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1
Lichterfeld, Yannick, Laura Kalinski, Sarah Schunk, et al. "Hypergravity Attenuates Reactivity in Primary Murine Astrocytes." Biomedicines 10, no. 8 (2022): 1966. http://dx.doi.org/10.3390/biomedicines10081966.
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Neuronal activity is the key modulator of nearly every aspect of behavior, affecting cognition, learning, and memory as well as motion. Hence, disturbances of the transmission of synaptic signals are the main cause of many neurological disorders. Lesions to nervous tissues are associated with phenotypic changes mediated by astrocytes becoming reactive. Reactive astrocytes form the basis of astrogliosis and glial scar formation. Astrocyte reactivity is often targeted to inhibit axon dystrophy and thus promote neuronal regeneration. Here, we aim to understand the impact of gravitational loading induced by hypergravity to potentially modify key features of astrocyte reactivity. We exposed primary murine astrocytes as a model system closely resembling the in vivo reactivity phenotype on custom-built centrifuges for cultivation as well as for live-cell imaging under hypergravity conditions in a physiological range (2g and 10g). We revealed spreading rates, migration velocities, and stellation to be diminished under 2g hypergravity. In contrast, proliferation and apoptosis rates were not affected. In particular, hypergravity attenuated reactivity induction. We observed cytoskeletal remodeling of actin filaments and microtubules under hypergravity. Hence, the reorganization of these key elements of cell structure demonstrates that fundamental mechanisms on shape and mobility of astrocytes are affected due to altered gravity conditions. In future experiments, potential target molecules for pharmacological interventions that attenuate astrocytic reactivity will be investigated. The ultimate goal is to enhance neuronal regeneration for novel therapeutic approaches.
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Yang, Sijie (Shirley), Svetlana Simtchouk, Julien Gibon, and Andis Klegeris. "Regulation of the phagocytic activity of astrocytes by neuroimmune mediators endogenous to the central nervous system." PLOS ONE 18, no. 7 (2023): e0289169. http://dx.doi.org/10.1371/journal.pone.0289169.
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The phagocytic activity of glial cells is essential for maintaining normal brain activity, and its dysfunction may contribute to the central nervous system (CNS) pathologies, including neurodegenerative diseases. Phagocytic activity is one of the well-established neuroimmune functions of microglia. Although emerging evidence indicates that astrocytes can also function as CNS phagocytes in humans and rodents, limited information is available about the molecular mechanism regulating this function. To address this knowledge gap, we studied modulation of the phagocytic activity of human U118 MG astrocytic cells and murine primary astrocytes by four CNS inflammatory mediators and bacterial endotoxin lipopolysaccharide (LPS). LPS and cytochrome c (CytC) upregulated, while interferon (IFN)-γ downregulated, phagocytosis of latex beads by human astrocytic cells and phagocytosis of synaptosomes by murine primary astrocytes. Interleukin (IL)-1β and tumor necrosis factor (TNF)-α had no effect on the phagocytic activity of human astrocytic cells but upregulated this function in murine astrocytes. Varying effects of combinations of the above inflammatory mediators were observed in these two cell types. LPS- and CytC-induced phagocytic activity of human astrocytic cells was partially mediated by activation of toll-like receptor 4 (TLR4). By monitoring other functions of astrocytes, we concluded there were no correlations between the effects of the mediators studied on astrocyte phagocytic activity and their secretion of cytokines, cytotoxins, or glutamate. Our study identified four candidate CNS regulators of astrocyte phagocytic activity. Future investigation of molecular mechanisms behind this regulation could identify novel therapeutic targets allowing modulation of this astrocyte-mediated clearance mechanism in CNS pathologies.
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Mitroshina, Elena V., Mikhail I. Krivonosov, Alexander M. Pakhomov, et al. "Unravelling the Collective Calcium Dynamics of Physiologically Aged Astrocytes under a Hypoxic State In Vitro." International Journal of Molecular Sciences 24, no. 15 (2023): 12286. http://dx.doi.org/10.3390/ijms241512286.
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Astrocytes serve many functions in the brain related to maintaining nerve tissue homeostasis and regulating neuronal function, including synaptic transmission. It is assumed that astrocytes are crucial players in determining the physiological or pathological outcome of the brain aging process and the development of neurodegenerative diseases. Therefore, studies on the peculiarities of astrocyte physiology and interastrocytic signaling during aging are of utmost importance. Calcium waves are one of the main mechanisms of signal transmission between astrocytes, and in the present study we investigated the features of calcium dynamics in primary cultures of murine cortical astrocytes in physiological aging and hypoxia modeling in vitro. Specifically, we focused on the assessment of calcium network dynamics and the restructuring of the functional network architecture in primary astrocytic cultures. Calcium imaging was performed on days 21 (“young” astrocyte group) and 150 (“old” astrocyte group) of cultures’ development in vitro. While the number of active cells and frequency of calcium events were decreased, we observed a reduced degree of correlation in calcium dynamics between neighboring cells, which was accompanied by a reduced number of functionally connected cells with fewer and slower signaling events. At the same time, an increase in the mRNA expression of anti-apoptotic factor Bcl-2 and connexin 43 was observed in “old” astrocytic cultures, which can be considered as a compensatory response of cells with a decreased level of intercellular communication. A hypoxic episode aggravates the depression of the connectivity of calcium dynamics of “young” astrocytes rather than that of “old” ones.
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Montgomery, D. L. "Astrocytes: Form, Functions, and Roles in Disease." Veterinary Pathology 31, no. 2 (1994): 145–67. http://dx.doi.org/10.1177/030098589403100201.
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Astrocytes, once relegated to a mere supportive role in the central nervous system, are now recognized as a heterogeneous class of cells with many important and diverse functions. Major astrocyte functions can be grouped into three categories: guidance and support of neuronal migration during development, maintenance of the neural microenvironment, and modulation of immune reactions by serving as antigen-presenting cells. The concept of astrocytic heterogeneity is critical to understanding the functions and reactions of these cells in disease. Astrocytes from different regions of the brain have diverse biochemical characteristics and may respond in different ways to a variety of injuries. Astrocytic swelling and hypertrophy-hyperplasia are two common reactions to injury. This review covers the morphologic and pathophysiologic findings, time course, and determinants of these two responses. In addition to these common reactions, astrocytes may play a primary role in certain diseases, including epilepsy, neurological dysfunction in liver disease, neurodegenerative disorders such as Parkinson's and Huntington's diseases, and demyelination. Evidence supporting primary involvement of astrocytes in these diseases will be considered.
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Lim, Dmitry, Lisa Mapelli, Pier Luigi Canonico, Francesco Moccia, and Armando A. Genazzani. "Neuronal Activity-Dependent Activation of Astroglial Calcineurin in Mouse Primary Hippocampal Cultures." International Journal of Molecular Sciences 19, no. 10 (2018): 2997. http://dx.doi.org/10.3390/ijms19102997.
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Astrocytes respond to neuronal activity by generating calcium signals which are implicated in the regulation of astroglial housekeeping functions and/or in modulation of synaptic transmission. We hypothesized that activity-induced calcium signals in astrocytes may activate calcineurin (CaN), a calcium/calmodulin-regulated protein phosphatase, implicated in neuropathology, but whose role in astroglial physiology remains unclear. We used a lentiviral vector expressing NFAT-EYFP (NY) fluorescent calcineurin sensor and a chemical protocol of LTP induction (cLTP) to show that, in mixed neuron-astrocytic hippocampal cultures, cLTP induced robust NY translocation into astrocyte nuclei and, hence, CaN activation. NY translocation was abolished by the CaN inhibitor FK506, and was not observed in pure astroglial cultures. Using Fura-2 single cell calcium imaging, we found sustained Ca2+ elevations in juxtaneuronal, but not distal, astrocytes. Pharmacological analysis revealed that both the Ca2+ signals and the nuclear NY translocation in astrocytes required NMDA and mGluR5 receptors and depended on extracellular Ca2+ entry via a store-operated mechanism. Our results provide a proof of principle that calcineurin in astrocytes may be activated in response to neuronal activity, thereby delineating a framework for investigating the role of astroglial CaN in the physiology of central nervous system.
