Dissertations / Theses on the topic 'Astrocytes Neuroinflammation'

Astrocytes are the most abundant glia in the central nervous system (CNS), classically identified by their high expression of the intermediate filament, glial fibrillary acidic protein (GFAP). Astrocytes participate in a number of biochemical events important for CNS functions and play a dynamic role in regulating CNS injury/repair processes. In chronic inflammatory conditions such as multiple sclerosis (MS), astrocytes undergo pathophysiological changes that lead to a feature termed astrogliosis (Liberto, Albrecht et al. 2004; Sidoryk-Wegrzynowicz, Wegrzynowicz et al. 2011). Astrogliosis is common to MS lesions, and a novel therapeutic agent for MS, FTY720 (fingolimod, Gilenya™) demonstrates neuroprotective potential by inhibiting astrogliosis development (Choi, Gardell et al. 2011). FTY720 is an oral therapy recently approved for the treatment of MS, and is shown to readily access the CNS. There, it binds directly to sphingosine-1-phosphate receptors (S1PR) on astrocytes and the dynamics of S1PR signaling is shown to modulate astrocytic cellular responses that closely relate to MS pathology. This thesis examines the signaling and functional effects of FTY720 on primary human astrocytes. We used astrocytes derived from the human fetal CNS to explore neuroinflammation-relevant responses mediated by chronic (repeated daily) FTY720 administrations. FTY720 is known to initially acts as an agonist, activating S1PRs but also functions as an antagonist by promoting S1PR internalization and degradation; we examined whether these effects occurred in tandem. We report that receptors internalized by FTY720 can persist and continue to signal for an extended time period (hours). A single addition of FTY720 desensitizes the extracellular receptor-regulated phosphorylation (pERK) signaling response for >24 hours. Such refractory period for pERK signal transduction was maintained in astrocytes treated repeatedly (daily) with FTY720, otherwise the return of pERK activation was achieved by 72 hours following initial treatment. Moreover, receptor desensitization patterns correlated with the loss of proliferative responses induced by the natural ligand sphingosine-1-phosphate (S1P). We show that even under the condition of receptor desensitization (repeated daily administrations) FTY720 attenuated the capacity of the pro-inflammatory cytokine IL-1β, to activate calcium sensitive pathways. Repeated FTY720 treatments did not inhibit serum-induced pERK responses or the secretions of IL-6 and IP-10 in response to IL-1β activation. Our results indicate that daily FTY720 exposures can be a relevant regulator of neuro-inflammation by acting as a functional antagonist for external stimuli (natural ligand S1P) while sustaining internalized receptor-dependent agonist functions (inhibit IL-1β induced calcium mobilization).
Les astrocytes sont les cellules gliales les plus abondantes du système nerveux central (SNC). Leur grande expression en filaments intermédiaires, la protéine acide fibrillaire gliale (GFAP), est une caractéristique permettant leur identification. Les astrocytes sont d'importants contributeurs aux événements biochimiques du SNC et jouent un rôle clé dans le processus de régulation des dommages et de la guérison du SNC. Sous des conditions d'inflammation chronique, tel la Sclérose en Plaques (SP), les astrocytes subissent des changements pathophysiologiques causant l'astrogliose (Liberto, Albrecht et al. 2004; Sidoryk-Wegrzynowicz, Wegrzynowicz et al. 2011). Ce mécanisme de cicatrisation est commun dans la SP et un nouvel agent thérapeutique, FTY720 (fingolimod, Gilenya™) démontre des effets protecteurs du SNC en prévenant l'évolution de l'astrogliose. (Choi, Gardell et al. 2011). FTY720 est un agent thérapeutique récemment approuvé pour traiter la SP. Il est administré oralement et a la capacité d'accéder au SNC. Une fois en place dans ce système, cet agent entre en contact direct avec le récepteur sphingosine-1-phosphate (S1PR) sur les astrocytes. Les réponses des astrocytes en réaction aux signaux générés par ce récepteur sont reliées à la pathologie de la SP. Cette thèse examine les signaux engendrés par FTY720 ainsi que ses fonctions sur les astrocytes humains primaires. Nous avons utilisé des astrocytes isolés à partir de SNC humains fœtaux pour examiner les réponses neuro-inflammatoires générées par l'administration quotidienne de FTY720. FTY720 agit initialement comme un agoniste en activant le récepteur S1P, mais il agit également comme un antagoniste en causant l'internalisation et la dégradation de ce récepteur. Nous avons examiné ces deux phénomènes de façon à savoir s'ils agissent en concert. Nous affirmons qu'un récepteur internalisé par FTY720 continue de générer des signaux pour une période de temps prolongée (heures). Une addition simple de FTY720 désensibilise l'astrocyte, pour une période de >24h, au signal de phosphorylation de ERK (pERK) qui est généré par le récepteur extracellulaire. Cette période réfractaire du signal de transduction de pERK fût maintenue dans les astrocytes traités quotidiennement avec FTY720, sinon le signal pERK reparaît 72 heures après le traitement initial. De plus, la désensibilisation du récepteur fût reliée à l'absence de réponse proliférative induite par le ligand naturel sphingosine-1-phosphate (S1P). Nous avons aussi démontré que le traitement quotidien des astrocytes avec FTY720 atténue la capacité de IL-1β à activer les voies moléculaires sensibles au calcium. Le traitement quotidien avec FTY720 n'inhibe pas les signaux de pERK lorsque les astrocytes sont stimulés à l'aide de sérum, ni la sécrétion de IL-6 ou de IP-10 lorsqu'ils sont stimulés avec IL-1β. Nos résultats suggèrent que l'exposition quotidienne à FTY720 agit comme un antagoniste aux stimuli extérieur (tel le ligand naturel S1P) ainsi qu'un agoniste lorsque le récepteur est internalisé (inhibe la mobilisation du calcium lorsqu'exposé à IL-1β).

In neurodegenerative diseases, the CNS becomes inflamed through activation of pathways, including the NF-B pathway. Some of the therapies for those diseases target neuroinflammatory pathways. Here, we explore the mechanisms for the upregulation of a subset of genes following a restimulation of the NF-B pathway. We discover that this upregulation occurs independent of IRF1 expression and type 1 interferon signaling. A knockdown of IRF1 using siRNA and an inhibition of JAK proteins using inhibitor AG490 both had no effect on priming. A secreted factor was found to upregulate the expression of both this subset of genes and genes encoding pro-inflammatory cytokines induced by NF-B activation. We also explored the role of IRF1 in a mouse model of multiple sclerosis. We found that the deletion of IRF1 from oligodendrocytes diminished EAE severity. A deletion of IRF1 from myeloid cells within mice did not diminish EAE severity, however showed a promising decrease in the expression of certain inflammatory genes. Thus, IRF1 plays a critical role in fine-tuning inflammatory responses in the brain.

Les astrocytes sont des cellules cruciales pour une variété de fonctions physiologiques cérébrales telles que l’homéostasie, le métabolisme, le couplage neurovasculaire ou la régulation de la neurotransmission. Lors de lésions cérébrales, les astrocytes deviennent réactifs et tiennent un rôle prépondérant dans la réponse neuroinflammatoire. Cette réactivité astrocytaire est hétérogène et dépend de nombreux paramètres tels que le type et la sévérité de la lésion, la proximité de l’astrocyte à la lésion, ou encore l’état de maturité du cerveau. Cependant, la réponse spécifique des astrocytes au traumatisme crânien (TC) léger dans un contexte développemental n’a encore jamais été explorée. Le TC léger est pourtant la première cause de visite aux urgences pour la population pédiatrique. Il est maintenant établi qu’une proportion significative de ces patients pédiatriques souffrira de troubles cognitifs et émotionnels durables suite au TC léger, mais les mécanismes moléculaires et cellulaires sous-jacents sont encore peu connus. Il est possible que les astrocytes prennent part à cette vulnérabilité et soient en partie responsables des conséquences sur le long-terme.Nous avons investigué la réponse astrocytaire au TC juvénile léger et avons émis l’hypothèse que (1) les astrocytes déploient une réactivité spécifique évoluant au cours du temps et du développement cérébral, et que (2) cette réactivité diffère lorsque le TC est précédé d’une inflammation systémique précoce induisant un priming des astrocytes, avec une réponse neuroinflammatoire et vasculaire différente au TC juvénile léger, impactant la vulnérabilité cérébrale et les conséquences à long terme.Nous avons montré que :(1) Les astrocytes expriment une réactivité spatiotemporelle spécifique au TC, même à distance de la lésion, en termes d’expression de filaments intermédiaires et d’évolution morphologique, et que des altérations structurelles surviennent à l’imagerie cérébrale sur le long terme après un TC juvénile léger.(2) Lorsque le TC juvénile léger est précédé d’une inflammation systémique périnatale, les astrocytes ont un phénotype de réactivité différent, correspondant à un état de transition en direction des astrocytes formant la cicatrice gliale, avec une sur-régulation de gènes impliqués dans le métabolisme et la matrice extracellulaire, associés à des altérations morphologiques persistantes et une surexpression de VEGF retardée, modifiant les changements vasculaires survenant après un TC seul.Ce travail apporte de nouvelles connaissances sur les spécificités de la réactivité astrocytaire et sur la pathophysiologie de la vulnérabilité induite par un TC léger juvénile, ouvrant des possibilités en termes de cible thérapeutique
Astrocytes are crucial for various physiological functions in the brain such as homeostasis, metabolism, neurovascular coupling or neurotransmission regulation. In injuries, astrocytes become reactive and have a crucial role in the neuroinflammatory response. This reactivity is heterogeneous and depends on many parameters such as the type and severity of insult, astrocyte proximity to insult, or state of brain maturity. However, the specific response of astrocytes to mild traumatic brain injury (TBI) in the developmental context has never been studied yet. Mild TBI is the leading cause of emergency department visits in the pediatric population. A significant proportion of mild TBI pediatric patients will suffer of long-lasting cognitive and emotional impairments but the underlying cellular and molecular mechanisms are still poorly understood. Astrocytes might take part to this vulnerability and be partly responsible for the long-term consequences.We investigated astrocyte response to juvenile mild TBI and hypothesized that: (1) astrocytes display a specific pattern of reactivity evolving over time and brain development; and that (2) astrocytes reactivity differs when the TBI is preceded by an early systemic inflammation inducing a priming of astrocytes, with a different neuroinflammatory and vascular response to juvenile mild TBI, impacting the brain vulnerability and long-term outcome.We have shown that:(1) Reactive astrocytes express a specific spatiotemporal reactivity pattern even at distance from the injury site, in terms of intermediate filaments expression and morphological evolution, and that structural alterations are observed in brain imaging on the long-term after juvenile mild TBI.(2) When the juvenile mild TBI is preceded by perinatal systemic inflammation, astrocytes express a different reactivity phenotype considered as a state of transition towards scar-forming astrocytes, with increased metabolism and extracellular matrix-related gene changes, associated to morphological alterations sustaining over time and delayed over-expression of VEGF, resulting in the absence of vascular alterations induced by TBI alone.This work brings new insights in the specificities of astrocyte reactivity and in the pathophysiology of vulnerability after juvenile mild TBI, opening possibilities for novel targets for therapeutics

Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterised by accumulation of ß-amyloid in extracellular plaques, intracellular neurofibrillary tangles composed of abnormally phosphorylated and aggregated tau, and widespread synaptic dysfunction and neuron loss that underlie the clinical symptoms of AD. Glial activation and a neuroinflammatory immune response is also a key aspect of the pathological progression of AD. The activation of astrocytes appears to be particularly associated with pathological changes in tau. This thesis aims to investigate the association between astrocyte activation and abnormal tau processing using primary cell culture and human post-mortem brain. Furthermore, it aims to explore possible regional differences in this role of astrocytes, and the molecular signalling pathways by which astrocytes exert their effects on tau. Experiments in primary astrocyte and neuron co-cultures demonstrated that astrocytes were involved in accelerating Aß-induced neurotoxicity in hippocampal cultures, but not cortical cultures although the differences were quite subtle. Interestingly, astrocytes were important for the neuronal release of tau from cortical neurons under basal conditions, suggesting that astrocytes may be important for pathological tau spread in AD. Analysis of human post-mortem brain showed differences in astrocytic changes in hippocampus and cortex as AD progresses. In addition, these experiments also suggested regional differences in mechanisms related to synaptic dysfunction and loss as disease progresses. These data suggest that different mechanisms may underlie the neurodegenerative effects of ß-amyloid and/or activated astrocytes in distinct brain regions; an important consideration when considering therapeutic strategies for AD. In addition, the potential benefits for tauopathy of repurposing an already licenced drug with anti-inflammatory action were investigated. Despite showing significant modulation of tau phosphorylation in primary cultures, dimethyl fumarate had little influence on disease-associated tau species when tested in vivo in a mouse model of tauopathy. Overall, the findings of this thesis suggest that there are regional differences in astrocyte activation during the development of AD, that are somewhat associated with AD-relevant changes in tau. This work also supports a role for astrocytes in physiological tau release. Further elucidating these differences will increase understanding of neurodegenerative mechanisms. Moreover, these data suggest that regional involvement at different disease stages could be an important consideration when targeting specific mechanisms for therapeutic development.

Neuroinflammation occurs in Alzheimer’s disease (AD) brain, and plays a role in neurodegeneration. The main aim of this study was to determine how treatments with exogenous apolipoprotein E (ApoE2, E3 and E4 isoforms), a genetic risk factor for AD, affects the amyloid-β (Aβ) induced inflammatory response in vitro in astrocytes. Recombinant, lipid-free ApoE4 was found not to affect Aβ-induced inflammation in rat astrocytes, while ApoE2 showed a protective effect. Mouse cells expressing human ApoE isoforms, which have similar lipidation and modification to native human ApoE, showed ApoE4 promoting inflammation, and no ApoE2 protective effect upon Aβ treatment. A Protein/DNA array was used to screen 345 transcription factors in rat astrocytes treated with Aβ and/or ApoE isoforms, in order to determine which contribute to the observed ApoE2 protection. Some candidates were validated by Western Blot or EMSA and/or by inhibition or activation. The findings suggest ApoE isoforms differentially regulate Aβ-induced inflammation, and multiple signalling pathways are involved in the process.

