GABAergic interneurons control spiking of adult-born hippocampal granule cells via nonlinear α5-GABAA receptors

Excitatory GABAergic synapses have been shown to promote development and maturation of newborn granule cells in the adult hippocampus. In addition, strong GABAergic synaptic inputs are known to generate effective shunting inhibition in these young neurons. However, the functional properties of the GABA receptors mediating excitation and inhibition are largely unknown. Here we analyzed GABA receptors in young neurons activated by soma-targeting parvalbumin and dendrite-targeting somatostatin inhibitory interneurons. The synaptic GABAA receptors in young neurons show a pronounced non-linear voltage dependence and are assembled in part by a5- subunits. As a consequence, synaptic conductance is 4-fold larger around the AP threshold (-35 mV) as compared to the resting potential (-80mV), independent of the interneuron subtype. By contrast, in mature granule cells, parvalbumin interneurons mediate linear GABAergic synaptic currents lacking a5-subunits. Blocking a5-GABAA- receptor-mediated synaptic currents in young neurons not only reduced GABAergic depolarization, but also effectively reduced shunting inhibition of AP generation. Taken together, this data shows that nonlinear GABAA receptors support both GABAergic depolarization and effective GABAergic shunting inhibition in newborn young granule cells of the adult hippocampus

Epigenetic regulation of adult neural stem cells: implications for Alzheimer's disease.

Published onlineJournal ArticleResearch Support, Non-U.S. Gov'tReviewExperimental evidence has demonstrated that several aspects of adult neural stem cells (NSCs), including their quiescence, proliferation, fate specification and differentiation, are regulated by epigenetic mechanisms. These control the expression of specific sets of genes, often including those encoding for small non-coding RNAs, indicating a complex interplay between various epigenetic factors and cellular functions.Previous studies had indicated that in addition to the neuropathology in Alzheimer's disease (AD), plasticity-related changes are observed in brain areas with ongoing neurogenesis, like the hippocampus and subventricular zone. Given the role of stem cells e.g. in hippocampal functions like cognition, and given their potential for brain repair, we here review the epigenetic mechanisms relevant for NSCs and AD etiology. Understanding the molecular mechanisms involved in the epigenetic regulation of adult NSCs will advance our knowledge on the role of adult neurogenesis in degeneration and possibly regeneration in the AD brain.Internationale Stichting Alzheimer Onderzoek (ISAO)Netherlands Organization for Scientific Research (NWO)Maastricht University Medical Centre 

O papel do fator de transcrição AP2γ na modulação da neurogénese glutamatérgica adulta em depressão