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Emerson, Jacen, Thomas Delgado, Peter Girardi, and Gail V. W. Johnson. "Deletion of Transglutaminase 2 from Mouse Astrocytes Significantly Improves Their Ability to Promote Neurite Outgrowth on an Inhibitory Matrix." International Journal of Molecular Sciences 24, no. 7 (2023): 6058. http://dx.doi.org/10.3390/ijms24076058.
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Astrocytes are the primary support cells of the central nervous system (CNS) that help maintain the energetic requirements and homeostatic environment of neurons. CNS injury causes astrocytes to take on reactive phenotypes with an altered overall function that can range from supportive to harmful for recovering neurons. The characterization of reactive astrocyte populations is a rapidly developing field, and the underlying factors and signaling pathways governing which type of reactive phenotype that astrocytes take on are poorly understood. Our previous studies suggest that transglutaminase 2 (TG2) has an important role in determining the astrocytic response to injury. Selectively deleting TG2 from astrocytes improves functional outcomes after CNS injury and causes widespread changes in gene regulation, which is associated with its nuclear localization. To begin to understand how TG2 impacts astrocytic function, we used a neuron-astrocyte co-culture paradigm to compare the effects of TG2−/− and wild-type (WT) mouse astrocytes on neurite outgrowth and synapse formation. Neurons were grown on a control substrate or an injury-simulating matrix comprised of inhibitory chondroitin sulfate proteoglycans (CSPGs). Compared to WT astrocytes, TG2−/− astrocytes supported neurite outgrowth to a significantly greater extent only on the CSPG matrix, while synapse formation assays showed mixed results depending on the pre- and post-synaptic markers analyzed. We hypothesize that TG2 regulates the supportive functions of astrocytes in injury conditions by modulating gene expression through interactions with transcription factors and transcription complexes. Based on the results of a previous yeast two-hybrid screen for TG2 interactors, we further investigated the interaction of TG2 with Zbtb7a, a ubiquitously expressed transcription factor. Co-immunoprecipitation and colocalization analyses confirmed the interaction of TG2 and Zbtb7a in the nucleus of astrocytes. Overexpression or knockdown of Zbtb7a levels in WT and TG2−/− astrocytes revealed that Zbtb7a robustly influenced astrocytic morphology and the ability of astrocytes to support neuronal outgrowth, which was significantly modulated by the presence of TG2. These findings support our hypothesis that astrocytic TG2 acts as a transcriptional regulator to influence astrocytic function, with greater influence under injury conditions that increase its expression, and Zbtb7a likely contributes to the overall effects observed with astrocytic TG2 deletion.
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Preato, André Maciel, Ester da Silva Pinheiro, Tatiana Rosado Rosenstock, and Isaias Glezer. "The Relevance of Astrocytic Cell Culture Models for Neuroinflammation in Neurodegeneration Research." Neuroglia 5, no. 1 (2024): 27–49. http://dx.doi.org/10.3390/neuroglia5010003.
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Astrocytes are the predominant glial cells that provide essential support to neurons and promote microenvironment changes in neuropathological states. Astrocyte and astrocytic-like cell culture have substantially contributed to elucidating the molecular pathways involved in key glial roles, including those relevant to neurodevelopment, brain physiology and metabolism, which are not readily accessible with traditional approaches. The in vitro methodology has also been applied to neuroinflammatory and neurodegeneration contexts, revealing cellular changes involved in brain dysfunction. Astrocytes studies in culture started with primary cell approaches using embryonic and postmortem tissue. Further developments included newborn rodent primary cells, cell lines and immortalized astrocytes, which resulted in homogeneous cell-type preparations grown on flat surfaces. To overcome some in vitro shortcomings, tridimensional bioprinted models and organoid culture enabled the mimicking of tissue cellular arrangements and, above these achievements, complex astrocyte cell culture can be generated from induced pluripotent stem cells (iPSCs) to model diseases. These unprecedented breakthroughs allowed the development of platforms to test new therapies in brain cells derived from human material noninvasively obtained from live patients. In this work, we reviewed the most studied astrocytic cell models for discussing limitations, advantages and reliable experimental readouts for neuroinflammation in neurodegeneration research.
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Padmashri, Ragunathan, Anand Suresh, Michael D. Boska, and Anna Dunaevsky. "Motor-Skill Learning Is Dependent on Astrocytic Activity." Neural Plasticity 2015 (2015): 1–11. http://dx.doi.org/10.1155/2015/938023.
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Motor-skill learning induces changes in synaptic structure and function in the primary motor cortex through the involvement of a long-term potentiation- (LTP-) like mechanism. Although there is evidence that calcium-dependent release of gliotransmitters by astrocytes plays an important role in synaptic transmission and plasticity, the role of astrocytes in motor-skill learning is not known. To test the hypothesis that astrocytic activity is necessary for motor-skill learning, we perturbed astrocytic function using pharmacological and genetic approaches. We find that perturbation of astrocytes either by selectively attenuating IP3R2 mediated astrocyte Ca2+signaling or using an astrocyte specific metabolic inhibitor fluorocitrate (FC) results in impaired motor-skill learning of a forelimb reaching-task in mice. Moreover, the learning impairment caused by blocking astrocytic activity using FC was rescued by administration of the gliotransmitter D-serine. The learning impairments are likely caused by impaired LTP as FC blocked LTP in slices and prevented motor-skill training-induced increases in synaptic AMPA-type glutamate receptorin vivo. These results support the conclusion that normal astrocytic Ca2+signaling during a reaching task is necessary for motor-skill learning.
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Billaud, Jean-Noel, Calvin Ly, Tom R. Phillips, and Juan Carlos de la Torre. "Borna Disease Virus Persistence Causes Inhibition of Glutamate Uptake by Feline Primary Cortical Astrocytes." Journal of Virology 74, no. 22 (2000): 10438–46. http://dx.doi.org/10.1128/jvi.74.22.10438-10446.2000.
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ABSTRACT Borna disease virus (BDV), a nonsegmented, negative-stranded (NNS) RNA virus, causes central nervous system (CNS) disease in a broad range of vertebrate species, including felines. Both viral and host factors contribute to very diverse clinical and pathological manifestations associated with BDV infection. BDV persistence in the CNS can cause neurobehavioral and neurodevelopmental abnormalities in the absence of encephalitis. These BDV-induced CNS disturbances are associated with altered cytokine and neurotrophin expression, as well as cell damage that is very restricted to specific brain regions and neuronal subpopulations. BDV also targets astrocytes, resulting in the development of prominent astrocytosis. Astrocytes play essential roles in maintaining CNS homeostasis, and disruption of their normal activities can contribute to altered brain function. Therefore, we have examined the effect of BDV infection on the astrocyte's physiology. We present here evidence that BDV can establish a nonlytic chronic infection in primary cortical feline astrocytes that is associated with a severe impairment in the astrocytes' ability to uptake glutamate. In contrast, the astrocytes' ability to uptake glucose, as well as their protein synthesis, viability, and rate of proliferation, was not affected by BDV infection. These findings suggest that, in vivo, BDV could also affect an important astrocyte function required to prevent neuronal excitotoxicity. This, in turn, might contribute to the neuropathogenesis of BDV.
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Gasque, P., P. Chan, C. Mauger, et al. "Identification and characterization of complement C3 receptors on human astrocytes." Journal of Immunology 156, no. 6 (1996): 2247–55. http://dx.doi.org/10.4049/jimmunol.156.6.2247.
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Abstract Astrocytes express C components and have been implicated as a major source of intrathecal C. To ascertain the effects of C activation on these cells, we have evaluated the expression of CR1, CR2, and CR3 (CD35, CD21, and CD11b/CD18) in primary fetal astrocytes and astrocyte cell lines. None of the astrocyte cells tested expressed CR3, whereas primary astrocytes and one of four astrocyte cell lines expressed CR1 (220 kDa), as assessed at the protein and mRNA level. Primary fetal astrocytes and all four astrocyte cell lines expressed CR2 (155 kDa). Expression of CR2 by astrocytes was confirmed at mRNA level by reverse-transcriptase PCR, using different combinations of seven specific CR2 oligonucleotides, and by partial sequencing of the astrocyte CR2 cDNA. Astrocyte CR2 cDNA presented 100% homology with the lymphocyte CR2 cDNA between the position 181 bp to 600 bp and position 1017 bp to 1347 bp. An alternative splicing pattern of exon 11, reported previously in B cells, was observed in astrocyte CR2 cDNA. Astrocyte CR2 was functional, in that it specifically bound C3d and the EBV surface protein gp340, and the binding was blocked specifically with polyclonal anti-CR2. Scatchard analysis of membrane expression of CR2 on astrocytes revealed 2000 functional sites per cell with a Kd (3 x 10(-7) M) identical with that of CR2 on B cell (Raji).
