Made available in DSpace on 2014-12-17T15:28:51Z (GMT). No. of bitstreams: 1 PriscilaTM_DISSERT.pdf: 2986259 bytes, checksum: b2713c3bec2fea6430b0f74437631d89 (MD5) Previous issue date: 2012-04-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The caffeine is a mild psychostimulant that has positive cognitive effects at low doses, while promotes detrimental effects on these processes at higher doses. The episodic-like memory can be evaluated in rodents through hippocampus-dependent tasks. The dentate gyrus is a hippocampal subregion in which neurogenesis occurs in adults, and it is believed that this process is related to the function of patterns separation, such as the identification of spatial and temporal patterns when discriminating events. Furthermore, neurogenesis is influenced spatial and contextual learning tasks. Our goal was to evaluate the performance of male Wistar rats in episodic-like tasks after acute or chronic caffeine treatment (15mg/kg or 30mg/kg). Moreover, we assessed the chronic effect of the caffeine treatment, as well as the influence of the hippocampus-dependent learning tasks, on the survival of new-born neurons at the beginning of treatment. For this purpose, we used BrdU to label the new cells generated in the dentate gyrus. Regarding the acute treatment, we found that the saline group presented a tendency to have better spatial and temporal discrimination than caffeine groups. The chronic caffeine group 15 mg/kg (low dose) showed the best discrimination of the temporal aspect of episodic-like memory, whereas the chronic caffeine group 30mg/kg (high dose) was able to discriminate temporal order, only in a condition of greater difficulty. Assessment of neurogenesis using immunohistochemistry for evaluating survival of new-born neurons generated in the dentate gyrus revealed no difference among groups of chronic treatment. Thus, the positive mnemonic effects of the chronic caffeine treatment were not related to neuronal survival. However, another plastic mechanism could explain the positive mnemonic effect, given that there was no improvement in the acute caffeine groups / A cafe?na ? um leve psicoestimulante que em baixas doses tem efeitos cognitivos e mnem?nicos positivos, enquanto em altas doses tende a possuir efeitos prejudiciais sobre esses processos. A mem?ria tipo-epis?dica em roedores pode ser avaliada com tarefas hipocampo-dependentes. O giro denteado ? uma subregi?o hipocampal onde ocorre neurog?nese no adulto, e acredita-se que esse processo esteja relacionado ? sua fun??o de separa??o de padr?es, ou seja, identifica??o de padr?es espa?o-temporais para discriminar eventos. Al?m disso, a neurog?nese ? influenciada pelo aprendizado de tarefas espaciais e contextuais. Nosso objetivo foi avaliar os efeitos comportamentais em tarefas tipo-epis?dicas, em ratos Wistar machos, submetidos a tratamentos agudo ou cr?nico com cafe?na, nas doses de 15mg/kg ou 30mg/kg. Al?m disso, procuramos avaliar as rela??es do efeito cr?nico da cafe?na, em doses baixa e elevada, bem como da influ?ncia do aprendizado de tarefas hipocampo-dependentes, sobre a sobreviv?ncia de neur?nios nascidos no in?cio do tratamento, fazendo uso de BrdU para marcar novas c?lulas geradas no giro denteado. Quanto ao tratamento agudo, vimos que o grupo salina tendeu a apresentar melhor discrimina??o temporal e espacial que os grupos cafe?na, nas tarefas executadas. Os resultados do tratamento cr?nico mostraram que houve melhor discrimina??o do grupo cafe?na 15 mg/kg (dose baixa) quanto ao aspecto temporal da mem?ria epis?dica; j? o grupo cafe?na 30mg/kg (dose alta) conseguiu discriminar melhor temporalmente em condi??o de maior dificuldade de execu??o em compara??o a menor dificuldade. Avalia??o da neurog?nese por meio de imunohistoqu?mica para contagem de novos neur?nios gerados no giro denteado n?o revelou nenhuma diferen?a entre os grupos do tratamento cr?nico. Assim, os efeitos positivos mnem?nicos do tratamento cr?nico com cafe?na n?o est?o relacionados com a sobreviv?ncia neuronal. Entretanto, outro mecanismo pl?stico deve explicar o efeito mnem?nico positivo, haja vista que n?o houve melhora nos grupos tratados com cafe?na administrada agudamente

The cerebral cortex is one of the most complex and divergent of all biological structures and is composed of hundreds of different types of highly interconnected neurons. This complexity underlies its ability to perform exceedingly complex neural processes. One of the most important questions in developmental neurobiology is how such a vast degree of diversity and specificity is achieved during embryogenesis. Furthermore, understanding the cellular and genetic basis of cortical development may yield insights into the mechanisms underlying human disorders such as mental retardation, autism, epilepsies and brain tumors. <p>During this Phd-project, we set out to identify novel transcription factors involved in cortical neurogenesis. Therefore, we initially took advantage of a model of in vitro embryonic stem cell (ESC)-derived corticogenesis that was previously established in the lab (Gaspard et al. 2008) and from several previously generated ESC lines that allow overexpression