Epidemiological studies report that infarct size is decreased and stroke outcomes are improved in young females when compared to males. However, mechanistic insight is lacking. We posit that sex-specific differences in glial cell functions occurring immediately after ischemic stroke are a source of dichotomous outcomes. In this study we assessed astrocyte Ca2+ dynamics, aquaporin 4 (AQP4) polarity, Sloop expression pattern, as well as, microglia morphology and phagocytic marker CD11b in male and female mice following 60 min of middle cerebral artery (MCA) occlusion. We reveal sex differences in the frequency of intracellular astrocyte Ca2+ elevations (F-(1,F-86) = 8.19, P = 0.005) and microglia volume (F-(1,F-40) = 12.47, P = 0.009) immediately following MCA occlusion in acute brain slices. Measured in fixed tissue, AQP4 polarity was disrupted (F-(5,F-86) = 3.30, P = 0.009) and the area of non-S100 beta immunoreactivity increased in ipsilateral brain regions after 60 min of MCA occlusion (F-(5,F-86) = 4.72, P = 0.007). However, astrocyte changes were robust in male mice when compared to females. Additional sex differences were discovered regarding microglia phagocytic receptor CD11b. In sham mice, constitutively high CD11b immunofluorescence was observed in females when compared to males (P = 0.03). When compared to sham, only male mice exhibited an increase in CD11b immunoreactivity after MCA occlusion (P = 0.006). We posit that a sex difference in the presence of constitutive CD11b has a role in determining male and female microglia phagocytic responses to ischemia. Taken together, these findings are critical to understanding potential sex differences in glial physiology as well as stroke pathobiology which are foundational for the development of future sex-specific stroke therapies. (C) 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

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Previous in vitro studies have demonstrated that AQP1, traditionally viewed as a water channel, also facilitates the passage of CO2 and ammonia across cell membranes. This thesis summarizes the first in vivo studies confirming a physiologically-relevant role for AQP1 in acid-base balance and nitrogenous waste excretion. Zebrafish embryos were microinjected with a translation-blocking morpholino oligonucleotide targeted to the zebrafish AQP1 paralog, AQP1a. Closed-system respirometry, total CO2 analysis, tritiated water fluxes and measurement of ammonia excretion were performed on larvae at 4 days post-fertilization (dpf). Knockdown of AQP1a significantly reduced rates of water, CO2 and ammonia excretion. Use of phenylhydrazine, a haemolytic agent, provided evidence that the yolk sac epithelium AQP1a (and not erythrocyte AQP1a) is the major site of CO2 and ammonia movements. Further, the hypothesis that AQP1a and the Rh glycoprotein Rhcg1, another multi-functional gas channel, act in concert to regulate CO2 and ammonia excretion was explored. Exposure to conditions impairing ammonia excretion (such as high external ammonia (HEA) or alkaline water) modulated AQP1a protein expression in 4 dpf zebrafish larvae experiencing knockdown of Rhcg1. Chronic HEA exposure triggered a significant compensatory increase in AQP1a protein abundance in Rhcg1 morphants. Exposure of Rhcg1 morphants to pH 10 water, however, caused a significant decrease in AQP1a protein expression. Interestingly, when AQP1a mRNA and protein levels were examined in Rhcg1 morphants and vice versa, no changes were observed. Overall, zebrafish AQP1a was found to be a multi-functional transporter of water, CO2 and ammonia, though the exact relationship it holds with other such gas channels bears further exploration.

Previous in vitro studies have demonstrated that AQP1, traditionally viewed as a water channel, also facilitates the passage of CO2 and ammonia across cell membranes. This thesis summarizes the first in vivo studies confirming a physiologically-relevant role for AQP1 in acid-base balance and nitrogenous waste excretion. Zebrafish embryos were microinjected with a translation-blocking morpholino oligonucleotide targeted to the zebrafish AQP1 paralog, AQP1a. Closed-system respirometry, total CO2 analysis, tritiated water fluxes and measurement of ammonia excretion were performed on larvae at 4 days post-fertilization (dpf). Knockdown of AQP1a significantly reduced rates of water, CO2 and ammonia excretion. Use of phenylhydrazine, a haemolytic agent, provided evidence that the yolk sac epithelium AQP1a (and not erythrocyte AQP1a) is the major site of CO2 and ammonia movements. Further, the hypothesis that AQP1a and the Rh glycoprotein Rhcg1, another multi-functional gas channel, act in concert to regulate CO2 and ammonia excretion was explored. Exposure to conditions impairing ammonia excretion (such as high external ammonia (HEA) or alkaline water) modulated AQP1a protein expression in 4 dpf zebrafish larvae experiencing knockdown of Rhcg1. Chronic HEA exposure triggered a significant compensatory increase in AQP1a protein abundance in Rhcg1 morphants. Exposure of Rhcg1 morphants to pH 10 water, however, caused a significant decrease in AQP1a protein expression. Interestingly, when AQP1a mRNA and protein levels were examined in Rhcg1 morphants and vice versa, no changes were observed. Overall, zebrafish AQP1a was found to be a multi-functional transporter of water, CO2 and ammonia, though the exact relationship it holds with other such gas channels bears further exploration.