Les astrocytes sont des éléments clés de la physiologie cérébrale. Dans les maladies neurodégénératives comme la maladie d’Alzheimer (MA), les astrocytes deviennent réactifs. Cette réactivité astrocytaire (RA) est essentiellement caractérisée par des changements morphologiques. En revanche, les effets de la réactivité sur les fonctions de support des astrocytes sont mal connus. De plus, les cascades de signalisation qui conduisent à la RA restent à déterminer. Les objectifs de ce projet étaient de : 1/ démontrer que la voie JAK2-STAT3 (Janus Kinase 2 - Signal Transducer and Activator of Transcription 3) joue un rôle central dans le contrôle de la RA au cours des maladies neurodégénératives ; 2/ comprendre quelle est l’implication de la RA dans les altérations moléculaires, cellulaires et fonctionnelles observées dans la MA. Nous avons montré que la voie JAK2-STAT3 est une cascade de signalisation centrale dans la RA (Ben Haim et al., 2015). Dans ce projet, nous démontrons en utilisant de nouveaux outils moléculaires basés sur des vecteurs viraux, que cette voie est nécessaire et suffisante à la RA. Nos résultats montrent également que la modulation de la RA dans deux modèles murins de la MA (souris APP/PS1dE9 et 3xTg-AD) influence certains index pathologiques, mais de façon contexte-dépendante. L’ensemble de ce travail a permis de valider de nouveaux outils pour étudier les astrocytes réactifs in situ et souligne l’importance et la complexité de leur fonctions au cours des maladies neurodégénératives
Astrocytes are emerging as key players in brain physiology. In Alzheimer’s disease (AD), astrocytes become reactive. Astrocyte reactivity (AR) is essentially characterized by morphological changes. But how the normal supportive functions of astrocytes are changed by their reactive state is unclear. Moreover, signaling cascades leading to AR are not yet determined. In this study, we aim to: 1/ demonstrate the JAK2-STAT3 pathway (Janus Kinase 2 - Signal Transducer and Activator of Transcription 3) is responsible for AR in neurodegenerative diseases ; 2/ understand the contribution of reactive astrocytes to molecular, cellular and functional alterations in AD. We already reported that the JAK2- STAT3 pathway is a central cascade for AR (Ben Haim et al., 2015). Here, we demonstrate, with new molecular tools based on viral vectors, that this pathway is necessary and sufficient to AR. Our results also show that the modulation of AR in two AD mouse models (APP/PS1dE9 and 3xTg-AD mice) influence several pathological hallmarks, but in a context-dependent manner. Overall, this work has generated new original tools to study reactive astrocytes in situ and it underlines the importance and complexity of their functions in neurodegenerative diseases

La sclérose en plaques (SEP) est une maladie autoimmune, démyélinisante et dégénérative du système nerveux central (SNC). La réponse T CD4 est impliquée dans le développement de la SEP et son modèle animal : l'encéphalomyélite autoimmune expérimentale (EAE). De récentes données montrent que les lymphocytes T CD8 anti-myéline peuvent être impliqués d'autant plus qu'ils sont présents abondamment dans les lésions SEP. Afin de mieux comprendre la contribution des T CD8 pathogéniques, deux récents modèles ont été évalués. Le premier consiste à immuniser des souris avec un épitope de la myéline T CD8 spécifique (MOG37-46). Les souris développent une EAE modéré avec une prépondérance de T CD4 dans le SNC. La réactivation périphérique des T CD8 augmente le rapport T CD8/T CD4 dans le SNC. Le second modèle est basé sur le transfert de T CD8 anti-hémagglutinine (HA) dans des souris DKI exprimant HA par les oligodendrocytes. L'irradiation (2Gy) des souris DKI permet une infiltration lymphocytaire sans symptôme apparent. Les résultats sont discutés au vu des données récentes de la littérature. En parallèle, nous avons caractérisé la réactivité astrocytaire dans le modèle classique EAE afin de mieux définir l'implication des astrocytes dans la maladie. Pour la première fois dans ce modèle, nous avons caractérisé le profil moléculaire des astrocytes de la substance blanche isolés par microdissection laser dans les lésions spinales mettant en évidence l'expression spécifique de médiateurs proinflammatoires et d'enzymes du métabolisme stéroïdien. Ces données ouvrent de nouvelles voies pour contrer la réactivité gliale dans les maladies neuroinflammatoires comme la SEP
Multiple sclerosis (MS) is an autoimmune, demyelinating and degenerative disease of the central nervous system (CNS), in which astrocyte reactivity is considered an important player. The CD4 T cell response is strongly associated with development of MS and its animal models such as experimental autoimmune encephalomyelitis (EAE). Recent data suggest that anti-myelin CD8 T cells may be also implicated as CD8 T cells are abundant in MS lesions. To better understand the contribution of pathogenic CD8 T cells, two animal models that have been described were evaluated. The first one consists of mice immunized with a specific CD8 T cell myelin epitope (MOG37-46). Mice develop mild EAE with CD4 T overwhelming CD8 T cells in CNS. Boosting the CD8 immune response increased slightly the CD8/CD4 ratio in the CNS. The second model is based on the adoptive transfer of anti-HemAgglutinin (HA) CD8 T cells in DKI transgenic mice, which express HA by oligodendrocytes. Only irradiation (2Gy) of DKI mice allowed CNS infiltration of CD8 T cells but without apparent clinical signs. These results are discussed in light of recent literature. In parallel, we characterized the astrocyte reactivity in a classical EAE to better define the implication of astrocytes in the pathology. For this, we used for the first time in this model laser-capture microdissection to isolate white matter astrocytes in spinal cord lesion. Selected transcript profiling analysis revealed astrocytic expression of pro-inflammatory mediators and enzymes involved in oestrogen metabolism. These results give new clues for targeting glial reactivity in neuroinflammatory disorders such as MS

Les astrocytes deviennent réactifs dans les maladies neurodégénératives (MND) comme la maladie d’Alzheimer (MA) et de Huntington (MH) mais les conséquences fonctionnelles de cette réactivité sont peu connues. Dans cette étude, nous avons évalué 1) les voies de signalisation impliquées dans la réactivité astrocytaire, 2) la contribution des astrocyte réactifs (AR) à la dysfonction neuronale dans des modèles de MND et 3) les caractéristiques fonctionnelles des AR.Nous avons montré que la voie JAK2/STAT3 est responsable de la réactivité astrocytaire dans des modèles murins de la MA et la MH. Nous avons développé de nouveaux vecteurs viraux ciblant cette voie dans les astrocytes, in vivo. Grâce à ces outils, nous avons étudié la contribution des AR à la dysfonction neuronale dans deux modèles murins de la MH. Nos résultats suggèrent que les AR ne jouent pas un rôle central dans ces modèles de pathologie. En ciblant la voie JAK2/STAT3, nous avons induit la réactivité astrocytaire chez la souris sauvage et avons montré que cette voie régule la transcription de gènes impliqués dans des fonctions cellulaires importantes. De plus, nous avons observé que l’activation des astrocytes conduit à une diminution de la plasticité synaptique dans le cerveau de souris.En conclusion, nous avons montré que la voie JAK2/STAT3 est une voie centrale dans les AR. Nous avons développé des vecteurs viraux innovants pour évaluer 1) la contribution des AR à la dysfonction neuronale dans des modèles de MND et 2) les propriétés fonctionnelles des AR in vivo. L’étude des AR permettra d’identifier de nouvelles cibles moléculaires pour manipuler ces cellules pléiotropes à des fins thérapeutiques
Astrocyte reactivity is a hallmark of pathological conditions in the CNS including neurodegenerative diseases (ND) such as Alzheimer’s (AD) and Huntington’s (HD) diseases. Reactive astrocytes (RA) are identified by morphological changes but their functional features and influence on neurons are poorly understood, especially in ND. Therefore, we aimed at 1) identifying the signaling cascades involved in astrocyte reactivity in ND, 2) evaluating RA contribution to disease phenotype in ND models and 3) deciphering RA functional features. The JAK2/STAT3 pathway is a known trigger of astrocyte reactivity in CNS injuries. Here, we show that this pathway is a common inducer of astrocyte reactivity in AD and HD models. We developed new viral vectors to target this cascade in astrocytes and manipulate astrocyte reactivity in vivo. We used these vectors to determine the contribution of RA to neuronal dysfunction in HD mouse models. We found that RA do not primarily influence disease phenotype in HD. Last, we targeted the JAK2/STAT3 pathway in WT mice to characterize RA functional features in vivo. We show RA undergo transcriptional changes of numerous genes involved in metabolism, protein degradation pathways and immune response. Moreover, we show that astrocyte reactivity alters synaptic plasticity in the mouse hippocampus. Our results identify the JAK2/STAT3 pathway as a central cascade for astrocyte reactivity. The viral vectors developed in this project represent powerful tools to decipher the roles of RA in various ND models and to characterize RA functional features in vivo. Better understanding RA functions may lead to the identification of new therapeutic targets for ND

La sclérose en plaques (SEP) est une maladie neuro-inflammatoire chronique, première cause de handicap chez le jeune adulte. Actuellement, aucun traitement ne freine l'aggravation des symptômes liée aux formes progressives. Bien que connue, l'implication des macrophages et de la microglie dans la démyélinisation et l'atteinte axonale doit être plus finement caractérisée. Ce d'autant plus que la plasticité fonctionnelle de ces cellules suggère une réponse spécifique selon la pathologie, la localisation des lésions et le stade évolutif de la maladie. Ce travail de thèse a consisté en une caractérisation moléculaire des programmes d'activation macrophagique/ microgliale dans deux types d'altérations tissulaires du système nerveux central des patients SEP : les zones partiellement démyélinisées bordant les plaques de la moelle épinière et les lésions corticales. Cette étude a été réalisée sur des tissus post-mortem de patients atteints de formes progressives, formes dans lesquelles les lésions médullaires et corticales sont nombreuses et impliquées dans le handicap progressif et irréversible. Nous avons identifié des spécificités moléculaires caractérisant l'activation macrophagique/microgliale au cours de la SEP en comparant, par une approche in silico, les profils caractérisés à ceux observés dans des pathologies neuro-dégénératives à composantes inflammatoires, la maladie d'Alzheimer et de Parkinson notamment. Dans l'ensemble, ces résultats suggèrent que l'activation chronique des macrophages/cellules microgliales contribue à l'extension à bas bruit des lésions médullaires et corticales pendant la phase progressive de la SEP et proposent de nouvelles cibles thérapeutiques

Alzheimer’s disease (AD) is a neurodegenerative disorder, with extensive evidence implicating the misfolding, aggregation and deposition of the amyloid-β (Aβ) peptide as central to the pathogenesis. Heparan sulfate (HS) is an interactive glycosaminoglycan, attached to core proteins as HS proteoglycans (HSPGs). HSPGs are present on cell surfaces and in the extracellular matrix where they facilitate multiple signaling functions, but HS is also consistently present in all amyloid deposits, including those of AD. In amyloidosis HS has been studied as an aggregation template, promoting fibril formation and serving a scaffold function in the resulting deposits. The objective of this thesis was to assess how cell surface HS is potentially implicated in Aβ amyloidosis and the associated neuroinflammation of AD.   In AD brain we determined that HS predominantly accumulated in Aβ deposits with dense cores and found glial-expressed HSPGs within these deposits. Aβ elevated HSPG levels in primary glial cultures, implicating activated glia as one source of the Aβ-associated HS. Next, we determined that microglial HSPGs are critical for the upregulation of interleukin-1β and tumor necrosis factor-α following exposure to lipopolysaccharide, an established inflammatory insult. Together these results raise the possibility that Aβ-induced expression of microglial HSPGs may promote neuroinflammation.   Multiple mechanisms of Aβ toxicity have been proposed and different Aβ assemblies exert their toxicity through alternative routes. We found that three different preparations of Aβ aggregates all exhibited HS-dependent cytotoxicity, which in part correlated with Aβ internalization. Furthermore, heparin treatment attenuated Aβ cytotoxicity and uptake. In Aβ-positive AD microvasculature, HS deposited with Apolipoprotein E (ApoE) and its receptor, the low density lipoprotein receptor-related protein 1 (LRP1). In cell culture, HS and LRP1 co-operated in Aβ interactions and the addition of ApoE increased the levels of cell-associated Aβ in a HS- and LRP1-dependent manner. This ApoE-mediated increase in cell-associated Aβ may promote toxicity and vascular degeneration, but equally HS-mediated internalization of Aβ could represent a clearance route across the blood-brain-barrier. The findings presented here illustrate multiple roles for cell-surface HSPGs in interactions relevant to the pathogenesis of AD.

A epilepsia do lobo temporal (ELT) é a um dos casos mais frequente epilepsia em humanos e de maior refratariedade nos pacientes. A maioria dos fármacos antiepilépticos são moduladores da atividade neuronal e atuam sobre canais iônicos do receptor GABAA. Estudos vêm demonstrando o papel das células gliais e da neuroinflamação na epileptogênese e a modulação desta resposta pode ser um alvo potencial para drogas adjuvantes aos fármacos anti-epilépticos. Astrócitos são células gliais participantes da sinapse tripartite, moduladores da atividade neuronal. Os astrócitos são capazes de promover a homeostase de íons e de neurotransmissores, são responsáveis pelo metabolismo energético e da produção de fatores neurotróficos, glutationa, glutamina, S100B e citocinas. Neste trabalho, induzimos status epilepticus (SE) em ratos jovens (PN28) através do modelo lítio-pilocarpina que mimetiza alterações neuronais, bioquímicas e morfológicas similares à ELT em humanos. Os animais foram divididos nos tempos 1, 14 e 56 dias após a indução de status epilepticus (SE). Estes períodos são caracterizados respectivamente como a fase aguda, latente e crônica da epilepsia. Inicialmente, analisamos as mudanças neuroquímicas e astrocitárias ao longo do tempo. Foi observada neuroinflamação inicial e transitória que promove morte neuronal e mudanças ao longo do tempo de astrogliose e disfunção astrocitária. Também foi observado que a proteína S100B, proteína ligante de cálcio, predominantemente astrocitária, pode ser considerado um marcador da disfunção neuronal e astrocitária promovida neste modelo de epilepsia. Em seguida, demonstramos que a modulação da secreção de S100B pelo anti-inflamatório dexametasona um dia após indução de SE reverte a neuroinflamação, astrogliose e disfunção astrocitária à curto e à longo prazo. Por conseguinte, observamos que a modulação do receptor GABAA através de agonistas e antagonistas GABAérgicos altera a secreção de S100B em fatias hipocampais agudas e em cultura de astrócitos. Portanto, pode-se sugerir que as alterações astrogliais e a neuroinflamação dependentes do tempo podem estar ligadas à excitabilidade neuronal e/ou à morte neuronal em ratos jovens em modelo de epilepsia; que a proteína S100B pode ser considerada um marcador deste modelo de epilepsia e que a modulação da sua secreção pode ser um possível alvo farmacológico no tratamento da epilepsia.
Temporal lobe epilepsy (TLE) is the most frequent type of epilepsy in humans and is more associated to refractory to anti-epileptic drugs (AED) in patients. The most AEDs are modulators of neuronal activity and act on ion channels, such as GABAA receptor. Studies have been demonstrating the role of glial cells and neuroinflammation in epileptogenesis. The modulation of this response may be a potential target for adjunctive drugs to anti-epileptic drugs. Astrocytes are glial cells that participated in the tripartite synapse and modulated neuronal activity. Astrocytes are able to promote homeostasis of ions and neurotransmitters, are responsible for energy metabolism and the production of neurotrophic factors, glutathione, glutamine, S100B and cytokines. In this work, we induced status epilepticus (SE) in young rats (PN28) through the lithiumpilocarpine model that mimics neuronal, biochemical and morphological alterations similar to ELT in humans. The animals were divided at times 1, 14 and 56 days after the induction of SE. These periods are characterized respectively as the acute, latent and chronic phase of epilepsy. Initially, we analyzed neurochemical and astrocytic changes over time. Initial and transient neuroinflammation was observed and promoted over time neuronal death, astrogliosis and astrocytic dysfunction. It has also been observed that the protein S100B, a calcium-binding protein, predominantly astrocytic, can be considered a marker of neuronal and astrocytic dysfunction promoted by this model of epilepsy. Next, we demonstrate that the modulation of S100B secretion by the antiinflammatory dexamethasone one day after SE induction reverses neuroinflammation, astrogliosis and astrocytic dysfunction in the acute and chronic time. Therefore, we analyzed that modulation of the GABAA receptor through GABAergics agonists and antagonists alters the secretion of S100B in acute hippocampal slices and in astrocyte culture. Therefore, it may be suggested that astroglial changes and time dependent neuroinflammation may be related to neuronal excitability and/or neuronal death in young rats in this epilepsy model; that S100B protein can be considered a marker of this epilepsy model and that the modulation of its secretion may be a possible pharmacological target in the treatment of epilepsy.

