O açaí é uma fruta da amazônia brasileira de consumo emergente no Brasil e outras regiões do mundo. Primordialmente consumida na sua forma de polpa e estabelecida como uma \"superfruta\" graças ao seu potencial antioxidante e anti-inflamatório. No entanto, pouco se sabe sobre sua composição em relação aos elementos químicos essenciais. No presente estudo, a análise sistemática de 12 polpas produzidas com frutos de diferentes localidades demonstrou que a polpa de açaí é naturalmente rica nos elementos Ca, Cu, Fe, Mg, Zn, com destaque para as concentrações encontradas de Mn que foram notoriamente maiores do que as comumente observadas em alimentos considerados como fonte principal deste elemento químico essencial na dieta. Cabe destacar que em valores médios, todos os elementos químicos estudados no açaí apresentaram-se em concentrações que contribuem significativamente para as suas necessidades diárias preconizadas. Entretanto, a análise do fracionamento químico da polpa de açaí demonstrou que o ferro está em uma forma potencialmente indisponível; enquanto que cerca de 40% dos elementos cálcio, magnésio, manganês e zinco estão quelados a um composto não fenólico de alta solubilidade em água e que pode aumentar suas biodisponibilidades. Diferentemente dos demais, o cobre interagiu significativamente com a fração fenólica solúvel em água do alimento. A fração fenólica eluída com metanol por SPE demonstrou ter considerável atividade antioxidante direta no ensaio de sequestro de DPPH com EC50 de 19,1 ?g/L e foi eficaz na atenuação da citotoxicidade, estresse oxidativo e alteração funcional induzidos por 500 ?M de MnCl2 em cultura primária de astrócitos, avaliados pelos ensaios de LDH e MTT, GSH/GSSG, F2-IsoPs, captação de glutamato e expressão de Nrf2. Este extrato de açaí em uma concentração ótima de 0,1 ?g/mL preveniu o estresse oxidativo induzido por Mn restaurando a razão GSH/GSSG, protegendo as membranas astrocitárias da lipoperoxidação e diminuindo a expressão de Nrf2. Uma concentração mais elevada de extrato de açaí exacerbou os efeitos do Mn nestes mesmos parâmetros, exceto na lipoperoxidação medida pela formação de F2-IsoPs. Assim, o pré-tratamento dos astrócitos com concentrações mais elevadas destas antocianinas com exposição ao Mn em sequência, pode predispor os astrócitos fazendo com que um efeito pró-oxidante prevaleça. Estes achados devem ser considerados em estudos futuros que explorem a potencialidade das antocianinas do açaí em formulações nutracêuticas para obtenção de efeitos antioxidantes e neuroprotetores ideais. Assim, conclui-se que o próprio açaí possui uma fração de compostos fenólicos capaz de atenuar os efeitos oxidativos do Mn e que portanto, pode indicar uma segurança alimentar do consumo do fruto em quantidades moderadas. / Açaí is a fruit from Brazilian Amazon with an exotic flavor possessing high antioxidant and anti-inflammatory properties. Based on these properties, the fruit is classified as one of the new \"super fruits\". However, few is known about its essential chemical elements content. In this study, the systematic analysis of 12 freeze-dried pulps processed with açaí of different locations showed that açaí pulp is naturally rich in Ca, Cu, Fe, Mg, Zn, and especially amounts of Mn, which was significantly greater than in foods considered Mn dietary source, were found. In average values, all determined essential chemical elements contribute significantly to their recommended daily intake requirements. However, fractionation analysis of açai showed that iron is potentially not bioavailable. While about 40% of calcium, magnesium, manganese, and zinc are binding to a non-phenolic compound high soluble in water, which may increase their bioavailabilities. Copper is significantly bound to water-soluble phenolic fraction in açaí. This phenolic fraction eluted with methanol by SPE provided considerable direct antioxidant activity on the DPPH scavenging assay with EC50 of 19.1 ?g/L and was effective attenuating cytotoxicity, oxidative stress, and functional alterations induced by 500 ?M of MnCl2 in primary cultured astrocytes, which was assessed by MTT and LDH assay, GSH/GSSG ratio, F2-IsoPs formation, glutamate uptake, and Nrf2 levels. This anthocyanin-rich açaí extract in an optimal concentration of 0.1 ?g/mL prevented oxidative stress induced by Mn restoring the GSH/GSSG ratio, protecting the astrocytic membrane from lipid peroxidation and decreasing levels of Nrf2. A higher concentration of the same açaí extract exacerbated the effects of Mn in these same parameters, except on lipid peroxidation assessed by F2-IsoPs. In constrast, pre-treating with high concentrations of açaí\'s anthocyanins followed by Mn exposure, pro-oxidant effects likely prevails. These findings should be considered in future studies regarding the potential of anthocyanins in açaí on nutraceutical formulations to obtain optimal antioxidant and neuroprotective effects. Thus, we conclude that the own açaí has a phenolic fraction capable of attenuates the oxidative effects of Mn. So, may indicates that moderate açaí consumption is dietary safe regarding Mn neurotoxicity.

In Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), aggregated α-synuclein deposit inside cells within the brain. Smaller soluble α-synuclein aggregates, oligomers, are present both intra- and extracellularly. The α-synuclein oligomers are known to be particularly harmful, although the underlying neurotoxic mechanisms are not fully understood. The aim of this thesis was to investigate the pathogenic roles of α-synuclein oligomers and the possibility to target such species with antibody treatment. Passive immunotherapy with α-synuclein antibodies can lead to reduced pathology and ameliorated symptoms in transgenic mice. However, it remains unknown whether the antibodies are taken up by cells or whether they act extracellularly. In Paper I, we assessed cellular internalization of various α-synuclein monoclonal antibodies. The oligomer selective mAb47 displayed the highest uptake, which was promoted by the extracellular presence of α-synuclein. Alpha-synuclein aggregates can be found in both neurons and glial cells, but the pathogenic role of glial deposits has only been sparsely investigated. In Paper II, co-cultures of neurons and glia were exposed to α-synuclein oligomers. The astrocytes in the cultures rapidly accumulated oligomers, which were only partially degraded by lysosomes. The sustained intracellular α-synuclein deposits were associated with mitochondrial stress reactions in the astrocytes.  In Paper III, we sought to explore whether the astrocytic pathology induced by α-synuclein oligomers could be ameliorated by antibody treatment. Pre-incubation of oligomers with mAb47 promoted α-synuclein clearance, reduced astrocytic accumulation and rescued cells from mitochondrial stress. We could demonstrate that binding of the antibody to its antigen in the extracellular space was crucial for these effects to occur. The progressive pathology in PD is believed to be driven by cell-to-cell spreading of α-synuclein aggregates, potentially via exosomes and other extracellular vesicles (EVs). In Paper IV, we found that either fusing α-synuclein to a non-physiological protein tag or introducing the PD-causing A53T mutation directed α-synuclein towards EV secretion. Also, EV-associated α-synuclein was particularly prone to induce toxicity in recipient cells. In conclusion, this thesis sheds new light on the cellular dysfunction related to α-synuclein pathology and on how the underlying pathogenic processes may be targeted by antibody treatment.

Cannabinoids have been shown to alter synaptic plasticity but the mechanism by which this occurs at hippocampal CA3-CA1 synapses in vivo is not yet known. Utilizing in vivo electrophysiological recordings of field excitatory postsynaptic potentials (fEPSP) on anesthetized rats and mice as well as three lines of conditional knockout mouse models, the objective was to show a two-part mechanistic breakdown of cannabinoid-evoked CA3-CA1 long-term depression (LTD) in its induction as well as early and later-phase expression stages. It was determined that this cannabinoid-induced in vivo LTD requires cannabinoid type-1 receptors (CB1Rs) on astrocytes, but not CB1Rs on glutamatergic or GABAergic neuronal axons/terminals. Pharmacological testing determined that cannabinoid-induced in vivo LTD also requires activation of NMDA receptors (NMDAR) and subsequent postsynaptic endocytosis of AMPA receptors (AMPAR). There exists a clear role for NR2B-containing NMDARs in a persistent, transitory form, potentially related to prolonged or delayed glutamate release (possibly as a result of the astrocytic network). A key determination of the expression phase is the involvement of new protein synthesis (using translation and transcription inhibitors) – further evidence of the long-term action of the synaptic plasticity from a single cannabinoid dose.