of specific transcription factors potentially involved in corticogenesis (van den Ameele et al. 2012). <p>Among the genes tested, Bcl6, a B-cell lymphoma oncogene known to be expressed during cortical development but without well-characterized function in this context, displayed a strong proneurogenic activity and thus became the main focus of this thesis. <p><p>During neurogenesis, neural stem/progenitor cells (NPCs) undergo an irreversible fate transition to become neurons. The Notch pathway is well known to be important for this process, and repression of Notch-dependent Hes genes is essential for triggering differentiation. However, Notch signalling often remains active throughout neuronal differentiation, implying a change in the transcriptional responsiveness to Notch during the neurogenic transition.<p>We showed that Bcl6 starts to be expressed specifically during the transition from progenitors to postmitotic neurons and is required for proper neurogenesis of the mouse cerebral cortex. Bcl6 promotes this neurogenic conversion by switching the composition of Notch-dependent transcriptional complexes at the Hes5 promoter. Bcl6 triggers exclusion of the co-activator Mastermind-like 1 and recruitment of the NAD+-dependent deacetylase Sirt1, which we showed to be required for Bcl6-dependent neurogenesis in vitro. The resulting epigenetic silencing of Hes5 leads to neuronal differentiation despite active Notch signalling. These findings thus suggest a role for Bcl6 as a novel proneurogenic factor and uncover Notch-Bcl6-Sirt1 interactions that may affect other aspects of physiology and disease (Tiberi et al. 2012a). <p><p>A subsequent yet unpublished part of this Phd-project focused on unraveling roles for Bcl6 in regionalization of the cerebral cortex. In all mammals, the three major areas of the neocortex are the motor, somatosensory and visual areas, each subdivided in secondary domains and complemented with species-specific additional areas. All these domains comprise of neurons with different functionality, molecular profiles, electrical activity and connectivity. Spatial patterning of the cortex is mainly under the control of diffusible molecules produced by organizing centers, but is also regulated by intrinsic, cell-autonomous programs (Tiberi et al. 2012b). <p>Since Bcl6 expression is confined to frontal and parietal regions of the developing cerebral cortex and remains high in postmitotic neurons, also after completion of neurogenesis, we hypothesized it would be involved in acquisition of motor and somatosensory identity. As expected from the neurogenesis defect in these regions, we observed a trend towards a reduced size of the frontal areas in the Bcl6 mutant cortex. Preliminary data from cDNA microarray profiling after gain- and loss-of-function of Bcl6 and from in situ hybridization on mouse cortex however do not show dramatic changes in molecular markers of different cortical areas. Similarly, the coarse-grained pattern of thalamocortical and efferent projections of motor and somatosensory neurons appears to be spared. These preliminary findings thus suggest that Bcl6 is not strictly required for proper acquisition of motor and somatosensory areal identity. / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Mes études doctorales ont permis de démontrer que les souris déficientes en VIP présentent une microcéphalie ayant principalement une origine maternelle qui affecte secondairement le développement de la substance blanche. Cette production placentaire par les lymphocytes T pourrait être affectée dans des pathologies du système immunitaire. De plus, nos données indiquent qu’une déficience en VIP prédispose à l'apparition de troubles sensoriels, en particulier de la nociception. Il est donc possible que les déficits précoces de développement du cerveau murin et l'apparition de l'hypersensibilité cutanée mécanique et thermique froide soient deux facettes d'une même pathologie. Des mesures d'activité de décharge spontanée des neurones dans le thalamus sensoriel chez des mâles adultes anesthésiés ont montré que les neurones des animaux KO sont hyper-excités, ce qui suggère un traitement aberrant des informations, notamment nociceptives, ou que l'activité inhibitrice des interneurones des réseaux locaux est réduite. / The studies carried out during my PhD demonstrate that VIP-deficient mice suffer from microcephaly and as well as white matter deficits mainly due to the absence of maternal VIP during embryogenesis, Placental secretion of VIP is dependent on T lymphocytes and could be altered in pathologies of the immune system. Moreover, our data links VIP deficiency to sensory alterations, specifically, the nociceptive system. Thus, it is possible that early developmental defects and hypersensitivity to mechanical and cold stimuli are two manifestations of the same pathology. This hypothesis was reinforced following analysis of spontaneous firing patterns of neurons in the sensory thalamus of anesthetized adult males. Neurons from VIP-KO mice are hyperactive, which suggests aberrant local processing of nociceptive input or that the inhibitory inputs from local interneuron networks is reduced.