Os objetivos principais desta tese foram avaliar parâmetros bioquímicos gliais, inflamatórios e comportamentais em pacientes com lesão medular (LM) submetidos a um protocolo de dança, e em ratos submetidos aos protocolos de exercício voluntário e forçado. Para tanto, foram realizados 4 experimentos. No primeiro, os ratos foram submetidos a 4 semanas de exercício moderado em esteira ergométrica (20 min por dia). No segundo, os animais foram submetidos à 4 semanas de exercício voluntário em rodas de correr (12 horas por dia). No terceiro experimento, os ratos foram expostos à esteira ergométrica durante 2 semanas (20 min/dia) e, após o último treino, receberam uma injeção intraperitoneal de LPS. Neste último, a memória e aprendizagem dos animais foram investigadas. Ao término do período de exercício, ou após a injeção de LPS, as análises bioquímicas do hipocampo foram realizadas. O quarto experimento foi realizado com indivíduos com LM que foram submetidos a 4 semanas de dança. Análises sorológicas e comportamentais foram efetuadas. Os resultados mostraram que o exercício forçado promoveu o aumento da glutamina sintetase (GS) e diminuição de proteína glial fibrilar ácida (GFAP) e óxido nítrico (NO) no hipocampo, além de aumentar os níveis de corticosterona, o que pode estar mediando os efeitos do exercício sobre os astrócitos. O exercício voluntário induziu o aumento da GS e BDNF. A aplicação de LPS promoveu aumento dos níveis de TNF-α no hipocampo dos animais, o que coincidiu com o aumento dos níveis de S100B no fluído cerebrospinal. Os indivíduos com LM submetidos à dança apresentaram melhora significativa nos escores da Medida de Independência Funcional, Índice de Barthel, Escala de Berg e Escala Hospitalar de Ansiedade e Depressão, e aumento dos níveis sorológicos de BDNF. A dança não teve efeito sobre os parâmetros gliais, metabólicos e inflamatórios periféricos. Estes resultados sugerem que diferentes tipos de exercício físico exercem diferentes efeitos sobre os astrócitos hipocampais, o que pode interferir na indicação de um ou outro dependendo do objetivo a ser alcançado. O exercício em esteira pode ser uma atividade indicada para prevenção de doenças que envolvem neuroinflamação, e a dança pode ser uma intervenção terapêutica eficaz para reabilitação de indivíduos com lesão medular uma vez que contribui para melhora física e psicológica desta população. Tomados juntos, os resultados desta Tese ressaltam a importância da prática de exercício físico para o metabolismo neural, e a relevância de estudar os astrócitos para compreensão dos mecanismos envolvidos no efeito do exercício físico no SNC.
The main aim of this Thesis was to evaluate the biochemical glial, inflammatory and behavioral parameters, in patient with spinal cord injury (SCI) submitted to a protocol of dance, and in rats submitted to voluntary and forced exercise. Four experiments were made. In the 1st, the rats were submitted to 4 weeks of moderate exercise on treadmill (20 min per day). In the 2nd, the animals were submitted to 4 weeks of voluntary exercise on wheel running (12 hours per day). In the 3rd experiment, the rats were exposed to treadmill during 2 weeks (20 min per day) and, after the last session, they received intraperitoneal injection of LPS. In this last experiment, the memory and learning were investigated. At the finish of the exercise period, or after the LPS injection, the biochemical analysis of the hippocampus was realized. The 4th experiment was realized with individuals with spinal cord injury that were submitted to 4 weeks of dance practice. Behavioral and serological analyses were performed. Data show that treadmill running increased glutamine synthetase (GS) activity and decreased hippocampal glial fibrillary acidic protein (GFAP) and nitric oxide (NO) , as well as increased corticosterone level, that can mediate the effects of the exercise on astrocytes. The voluntary exercise increased GS and BDNF. The LPS administration increased hippocampal TNF-α level in rats concomitantly with the increase in the S100B levels in cerebrospinal fluid. The individuals with spinal cord injury submitted to dance showed a significant improve in the scores of Functional Independency Measure, Barthel Index, Berg Scale and Ansiety and Depression Hospitalar Scale, and a increase in the serologic levels of BDNF. The dance had no effect on glial, metabolic and inflammatory parameters. These results suggest that different types of exercise exert different effects on hippocampal astrocytes, which may interfere with the appointment of one or the other depending on the objective to be achieved. The treadmill exercise can be a good strategy in the prevention of neuroinflammatory diseases, and dance can be an effective therapeutic intervention for rehabilitation of individuals with SCI as it helps to improve physical and psychological conditions in this population. Taken together, the present data highlight the importance of physical exercise for neural functions, and the relevance of studying astrocytes to understand the mechanisms involved in the effect of exercise on CNS.

Nous avons exploré les altérations cérébrales liées à l’âge, dans des conditions physiologiques ou pathologiques (maladie d’Alzheimer), à partir de deux modèles murins, SAM-P8 (souris à sénescence accélérée) et 5xFAD..Dans les deux cas, les animaux montrent des signes de neuroinflammation avec libération de la principale cytokine pro-inflammatoire IL-1β. Ces troubles sont sans conteste d’origine endogène, puisqu’ils n’ont pas été observés chez les témoins. Il faut souligner que les astrocytes 5xFAD ont été prélevés chez des nouveau-nés et par conséquent leur état inflammatoire signifie que la neuroinflammation est une des toutes premières manifestations de la pathologie, bien en amont de la formation des plaques amyloïdes.Dans toute pathologie complexe, les troubles sont la résultante d’un ensemble de processus pathologiques. Nous avons donc recherché les effets protecteurs de la pantéthine. Cette molécule naturelle de faible taille moléculaire, a démontré une action de protection dans plusieurs pathologies cérébrales. Dans la présente étude, le traitement des astrocytes et des souris par la pantéthine modère les altérations observées dans les deux modèles. En particulier, le traitement stimule l’expression de HIF-1α, via son action sur le cycle de Krebs et sur l’activité du protéasome.En conclusion, nous avons montré que la neuroinflammation est à la base du processus pathologique conduisant à la maladie d’Alzheimer et qu’elle se manifeste également au cours du vieillissement. La pantéthine est donc susceptible de contrecarrer le développement de la maladie, comme de tempérer les effets du vieillissement cérébral
We used two mouse models to explore the age-related cerebral alterations, under physiological and pathological conditions (Alzheimer’s disease), i.e. senescence accelerated SAM-P8 and transgenic 5xFAD mice.In the two models, mice showed signs of neuroinflammation with release of the major inflammatory cytokine IL-1β. Such events were undoubtedly of endogenous origin as they did not occur in the controls. It should be underlined that, since 5xFAD astrocytes were collected in newborns, their inflammatory status means that neuroinflammation is a very early step of Alzheimer’s disease pathological process, upstream of β-amyloid accumulation.Since in a complex disease such as Alzheimer’s brain insults result not from a single cause but from multiple pathological processes, we explored the protective effects of pantethine, a low-molecular-weight, multifunctional agent which has been shown to exert protective effects in several neurodegenerative diseases through multiple convergent mechanisms. In our study, pretreatment of astrocytes and treatment of mice with pantethine moderated age-related alterations. Moreover, it enhanced HIF-1α expression via the modulation of the Krebs’ cycle and proteasomal activities. In addition, a genome wide transcriptomic analysis from hippocampus samples of 5xFAD mice showed that pantethine attenuated most of gene overexpression in transgenic vs WT mice.In conclusion, we found that neuroinflammation lays at the root of Alzheimer’s disease pathological process and is also present in aging mice. Pantethine, this natural and well-tolerated compound could therefore prevent the disease development and temper the deleterious aging effects

La Interleucina 6 (IL-6) és una citocina altament multifuncional, amb moltes accions pleiotròpiques, considerada una de les principals citocines controlant el sistema immune i coordinant-lo amb els sistemes nerviós i endocrí. La IL-6 es produeix en molts tipus cel·lulars dins del sistema nerviós central (SNC) i al seu torn, múltiples cèl·lules hi poden respondre. Per tant, és necessari caracteritzar quina és la contribució específica de cada tipus cel·lular en el paper global de la IL-6, tant en condicions fisiològiques com patològiques. Com que els astròcits tenen una important resposta enfront la IL-6 i, a més, són un dels principals productors de IL-6, hem produït per primera vegada ratolins amb una deficiència de IL-6 específica als astròcits (ratolins Ast-IL-6 KO) i ratolins amb una supressió del receptor de IL-6 en astròcits (ratolins Ast-IL-6R KO). Els nostres resultats indiquen que IL-6 i IL-6R astrocitaris influeixen en la supervivència inicial, presumptament per mitjà de la mortalitat intrauterina, i que estan involucrats en diferents graus en el control del pes corporal en adults i en el comportament (activitat locomotora, ansietat, exploració, agressivitat, aprenentatge i memòria), entre d’altres. A més, per tal d’estudiar el seu paper durant la neuro-inflamació, hem usat el model animal àmpliament usat de l’Esclerosis múltiple, la encefalitis autoimmune experimental (EAE), i un model de lesió traumàtica en escorça encefàlica (criolesió) en ratolins Ast-IL-6 KO i Ast-IL-6R KO. En referència a la EAE, els resultats indiquen que la deficiència en IL-6 i IL-6R astrocitàries no prevé completament la simptomatologia típica de paràlisis ascendent de la EAE però que si que modifica la seva simptomatologia d’una manera diferent segons el sexe. En referència a la lesió traumàtica a l’escorça, resultats inicials suggereixen un paper de la deficiència de IL-6 astrocitària en la resolució de la lesió traumàtica. En algunes ocasions, eliminar el IL-6R astrocitari imita el fenotip dels ratolins Ast-IL-6 KO, mentre que en altres casos s’observava el contrari, suggerint accions autocrines i paracrines de la IL-6 astrocitaria. Els nostres resultats indiquen importants funcions de la IL-6 i el IL-6R astrocitaris, en alguns casos totalment diferent d’aquelles que s’havien vist amb els animals IL-6 KO totals. Finalment, com que la IL-6 és capaç de senyalitzar sense unir-se al receptor de membrana mIL-6R sinó unint-se al receptor soluble (sIL-6R) (trans-senyalització), és necessari estudiar la importància d’aquesta via en les accions de la IL-6 al SNC, la qual cosa s’ha fet en ratolins bigènics (GFAP-IL6/sgp130) amb expressió restringida als astròcits de IL-6 i d’un inhibidor específic de la trans-senyalitazació, el sgp130. El bloqueig de la trans-senyalització en SNC redueix varis dels efectes perjudicials que té la IL-6 en un model animal de neuroinflamació (ratolins GFAP-IL6) com la gliosis severa, alteracions vasculars, neurogènesis malmesa i neurodegeneració.
Interleukin-6 (IL-6) is a highly plurifunctional cytokine, with many pleiotropic actions, considered one of the main cytokines controlling the immune system and coordinating it with the nervous and endocrine systems. IL-6 is produced in multiple cell types in the central nervous system (CNS), and in turn many cells do respond to it. It is therefore important to ascertain which the contribution of each cell type is in the overall role of IL-6, during both physiological and pathological conditions. As astrocytes are major responders to IL-6 as well as one of the main CNS producers of IL-6, we have produced for the first time mice with astrocyte-derived IL-6 deficiency (Ast-IL-6 KO mice) and mice with deletion of IL-6 receptor in astrocytes (Ast-IL-6R KO mice). Our results indicate that astrocyte IL-6 system influenced the early survival, presumably due to intrauterine death, and was also involved to various degrees in the control of adult body weight and behavior (such as locomotor activity, anxiety, exploration, aggressiveness, learning and memory), among others. Also, in order to test its role during neuroinflammation, we studied an extensively used animal model of Multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), and a traumatic brain injury model (cryolesion) in Ast-IL-6 KO and Ast-IL-6R KO mice. Regarding EAE, results indicate that lack astrocytic IL-6 and IL-6R deficiency did not fully prevent EAE's prototypical ascending paralysis course but they modified its symptomatology in a gender-dependent manner. Regarding traumatic injury to the cortex, preliminary results obtained suggest a role of astrocytic IL-6 deficiency in traumatic brain injury resolution. In some occasions deleting IL-6R in astrocytes mimicked the phenotype of Ast-IL-6 KO mice, while in others the opposite was observed, suggesting autocrine and paracrine roles of astrocytic IL-6. Our results suggest important roles of the astrocyte IL-6 system, in some cases totally unexpected from previous results with animals with total deletion in IL-6 (IL-6 KO mice). Finally, as IL-6 is able to signal without binding to the membrane bound mIL-6R but by binding to a soluble receptor (sIL-6R) (trans-signaling), it is necessary to assess the importance of this pathway in mediating IL-6 actions in CNS. Bigenic mice (GFAP-IL6/sgp130 mice) with astrocyte-targeted production of IL-6 and coproduction of the specific inhibitor of IL-6 trans-signaling, human sgp130 were studied. Blockade of trans-signaling in the CNS reduced many of the detrimental effects that IL-6 have in the GFAP-IL-6 mice model of neuroinflammation; such as the severity of the gliosis, vascular alterations, impaired neurogenesis and neurodegeneration.