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Norante, Peggion, Rossi, et al. "ALS-Associated SOD1(G93A) Decreases SERCA Pump Levels and Increases Store-Operated Ca2+ Entry in Primary Spinal Cord Astrocytes from a Transgenic Mouse Model." International Journal of Molecular Sciences 20, no. 20 (2019): 5151. http://dx.doi.org/10.3390/ijms20205151.
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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective death of motor neurons (MNs), probably by a combination of cell- and non-cell-autonomous processes. The past decades have brought many important insights into the role of astrocytes in nervous system function and disease, including the implication in ALS pathogenesis possibly through the impairment of Ca2+-dependent astrocyte-MN cross-talk. In this respect, it has been recently proposed that altered astrocytic store-operated Ca2+ entry (SOCE) may underlie aberrant gliotransmitter release and astrocyte-mediated neurotoxicity in ALS. These observations prompted us to a thorough investigation of SOCE in primary astrocytes from the spinal cord of the SOD1(G93A) ALS mouse model in comparison with the SOD1(WT)-expressing controls. To this purpose, we employed, for the first time in the field, genetically-encoded Ca2+ indicators, allowing the direct assessment of Ca2+ fluctuations in different cell domains. We found increased SOCE, associated with decreased expression of the sarco-endoplasmic reticulum Ca2+-ATPase and lower ER resting Ca2+ concentration in SOD1(G93A) astrocytes compared to control cells. Such findings add novel insights into the involvement of astrocytes in ALS MN damage.
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Rogers, Richard C., David H. McDougal, Sue Ritter, Emily Qualls-Creekmore, and Gerlinda E. Hermann. "Response of catecholaminergic neurons in the mouse hindbrain to glucoprivic stimuli is astrocyte dependent." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 315, no. 1 (2018): R153—R164. http://dx.doi.org/10.1152/ajpregu.00368.2017.
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Hindbrain catecholaminergic (CA) neurons are required for critical autonomic, endocrine, and behavioral counterregulatory responses (CRRs) to hypoglycemia. Recent studies suggest that CRR initiation depends on hindbrain astrocyte glucose sensors (McDougal DH, Hermann GE, Rogers RC. Front Neurosci 7: 249, 2013; Rogers RC, Ritter S, Hermann GE. Am J Physiol Regul Integr Comp Physiol 310: R1102–R1108, 2016). To test the proposition that hindbrain CA responses to glucoprivation are astrocyte dependent, we utilized transgenic mice in which the calcium reporter construct (GCaMP5) was expressed selectively in tyrosine hydroxylase neurons (TH-GCaMP5). We conducted live cell calcium-imaging studies on tissue slices containing the nucleus of the solitary tract (NST) or the ventrolateral medulla, critical CRR initiation sites. Results show that TH-GCaMP5 neurons are robustly activated by a glucoprivic challenge and that this response is dependent on functional astrocytes. Pretreatment of hindbrain slices with fluorocitrate (an astrocytic metabolic suppressor) abolished TH-GCaMP5 neuronal responses to glucoprivation, but not to glutamate. Pharmacologic results suggest that the astrocytic connection with hindbrain CA neurons is purinergic via P2 receptors. Parallel imaging studies on hindbrain slices of NST from wild-type C57BL/6J mice, in which astrocytes and neurons were prelabeled with a calcium reporter dye and an astrocytic vital dye, show that both cell types are activated by glucoprivation but astrocytes responded significantly sooner than neurons. Pretreatment of these hindbrain slices with P2 antagonists abolished neuronal responses to glucoprivation without interruption of astrocyte responses; pretreatment with fluorocitrate eliminated both astrocytic and neuronal responses. These results support earlier work suggesting that the primary detection of glucoprivic signals by the hindbrain is mediated by astrocytes.
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Sun, Yuanhong, Ali Winters, Linshu Wang, et al. "Metabolic Heterogeneity of Cerebral Cortical and Cerebellar Astrocytes." Life 13, no. 1 (2023): 184. http://dx.doi.org/10.3390/life13010184.
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Astrocytes play critical roles in regulating neuronal synaptogenesis, maintaining blood–brain barrier integrity, and recycling neurotransmitters. Increasing numbers of studies have suggested astrocyte heterogeneity in morphology, gene profile, and function. However, metabolic phenotype of astrocytes in different brain regions have not been explored. In this paper, we investigated the metabolic signature of cortical and cerebellar astrocytes using primary astrocyte cultures. We observed that cortical astrocytes were larger than cerebellar astrocytes, whereas cerebellar astrocytes had more and longer processes than cortical astrocytes. Using a Seahorse extracellular flux analyzer, we demonstrated that cortical astrocytes had higher mitochondrial respiration and glycolysis than cerebellar astrocytes. Cerebellar astrocytes have lower spare capacity of mitochondrial respiration and glycolysis as compared with cortical astrocytes. Consistently, cortical astrocytes have higher mitochondrial oxidation and glycolysis-derived ATP content than cerebellar astrocytes. In addition, cerebellar astrocytes have a fuel preference for glutamine and fatty acid, whereas cortical astrocytes were more dependent on glucose to meet energy demands. Our study indicated that cortical and cerebellar astrocytes display distinct metabolic phenotypes. Future studies on astrocyte metabolic heterogeneity and brain function in aging and neurodegeneration may lead to better understanding of the role of astrocyte in brain aging and neurodegenerative disorders.
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Filippova, Svetlana Yu, Aleksandr K. Logvinov, and Evgeniya Yu Kirichenko. "Uneven Distribution of Astrocyte Membranes over the Layers of the Rat Primary Somatosensory Cortex." Journal of Medical and Biological Research, no. 4 (November 15, 2020): 409–18. http://dx.doi.org/10.37482/2687-1491-z034.
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Astrocytes are the main glial cells maintaining water-electrolyte and energy balance in the brain. Today, astroglia is also believed to take a direct part in the regulation of synaptic transmission and in enabling synchronous operation of neurons at large distances. Astrocytes fulfil their functions through numerous processes that penetrate the entire neuropil. The authors believe that changes in the astrocyte membrane surface area per unit volume of neuropil directly reflect changes in the intensity of the astrocyte–neuron interaction. Strengthening or weakening of astrocyte regulation, undoubtedly, affect the functioning of neural circuits. Nevertheless, in spite of the growing popularity of research into the glia–neuron relations, this aspect remains insufficiently studied when it comes to the cerebral cortex. The purpose of this study was to layer-by-layer determine the astrocyte membrane surface per unit volume in the neuropil of the rat primary somatosensory cortex. The research was conducted on samples of the primary somatosensory cortex obtained from 5 white male rats (P60–80). After immune labeling against astrocytic marker S100B using the pre-embedding method, the samples were prepared for transmission electron microscopy according to the standard technique. In total, 250 electron micrographs were obtained for each layer of the primary somatosensory cortex, which were then used to determine the astrocyte membrane surface area per unit volume in the neuropil by means of the random secant method. The research found that this indicator is the minimum in the first and maximum in the fifth layers of the cortical column. In addition, the article discusses the possible functional consequences of uneven distribution of astrocytic membranes in the neocortex.
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Wakida, Nicole M., Alice L. Lau, Jessica Nguyen, et al. "Diminished LC3-Associated Phagocytosis by Huntington’s Disease Striatal Astrocytes." Journal of Huntington's Disease 11, no. 1 (2022): 25–33. http://dx.doi.org/10.3233/jhd-210502.