Les processus compensatoires qui accompagnent les atteintes du système dopaminergique (DA-ergic) nigrostrié illustrent les capacités adaptatives du cerveau adulte. Cette neuroplasticité permet le maintien de la transmission dopaminergique pendant un certain temps de sorte que les symptômes moteurs cardinaux de la Maladie de Parkinson (MP), qui se caractérise par une dégénérescence progressive des neurones DA-ergiques de la substantia nigra (SN), ne se manifestent qu'après une perte neuronale très importante. De ce fait, le diagnostic présymptomatique est une question cruciale pour le développement de traitements neuroprotecteurs. Un autre exemple de neuroplasticité est illustré par la production de nouveaux neurones dans le cerveau adulte (neurogenèse adulte). Cette neurogenèse s’observe principalement dans deux zones: le continuum zone sous-ventriculaire (SVZ)-bulbe olfactif (OB) et le gyrus denté (DG) de l'hippocampe, et se trouve altérée chez les patients parkinsoniens. Ces dernières années, le traitement chirurgical par la stimulation à haute fréquence (SHF) du noyau sous-thalamique (NST) s'est avéré être une option thérapeutique très efficace pour ces patients. Dans ce contexte, mon travail de thèse a été axé sur l’étude de la neuroplasticité dans différents modèles de la maladie de Parkinson et de son traitement avec les objectifs principaux: 1) Développer un modèle de MP présymptomatique; 2) étudier les mécanismes compensatoires impliquant le système nigrostrié; 3) Déterminer les effets de la SHF-NST sur la neurogenèse adulte dans la SVZ-OB et le DG.Dans la première étude, nous avons développé des modèles expérimentaux de la MP à différents stades, basés sur l’administration de MPTP chez la souris. Nous avons montré que le passage du stade avancé présymptomatique au stade symptomatique précoce correspondant au seuil d’atteinte des systèmes DA-ergiques associé à l’apparition des déficits moteurs, se caractérise par : (a) une diminution de DA dans les terminaisons striatales épargnées par la lésion; (b) une augmentation de DA et d’expression de la tyrosine hydroxylase dans les cellules de la SN; (c) une augmentation du renouvellement de la DA dans le striatum et une augmentation moindre dans la SN.La deuxième étude est basée sur un modèle de lésion DA-ergique extensive par injection intranigrale de 6-hydroxydopamine chez le rat, imitant les stades tardifs de la MP. Nous avons étudié séparément les étapes de prolifération et de survie des nouvelles cellules sur des animaux non lésés et des animaux lésés avec ou sans SHF subchronique (8 jours) du NST. Nous avons pu montrer une régulation spécifique des étapes de prolifération et de survie suite à la lésion dopaminergique, et des effets stimulateurs de la SHF du NST sur la survie des cellules néoformées, suggérant un effet neuroprotecteur de ce traitement. / The compensatory processes that accompany a lesion of the nigrostriatal dopaminergic (DA-ergic) system serve to maintain its function and illustrate adult brain neuroplasticity. The typical motor symptoms of Parkinson’s diseases (PD), characterized by progressive degeneration of DA-ergic neurons of substantia nigra (SN), appear only after substantial neuronal loss. Therefore presymptomatic diagnosis is a crucial issue for future neuroprotective therapies. Another good manifestation of neuroplasticity is adult neurogenesis, known to persist in two areas: the subventricular zone (SVZ) – the olfactory bulb (OB) continuum, and the dentate gyrus (DG) of the hippocampus, and to be altered in PD. In recent years, the surgical treatment by high frequency stimulation (HFS) of the subthalamic nucleus (STN) has proven to be an efficient therapeutic option for PD patients. In this context, my PhD work was focused on neuroplasticity under the functional deficiency of the nigrostriatal DA-ergic system (parkinsonism) and its treatment with the following main objectives: 1 - Develop a model of presymptomatic parkinsonism; 2 - study compensatory mechanisms in nigrostriatal system; 3 - Characterize the effects of subchronic STN HFS on adult neurogenesis. In the first part, we have developed models of presymptomatic parkinsonism based on MPTP administration in mice, as defined by