La inflamació és essencial en les respostes a les infeccions i danys perifèrics. Es poden obtenir beneficis de la inflamació si es controla durant un període de temps definit. La inflamació no regulada, sostinguda o excessiva és la causa principal de diferents neuropatologies. Al sistema nerviós central (SNC), la resposta neuroinflamatòria cerebral després d’una lesió cerebral traumàtica (TBI) s’ha caracteritzat en pacients i en diferents models animals experimentals com una de les principals causes de lesions secundàries que condueixen a la degeneració neuronal. La neuroinflamació es caracteritza per l’activació de cèl·lules glials, el reclutament de leucòcits i la regulació i secreció de citocines i mediadores de quimiocines. A més, la deficiència de la barrera hematoencefàmica (BBB) té un paper important en el temps agut després del TBI, permetent l’entrada de neutròfils, monòcits i limfòcits T al lloc de la lesió, afectant així la mort neuronal. En diferents estudis s’ha demostrat el paper crucial de les citocines antiinflamatòries en la reducció de les respostes neuroinflamatòries. En aquest context, l’objectiu de la present tesis doctorat era caracteritzar els efectes de la producció local d’IL-10 sobre la resposta neuroinflamatòria característica del TBI. Per aconseguir aquest model la lesió criogènica de TBI es va aplicar a animals transgènics que produïen IL-10 sota el promotor d[astròcits GFAP (animals GFAP-IL10Tg) i els seus homòlegs de tipus salvatge, i es van analitzar diferents característiques de la resposta neuroinflamatòria. Aquest treball va revelar que IL-10 promou la supervivència de les neurones a les primeres hores després de lesionar-se mitjançant la inhibició de l’activació de cèl·lules glia, el reclutament de neutròfils, la interrupció del BBB i els nivells d’expressió de la citocina proinflamatòria IL-1β, associada a l’infiltració augmentada dels limfòcits T. Aquests resultats assenyalen IL-10 com a bon candidat per millorar la resposta neuroinflamatòria després del TBI i evitar danys secundaris en el teixit cerebral.
La inflamación es esencial en las respuestas a las infecciones y daños periféricos. Se pueden obtener beneficios de la inflamación si se controla durante un período de tiempo definido. La inflamación no regulada, sostenida o excesiva es la causa principal de diferentes neuropatologías. En el sistema nervioso central (SNC), la respuesta neuroinflamatoria cerebral tras una lesión cerebral traumática (TBI) se ha caracterizado en pacientes y en diferentes modelos animales experimentales como una de las principales causas de lesiones secundarias que conducen a la degeneración neuronal. La neuroinflamación se caracteriza por la activación de células gliales, el reclutamiento de leucocitos y la regulación y secreción de citocinas y mediadoras de quimiocinas. Además, la deficiencia de la barrera hematoencefàmica (BBB) tiene un papel importante en el tiempo agudo después del TBI, permitiendo la entrada de neutrófilos, monocitos y linfocitos T en el lugar de la lesión, afectando así la muerte neuronal. En diferentes estudios se ha demostrado el papel crucial de las citocinas antiinflamatorias en la reducción de las respuestas neuroinflamatorias. En este contexto, el objetivo de la presente tesis doctoral era caracterizar los efectos de la producción local de IL-10 sobre la respuesta neuroinflamatoria característica del TBI. Para conseguir este modelo la lesión criogénica de TBI se aplicó a animales transgénicos que producían IL-10 bajo el promotor de [astrocitos GFAP (animales GFAP-IL10Tg) y sus homólogos de tipo salvaje, y se analizaron diferentes características de la respuesta neuroinflamatoria. Este trabajo reveló que IL-10 promueve la supervivencia de las neuronas en las primeras horas después de lesionarse mediante la inhibición de la activación de células glía, el reclutamiento de neutrófilos, la interrupción del BBB y los niveles de expresión de la citoquina proinflamatoria IL-1β, asociada a la infiltración aumentada de los linfocitos T. Estos resultados señalan IL-10 como buen candidato para mejorar la respuesta neuroinflamatoria después del TBI y evitar daños secundarios en el tejido cerebral.
Inflammatory is essential in responses to peripheral infections and damage. Benefits of inflammation can be achieved if this phenomenon is controlled and for a defined period of time. Unregulated, sustained or excessive inflammation is the major cause of different neuropathologies. In the central nervous system (CNS), cerebral neuroinflammatory response after traumatic brain injury (TBI) has been characterized in patients and in different experimental animal models as one of the main causes of secondary injury leading to neuronal degeneration. Neuroinflammation is characterized by glial cell activation, leukocyte recruitment, and upregulation and secretion of cytokines and chemokines mediators. In addition, blood brain barrier (BBB) impairment has an important role in the acute time after TBI, allowing the entry of circulating neutrophils, monocytes, and T lymphocytes to the site of injury, and thus affecting neuronal death. In different studies the crucial role of anti-inflammatory cytokines in reduction of neuroinflammatory responses have been demonstrated. In this context, the objective of the present PhD was to characterize the effects of local production of IL-10 on the neuroinflammatory response characteristic of TBI. To achieve that cryogenic lesion model of TBI was applied to transgenic animals producing IL-10 under the astrocytic promoter GFAP (GFAP-IL10Tg animals) and their wild type counterparts, and different features of the neuroinflammatory response analyzed. This work revealed that IL-10 promotes the survival of neurons in the early hours after injury by inhibiting glia cell activation, neutrophil recruitment, disruption of BBB and the expression levels of the proinflammatory cytokine IL-1β, associated with increased T lymphocytes infiltration. These results point to IL-10 as a good candidate to ameliorate the neuroinflammatory response after TBI and prevent secondary tissue damage.

Alzheimers disease (AD) is pathologically characterized by amyloid plaques, neurofibrillary tangles, inflammation, and neurodegeneration. According to the amyloid hypothesis of AD, the central mediating event of the disease is the deposition of amyloid. The inflammation hypothesis of AD states that it is the inflammatory response to plaques and tangles, rather than the actual lesions, which causes the disease. Studies described here combine the two approaches into a single model. Four studies are presented using a basic protocol of intrahippocampal lipopolysaccharide (LPS) injection to stimulate inflammation in transgenic mice. The first study looked at alpha7 nicotinic receptors during the glial response to Abeta deposits and LPS. Reactive astrocytes which immunolabeled for alpha7 were co-localized with Congophilic deposits in APP and APP+PS1 mice, and increased after LPS injection. Unfortunately, LPS injection into alpha7 knock out mice revealed the alpha7 labeling to be nonspecific. The second study evaluated the time course of protein and gene expression after LPS injection into nontransgenic mice. This experiment identified both a transient and chronic microglial inflammatory response, with changes in cell morphology. The third study evaluated a similar time course in APP mice. Concurrent with the inflammatory response, transient reductions in Abeta burden were seen, though compact plaque load was unaffected. The fourth and final study used dexamethasone to inhibit LPS-induced inflammation in APP mice. LPS injection reduced Abeta burden, but was completely blocked by dexamethasone co-treatment. Though dexamethasone inhibited LPS-induced CD45 and complement receptor 3 levels (markers of general microglial activation), dexamethasone had no effect on scavenger receptor A or Fc gamma receptor II/III levels. An overall hypothesis regarding LPS mediated reductions in Abeta can be proposed: It is not the presence of the LPS molecule, nor the upregulation of receptors involved in phagocytosis, but rather general glial cell activation that mediates Abeta removal. Thus, a phagocytic cell must not only bind Abeta (by various receptors) but must also be capable of engulfing the material (via general cell activation). Taken together, these studies suggest that some level of inflammation in AD is beneficial and responsible for maintaining a balance between amyloid deposition and removal.

博士
長庚大學
生物醫學研究所
98
Emerging evidence suggests that astrocytes undergo large morphologic and gene expression changes in response to central nervous system (CNS) injury. In the CNS pathology, uncontrolled metalloproteinase-9 (MMP-9) activity and nitric oxide (NO) production are implicated in the increase of blood–brain barrier (BBB) permeability, the entry of leukocytes into the CNS, and sustained inflammatory responses. After traumatic and ischemic brain insults, disruption of the BBB raises the possibility of exposing the CNS to oxidized low-density lipoprotein (oxLDL), a risk factor implicated in neurodegenerative diseases. Hypoxic/ischemic injury also elicits endothelin-1 (ET-1) release in the CNS, behaving through G-protein coupled ET receptors. However, the detailed mechanisms of oxLDL and ET-1 action related to MMP-9 and iNOS expression and NO release on rat brain astrocytes remain largely unknown. Thus, this thesis focuses on investigating the signaling pathways by which oxLDL and ET-1 induced MMP-9 and iNOS expression and the functional consequences in astrocytes. Our results concluded that up-regulation of MMP-9 and iNOS induced by oxLDL and ET-1 may contribute to reactive responses of astrocytes after brain insults. First, the mechanisms underlying oxLDL-induced MMP-9 expression were investigated. We found that oxLDL induces expression of proMMP-9 via a mitogen-activated protein kinase (MAPK)-dependent activator protein-1 (AP-1) activation in rat brain astrocyte (RBA)-1 cells. Results revealed by gelatin zymography, RT-PCR, and Western blotting analyses showed that oxLDL-induced proMMP-9 gene expression was mediated through Akt, c-Jun N-terminal kinase (JNK1/2), and p42/p44 MAPK phosphorylation in RBA-1 cells. Moreover, the regulation of MMP-9 gene transcription by AP-1 was confirmed by chromatin immunoprecipitation (ChIP) assay which indentified the in vivo binding of c-Fos and c-Jun to the MMP-9 promoter, and by MMP-9 luciferase activity which was totally lost in cells transfected with the AP-1 binding site-mutated MMP-9 promoter construct (mt-AP1-MMP-9). These results suggest that oxLDL-induced proMMP-9 expression is mediated through phosphoinositide 3-kinase (PI3-K)/Akt, JNK1/2, and p42/p44 MAPK leading to AP-1 activation. Next, the roles of protein kinase C- (PKC-) and p42/p44 MAPK/Elk-1 cascades in oxLDL-induced MMP-9 expression were investigated. OxLDL induced MMP-9 expression via a PKC-/p42/p44 MAPK-dependent Elk-1 activation in RBA-1 cells. In vivo binding of Elk-1 to the MMP-9 promoter was evaluated by ChIP assay, revealing that oxLDL stimulated a time-dependent increase in binding of Elk-1 and p300 histone acetyltransferase (HAT), and sequential acetylated histone-4 to the MMP-9 promoter. Elk-1-mediated MMP-9 gene transcription was confirmed by transfection with an Elk-1 binding site-mutated MMP-9 promoter construct (mt-Ets-MMP9), which blocked oxLDL-stimulated MMP-9 luciferase activity. For astrocytic migration, the results suggested that the PKC-/p42/p44 MAPK/Elk-1-dependent proMMP-9 up-regulation is essential for the initiation of cell migration by oxLDL in RBA-1 cells. Furthermore, the participation of Elk-1, nuclear factor-κB (NF-B), and AP-1 in ET-1-induced proMMP-9 expression was proved. The data showed that ET-1-induced proMMP-9 expression was mediated through an ETB-dependent transcriptional activation. Engagement of Gi/o- and Gq-coupled ETB receptor by ET-1 led to activation of p42/p44 MAPK and then activated transcription factors including Elk-1, NF-B, and AP-1 (c-Jun/c-Fos). These activated transcription factors translocated into nucleus and bound to their corresponding binding sites in MMP-9 promoter, thereby turning on MMP-9 gene transcription. Functionally, up-regulation of proMMP-9 by ET-1 enhanced the migration of astrocytes. Taken together, these results suggested that in astrocytes, activation of Elk-1, NF-B, and AP-1 by ETB-dependent p42/p44 MAPK signaling is necessary for ET-1-induced MMP-9 gene up-regulation. Moreover, we demonstrated that exposure of astrocytes to ET-1 results in the iNOS up-regulation, NO production, and MMP-9 activation in astrocytes. Our data showed that ET-1-induced iNOS expression and NO production were mediated through an ETB-dependent transcriptional activation. ET-1 exerted the activation of c-Src-dependent PI3K/Akt and p42/p44 MAPK and NF-B, thereby promoting iNOS gene transcription. Sequential NO production enhanced astrocytic migration through the tyrosine nitration of MMP-9. These results suggested that in astrocytes, activation of NF-B by ETB-dependent c-Src, PI3K/Akt, and p42/p44 MAPK signalings is necessary for ET-1-induced iNOS gene up-regulation. Inclusion, we provide the mechanisms of oxLDL and ET-1 action on astrocytes, supporting the hypothesis that oxLDL and ET-1 contribute to the migration of astrocytes and the increased NO level involved in expression of MMP-9 leading to the development of CNS diseases. Understanding the mechanisms of MMP-9 and iNOS expression and functional changes regulated by oxLDL and ET-1/ETB system on astrocytes may provide rational therapeutic interventions for brain injury associated with the increased levels of MMP-9, iNOS, and NO.

Dissertação de Mestrado em Investigação Biomédica apresentada à Faculdade de Medicina
Neuroinflammation is an essential mechanism of innate immune defense in the CNS, but when unregulated it has been associated with the onset and progression of several pathologies, as neurodegenerative diseases. Astrocytes and microglia are the main immune effectors of the CNS and can become reactive when brain homeostasis is disrupted. In this way, these cells can contribute to the development of an inflammatory environment through the secretion of pro-inflammatory cytokines, like IL-1beta. Furthermore, astrocytes and microglia are in constant communication and can regulate each-other’s activity. Specifically, reactive microglia can secrete factors that induce a pro-inflammatory phenotype in astrocytes, the A1 phenotype.The main mechanism of IL-1beta production and secretion it’s the NLRP3 inflammasome, a multiprotein cytosolic complex that senses interferences in cell homeostasis. This inflammasome is activated through a two-step mechanism: a priming stimulus (like LPS), which induces an increased production of NLRP3 protein and pro-IL-1beta, and a second stimulus (like ATP), responsible for inflammasome oligomerization and consequent maturation and secretion of IL-1beta. The NLRP3 inflammasome assembly is well established in microglia, but it’s presence in astrocytes remains controversial.This project aimed to characterize NLRP3 inflammasome expression and function in A1 astrocytes and assess the role of microglia in these events. For this purpose, primary cultures of astrocytes isolated from newborn Sprague-Dawley rats were used. After 19 days in vitro, pure astrocytic cultures were incubated with LPS (100 ng/mL), ATP (1mM) or both. To simulate microglia signaling, astrocytes were also stimulated with a cocktail of factors: TNF-alpha (30 ng/mL), IL-1alpha (3 ng/mL) and C1q (400 ng/mL).Our results show that under LPS/ATP stimulation, astrocytes become reactive and secrete IL-1beta, mostly through NLRP3 inflammasome activity. Furthermore, TNF-alpha, IL-1alpha and C1q, besides activating astrocytes, act as a priming event capable of inducing NLRP3 and pro-IL-1beta production. However, astrocytes still require a second stimuli (as ATP) for inflammasome oligomerization and subsequent cytokine maturation.Overall, our work shows a preponderant role for astrocytes during neuroinflammation and reinforce the importance of microglia-astrocytes communication for CNS homeostasis and pathology.
A neuroinflamação é um mecanismo essencial do sistema imunitário inato no Sistema Nervoso Central, mas, quando desregulada, está associada ao desenvolvimento e progressão de várias patologias, como as doenças neurodegenerativas. Os astrócitos e a microglia são os principais efetores imunitários do SNC e têm a capacidade, quando há disrupções na homeostasia cerebral, de se tornarem reativos. Desta forma, estas células contribuem para o desenvolvimento de um ambiente inflamatório no SNC através da secreção de citocinas pro-inflammatórias, como a IL-1beta. Para além disto, os astrócitos e a microglia encontram-se em constante comunicação e podem regular a sua atividade mutuamente. Mais especificamente, a microglia reativa pode secretar fatores que induzem um fenótipo pro-inflamatório nos astrócitos, o fenótipo A1.O principal mecanismo de produção e secreção de IL-1beta é o inflamassoma NLRP3, um complexo citosólico multiproteico que deteta interferências na homeostasia celular. O inflamassoma é ativado através de dois passos: primeiro, um estímulo de priming (como LPS) induz um aumento na produção da proteína NLRP3 e de pro- IL-1beta. Um segundo estímulo (como ATP) induz a oligomerização do inflamassoma e consequente maturação e secreção de IL-1beta. Sabe-se que a microglia consegue expressar o inflamassoma NLRP3 ativo, mas a presença deste em astrócitos ainda é debatida.Este projeto tem como objetivo caracterizar a expressão e as funções do inflamassoma NLRP3 em astrócitos A1 e determinar o papel da microglia nestes eventos. Para isto, culturas primárias de astrócitos foram isoladas de ratos Sprague-Dawley recém-nascidos. Após 19 dias in vitro, as culturas puras de astrócitos foram incubadas com LPS (100 ng/mL), ATP (1mM) ou ambos. Para simular os sinais secretados pela microglia, os astrócitos foram estimulados com um cocktail de fatores: TNF-alpha (30 ng/mL), IL-1alpha (3 ng/mL) e C1q (400 ng/mL).Os nossos resultados demonstram que, sob estimulação com LPS/ATP, os astrócitos tornam-se reativos e secretam IL-1beta, maioritariamente devido à atividade do inflamassoma NLRP3. Para além do mais, TNF-alfa, IL-1alfa e C1q ativam os astróctios, atuando como um estímulo de priming capaz, de induzir a produção de NLRP3 e pro-IL-1beta. No entanto, os astrócitos necessitam de um segundo estímulo (por exemplo, o ATP) para que ocorra oligomerização do inflamassoma e consequente maturação de citocinas.Em resumo, este trabalho demonstra o papel preponderante dos astrócitos na neuroinflamação e reforça a importância da comunicação microglia-astrócitos para a homeostasia e patologia no SNC.
Outro - Project FCT, PTDC/MED-FAR/30933/2017