Astrocytes are emerging as important regulators of neural circuit function and behavior in the healthy and diseased nervous system. In a screen for astrocyte molecules that modulate neuronal hyperexcitability we identified multiple components of focal adhesion complexes (FAs) as potent suppressors of genetically- or pharmacologically-induced seizure-like activity. Depletion of astrocytic Tensin, b-integrin, Talin, Focal adhesion kinase (FAK), or matrix metalloproteinase 1 (Mmp1), which degrades extracellular matrix to activate b-integrin receptors, resulted in enhanced recovery from, or resistance to seizure activity. Reciprocally, promoting FA signaling by overexpression of Mmp1 in astrocytes led to enhanced-seizure severity. Blockade of FA signaling in astrocytes led to reduced-astrocytic coverage of the synaptic neuropil and reduced expression of the excitatory amino acid transporter EAAT1. However, upon seizure induction, depletion of FA signaling components resulted in enhanced astrocyte coverage of the synaptic neuropil and a ~2-fold increase in EAAT1 levels compared to controls. Our data indicate that FAs promote astrocyte coverage in neuropil and EAAT1 expression under normal physiological conditions, but in the context of hyperexcitability, FAs negatively regulate the extent of astrocytic processes within neuropil and EAAT1 expression, thereby inhibiting a more rapid recovery from conditions of excessive neuronal activity.

No description available.

No description available.

<p>Human pluripotent stem cells (hPSCs) are an attractive tool for the study of different neurodegenerative diseases due to their potential to form any cell type of the body. Due to their versatility and self-renewal capacity, they have different applications such as disease modeling, high throughput drug screening and transplantation. Different animal models have helped answer broader questions related to the physiological functioning of various pathways and the phenotypic effects of a particular neurodegenerative disease. However, due to the lack of success recapitulating some targets identified from animal models into successful clinical trials, there is a need for a direct translational disease model. Since their advent, hPSCs have helped understand various disease effectors and underlying mechanisms using genetic engineering techniques, omics studies and reductionist approaches for the recognition of candidate molecules or pathways required to answer questions related to neurodevelopment, neurodegeneration and neuroregeneration. Due to the simplified approach that iPSC models can provide, some <em>in vitro</em> approaches are being developed using microphysiological systems (MPS) that could answer complex physiological questions. MPS encompass all the different <em>in vitro</em> systems that could help better mimic certain physiological systems that tend to not be mimicked by <em>in vivo</em> models. In this dissertation, efforts have been directed to disease model as well as to understand the intrinsic as well as extrinsic cues using two different MPS. First, we have used hPSCs with Alzheimer’s disease (AD)-related mutations to differentiate into retinal organoids and identify AD related phenotypes for future studies to identify retinal AD biomarkers. Using 5 month old retinal organoids from AD cell lines as well as controls, we could identify retinal AD phenotypes such as an increase in Aβ42:Aβ40 ratio along with increase in pTau:Tau. Nanostring analyses also helped in identification of potential target genes that are modulated in retinal AD that were related to synaptic dysfunction.  Thus, using retinal organoids for the identification of retinal AD phenotypes could help delve deeper into the identification of future potential biomarkers in the retina of AD patients, with the potential to serve as a means for early identification and intervention for patients. The next MPS we used to serve to explore non-cell autonomous effects associated with glaucoma to explore the neurovascular unit. Previous studies have demonstrated the degeneration of RGCs in glaucoma due to a point mutation OPTN(E50K) that leads to the degeneration of RGCs both at morphological and functional levels. Thus, using the previous studies as a basis, we wanted to further unravel the impact of this mutation using the different cell types of the neurovascular unit such as endothelial cells, astrocytes and RGCs. Interestingly, we observed the barrier properties being impacted by the mutation present in both RGCs and astrocytes demonstrated through TEER, permeability and transcellular transport changes. We also identified a potential factor TGFβ2 that was observed to be overproduced by the OPTN E50K astrocytes to demonstrate similar effects with the exogenous addition of TGFβ2 on the barrier. Furthermore, the inhibition of TGFβ2 helped rescue some of the barrier dysfunction phenotypes. Thus, TGFβ2 inhibition can be used as a potential candidate that can be used to further study its impact in <em>in vivo</em> models and how that can be used in translational applications. Thus, MPS systems have a lot of applications that can help answer different physiologically relevant questions that are hard to approach using <em>in vivo</em> models and the further development of these systems to accentuate the aspects of neural development and how it goes awry in different neurodegenerative diseases.  </p>

No description available.

