation to identify whether specific neural lineages are affected. Next, using epigenomic and transcriptomic profiling we will study whether binding between ZMYND11 and H3K36me3 is disrupted, and the downstream impact on Post-Translational Modifications of histones (PTMs) and transcription. This project will lead to a better understanding of the crucial role of the chromatin reader ZMYND11 on chromatin remodeling and the biological function during neural development.

Während der embryonalen Neurogenese spielt die Repression neuraler Gene in nicht neuralen Zellen, sowie in neuralen Vorläuferzellen durch den REST (repressor element silencing transcription factor)-Komplex eine wichtige Rolle. Durch die schrittweise Inaktivierung diese Komplexes im Verlauf der Differenzierung werden neurale Genexpressionsprogramme gesteuert. Zusätzlich kommt bei der Kontrolle der räumlichen und zeitlichen Regulation der Genexpression während der Neurogenese verschiedenen miRNAs eine wichtige Rolle zu. So konnte in vorangegangenen Arbeiten im Zebrafischen gezeigt werden, dass miR-26b die Transkription eines wichtigen Effektorproteins des REST-Komplexes, CTDSP2 (C-terminal domain small phosphatases), während der Neurogenese negativ reguliert. Da darüber hinaus die miR-26 Repression zu einer stark verminderten neuronalen Differenzierung führte, kommt diesem regulatorischen Schaltkreis eine zentrale Rolle bei der Neurogenese im Zebrafisch zu. Die zusammen mit ihren Ctdsp-Wirtsgenen koexprimierte miR-26 Familie liegt in Vertebraten evolutionär hoch konserviert vor. Analog zum Zebrafisch konnte im murinen in vitro ES-Zell Differenzierungssystem gezeigt werden, dass miR-26 die Expression von Ctdsp2 reprimiert. Weiterhin konnte in diesem System gezeigt werden, dass auch Rest ein miR-26 Zielgen ist und dass der Verlust der miR-26 zu einem Arrest der differenzierenden Zellen im neuronalen Vorläuferstadium führt. Zusammengenommen deuten diese vorangegangenen Arbeiten auf eine zentrale Rolle der miR-26 während der Neurogenese hin. Die hier vorgestellte Arbeit zielte zunächst darauf ab die Regulation des REST-Komplexes durch die miR-26 auf molekularer Ebene besser zu verstehen. Der Verlust der miR-26 Bindestelle in der Ctdsp2 mRNA führte zu einer erhöhten Ctdsp2 Expression, beeinflusste aber nicht die terminale Differenzierung zu Neuronen. Im Gegensatz hierzu führte der Verlust der miR-26 Bindestelle in der Rest mRNA zu einem Arrest der Differenzierung im neuralen Vorläuferzellstadium. Zellen in denen die miR-26 Bindestelle in Rest deletiert war, zeigten zudem, genau wie miR-26 knockout (KO) Zellen, eine erhöhte Expression von REST-Komplex Komponenten, sowie eine verringerte Expression von REST-regulierten miRNAs. Zusammengenommen weisen diese Daten daraufhin, dass während der Neurogenese im Säugersystem die Inaktivierung von Rest durch miR-26 für die Maturierung von Neuronen eine zentrale Rolle spielt. Ein weiterer Fokus dieser Arbeit lag auf der Regulation der miR-26 Expression während der Neurogenese. Vorangegangene Arbeiten in nicht-neuronalen Zelltypen identifizierten die lnc (long-non-coding) RNA Malat1 als eine ce (competitive endogenous) RNA der miR-26. Um den Einfluss von Malat1 auf die miR-26 Expression während der Neurogenese zu untersuchen, wurde zunächst mittels CRISPR/Cas9 der vollständige Malat1-Lokus in ESCs deletiert. Der Verlust von Malat1 führte zu einer erhöhten Expression der miR-26 Familienmitglieder sowie deren Ctdsp-Wirtsgene. Weiterhin war die Proliferation von Malat1 KO neuronalen Vorläuferzellen stark vermindert, was mit einer Erhöhung der Frequenz seneszenter Zellen einherging. Durch die Inaktivierung von miR-26 in differenzierenden Malat1 KO ESCs konnte dieser proliferative Phänotyp aufgehoben werden. Darüber hinaus konnte eine verstärkte neuronale Differenzierung dieser Zellen beobachtet werden. Zusammenfassend zeigen diese Daten, dass neben der Regulation des REST-Komplexes durch miR-26 auch die Kontrolle des Zellzyklus über die Malat1-vermittelte Regulation der miR-26 in neuronalen Vorläuferzellen einen kritischen Schritt bei der Differenzierung von neuronalen Vorläuferzellen zu maturen Neuronen darstellt., During embryonic neurogenesis, repression of neural genes in non-neural cells, as well as in neural progenitor cells by the REST (repressor element silencing transcription factor) complex, plays an important role. The gradual inactivation of this complex during differentiation controls neural gene expression programs. In addition, different miRNAs play important roles in controlling the spatial and temporal regulation of gene expression during neurogenesis. For example, previous work in zebrafish demonstrated that miR-26b negatively regulates the transcription of a key effector protein of the REST complex, CTDSP2 (C-terminal domain small phosphatases), during neurogenesis. Since miR-26 repression also resulted in severely reduced neuronal differentiation, this regulatory circuit plays a central role in zebrafish neurogenesis. The miR-26 family, co-expressed with its Ctdsp host genes, is evolutionarily highly conserved in vertebrates. Analogous to zebrafish, miR-26 was shown to repress Ctdsp2 expression in a murine in vitro ESC differentiation system. Furthermore, in this system, it was shown that Rest is also a miR-26 target and that loss of miR-26 leads to arrest of differentiating cells at the neuronal progenitor stage. Taken together, these previous analyses suggest a central role for miR-26 during neurogenesis. The work presented here first aimed to better understand the regulation of the REST complex by miR-26 at the molecular level. Loss of the miR-26 binding site in Ctdsp2 mRNA increased Ctdsp2 expression but did not affect terminal differentiation into neurons. In contrast, loss of the miR-26 binding site in the Rest mRNA resulted in arrest of differentiation at the neural progenitor cell stage. Cells in which the miR-26 binding site was deleted in Rest also showed increased expression of REST complex components, as well as decreased expression of RESTregulated miRNAs, just like miR-26 knockout (KO) cells. Taken together, these data indicate that during mammalian neurogenesis, inactivation of REST by miR-26 plays a central role in the maturation of mammalian neurons. Another focus of this work was on the regulation of miR-26 expression during neurogenesis. Previous analyses in non-neuronal cell types identified the lnc(long-non-coding)RNA Malat1 as a ce(competitive endogenous)RNA of miR-26. To investigate the effect of Malat1 on miR-26 expression during neurogenesis, the complete Malat1 locus was deleted in ESCs using CRISPR/Cas9. Loss of Malat1 resulted in increased expression of miR-26 family members as well as their Ctdsp host genes. Furthermore, proliferation of Malat1 KO neural progenitor cells was greatly reduced, which was accompanied by an increase in the frequency of senescent cells. Inactivation of miR-26 in differentiating Malat1 KO ESCs abrogated this proliferative phenotype. In addition, increased neuronal differentiation of these cells was observed. In conclusion, these data demonstrate that in addition to regulation of the REST complex by miR-26, cell cycle control via Malat1-mediated regulation of miR-26 in neuronal progenitor cells is a critical step for the differentiation of neuronal progenitor cells into mature neurons.

Tese de doutoramento em Ciências da Saúde, A génese celular no cérebro de mamífero adulto desafiou o dogma de longa data de que o sistema nervoso central adulto era imutável e incapaz de gerar novas células. É amplamente aceite que o cérebro adulto possui habilidades dinâmicas de remodelação axodendrítica, bem como a capacidade de produzir novas células neuronais e gliais ao longo da vida em regiões restritas do cérebro, processo conhecido como neuroe gliogénese, respetivamente. O giro denteado (GD) do hipocampo adulto constitui uma das regiões do cérebro onde a citogénese ocorre ao longo da vida. Desde a descoberta da citogénese adulta no GD hipocampal, muito progresso foi feito na compreensão da capacidade de plasticidade neural cerebral, bem como as suas implicações comportamentais funcionais em contextos saudáveis e neuropatológicos, como na Depressão Maior. No entanto, ainda não compreendemos completamente os mecanismos moleculares subjacentes que controlam a formação e integração funcional de células recém-formadas. Algumas questões sobre a citogénese adulta ainda precisam de ser respondidas e totalmente compreendidas, tanto na Saúde quanto na Depressão Maior. Assim, nesta tese, procuramos entender o impacto funcional da citogénese hipocampal, bem como defeitos neste processo podem afetar a aquisição do desenvolvimento comportamental no cérebro saudável e deprimido. Para tal, estudamos como as deficiências da neurogénese hipocampal causadas pelo défice do fator de transcrição da proteína ativadora dois gama (AP2g) podem afetar o desenvolvimento de estados emocionais e processos de memória, bem como o seu impacto em um modelo de depressão induzida por stress. Ademais, desenvolvemos novas ferramentas genéticas virais para entender o envolvimento específico de astrócitos e astrogliogénese em contextos saudáveis e deprimidos. As evidências apresentadas nesta tese forneceram uma caracterização adicional da orquestração transcricional da neurogénese hipocampal pós-natal e a sua relevância comportamental e eletrofisiológica funcional em contextos basais e depressivos. Observamos um impacto progressivo e cumulativo da deficiência constitutiva de AP2g na neurogénese hipocampal, bem como alterações na conectividade límbicocortical, juntamente com prejuízos comportamentais funcionais em estados emocionais e processos de memória. Em um modelo de camundongo de depressão induzida por stress, mostramos que AP2g medeia o comportamento cognitivo, apontando para o surgimento de um fenótipo resiliente anteriormente desconhecido após deleção de AP2g em animais expostos ao stress. Ainda, o trabalho nesta tese permitiu a geração de novas ferramentas genético-virais para comprometer especificamente a astrogliogénese e os astrócitos no cérebro adulto. Prevemos que os conhecimentos aqui adquiridos abrirão caminho para futuros trabalhos, nomeadamente na área da citogénese hipocampal adulta no contexto da depressão., Cell genesis in the adult mammalian brain defied the long-held dogma that the adult central nervous system was immutable and incapable of generating new cells. It is widely accepted that the adult brain has dynamic axodendritic remodeling abilities, as well as the capacity of producing new neuronal and glial cells throughout life in restricted brain regions, a process known as neuro- and gliogenesis, respectively. The adult hippocampal dentate gyrus (DG) constitutes one of the brain regions where cytogenesis occurs throughout life. Since the discovery of adult cytogenesis in the hippocampal DG, much progress has been made in understanding brain neural plasticity capacity, as well as its functional behavioral implications in healthy and neuropathological contexts such as in Major Depression. Nonetheless, we still do not fully comprehend the underlying molecular mechanisms that control the formation and functional integration of newly formed cells. Some questions regarding adult cytogenesis remain to be answered and fully understood, both in Health and in Major Depression. As so, in this thesis, we sought to understand the functional impact of hippocampal cytogenesis, as well as how defects in this process might affect behavioral development acquisition in the healthy and depressed-like brain. To do so, we studied how hippocampal neurogenesis impairments caused by deficiency of the activating protein two gamma (AP2g) transcription factor can affect the development of emotional states and memory processes, as well as its impact on a stress-induced model of depression. Moreover, we developed new genetic viral tools to understand the specific involvement of astrocytes and astrogliogenesis in healthy and depressed-like contexts. Taken together, the evidence presented in this thesis provided further characterization of the transcriptional orchestration of postnatal hippocampal neurogenesis and its functional behavioral and electrophysiological relevance in basal and depressive-like contexts. We observed a progressive and cumulative impact of constitutive AP2g deficiency on hippocampal neurogenesis, as well as alterations on limbic-cortical connectivity, together with functional behavioral impairments in emotional states and memory processes. In a mouse model of stress-induced depression, we showed that AP2g mediates cognitive behavior, pointing to the emergence of a previously unknown resilient phenotype after deletion of AP2g in stress-exposed animals. Moreover, the work in this thesis enabled the generation of new genetic-viral constructs to specifically compromise astrogliogenesis and astrocytes in the adult brain. We anticipate that the knowledge acquired here will pave the way for future works, particularly in the field of adult hippocampal cytogenesis in the context of depression., Financial support of this work was provided by grants from the Foundation for Science and Technology (FCT/SFRH/BD/131278/2017 and COVID/BD/152412/2022 to Eduardo Campos; IF/01079/2014). Financial support was provided by grants from the BIAL Foundation (427/014), Nature Research Award for Driving Global Impact - 2019 Brain Sciences; the projects NORTE-01-0145-FEDER-000013 and NORTE-01-0145-FEDER-000023, supported by the Norte Portugal Region Operational Program (NORTE 2020), under the Portugal 2020 partnership agreement, through the European Regional Development Fund (ERDF); by National funds, through the Foundation for Science and Technology (FCT) - project UIDB/50026/2020 and UIDP/50026/2020; by ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI - Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122); by National funds, through the Foundation for Science and Technology (FCT) - project UIDB/50026/2020 and UIDP/50026/2020.