Dissertação de mestrado em Ciências da SaúdeMajor depressive disorder (MDD) is a multidimensional psychiatric disease, considered by the World Health Organization as one of the leading causes of disability. Despite the importance of this disease in modern societies and the large investment of resources already made in its study, the processes underlying its pathophysiology remain poorly understood. Several hypotheses have been proposed to clarify the neurobiological mechanisms underlying this psychiatric disorder, being the link between adult hippocampal neurogenesis and MDD a central topic in the past decades. Previous studies have identified AP2γ as a key regulator of adult hippocampal neurogenesis in mice, being expressed in a subpopulation of adult transient amplifying progenitors, and acting as a regulator of basal progenitors, promoting proliferation and glutamatergic neuronal differentiation. Thus, we wanted to further explore the impact of AP2γ in brain neurophysiology and behavior during development and at adult stages, dissecting also its mechanisms both in healthy and depressive states. With this study, we were able to understand the impact of AP2γ in post-natal development and during juvenile age, through the AP2γ constitutive knockout (KO) model. In the developmental milestones assessment we did not find any major impairment in the behavioral performance of AP2γ KO mice, since all parameters analyzed, including the ones where we found differences, were within the typical range for appearance of the developmental milestones. However, in the juvenile behavior assessment and in the hippocampal glutamatergic neurogenesis process, impairments were found, since AP2γ KO mice showed anxious-like behavior and decreased proliferation of immature neurons. To study the impact of modulating the transcription factor AP2γ in depression we exposed both constitutive and conditional KO animal models to a chronic stress protocol, which efficiently induced core depressive-like symptoms. Through the conditional AP2γ KO mice, we were able to elucidate the impact of deleting AP2γ on behavior and neurogenesis in depressive-like conditions specifically in adult age, without the interference of potential functions of the gene during early development that may appear in the constitutive AP2γ model. Through a multidimensional behavioral analysis, we observed that both models presented similar results in the three most affected behavioral dimensions in depression, namely anxiety, mood and cognition. Regarding anxiety and mood no major differences were found between genotypes in both animal models. Moreover, AP2γ KO mice presented cognitive deficits in basal conditions, but when exposed to chronic mild stress no detrimental effects of deletion of the gene were observed. In this work, we also identified, through a broad analysis of the dentate gyrus neurogenic niche, alterations of epigenetic regulators in the AP2γ constitutive KO mice after uCMS exposure. The reported results not only support the involvement of AP2γ in the transcriptional network that modulates the juvenile and adult neurogenic process, but also highlight the potential of this molecule as a future therapeutical tool in neuropsychiatric disorders, in which neurogenesis is impaired.O transtorno depressivo persistente é uma doença psiquiátrica multidimensional, considerada pela Organização Mundial de Saúde como uma das principais causas de incapacidade. Apesar da importância desta doença na sociedade moderna, e do largo investimento de recursos já feitos no seu estudo, os processos subjacentes à sua patofisiologia continuam pouco percebidos. Várias hipóteses foram propostas para clarificar os mecanismos neurobiológicos implícitos nesta doença psiquiátrica, tendo sido o vínculo entre a neurogénese hipocampal adulta e a depressão um tópico central nas décadas passadas. Estudos anteriores identificaram o AP2γ como um regulador chave da neurogénese hipocampal adulta em ratinhos, sendo expresso numa subpopulação de células progenitoras de rápida amplificação adultas, e atuando como regulador de progenitores basais, promovendo a proliferação e a diferenciação neuronal glutamatérgica. Deste modo, propusemos continuar a explorar o impacto do AP2γ na neurofisiologia cerebral e no comportamento, durante a fase de desenvolvimento e na idade adulta, procurando entender também os seus mecanismos tanto no estado saudável como em depressão. Com este trabalho, fomos capazes de entender o impacto do AP2γ no desenvolvimento pós-natal e em idade juvenil, a partir do modelo animal de deleção constitutiva do AP2γ. Na avaliação dos marcos de desenvolvimento, não encontramos nenhuma alteração no desempenho comportamental nos animais com deleção de AP2γ, visto que todos os parâmetros analisados, incluindo os que encontramos alguma diferença, se encontravam dentro dos intervalos típicos de aparecimento dos marcos de desenvolvimento. Contudo, na avaliação do comportamento juvenil e no processo de neurogénese glutamatérgica hipocampal observamos défices, visto que os animais com deleção de AP2γ apresentaram comportamento ansioso e uma diminuição da proliferação de neurónios imaturos. Para estudar o impacto da modulação do fator de transcrição AP2γ em depressão expusemos tanto o modelo animal com deleção constitutiva bem como o modelo animal condicional do gene a um protocolo de stress crónico, o qual eficientemente induziu sintomas primários de depressão. Através do modelo animal condicional do AP2γ, conseguimos compreender o impacto da deleção do AP2γ na modulação do comportamento e neurogénese em condições depressivas especificamente em idade adulta, sem interferência das potenciais funções do gene durante o período de desenvolvimento dos animais, que poderão surgir no modelo animal constitutivo do AP2γ. Através, de uma análise comportamental multidimensional, observamos que ambos os modelos apresentaram resultados similares nas dimensões comportamentais mais afetadas na depressão, nomeadamente a ansiedade, o humor e a cognição. Relativamente à ansiedade e ao humor não encontramos grandes diferenças entre genótipos em ambos os modelos animais. Além disso, os modelos animais de deleção do AP2γ apresentaram défices cognitivos em condições basais, mas após exposição ao stress crónico não foram observados os efeitos prejudiciais da deleção do gene. Neste trabalho, também identificamos, através de uma análise abrangente do nicho neurogénico girus denteado, um reguladores epigenéticos alterados no animal constitutivo do AP2γ. Os resultados apresentados não só suportam o envolvimento do AP2γ na rede transcripcional responsável pela modulação do processo neurogénico juvenil e adulto, como também destacam o potencial desta molécula em abordagens terapêuticas futuras em doenças neuropsiquiátricas, nas quais a neurogénese se encontra afetada