Intracerebral hemorrhage (ICH) is a devastating form of stroke that results from rupture of a blood vessel in the brain. Tissue inside the hematoma is irreversibly damaged soon after ICH onset and when this thesis research began, there was a dearth of information regarding pathological changes outside the hematoma. Inflammation is often proposed as a mechanism of injury, but very little information was available to show that inflammatory cells were in the right place at the right time to cause secondary brain injury. Using the collagenase-induced model of ICH, this work sought to better define spatial and temporal relationships between secondary brain injury and the inflammatory response after ICH. To test the hypothesis that reducing inflammation can protect the brain from secondary injury, minocycline, an antibiotic with established anti-inflammatory effects, was administered 6 hours after ICH onset. A small number of neurons die in the parenchyma bordering the hematoma between 6 hours and 3 days after ICH onset. This area was not associated with neutrophil infiltration, and most activated microglia/macrophages did not accumulate until after most neuron death had occurred. Despite a pronounced microglial response and prolonged increase in expression of many inflammatory genes, including complement receptor-3, interleukin-1 beta, interleukin-6, and interleukin-1 converting enzyme, no dying neurons were observed further outside the hematoma at any time. Interestingly, less early neuron death was observed in aged than in young animals, without a concomitant difference in the amount of tissue lost at 28 days. However, aged animals had less early microglial activation and a larger residual lesion, which might have resulted from impaired phagocytosis by activated microglia/macrophages. Minocycline was less effective in reducing microglial activation in aged animals, and did not reduce neuron death in either young or aged animals. Edema and BBB disruption was associated with degradation of the basal lamina protein, collagen type IV, and that damaged vessels are associated with tumor necrosis factor-alpha (TNFα)-positive neutrophils and active matrix metalloprotease-12 (MMP-12), all of which were reduced by delayed minocycline treatment. In contrast to ischemic stroke, there is a limited ‘penumbra’ outside the hematoma. Nevertheless, BBB damage in this region appears to be a potential target for protection. Furthermore, the prominent inflammatory response that continues for days after ICH does not appear to be associated with damage to other areas of the brain. Minocycline appears to protect the BBB by reducing neutrophil infiltration and the MMP-12 mediated basal lamina degradation. Future studies should investigate other targets for protection (i.e., white matter injury), and seek drugs that modulate the inflammatory response in aged animals and promote lesion resolution.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder, characterized by motor neurons (MN) loss in the brain and spinal cord (SC). Astrocyte reactivity and microglia activation account for neurodegeneration through the release of neurotoxins. Cytokines from activated microglia were shown to activate astrocytes and the inhibition of microglial microRNA(miRNA)-155 in ALS mice with G93A mutation in SOD1 gene (mSOD1) to recover their steady state phenotype and to attenuate disease progression. This thesis explored the reactive/inflammatory signature of ALS SC mSOD1 astrocytes, their response to microglial cytokines and the efficacy of anti-miR-155 modulation in recovering astrocyte neuroprotective properties. Astrocytes were isolated from the SC of 8-day-old mSOD1 mice pups and after 11 days in vitro, cells were incubated with cytokines that mimic A1-microglial activation (IL-1α/TNF-α/C1q) during 48h. Mutated astrocytes were transfected (or not) with anti-miRNA-155 and their secretome added to mSOD1-NSC-34 MN-like cells for 48h. We demonstrated that mSOD1 astrocytes have an inflammatory profile (elevated miR-21, miR-124, miR-155, IL-1β, Cx43 and HMGB1), further upregulated by A1-stimulation. Though a similar elevation of miR-155, iNOS and TNF-α was observed in both WT and mutated cells upon stimulation, increased levels of miR-21, miR-124, Cx43 and IL-6 mRNA, together with GFAP, S100B and vimentin protein were more evident in mSOD1 astrocytes, while IL-1β and IL-10 mRNAs remained unchanged and lower miR-146a upregulation was found. Downregulation of miR-155 in mSOD1 astrocytes reduced IL-1β, TNF-α and HMGB1, while upregulated IL-6, IL-10, SOCS1 and miR-146a. Importantly, secretome from astrocytes transfected with anti-miR-155 increased cell survival, mitochondria viability and neurite number in degenerating mSOD1-NSC-34 MN-like cells, in part mediated by miR-124 downregulation. Our results highlight the inflammatory/reactive/aberrant phenotype of mSOD1 SC astrocytes and its exacerbation by activated microglia as players in ALS neuroinflammation. Importantly, treatment with anti-miR-155 reduces the pro-inflammatory pattern of ALS astrocytes and restores their neuroprotective properties.

Neuropathic pain is one of the most debilitating disorders. Currently available treatments for neuropathic pain are still unsatisfactory as they have only limited treatment effect and patients may suffer from unwanted side effects. Mechanism-based approaches to neuropathic pain treatment are considered to be more effective. Therefore multiple studies are dedicated to study the pathophysiological mechanisms of neuropathic pain. One of the possible underlying mechanism that causes neuropathic pain is neuroinflammation. Recent studies suggested that angiotensin II ( main effector molecule of the renin-angiotensin system) via its receptors in the central nervous system may be involved in the neuroinflammatory processes. The aim of this study was to investigate the role of angiotensin receptor type 1 in the developement and maintenance of neuropathic pain induced in animal model. Spinal nerve ligation (L5) was used as a model of peripheral neuropathy. Our results showed that treatment with AT1R blocker losartan markedly reduced thermal hyperalgesia and reduced increased sensitivity to mechanical stimuli in the SNL-operated rats.This indicates a possibly significant role of AT1 receptors in the development of neuropathic pain, probably due to reduction of neuroinflammation in the nervous system. These findings...

Dysregulated cross-talk between activated glia and neurons, mediated by exosomal trafficking, is a key mechanism in the spreading of neuroinflammation and in the exacerbation of the Alzheimer’s disease (AD) pathology. In AD patients, A1 reactive astrocytes are found in brain regions affected by neurodegeneration, and display a neurotoxic profile that is crucial for the disease progression. Despite its pivotal role in AD progression, the astroglial population remains strikingly under-investigated, mainly due to the lack of reliable experimental biological platforms to study glial cell function in a disease context. In this thesis, our goal was to develop a human model able to recapitulate the pathological potential of AD astrocytes, thus surpassing species-specific differences in the study of neurodegenerative mechanisms. For that we differentiated astrocytes from iPSCs generated from the fibroblasts of AD patients and healthy matched controls. After a time-consuming process of differentiation, iPSCs-derived astrocytes from AD patients with PSEN1ΔE9 mutation showed a decrease in cellular HMGB1, S100B and microRNA (miR)-155 expression, together with a reduction in the number of GFAP-positive cells, a finding sustained after A1 induction for 48 h exposure to microglial cytokines (C1q/IL-1α/TNF-α), relatively to matched controls. Such treatment decreased cell arborisation leading to polarization as fibroblast-like and rounded cells, with increased mean surface area, and triggered the release of sAPPβ. A1 stimulation increased miR-155 in cells and exosomes, while diminished the cellular increase in miR-21 and miR-125b by inducing their package in exosomes, thus favouring the dissemination of inflammation to far and near cells. As observed in vivo, our cell population showed astrocyte heterogeneity, mainly for alarmins and miRNAs. In sum, astrocytic atrophy and abnormal distribution of inflammatory-miRNA in iPSCs-derived astrocytes carrying the PSEN1ΔE9 mutation and in their exosomes may provide important tools in targeting discovery, therapeutic development and personalised medicine for AD intervention.

L’insuffisance hépatique aiguë (IHA) se caractérise par la perte soudaine de la fonction hépatique résultant de la nécrose massive des hépatocytes en l’absence de pathologie hépatique préexistante. L’IHA s’accompagne de perturbations métaboliques et immunologiques qui peuvent entraîner l’apparition de complications périphériques et cérébrales telles qu’un syndrome de réponse inflammatoire systémique (SIRS), une encéphalopathie hépatique (EH), un œdème cérébral, une augmentation de la pression intracrânienne, et la mort par herniation du tronc cérébral. Les infections sont une complication fréquente de l’IHA et elles sont associées à un risque accru de développer un SIRS et une aggravation subséquente de l’EH avec un taux de mortalité augmenté. L’ammoniaque joue un rôle majeur dans les mécanismes physiopathologiques qui mènent au développement de l’EH et de l’œdème cérébral, et des études récentes suggèrent que les cytokines pro-inflammatoires sont également impliquées. Le but de cette thèse est d’étudier le rôle des cytokines pro-inflammatoires circulantes et cérébrales dans le développement de l’EH et de l’œdème cérébral lors d’IHA. Dans l’article 1, nous démontrons que l’inhibition périphérique du facteur de nécrose tumorale-α (TNF-α) par l’etanercept retarde la progression de l’EH en diminuant le dommage hépatocellulaire, réduisant l’inflammation périphérique et centrale ainsi que le stress oxydatif/nitrosatif hépatique et cérébral associé chez la souris avec une IHA induite par l’azoxyméthane (AOM). Ces résultats démontrent un rôle important du TNF-α dans la physiopathologie de l’EH lors d’IHA d’origine toxique et suggèrent que l’etanercept pourrait constituer une approche thérapeutique dans la prise en charge des patients en attente de transplantation hépatique. Dans l’article 2, nous simulons la présence d’une infection chez la souris avec une IHA induite par l’AOM pour mettre en évidence une éventuelle augmentation de la réponse inflammatoire. Nous démontrons que l’endotoxémie induite par le lipopolysaccharide (LPS) précipite la survenue du coma et aggrave la pathologie hépatique. Les cytokines pro-inflammatoires systémiques et cérébrales sont augmentées de façon synergique par le LPS lors d’IHA et résultent en une activation accrue de la métalloprotéinase matricielle-9 cérébrale qui s’accompagne d’une extravasation d’immunoglobulines G (IgG) dans le parenchyme cérébral. Ces résultats démontrent une augmentation majeure de la perméabilité de la barrière hémato-encéphalique (BHE) qui contribue à la pathogenèse de l’EH lors d’IHA en condition infectieuse. Les résultats de l’article 3 démontrent que l’augmentation de la perméabilité de la BHE lors d’IHA induite par l’AOM en condition non infectieuse ne résulte pas de l’altération de l’expression des protéines constitutives de la BHE. Dans l’article 4, nous démontrons que l’exposition d’astrocytes en culture à des concentrations physiopathologiques d’ammoniaque ou d’interleukine-1β résulte en l’altération de gènes astrocytaires impliqués dans la régulation du volume cellulaire et dans le stress oxydatif/nitrosatif. Un effet additif est observé dans le cas d’un traitement combiné au niveau des gènes astrocytaires impliqués dans le stress oxydatif/nitrosatif. L’ensemble des résultats de cette thèse démontre un rôle important de l’inflammation périphérique et cérébrale dans la survenue des complications neurologiques lors d’IHA et une meilleure compréhension des mécanismes physiopathologiques impliqués pourrait contribuer à la mise en place de stratégies thérapeutiques chez les patients atteints d’IHA en attente de transplantation.
Acute liver failure (ALF) is the clinical manifestation of an abrupt loss of hepatic function resuting from a massive hepatocyte necrosis in a patient with no preexisting liver disease. ALF is associated with metabolic and immunological disturbances that may lead to peripheral and cerebral complications such as systemic inflammatory response syndrome (SIRS), hepatic encephalopathy (HE), brain edema, increased intracranial pressure (ICP) and ultimately death by cerebral herniation. ALF is frequently complicated by infections, which are known to increase the risk of developing a SIRS with a subsequent worsening of HE and higher mortality rates. Ammonia plays a pivotal role in the pathophysiological mechanisms leading to HE and brain edema, and recent studies suggest that pro-inflammatory cytokines may also be involved. The aim of this thesis is therefore to investigate the role of circulating and cerebral pro-inflammatory cytokines in the setting of HE and brain edema during ALF. In article No. 1, we demonstrated that peripheral inhibition of tumor necrosis factor-alpha (TNF-α) by etanercept delays the progression of HE by reducing hepatocellular damage, decreasing peripheral and cerebral inflammation as well as associated oxidative/nitrosatif stress in mice with ALF induced by azoxymethane (AOM). These findings demonstrate an important role of TNF-α in the pathophysiology of HE during toxic liver injury and suggest that etanercept may provide a therapeutic approach in the management of patient awaiting liver transplantation. In article No. 2, we mimicked infection in mice with AOM-induced ALF in order to better understand the effects of an increased inflammatory response. We demonstrated that endotoxemia induced by lipopolysaccharide (LPS) precipitates the onset of coma and worsens the liver pathology. Peripheral and brain pro-inflammatory cytokines are synergistically raised by LPS during ALF and result in a large increase in cerebral matrix metalloprotease-9 (MMP-9) activity that was associated with immunoglobulin G (IgG) extravasation in the brain parenchyma. These results demonstrate a major increase of blood-brain barrier (BBB) permeability that contributes to the pathogenesis of HE during ALF with superimposed infection. Results from article No. 3 demonstrate that increase of BBB permeability during AOM-induced ALF without superimposed infection is not due to alteration of BBB constitutive proteins. In article No. 4, we demonstrated that exposure of cultured astrocytes to pathophysiological concentrations of ammonia or interleukin-1β results in an alteration of the expression of astrocytic genes implicated in cell volume regulation and oxidative/nitrosative stress. An additive effect on astrocytic genes implicated in oxidative/nitrosative was made evident in case of co-treatment. Taken together, results of the present thesis demonstrate a major role of peripheral and cerebral inflammation in the onset of neurological complications during ALF and a better understanding of the pathophysiological mechanisms implicated may contribute to new therapeutic strategies for ALF patients awaiting transplantation.