The glial-lymphatic or glymphatic fluid clearance system promotes the exchange of interstitial fluid (ISF) and cerebrospinal fluid through the arterial perivascular spaces into the brain. This process is facilitated in part by aquaporin-4 (AQP4) water channels located primarily on astrocyte end feet abutting endothelial cells of the blood brain barrier. Changes in expression levels or mislocalization of AQP4 from astrocytic end feet to the soma can lead to decreased ISF flow leading to buildup of extracellular waste products like hyperphosphorylated Tau (pTau). pTau accumulation is a neuropathological hallmark in Alzheimer’s disease (AD) and in some people with human immunodeficiency virus (HIV). Approximately 50% of people with HIV (PWH) suffer from HIV-associated neurocognitive disorders (HAND), which is a spectrum disorder linked to cognitive and motor decline in PWH. Limited studies have shown that in HIV CNS infection that expression levels of AQP4 in brain homogenates from the mid-frontal gyrus of PWH with symptomatic HAND were significantly increased compared to those with asymptomatic HAND, which raises the question if AQP4 function and subcellular localization may contribute to cognitive status. Studies in other neuroinflammatory diseases have shown dysregulation of AQP4 through the adenosine A2aR (A2aR) signaling. A2aR activation leads to PKA/PKC-mediated inhibitory phosphorylation of AQP4 (Ser180, Ser276) that is proposed to contribute to channel internalization, mislocalization and decreased expression. In addition, common single nucleotide polymorphisms in aqp4 have been associated with more rapid cognitive decline some neurodegenerative diseases. Therefore, it is possible that common mutations in aqp4, subcellular mislocalization, dysfunction, expression levels or post-translational modifications contribute to HAND. Therefore, the cognitive changes we see in HAND maybe due to changes in AQP4 may contribute by decreasing clearance of toxic aberrant proteins and HIV mechanistically alters AQP4 in part via dysregulation of A2aR. / Biomedical Sciences

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En tant qu’acteurs moléculaires impliqués dans le transport d’eau au travers des membranes biologiques, les aquaporines (AQP) jouent un rôle et sont régulées en réponse à des stress. Ils sont soumis à d’intenses recherches, en particulier chez le riz. En utilisant une approche de génomique fonctionnelle, nous avons généré 5 lignées transgéniques de riz dans le fond génétique Nipponbare, en sur-exprimant des AQP uniques fusionnées à un marqueur fluorescent ; parmi celles-là, figurent trois AQP de la membrane plasmique (OsPIP1;1, OsPIP2;4, OsPIP2;5) fusionnées à la GFP et deux AQP de la membrane tonoplastique (OsTIP1;1 and OsTIP2;2) fusionnées à la mCherry. Leurs localisations subcellulaires en condition contrôle ont été observées. Chez le riz, les isoformes OsPIP présentaient un marquage homogène typique de la membrane plasmique, tandis que les isoformes OsTIP ont été trouvées avec un marquage des invaginations intracellulaires qui entourent le noyau, typique du tonoplaste. Le comportement des AQP de la membrane plasmique a été testé en conditions de stress salin et osmotique. Les stress abiotiques ont provoqué une relocalisation des AQP et le stress salin a augmenté l’endocytose de l’isoforme OsPIP2;5 dans les cellules de la racine. Par ailleurs, la sur-expression de tels transgènes ne semblait pas affecter la morphologie des plantes et ne conférait pas un effet bénéfique sur la production de graines, aussi bien en condition contrôle que stressée. Enfin, nous nous sommes focalisés sur la contribution des AQP dans la racine de riz en relation avec la morphologie racinaire. Nous avons trouvé que les AQP contribuaient à un pourcentage relativement important dans le transport de l’eau dans la racine entière (44-58%) et que cette contribution semblait plus importante dans les racines primaires que latérales. / As molecular players involved into the water transport through biological membranes, aquaporins (AQPs) have a role and are regulated in stress response. They were deeply investigated in plants and particularly in rice. Using functional genetic approach, we generated 5 transgenic rice lines based on Nipponbare cultivar, by overexpressing a single AQP in fusion with a fluorescent marker; among them, 3 plasma membrane AQPs (OsPIP1;1, OsPIP2;4, OsPIP2;5) fused with GFP and 2 tonoplast AQPs (OsTIP1;1 and OsTIP2;2) fused with mCherry. Their subcellular localizations in resting condition were investigated. In rice, OsPIP isoforms showed typical homogeneous labelling of the plasma membrane, whereas OsTIP isoforms were observed localized in the tonoplast with a typical labelling of intracellular invaginations that skirted the nucleus. The behaviors of plasma membrane AQPs were tested in salt and drought stress-mimicked-conditions. Abiotic stresses triggered a re-localization of plasma membrane AQPs and salt stress enhanced endocytosis process of OsPIP2;5 in rice root cells. Overexpressing such transgenes did not seem to affect the plant morphology and showed no beneficial effects for grain yield in both non-stress and stress conditions. We took more focus on the contribution of AQPs in rice root water transport in link with root morphology. AQPs contributed a relative high percentage of water transport in whole root system (44-58%) and seemed to contribute more in primary roots rather than in lateral roots.