Environmental and genetic correlates of neuropsychiatric diseases and the role of erythropoietin/hypoxia in the brain as potential treatment targets

Neuropsychiatric disorders are relatively frequent and present a considerable burden to affected individuals and society. Disease etiology is often complex and patients exhibit large heterogeneity regarding disease causing factors, presentation and progression. Available treatment options are often ineffective and/or linked to unwanted side-effects. On the other hand, availability of successful preventive actions is limited and requires a detailed study of risk factors for neuropsychiatric disease and its specific symptoms. The first part of this thesis aimed to investigate environmental and genetic risk factors for polytoxicomania, i.e. multiple drug (ab)use, as a frequent comorbidity of schizophrenia. In a sample of $\sim$2000 schizophrenia/schizoaffective patients, we addressed the question if the exposure to accumulated environmental risk in early life increases susceptibility to polytoxicomania. Indeed, the accumulation of environmental risk was strongly associated with polytoxicomania throughout life and in particular in pre-adulthood. Moreover, the development of a novel GWAS-PGAS approach led to the identification of 41 common genetic variants potentially conferring risk to preadult polytoxicomania. The objective of the second part of this thesis work was to further investigate brain-directed effects of hypoxia and erythropoietin (EPO) - a central hypoxia-inducible gene - as potential treatment option for neuropsychiatric disorders. Using a hypoxia reporter mouse line (CAG-CreERT2-ODD::R26R-tdTomato), we showed that both inspiratory hypoxia and motor-cognitive challenge, which causes endogenous hypoxia as a result of extensive neuronal activation (termed "functional hypoxia"), increased the number of hypoxic cells in the brain and the expression of hypoxia-inducible genes in the hippocampus. Interestingly, cell types showed variable responsivity to hypoxia: While neurons and endothelial cells were frequently labelled, hypoxia-labelling in microglia was entirely absent. Technical artifacts explaining this phenomenon were excluded by comparing construct mRNA levels across all cell types. Hexokinase 2 (Hk2) was identified as a mediator of cell type-specific hypoxia responsivity. In addition, we report rapid effects of EPO on adult neurodifferentiation in the CA1 (6 hours after injection). Enhanced neuronal differentiation continued under EPO treatment, driving immature neurons (\textit{Tbr1}+, \textit{Tle4}+ and later \textit{Zbtb20}+) towards maturity, and resulted in $\sim$20\% more neurons in the CA1 after 3 weeks of treatment. Simultaneously, the number of microglia in this region declined by an initial wave of apoptosis, followed by attenuated proliferation. This reduction was necessary for the increase in new neurons. Expression data further indicated that the microglial-neuron balance was maintained by signalling of microglial Colony-Stimulating Factor 1 Receptor (CSF1R) and its neuronally expressed ligand Interleukin 34 (IL34).2022-06-0

Mechanisms underlying activation of neural stem cells in the adult central nervous system