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Background: In recent years the functions of astrocytes have shifted from conventional supportive roles to also include active roles in altering synapses and engulfment of cellular debris. Recent studies have implicated astrocytes in both protective and pathogenic roles impacting Huntington’s disease (HD) progression. Objective: The goal of this study is to determine if phagocytosis of cellular debris is compromised in HD striatal astrocytes. Methods: Primary adult astrocytes were derived from two HD mouse models; the fast-progressing R6/2 and slower progressing Q175. With the use of laser nanosurgery, a single astrocyte was lysed within an astrocyte network. The phagocytic response of astrocytes was observed with phase contrast and by fluorescence microscopy for GFP-LC3 transiently transfected cells. Results: Astrocyte phagocytosis was significantly diminished in primary astrocytes, consistent with the progression of HD in R6/2 and Q175 mouse models. This was defined by the number of astrocytes responding via phagocytosis and by the average number of vesicles formed per cell. GFP-LC3 was found to increasingly localize to phagocytic vesicles over a 20-min imaging period, but not in HD mice, suggesting the involvement of LC3 in astrocyte phagocytosis. Conclusion: We demonstrate a progressive decrease in LC3-associated phagocytosis in HD mouse striatal astrocytes.
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Hutyrová, Paulína, Jan Stangelj, and Metoda Lipnik-Stangelj. "The effects of gabapentin and ethanol on the regulated cell death of astrocytes in primary culture." Acta Medico-Biotechnica 12, no. 2 (2021): 81–88. http://dx.doi.org/10.18690/actabiomed.189.
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Purpose: In this study, the effect of gabapentin on the regulated cell death of astrocytes in primary culture was examined. Because astrocytes are relatively resistant to decay by apoptotic pathways, the effect of different concentrations of gabapentin on apoptosis in necroptosis was tested as another form of regulatedcell death. In addition, the impact of gabapentin on the death of astrocytes that were exposed to ethanol was also examined.
 Methods: Primary cultures of astrocytes that were obtained from the brain cortex of newborn rats were used as the experimental model. Cells were exposed to different concentrations of gabapentin only, ethanol only or to a combination of ethanol and gabapentin. Using flow cytometry, the proportions of viable, early apoptotic, necroptotic, and secondary dead cells were determined.
 Results: The effect of gabapentin on early astrocytic apoptosis and necroptosis was dependent on concentration. In concentrations of up to 10 μg/mL, gabapentin did not affect astrocyte deaths; whereas at higher concentrations, the proportion of necroptotic cells increased. The concomitant exposure of the cells to gabapentin (10 μg/mL) and ethanol (100 mM) for 24 hours did not significantly affect cell death caused by ethanol. For cells that are exposed to 50 mM ethanol for 7 days, gabapentin slightly reduced the proportion of necrotic cells.
 Conclusion: Gabapentin did not affect the viability of astrocytes in concentrations up to 10 μg/mL. The concomitant exposure of astrocytes to ethanol and gabapentin for 24 hours did not reduce the toxicity of ethanol. In astrocytes that are chronically exposed to ethanol, gabapentin slightly reduced the effect of ethanol on necroptosis.
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Song, Yizhi, Zunshu Du, Xinyue Chen та ін. "Astrocytic N-Methyl-D-Aspartate Receptors Protect the Hippocampal Neurons Against Amyloid-β142-Induced Synaptotoxicity by Regulating Nerve Growth Factor". Journal of Alzheimer's Disease 85, № 1 (2022): 167–78. http://dx.doi.org/10.3233/jad-210730.
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Background: Soluble oligomeric amyloid-β (Aβ)-induced synaptic dysfunction is an early event in Alzheimer’s disease (AD) pathogenesis. Mounting evidence has suggested N-methyl-D-aspartate receptors (NMDARs) play an important role in Aβ-induced synaptotoxicity. Originally NMDARs were believed to be expressed exclusively in neurons; however, recent two decades studies have demonstrated functional NMDARs present on astrocytes. Neuronal NMDARs are modulators of neurodegeneration, while our previous initial study found that astrocytic NMDARs mediated synaptoprotection and identified nerve growth factor (NGF) secreted by astrocytes, as a likely mediator, but how astrocytic NMDARs protect neurons against Aβ-induced synaptotoxicity through regulating NGF remains unclear. Objective: To achieve further insight into the mechanism of astrocytic NMDARs oppose Aβ-induced synaptotoxicity through regulating NGF. Methods: With the primary hippocampal neuronal and astrocytic co-cultures, astrocytes were pretreated with agonist or antagonist of NMDARs before Aβ142 oligomers application to neuron-astrocyte co-cultures. Western blot, RT-PCR, etc., were used for the related proteins evaluation. Results: Activation of astrocytic NMDARs can significantly mitigate Aβ142-induced loss of PSD-95 and synaptophysin through increasing NGF release. Blockade of astrocytic NMDARs inhibited Aβ-induced compensatory protective NGF increase in protein and mRNA levels through modulating NF-κB of astrocytes. Astrocytic NMDARs activation can enhance Aβ-induced Furin increase, and blockade of astrocytic NMDARs inhibited Aβ-induced immunofluorescent intensity elevation of vesicle trafficking protein VAMP3 and NGF double-staining. Conclusion: Astrocytic NMDARs oppose Aβ-induced synaptotoxicity through modulating the synthesis, maturation, and secretion of NGF in astrocytes. This new information may contribute to the quest for specific targeted strategy of intervention to delay the onset of AD.
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Tedeschi, B., J. N. Barrett, and R. W. Keane. "Astrocytes produce interferon that enhances the expression of H-2 antigens on a subpopulation of brain cells." Journal of Cell Biology 102, no. 6 (1986): 2244–53. http://dx.doi.org/10.1083/jcb.102.6.2244.
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Using primary culture methods, we show that purified astrocytes from embryonic mouse or rat central nervous system (CNS) can be induced to produce interferon (IFN) activity when pretreated with a standard IFN-superinducing regimen of polyribonucleotide, cycloheximide, and actinomycin D, whereas IFN activity was not inducible in neuronal cultures derived from mouse CNS. Astrocyte IFN displays inductive, kinetic, physicochemical, and antigenic properties similar to those of IFN-alpha/beta, but is dissimilar to lymphocyte IFN (IFN-gamma). Treatment of pure astrocytic cultures or astrocytes cultured with neurons with astrocyte IFN or IFN-alpha/beta induced a dramatic increase in the expression of H-2 antigens on a subpopulation of astrocytes. Neither neurons nor oligodendroglia expressed detectable levels of H-2 antigens when exposed to astrocyte IFN, IFN-alpha/beta, or to IFN-beta. Injection of astrocyte IFN or IFN-alpha/beta directly into brains of newborn mice indicated that H-2 antigens were also induced in vivo. None of the IFNs (astrocyte, alpha/beta, or beta) tested induced Ia antigens on CNS cells in vitro or in vivo. Since H-2 antigens have a critical role in immune responses, astrocyte IFN may initiate and participate in immune reactions that contribute to immunoprotective and immunopathological responses in the CNS.
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Lin, Chia-Ho, Han-Yu Chen, and Kai-Che Wei. "Role of HMGB1/TLR4 Axis in Ischemia/Reperfusion-Impaired Extracellular Glutamate Clearance in Primary Astrocytes." Cells 9, no. 12 (2020): 2585. http://dx.doi.org/10.3390/cells9122585.
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(1) Background: Abnormal accumulation of extracellular glutamate can occur as dysfunction of astrocytic glutamate transporters, which has been linked to ischemic brain injury. Excessive extracellular glutamate-induced abnormal excitotoxicity is the major cause of secondary neuronal damage after cerebral ischemia/reperfusion. However, the definite mechanism of impaired astrocytic glutamate reuptake remains unclear. (2) Methods: We investigated the mechanism of the HMGB1/TLR4 axis in extracellular glutamate clearance in primary astrocytes exposed to ischemia/reperfusion by using OGD/R (oxygen-glucose deprivation/reoxygenation) model. (3) Results: OGD/R insult activated the HMGB1/TLR4 axis for reducing the activity of glutamate clearance by inhibiting GLAST (glutamate aspartate transporter) expression in primary astrocytes. Interestingly, OGD/R-untreated astrocytes showed impairment of glutamate clearance after exposure to exogenous HMGB1 or conditioned medium from OGD/R-treated astrocytes culture. Inhibition of HMGB1 or TLR4 effectively prevented impaired glutamate clearance, which was induced by OGD/R, exogenous HMGB1, or conditioned medium from OGD/R-treated astrocytes. Furthermore, glycyrrhizic acid attenuated OGD/R-induced impairment of astrocytic glutamate clearance mediated by the HMGB1-TLR4 axis. (4) Conclusion: The HMGB1/TLR4 axis is a potential target for the treatment of post-ischemic excitotoxicity caused by GLAST dysfunction in astrocytes.