sub-threshold DA depletion and degeneration of DA-ergic axons in the striatum followed by a loss of DA-ergic cell bodies in the SN (advanced presymptomatic stage). In the early symptomatic stage, these parameters reach a threshold that is associated with the appearance of motor deficiency. We have shown that the transition from the advanced presymptomatic stage to the early symptomatic stage is characterized by: (a) a decrease of DA content in surviving DA-ergic axons in the striatum; (b) an increase of DA content and TH-expression in surviving neuronal cell bodies in the SN; (c) an increase of DA turnover in the striatum and much less increase in the SN. The last part of my work is based on extensive DA lesion in rats, using intranigral 6-hydroxydopamine injection mimicking late PD stages, to determine a possible effect of STN-HFS on adult neurogenesis. We have completed series of animals with DA lesion either sham implanted or subsequently treated for 8 days by STN-HFS to be compared with unlesioned rats, and studied selective phases of neurogenesis: proliferation and survival. This study demonstrates selective regulation of cell proliferation and survival following DA depletion and provides the first evidence that prolonged STN-HFS might have a neuroprotective action as shown by the selective increase in survival of newly formed cells following this treatment.

La neurogenèse est présente, dans le cerveau adulte, dans la zone sous-ventriculaire (ZSV) encadrant les ventricules latéraux et dans le gyrus dentelé (GD) de l’hippocampe, permettant l’apprentissage, la mémoire et la fonction olfactive. Ces micro-environnements possèdent des signaux contrôlant l’auto-renouvellement des cellules souches neurales (CSN), leur prolifération, leur destin et leur différenciation. Or, lors du vieillissement, les capacités régénératives et homéostatiques et la neurogenèse déclinent. Les patients atteints de la maladie d’Alzheimer (MA), comme le modèle animal reproduisant cette maladie (3xTg-AD), montrent une accélération des phénotypes liés au vieillissement dont une diminution de la neurogenèse. Notre hypothèse est que la découverte des mécanismes affectant la neurogenèse, lors du vieillissement et de la MA, pourrait fournir de nouvelles cibles thérapeutiques pour prévenir le déclin cognitif. Les études sur l’âge d’apparition et les mécanismes altérant la neurogenèse dans la MA sont contrastées et nous ont guidé vers deux études. L’examen des changements dans les étapes de la neurogenèse lors du vieillissement et du développement de la neuropathologie. Nous avons étudié la ZSV, les bulbes olfactifs et le GD de souris femelles de 11 et 18 mois, et l’apparition des deux pathologies associées à la MA : les plaques amyloïdes et les enchevêtrements neurofibrillaires. Nous avons découvert que les souris 3xTg-AD possèdent moins de cellules en prolifération, de progéniteurs et de neuroblastes, induisant une diminution de l’intégration de nouvelles cellules dans le GD et les bulbes olfactifs. Notons que le taux de neurogenèse chez ces souris de 11 mois est similaire à celui des souris de phénotype sauvage de 18 mois, indiquant une accélération des changements liés au vieillissement dans la MA. Dans la ZSV, nous avons aussi démontré une accumulation de gouttelettes lipidiques, suggérant des changements dans l’organisation et le métabolisme de la niche. Enfin, nous avons démontré que le déficit de la neurogenèse apparait lors des premières étapes de la MA, avant l’apparition des plaques amyloïdes et des enchevêtrements neurofibrillaires. A l’examen des mécanismes inhibant la neurogenèse lors de la MA, nous voyons que chez des souris de 5 mois, le déficit de la neurogenèse dans la ZSV et le GD est corrélé avec l’accumulation de lipides, qui coïncide avec l’apparition du déclin cognitif. Nous avons aussi découvert que dans le cerveau humain de patients atteints de la MA et dans les 3xTg-AD, des gouttelettes lipidiques s’accumulaient dans les cellules épendymaires, représentant le principal soutien des CSN de la niche. Ces lipides sont des triglycérides enrichis en acide oléique qui proviennent de la niche et pas d’une défaillance du système périphérique. De plus, l’infusion locale d’acide oléique