Die embryonale Neurogenese in Drosophila ist eine hochgradig koordinierte Abfolge von Zellschicksalsentscheidungen, die viele Ähnlichkeiten mit der Entwicklung des Nervensystems in Wirbeltieren aufweist. Diese Zellschicksalsentscheidungen sind räumlich und zeitlich koordiniert. Diese Zellen entstehen an stereotypen Positionen in jedem Segment und sind entlang zweier räumlicher Achsen angeordnet: der dorsoventralen und der anteroposterioren Achse. Neuroblasten teilen sich, um stereotype Zelllinien zu bilden, und die Zellen weisen charakteristische Zellmorphologien und -ziele auf, wobei die molekularen Mechanismen, die diese Merkmale bestimmen, noch weitgehend unbekannt sind. Jahrzehnte der Genetik haben einige Faktoren aufgedeckt, die für viele dieser Entscheidungen notwendig sind, aber ein Verständnis der einzelnen neurogenen Linien auf Genomebene war bis vor kurzem in vivo unmöglich. Ich habe mRNA aus Einzelzellen verwendet, um die Transkriptomdynamik von Schicksalsentscheidungen in der frühen Entwicklung des Nervensystems zu untersuchen. Mein Ziel ist es, zu entschlüsseln, wie sich Zellen unterscheiden, wenn Entscheidungen getroffen werden, die für die Entwicklung des Nervensystems wesentlich sind. Ich habe Transkriptomdaten von einzelnen Zellen aus Zehntausenden von Neuroblasten während der gesamten embryonalen Neurogenese erstellt. Es gelang mir, spezifische neurogene Populationen und ihre Genexpressionsprofile entlang ihrer Differenzierungswege zu identifizieren. Ich konnte die komplizierten zeitlichen Achsen, die das sich entwickelnde embryonale Nervensystem formen, teilweise entschlüsseln - ein Prozess, der von der Fliege bis zum Menschen konserviert ist. Diese Arbeit hat die Identifizierung lokalisierter Marker und sogar spezifischer Neuroblasten ermöglicht. Dieses Verständnis kann nun mit Informationen über die einzelnen Zellschicksale kombiniert werden, aus denen diese Neuroblasten hervorgehen, wie z. B. ihre spezifischen neuronalen und glialen Schicksale., Embryonic neurogenesis in Drosophila is a highly coordinated sequence of cell fate decisions that bears many similarities to the development of the nervous system in vertebrates. These cell fate decisions are spatially and temporally coordinated. These cells arise at stereotypic positions in each segment and are arranged along two spatial axes: the dorsoventral axis and the anteroposterior axis. Neuroblasts divide to give rise to stereotypic lineages and the cells exhibit characteristic cell morphologies, branching patterns, and targets, the molecular mechanisms that determine these characteristics are still largely unknown. Decades of genetics have uncovered some factors necessary for many of these decisions, but understanding individual neurogenic lineages at the genome level has been impossible in vivo until recently. I have used Single cell mRNA to study the transcriptome dynamics that accompany important fate decisions in early nervous system development. My goal is to decipher how cells differ when decisions are made that are essential for nervous system development. This knowledge is invaluable for developing models for the in vivo mechanisms that allow individual cells in the nervous system to specify and differentiate. I have generated transcriptome data of single cells from tens of thousands of neuroblasts throughout embryonic neurogenesis. I was able to identify specific neurogenic populations and their gene expression profiles along their differentiation pathways. I was able to partially decipher the intricate temporal axes that shape the developing embryonic nervous system, a process that is conserved from fly to human. Single-cell transcriptomics has enabled the identification of localized markers and even specific neuroblasts. This understanding can now be combined with information about the individual cell fates that give rise to these neuroblasts, such as their specific neuronal and glial fates.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES Toxoplasmosis is a parasitic disease caused by the obligate intracellular protozoan Toxoplasma gondii. Its characteristics of encystment and persistence, preferably in the central nervous system of mammals, are worrying, in addition to placental transmission deserves attention due to the possibility of causing miscarriages, malformations and behavioural disturbances. Brain development during embryogenesis is highly compromised by parasite, interfering with cell quantity, migration and differentiation of neural precursor cells. The role of the purinergic system in immune defence and neuroprotection in toxoplasmosis has been gaining attention, in addition to the fact that purinergic components are found during neurogenesis. Despite the serious pathogenicity of the parasite, with a wide distribution worldwide and a high prevalence of vertical transmission, there are no effective therapies in the chronic phase of the disease, as available drugs do not act on tissue cysts. The problems presented and the epidemiological data motivate this study and the search for new therapies to minimize the damage caused by the parasite in congenital infection, here we propose the use of curcumin as a possible treatment. Curcumin is a polyphenol found in higher concentrations in the roots of the Curcuma longa plant and is widely used in scientific literature due to its antioxidant and anti-inflammatory properties, in addition to its neuroprotective action in disorders involving the central nervous system and promising results in a few studies on toxoplasmosis. Despite these beneficial characteristics, curcumin has low bioavailability, with rapid metabolism and certain toxicity, mainly seen at the hepatobiliary and reproductive systems. In the case of the central nervous system, no studies are demonstrating safe concentrations/doses in healthy models. Since our general objective was to evaluate the effects of curcumin on purinergic signalling and neurogenesis of neural precursor cells in the telencephalon of embryos from infected and uninfected mice with Toxoplasma gondii, first of all, it was essential to study the impact of the use of this polyphenol and two curcumin nanocapsules in cells originating from the central nervous system. Exposure of treatments in microglia and neuron cell lines revealed that concentrations between 1-5 μM of curcumin, NC-PCL, and NC-EDG can be used in in vitro models of brain diseases, as they do not significantly affect cell homeostasis. Given this finding, we continued with the isolation of neural precursor cells infected with the VEG strain of Toxoplasma gondii. Our results complement the parasite interference in neural differentiation, but the use of curcumin was able to restore gliogenesis and further increase neurogenesis, with cell size recovery. Changes in the expression of purinergic receptors were also seen in the infection, the use of curcumin prevented an excessive immune response, with control of cell damage, by decreasing the expression of P2X7 and A2A receptors, as well as its neuroprotective action, was increased against receptor activation purinergic A1. The ERK ½ signalling pathway, responsible for the production of inflammatory mediators and cell proliferation, was regulated by curcumin to attenuate pro-inflammatory responses and control cell proliferation. Together, these results show promise for the use of curcumin for the treatment of congenital toxoplasmosis. A toxoplasmose é uma doença parasitária causada pelo protozoário intracelular obrigatório Toxoplasma gondii. Suas características de encistamento e persistência, preferencialmente no sistema nervoso central de mamíferos, são preocupantes, além disso a transmissão placentária merece atenção pela possibilidade de causar abortos, malformações e distúrbios comportamentais. O desenvolvimento cerebral durante a embriogênese é altamente comprometido pelo parasito, interferindo na quantidade celular, migração e diferenciação de células precursoras neurais. A participação do sistema purinérgico na defesa imune e neuroproteção na toxoplasmose, vem ganhando destaque, além de que componentes purinérgicos são encontrados na neurogênese. Apesar da grave patogenicidade do parasito, com ampla distribuição mundial e alta prevalência de transmissão vertical, não existem terapias eficazes diante da fase crônica da doença, uma vez que medicamentos disponíveis não atuam no cisto tecidual. As problemáticas apresentadas e os dados epidemiológicos motivam o estudo e a busca por novas terapias a fim de minimizar os danos gerados pelo parasito em uma infecção congênita, para isso propomos o uso da curcumina como um possível tratamento. A curcumina é um polifenol encontrado em maiores concentrações nas raízes da planta Curcuma longa e amplamente utilizado na literatura científica devido a suas capacidades antioxidante e anti-inflamatória, além da atuação neuroprotetora em distúrbios envolvendo o sistema nervoso central e resultados promissores em poucos estudos na toxoplasmose. Apesar dessas características benéficas, a curcumina apresenta baixa biodisponibilidade, com rápido metabolismo e certa toxicidade, vista principalmente a nível hepatobiliar e reprodutor. No caso do sistema nervoso central, não existem estudos demonstrando concentrações/doses seguras em modelos saudáveis. Uma vez que nosso objetivo geral foi avaliar os efeitos da curcumina na sinalização purinérgica e na neurogênese de células precursoras neurais do telencéfalo de embriões oriundos de camundongos infectados e não infectados com Toxoplasma gondii, antes de mais nada foi imprescindível estudar o impacto da utilização deste polifenol e de duas nanocápsulas de curcumina em células originárias do sistema nervoso central. A exposição dos tratamentos nas linhagens celulares de micróglia e neurônios revelou que as concentrações entre 1-5 μM, da curcumina, NC-PCL e NC-EDG, podem ser usadas em modelos in vitro de doenças cerebrais, pois não afetam consideravelmente a homeostase celular. Diante deste achado, seguimos com o isolamento das células precursoras neurais infectadas com a cepa VEG do Toxoplasma gondii. Nossos resultados complementam a interferência do parasito na diferenciação neural, mas o uso da curcumina conseguiu restaurar a gliogênese e aumentar ainda mais a neurogênese, com recuperação do tamanho celular. Alteração na expressão dos receptores purinérgicos também foram visualizados na infecção, o uso da curcumina impediu a resposta imune excessiva, com controle do dano celular, pela diminuição na expressão dos receptores P2X7 e A2A, assim como sua ação neuroprotetora foi aumentada frente a ativação do receptor purinérgico A1. A via de sinalização ERK ½, responsável pela produção de mediadores inflamatórios e proliferação celular, foi regulada pela curcumina a fim de atenuar as respostas pró-inflamatórias e controle da proliferação celular. Juntos, esses resultados demonstram-se promissores quanto ao uso da curcumina para tratamento da toxoplasmose congênita.