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Silva, Ellen A., Ana P. Dalla Costa, Juliana S. Ruas, Edilene S. Siqueira-Santos, Annelise Francisco, and Roger F. Castilho. "Proliferating Astrocytes in Primary Culture Do Not Depend upon Mitochondrial Respiratory Complex I Activity or Oxidative Phosphorylation." Cells 12, no. 5 (2023): 683. http://dx.doi.org/10.3390/cells12050683.
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Understanding the role of astrocytes in the development of the nervous system and neurodegenerative disorders implies a necessary knowledge of the oxidative metabolism of proliferating astrocytes. The electron flux through mitochondrial respiratory complexes and oxidative phosphorylation may impact the growth and viability of these astrocytes. Here, we aimed at assessing to which extent mitochondrial oxidative metabolism is required for astrocyte survival and proliferation. Primary astrocytes from the neonatal mouse cortex were cultured in a physiologically relevant medium with the addition of piericidin A or oligomycin at concentrations that fully inhibit complex I-linked respiration and ATP synthase, respectively. The presence of these mitochondrial inhibitors for up to 6 days in a culture medium elicited only minor effects on astrocyte growth. Moreover, neither the morphology nor the proportion of glial fibrillary acidic protein-positive astrocytes in culture was affected by piericidin A or oligomycin. Metabolic characterization of the astrocytes showed a relevant glycolytic metabolism under basal conditions, despite functional oxidative phosphorylation and large spare respiratory capacity. Our data suggest that astrocytes in primary culture can sustainably proliferate when their energy metabolism relies only on aerobic glycolysis since their growth and survival do not require electron flux through respiratory complex I or oxidative phosphorylation.
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Lindman, Marissa, Juan Angel, Kimberly Newman, Colm Atkins, and Brian Daniels. "Astrocytic RIPK3 confers protection against deleterious neuroinflammation during Zika virus infection." Journal of Immunology 208, no. 1_Supplement (2022): 163.27. http://dx.doi.org/10.4049/jimmunol.208.supp.163.27.
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Abstract This study aims to identify the function(s) of RIPK3 signaling in astrocytes following Zika virus infection. Previous work found that RIPK3 signaling in Zika virus-infected neurons activates inflammatory transcription factors such as NFκB and IRF1, leading to the upregulation of inflammation-associated transcripts. We were thus interested in determining the role of RIPK3 signaling in astrocytes, which are critical regulators of neuroinflammation. Using mice with an astrocyte-specific conditional Ripk3 deletion, we found that intracranial Zika virus infection was significantly more lethal in mice deficient in astrocytic Ripk3 than in littermate controls. To identify mechanisms underlying this difference, we isolated and infected primary fore- and hind-brain astrocytes with Zika virus to determine the transcriptional consequences of genetic Ripk3 ablation. Surprisingly, we found increased expression of several chemokines, cytokines and ISGs in Ripk3−/− hindbrain astrocytes, in contrast to our previous findings in neurons. Subsequent leukocyte profiling from the brains of Zika virus-infected mice revealed increased numbers of CD4+ and CD8+ T cells, natural killer cells, and monocytes in mice deficient in astrocytic Ripk3 compared to those found in littermate controls. As previous work has demonstrated that astrocytic type I interferon signaling in the hindbrain is responsible for downregulating proinflammatory molecules to prevent lethal neuroinflammation, our data suggest that synergistic signaling between type I IFN and RIPK3 in hindbrain astrocytes suppresses deleterious neuroinflammation and promotes host survival in the setting of Zika virus encephalitis. Supported by a grant from NIH (R01 NS120895).
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Grabarczyk, Mikolaj, Dominika Ksiazek-Winiarek, Andrzej Glabinski, and Piotr Szpakowski. "Dietary Polyphenols Decrease Chemokine Release by Human Primary Astrocytes Responding to Pro-Inflammatory Cytokines." Pharmaceutics 15, no. 9 (2023): 2294. http://dx.doi.org/10.3390/pharmaceutics15092294.
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Astrocytes are considered to be the dominant cell fraction of the central nervous system. They play a supportive and protective role towards neurons, and regulate inflammatory processes; they thus make suitable targets for drugs and supplements, such as polyphenolic compounds. However, due to their wide range, knowledge of their anti-inflammatory potential remains relatively incomplete. The aim of this study was therefore to determine whether myricetin and chrysin are able to decrease chemokine release in reactive astrocytes. To assess the antioxidant and anti-inflammatory potential of polyphenols, human primary astrocytes were cultured in the presence of a reactive and neurotoxic astrocyte-inducing cytokine mixture (TNF-α, IL-1a, C1q), either alone or in the presence of myricetin or chrysin. The examined polyphenols were able to modify the secretion of chemokines by human cortical astrocytes, especially CCL5 (chrysin), CCL1 (myricetin) and CCL2 (both), while cell viability was not affected. Surprisingly, the compounds did not demonstrate any antioxidant properties in the astrocyte cultures.
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Imraish, Amer, Tuqa Abu Thiab, Mohammad Alsalem, et al. "The neuroprotective effect of human primary astrocytes in multiple sclerosis: In vitro model." PLOS ONE 19, no. 4 (2024): e0300203. http://dx.doi.org/10.1371/journal.pone.0300203.
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Recent studies highlighted the role of astrocytes in neuroinflammatory diseases, particularly multiple sclerosis, interacting closely with other CNS components but also with the immune cells. However, due to the difficulty in obtaining human astrocytes, their role in these pathologies is still unclear. In this study we develop an astrocyte in vitro model to evaluate their role in multiple sclerosis after being treated with CSF isolated from both healthy and MS diagnosed patients. Gene expression and ELISA assays reveal that several pro-inflammatory markers IL-1β, TNF-α and IL-6, were significantly downregulated in astrocytes treated with MS-CSF. In contrast, neurotrophic survival, and growth factors, and GFAP, BDNF, GDNF and VEGF, were markedly elevated upon the same treatment. In summary, this study supports the notion of the astrocyte involvement in MS. The results reveal the neuroprotective role of astrocyte in MS pathogenicity by suppressing excessive inflammation and increasing the expression of tropic factors.
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Elorza-Vidal, Xabier, Héctor Gaitán-Peñas, and Raúl Estévez. "Chloride Channels in Astrocytes: Structure, Roles in Brain Homeostasis and Implications in Disease." International Journal of Molecular Sciences 20, no. 5 (2019): 1034. http://dx.doi.org/10.3390/ijms20051034.
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Astrocytes are the most abundant cell type in the CNS (central nervous system). They exert multiple functions during development and in the adult CNS that are essential for brain homeostasis. Both cation and anion channel activities have been identified in astrocytes and it is believed that they play key roles in astrocyte function. Whereas the proteins and the physiological roles assigned to cation channels are becoming very clear, the study of astrocytic chloride channels is in its early stages. In recent years, we have moved from the identification of chloride channel activities present in astrocyte primary culture to the identification of the proteins involved in these activities, the determination of their 3D structure and attempts to gain insights about their physiological role. Here, we review the recent findings related to the main chloride channels identified in astrocytes: the voltage-dependent ClC-2, the calcium-activated bestrophin, the volume-activated VRAC (volume-regulated anion channel) and the stress-activated Maxi-Cl−. We discuss key aspects of channel biophysics and structure with a focus on their role in glial physiology and human disease.
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Stella, Roberto, Raphael Severino Bonadio, Stefano Cagnin, Maria Lina Massimino, Alessandro Bertoli, and Caterina Peggion. "Perturbations of the Proteome and of Secreted Metabolites in Primary Astrocytes from the hSOD1(G93A) ALS Mouse Model." International Journal of Molecular Sciences 22, no. 13 (2021): 7028. http://dx.doi.org/10.3390/ijms22137028.