chez des souris de phénotype sauvage permet de reproduire l’accumulation de triglycérides dans les cellules épendymaires, comme dans la MA. Ces gouttelettes induisent un dérèglement de la voie de signalisation Akt-FoxO3 dans les CSN, menant à l’inhibition de leur activation in vitro et in vivo. Ces résultats permettent une meilleure compréhension de la régulation de la neurogenèse par le métabolisme lipidique. Nous avons démontré un nouveau mécanisme par lequel l’accumulation des lipides dans la ZSV induit une inhibition des capacités de prolifération et de régénération des CSN lors de la MA. Les travaux futurs permettront de comprendre comment et pourquoi le métabolisme lipidique du cerveau est altéré dans la MA, ce qui pourrait offrir de nouvelles voies thérapeutiques pour la prévention et la régénération. / In the adult brain, neurogenesis continues in the subventricular zone (SVZ) surrounding the lateral ventricles and the dentate gyrus (DG) of the hippocampus where it plays a critical role in learning, memory, and olfactory function. Within these microenvironments, combinatorial signals control neural stem cell (NSC) self-renewal, proliferation, fate determination and differentiation. Unfortunately, during aging, neurogenesis declines along with many other homeostatic and regenerative capabilities. Furthermore, Alzheimer’s disease (AD) patients and many AD models show an acceleration of several aging related phenotypes including neurogenesis. We hypothesize that uncovering how neurogenesis is affected during aging and in AD will provide novel targets for prevention of cognitive decline. However, conflicting findings including the age of onset and the mechanisms altering neurogenesis in AD propelled us to perform the two following studies. First, we examined the various steps of neurogenesis and how they change as a result of aging, neuropathology development, and neurogenic niches. We studied neurogenesis in the SVZ, olfactory bulb, and DG of 11- and 18-month-old female mice and simultaneously measured the stages of the two major AD-associated pathologies, amyloid plaques and neurofibrillary tangles. We found that, 3xTg-AD mice had fewer proliferating cells, neural progenitors and neuroblasts, resulting in decreased numbers of adult-born cells added to the dentate granule cell layer and the olfactory bulbs. Interestingly, the levels of neurogenesis in 11-month-old 3xTg mice were similar to those in 18-month-old WT mice, indicating an acceleration of aging-related changes in neurogenesis. Interestingly, we found that the deficits in neurogenesis appear at early stages of AD-associated pathologies, before the appearance of the hallmark Aβ plaques and neurofibrillary tangles. Instead, we found a pronounced accumulation of large lipid droplets, suggestive of significant organizational and metabolic changes (Chapter 2). Second, we examined the mechanisms inhibiting neurogenesis in AD. Studying young 5-month-old mice, we found that deficits in SVZ and DG neurogenesis still correlated with extensive lipid accumulation coinciding with the onset of cognitive decline. Importantly, we found that postmortem AD brains and 3xTg-AD mice accumulate neutral lipids within ependymal cells, the main support cell of the SVZ niche. Using novel mass spectrometry techniques, we identified these lipids as oleic acid-enriched triglycerides. Moreover, analysis of plasma, cerebrospinal fluid, and microdissected SVZs showed that these lipids originate from niche-derived rather than peripheral lipid metabolism defects. Remarkably, locally increasing oleic acid in wild-type mice was sufficient to recapitulate the AD-associated ependymal triglyceride phenotype and led to a de-regulation of the Akt-FoxO3 NSC preservation pathway, and inhibition of NSC activation in vitro and in vivo (Chapter 3). Together, this work suggests a novel mechanism of cognitive defects. Specifically, lipid accumulation within SVZ niche cells during early adulthood inhibits the proliferative and regenerative capacity of NSCs in AD. Future work aimed at understanding how and why brain lipid metabolism is altered in AD could provide therapeutic targets for preventative and regenerative strategies for those suffering from AD.