Obesity is a worldwide epidemic leading to many health concerns and is a risk factor for the development of type 2 diabetes. In Reunion Island, obesity and diabetes are widely spread among the population. Both diseases share several metabolic disorders and have been recently implicated in deteriorating brain health, contributing to cognitive impairments. The mechanisms behind the onset of altered brain homeostasis are not well understood. Besides, no therapy has yet been established to prevent brain disruptions. The aim of this thesis is to study the impact of obesity/prediabetes on brain homeostasis and cerebral plasticity, and then to alleviate these deleterious effects using medicinal plants from Reunion Island. To this end, we set up a diet-induced obesity (DIO) protocol in zebrafish (Danio rerio). Zebrafish recently emerges as a relevant model to mimic metabolic diseases (obesity and diabetes), and to investigate brain homeostasis and plasticity (i.e., blood-brain barrier (BBB) and neurogenesis). Our DIO model, established by overfeeding adult zebrafish for 4 weeks, resulted in metabolic disorders and loss of central nervous system (CNS) homeostasis. Indeed, DIO fish displayed increased body weight and body mass index (BMI), hyperglycemia, liver steatosis and disturbed redox balance. In the central nervous system, overfeeding led to BBB leakage, neuro-inflammation, cerebral oxidative stress and decreased neurogenesis. As well, a change in the locomotor behavior was observed in obese fish. In a next step, we tested the potential beneficial properties of two Reunionese plants registered in the French pharmacopeia: Antirhea borbonica (A. borbonica) and Psiloxylon mauritianum (P. mauritianum). Both plants were traditionally used for their positive effects on metabolic disruptions as “anti-diabetic” effects for A. borbonica and “anti-lipidemic” for P. mauritianum. However, the scientific data supporting these properties are lacking. First, the chemical analysis of aqueous extract of A. borbonica and P. mauritianum revealed their abundance in polyphenols, correlated to their antioxidant properties. LC-MS/MS analysis was used to determine the nature of the polyphenolic compounds in each extract. Next, we performed toxicity assays using OECD guidelines 36 (Organization for Economic Co-operation and Development) (OECD, 2013) and defined a maximum non-toxic concentration for each extract. The overnight treatment with aqueous extract of A. borbonica (0.5g/L) during the DIO protocol demonstrated its preventive properties against the deleterious effects on the CNS induced by overfeeding. Indeed, A. borbonica preserved the BBB function, prevented the increase in cerebral oxidative stress, neuro-inflammation and normalized neurogenesis. Similarly, the aqueous extract of P. mauritianum (0.25 g/L) was tested on adult DIO zebrafish and in high-fat diet (HFD) treated larvae. The treatment avoided lipid accumulation in HFD larvae. It also prevented body weight increase, BMI, hyperglycemia and liver steatosis in adult DIO zebrafish. Furthermore, brain homeostasis seems to be preserved probably through P. mauritianum anti-weight gain properties. We suggested that P. mauritianum could significantly affect lipid absorption and metabolism possibly through the modulation of gut microbiota. In conclusion, during this thesis, we have developed a simple and rapid overfeeding (DIO) model inducing peripheral and CNS disruptions similar to those encountered in mammals. For the first time, we studied the toxicity of aqueous extract of the two medicinal plants A. borbonica and P. mauritianum. We confirmed their beneficial effects on different metabolic parameters and on the brain using zebrafish model of obesity and prediabetes. Together, these data highlight the use of zebrafish to mimic metabolic diseases and to screen the beneficial properties of medicinal plants extracts., L'obésité est une épidémie mondiale entraînant de nombreux problèmes de santé et constitue un facteur de risque pour le développement du diabète de type 2. Les deux maladies partagent plusieurs troubles métaboliques et ont récemment été impliquées dans la détérioration de la santé cérébrale, contribuant aux troubles cognitifs. L'objectif de cette thèse est d'étudier l'impact de l'obésité/prédiabète sur l'homéostasie cérébrale et la plasticité cérébrale, puis d'atténuer ces effets délétères à l'aide de plantes médicinales réunionnaises. Pour cela, nous avons mis en place un protocole d'obésité induite par l'alimentation (DIO) chez le poisson zèbre (Danio rerio). Notre modèle DIO, établi en suralimentant des poissons zèbres adultes pendant 4 semaines, a entraîné des troubles métaboliques et une perte d'homéostasie du système nerveux central (SNC). Dans un second temps, nous avons testé les propriétés bénéfiques potentielles de deux plantes réunionnaises inscrites à la pharmacopée française : Antirhea borbonica (A. borbonica) et Psiloxylon mauritianum (P. mauritianum). Dans un premier temps, l'analyse chimique d'extraits aqueux d'A. borbonica et de P. mauritianum a révélé leur abondance en polyphénols, corrélée à leurs propriétés antioxydantes. Les deux plantes reflètent des effets préventifs luttant contre les effets DIO sur la périphérie et le cerveau. En conclusion, au cours de cette thèse, nous avons développé un modèle simple et rapide de suralimentation (DIO) induisant des perturbations périphériques et du SNC similaires à celles rencontrées chez les mammifères. Pour la première fois, nous avons étudié la toxicité de l'extrait aqueux des deux plantes médicinales A. borbonica et P. mauritianum. Nous avons confirmé leurs effets bénéfiques sur différents paramètres métaboliques et sur le cerveau en utilisant le modèle d'obésité et de prédiabète du poisson zèbre. Ensemble, ces données mettent en évidence l'utilisation du poisson zèbre pour imiter les maladies métaboliques et pour dépister les propriétés bénéfiques des extraits de plantes médicinales.

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.

Obesity is a worldwide epidemic leading to many health concerns and is a risk factor for the development of type 2 diabetes. In Reunion Island, obesity and diabetes are widely spread among the population. Both diseases share several metabolic disorders and have been recently implicated in deteriorating brain health, contributing to cognitive impairments. The mechanisms behind the onset of altered brain homeostasis are not well understood. Besides, no therapy has yet been established to prevent brain disruptions. The aim of this thesis is to study the impact of obesity/prediabetes on brain homeostasis and cerebral plasticity, and then to alleviate these deleterious effects using medicinal plants from Reunion Island. To this end, we set up a diet-induced obesity (DIO) protocol in zebrafish (Danio rerio). Zebrafish recently emerges as a relevant model to mimic metabolic diseases (obesity and diabetes), and to investigate brain homeostasis and plasticity (i.e., blood-brain barrier (BBB) and neurogenesis). Our DIO model, established by overfeeding adult zebrafish for 4 weeks, resulted in metabolic disorders and loss of central nervous system (CNS) homeostasis. Indeed, DIO fish displayed increased body weight and body mass index (BMI), hyperglycemia, liver steatosis and disturbed redox balance. In the central nervous system, overfeeding led to BBB leakage, neuro-inflammation, cerebral oxidative stress and decreased neurogenesis. As well, a change in the locomotor behavior was observed in obese fish. In a next step, we tested the potential beneficial properties of two Reunionese plants registered in the French pharmacopeia: Antirhea borbonica (A. borbonica) and Psiloxylon mauritianum (P. mauritianum). Both plants were traditionally used for their positive effects on metabolic disruptions as “anti-diabetic” effects for A. borbonica and “anti-lipidemic” for P. mauritianum. However, the scientific data supporting these properties are lacking. First, the chemical analysis of aqueous extract of A. borbonica and P. mauritianum revealed their abundance in polyphenols, correlated to their antioxidant properties. LC-MS/MS analysis was used to determine the nature of the polyphenolic compounds in each extract. Next, we performed toxicity assays using OECD guidelines 36 (Organization for Economic Co-operation and Development) (OECD, 2013) and defined a maximum non-toxic concentration for each extract. The overnight treatment with aqueous extract of A. borbonica (0.5g/L) during the DIO protocol demonstrated its preventive properties against the deleterious effects on the CNS induced by overfeeding. Indeed, A. borbonica preserved the BBB function, prevented the increase in cerebral oxidative stress, neuro-inflammation and normalized neurogenesis. Similarly, the aqueous extract of P. mauritianum (0.25 g/L) was tested on adult DIO zebrafish and in high-fat diet (HFD) treated larvae. The treatment avoided lipid accumulation in HFD larvae. It also prevented body weight increase, BMI, hyperglycemia and liver steatosis in adult DIO zebrafish. Furthermore, brain homeostasis seems to be preserved probably through P. mauritianum anti-weight gain properties. We suggested that P. mauritianum could significantly affect lipid absorption and metabolism possibly through the modulation of gut microbiota. In conclusion, during this thesis, we have developed a simple and rapid overfeeding (DIO) model inducing peripheral and CNS disruptions similar to those encountered in mammals. For the first time, we studied the toxicity of aqueous extract of the two medicinal plants A. borbonica and P. mauritianum. We confirmed their beneficial effects on different metabolic parameters and on the brain using zebrafish model of obesity and prediabetes. Together, these data highlight the use of zebrafish to mimic metabolic diseases and to screen the beneficial properties of medicinal plants extracts., L'obésité est une épidémie mondiale entraînant de nombreux problèmes de santé et constitue un facteur de risque pour le développement du diabète de type 2. Les deux maladies partagent plusieurs troubles métaboliques et ont récemment été impliquées dans la détérioration de la santé cérébrale, contribuant aux troubles cognitifs. L'objectif de cette thèse est d'étudier l'impact de l'obésité/prédiabète sur l'homéostasie cérébrale et la plasticité cérébrale, puis d'atténuer ces effets délétères à l'aide de plantes médicinales réunionnaises. Pour cela, nous avons mis en place un protocole d'obésité induite par l'alimentation (DIO) chez le poisson zèbre (Danio rerio). Notre modèle DIO, établi en suralimentant des poissons zèbres adultes pendant 4 semaines, a entraîné des troubles métaboliques et une perte d'homéostasie du système nerveux central (SNC). Dans un second temps, nous avons testé les propriétés bénéfiques potentielles de deux plantes réunionnaises inscrites à la pharmacopée française : Antirhea borbonica (A. borbonica) et Psiloxylon mauritianum (P. mauritianum). Dans un premier temps, l'analyse chimique d'extraits aqueux d'A. borbonica et de P. mauritianum a révélé leur abondance en polyphénols, corrélée à leurs propriétés antioxydantes. Les deux plantes reflètent des effets préventifs luttant contre les effets DIO sur la périphérie et le cerveau. En conclusion, au cours de cette thèse, nous avons développé un modèle simple et rapide de suralimentation (DIO) induisant des perturbations périphériques et du SNC similaires à celles rencontrées chez les mammifères. Pour la première fois, nous avons étudié la toxicité de l'extrait aqueux des deux plantes médicinales A. borbonica et P. mauritianum. Nous avons confirmé leurs effets bénéfiques sur différents paramètres métaboliques et sur le cerveau en utilisant le modèle d'obésité et de prédiabète du poisson zèbre. Ensemble, ces données mettent en évidence l'utilisation du poisson zèbre pour imiter les maladies métaboliques et pour dépister les propriétés bénéfiques des extraits de plantes médicinales.