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Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease whose pathophysiology is largely unknown. Despite the fact that motor neuron (MN) death is recognized as the key event in ALS, astrocytes dysfunctionalities and neuroinflammation were demonstrated to accompany and probably even drive MN loss. Nevertheless, the mechanisms priming astrocyte failure and hyperactivation are still obscure. In this work, altered pathways and molecules in ALS astrocytes were unveiled by investigating the proteomic profile and the secreted metabolome of primary spinal cord astrocytes derived from transgenic ALS mouse model overexpressing the human (h)SOD1(G93A) protein in comparison with the transgenic counterpart expressing hSOD1(WT) protein. Here we show that ALS primary astrocytes are depleted of proteins—and of secreted metabolites—involved in glutathione metabolism and signaling. The observed increased activation of Nf-kB, Ebf1, and Plag1 transcription factors may account for the augmented expression of proteins involved in the proteolytic routes mediated by proteasome or endosome–lysosome systems. Moreover, hSOD1(G93A) primary astrocytes also display altered lipid metabolism. Our results provide novel insights into the altered molecular pathways that may underlie astrocyte dysfunctionalities and altered astrocyte–MN crosstalk in ALS, representing potential therapeutic targets to abrogate or slow down MN demise in disease pathogenesis.
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Sočan, Vesna, Klemen Dolinar, and Mojca Kržan. "Cortical and Striatal Astrocytes of Neonatal Rats Display Distinct Molecular and Pharmacological Characteristics of Dopamine Uptake." International Journal of Molecular Sciences 25, no. 2 (2024): 911. http://dx.doi.org/10.3390/ijms25020911.
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Astrocytes are crucial in the regulation of neurotransmitter homeostasis, and while their involvement in the dopamine (DA) tripartite synapse is acknowledged, it necessitates a more comprehensive investigation. In the present study, experiments were conducted on primary astrocyte cultures from the striatum and cortex of neonatal rats. The pharmacological intricacies of DA uptake, including dependence on time, temperature, and concentration, were investigated using radiolabelled [3H]-DA. The mRNA expression of transporters DAT, NET, PMAT, and OCTs was evaluated by qPCR. Notably, astrocytes from both brain regions exhibited prominent mRNA expression of NET and PMAT, with comparatively lower expression of DAT and OCTs. The inhibition of DA uptake by the DAT inhibitor, GBR12909, and NET inhibitors, desipramine and nortriptyline, impeded DA uptake in striatal astrocytes more than in cortical astrocytes. The mRNA expression of NET and PMAT was significantly upregulated in cortical astrocytes in response to the DA receptor agonist apomorphine, while only the mRNA expression of NET exhibited changes in striatal astrocytes. Haloperidol, a DA receptor antagonist, and L-DOPA, a DA precursor, did not induce significant alterations in transporter mRNA expression. These findings underscore the intricate and region-specific mechanisms governing DA uptake in astrocytes, emphasizing the need for continued exploration to unravel the nuanced dynamics of astrocytic involvement in the DA tripartite synapse.
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Matthiesen, Isabelle, Rohollah Nasiri, Alessandra Tamashiro Orrego, Thomas E. Winkler, and Anna Herland. "Metabolic Assessment of Human Induced Pluripotent Stem Cells-Derived Astrocytes and Fetal Primary Astrocytes: Lactate and Glucose Turnover." Biosensors 12, no. 10 (2022): 839. http://dx.doi.org/10.3390/bios12100839.
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Astrocytes represent one of the main cell types in the brain and play a crucial role in brain functions, including supplying the energy demand for neurons. Moreover, they are important regulators of metabolite levels. Glucose uptake and lactate production are some of the main observable metabolic actions of astrocytes. To gain insight into these processes, it is essential to establish scalable and functional sources for in vitro studies of astrocytes. In this study, we compared the metabolic turnover of glucose and lactate in astrocytes derived from human induced pluripotent stem cell (hiPSC)-derived Astrocytes (hiAstrocytes) as a scalable astrocyte source to human fetal astrocytes (HFAs). Using a user-friendly, commercial flow-based biosensor, we could verify that hiAstrocytes are as glycogenic as their fetal counterparts, but their normalized metabolic turnover is lower. Specifically, under identical culture conditions in a defined media, HFAs have 2.3 times higher levels of lactate production compared to hiAstrocytes. In terms of glucose, HFAs have 2.1 times higher consumption levels than hiAstrocytes at 24 h. Still, as we describe their glycogenic phenotype, our study demonstrates the use of hiAstrocytes and flow-based biosensors for metabolic studies of astrocyte function.
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Leffler, Charles W., Helena Parfenova, Alexander L. Fedinec, Shyamali Basuroy, and Dilyara Tcheranova. "Contributions of astrocytes and CO to pial arteriolar dilation to glutamate in newborn pigs." American Journal of Physiology-Heart and Circulatory Physiology 291, no. 6 (2006): H2897—H2904. http://dx.doi.org/10.1152/ajpheart.00722.2006.
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Astrocytes can act as intermediaries between neurons and cerebral arterioles to regulate vascular tone in response to neuronal activity. Release of glutamate from presynaptic neurons increases blood flow to match metabolic demands. CO is a gasotransmitter that can be related to neural function and blood flow regulation in the brain. The present study addresses the hypothesis that glutamatergic stimulation promotes perivascular astrocyte CO production and pial arteriolar dilation in the newborn brain. Experiments used anesthetized newborn pigs with closed cranial windows, piglet astrocytes, and cerebrovascular endothelial cells in primary culture and immunocytochemical visualization of astrocytic markers. Pial arterioles and arteries of newborn pigs are ensheathed by astrocytes visualized by glial fibrillary acidic protein staining. Treatment (2 h) of astrocytes in culture with l-2-α-aminoadipic acid (l-AAA), followed by 14 h in toxin free medium, dose-dependently increased cell detachment, suggesting injury. Conversely, 16 h of continuous exposure to l-AAA caused no decrease in endothelial cell attachment. In vivo, topical l-AAA (2 mM, 5 h) disrupted the cortical glia limitans histologically. Such treatment also eliminated pial arteriolar dilation to the astrocyte-dependent dilator ADP and to glutamate but not to isoproterenol or CO. Glutamate stimulated CO production by the brain surface that also was abolished following l-AAA. In contrast, tetrodotoxin blocked dilation to N-methyl-d-aspartate but not to glutamate, isoproterenol, or CO or the glutamate-induced increase in CO. The concurrent loss of CO production and pial arteriolar dilation to glutamate following astrocyte injury suggests astrocytes may employ CO as a gasotransmitter for glutamatergic cerebrovascular dilation.
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Fan, Yu-Ying, Jun-Mei Zhang, Hua Wang, Xue-Yan Liu, and Feng-Hua Yang. "Leukemia inhibitory factor inhibits the proliferation of primary rat astrocytes induced by oxygen-glucose deprivation." Acta Neurobiologiae Experimentalis 73, no. 4 (2013): 485–94. http://dx.doi.org/10.55782/ane-2013-1954.
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Leukemia inhibitory factor (LIF) is a neuroprotective cytokine that is necessary for the normal development of astrocytes. Oxygen-glucose deprivation (OGD) can induce astrocyte proliferation by increasing hypoxia-inducible factor alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF). Here, we studied whether LIF affects the proliferation of cultured primary rat astrocytes under OGD conditions by measuring EdU incorporation into astrocyte DNA and the expression of proliferating cell nuclear antigen (PCNA) mRNA and protein. Our findings show that low concentrations of LIF (5 and 10 ng/mL) significantly decreased EdU incorporation and downregulated the expression of PCNA mRNA and PCNA protein in astrocytes subjected to OGD. A low concentration of LIF (10 ng/mL) clearly inhibited astrocyte proliferation induced by OGD, while a higher concentration (50 ng/mL) had no effect. To investigate the mechanism of this inhibition by LIF (10 ng/mL), the expression of 3 related genes (LIF receptor, HIF-1alpha, and VEGF) was assessed using real-time PCR; VEGF protein expression was measured by Western blot. Our results indicate that LIFR mRNA was downregulated in astrocytes subjected to OGD. Interestingly, treatment with LIF further reduced LIFR mRNA expression in these cells. LIF treatment also decreased the expression of HIF-1alpha mRNA, VEGF mRNA, and VEGF protein induced by OGD. Low concentrations of LIF were observed to inhibit astrocyte proliferation induced by OGD.