Dissertação de mestrado em Ciências da Saúde
Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS), characterized by the presence of demyelination plaques, inflammation and gliosis that consequently lead to axonal damage. The sequence of events that leads to demyelination remains unclear and the pathophysiological mechanisms are diverse. Also, although this is a disease of the CNS, there is no doubt that, in terms of peripheral organs, the thymus, as the organ of T cell differentiation and maturation, plays an important role in the pathophysiology of the disease. Recently, the levels of lipocalin 2 (LCN2), an acute phase protein that is part of the defense system against bacteria, by binding to iron-loaded siderophores, were found to be increased in cerebrospinal fluid (CSF) and serum of MS patients, when compared to control subjects. Similarly, using the MS animal model of experimental autoimmune encephalomyelitis (EAE), LCN2 was detected in brain parenchyma astrocytes, in regions typically affected in MS patients. This expression by astrocytes, together with an increased LCN2 level in the CSF, occurs during the active phases of the disease, which could point towards a role for LCN2 secreted by astrocytes in the mediation of inflammatory responses in the EAE model. Altogether, these findings support LCN2 as a valuable molecule for the diagnostic/monitoring of MS and suggest its potential involvement as a disease modulator. Of relevance, the exact role of LCN2 in the pathophysiology of the disease remains largely unknown and contradictory data exists on its potential protective or deleterious effect. Therefore, we sought to investigate the role of LCN2 in the onset and progression of the disease. Herein, we tackled the disease, by evaluating the role of LCN2, not only in the perspective of the CNS, but also on the perspective of peripheral organs such as the thymus. First we intended to perform a characterization of the thymus regarding thymocyte populations and histological morphology, in wild-type (WT) animals induced with EAE, in the onset and chronic phases of disease. Next, to further understand the role of LCN2 in MS pathology, we induced EAE both in LCN2- null mice and in WT littermates. Non-induced EAE animals were used as controls. The thymus of EAE animals was atrophied, as assessed by its weight, normalized for total body weight, and by the number of total cells. Also, we found a decrease in total cell number of all thymocyte populations, during the onset and chronic phases of EAE. In relative terms, the percentage of double positive cells was decreased, and the percentages of the cluster of differentiation (CD)4 and CD8 single positive cells were increased, during the onset phase. At the chronic phase, the proportions between the different populations were restored. LCN2-null mice induced with EAE did not present major alterations in terms of the clinical score, when compared with WT littermates also induced with EAE. Likewise, their thymic alterations were similar to the ones observed in WT EAE animals. Of relevance, as for the inflammatory profile in the cerebellum, LCN2-null mice presented less inflammation, as assessed by decreased expression levels of proinflammatory cytokines interferon (Ifn)-gamma, interleukin (Il)12a and Il17a. Also of interest, the cerebellum of LCN2-null mice presented a decrease in the percentage of lesioned areas. Finally, EAE animals, from both genotypes, presented an increase in the area positive for glial fibrillary acidic protein (GFAP), in the white matter of the cerebellum, in both the onset and chronic phases of disease. On the contrary, the expression levels of Gfap in the cerebellum were only increased at the onset phase of disease.
A Esclerose Múltipla (EM) é uma doença autoimune desmielinizante do sistema nervosa central (SNC), caracterizada pela presença de placas de desmielinização, inflamação e gliose, que tem como consequência dano axonal. A sequência de eventos que induzem desmielinização permanecem desconhecidos, e os mecanismos patofisiológicos são diversos. Embora esta seja uma doença do SNC, não há dúvidas que, em termos de órgãos periféricos, o timo, sendo o órgão de maturação e diferenciação das células T, desempenha um papel importante na patofisiologia da doença. Recentemente, os níveis de lipocalin-2 (LCN2), uma proteína de fase aguda que participa no sistema de defesa contra infeções bacterianas, através da ligação a sideróforos, foram encontrados como estando elevados no líquido cefalorraquidiano (LCR) e no soro de doentes com EM, comparativamente aos controlos. Da mesma maneira, usando o modelo animal de EM de encefalomielite autoimune experimental (EAE), a LCN2 foi detetada em astrócitos do parênquima, em regiões tipicamente afetadas em doentes com EM. Esta expressão pelos astrócitos, associada a um aumento de LCN2 no LCR, ocorreu durante as fases ativas da doença, o que aponta para um papel da LCN2 secretada pelos astrócitos na mediação da response inflamatória no modelo de EAE. No seu conjunto, estas evidências suportam o papel da LCN2 como uma molécula importante no diagnóstico e/ou monitorização da EM, e sugere o seu possível envolvimento como moduladora da doença. É relevante dizer que o papel exato da LCN2 na patofisiologia da doença permanece desconhecido, e existem dados contraditórios no que diz respeito ao seu potencial efeito protetor ou deletério. Por isso, nós procurámos perceber o papel da LCN2 no onset e na progressão da doença. Assim, nós investigámos o papel da LCN2 na doença, não só na perspetiva do SNC, mas também dos órgãos periféricos, nomeadamente do timo. Primeiro pretendemos caracterizar o timo em relação às populações de timócitos e morfologia histológica, em animais wild-type (WT) induzidos com EAE, no onset e na fase crónica da doença. De seguida, para melhor entender o papel da LCN2 na patologia da EM, induzimos EAE em animais LCN2- null e em WT da mesma ninhada. Para além disso, usámos animais não induzidos como controlos. Nós observámos que o timo dos animais induzidos com EAE estava atrofiado, com base no seu peso, após normalização para o peso total do animal, e no número total de células. Para além disso, encontrámos uma diminuição no número total de células de todas as principais populações de timócitos, durante o onset e fase crónica da doença. No que diz respeito à percentagem de cada uma das populações de timócitos, durante o onset da doença, a percentagem de células duplas positivas encontrava-se diminuída, enquanto as percentagens das populações CD4+CD8- e CD4-CD8+ se encontrava aumentada. Na fase crónica da doença, as proporções entre as diferentes populações foram reestabelecidas. Os animais LCN2-null induzidos com EAE não apresentaram grandes alterações em termos de score clínico, quando comparados com os animais WT da mesma ninhada também induzidos com EAE. Para além disso, as alterações observadas no timo foram semelhantes às encontradas nos animais WT EAE. De relevância, no que diz respeito ao perfil inflamatório no cerebelo, os animais LCN2-null apresentaram menos inflamação, o que é suportado por níveis diminuídos dos níveis de expressão das citoquinas pró-inflamatórias interferão-gama, e interleucinas 12 e 17. É importante também referir que os cerebelos de animais LCN2-null apresentaram uma diminuição na percentagem de áreas com lesões. Os animais EAE, de ambos os genótipos, apresentaram um aumento na área positiva para GFAP, na substância branca do cerebelo, no onset e na fase crónica da doença. Pelo contrário, os níveis de expressão de Gfap no cerebelo só foram encontrados elevados no onset da doença.

Microglia are a population of specialized, tissue-resident immune cells that make up around 10% of total cells in our brain. They actively prune neuronal synapses, engulf cellular debris, and misfolded protein aggregates such as the Alzheimer’s Disease (AD)-associated amyloid-beta (Aβ) by the process of phagocytosis. During AD, microglia are unable to phagocytose Aβ, perhaps due to the several disease-associated changes affecting their normal function. Functional molecules such as lipids and metabolites also influence microglial behavior but have primarily remained uncharacterized to date. The overarching question of this work is, How do microglia become dysfunctional in chronic inflammation? To this end, we developed new chemical tools to better understand and investigate the microglial response to Aβ in vitro and in vivo. Specifically, we introduce three new tools. (1) Recombinant human Aβ was developed via a rapid, refined, and robust method for expressing, purifying, and characterizing the protein. (2) A pH-sensitive fluorophore conjugate of Aβ (called Aβ^pH) was developed to identify and separate Aβ-specific phagocytic and non-phagocytic glial cells ex vivo and in vivo. (3) New lysosomal, mitochondrial, and nuclei-targeting pH-activable fluorescent probes (called LysoShine, MitoShine, and NucShine, respectively) to visualize subcellular organelles in live microglia. Next, we asked, What changes occur to the global lipid and metabolite profiles of microglia in the presence of Aβ in vitro and in vivo? We screened 1500 lipids comprising 10 lipid classes and 700 metabolites in microglia exposed to Aβ. We found significant changes in specific lipid classes with acute and prolonged Aβ exposure. We also identified a lipid-related protein that was differentially regulated due to Aβ in vivo. This new lipid reprogramming mechanism “turned on” in the presence of cellular stress was also present in microglia in the brains of the 5xFAD mouse model, suggesting a generic response to inflammation and toxicity. It is well known that activated microglia induce reactive astrocytes during inflammation. Therefore, we asked, What changes in proteins, lipids, and metabolites occur in astrocytes due to their reactive state? We provide a comprehensive characterization of reactive astrocytes comprising 3660 proteins, 1500 lipids, and 700 metabolites. These microglia and astrocytes datasets will be available to the scientific community as a web application. We propose a final model wherein the molecules secreted by reactive astrocytes may also induce lipid-related changes to the microglial cell state in inflammation. In conclusion, this thesis highlights chemical neuroimmunology as the new frontier of neuroscience propelled by the development of new chemical tools and techniques to characterize glial cell states and function in neurodegeneration.

博士
國立成功大學
基礎醫學研究所
105
Neuroinflammation has been suggested to play a critical role in the pathogenesis of neurodegenerative disorders and central nervous system injury such as Alzheimer’s disease (AD) and spinal cord injury. Neuroinflammation can cause glia cell activation and produce inflammatory cytokines, especially astrocyte. Astrocytes are the principal cell type in the central nervous system (CNS) and are essential in normal brain tissue. They can secrete neurotrophic factors that support normal neuron function and survival. In contrast, astrocytes can be activated and cause inflammation in a neuroinflammatory environment. Further, activated astrocytes can cause astrogliosis, which is characterized by cell anti-apoptosis, proliferation and the migration of astrocytes, and eventually leads to scar formation. The CCAAT/enhancer binding protein delta (CEBPD) protein is an inflammatory transcription factor and participates in both chronic and acute inflammation in CNS. Previous studies have shown that the CEBPD protein contributes to expression of Alzheimer’s disease. However, its role in Alzheimer’s disease remains unclear. Our data show that CEBPD is highly expressed in astrocytes in AD mouse models. In AD, a lack of CEBPD in AppTg mice can decrease astrocyte activation and reduce ROS formation. Further, I found that astrocytic CEBPD can activate ZNF179 expression, and ZNF179 can interact with PLZF to repress pro-apoptosis gene expression, including IGFBP3 and BIK. Furthermore, astrocytic CEBPD can induce ROS formation through p47phox and p67phox expression. Cu/Zn superoxide dismutase (SOD1), an antioxidant enzyme, is also up-regulated by CEBPD in astrocytes, which may contribute to the astrocyte resistance in an oxidative stress. Taken together, astrocytic CEBPD contributes to astrogliosis, has pathogenic consequences in neuroinflammatory disease, and provides new insights into the functional role of CEBPD in astrocytes that may lead to novel therapeutic strategies for the treatment.

Mild traumatic brain injury (mTBI), often referred to as concussion, has become increasingly recognized as a serious health issue in the general population. The prevalence of mTBI in athletes, particularly repeated injuries in young athletes, is of great concern as injuries to the developing brain can have long-term detrimental effects. In this study we used a novel awake closed-head injury (ACHI) model in rodents to examine repeated mTBI (rmTBI), to determine if repeated injuries produced the neurological and molecular changes evident with human concussion. Animals were administered 4, 8, and 16 rmTBIs and acute neurological assessments were performed after the injuries. Changes in glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba-1) levels were assessed using Western blot analysis at one day following rmTBI in the ipsilateral dentate gyrus (DG) and the cornu ammonis (CA) regions of the hippocampus and the cortex (CX) indicative of astrocyte and microglial cell reactivity. Results indicated that the ACHI model produces neurological deficits immediately after the injuries, with the most deficits arising in the rmTBI16 group. Despite deficits in all injury groups, histological staining with cresyl violet revealed no significant morphological tissue damage to the brain. Western blot analysis, however, showed a significant increase in DG and CX GFAP expression in the rmTBI16 group with no changes in Iba-1 levels. This suggests an acute activation of astrocytes in response to injury, with a delay or absence of microglial activation. Our findings show that with repetitive concussions, we are able to detect acute neurological and molecular changes in the juvenile female brain. However, further investigation is necessary to determine if these are transient changes.
Graduate