À la fin du 19e siècle, Dr. Ramón y Cajal, un pionnier scientifique, a découvert les éléments cellulaires individuels, appelés neurones, composant le système nerveux. Il a également remarqué la complexité de ce système et a mentionné l’impossibilité de ces nouveaux neurones à être intégrés dans le système nerveux adulte. Une de ses citations reconnues : “Dans les centres adultes, les chemins nerveux sont fixes, terminés, immuables. Tout doit mourir, rien ne peut être régénérer” est représentative du dogme de l’époque (Ramón y Cajal 1928). D’importantes études effectuées dans les années 1960-1970 suggèrent un point de vue différent. Il a été démontré que les nouveaux neurones peuvent être générés à l’âge adulte, mais cette découverte a créé un scepticisme omniprésent au sein de la communauté scientifique. Il a fallu 30 ans pour que le concept de neurogenèse adulte soit largement accepté. Cette découverte, en plus de nombreuses avancées techniques, a ouvert la porte à de nouvelles cibles thérapeutiques potentielles pour les maladies neurodégénératives. Les cellules souches neurales (CSNs) adultes résident principalement dans deux niches du cerveau : la zone sous-ventriculaire des ventricules latéraux et le gyrus dentelé de l’hippocampe. En condition physiologique, le niveau de neurogenèse est relativement élevé dans la zone sous-ventriculaire contrairement à l’hippocampe où certaines étapes sont limitantes. En revanche, la moelle épinière est plutôt définie comme un environnement en quiescence. Une des principales questions qui a été soulevée suite à ces découvertes est : comment peut-on activer les CSNs adultes afin d’augmenter les niveaux de neurogenèse ? Dans l’hippocampe, la capacité de l’environnement enrichi (incluant la stimulation cognitive, l’exercice et les interactions sociales) à promouvoir la neurogenèse hippocampale a déjà été démontrée. La plasticité de cette région est importante, car elle peut jouer un rôle clé dans la récupération de déficits au niveau de la mémoire et l’apprentissage. Dans la moelle épinière, des études effectuées in vitro ont démontré que les cellules épendymaires situées autour du canal central ont des capacités d’auto-renouvellement et de multipotence (neurones, astrocytes, oligodendrocytes). Il est intéressant de noter qu’in vivo, suite à une lésion de la moelle épinière, les cellules épendymaires sont activées, peuvent s’auto-renouveller, mais peuvent seulement ii donner naissance à des cellules de type gliale (astrocytes et oligodendrocytes). Cette nouvelle fonction post-lésion démontre que la plasticité est encore possible dans un environnement en quiescence et peut être exploité afin de développer des stratégies de réparation endogènes dans la moelle épinière. Les CSNs adultes jouent un rôle important dans le maintien des fonctions physiologiques du cerveau sain et dans la réparation neuronale suite à une lésion. Cependant, il y a peu de données sur les mécanismes qui permettent l'activation des CSNs en quiescence permettant de maintenir ces fonctions. L'objectif général est d'élucider les mécanismes sous-jacents à l'activation des CSNs dans le système nerveux central adulte. Pour répondre à cet objectif, nous avons mis en place deux approches complémentaires chez les souris adultes : 1) L'activation des CSNs hippocampales par l'environnement enrichi (EE) et 2) l'activation des CSNs de la moelle épinière par la neuroinflammation suite à une lésion. De plus, 3) afin d’obtenir plus d’information sur les mécanismes moléculaires de ces modèles, nous utiliserons des approches transcriptomiques afin d’ouvrir de nouvelles perspectives. Le premier projet consiste à établir de nouveaux mécanismes cellulaires et moléculaires à travers lesquels l’environnement enrichi module la plasticité du cerveau adulte. Nous avons tout d’abord évalué la contribution de chacune des composantes de l’environnement enrichi à la neurogenèse hippocampale (Chapitre II). L’exercice volontaire promeut la neurogenèse, tandis que le contexte social augmente l’activation neuronale. Par la suite, nous avons déterminé l’effet de ces composantes sur les performances comportementales et sur le transcriptome à l’aide d’un labyrinthe radial à huit bras afin d’évaluer la mémoire spatiale et un test de reconnaissante d’objets nouveaux ainsi qu’un RNA-Seq, respectivement (Chapitre III). Les coureurs ont démontré une mémoire spatiale de rappel à court-terme plus forte, tandis que les souris exposées aux interactions sociales ont eu une plus grande flexibilité cognitive à abandonner leurs anciens souvenirs. Étonnamment, l’analyse du RNA-Seq a permis d’identifier des différences claires dans l’expression des transcripts entre les coureurs de courte et longue distance, en plus des souris sociales (dans l’environnement complexe). iii Le second projet consiste à découvrir comment les cellules épendymaires acquièrent les propriétés des CSNs in vitro ou la multipotence suite aux lésions in vivo (Chapitre IV). Une analyse du RNA-Seq a révélé que le transforming growth factor-β1 (TGF-β1) agit comme un régulateur, en amont des changements significatifs suite à une lésion de la