Neurotoxlns represent unique chemical tools, providing a means to 1) gain insight into cellular mechanisms of apopotosis and necrosis, 2) achieve a morphological template for studies otherwise unattainable, 3) specifically produce a singular phenotype of denervation, and 4) provide the starting point to delve into processes and mechanisms of nerve regeneration and sprouting. There are many other notable uses of neurotoxins in neuroscience research, and ever more being discovered each year. The objective of this review paper is to highlight the broad areas of neuroscience in which neurotoxins and neurotoxicity mechanism come into play. This shifts the focus away from neurotoxins per se, and onto the major problems under study today. Neurotoxins broadly defined are used to explore neurodegenerative disorders, psychiatric disorders and substance use disorders. Neurotoxic mechanisms relating to protein aggregates are indigenous to Alzheimer disease, Parkinson's disease. NeuroAIDS is a disorder in which microglia and macrophages have enormous import. The gap between the immune system and nervous system has been bridged, as neuroinflammation is now considered to be part of the neurodegenerative process. Related mechanisms now arise in the process of neurogenesis. Accordingly, the entire spectrum of neuroscience is within the purview of neurotoxins and neurotoxicity mechanisms. Highlights on discoveries in the areas noted, and on selective neurotoxins, are included, mainly from the past 2 to 3 years.

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.

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.

[ES] El envejecimiento se concibe como un proceso progresivo de deterioro funcional que se produce a lo largo de la vida. En general, el avance de la edad va acompañado del deterioro de múltiples tejidos, la alteración de la homeostasis y el aumento de la fragilidad. La edad se considera el principal factor de riesgo de un gran número de enfermedades con elevadas tasas de mortalidad, como las patologías cardiovasculares, el cáncer, la fibrosis pulmonar, la esteatosis hepática y los trastornos neurodegenerativos, entre otros. A medida que aumenta la población de edad avanzada, estas patologías son cada vez más frecuentes en nuestra sociedad y constituyen actualmente una de las principales preocupaciones sanitarias en atención primaria. Por consiguiente, comprender los mecanismos que causan el envejecimiento y el consiguiente deterioro de la salud es un reto importante para la comunidad científica, respaldado por un significativo interés socioeconómico. Alcanzar este objetivo permitiría realizar intervenciones específicas contra las enfermedades relacionadas con la edad, promover un envejecimiento saludable y prolongar la vida humana. En este contexto, la senescencia celular se ha convertido en un importante objetivo de investigación, ya que este destino celular se considera un factor que contribuye al deterioro funcional y estructural de los tejidos con el aumento de la edad, dando lugar a enfermedades crónicas. Los trastornos neurodegenerativos son en su mayoría de etiología desconocida, y en la actualidad sólo se dispone de tratamientos paliativos. La neurodegeneración puede deberse a la disminución de la neurogénesis que se produce con el envejecimiento. A este respecto, la vasculatura cerebral representa un componente esencial de los nichos neurogénicos, donde residen las células con capacidad para generar nuevas neuronas. Los microvasos cerebrales contribuyen sustancialmente a preservar la homeostasis y el potencial neurogénico de estas regiones, pero también experimentan cambios estructurales y funcionales con la edad. Sin embargo, aún no está claro si estas alteraciones están relacionadas con la senescencia celular. De ahí que el propósito de esta tesis sea profundizar en los entresijos de la senescencia relacionada con la edad y su impacto en el proceso de envejecimiento cerebral. En este sentido, hemos desarrollado una sonda fluorogénica para la detección in vivo de la actividad ß-Gal, un marcador ampliamente utilizado de senescencia celular. Hemos estimado la carga de células senescentes de forma no invasiva en estudios longitudinales utilizando esta sonda durante el envejecimiento natural y acelerado o durante tratamientos senolíticos. Estos resultados nos han permitido establecer una correlación real entre envejecimiento y senescencia. Por otro lado, hemos centrado nuestros esfuerzos en crear estrategias para detectar la senescencia en las células endoteliales cerebrales y estudiar sus efectos sobre la neurogénesis durante el envejecimiento. Los resultados mostraron que las células endoteliales senescentes a nivel cerebral tienen un impacto perjudicial sobre la neurogénesis y las células madre neurales, lo que sitúa a estas células como diana para futuros estudios sobre el envejecimiento cerebral. / [CA] L'envelliment es concep com un procés progressiu de deterioració funcional que es produeix al llarg de la vida. En general, l'avanç de l'edat va acompanyat de la deterioració de múltiples teixits, l'alteració