Tese de doutoramento do Programa Interuniversitário de Doutoramento em Envelhecimento e Doenças Crónicas, apresentada à Faculdade de Medicina da Universidade de Coimbra
Methylphenidate (MPH) is an amphetamine-like psychostimulant that has become the primary drug of choice for treating attention-deficit hyperactivity disorder (ADHD), a highly prevalent neuropsychiatric disorder with an onset in early childhood. ADHD includes behavioral and cognitive symptoms, such as inattention and/or impulsivity/hyperactivity. In more than 50% of children with ADHD, the symptoms persist into adolescence and adulthood. Moreover, the diagnosis of ADHD itself is non-consensual and several reports claim that physicians tend to be preoccupied with reducing symptoms via medication but often fail to recognize the context in which the behavior exhibits, which suggest that ADHD is being overdiagnosed in children. Despite being beneficial under ADHD conditions, MPH misuse is nowadays a problem with high impact on society. The full comprehension of the cellular/molecular mechanisms trigger by MPH use is still elusive. To date, most of the studies that explain the underlying MPH effects on central nervous system have focused on intra-neuronal events, particularly on dopaminergic alterations. The impact of MPH on blood brain-barrier (BBB) function has never been addressed before neither in non-pathological conditions nor in ADHD. The BBB is a barrier highly selective responsible for the regulation and maintenance of brain homeostasis creating a proper microenvironment for neural function. Given the evidence of BBB damage as an early event in many neurological conditions, it is not surprising that there is a growing interest in the BBB as a therapeutic target. Thus, the major goal of the present thesis was to clarify the effects of MPH on the BBB function in both physiological and ADHD conditions. Specifically, we aimed to investigate the direct impact of MPH on human brain microvascular endothelial cells (HBMVECs), and to evaluate the effect of chronic MPH use during normal and ADHD rat neurodevelopmental on the neurogliovascular unit, brain immune surveillance neuroinflammation, and behavior. This thesis is organized in 5 chapters. In chapter 1 is presented a review of the literature about the topics explored in this work with emphasis in ADHD, MPH and BBB. In chapter 2, we evaluated the direct effect of MPH on HBMVEC. It was found that MPH increases brain endothelial cell permeability by promoting the vesicular transport. Specifically, we identified the molecular event critical to MPH-induced transcytosis, showing that c-Src is activated in result of intracellular reactive oxygen species generated by Rac1/NOX that interacts and phosphorylates Caveolin1, resulting in caveolae formation and consequent vesicular permeability. Next in chapter 3, we unraveled MPH chronic outcome in the healthy brain as well as cognitive concerns that might arise from its misuse. Since hippocampus is highly involved in memory/learning processes we focused on this brain region. Our data revealed that higher doses of MPH cause endothelial dysfunction and activation with a consequent increase of leukocyte infiltration by transcytosis. In addition to disruption of hippocampal vasculature, alterations in astrocytic morphology and synaptic machinery were also observed. These neurogliovascular disturbances culminated in memory deficit. Contrariwise, a lower dose of MPH improved cognition in parallel with an increase of astrocytic processes but with no major effect on BBB permeability. Furthermore, we showed that MPH misuse modulated AKT/CREB signaling pathway, which can explain alterations on cognitive performance. Afterwards, in chapter 4, we dissected the impact of early-life chronic consumption of MPH on brain immune surveillance and neuroinflammation in both physiological and ADHD conditions. This work was focused on the prefrontal cortex that is a brain region with a prominent role in the pathophysiology of ADHD. Our results show that chronic exposure to a higher dose of MPH caused BBB leakage and elicited an anxious-like behavior in both healthy and ADHD conditions. Nevertheless, BBB dysfunction was more prominent in control animals, which was proved by a downregulation and disruption of intercellular junctions, an increase of microvessels vesicles, and endothelium activation concomitant with infiltration of peripheral immune cells. Moreover, independently of the dose used, MPH triggered a robust oxidative and neuroinflammatory response in healthy rats shown by astrocytes and microglial cells activation and upregulation of pro-inflammatory cytokines. On the contrary, in ADHD animal model, MPH at the lower dose restored brain homeostasis by decreasing the inflammatory and oxidative status. Moreover, the MPH immune-modulatory effects in both animal conditions seem to be mediated through NF-κB/NLRP3 signaling pathways. Finally, in chapter 5, a general discussion with final remarks is presented. Overall, this thesis provides evidence that MPH acts directly on endothelial cells impairing BBB integrity, which may favor the access of peripheral cells into the brain. Additionally, MPH has an effect on glial cells leading to a neuroinflammatory response and oxidative status. Behavior alterations were also observed, including spatial working memory deficit and anxiety-like behavior. Herein, we improved our understanding about neurogliovascular unit alterations associated with chronic MPH treatment during development, and how these changes depend on the dose of MPH and (non)-pathological conditions, which highlights the importance of an appropriate MPH dose regimen for ADHD and an adequate diagnosis to avoid MPH misuse.
O metilfenidato (MFD) é um psicoestimulante do grupo das anfetaminas usado como primeira linha de tratamento da perturbação de hiperatividade e défice de atenção (PHDA). PHDA é uma perturbação neuropsiquiátrica altamente prevalente com inicio na infância e que inclui sintomas comportamentais e cognitivos, nomeadamente falta de atenção e/ou impulsividade/hiperatividade. Normalmente, em mais de 50% das crianças com PHDA os sintomas persistem até à adolescência e fase adulta. Além disso, o diagnóstico desta patologia não é consensual com vários relatórios a afirmar que os médicos tendem a estar mais preocupados com a redução dos sintomas através da medicação e muitas vezes não conseguem reconhecer o contexto em que o comportamento é exibido, o que sugere que a PHDA está a ser sobrediagnosticada em crianças. Apesar de ser benéfico em condições de PHDA, o uso indevido de MFD é hoje em dia um problema com alto impacto na sociedade. Os mecanismos celulares/moleculares desencadeados pelo uso de MFD ainda são largamente desconhecidos. Até à data, a maioria dos estudos que explicam os efeitos no sistema nervoso central subjacentes ao uso do MFD têm-se focado nos eventos intraneuronais, particularmente nas alterações dopaminérgicas. Neste sentido, o impacto do MFD na função de barreira hematoencefálica (BHE) nunca foi abordado antes, quer em condições não patológicas quer em situações de PHDA. A BHE é uma barreira altamente seletiva responsável pela regulação e manutenção da homeostasia cerebral, criando um microambiente adequado para a função neuronal. Visto que a disfunção na BHE é considerada um evento precoce em muitas patologias do cérebro, não é surpreendente que haja um interesse crescente na BHE como um alvo terapêutico. Desta forma, o principal objetivo da presente tese foi esclarecer os efeitos do MFD na função da BHE em condições fisiológicas e de PHDA. Especificamente, pretendeu-se investigar o efeito direto do MFD nas células endoteliais da microvasculatura do cérebro humano (CEs) e avaliar o efeito do uso crónico de MFD durante o desenvolvimento neurológico normal e numa condição de PDHA, tendo como principais focos a unidade neurogliovascular, a resposta imunitária e neuroinflamatória, bem como as alterações comportamentais. Esta tese encontra-se organizada em 5 capítulos. No capítulo 1, é apresentada uma revisão da literatura onde se exploram tópicos essenciais à compreensão deste trabalho dando ênfase à PHDA, MFD e BHE. No capítulo 2, avaliamos o efeito direto do MFD nas CEs. Verificou-se que o MFD aumenta a permeabilidade das CEs promovendo o transporte vesicular. Especificamente, identificámos a via de sinalização intracelular responsável pela transcitose induzida por MFD, mostrando que a c-Src é ativada por espécies reativas de oxigénio intracelular gerados pelo complexo Rac1/NOX que por sua vez interage e fosforila a caveolina 1, resultando na formação de cavéolas e consequente permeabilidade vesicular. Em seguida, no capítulo 3, desvendámos os efeitos resultantes do consumo crónico do MFD no cérebro saudável, bem como as consequências cognitivas que podem surgir do seu uso indevido. Tendo em consideração que o hipocampo está amplamente envolvido em processos de memória/aprendizagem, focámos o nosso estudo nesta região cerebral. Concluímos que doses mais altas de MFD causam disfunção e ativação endotelial com o consequente aumento da infiltração de leucócitos por transcitose. Além da disfunção da vasculatura do hipocampo, também foram observadas alterações na morfologia dos astrocítos e na maquinaria sináptica. Estas alterações neurogliovasculares culminaram em défice de memória. Por outro lado, uma menor dose de MFD melhorou a memória em simultâneo com um aumento dos prolongamentos astrocíticos, mas sem efeito significativo na permeabilidade da BHE. Adicionalmente, mostrámos que o uso indevido do MFD é capaz de modular a via de sinalização da AKT/CREB, o que pode explicar as alterações no desempenho cognitivo. Posteriormente, no capítulo 4, avaliámos o impacto do consumo crónico de MFD no período correspondente desde a infância até à adolescência tardia na vigilância imunológica do cérebro e na neuroinflamação em condições fisiológicas e de PHDA. Este trabalho foi focado no córtex pré-frontal, região cerebral com um papel proeminente na fisiopatologia da PHDA. Os nossos resultados mostram que a exposição crónica a uma dose mais elevada de MFD causou um aumento da permeabilidade da BHE e incitou um comportamento do tipo ansioso em ambas as condições estudadas, saudável e PHDA. No entanto, a disfunção da BHE foi mais proeminente nos animais sem patologia, o que foi evidenciado por uma diminuição dos níveis e rutura das junções intercelulares, um aumento de vesículas nos microvasos e ativação do endotélio, concomitante com a infiltração de células do sistema imunitário periférico para o cérebro. Para além disso, independentemente da dose utilizada, o MFD desencadeou uma resposta oxidativa e neuroinflamatória robusta nos ratos saudáveis comprovado pela ativação dos astrócitos e das células da microglia, assim como pelo aumento de citocinas pró-inflamatórias. Pelo contrário, no modelo animal de PHDA, o MFD na dose mais baixa restaurou a homeostasia cerebral, diminuindo o estado inflamatório e oxidativo. Além disso, os efeitos imunomoduladores do MFD em ambos os modelos animais parecem ser mediados pela via de sinalização do NF-κB/NLRP3. Finalmente, no capítulo 5, uma discussão geral com considerações finais é apresentada. Em conclusão, esta tese dá-nos evidências de que o MFD atua diretamente nas CEs prejudicando a integridade da BHE, o que pode favorecer o acesso das células periféricas ao cérebro. De realçar que o MFD tem também um efeito nas células da glia, desencadeando uma resposta neuroinflamatória e causando stress oxidativo. Alterações comportamentais também foram observadas, incluindo défice de memória de trabalho e comportamento do tipo ansioso. Desta forma, contribuímos para uma melhor compreensão das alterações que ocorrem na unidade neurogliovascular devido a um tratamento crónico com MFD durante o desenvolvimento numa condição fisiológica versus PHDA, e como essas alterações dependem ainda da dose de MFD. Estas observações evidenciam a importância da dose terapêutica de MFD que é prescrita e de um diagnóstico mais objetivo para evitar o uso indevido de MFD.
FCT - PTDC/NEU-OSD/0312/2012 (COMPETE e FEDER ) ; Pest-C/SAU/UI3282/2013-2014 e CNC.IBILI UID/NEU/04539/2013 ; PT2020/COMPETE 2020 e Operational Program for Competitiveness and Internalization (POCI) - FCOMP-01-0124-FEDER-028417 e POCI-01-0145-FEDER-007440

Tese de doutoramento em Engenharia Biomédica, apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra
Methamphetamine (METH) is a powerful psychostimulant drug of abuse that has gained worldwide popularity, and its use originates severe health problems. Despite extensive characterization of METH-induced neurotoxicity over the last years, many questions remain unanswered. Several reports have demonstrated that oxidative stress, mitochondrial dysfunction, and neuroinflammation are some of the neurotoxic features of METH. More recently, it was shown that METH compromises the blood-brain barrier (BBB) and causes a disturbance in the water homeostasis leading to brain edema. Additionally, it is well known that astrocytes play a crucial role in modulating BBB structure and function, as well as in regulating brain water content. However, the effect of METH on the crosstalk between brain endothelial cells (ECs) and astrocytes has never been addressed before. Also, water fluxes that take place between the different compartments of the brain, and between brain parenchyma and the blood are highly controlled. Thus, disturbances in this well-regulated homeostasis cause brain edema, which will have deleterious effects on brain function. Importantly, the water transport at BBB is regulated by water channels, aquaporins (AQPs), and AQP4 is the most important at the Central Nervous System, being express on astrocytic endfeet in contact with brain vessels. Brain edema is a hallmark of several neuropathologies, and METH consumption is not an exception. Yet, to date, nothing is known about the role of AQP4 under METH conditions. Furthermore, AQP4 has two isoforms, M1 and M23, and the ratio M1/M23 regulates water homeostasis since M23 stabilizes the channel function but M1 disrupts the AQP4 structure. Taking into consideration all the gaps in this field, it is urgent to clarify the role of AQP4 in METH-induced BBB dysfunction and brain edema formation. The present thesis is divided into 5 chapters. In chapter 1 is presented a review of the literature about the different themes that were explored in the laboratory and detailed in the following chapters. In chapter 2, the impact of METH on astrocytes-ECs crosstalk was investigated with a particular interest in the role of tumor necrosis factor alpha (TNF-α). After observing that METH increased TNF-α released by both astrocytes and ECs, it was also proved that this proinflammatory cytokine was responsible for endothelial permeability through the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. These in vitro results were corroborated by animal studies showing an increase of BBB permeability and TNF-α levels in the mice striatum, which was prevented by NF-κB pathway blockade. Overall, it was shown that TNF-α/NF-κB signaling pathway has a key role in METH-induced BBB dysfunction. Next, in chapter 3, it was investigated the direct effect of METH on AQP4 system concluding that METH, besides BBB dysfunction, is also able to induce a cytotoxic brain edema and depressive-like behavior. Curiously, AQP4 was shown to have a predominant role of such alterations since its inhibition prevented all the effects observed in mice. Moreover, AQP4 via reactive oxygen species (ROS) production was involved in cell swelling and altered astrocyte morphology triggered by METH since AQP4 knockdown or its pharmacological blockade, as well as an antioxidant treatment (namely vitamin C) were able to prevent METH effects in astrocytes. In conclusion, AQP4 was identified as a new target against METH-induced neurogliovascular dysfunction and depressive-like behavior. Following the results observed in chapter 2 and 3, a new strategy to counteract the negative effects of METH was applied by using a natural flower product. Thus, in chapter 4, it was proved that parthenolide (PTL), a feverfew plant extract, has an anti-inflammatory role and prevented METH-induced BBB permeability and brain edema. Additionally, TNF-α via activation of its receptor 1 (TNFR1) was involved in astrocytic swelling induced by METH. In sum, PTL plays a beneficial role against neuroinflammation and neurogliovascular dysfunction triggered by METH. Finally, in chapter 5, a general discussion is presented. Overall, the present work shows that METH interferes with brain water homeostasis and BBB function, culminating in behavioral abnormalities. Moreover, both neuroinflammation and oxidative stress are involved in such negative effects of METH, and new strategies to counteract these deleterious consequences were identified, such as AQP4 blockade and the use of PTL.
A metanfetamina (MET) é uma droga de abuso muito viciante com grande popularidade mundial, e que causa sérios problemas de saúde. Apesar da extensa caracterização da sua neurotoxicidade nos últimos anos, muitas questões continuam sem resposta. Alguns estudos têm mostrado que o stresse oxidativo, a disfunção mitocondrial e a neuroinflamação são alguns dos efeitos nefastos da MET. Mais recentemente demonstrou-se que a MET interfere com a função normal da barreira hematoencefálica (BHE), causando alterações na homeostase da água o que pode levar a uma situação de edema cerebral. Para além disso, sabe-se também que os astrócitos têm um papel muito importante na modulação da estrutura e função da BHE, bem como na regulação do conteúdo de água cerebral. No entanto, o efeito da MET na comunicação entre as células endoteliais (CEs) e os astrócitos nunca foi estudado anteriormente. Por outro lado, o movimento de moléculas de água entre os diferentes compartimentos do cérebro e entre o parênquima cerebral e a corrente sanguínea ocorre de forma controlada. Assim, distúrbios nesta homeostase irão causar uma situação de edema, o qual terá um impacto negativo na função cerebral. O transporte de água na BHE é regulado por canais de água, denominados aquaporinas (AQPs), sendo que a AQP4 é a mais importante no Sistema Nervoso Central, e encontra-se expressa nas terminações dos astrócitos que contactam com os vasos cerebrais. De facto, o edema cerebral ocorre em muitas neuropatologias, e o consumo de MET não é exceção. No entanto, o papel da AQP4 nos efeitos da MET é ainda desconhecido. Além disso, a AQP4 tem duas isoformas, a M1 e a M23, e é a sua proporção que regula a homeostase da água, uma vez que a presença da isoforma M23 estabiliza a função do canal de água enquanto a isoforma M1 causa alterações na função da AQP4. Deste modo, é importante esclarecer o papel da AQP 4 na disfunção da barreira hematoencefálica e na formação do edema cerebral induzidos por MET. A presente tese está dividida em 5 capítulos. No capítulo 1 é apresentada uma revisão da literatura sobre os diversos temas estudados no laboratório e detalhados nos capítulos seguintes. No capítulo 2 investigou-se o efeito da MET na comunicação entre astrócitos e CEs com particular interesse no papel do fator de necrose tumoral alfa (TNF-α). Depois de mostrar um aumento da libertação de TNF-α induzido por MET, quer pelos astrócitos quer pelas CEs, provou-se que esta citocina pró-inflamatória estava envolvida no aumento da permeabilidade das CEs através da ativação da via de sinalização do fator nuclear kappa B (NF-κB). Estes resultados foram corroborados por estudos em animais onde se observou um aumento da permeabilidade da BHE e dos níveis de TNF-α no estriado de murganho, efeitos estes que foram prevenidos pelo bloqueio da via do NF-κB. Deste modo, conclui-se que a via de sinalização do TNF-α/NF-κB está envolvida na disfunção da BHE induzida por MET. De seguida, no capítulo 3 avaliou-se o impacto direto da MET no sistema da AQP4 e foi possível demonstrar que esta droga de abuso, para além de induzir uma disfunção da BHE, também originou um edema cerebral citotóxico e comportamento do tipo depressivo. Curiosamente, a AQP4 teve um papel predominante nestas alterações já que o seu bloqueio preveniu todos os efeitos observados nos murganhos. In vitro foi também possível comprovar o papel importante da AQP4 via produção de espécies reactivas de oxigénio já que o silenciamento deste canal de água ou a sua inibição farmacológica, bem como a exposição a um antioxidante (vitamina C) preveniram as alterações morfológicas induzidas pela MET nos astrócitos. Em conclusão, a AQP4 foi identificada como um alvo importante para prevenir as alterações neurogliovasculares e comportamento depressivo induzidos por MET. Na sequência dos efeitos negativos da MET observados nos capítulos 2 e 3, colocou-se a hipótese de uma nova abordagem com um produto natural de origem vegetal. Deste modo, no capítulo 4 concluíu-se que o partenolídeo (PTL), um extrato obtido da artemísia dos prados (Tanacetum parthenium), tem um papel anti-inflamatório e preveniu o aumento da permeabilidade da BHE e formação de edema cerebral induzidos por MET. Mais ainda, foi possível demonstrar que o TNF-α, através da ativação do seu recetor TNFR1, estava envolvido no aumento de volume dos astrócitos observado na presença de MET. Assim, este trabalho permitiu concluir que o PTL tem um feito benéfico em condições de neuroinflamação e disfunção neurogliovascular induzidos por MET. Por último, o capítulo 5 inclui uma discussão geral sobre os resultados obtidos nos capítulos anteriores. Em conclusão, esta tese permitiu mostrar que a MET interfere não só com a homeostase da água no cérebro, mas também com a função da BHE, e que estes efeitos podem conduzir a alterações comportamentais. Para além disso, demonstrou-se ainda que a neuroinflamação e o stresse oxidativo estão subjacentes aos efeitos negativos causados pela MET e foram identificadas duas abordagens para prevenir estes efeitos, tais como o bloqueio da AQP4 e o uso do partenolídeo.