moelle épinière. Nous avons alors confirmé la présence de cette cytokine suite à la lésion et caractérisé son rôle sur la prolifération, différentiation, et survie des cellules initiatrices de neurosphères de la moelle épinière. Nos résultats suggèrent que TGF-β1 régule l’acquisition et l’expression des propriétés de cellules souches sur les cellules épendymaires provenant de la moelle épinière.At the end of the 19th century, Dr. Ramón y Cajal, a scientific pioneer, discovered that the nervous system was composed of individual cellular elements, later called neurons. He also noticed the complexity of this system and mentioned the impossibility of new neurons to be integrated into the adult nervous system. One of his famous quotes: “In adult centers the nerve paths are something fixed, ended, immutable. Everything may die, nothing may be regenerated” is representative of the prevalent dogma at the time (Ramón y Cajal 1928). Key studies conducted in the 1960-1970s suggested a different point of view. It was demonstrated that new neurons could be born during adulthood, but this discovery created an omnipresent skepticism in the scientific community. It took 30 years for the concept of adult neurogenesis to become widely accepted. This discovery, along with more advanced techniques, opened doors to potential therapeutic avenues for neurodegenerative diseases. Adult neural stem cells (NSCs) reside mainly in two niches in the brain: the subventricular zone of the lateral ventricles and the dentate gyrus of the hippocampus. Under normal conditions, neurogenesis level is relatively high in the SVZ whereas some steps are rate-limiting in the hippocampus. In contrast, the spinal cord is rather defined as a quiescent environment. One of the main questions that arose from these discoveries is: how do you activate adult NSCs in order to increase neurogenesis levels? In the hippocampus, environmental enrichment (including cognitive stimulation, exercise and social interactions) has been shown to promote hippocampal neurogenesis. The plasticity potential of this region is important as it could play a crucial role in rescuing learning and memory deficits. In the spinal cord, studies conducted in vitro demonstrated that ependymal cells found around the central canal have self-renewal and multipotency capacities (neurons, astrocytes, oligodendrocytes). Interestingly, it turns out that in vivo, following a spinal cord lesion, ependymal cells become activated, can self-replicate, but can only give rise to glia cell fate (astrocytes and oligodendrocytes). This new post-injury function shows that plasticity can still occur in a quiescent environment and could be exploited to develop endogenous spinal cord repair strategies. v As mentioned above, NSCs play important roles in normal brain function and neural repair following injury. However, little information is known about how a quiescent NSC becomes activated in order to perform these functions. The general objective of this project was to investigate the mechanisms underlying activation of neural stem cells in the adult central nervous system. My specific aims were to address this question using adult mice in two complementary models: 1) activation of hippocampal NSCs by environmental enrichment, and 2) activation of spinal cord NSCs by injury-induced neuroinflammation. Moreover, 3) to gain new insights into the molecular mechanisms of these models, we will perform transcriptomics studies to open new lines of investigation. The first project is expected to provide us with new insights into the basic cellular and molecular mechanisms through which environmental enrichment modulates adult brain plasticity. We first evaluated the contribution of individual environmental enrichment components to hippocampal neurogenesis (Chapter II). Voluntary exercise promotes neurogenesis, whereas a social context increases neuronal activation. We then determined the effect of these components on behavioural performances and transcriptome using an eight-arm radial maze to assess spatial memory, novel object recognition, and RNA-Seq, respectively (Chapter III). Runners show stronger spatial short-term memory recall, whereas mice exposed to social interactions had a better cognitive flexibility to abandon old memory. Surprisingly, RNA-Seq analysis indicated clear differences in the expression of modified transcripts between low runners and high runners, as well as for social interacting mice (within the complex environment). The second project consists of discovering how ependymal cells acquire NSC properties in vitro or multipotentiality following lesions in vivo. A RNA-Seq analysis revealed that the transforming growth factor-β1 (TGF-β1) acts as an upstream regulator of significant changes following spinal cord injury (Chapter IV). We therefore confirmed the presence of this cytokine after lesion and investigated its role on proliferation, differentiation, and survival of neurosphere-initiating cells from the spinal cord. Our results suggest that TGF-β1 regulates the acquisition and expression of stem cell properties of spinal cord-derived ependymal cells