de l'homeòstasi i l'augment de la fragilitat. L'edat es considera el principal factor de risc d'un gran nombre de malalties amb elevades taxes de mortalitat, com les patologies cardiovasculars, el càncer, la fibrosi pulmonar, la esteatosis hepàtica i els trastorns neurodegeneratius, entre altres. A mesura que augmenta la població d'edat avançada, aquestes patologies són cada vegada més freqüents en la nostra societat i constitueixen actualment les principals preocupacions sanitàries en atenció primària. Per consegüent, comprendre els mecanismes que causen l'envelliment i la consegüent deterioració de la salut és un repte important per a la comunitat científica, recolzat per un significatiu interés socioeconòmic. Aconseguir aquest objectiu permetria realitzar intervencions específiques contra les malalties relacionades amb l'edat, promoure un envelliment saludable i prolongar la vida humana. En aquest context, la senescència cel·lular s'ha convertit en un important objectiu d'investigació, ja que aquest destí cel·lular es considera un factor que contribueix a la deterioració funcional i estructural dels teixits amb l'augment de l'edat, donant lloc a malalties cròniques. Els trastorns neurodegeneratius són en la seua majoria d'etiologia desconeguda, i en l'actualitat només es disposa de tractaments pal·liatius. La neurodegeneración pot deure's a la disminució de la neurogènesi que es produeix amb l'envelliment. Referent a això, la vasculatura cerebral representa un component essencial dels nínxols neurogènics, on resideixen les cèl·lules amb capacitat per a generar noves neurones. Els microvasos cerebrals contribueixen substancialment a preservar l'homeòstasi i el potencial neurogènic d'aquestes regions, però també experimenten canvis estructurals i funcionals amb l'edat. No obstant això, encara no és clar si aquestes alteracions estan relacionades amb la senescència cel·lular. D'aquí ve que el propòsit d'aquesta tesi siga aprofundir en els secrets de la senescència relacionada amb l'edat i el seu impacte en el procés d'envelliment cerebral. En aquest sentit, hem desenvolupat una sonda fluorogénica per a la detecció in vivo de l'activitat ß-Gal, un marcador àmpliament utilitzat de senescència cel·lular. Hem estimat la càrrega de cèl·lules senescentes de forma no invasiva en estudis longitudinals utilitzant aquesta sonda durant l'envelliment natural i accelerat o durant tractaments senolíticos. Aquests resultats ens han permés establir una correlació real entre envelliment i senescència. D'altra banda, hem centrat els nostres esforços a crear estratègies per a detectar la senescència en les cèl·lules endotelials cerebrals i estudiar els seus efectes sobre la neurogènesi durant l'envelliment. Els resultats van mostrar que les cèl·lules endotelials *senescentes a nivell cerebral tenen un impacte perjudicial sobre la neurogènesi i les cèl·lules mare *neurales, la qual cosa situa a aquestes cèl·lules com a diana per a futurs estudis sobre l'envelliment cerebral. / [EN] Ageing is conceived as a progressive process of functional decline that occurs over the course of life. In general, advancing age is accompanied by the deterioration of multiple tissues, altered homeostasis and increased frailty. Age is considered to be the main risk factor for a large number of diseases with high mortality rates, such as cardiovascular pathologies, cancer, pulmonary fibrosis, hepatic steatosis, and neurodegenerative disorders, among others. As the elderly population grows, these pathologies are becoming increasingly prevalent in our society and are now the leading health concerns in primary care. Consequently, comprehending the mechanisms that cause ageing and resulting health decline is a major challenge for the scientific community, backed by significant socio-economic interest. Achieving this goal would allow for targeted interventions against age-related diseases, promote healthy ageing, and extend human lifespan. In this context, cellular senescence has emerged as an important research target, as this cellular fate is considered a contributing factor to the functional and structural deterioration of tissues with increasing age, leading to chronic diseases. Neurodegenerative disorders are mostly of unknown etiology, and only palliative treatments are currently available. Neurodegeneration may be prompted by the decline in neurogenesis that occurs with ageing. In this regard, the brain vasculature represents an essential component of the neurogenic niches, where cells with the capacity to generate new neurons reside. Brain microvessels contribute substantially to preserving the homeostasis and neurogenic potential of these regions, but they also undergo structural and functional changes with age. However, whether these alterations are linked to cellular senescence it is not yet clear. Hence, the purpose of this thesis is to delve deeper into the intricacies of age-related senescence and its impact on the process of brain ageing. In this regard, we have developed a fluorogenic probe for the in vivo detection of ß-Gal activity, a