La barrière hémato-encéphalique (BHE) protège le système nerveux central (SNC) en contrôlant le passage des substances sanguines et des cellules immunitaires. La BHE est formée de cellules endothéliales liées ensemble par des jonctions serrées et ses fonctions sont maintenues par des astrocytes, celles ci sécrétant un nombre de facteurs essentiels. Une analyse protéomique de radeaux lipidiques de cellules endothéliales de la BHE humaine a identifié la présence de la voie de signalisation Hedgehog (Hh), une voie souvent liées à des processus de développement embryologique ainsi qu’au niveau des tissus adultes. Suite à nos expériences, j’ai déterminé que les astrocytes produisent et secrètent le ligand Sonic Hh (Shh) et que les cellules endothéliales humaines en cultures primaires expriment le récepteur Patched (Ptch)-1, le co-récepteur Smoothened (Smo) et le facteur de transcription Gli-1. De plus, l’activation de la voie Hh augmente l’étanchéité des cellules endothéliales de la BHE in vitro. Le blocage de l’activation de la voie Hh en utilisant l’antagoniste cyclopamine ainsi qu’en utilisant des souris Shh déficientes (-/-) diminue l’expression des protéines de jonctions serrées, claudin-5, occcludin, et ZO-1. La voie de signalisation s’est aussi montrée comme étant immunomodulatoire, puisque l’activation de la voie dans les cellules endothéliales de la BHE diminue l’expression de surface des molécules d’adhésion ICAM-1 et VCAM-1, ainsi que la sécrétion des chimiokines pro-inflammatoires IL-8/CXCL8 et MCP-1/CCL2, créant une diminution de la migration des lymphocytes CD4+ à travers une monocouche de cellules endothéliales de la BHE. Des traitements avec des cytokines pro-inflammatoires TNF-α and IFN-γ in vitro, augmente la production de Shh par les astrocytes ainsi que l’expression de surface de Ptch-1 et de Smo. Dans des lésions actives de la sclérose en plaques (SEP), où la BHE est plus perméable, les astrocytes hypertrophiques augmentent leur expression de Shh. Par contre, les cellules endothéliales de la BHE n’augmentent pas leur expression de Ptch-1 ou Smo, suggérant une dysfonction dans la voie de signalisation Hh. Ces résultats montrent que la voie de signalisation Hh promeut les propriétés de la BHE, et qu’un environnement d’inflammation pourrait potentiellement dérégler la BHE en affectant la voie de signalisation Hh des cellules endothéliales. / The blood-brain barrier (BBB), composed of tightly bound endothelial cells (ECs), regulates the entry of blood-borne molecules and immune cells into the CNS. Recent studies indicate that the Hedgehog (Hh) signaling pathway in adult tissues plays an important role in vascular proliferation, differentiation and tissue repair. Using a lipid membrane raft-based proteomic approach, I have identified the Hedgehog (Hh) pathway as a signaling cascade involved in preserving and upkeeping BBB functions. My study shows that human astrocytes express and secrete Sonic Hh (Shh) and conversely, that human BBB-ECs bear the Hh receptor Patched-1 (Ptch-1), the signal transducer Smoothened (Smo) as well as transcription factors of the Gli family. Furthermore, activation of the Hh pathway in BBB-ECs restricts the passage of soluble tracers in vitro. By blocking the Hh signaling in vitro and by using Shh knock-out (-/-) embryonic mice, I demonstrate a reduced expression of TJ molecules claudin-5, occludin and ZO-1. Hh activation also decreases the surface expression of cell adhesion molecules ICAM-1 and VCAM-1, and decreases BBB-ECs secretion of pro-inflammatory chemokines IL-8/CXCL8 and monocytes chemoattractant protein 1 MCP-1/CCL2, resulting in a reduction of migrating CD4+ lymphocytes across human BBB-EC monolayers. In vitro treatment with inflammatory cytokines TNF-α and IFN-γ, upregulates the production of astrocytic Shh and the BBB-EC surface expression of Ptch-1 and Smo. In active Multiple Sclerosis (MS) lesions, in which the BBB is disrupted, Shh expression is drastically upregulated in hypertrophic astrocytes, while Ptch-1 and Smo expression is down-regulated or left unchanged, suggesting that a deregulation in the Hh signaling pathway may prevent the barrier stabilizing properties of Hh. Our data demonstrate an anti-inflammatory and BBB-promoting effect of astrocyte-secreted Hh and suggest that a pro-inflammatory environment disrupt the BBB by impacting, at least in part, on Hh signaling in brain ECs.