Research Doctorate - Doctor of Philosophy (PhD)
Schizophrenia is a debilitating disorder of neurodevelopmental origins that likely stems from the cumulative action of a range of genetic and environmental factors. Epidemiological evidence has identified maternal infection during gestation as one significant environmental risk factor for the development of the disorder. Evidence from animal models has further validated the link between maternal immune activation (MIA) in the absence of an active infection and the later life development of schizophrenia-like pathology in the offspring. In particular, work in mouse models has suggested that the gestational time at which MIA occurs can alter the behavioural and neurobiological phenotype displayed. Specifically, that MIA in late gestation is involved in schizophrenia-relevant cognitive dysfunction and altered NMDA receptor expression, whereas MIA in early gestation is more closely associated with behavioural deficits reminiscent of positive symptomology and dopaminergic neurotransmission. The aim of the current thesis was to extend the mouse findings to another species, the rat, and further explore the effects of MIA. In addition to producing a reliable rat model of schizophrenia where distinct behavioural and neurological phenotypes associated with schizophrenia are produced following MIA at either early or late gestational time-points (gestational day 10 or 19, respectively), the current thesis extends on previous work by examining the schizophrenia biomarker of mismatch negativity and assessing the neuroinflammatory state of offspring. Behavioural assessments revealed that MIA in either early or late gestation produced transient impairments in working memory and reductions in PPI. In these behavioural studies, there was no clear distinction between a dopamine and glutamate-related behavioural phenotype based on the gestational timing of exposure. However, early but not late gestation MIA did produce alterations in the dopaminergic system of males, as indicated by increased dopamine 1 receptor mRNA in the nucleus accumbens. EEG experiments demonstrated that although the male rat brain is able to generate human-like (adaptation-independent) mismatch responses (MMRs), and although MIA (regardless of gestational timing) does alter MMRs, it does not do so in a manner comparable with schizophrenia. Immunohistochemical techniques revealed that MIA does result in subtle neuro-immune changes in adult offspring, with an increase in microglial immunoreactivity identified in the frontal white matter of late, but not early, gestation MIA animals. Furthermore, a strong trend towards increased astrocyte immunoreactivity that approached significance was identified in the prefrontal cortex of late, but not early MIA offspring. The combined results have demonstrated that MIA during the chosen gestational time-points are sufficient to disrupt neurodevelopmental processes producing long-term alterations in behavioural and neuropathological measures relevant to schizophrenia. However, the phenotype characterised here deviates slightly from previous findings from mouse models indicating potential differences in the critical periods of neurodevelopmental susceptibility to MIA exposure between the rat and mouse. Importantly this research has provided insights into the underlying neuro-immune changes which may contribute to the behavioural abnormalities seen in adult MIA offspring and has provided evidence that MIA in rats can alter the prominent schizophrenia relevant electrophysiological biomarker of adaptation-independent MMRs, providing a basis to further investigate these measures and their underlying mechanisms.

La Sclérose en plaques (SEP) est une maladie auto-immune inflammatoire démyélinisante du système nerveux central (SNC), lors de laquelle des cellules inflammatoires du sang périphérique infiltrent le SNC pour y causer des dommages cellulaires. Dans ces réactions neuroinflammatoires, les cellules immunitaires traversent le système vasculaire du SNC, la barrière hémo-encéphalique (BHE), pour avoir accès au SNC et s’y accumuler. La BHE est donc la première entité que rencontrent les cellules inflammatoires du sang lors de leur migration au cerveau. Ceci lui confère un potentiel thérapeutique important pour influencer l’infiltration de cellules du sang vers le cerveau, et ainsi limiter les réactions neuroinflammatoires. En effet, les interactions entre les cellules immunitaires et les parois vasculaires sont encore mal comprises, car elles sont nombreuses et complexes. Différents mécanismes pouvant influencer la perméabilité de la BHE aux cellules immunitaires ont été décrits, et représentent aujourd’hui des cibles potentielles pour le contrôle des réactions neuro-immunes. Cette thèse a pour objectif de décrire de nouveaux mécanismes moléculaires opérant au niveau de la BHE qui interviennent dans les réactions neuroinflammatoires et qui ont un potentiel thérapeutique pour influencer les interactions neuro-immunologiques. Ce travail de doctorat est séparé en trois sections. La première section décrit la caractérisation du rôle de l’angiotensine II dans la régulation de la perméabilité de la BHE. La seconde section identifie et caractérise la fonction d’une nouvelle molécule d’adhérence de la BHE, ALCAM, dans la transmigration de cellules inflammatoires du sang vers le SNC. La troisième section traite des propriétés sécrétoires de la BHE et du rôle de la chimiokine MCP-1 dans les interactions entre la BHE et les cellules souches. Dans un premier temps, nous démontrons l’importance de l’angiotensinogène (AGT) dans la régulation de la perméabilité de la BHE. L’AGT est sécrété par les astrocytes et métabolisé en angiotensine II pour pouvoir agir au niveau des CE de la BHE à travers le récepteur à l’angiotensine II, AT1 et AT2. Au niveau de la BHE, l’angiotensine II entraîne la phosphorylation et l’enrichissement de l’occludine au sein de radeaux lipidiques, un phénomène associé à l’augmentation de l’étanchéité de la BHE. De plus, dans les lésions de SEP, on retrouve une diminution de l’expression de l’AGT et de l’occludine. Ceci est relié à nos observations in vitro, qui démontrent que des cytokines pro-inflammatoires limitent la sécrétion de l’AGT. Cette étude élucide un nouveau mécanisme par lequel les astrocytes influencent et augmentent l’étanchéité de la BHE, et implique une dysfonction de ce mécanisme dans les lésions de la SEP où s’accumulent les cellules inflammatoires. Dans un deuxième temps, les techniques établies dans la première section ont été utilisées afin d’identifier les protéines de la BHE qui s’accumulent dans les radeaux lipidiques. En utilisant une technique de protéomique nous avons identifié ALCAM (Activated Leukocyte Cell Adhesion Molecule) comme une protéine membranaire exprimée par les CE de la BHE. ALCAM se comporte comme une molécule d’adhérence typique. En effet, ALCAM permet la liaison entre les cellules du sang et la paroi vasculaire, via des interactions homotypiques (ALCAM-ALCAM pour les monocytes) ou hétérotypiques (ALCAM-CD6 pour les lymphocytes). Les cytokines inflammatoires augmentent le niveau d’expression d’ALCAM par la BHE, ce qui permet un recrutement local de cellules inflammatoires. Enfin, l’inhibition des interactions ALCAM-ALCAM et ALCAM-CD6 limite la transmigration des cellules inflammatoires (monocytes et cellules T CD4+) à travers la BHE in vitro et in vivo dans un modèle murin de la SEP. Cette deuxième partie identifie ALCAM comme une cible potentielle pour influencer la transmigration de cellules inflammatoires vers le cerveau. Dans un troisième temps, nous avons pu démontrer l’importance des propriétés sécrétoires spécifiques à la BHE dans les interactions avec les cellules souches neurales (CSN). Les CSN représentent un potentiel thérapeutique unique pour les maladies du SNC dans lesquelles la régénération cellulaire est limitée, comme dans la SEP. Des facteurs qui limitent l’utilisation thérapeutique des CSN sont le mode d’administration et leur maturation en cellules neurales ou gliales. Bien que la route d’administration préférée pour les CSN soit la voie intrathécale, l’injection intraveineuse représente la voie d’administration la plus facile et la moins invasive. Dans ce contexte, il est important de comprendre les interactions possibles entre les cellules souches et la paroi vasculaire du SNC qui sera responsable de leur recrutement dans le parenchyme cérébral. En collaborant avec des chercheurs de la Belgique spécialisés en CSN, nos travaux nous ont permis de confirmer, in vitro, que les cellules souches neurales humaines migrent à travers les CE humaines de la BHE avant d’entamer leur différenciation en cellules du SNC. Suite à la migration à travers les cellules de la BHE les CSN se différencient spontanément en neurones, en astrocytes et en oligodendrocytes. Ces effets sont notés préférentiellement avec les cellules de la BHE par rapport aux CE non cérébrales. Ces propriétés spécifiques aux cellules de la BHE dépendent de la chimiokine MCP-1/CCL2 sécrétée par ces dernières. Ainsi, cette dernière partie suggère que la BHE n’est pas un obstacle à la migration de CSN vers le SNC. De plus, la chimiokine MCP-1 est identifiée comme un facteur sécrété par la BHE qui permet l’accumulation et la différentiation préférentielle de cellules souches neurales dans l’espace sous-endothélial. Ces trois études démontrent l’importance de la BHE dans la migration des cellules inflammatoires et des CSN vers le SNC et indiquent que de multiples mécanismes moléculaires contribuent au dérèglement de l’homéostasie du SNC dans les réactions neuro-immunes. En utilisant des modèles in vitro, in situ et in vivo, nous avons identifié trois nouveaux mécanismes qui permettent d’influencer les interactions entre les cellules du sang et la BHE. L’identification de ces mécanismes permet non seulement une meilleure compréhension de la pathophysiologie des réactions neuroinflammatoires du SNC et des maladies qui y sont associées, mais suggère également des cibles thérapeutiques potentielles pour influencer l’infiltration des cellules du sang vers le cerveau
Multiple Sclerosis is an inflammatory demyelinating disease in which immune cells from the peripheral blood infiltrate the central nervous system (CNS) to cause a pathologic neuroinflammatory reaction. Blood borne leucocytes cross the restrictive cerebral endothelium, the blood brain barrier (BBB), to gain access to the CNS parenchyma and cause cellular damage leading to the characteristic demyelinating lesions. The BBB is the interface between the blood and the CNS and as such is a critical mediator of neuro-immune reactions and an important therapeutic target to modulate neuroinflammation. It is essential to have a better understanding of the molecular mechanisms that regulate the BBB properties to elaborate new therapeutic strategies to modulate the BBB and thus the local neuroinflammation reaction. This Ph.D. thesis describes three distinct molecular mechanisms which regulate key BBB properties. The first section describes a novel role for the renin-angiotensin system (RAS) in the neuro-vascular unit (NVU) as a regulator of paracellular permeability. The second part of this thesis characterises the role of a novel adhesion molecule of the BBB, ALCAM. The third part of this work studies the interactions between neural stem cells (NSC) and the BBB and identifies MCP-1 as a critical factor involved in NSC recruitment to the CNS. In the first experimental section we provide evidence that angiotensinogen (AGT) produced and secreted by astrocytes, is cleaved into angiotensin II (AngII) and acts on type 1 angiotensin receptors (AT1) expressed by BBB endothelial cells (ECs). Activation of AT1 restricts the passage of molecular tracers across human BBB-derived ECs through threonine-phosphorylation of the tight junction protein occludin and its mobilization to lipid raft membrane microdomains. We also show that AGT knockout animals have disorganized occludin strands at the level of the BBB and a diffuse accumulation of the endogenous serum protein plasminogen in the CNS, as compared to wild type animals. Finally, we demonstrate a reduction in the number of AGT-immunopositive perivascular astrocytes in multiple sclerosis (MS) lesions, which correlates with a reduced expression of occludin similarly seen in the CNS of AGT knockout animals. Such a reduction in astrocyte-expressed AGT and AngII is dependent, in vitro, on the pro-inflammatory cytokines tumor necrosis factor-α and interferon-γ. Our study defines a novel physiological role for AngII in the CNS and suggests that inflammation-induced downregulation of AngII production by astrocytes is involved in BBB dysfunction in MS lesions. In the second experimental part we focus on adhesion molecules of the BBB. Using a lipid raft-based proteomic approach, we identified ALCAM (Activated leukocyte cell adhesion molecule) as an adhesion molecule involved in leukocyte migration across the BBB. ALCAM expressed on BBB endothelium co-localized with CD6 expressed on leukocytes and with BBB endothelium transmigratory cups. ALCAM expression on BBB cells was up-regulated in active multiple sclerosis and experimental auto-immune encephalomyelitis (EAE) lesions. Moreover, ALCAM blockade restricted transmigration of CD4+ lymphocytes and monocytes across BBB endothelium in vitro and in vivo, and reduced the severity and time of onset of EAE. Our findings point to an important role for ALCAM in leukocyte recruitment into the brain and identify ALCAM as a potential therapeutic target to dampen neuroinflammation. The third experimental part of this thesis studies the interactions between NCS and BBB. NCS represent an attractive source for cell transplantation and neural tissue repair. After systemic injection, NCS are confronted with the specialized BBB endothelial cells before they can enter the brain parenchyma. We investigated the interactions of human fetal neural precursor cells with human brain endothelial cells in an in vitro model using primary cultures. We demonstrated that human fetal neural precursor cells efficiently and specifically migrate to sub-endothelial space of human BBB-endothelium, but not pulmonary artery endothelial cells. When migrated across BBB-endothelial cells, fetal neural precursor cells spontaneously differentiate to neurons, astrocytes and oligodendrocytes. Effective migration and subsequent differentiation was found to be dependant on the chemokine CCL2/MCP-1, but not CXCL8/IL-8. Our findings suggest that an intact blood-brain barrier is not an intrinsic obstacle to neural stem cell migration into the brain and that differentiation of neural precursor cells occur in a sub-endothelial niche, under the influence of the chemokine CCL2/MCP-1. These three experimental sections demonstrate the crucial roles that the BBB plays in regulating the CNS homeostasis. Under pathological conditions, such as during neuro-immune reactions, the BBB is altered and becomes an important local player. The three different molecular mechanisms described in this thesis, contribute to our understanding of the BBB and may allow for the development of novel therapeutic strategies to limit neuroinflammation.