widely used marker of cellular senescence. We have estimated the senescent cell burden non-invasively in longitudinal studies using this probe during natural and accelerated ageing or during senolytic treatments. These results have allowed us to establish a real correlation between ageing and global senescence. On the other hand, we have focused our efforts on creating strategies to detect senescence in brain endothelial cells and to study its effects on neurogenesis during ageing. The results showed that senescent endothelial cells at the brain level have a detrimental impact on neurogenesis and neural stem cells, positioning these cells as a target for future studies on brain ageing. / This PhD thesis has been supported by a pre-doctoral scholarship from the Spanish Ministry of Universities, “Programa de Formación del Profesorado Universitario (FPU)”, and a technical expert contract funded by the European Commission. The research has been funded by the following research projects led by Ramón Mártinez Máñez: - Gobierno de España (RTI2018-100910-B-C41 and PID2021-126304OB-C41). - Generalitat Valenciana (PROMETEO 2018/024 and CIPROM/2021/007). - CIBER-BBN- Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (CB06/01/2012). And by the following research projects led by Isabel Fariñas: - 2014-2018. Estudio de células madre en el ámbito de las investigaciones básicas en terapia celular. Fundación Botín-Banco de Santander. - 2018-2021. Regulación del comportamiento de las células madre neurales por el medio sistémico: el nicho extendido. MINECO (SAF2017-86690-R). - 2016-ongoing. CIBER en Enfermedades Neurodegenerativas (CIBERNED). ISCIII (Programa de Investigación Cooperativa, CB06/05/0086). - 2017-2021. Efectos directos y remotos de la respuesta inflamatoria sobre las células madre neurales. Generalitat Valenciana (Proyectos de Excelencia, PROMETEO/2017/030). - 2017-2021. RETIC de terapia celular ISCIII (RD16/0011/0017). - 2021-2024 Regulación molecular de la heterogeneidad celular en los nichos neurogénicos adultos MICINN (PID2020- 117937GB-I00). - 2021-2025. Una aproximación multidisciplinar al estudio de la respuesta al daño genómico en células madre neurales: de levadura a mamíferos y vuelta. Generalitat Valenciana. Programa Prometeo de Proyectos de Excelencia (PROMETEO/2021/028). / Rojas Vázquez, S. (2023). Impact of Brain Endothelial Senescence on Neurogenesis: Application of Novel Strategies for Age-Related Senescence Detection [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/194629

The risks of damage to the developing nervous system of many chemicals are not known because these studies often require costly and time-consuming multi-generational animal experiments. Pluripotent stem cell-based systems can facilitate developmental neurotoxicity studies because disturbances in nervous system development can be modeled in vitro. In this study, neurons derived from embryonal carcinoma (EC) and induced pluripotent stem (iPS) cells, were first characterized to establish their suitability for developmental neurotoxicity studies. The EC stem cell line, TERA2.cl.SP-12, was differentiated into neurons that expressed voltage-gated sodium and potassium channels as well as ionotropic GABA and glutamate receptors. These cells could also fire action potentials when stimulated electronically. However spontaneous action potentials were not observed. In contrast, pre-differentiated neurons derived from iPS cells fired evoked and spontaneous action potentials. Furthermore, iPS cell-derived neurons also expressed a wide array of functional voltage- and ligand-gated ion channels. Antiepileptic drugs (AEDs) are associated with developmental neurotoxicity. These agents can cause congenital malformations, cognitive deficits and behavioral impairment in children as a result of in utero exposure. The impact of four major AEDs, namely phenobarbital, valproic acid, carbamazepine and lamotrigine, on cell viability, cell cycle and differentiation of TERA2.cl.SP-12 into neurons was studied. All AEDs tested reduced differentiating stem cell viability. Valproic acid and carbamazepine increased apoptosis and reduced cell proliferation. A brief exposure to phenobarbital, valproic acid and lamotrigine at the start of differentiation impaired the subsequent generation of neurons. Additionally, the effect of transient exposure to phenobarbital and carbamazepine on neuronal maturation of iPS-derived neurons was investigated. Exposure to both AEDs resulted in diminished membrane potentials and reduced the proportion of cells that were able to fire action potentials spontaneously in culture. The data from these studies suggest that impairments in proliferation, differentiation and maturation of neurons derived from human stem cells may be sensitive indicators of neurodevelopmental disruption by these drugs that can result from in utero exposure. Furthermore, these findings suggest that the use of human pluripotent stem cells and neurons derived from them can reduce the time, cost and the number of animals used in toxicological research.