Ich verwendete in meiner Arbeit das Pilocarpin-Mausmodell der Epilepsie, welches das Krankheitsbild der Temporallappen Epilepsie (TLE) auf molekularer und histopathologischer Ebene gut nachahmt. Die Rolle des Transkriptionsfaktors Serum response factor (SRF) war hierbei von Interesse. Ich untersuchte hippocampales Gewebe anhand RT-qPCR Analysen auf SRF abh��ngige Ver��nderung bekannter in der Epilepsie aberranter Signaltransduktions-Molek��le, sowie auf SRF abh��ngige Unterschiede der Neurogenese in der Subgranul��ren Zone (SGZ) des Hippocampus (HC). Die TLE stellt eine der h��ufigsten und eine sehr schwer therapierbare Entit��t der Epilepsien dar und da ihre zugrundeliegenden Patho-Mechanismen noch nicht endg��ltig gekl��rt sind ist ein Verst��ndnis solcher f��r zielgerichtete Therapieans��tze essenziell. Eine Analyse von SRF im Zusammenhang mit TLE liegt nahe, da SRF ein bekannter transkriptioneller Regulator neuronaler Plastizit��t, sowie aktivit��tsabh��ngiger Geninduktion ist und diese Hauptmerkmale der TLE sind. Im Vorfeld dieser Arbeit konnte meine Arbeitsgruppe erstmals eine erh��hte Status Epilepticus (SE)-Schwelle in Srf-Knockout Tieren im Pilocarpin Modell der Epilepsie beschreiben. Zudem beobachtete meine Arbeitsgruppe eine erh��hte Frequenz spontaner epileptischer Anf��lle (SRS) nach SRF Verlust in der chronischen Phase der Epilepsie und konnte SRF als wichtigen Regulator SE induzierter Genregulation nach Pilocarpin-Injektion identifizieren. Eine erh��hte SE Anf��lligkeit bei verminderter Neurogeneserate konnte bisher in Kainat und Pilocarpin Modellen der Epilepsie beschrieben werden. In meiner Arbeit konnte ich eine m��gliche neuroprotektive Rolle von SRF vor SE durch verst��rkte basale Neurogenese identifizieren. Nach SE blieb eine Induktion der Neurogenese in SRF defizienten Tieren aus. Ein pro-epileptogene Effekte durch fehlenden Ersatz nach Status Epilepticus apoptotischer inhibitorischer Interneurone ist m��glich. Dies k��nnte die erh��hte SRS Frequenz der SRF defizienten Tiere erkl��ren. In der chronischen Phase der Epilepsie konnte ich keine SRF abh��ngige Regulation der Neurogenese beobachten. Durch RT-qPCR Analysen konnte ich eine verminderte Expression des Calcium-Kanals Ryr1 in SRF defizienten Tieren beobachten. Eine gest��rte Calcium Hom��ostase k��nnte Einfluss auf Langzeitver��nderungen der synaptischen Plastizit��t haben. Der GABA-Transporter Gat1 wurde in Wildtyp-Tieren nach SE herunterreguliert, in SRF defizienten Tieren jedoch ��berexprimiert. Diese Beobachtung k��nnte zum einen den verminderten Verlust inhibitorischer Interneurone indizieren, zum anderen jedoch ein Hinweis auf neuroprotektive Gegenregulation des HC darstellen. Diese kompensatorische Reaktion des HC k��nnte und mit der von meiner Arbeitsgruppe beobachteten verl��ngerten Latenzzeit der SRF defizienten Tiere bis zu ersten spontanen Anf��llen zusammenh��ngen. Eine verminderte Expression des Ionentransporters NKCC1 nach SRF Verlust k��nnte mit der von mir beobachteten verminderten reaktiven Neurogenese nach SE in Zusammenhang stehen. SRF scheint also eine duale Rolle in der Epileptogenese einzunehmen. Zum einen beobachtete meine Arbeitsgruppe eine erh��hte SE Resistenz in SRF defizienten Tieren, welche mit von mir beobachteten erh��hten, neuroprotektiven basalen Neurogenese einhergeht. Zum anderen konnte eine, vom Epilepsie-induzierenden Agens unabh��ngige, erh��hte Frequenz spontaner Anf��lle in SRF defizienten Tieren beobachtet werden und weist SRF nach SE eine anti-epileptogene Rolle zu.

La neurogenèse est maintenue dans le cerveau adulte dans des régions restreintes du cerveau appelées niches neurogéniques. L’une des niches principales est la zone ventriculaire/sous-ventriculaire (V-SVZ) dans laquelle résident des cellules souches neurales (NSCs). Les NSCs sont à l’origine de la formation des nouveaux neurones en donnant naissance aux progéniteurs puis aux neuroblastes. Les études récentes sur la neurogenèse ont mis en évidence l’existence des NSCs quiescentes (qNSCs, aussi appelées cellules B1) et des NSCs actives (aNSCs). Le modèle actuel de la neurogenèse adulte place les qNSCs B1 en amont des aNSCs. L’hypothèse étant que cette population dormante constitue une « réserve », afin de maintenir les aNSCs tout au long de la vie. Les techniques actuelles ne permettent pas de cibler les qNSCs spécifiquement in vivo et donc, d’analyser leurs propriétés biologiques, leurs mécanismes d’activation ainsi que leur relation avec les aNSCs. Cette compréhension est nécessaire pour la mise au point de stratégies thérapeutiques pouvant utiliser le potentiel des cellules souches pour restaurer la neurogenèse dans les contextes de vieillissement et de maladies neurodégénératives. Afin de caractériser les qNSCs de la V-SVZ, nous avons utilisé l’électroporation de plasmides dans un modèle de souris rapportrice Rosa26-stop-EYFP. Dans celle-ci, la séquence codant pour la protéine EYFP précédée par un codon STOP floxé, est inséré au locus Rosa26. L’excision du codon STOP par une recombinase permet l’expression du rapporteur dans les cellules électroporées ainsi que leur descendance. Cette technique nous a permis de cibler spécifiquement une population d’astrocytes en contact avec le ventricule et d’étudier leur contribution à la neurogenèse adulte. À la différence des approches virales et transgéniques, l’électroporation peut cibler les cellules quiescentes et l’expression du plasmide est limité aux cellules en contact avec le ventricule. Grâce à cette technique nous avons mis en évidence des éléments surprenants : i) cette population est majoritairement quiescente et ne contribuent à la neurogenèse que de manière minimale, ii) cette population ne participe pas à la régénération de la niche in vivo, iii) elles ne génèrent pas les aNSCs à l’origine des neurosphères in vitro et iv) son activité neurogénique peut être augmentée en exprimant le gène pro-neural Mash1. Ensuite, nous nous sommes intéressés au rôle de la signalisation EGFR dans la régulation de l’activité des cellules souches/progéniteurs neuraux (NSPCs). Dans cette seconde étude, nous montrons que i) la signalisation EGFR est réduite avec l’âge, ii) PI3K/AKT, MEK/ERK et mTOR régulent différemment la prolifération, la différenciation et la survie des NSPCs et iii) l’activation d’EGFR dans les qNSCs permet d’augmenter la neurogenèse sous ventriculaire à 3 mois, mais pas à 6 mois ou dans un modèle de la maladie d’Alzheimer. Nos données suggèrent donc que les qNSCs représentent une population hétérogène et/ou présentant 2 voies neurogéniques distinctes. De plus, nous avons montré que les voies de signalisation associées à EGFR exercent un contrôle différentiel sur l’activité des NSPCs. Enfin, nos résultats indiquent que les facteurs présents dans la niche sous-ventriculaire lors du vieillissement inhibent de manière dominante l’activation des NSCs., Neurogenesis is maintained in restricted regions of the adult brain called neurogenic niches. One of the main neurogenic niches is the ventricular-subventricular zone (V-SVZ) in which neural stem cells (NSCs) reside. NSCs produce neurons through the generation of transit amplifying progenitors and neuroblasts. Recent studies on adult neurogenesis revealed the existence of quiescent NSCs (qNSCs, also called B1) and activated NSCs (aNSCs). The current model of adult neurogenesis places qNSCs (B1) upstream of aNSCs in the lineage. The hypothesis is that the qNSC population constitutes a “reserve” pool to maintain aNSC pool throughout life. So far, the techniques used do not allow specific targeting of the qNSCs in vivo. Therefore, it is not possible to analyze their biological properties, activation mechanisms and relationship with aNSCs. This understanding is also necessary to establish therapeutic strategies that could utilize the potential of stem cells to restore neurogenesis in contexts of aging and neurodegenerative diseases. In order to characterize qNSCs in the ventricular zone, we took advantage of plasmid electroporation in a reporter mouse model, Rosa26-stop-EYFP. In this model, the sequence coding for EYFP preceded by a floxed STOP codon is inserted at the Rosa26 locus. Excision of the STOP codon by a recombinase enables expression of the reporter in electroporated cells and their progeny. This technique enabled the specific targeting of a ventricle-contacting astrocytes population and to study their contribution to adult neurogenesis. Unlike transgenic or viral approaches, electroporation can target quiescent cells and the expression of the plasmid is restricted to the ventricle-contacted cells. Using this approach, we made surprising observations: i) this population is mostly quiescent and only minimally contributes to adult neurogenesis, ii) this population does not participate in niche regeneration in vivo and iv) their neurogenic output can be increased by expressing the pro-neural gene Mash1. Next, we investigated the role of EGFR signaling in the regulation neural stem and progenitor cells (NSPCs) activity. In this second study, we show that i) EGFR signaling decreases during aging, ii) PI3K/AKT, MEK/ERK and mTOR exert different regulation proliferation, differentiation and survival of NSPCs and iii) activation of EGFR in the qNSCs increases V-SVZ neurogenesis in 3-months-old animals but not in 6-months-old or Alzheimer’s disease model animals. Our data suggests that the NSC population is heterogeneous, with variable neurogenic output from the different sub-populations, as well as different activation modalities. We also showed that EGFR-associated signalling pathways differentially regulate NSPCs activity. Finally, our results indicate that the factors present in the V-SVZ niche during aging dominantly inhibit activation of NSCs.

Normal aging is accompanied by cognitive and sensory deficits, including olfactory deficits, which affect the quality of life and health of elderly subjects. However, normal aging (independently of any degenerative pathology) is poorly explored and the cellular mechanisms underlying these deficits remain largely unknown. This lack of knowledge limits the care of elderly subjects as well as the development of remediation strategies to counteract olfactory and cognitive aging. The objectives of this thesis were first to improve the understanding of olfactory aging and the neural mechanisms underlying these deficits in mice, and second to improve the functioning of the aging brain. In a first study, we used the olfactory perceptual learning model to identify the cellular mechanisms underlying olfactory aging under basal and plasticity-requiring conditions. This learning improves discrimination between two perceptually similar odorants following repeated exposure to these odorants. In rodents, it requires the contribution of adult-born neurons in the olfactory bulb. We finely studied the kinetics of the appearance of olfactory perceptual learning deficits over the mouse lifespan (from 2 to 18 months) and revealed that the long-term retention of olfactory perceptual learning is impaired as early as 12 months of age, before the total loss of learning abilities at 18 months. These learning retention deficits are associated with an alteration of the structural plasticity of adult-born granule cells in the olfactory bulb. In a second study, we developed a remediation strategy to improve olfactory and cognitive aging, based on olfactory stimulation with a new odor every day (enrichment). These olfactory enrichment sessions were repeated throughout the life of the animal to allow the constitution of a cognitive reserve. This concept is associated in humans with a high level of cognitive stimulation and resistance to the deleterious effects of aging on cognitive performances and could be an effective strategy to promote better aging. Mice subjected to such olfactory enrichment displayed improved olfactory discrimination performance at late ages, as well as spatial memory and cognitive flexibility, indicating that the benefits of lifelong olfactory enrichment extend beyond the olfactory sphere and include broader cognitive benefits. At the cellular level, we did not observe any changes in adult neurogenesis in the olfactory bulb, a mechanism known to underlie olfactory learning and memory in the young adult. However, we revealed an increase in noradrenergic innervation that correlates with behavioral performances. Finally, we identified remodeling of brain networks implicated in behavioral tasks, which may underlie the improvement of cognitive performances in aged mice subjected to long-run olfactory enrichment. Overall, our data contribute to deepen our understanding of age-related alterations in brain plasticity mechanisms and their implication in learning and memory deficits. Moreover, this work proposes repeated olfactory enrichment throughout life as an effective strategy to promote better aging by improving not only olfactory, but also cognitive aging in general, inducing structural and functional remodeling in the brains of aged mice., Le vieillissement normal s’accompagne de déficits cognitifs et sensoriels, notamment olfactifs qui affectent la qualité de vie et la santé des individus âgés. Cependant, le vieillissement normal (indépendamment de toute pathologie dégénérative) reste peu étudié et les changements de la structure et du fonctionnement cérébraux qui sous-tendent ces déficits sont encore largement méconnus. Ce manque de connaissances limite la prise en charge des sujets âgés ainsi que le développement de stratégies de remédiation pour lutter contre le vieillissement olfactif et cognitif. Les objectifs de cette thèse étaient premièrement d’avancer dans la compréhension du vieillissement olfactif et des mécanismes neuronaux qui sous-tendent ces déficits chez la souris, puis dans un second temps, d’améliorer le fonctionnement du cerveau vieillissant. Dans une première étude, nous avons utilisé le modèle de l’apprentissage olfactif perceptif pour identifier des mécanismes cellulaires sous-tendant le vieillissement olfactif en conditions basales et dans des conditions sollicitant la plasticité. Cet apprentissage permet l’amélioration de la discrimination de deux odorants proches d’un point de vue perceptif, après une exposition répétée à ceux-ci. Chez le rongeur, il nécessite l'apport de nouveaux neurones dans le bulbe olfactif adulte. Nous avons étudié finement la cinétique d’apparition des déficits d’apprentissage olfactif perceptif au cours de la vie de la souris (de 2 à 18 mois) et révélé que la rétention à long terme de l’apprentissage olfactif perceptif est altérée dès l’âge de 12 mois, avant la perte totale des capacités d’apprentissage à 18 mois. Ces déficits de rétention sont associés à une altération de la plasticité structurale des nouveaux neurones granulaires du bulbe olfactif. Dans une deuxième étude, nous avons développé une stratégie de remédiation du vieillissement olfactif et cognitif, basée sur une stimulation olfactive avec une nouvelle odeur chaque jour (enrichissement). Des sessions d'enrichissement olfactif ont été répétées tout au long de la vie de l'animal afin de permettre la constitution d’une réserve cognitive. Ce concept est associé chez l’humain à un niveau de stimulation cognitive élevé et à une résistance face aux effets délétères du vieillissement sur les performances cognitives et pourrait constituer une stratégie efficace pour promouvoir le bien-vieillir. Les souris soumises à un tel enrichissement olfactif présentent de meilleures performances de discrimination olfactive à un âge avancé, ainsi que de mémoire spatiale et de flexibilité cognitive, indiquant que les bénéfices d'un enrichissement olfactif tout au long de la vie s’étendent au-delà de la sphère olfactive et incluent des bénéfices cognitifs plus larges. Au niveau cellulaire, nous n’avons pas observé de modifications de la neurogenèse adulte bulbaire, un mécanisme connu pour sous-tendre l’apprentissage et la mémoire olfactifs dans le bulbe olfactif jeune adulte. En revanche, nous révélons une augmentation de l’innervation noradrénergique qui corrèle avec les performances comportementales. Enfin, nous mettons en évidence des remaniements des réseaux cérébraux mis en jeu par les tâches comportementales, qui pourraient sous-tendre l’amélioration des performances cognitives chez les souris âgées soumises à l’enrichissement olfactif tout au long de la vie. Dans l’ensemble, nos données contribuent à approfondir notre connaissance des altérations des mécanismes de plasticité cérébrale liées au vieillissement et leur implication dans les déficits d’apprentissage et de mémoire. De plus, ce travail propose l’enrichissement olfactif répété tout au long de la vie comme une stratégie efficace de lutte contre le vieillissement non seulement olfactif, mais cognitif en général, induisant des remaniements structuraux et fonctionnels dans le cerveau des souris âgées.

