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.

No description available.

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

No description available.

No description available.

Lung cancer is the leading cause of cancer-related deaths worldwide. One of the most common genetic aberrations in lung cancer patients is Kirsten rat sarcoma viral oncogene homolog (KRAS). The KRAS protein is a Ras superfamily GTPase that switches between an active GTP-bound form and an inactive GDP-bound form. The consequence of KRAS mutations results in constitutively active downstream pathways involved in uncontrolled cell proliferation and survival. Fortunately, there has been a recent development of KRASG12C inhibitors that directly target mutant KRAS, thereby arresting its proliferative effects. A recently FDA-approved KRASG12C inhibitor for the treatment of non-small cell lung cancer, Sotorasib (AMG 510), has been shown to produce insubstantial clinical response rates and a short duration of response. Similar to other targeted therapies, the limitations of this treatment are primarily due to the emergence of drug resistance. Drug resistance has been studied extensively regarding other anticancer treatments; however, the underlying molecular mechanisms remain poorly characterized. Our investigation begins by establishing and analyzing a subpopulation of cancer cells that evolve and mediate drug resistance, known as drug-tolerant persister cells (DTPCs), in KRASG12C mutant cells using AMG 510. First, we observed the reactivation of a pro-proliferative kinase, ERK, in AMG 510 DTPCs. Additionally, whole transcriptomics analysis, RT-qPCR, and immunofluorescent staining demonstrated significant upregulation of AQP4 in AMG 510 DTPCs compared to drug sensitive cells (DSCs). Aquaporin 4 (AQP4) is a water-selective transmembrane protein that regulates fluid homeostasis in many organ systems, including the lungs, and is involved in intracellular calcium signaling. We aim to explore the connections between AQP4, ERK, and calcium signaling in promoting drug tolerance to AMG 510. The insights gained from this research could lead to improved targeted therapies and clinical outcomes by identifying AQP4 as a resistance-driving biomarker.

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.

The response of cells to their environment is driven by a variety of proteins and messenger molecules. In eukaryotes, their distribution and location in the cell are regulated by the vesicular transport system. The transport of aquaporin 2 between membrane and storage region is a crucial part of the water reabsorption in renal principal cells, and its malfunction can lead to Diabetes insipidus. To understand the regulation of this system, we aggregated pathways and mechanisms from literature and derived three models in a hypothesis-driven approach. Furthermore, we combined the models to a single system to gain insight into key regulatory mechanisms of Aquaporin 2 recycling. To achieve this, we developed a multiscale computational framework for the modeling and simulation of cellular systems. The analysis of the system rationalizes that the compartmentalization of cAMP in renal principal cells is a result of the protein kinase A signalosome and can only occur if specific cellular components are observed in conjunction. Endocytotic and exocytotic processes are inherently connected and can be regulated by the same protein kinase A signal.