La barrière hémato-encéphalique (BHE) protège le système nerveux central (SNC) en contrôlant le passage des substances sanguines et des cellules immunitaires. La BHE est formée de cellules endothéliales liées ensemble par des jonctions serrées et ses fonctions sont maintenues par des astrocytes, celles ci sécrétant un nombre de facteurs essentiels. Une analyse protéomique de radeaux lipidiques de cellules endothéliales de la BHE humaine a identifié la présence de la voie de signalisation Hedgehog (Hh), une voie souvent liées à des processus de développement embryologique ainsi qu’au niveau des tissus adultes. Suite à nos expériences, j’ai déterminé que les astrocytes produisent et secrètent le ligand Sonic Hh (Shh) et que les cellules endothéliales humaines en cultures primaires expriment le récepteur Patched (Ptch)-1, le co-récepteur Smoothened (Smo) et le facteur de transcription Gli-1. De plus, l’activation de la voie Hh augmente l’étanchéité des cellules endothéliales de la BHE in vitro. Le blocage de l’activation de la voie Hh en utilisant l’antagoniste cyclopamine ainsi qu’en utilisant des souris Shh déficientes (-/-) diminue l’expression des protéines de jonctions serrées, claudin-5, occcludin, et ZO-1. La voie de signalisation s’est aussi montrée comme étant immunomodulatoire, puisque l’activation de la voie dans les cellules endothéliales de la BHE diminue l’expression de surface des molécules d’adhésion ICAM-1 et VCAM-1, ainsi que la sécrétion des chimiokines pro-inflammatoires IL-8/CXCL8 et MCP-1/CCL2, créant une diminution de la migration des lymphocytes CD4+ à travers une monocouche de cellules endothéliales de la BHE. Des traitements avec des cytokines pro-inflammatoires TNF-α and IFN-γ in vitro, augmente la production de Shh par les astrocytes ainsi que l’expression de surface de Ptch-1 et de Smo. Dans des lésions actives de la sclérose en plaques (SEP), où la BHE est plus perméable, les astrocytes hypertrophiques augmentent leur expression de Shh. Par contre, les cellules endothéliales de la BHE n’augmentent pas leur expression de Ptch-1 ou Smo, suggérant une dysfonction dans la voie de signalisation Hh. Ces résultats montrent que la voie de signalisation Hh promeut les propriétés de la BHE, et qu’un environnement d’inflammation pourrait potentiellement dérégler la BHE en affectant la voie de signalisation Hh des cellules endothéliales. / The blood-brain barrier (BBB), composed of tightly bound endothelial cells (ECs), regulates the entry of blood-borne molecules and immune cells into the CNS. Recent studies indicate that the Hedgehog (Hh) signaling pathway in adult tissues plays an important role in vascular proliferation, differentiation and tissue repair. Using a lipid membrane raft-based proteomic approach, I have identified the Hedgehog (Hh) pathway as a signaling cascade involved in preserving and upkeeping BBB functions. My study shows that human astrocytes express and secrete Sonic Hh (Shh) and conversely, that human BBB-ECs bear the Hh receptor Patched-1 (Ptch-1), the signal transducer Smoothened (Smo) as well as transcription factors of the Gli family. Furthermore, activation of the Hh pathway in BBB-ECs restricts the passage of soluble tracers in vitro. By blocking the Hh signaling in vitro and by using Shh knock-out (-/-) embryonic mice, I demonstrate a reduced expression of TJ molecules claudin-5, occludin and ZO-1. Hh activation also decreases the surface expression of cell adhesion molecules ICAM-1 and VCAM-1, and decreases BBB-ECs secretion of pro-inflammatory chemokines IL-8/CXCL8 and monocytes chemoattractant protein 1 MCP-1/CCL2, resulting in a reduction of migrating CD4+ lymphocytes across human BBB-EC monolayers. In vitro treatment with inflammatory cytokines TNF-α and IFN-γ, upregulates the production of astrocytic Shh and the BBB-EC surface expression of Ptch-1 and Smo. In active Multiple Sclerosis (MS) lesions, in which the BBB is disrupted, Shh expression is drastically upregulated in hypertrophic astrocytes, while Ptch-1 and Smo expression is down-regulated or left unchanged, suggesting that a deregulation in the Hh signaling pathway may prevent the barrier stabilizing properties of Hh. Our data demonstrate an anti-inflammatory and BBB-promoting effect of astrocyte-secreted Hh and suggest that a pro-inflammatory environment disrupt the BBB by impacting, at least in part, on Hh signaling in brain ECs.