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...

Serotonin gilt als Hauptangriffsstelle gängiger Antidepressiva bei schweren Depressionen, wie bspw. selektive Serotonin-Wiederaufnahmehemmer (SSRI), und -Enhancer (SSRE). Es bleibt offen, ob SSRI / E ausschließlich über die Manipulation des Serotoninspiegels wirken, oder ob alternative Signalwege daran beteiligt sind. Ansatzpunkte hierfür sind beispielsweise die neurotrophen Signalwege (spez. Brain derived neurotophic factor, BDNF) oder die Hypothalamus-Hypophysen-Nebennieren- (HPA) – Signalwege des Stressachsensystems. Ebenfalls wurde in Nagetiermodellen beobachtet, dass mit der Dysregulation des zentralen Serotoninsystems bei schweren Depressionen, ein Rückgang der Neurogenese im Gyrus dentatus des Hippocampus einhergeht. Ziel dieser Arbeit war, das Zusammenspiel von Serotonin, BDNF, adulter Neurogenese und der Stressachse zu untersuchen. Zentrum der Studien ist ein Mausmodell, mit einer genetischen Depletion des zentralen Serotonin-synthetisierenden Enzyms Tryptophanhydroxylase 2 (sog. Tph2-/- Mäuse). Es wurden die physiologische Reaktionen auf die Behandlung mit gängigen Antidepressiva abhängig von der Abwesenheit von Serotonin untersucht, um mögliche alternative Signalwege aufzeigen zu können. Die bekannte Zunahme der Neurogenese nach SSRI/SSRE-Behandlung wurde in Wildtyptieren beobachtet, während die Therapie in Tph2-/- Mäusen keine direkte kausale Wirkung zeigte. Im Gegensatz dazu waren die BDNF-Spiegel in depressionsrelevanten Hirnregionen in Tph2-/- Mäusen nach SSRI, signifikant verringert. Auch zeigen die Studien eine neurobiologische Relevanz von Serotonin im ZNS, bei den antidepressiven Mechanismen einer Elektrokonvulsiven Krampftherapie. Ebenfalls deuten erhöhte Neurogeneseraten bei lebenslanger Abwesenheit von Serotonin im ZNS, Therapiemethoden-unabhängig, möglicherweise auf eine modulierte Stressreaktion hin. Untersuchungen der Parameter des HPA-Stressachsensystems, wiesen auf einen grundlegend veränderten Stresshormonspiegel in Tph2-/- Mäusen hin. Serotonin, the "molecule of happiness" is an important target for antidepressants. The mainly prescribed drugs in major depression are selective serotonin re-uptake inhibitors (SSRI); but recently, SSR-enhancer (SSRE) have also attracted clinical attention. However, only a quarter of patients responds to treatment. It needs to be determined, whether SSRI/E act solely via manipulating serotonin levels or whether other pathways are involved, e.g. neurotrophic signaling (brain-derived neurotrophic factor, BDNF) or the hypothalamus-pituitary-adrenal (HPA)-axis. Furthermore, in major depression, dysregulation of central serotonin signaling is accompanied with a decline in hippocampal neurogenesis, as has been observed in rodent models. At the center of this thesis is a mouse model deficient in the central serotonin-synthesizing enzyme, tryptophan hydroxylase 2 (Tph2-/- mice). I have investigated physiological responses to antidepressant treatment in the absence of brain serotonin, and the possible role of alternative pathways. I observed the typical increase in neurogenesis upon SSRI treatment in WT mice, while it had no effect in Tph2-/- mice. In contrast, BDNF levels were significantly decreased in Tph2-/- mice after treatment with no effect in WT control mice. Furthermore, my results show a critical role of brain serotonin in the neurobiological effects of electroconvulsive seizure. Surprisingly, in animals lacking central serotonin, increased neurogenesis was observed independently of the treatment. The gathered data indicated an altered stress response; therefore, parameters of the HPA-axis have been studied, indicating a downregulated HPA system in Tph2-/-animals in baseline state, but showed no difference in treatment or feedback control. This thesis gives insight into the mechanisms of antidepressant action and reveals ideas for novel pathways involved in the process that could be used as targets in therapeutic approaches and further research in major depression.