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Yanagida, Takashi, Jun Tsushima, Yoshihisa Kitamura, et al. "Oxidative Stress Induction of DJ-1 Protein in Reactive Astrocytes Scavenges Free Radicals and Reduces Cell Injury." Oxidative Medicine and Cellular Longevity 2, no. 1 (2009): 36–42. http://dx.doi.org/10.4161/oxim.2.1.7985.
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Astrocytes, one of the predominant types of glial cells, function as both supportive and metabolic cells for the brain. Under cerebral ischemia/reperfusion-induced oxidative conditions, astrocytes accumulate and activate in the ischemic region. DJ-1 has recently been shown to be a sensor of oxidative stress in living cells. However, the function of astrocytic DJ-1 is still unknown. In the present study, to clarify the effect of astrocytic DJ-1 protein under massive oxidative insult, we used a focal ischemic rat model that had been subjected to middle cerebral artery occlusion (MCAO) and reperfusion. We then investigated changes in the distribution of DJ-1 in astrocytes, DJ-1 release from cultured astrocytes, and the effects of recombinant DJ-1 protein on hydrogen peroxide (H2O2)-induced death in normal and DJ-1-knockdown SH-SY5Y cells and on in vitro scavenging of hydroxyl radicals (•OH) by electron spin resonance spectrometry. At 24 h after 2-h MCAO and reperfusion, an infarct lesion was markedly observed using magnetic resonance imaging and 2,3,5-triphenyltetrazolium chloride staining. In addition, reactive astrocytes enhanced DJ-1 expression in the penumbral zone of the ischemic core and that DJ-1 protein was extracellularly released from astrocytes by H2O2 in in vitro primary cultures. Although DJ-1-knockdown SH-SY5Y cells were markedly vulnerable to oxidative stress, treatment with glutathione S-transferase-tagged recombinant human DJ-1 protein (GST-DJ-1) significantly inhibited H2O2-induced cell death. In addition, GST-DJ-1 protein directly scavenged•OH. These results suggest that oxidative stress induces the release of astrocytic DJ-1 protein, which may contribute to astrocyte-mediated neuroprotection.
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Zveik, Omri, Ariel Rechtman, Nitzan Haham, et al. "Sera of Neuromyelitis Optica Patients Increase BID-Mediated Apoptosis in Astrocytes." International Journal of Molecular Sciences 23, no. 13 (2022): 7117. http://dx.doi.org/10.3390/ijms23137117.
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Neuromyelitis optica (NMO) is a rare disease usually presenting with bilateral or unilateral optic neuritis with simultaneous or sequential transverse myelitis. Autoantibodies directed against aquaporin-4 (AQP4-IgG) are found in most patients. They are believed to cross the blood–brain barrier, target astrocytes, activate complement, and eventually lead to astrocyte destruction, demyelination, and axonal damage. However, it is still not clear what the primary pathological event is. We hypothesize that the interaction of AQP4-IgG and astrocytes leads to DNA damage and apoptosis. We studied the effect of sera from seropositive NMO patients and healthy controls (HCs) on astrocytes’ immune gene expression and viability. We found that sera from seropositive NMO patients led to higher expression of apoptosis-related genes, including BH3-interacting domain death agonist (BID), which is the most significant differentiating gene (p < 0.0001), and triggered more apoptosis in astrocytes compared to sera from HCs. Furthermore, NMO sera increased DNA damage and led to a higher expression of immunological genes that interact with BID (TLR4 and NOD-1). Our findings suggest that sera of seropositive NMO patients might cause astrocytic DNA damage and apoptosis. It may be one of the mechanisms implicated in the primary pathological event in NMO and provide new avenues for therapeutic intervention.
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Jha, Mithilesh Kumar, Myungjin Jo, Jae-Hong Kim, and Kyoungho Suk. "Microglia-Astrocyte Crosstalk: An Intimate Molecular Conversation." Neuroscientist 25, no. 3 (2018): 227–40. http://dx.doi.org/10.1177/1073858418783959.
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Microglia-astrocyte crosstalk has recently been at the forefront of glial research. Emerging evidence illustrates that microglia- and astrocyte-derived signals are the functional determinants for the fates of astrocytes and microglia, respectively. By releasing diverse signaling molecules, both microglia and astrocytes establish autocrine feedback and their bidirectional conversation for a tight reciprocal modulation during central nervous system (CNS) insult or injury. Microglia, the constant sensors of changes in the CNS microenvironment and restorers of tissue homeostasis, not only serve as the primary immune cells of the CNS but also regulate the innate immune functions of astrocytes. Similarly, microglia determine the functions of reactive astrocytes, ranging from neuroprotective to neurotoxic. Conversely, astrocytes through their secreted molecules regulate microglial phenotypes and functions ranging from motility to phagocytosis. Altogether, the microglia-astrocyte crosstalk is fundamental to neuronal functions and dysfunctions. This review discusses the current understanding of the intimate molecular conversation between microglia and astrocytes and outlines its potential implications in CNS health and disease.
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Murray, Taryn E., Tyler J. Wenzel, Svetlana Simtchouk, Bridget K. Greuel, Julien Gibon, and Andis Klegeris. "Extracellular Cardiolipin Modulates Select Immune Functions of Astrocytes in Toll-Like Receptor (TLR) 4-Dependent Manner." Mediators of Inflammation 2022 (March 25, 2022): 1–14. http://dx.doi.org/10.1155/2022/9946439.
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Alzheimer’s disease (AD) is characterized by chronic neuroinflammation, which is partially mediated by dysregulated functions of glial cells. Cardiolipin (CL) is a phospholipid normally confined to the inner mitochondrial membrane; however, it has been detected in human sera, indicating that it can exist in the extracellular space where it may interact with nearby cells. Although CL has been shown to modulate several functions of microglia in a toll-like receptor (TLR) 4-dependent manner, the effects of extracellular CL on astrocytes are unknown. In addition to their homeostatic functions, astrocytes participate in neuroimmune responses of the brain and express TLR 4. Therefore, we hypothesized that extracellular CL (1) modulates the secretion of cytokines and cytotoxins by astrocytes, as well as their phagocytic activity, and (2) acts by interacting with astrocyte TLR 4. We demonstrate that CL inhibits the lipopolysaccharide- (LPS-) induced secretion of cytotoxins and expression of glial fibrillary acidic protein (GFAP) by human U118 MG astrocytic cells. CL alone upregulates the phagocytic activity of human astrocytic cells and primary murine astrocytes. CL in combination with LPS upregulates secretion of interleukin (IL)-1β by astrocytic cells. Furthermore, CL alone increases the secretion of monocyte chemoattractant protein (MCP)-1 by astrocytic cells, which is blocked by the TLR 4-specific antagonist TAK-242. We demonstrate that CL upregulates MCP-1 secretion in the absence of its natural carrier protein, β2-glycoprotein 1, indicating that CL may be bioactive in the brain where this protein is not present. Lastly, we show that CL downregulates the expression of astrocytic TLR 4, implying that CL engages this receptor, as its activation has been shown to lead to its degradation. Overall, our study extends the list of cell type functions of which CL modulates and provides evidence that CL, or liposomes containing this phospholipid can be used to modulate specific neuroimmune functions of astrocytes.
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Liu, J., M. L. Zhao, C. F. Brosnan, and S. C. Lee. "Expression of type II nitric oxide synthase in primary human astrocytes and microglia: role of IL-1beta and IL-1 receptor antagonist." Journal of Immunology 157, no. 8 (1996): 3569–76. http://dx.doi.org/10.4049/jimmunol.157.8.3569.