Cognitive Impairment and Aberrant Plasticity in the Kindling Model of Epilepsy

Epilepsy is a neurological disorder that affects approximately 1% of the population worldwide. Although motor seizures are the best known feature of epilepsy, many patients also experience severe interictal (between-seizure) behavioral and cognitive comorbidities that have a greater negative influence on quality of life than seizure control or frequency. To study the characteristics of these interictal comorbidities and the neural mechanisms that underlie them, I use the kindling model of epilepsy. Kindling refers to the brief electrical stimulation of a discrete brain site that produces a gradual and permanent increase in the severity of elicited seizure activity. The repeated seizures associated with kindling induce robust structural and functional plasticity that appears to be primarily aberrant. Importantly, the aberrant plasticity evoked by repeated seizures is thought to contribute to the pathophysiology of epilepsy and its associated behavioral and cognitive comorbidities. Unfortunately, the relationship between aberrant plasticity and cognition dysfunction following repeated seizures remains poorly understood. The aim of this dissertation is to gain a better understanding of the effects of repeated convulsions on aberrant neural plasticity and interictal behavior. In Chapter 2, I will examine the effect of short- and long-term amygdala kindling on amygdala- and hippocampal-dependent forms of operant fear conditioning. To evaluate whether kindling alters neural circuits important in memory, I will analyze post-mortem measures of neural activity following the retrieval of fearful memories. In Chapter 3, I will evaluate whether deficits in operant fear learning and memory are a general consequence of convulsions induced by kindling stimulations or whether these deficits occur following kindling of specific brain regions. To evaluate whether aberrant plasticity following kindling of different brain regions contributes to learning and memory deficits, I will make post-mortem examinations of the inhibitory neurotransmitter neuropeptide Y and its Y2 receptor. In Chapter 4, I will investigate the relationship between hippocampal neurogenesis and cognition. Specifically, I will determine whether kindling of different brain regions induces an aberrant form of hippocampal neurogenesis that contributes to cognitive dysfunction. In Chapter 5, I will investigate whether kindling of different brain regions alters different subpopulations of hippocampal GABAergic interneurons, in terms of number and morphological features. Finally, Chapter 6 will provide preliminary evidence that the cognitive impairments associated with kindling can be ameliorated through intrahippocampal infusions of recombinant reelin. The collection of studies in this dissertation improves our understanding of the relationship between aberrant plasticity and cognitive impairments associated with repeated convulsions

Poor cognitive ageing: Vulnerabilities, mechanisms and the impact of nutritional interventions

Ageing is a highly complex process marked by a temporal cascade of events, which promote alterations in the normal functioning of an individual organism. The triggers of normal brain ageing are not well understood, even less so the factors which initiate and steer the neuronal degeneration, which underpin disorders such as dementia. A wealth of data on how nutrients and diets may support cognitive function and preserve brain health are available, yet the molecular mechanisms underlying their biological action in both normal ageing, age-related cognitive decline, and in the development of neurodegenerative disorders have not been clearly elucidated. Objectives: This review aims to summarise the current state of knowledge of vulnerabilities that predispose towards dysfunctional brain ageing, highlight potential protective mechanisms, and discuss dietary interventions that may be used as therapies. A special focus of this paper is on the impact of nutrition on neuroprotection and the underlying molecular mechanisms, and this focus reflects the discussions held during the 2nd workshop ‘Nutrition for the Ageing Brain: Functional Aspects and Mechanisms’ in Copenhagen in June 2016. The present review is the most recent in a series produced by the Nutrition and Mental Performance Task Force under the auspice of the International Life Sciences Institute Europe (ILSI Europe). Conclusion: Coupling studies of cognitive ageing with studies investigating the effect of nutrition and dietary interventions as strategies targeting specific mechanisms, such as neurogenesis, protein clearance, inflammation, and non-coding and microRNAs is of high value. Future research on the impact of nutrition on cognitive ageing will need to adopt a longitudinal approach and multimodal nutritional interventions will likely need to be imposed in early-life to observe significant impact in older age