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

Thyroid hormones (TH) are essential for proper embryonic development of the central nervous system. During this period maternal supply of TH is the only source of these hormones to the embryo. Using a zebrafish MCT8 knockdown model, with consequent inhibition of maternal thyroid hormones (MTH) uptake to the target cells, the aim of this thesis is to start a comprehensive understanding of the role of MTH during embryonic neural development. We characterised the transcriptome in 25hpf CTRL and MCT8MO zebrafish embryos and found 4,343 differentially expressed genes. Reactome analysis show that MTH regulate the expression of core developmental pathways such as NOTCH, SHH and WNT. The cellular distribution of neural MTH-target genes demonstrated their cell specific action on neural stem cells and differentiated neuron classes. We identified a series of genes involved in several key neurogenic processes to be modulated by MTH. By analysing these genes by qPCR in a temporal series, from the start of segmentation through hatching, we determined the developmental time-window where MTH are required for appropriate CNS development. We show MTH are involved in the regulation of NOTCH pathway components such as notch1a, dla, dld, her2 and her4 during neurogenesis, whereas neuroectodermal genes are not affected. Response to MTH begins at 12hpf, and the time window between 22-25hpf is particularly sensitive to MTH action. Overall, these results, show that MTH is not involved in neuroectoderm specification nor CNS compartmentalisation but stress the involvement of MTH in the early stages of neurogenesis by promoting the maintenance of specific neural progenitor populations. Analyzing the cytoarchitecture of the spinal cord we found that by the end of embryogenesis cells populating the spinal cord of control and MCT8 MO zebrafish are substantially different. Lack of thyroid hormone uptake leads to a generalized disorganization of the neural tissue, together with a decrease in: neural stem cells population, subpopulations of neuron progenitor cells, radial glial cells, mature glial cells and oligodendrocyte precursors, while the primary motor neuron domain was maintained. Colocalization analysis of neural progenitors with thraa, thrab and mct8 allowed identifying cells under the regulation of MTH via MCT8. Survival and proliferation of neural progenitor cells are compromised in MCT8MO, which could later impact on the diversity of neural cell populations obtained in the end of embryogenesis. Analysis of cell autonomous Notch activation showed it cannot rescue the phenotype induced by the lack of MTH demonstrating the niche importance in the regulation of TH action. Given that MTH regulate several important morphogenetic pathways it is likely that its action occurs as an integrator enabling an adequate equilibrium between all these signals in a time a context dependent manner. MTH actions are reflected on the timely development of neurons and glial cells. It is of great interest to continue to explore the significance of these findings to further clarify the genetic and cellular causes underlying human AHDS syndrome. In conclusion with this work, we show that thyroid hormone transferred from the mother to the embryo allows the enrichment of neural progenitor pools and the generation of cell diversity necessary to produce a fully functional central nervous tissue. As hormonas da tiróide (TH) são essenciais para o correto desenvolvimento embrionário do sistema nervoso central (CNS). Durante este período o fornecimento de hormona da tiróide pela via materna é a única fonte destas hormonas para o embrião, uma vez que a produção endógena desta hormona é iniciada apenas numa fase posterior do desenvolvimento. Evidências mostram que mesmo níveis baixos de deficiência de hormona da tiróide na mãe estão associados a desordens neurológicas e psiquiátricas nos filhos. No entanto os mecanismos moleculares envolvidos na ação desta hormona no embrião e feto continuam amplamente desconhecidos. Em humanos, mutações no principal transportador celular de TH, MCT8, causa a síndrome de Herndon-Dudley (AHDS). Esta síndrome é caracterizada pelo atraso mental, atraso global no desenvolvimento, impossibilidade de falar e uma deficiência neuromotora severa. O MCT8 é o principal transportador de hormona da tiróide presente no embrião. No presente trabalho utilizamos o “knockdown” deste transportador, inibindo a sua tradução, no modelo de peixe zebra. Este modelo foi previamente estabelecido e possui características semelhantes à síndrome AHDS humana. Neste modelo o transporte da hormona da tiróide materna (MTH) para as células alvo é bloqueado, uma vez que o MCT8 está ausente, inibindo a ação da hormona. O objetivo desta tese é começar a compreender o papel destas hormonas durante o desenvolvimento neural embrionário. Para isso caracterizamos o transcriptoma de peixe-zebra CTRL e MCT8MO às 25 horas pósfertilização (hpf) por RNA-seq. Foram encontrados 4343 genes diferencialmente expressos. A análise utilizando o Reactome revelou que a MTH está envolvida direta ou indiretamente na expressão de genes pertencentes a importantes vias de sinalização, incluindo Notch, Shh e Wnt. A análise da distribuição celular de genes alvo da MTH por hibridação in-situ revelou uma ação celular específica em células estaminais neurais, mas também sobre várias classes de neurónios diferenciados. A análise transcriptómica revelou também que uma série de genes envolvidos em vários passos de processos-chave da neurogénese são modulados pela MTH. Analisando alguns destes genes numa série temporal desde a fase da segmentação até à eclosão do embrião de peixe-zebra por qPCR, determinamos a janela temporal na qual a MTH é necessária para um correto desenvolvimento do sistema nervoso central no peixezebra. Mostramos que a MTH está envolvida na regulação de componentes da sinalização Notch, tais como notch1a, dla, dld, her2 e her4 durante o processo de neurogénese, enquanto que genes envolvidos na formação e manutenção da neuroectoderme não são regulados pela MTH. A resposta à ausência de MTH inicia-se às 12hpf, sendo que a janela de desenvolvimento entre as 22 e 25hpf parece ser particularmente sensível à ação da MTH. Globalmente estes resultados mostram que a MTH não está envolvida na indução neural, nem na compartimentalização do sistema nervoso central. No entanto é demonstrada a importância da MTH na fase inicial da neurogénese pela promoção da manutenção de populações de células progenitoras neurais. A análise à citoarquitectura da medula espinhal revelou que na fase final do desenvolvimento embrionário as células neurais presentes em embriões CTRLMO e MCT8MO diferem substancialmente. A diminuição de transporte de MTH para as células alvo leva a uma desorganização geral do tecido neural, para além duma redução em células estaminais neurais, subpopulações de células progenitoras de neurónios, células radiais gliais, células da glia maduras e progenitoras de oligodendrócitos; enquanto que a população de neurónios motores primários é mantida na ausência de MTH. Análise de colocalização de células progenitoras neurais her2(+), dla(+) e fabp7a(+) com RNAm de componentes do metabolismo da hormona da tiroide, nomeadamente recetores (thraa e thrab) e o transportador mct8, permitiu a identificação de células que estão sob a regulação da MTH. Um dos papeis clássicos da TH envolve a sobrevivência a proliferação celulares, no entanto a sua função varia com o tipo celular, o estadio de desenvolvimento e contexto celular. Verificámos que em embriões MCT8MO a sobrevivência e proliferação de células progenitoras neurais her2(+) e dla(+) estava comprometida, tanto na medula espinhal como no romboencéfalo. Este facto pode impactar a diversidade das células neurais obtidas no final da embriogénese. Na verdade, a divisão assimétrica de células progenitoras originando células da glia ou progenitoras intermediárias está diminuída nos embriões MCT8MO, mostrando um possível papel da MTH na sua formação. Com vista a esclarecer o papel da MTH sobre a sinalização Notch procedeu-se à ativação da sinalização Notch em células neurais da espinhal medula de embriões MCT8MO e CTRLMO. Esta sobre-expressão celular isolada da sinalização Notch em células individuais mostrou ser insuficiente para recuperar o fenótipo induzido pela falta de MTH, demonstrando a importância do nicho celular na regulação da ação da MTH. A ação da MTH parece estar envolvida no memanismo de divisão assimétrica de células neuroepiteliais. Este tipo de divisão está na origem da diversidade de células neurais obtidas. Um reflexo da perda deste tipo de divisões na espinhal medula pode ser a perda de progenitores intermediários que expressam neurogenina 1 e neurod6b. Dado que a MTH regula várias vias morfogenéticas é provável que a sua ação seja a de integradora entre estas vias, permitindo o equilíbrio entre estes sinais num determinado tempo e contexto celular específicos. As ações da MTH refletem-se no desenvolvimento correto e atempado de neurónios e células da glia. É de grande interesse continuar a explorar o significado destas descobertas, nomeadamente para esclarecer as causas genéticas e celulares que causam a síndrome de Herdnon-Dudley (AHDS). Foi desenvolvido um modelo de ação da MTH durante o desenvolvimento no peixe zebra, em que em subpopulações de células progenitoras contendo MCT8 e recetores da hormona da tiroide (sensíveis a TH) na ausência desta sofrem apoptose e/ou redução na proliferação em momentos distintos do desenvolvimento neural. As células progenitoras não responsivas à TH prosseguem o seu desenvolvimento. O balanço final será uma diminuição da diversidade de progenitores neurais e consequente perda na variedade de neurónios e células da glia maduros. Este efeito a nível celular terá efeito na função do CNS. Em conclusão este trabalho mostra que a hormona da tiroide transmitida da mãe para o embrião e que dá entrada nas células alvo através do MCT8 permite o desenvolvimento de populações de progenitores neurais e o consequente enriquecimento da diversidade celular, necessária à formação de um sistema nervoso central funcional. This study received Portuguese national funds from operational programmes CRESC Algarve 2020 and COMPETE 2020 through project EMBRC.PT ALG-01-0145-FEDER- 022121. The author was a recipient of a FCT PhD grant SFRH/BD/111226/2015

Die hypoxisch-ischämische Enzephalopathie ist eine der häufigsten Ursachen für neonatale Sterblichkeit und geht bei Überlebenden mit teils gravierenden körperlichen und geistigen Behinderungen einher. In der Behandlung zeigt die milde Hypothermie die besten Erfolge. Durch die Kühlung kann nur einem von neun Patienten geholfen werden, sodass adjuvante Therapien benötigt werden. Die Sildenafiltherapie stellt hier eine vielversprechende Behandlungsmöglichkeit dar, weil in tierexperimentellen Studien neuroprotektive Eigenschaften und eine verstärkende Wirkung auf neuronale Regeneration im adulten Gehirn beschrieben wurden. Diese Eigenschaften sind für das neonatale Hypoxie-Ischämie Modell bisher noch nicht untersucht. Die vorliegende Arbeit beschäftigt sich daher mit der Frage, ob es gemessen am zerebralen zyklischen Guanosinmonophosphat (cGMP) eine Wirkung durch Sildenafil gibt und ob sich neuroprotektive oder neuroregenerative Effekte nach Hypoxie-Ischämie bei neonatalen Mäusen durch eine Sildenafilbehandlung nachweisen lassen. Zur Induktion der neonatalen Hirnschädigung wurde das modifizierte Hypoxie-Ischämie Modell nach Rice und Vannucci an 9 Tage alten C57/Bl6 Mäusen verwendet. Es erfolgten Sildenafil Applikationen nach 2 Stunden einmalig oder alle 24 Stunden für 5 Tage. An P10 und P14 wurden die Tiere euthanasiert, um histologische Untersuchungen mittel Kresylviolett- und Immunfluoreszenz- Färbungen sowie ELISA zur Detektion von cGMP durchzuführen. Nach 24 Stunden zeigte sich ein Anstieg des zerebralen cGMP bei Sildenafil behandelten Tieren in der Schadensgruppe, was möglicherweise indirekt auf eine Sildenafilwirkung im Gehirn hinweisen könnte. Allerdings bleibt unklar, ob und über welchen Zeitraum Sildenafil die Blut-Hirn-Schranke überschreitet und wie lange eine mögliche Wirkung vorhält. Ein neuroprotektiver Effekt konnte durch Sildenafil nicht nachgewiesen werden, was auch in der Literatur kontrovers diskutiert wird. Hierbei spielen möglicherweise der Applikationszeitpunkt und das Applikationsintervall eine wichtige Rolle. Ein Anstieg neuronaler Vorläufer konnte hier im neonatalen Mausmodell gezeigt werden, was sich mit Untersuchungen an erwachsenen Nagern deckt. Es bleibt zu untersuchen, ob diese Vorläufer auch zu reifen Neuronen differenzieren und sich in das Gehirn integrieren. Dissertation, Universität Duisburg-Essen, 2021

Stroke significantly stimulates neurogenesis in the adult dentate gyrus, though the functional role of this postlesional response is mostly unclear. Recent findings suggest that new-born neurons generated in the context of stroke may fail to correctly integrate into pre-existing networks. We hypothesized that increased neurogenesis in the dentate gyrus following stroke is associated with aberrant neurogenesis and impairment of hippocampus-dependent memory. To address these questions, we used the middle cerebral artery occlusion model (MCAO) in mice. Animals were housed either under standard conditions or with free access to running wheels. New-born granule cells were labelled with the thymidine analogue EdU and retroviral vectors. To assess memory performance, we employed a modified version of the Morris water maze (MWM) allowing differentiation between hippocampus dependent and independent learning strategies. New-born neurons were morphologically analysed using confocal microscopy and Neurolucida system at 7 weeks. We found that neurogenesis was significantly increased following MCAO. Animals with MCAO needed more time to localize the platform and employed less hippocampus-dependent search strategies in MWM versus controls. Confocal studies revealed an aberrant cell morphology with basal dendrites and an ectopic location (e.g. hilus) of new granule cells born in the ischemic brain. Running increased the number of new neurons but also enhanced aberrant neurogenesis. Running, did not improve the general performance in the MWM but slightly promoted the application of precise spatial search strategies. In conclusion, ischemic insults cause hippocampal-dependent memory deficits which are associated with aberrant neurogenesis in the dentate gyrus indicating ischemia-induced maladaptive plasticity in the hippocampus.