Introduction. The glial cell water channel aquaporin-4 (AQP4) plays an important ro le in brain oedema, astrocyte migration and neuronal excitability. Current theories of AQP4 function are based largely on experiments using AQP4 -1- mice. These mice have only been partially characterized. I therefore undertook a detailed investigation of baseline brain properties in AQP4 -1- mice. In the second part of my experiments I investigated the role of AQP4 in brain oedema in a mouse model of subarachnoid haemorrhage. Method. Gross anatomical measurements included estimates of brain and ventricle size. Neurons, astrocytes and oligodendrocytes were assessed using the neuronal nuclear marker NeuN, the astrocyte marker GFAP, and the myelin stain Luxol Fast Blue. The blood brain barrier was studied by electron microscopy and the horseradish peroxidase extravasation technique. A mouse model in which 30~1 of autologous blood was injected into the basal cisterns was used to reproduce subarachnoid haemorrhage. Brain water content, intracranial pressure and neurological score were compared in wildtype and AQP4 -/- mice. I also measured blood brain barrier permeability and the osmotic permeability of the glia lim itans, one of the routes of oedema elimination.

O nematoide de vida livre Panagrolaimus superbus é uma espécie anidrobiótica, ou seja, possui a capacidade de sobreviver ao estresse hídrico extremo adentrando no estado de anidrobiose. Durante tal estado adquiri tolerância a extremos de temperatura (~0 K a +151 °C), pressões hidrostáticas (1,2 GPa) e radiação ionizante. Entretanto, não se sabe qual a tolerância a água deuterada, molécula que possui dois átomos de deutério (isótopo estável e natural do hidrogênio) ao invés do hidrogênio, que em altas concentrações afeta negativamente sistemas biológicos. Além disso, uma vez que o processo de anidrobiose depende do movimento de moléculas de água pela membrana plasmática da célula, não se sabe se os canais responsáveis por este transporte, as aquaporinas, de espécies anidrobióticas possuem alguma particularidade na sua estrutura. Em vista disso, o objetivo deste trabalho visa investigar se em concentrações de 10%, 40% e 99,9% D2O, tanto de modo crônico quanto agudo, P. superbus tem sua viabilidade, crescimento populacional e desenvolvimento alterados. Além disso, a comparação in silico da sequência de aminoácidos (estrutura primária) entre aquaporinas de espécies anidrobióticas e não anidrobióticas, com ênfase nas sequências genéticas de P. superbus foi feita. Os resultados encontrados demonstram a alta tolerância de P. superbus a concentrações relativamente elevadas de D2O, não tendo sua viabilidade e desenvolvimento alterados em nenhum cenário, mesmo com uma troca total, homogênea e instantânea de sua matriz aquosa. Efeitos negativos foram encontrados apenas no crescimento populacional após exposição 10%, 40% e 99,9% D2O, contudo não o inviabilizaram. Ademais, não foram encontradas grandes diferenças entre as sequências primárias de aquaporinas de anidrobiotos e não anidrobiotos, sugerindo que estes canais de água não divergem em estrutura terciária entre tais grupos. Dos dois ESTs encontrados em P. superbus (números de acesso no NCBI: GW411914.1 e GW408200.1) o primeiro deles é o provável representante do gene da aquaporina na espécie, enquanto que o segundo aparenta ser um transcrito não codificante de proteínas. / The free-living nematode Panagrolaimus superbus is an anhydrobiotic species, it means that this species has the capacity to survive extreme water stress entering into the state of anhydrobiosis. During such a state, it acquires tolerance to extremes of temperature (~ 0 K to +151 °C), hydrostatic pressures (1.2 GPa) and ionizing radiation. However, the tolerance to deuterium oxide is poorly investigated. This molecule has two atoms of deuterium (natural and stable isotope of hydrogen) rather than hydrogen and in high concentrations negatively affects biological systems. Furthermore, since the process of anhydrobiosis depends on the movement of water molecules across the cell membrane, it is unclear whether the channels responsible for this transport, aquaporins, of anhydrobiotic species have some particularity in their structures. In view of this, the work aims to investigate whether P. superbus has its viability, population growth and development altered at concentrations of 10%, 40% and 99.9% D2O in chronic and acute expositions. In addition, the in silico analyses of amino acid sequence (primary structure) of aquaporins between anhydrobiotic and non-anhydrobiotic species, with emphasis at the P. superbus genetic sequences, were performed. The results demonstrated the high tolerance of P. superbus at high concentrations of D2O, their viability and development did not change in any scenario, even with a total, homogeneous and instantaneous exchange of their aqueous milieu. Negative effects were found only on population growth after exposure to 10%, 40% and 99.9% D2O, although not hindering the procedure. Furthermore, no significant differences were found between the primary aquaporin sequences of anhydrobiotic and non-anhydrobiotic species, suggesting that these water channels do not differ in tertiary structure between such groups. Two ESTs found in P. superbus, (NCBI access numbers: GW411914.1 and GW408200.1): the first likely corresponds to the aquaporin gene in the species, while the second appears to be a noncoding transcript.