Parkinson's disease (PD) is caused by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), which results in dopamine (DA) depletion in the brain of affected patients. Today's therapy options are mostly symptomatic and limited to pharmacological replacement therapy of DA. Because medication is associated with several side-effects and efficacy of the treatment subsides with time, alternative therapy options are studied since many years. Especially transplantation of endogenous and exogenous dopaminergic stem cells as a possible cure for PD is increasingly under investigation. The adult mammalian brain contains only two regions that maintain the lifelong capacity to generate new neurons, a process which is called neurogenesis. The first region is the subgranular layer (SGZ) which is located in the dentate gyrus of the hippocampus. The second region is the subventricular zone (SVZ) adjacent to the walls of the lateral ventricles of the telencephalon. Neurons deriving from neural stem cells (NSCs) of the SVZ migrate along the rostral migratory stream (RMS) towards the olfactory bulb (BO). After reaching the BO these neurons differentiate into mature interneurons before integrating into the granular (GCL) or periglomerular cell layer (PGL). Approximately 40% of the newly integrated neurons of the PGL show a dopaminergic phenotype. The aim of the present study was to investigate the localization of the dopaminergic NSCs in the SVZ-RMS-BO-system. It is of special interest to find the exact position of these neurons because they could be used for transplantation projects in clinical studies. To elucidate this question we used a mouse model consisting of 11 animals and implanted a physical barrier (PB) to mechanically interrupt the RMS. Depletion of neural progenitors in the SVZ and RMS was achieved by a continuous infusion of AraC (cytosine β-D-arabinofuranoside) administered for one week. Subsequently newly proliferating neurons were labeled by BrdU (5-bromo-2'- deoxyuridine), an analogue of thymidine. The experimental group consisted of 8 animals which were sacrificed 55 (n=4) and 105 days (n=4) after stopping the AraC application. The three animals of the control group received neither a surgery for PB implantation nor AraC treatment. They only obtained BrdU injections and were sacrificed on day 105. For further analysis the mouse brains were removed from the skull. The brain tissue was processed and immunhistochemically stained. The proper function of the PB, as required for the experiment, was confirmed by investigation of the SVZ. As expected after depletion of the neuroblasts neurogenesis was regenerating after treatment, which was confirmed by BrdU immunostaining. Furthermore a distinct accumulation of neuroblasts in the SVZ on the interrupted side indicated a proper function of the PB. Interestingly, the BrdU staining of the BO on the interrupted side showed a significant reduction of new born neurons in the GCL, but not in the PGL. This result clearly implies that NSCs, which produce interneurons destined for the GCL are located in the SVZ, whereas NSCs which provide interneurons for the PGL (including dopaminergic interneurons) must be located in the RMS. With the help of a BrdU/NeuN-double staining of the PGL it was confirmed that the cell type of the new born cells were mainly neurons instead of reactive glial cells. Furthermore, various stainings, especially the TH-staining of the PGL validated a constant level of dopaminergic neurons. To the best of our knowledge, this is the first work detecting dopaminergic stem cells in the RMS of a mouse model by mechanical interruption of the RMS. These findings are congruent with the results of different scientific groups and support the hypothesis that the SVZ-RMS-BO system is a complex proliferative zone. In addition, it becomes obvious that NSCs are an inhomogenous cell population in which NSCs of distinct regions produce different subtypes of neurons. This diverse character of NSCs is described as regionalization by different authors. Stem cell therapy of the central nervous system is a promising new approach for the treatment of PD as well as for other neurodegenerative and neurooncological diseases. Previously described sources of dopaminergic stem cells comprise several disadvantages and clinical limitations. The extraction of dopaminergic NSCs from the RMS could be a promising alternative to overcome current objections. First preliminary studies on rodents and humans were already successfully accomplished., Ursache des idiopathischen Parkinson-Syndroms (IPS) ist ein fortschreitender Verlust dopaminerger Neurone der Substantia nigra pars compacta (SNpc), welcher im Gehirn betroffener Patienten zu einem Dopaminmangel führt. Heutige Therapieoptionen sind rein symptomatisch und zumeist auf den medikamentösen Ersatz des Neurotransmitters Dopamin (DA) beschränkt. Da die medikamentöse Therapie mit zahlreichen Nebenwirkungen assoziiert ist und ihre Wirksamkeit mit der Zeit nachlässt, wird seit Jahren nach alternativen Therapieoptionen gesucht. Unter anderem stellt die Implantation von körpereigenen oder körperfremden dopaminergen Stammzellen einen möglichen kurativen und somit interessanten Therapieansatz dar. Im Gehirn adulter Säugetiere bleiben nur zwei Hirnregionen lebenslang zur Neubildung von Neuronen (Neurogenese) fähig. Dies ist neben der Subgranulärzone (SGZ) des Gyrus dentatus des Hippocampus die Subventrikulärzone (SVZ) der Seitenventrikel des Telencephalons. Die in der SVZ neugebildeten Neurone gehen aus neuralen Stammzellen (NSCs) hervor und wandern entlang des rostralen Migrationsstroms (RMS) in den olfaktorischen Bulbus (BO) ein. Im BO kommt es zur Ausdifferenzierung und Integration der jungen Neurone entweder in die granuläre Zellschicht (GCL) oder periglomeruläre Zellschicht (PGL), wobei circa 40% der neu eingewanderten Neurone der PGL einen dopaminergen Phänotyp annehmen. Ziel der vorliegenden Arbeit war die Identifikation dopaminerger Stammzellen im SVZ-RMS-BO-System, da diese in zukünftigen klinischen Studien zu Transplantationszwecken eingesetzt werden könnten. Zur Klärung dieser Fragestellung diente ein Mausmodell bestehend aus 11 Tieren, bei dem der RMS der Versuchsgruppe mittels einer physikalischen Barriere (PB) mechanisch unterbrochen wurde. Teilungsaktive Vorläuferzellen konnten mit Hilfe einer kontinuierlichen einwöchigen Infusion des Zytostatikums AraC (Cytarabin) aus der SVZ und dem RMS eliminiert werden. Anschließend wurden die durch Regeneration des Systems neugebildete Nervenzellen durch Applikation des Thymidin-Analogons BrdU (Bromodeoxyuridin) markiert. Die Versuchstiere wurden 55 (n=4) beziehungsweise 105 Tage (n=4) nach Beendigung der AraC-Infusion geopfert. Die mitgeführten, nichtbehandelten Tiere der Kontrollgruppe (n=3) erhielten nur BrdU-Applikationen und wurden ebenfalls an Tag 105 geopfert. Post mortem erfolgte die Gehirnentnahme und Gewebeaufarbeitung. Mit Hilfe einer immunhistochemischen BrdU-Färbung konnten die neugebildeten Neurone nachgewiesen werden. Die Funktionstüchtigkeit der PB als Grundlage des Versuchsaufbaus konnte anhand der Untersuchung der SVZ bestätigt werden. Es zeigte sich erwartungsgemäß, dass nach Ablation der Neuroblasten die Neurogenese in der SVZ wieder aufgenommen wurde. Ebenso ließ sich ein vermehrter Rückstau an neugebildeten Neuronen auf der unterbrochenen Seite in die SVZ feststellen, wodurch die gewünschte Funktion der PB gezeigt werden konnte. Anhand der immunhistochemischen Untersuchungen der GCL und PGL des BO ließ sich eine signifikante Reduktion von neugebildeten Neuronen auf der unterbrochenen Seite in der GCL, nicht jedoch in der PGL, feststellen. Damit konnte gezeigt werden, dass diejenigen NSCs, welche Interneurone der GCL hervorbringen, in der SVZ liegen, während NSCs, welche Interneurone (inklusive dopaminerger Interneurone) für die PGL produzieren, im RMS rostral der PB lokalisiert sein müssen. Mit Hilfe einer BrdU/NeuN-Doppelfärbung der PGL konnte sichergestellt werden, dass es sich bei den neugebildeten Zellen tatsächlich um Neurone und nicht etwa um reaktive Gliazellen handelte. Anhand zusätzlicher Färbungen, speziell der dopaminergen Zellen in der PGL, ließ sich eine gleichbleibende Anzahl an dopaminergen Neuronen bestätigen. In der vorliegenden Arbeit konnte somit erstmals mit Hilfe einer mechanischen Unterbrechung des RMS in einem Mausmodell die Lokalisation dopaminerger Stammzellen im RMS nachgewiesen werden. Die Ergebnisse stimmen mit denjenigen von verschiedenen Arbeitsgruppen überein und stützen die Hypothese, dass es sich beim SVZ-RMS-BO-System um eine komplexe proliferative Zone handelt. Es wird deutlich, dass NSCs eine inhomogene Zellpopulation darstellen und somit abhängig von ihrer Regionalisierung unterschiedliche Typen von Neuronen hervorbringen. Therapeutisch stellt die Stammzelltherapie des zentralen Nervensystems (ZNS) einen neuen Ansatz zur Behandlung des IPS sowie anderer neurodegenerativer Erkrankungen dar. Da bisherige Quellen von dopaminergen NSCs unterschiedliche klinische und ethische Einschränkungen aufweisen, stellt die Gewinnung dieser Zellen aus dem RMS einen vielversprechenden neuen Therapieansatz dar, der in Zukunft sowohl am Nagetier als auch am Menschen genauer untersucht werden sollte.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul - FAPERGS Depression is a common disease worldwide and although there are several drugs for the treatment of depression, the effectiveness of this pharmacotherapy still presents limitations, possibly due to the difficulty of understanding its pathophysiology. Tactile stimulation (TS) is a manual therapy applied both in early and in adult life. Studies have shown the benefits of TS in rodents in models of psychiatric disorders, including anxiety and addiction. In this study, our objective was to evaluate the influence of TS applied in the neonatal period or adult life in different animal models of depression. The first experiment of this study evaluated the possible positive response of TS on the reversion of depression-like behaviors in adulthood after reserpine administration. Adult female Wistar rats received reserpine once daily for three consecutive days (1mg/kg s.c.). The antidepressant imipramine (10mg/kg i.p.) was used as a positive control. Immediately after the reserpine last administration, the TS protocol started (15min/3xday, 8 days). Afterward, depression behavioral tests were performed and then the animals were euthanized for blood collection, for analysis of corticosterone and adrenocorticotrophic hormone (ACTH) levels in plasma, besides of and prefrontal cortex removal for quantification of BDNF, proBDNF, TrkB, GDNF, GFAP and glucocorticoid receptor (GR) immunoreactivity analyzed by western blotting. We observed that TS reverse the depression-like behaviors induced by reserpine. Besides, our results showed that TS reduced plasma levels of corticosterone and ACTH, and reduced adrenal weight, parameters which were increased by reserpine administration. In the PFC, TS increased BDNF, TrkB, GFAP, and GR immunoreactivity and reduced proBDNF levels. The second experiment of this study aimed to evaluate the effect of neonatal TS (10min/1xday, 8 days) on heterozygous serotonin transporter rats (SERT+/-) and evaluate anxiety and depression-like behaviors in adulthood. After the behavioral analyzes the animals were euthanized, and the basolateral amygdala was removed for the expression of BDNF and its isoforms, glutamatergic and gabaergic components as well as glucocorticoid (GR) and mineralocorticoid (MR) receptors and glucocorticoid responsive-genes. We observed that TS improved the anxiety, depressive-like and social behaviors in SERT+/- rats. In the molecular analyzes, only TS per se showed modifications in in the basolateral amygdala, which decreased BDNF and its isoforms IV and VI, the ratio of GR/MR, the glucocorticoid responsive-genes, as well as the ratio of VGLUT/VGAT, while TS increased the GAD67 levels. As a last section of the thesis, a systematic review was performed to evaluate the impact of TS in rats. A total of 55 studies were identified and we observed that TS in rats showed beneficial influences on behaviors, mainly on emotional behaviors. Also, the effects on the peripheric and central nervous system were observed, in which TS improved important stress markers, such as corticosterone levels and also improved neuroplasticity markers, hypothesizing that TS is an effective tool for the improvement of neurobiological and behavioral response in rats. Considering the results found in this thesis, we can conclude that TS exerts a positive influence on anxiety and depression-like behaviors, confirming the hypothesis that these beneficial effects are associated with HPA axis and neuroplasticity. Besides, the systematic review confirmed these hypotheses of the beneficial effects of TS in rats, especially those observed on neuropsychiatric disorders, such as depression and anxiety. A depressão é um transtorno mental que afeta milhões de pessoas em todo o mundo, e embora diferentes fármacos sejam utilizados para tratar a doença, a eficácia destes tratamentos ainda apresenta limitações. A estimulação tátil (ET) é uma terapia manual que pode ser aplicada desde o nascimento até a vida adulta, e evidências experimentais têm mostrado seus benefícios em diferentes modelos animais. O objetivo dos estudos que compõem esta tese foi avaliar a influência da ET aplicada durante o período neonatal ou na vida adulta de ratos expostos à diferentes modelos de depressão. O protocolo experimental 1 avaliou a influência da ET na idade adulta sobre comportamentos tipo depressivos em ratas. As fêmeas receberam administração de reserpina (1mg/kg/dia, s.c. 3 dias) para a indução de sinais de depressão. O fármaco antidepressivo imipramina (10mg/kg, i.p.) foi utilizado como controle positivo. Após a última administração de reserpina, o protocolo de ET foi realizado (15min/3xdia, 8 dias). Subsequentemente à eutanásia, biomarcadores de estresse foram quantificados no sangue, enquanto a expressão de BDNF, proBDNF, TrkB, GDNF, GFAP e GR foram quantificados através de eletroforese em gel em amostras do córtex pré-frontal (CPF). Os resultados mostraram que a ET reverteu os comportamentos tipo depressivo induzidos pela reserpina, reduzindo também os níveis plasmáticos de corticosterona e do hormônio adrenocorticotrófico, juntamente com menor peso das adrenais. No CPF, a ET aumentou a expressão do BDNF, TrkB, GFAP e GR, e reduziu os níveis de proBDNF. No segundo protocolo experimental avaliamos a influência da ET neonatal (10min/1xdia, 8 dias) aplicada em ratos heterozigotos para o transportador de serotonina (SERT+/-) a fim de determinar a influência deste manuseio sobre o desenvolvimento de comportamentos social, ansiedade e anedonia. Após a eutanásia, a amígdala basolateral foi coletada e a expressão de BDNF e suas isoformas, transportador vesicular de glutamato (VGLUT) e do GABA (VGAT), enzima GAD67, GR e MR, assim como genes responsivos aos glicocorticoides foram mensurados por RT-PCR. Observamos que em animais SERT+/-, a ET melhorou os comportamentos social e afetivos. Em nível molecular, somente a ET per se mostrou alterações na amígdala basolateral, na qual reduziu os níveis de BDNF e das isoformas IV e VI, a razão dos receptores GR/MR, dos genes responsivos aos glicocorticoides, e a razão VGLUT/VGAT, enquanto que aumentou os níveis da enzima GAD67. Por fim, uma revisão sistemática foi realizada para avaliar o impacto da ET em ratos. Após busca em um banco de dados, 55 estudos foram selecionados dentro dos critérios exigidos. De modo geral, observamos que a ET exerceu influências benéficas em diferentes comportamentos, modificando principalmente, comportamentos relacionados à cognição e à emoção. Além disto, alterações periféricas, observadas principalmente através dos níveis séricos de corticosterona como também sobre o SNC, sugerindo que a aplicação da ET constitui um protocolo de manipulação não invasivo e eficaz para melhorar funções neurobiológicas dos animais. A partir dos resultados obtidos nos protocolos experimentais descritos nesta tese, observavamos que a ET exerceu influência favorável sobre comportamentos do tipo depressivo e de ansiedade. Estes resultados confirmam nossa hipótese inicial sobre os benefícios da ET na redução da hiperatividade do eixo hipotalâmico-pituitária-adrenal, minimizando assim a liberação de corticosterona, e favorecendo a ativação de fatores neurotróficos e de outros sistemas relacionados à emocionalidade. Por fim, a revisão sistemática corroborou com os resultados experimentais apresentados, os quais em conjunto, confirmam os benefícios da ET em diferentes períodos da vida sobre aspectos neuroquímico-moleculares e comportamentais relacionados a diferentes transtornos psiquiátricos, tais como ansiedade e depressão.

Introdução: A tiamina (vitamina B1) é cofator de enzimas do metabolismo energético central e sua deficiência (DT) compromete a fosforilação oxidativa, aumenta o estresse oxidativo e ativa processos inflamatórios que podem levar à neurodegeneração. A síndrome de Wernicke-Korsakoff (SWK) é uma manifestação da DT crônica, que leva à uma extensiva morte neuronal, e está associada a distúrbios neuropatológicos, incluindo déficits cognitivos e amnésia. O hipocampo é uma das áreas cerebrais mais afetadas pela SWK. A reposição de tiamina pode não ser suficiente para prevenir o déficit cognitivo irreversível associado à SWK. Materiais e métodos: Foi desenvolvido um modelo de cultura organotípica de fatias de hipocampo (COH) para investigar os mecanismos moleculares subjacentes ao desequilíbrio entre morte neuronal e neurogênese associadas à DT. Os principais efeitos da privação de tiamina nas COHs foram avaliados por imunofluorescência e microscopia confocal e análise do transcriptoma. Resultados: Nas COHs cultivadas sem tiamina, foi observada uma redução significativa na densidade neuronal após cinco dias e, no sétimo dia, as marcas epigenéticas associadas à modulação do estado da cromatina, H3K4me3 e H3K9me3, apresentavam-se alteradas em neurônios maduros, favorecendo a transcrição gênica. Surpreendentemente, entre o sétimo e o décimo quarto dia de cultivo sem tiamina, foi observado um pulso de neurogênese seguido de nova perda massiva de neurônios. O contraste entre os transcriptomas de COHs cultivadas com ou sem tiamina por nove dias revelou 89 genes diferencialmente expressos. Análises de enriquecimento funcional evidenciaram o aumento da expressão de genes das vias KEGG do metabolismo de triptofano e degradação de lisina e de genes com anotações Gene Ontology (GO) relacionadas à organização da matriz extracelular, adesão celular e regulação positiva da transmissão sináptica. Vários genes das vias de sinalização de TNF e FoxO e genes com anotações GO relacionadas à resposta inflamatória estavam inibidos. A ativação de Nsd1, cujo produto metila a histona H3 na lisina 36, também foi observada no grupo DT. O aumento de H3K36me3 em neurônios foi confirmado por imunofluorescência. Finalmente, o tratamento das COHs em privação de tiamina com anti-inflamatório foi eficaz para a antecipação do pulso de neurogênese, demonstrando o papel fundamental da resposta imune na regulação da neurogênese hipocampal durante a DT. Conclusão: A regulação positiva de genes das vias relacionadas à síntese de acetil-CoA a partir de aminoácidos sugere uma adaptação metabólica capaz de permitir que células progenitoras neurais utilizem a fosforilação oxidativa como fonte de energia para a neurogênese. O conjunto de genes diferencialmente expressos no nono dia de privação de tiamina aponta para uma programação genética favorável à maturação dos neurônios e a regulação negativa da neurogênese mediada pela marca H3K36me3 em neurônios já diferenciados. Sobretudo, a redução da expressão de genes inflamatórios antes do repovoamento das COHs com novos neurônios sugere uma relação causal, que é apoiada por estudos anteriores relatando a inflamação como um fator supressor da neurogênese. Portanto, a neuroinflamação parece ser crucial na neurodegeneração associada à DT, podendo ter um papel também na fisiopatologia da SWK. Introduction: Thiamine (vitamin B1) is a cofactor for enzymes of central energy metabolism and its deficiency (TD) impairs oxidative phosphorylation, increases oxidative stress, and activates inflammatory processes that can lead to neurodegeneration. Wernicke-Korsakoff syndrome (SWK) is a consequence of chronic TD, which leads to extensive neuronal death, and is associated with neuropathological disorders, including cognitive deficits and amnesia. The hippocampus is one of the brain areas most affected by SWK. B1 replacement may not be enough to prevent the irreversible cognitive deficit associated with SWK. Materials and methods: An organotypic hippocampal slice culture (OHC) model was developed to investigate the molecular mechanisms underlying the imbalance between neuronal death and neurogenesis associated with TD. The main effects of B1 deprivation on OHCs were assessed by immunofluorescence and confocal microscopy and transcriptome analysis. Results: In OHCs cultured without B1, a significant reduction in neuronal density was observed after five days and, on the seventh day, the epigenetic marks H3K4me3 and H3K9me3, capable of modulating the chromatin state, were altered in mature neurons favoring gene transcription. Surprisingly, between the seventh and the fourteenth day of B1 deprivation, a pulse of neurogenesis was observed followed by a further massive neuron loss. The contrast between the transcriptomes of OHCs cultured with or without B1 for nine days revealed 89 differentially expressed genes. Functional enrichment analyzes showed increased expression of genes in the KEGG pathways of tryptophan metabolism and lysine degradation, and genes with Gene Ontology (GO) annotations related to the organization of the extracellular matrix, cell adhesion, and positive regulation of synaptic transmission. Several genes of the TNF and FoxO signaling pathways and genes with GO terms related to the inflammatory response were inhibited. The activation of Nsd1, whose product methylates histone H3 at lysine 36, was also observed in the TD group. Increased H3K36me3 in neurons was confirmed by immunofluorescence. Finally, the treatment of COHs in B1 deprivation with anti-inflammatory was effective in anticipating the pulse of neurogenesis, demonstrating the fundamental role of the immune response in the regulation of hippocampal neurogenesis during TD. Conclusion: The positive regulation of genes from pathways related to the synthesis of acetyl-CoA from amino acids suggests a metabolic adaptation capable of allowing neural progenitor cells to use oxidative phosphorylation as energy source for neurogenesis. The set of genes differentially expressed at the ninth day of B1 deprivation points to a genetic programming favorable to the maturation of neurons and the negative regulation of neurogenesis mediated by the H3K36me3 mark in already differentiated neurons. Above all, the reduction in the expression of inflammatory genes before repopulation of OHCs with new neurons suggests a causal relationship, which is supported by previous studies reporting neuroinflammation as a suppressive factor in neurogenesis. Therefore, neuroinflammation seems to be crucial in neurodegeneration associated with TD, and may also have a role in the pathophysiology of SWK.

Dans le système nerveux central adulte, la glycine est principalement connue pour son rôle de transmission d’un signal inhibiteur à l'intérieur des neurones matures, régulant ainsi l'activité du réseau neuronal. Paradoxalement, durant l'embryogenèse, ce même neurotransmetteur génère une transmission excitatrice produisant ainsi le premier signal électrique dans les neurones immatures. Le rôle et la signification fonctionnelle de ce changement d’activité durant le développement neurologique restent toujours inconnus. En utilisant l’embryon du poisson-zèbre comme modèle, nous avons exploré les mécanismes moléculaires et cellulaires dépendants de la signalisation de glycine dans les cellules souches neuronales (CSNs). En premier lieu, nous avons développé un outil d’analyse basé sur une combinaison de deux éléments: une lignée transgénique qui exprime du GFP dans les CSNs et la technique de séquençage de l’ARN total. Nous avons utilisé cette technique pour isoler et déterminer les mécanismes moléculaires régulés par la glycine dans les CSNs. Ceci a permis d’identifier plusieurs gènes candidats dont l’expression est modulée par l’activité glycinergique. Ces gènes appartiennent principalement à cinq différentes voies de signalisation canoniques incluant la voie de signalisation du calcium, TGF-bêta, Shh, Wnt et p53. Pour en apprendre davantage sur ces mécanismes moléculaires, nous avons exploré l’un d’entre eux soit la régulation de la signalisation p53 par l’activité glycinergique. En effet, nous avons démontré que l’activité glycinergique favorise la survie des CSNs par la régulation de la signalisation de p53 et agit spécifiquement sur la sous-population CSN-nestin+ durant la neurogenèse. Dans un autre projet, nous avons examiné la régulation de l’expression de lnx1 par l’activité glycinergique. Nous avons démontré que la signalisation de glycine/lnx1 régule la prolifération des CSNs via la modulation de l’activité de Notch durant la neurogenèse. En conclusion, dans ce projet de thèse, j’ai mis en lumière plusieurs mécanismes moléculaires et cellulaires modulés par l’activité glycinergique dans les CSNs. Ceci peut contribuer dans le futur à la compréhension de la physiopathologie liée au dysfonctionnement de cette dernière ainsi qu’à l’identification de nouvelles cibles thérapeutiques., In the adult central nervous system, glycine is mainly known as an inhibitory neurotransmitter in mature neurons, thereby regulating the neural network activity. Paradoxically, during embryogenesis, the same neurotransmitter generates excitatory transmission and induces the first electrical signal in immature neurons. The role and functional significance of this change in glycinergic activity during neurogenesis are still unknown. In this study, we used zebrafish embryos as a model to explore the glycine-dependent molecular and cellular mechanisms in neural stem cells (NSCs). First, we developed an in vivo analysis method based on two main elements: a transgenic line that expresses GFP within NSCs and the RNA sequencing technique. This method of analysis was used to determine glycine-dependent molecular mechanisms in NSCs. We identified several candidate genes whose expression is modulated by the glycinergic activity. These genes participate in five different canonical signaling pathways including the calcium signaling pathway, TGF-beta, Shh, Wnt and p53. To further understand these molecular mechanisms, we focused our investigation on the regulation of p53 signaling by the glycinergic activity. Indeed, we have demonstrated that glycinergic activity promotes the survival of NSCs by regulating p53 signaling and more specifically acting on NSC-nestin + subpopulation during neurogenesis. Finally, we explored the regulation of lnx1 expression by glycinergic activity. We have demonstrated that glycine/lnx1 signaling regulates the proliferation of NSCs via the modulation of Notch activity during neurogenesis. In conclusion, during this thesis project, I highlighted several molecular and cellular mechanisms modulated by the glycinergic activity in NSCs. These relevant results may contribute in the future to the understanding of the physiopathology related to glycinergic activity dysfunctions and the identification of new therapeutic targets.