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Abstract In this work, we studied the expression of type II nitric oxide synthase (NOS) in primary cultures of human astrocytes and microglia. Cytokine-activated human fetal astrocytes expressed a 4.5-kb type II NOS mRNA that was first evident at 8 h, steadily increased through 48 h, and persisted through 72 h. The inducing signals for astrocyte NOS II mRNA expression were in the order IL-1beta + IFN-gamma &gt; IL-1beta + TNF-alpha &gt; IL-1beta. SDS-PAGE analysis of cytokine-stimulated astrocyte cultures revealed an approximately 130-kDa single NOS II band that was expressed strongly at 48 and 72 h (72 h &gt; 48 h). Specific NOS II immunoreactivity was detected in cytokine-treated astrocytes, both in the cytosol and in a discrete paranuclear region, which corresponded to Golgi-like membranes on immunoelectron microscopy. In human microglia, cytokines and LPS failed to induce NOS II expression, while the same stimuli readily induced TNF-alpha expression. In cytokine-treated human astrocytes, neither NOS II mRNA/protein expression nor nitrite production was inhibited by TGF-beta, IL-4, or IL-10. In contrast, IL-1 receptor antagonist exerted near complete inhibition of NOS II mRNA and nitrite induction. Monocyte chemoattractant peptide-1 mRNA was induced in TGF-beta-treated astrocytes, demonstrating the presence of receptors for TGF-beta in astrocytes. These results confirm that in humans, cytokines stimulate astrocytes, but not microglia, to express NOS II belonging to the high output nitric oxide system similar to that found in rodent macrophages. They also show that the regulation of type II NOS expression in human glia differs significantly from that in rodent glia. A crucial role for the IL-1 pathway in the regulation of human astrocyte NOS II is shown, suggesting a potential role for IL-1 as a regulator of astrocyte activation in vivo.
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Xing, Yuan, Nan Zhang, Wei Zhang, and Lei-Ming Ren. "Bupivacaine Indirectly Potentiates Glutamate-induced Intracellular Calcium Signaling in Rat Hippocampal Neurons by Impairing Mitochondrial Function in Cocultured Astrocytes." Anesthesiology 128, no. 3 (2018): 539–54. http://dx.doi.org/10.1097/aln.0000000000002003.
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Abstract Background Bupivacaine induces central neurotoxicity at lower blood concentrations than cardiovascular toxicity. However, central sensitivity to bupivacaine is poorly understood. The toxicity mechanism might be related to glutamate-induced excitotoxicity in hippocampal cells. Methods The intracellular free Ca2+ concentration ([Ca2+]i), mitochondrial membrane potential, and reactive oxygen species generation were measured by fluorescence and two-photon laser scanning microscopy in fetal rat hippocampal neurons and astrocytes. Results In astrocyte/neuron cocultures, 300 μM bupivacaine inhibited glutamate-induced increases in [Ca2+]i in astrocytes by 40% (P &lt; 0.0001; n = 20) but significantly potentiated glutamate-induced increases in [Ca2+]i in neurons by 102% (P = 0.0007; n = 10). Ropivacaine produced concentration-dependent effects similar to bupivacaine (0.3 to 300 μM). Tetrodotoxin did not mimic bupivacaine’s effects. In pure cell cultures, bupivacaine did not affect glutamate-induced increases in [Ca2+]i in neurons but did inhibit increased [Ca2+]i in astrocytes. Moreover, bupivacaine produced a 61% decrease in the mitochondrial membrane potential (n = 20) and a 130% increase in reactive oxygen species generation (n = 15) in astrocytes. Cyclosporin A treatment suppressed bupivacaine’s effects on [Ca2+]i, mitochondrial membrane potential, and reactive oxygen species generation. When astrocyte/neuron cocultures were incubated with 500 μM dihydrokainic acid (a specific glutamate transporter–1 inhibitor), bupivacaine did not potentiate glutamate-induced increases in [Ca2+]i in neurons but still inhibited glutamate-induced increases in [Ca2+]i in astrocytes. Conclusions In primary rat hippocampal astrocyte and neuron cocultures, clinically relevant concentrations of bupivacaine selectively impair astrocytic mitochondrial function, thereby suppressing glutamate uptake, which indirectly potentiates glutamate-induced increases in [Ca2+]i in neurons.
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Patton, Holly K., Zhen-Hong Zhou, James K. Bubien, Etty N. Benveniste, and Dale J. Benos. "gp120-induced alterations of human astrocyte function: Na+/H+exchange, K+conductance, and glutamate flux." American Journal of Physiology-Cell Physiology 279, no. 3 (2000): C700—C708. http://dx.doi.org/10.1152/ajpcell.2000.279.3.c700.
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Many human immunodeficiency virus (HIV)-infected patients suffer from impaired neurological function and dementia. This facet of the disease has been termed acquired immunodeficiency syndrome (AIDS)-associated dementia complex (ADC). Several cell types, including astrocytes and neurons, are not productively infected by virus but are involved in ADC pathophysiology. Previous studies of rat astrocytes showed that an HIV coat protein (gp120) accelerated astrocyte Na+/H+exchange and that the resultant intracellular alkalinization activated a pH-sensitive K+conductance. The present experiments were conducted to determine whether gp120 affected human astrocytes in the same fashion. It was found that primary human astrocytes express a pH-sensitive K+conductance that was activated on intracellular alkalinization. Also, gp120 treatment of whole cell clamped human astrocytes activated this conductance specifically. Furthermore, gp120 inhibited glutamate uptake by primary human astrocytes. These altered physiological processes could contribute to pathophysiological changes in HIV-infected brains. Because the gp120-induced cell physiological changes were partially inhibited by dimethylamiloride (an inhibitor of Na+/H+exchange), our findings suggest that modification of human astrocyte Na+/H+exchange activity may provide a means of addressing some of the neurological complications of HIV infection.
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Selmaj, K. W., M. Farooq, W. T. Norton, C. S. Raine, and C. F. Brosnan. "Proliferation of astrocytes in vitro in response to cytokines. A primary role for tumor necrosis factor." Journal of Immunology 144, no. 1 (1990): 129–35. http://dx.doi.org/10.4049/jimmunol.144.1.129.
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Abstract The effect of cytokines on astrocytes cultured from mature bovine brain was determined both in a serum-containing medium and in a chemically-defined medium. The results showed that in serum-free medium, human TNF and, to a lesser degree, IL-6 and lymphotoxin, were mitogenic for astrocytes. Increased uptake of [3H]thymidine could be detected within 36 h in vitro and its presence in astrocytes was confirmed by autoradiography. In contrast, neither IL-1 alpha nor IL-1 beta induced astrocyte proliferation in serum-free medium but showed some synergistic effect with serum after 72 h. The proliferative effect of TNF and IL-6 was confirmed by cell counting. None of the cytokines tested was toxic for astrocytes as measured by 51Cr release. No mitogenic effect for oligodendroglia, purified from the same source, was detected. The results support a role for products of activated inflammatory cells in the development of astrocyte proliferation that may contribute to the reactive gliosis found in white matter diseases of the central nervous system such as multiple sclerosis.
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Lanciotti, Angela, Maria Stefania Brignone, Pompeo Macioce, Sergio Visentin, and Elena Ambrosini. "Human iPSC-Derived Astrocytes: A Powerful Tool to Study Primary Astrocyte Dysfunction in the Pathogenesis of Rare Leukodystrophies." International Journal of Molecular Sciences 23, no. 1 (2021): 274. http://dx.doi.org/10.3390/ijms23010274.
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Astrocytes are very versatile cells, endowed with multitasking capacities to ensure brain homeostasis maintenance from brain development to adult life. It has become increasingly evident that astrocytes play a central role in many central nervous system pathologies, not only as regulators of defensive responses against brain insults but also as primary culprits of the disease onset and progression. This is particularly evident in some rare leukodystrophies (LDs) where white matter/myelin deterioration is due to primary astrocyte dysfunctions. Understanding the molecular defects causing these LDs may help clarify astrocyte contribution to myelin formation/maintenance and favor the identification of possible therapeutic targets for LDs and other CNS demyelinating diseases. To date, the pathogenic mechanisms of these LDs are poorly known due to the rarity of the pathological tissue and the failure of the animal models to fully recapitulate the human diseases. Thus, the development of human induced pluripotent stem cells (hiPSC) from patient fibroblasts and their differentiation into astrocytes is a promising approach to overcome these issues. In this review, we discuss the primary role of astrocytes in LD pathogenesis, the experimental models currently available and the advantages, future evolutions, perspectives, and limitations of hiPSC to study pathologies implying astrocyte dysfunctions.
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Tao, Fumiya, Keita Kitamura, Sanshiro Hanada, Kazuyuki Sugimoto, Tomomi Furihata, and Nobuhiko Kojima. "Rapid and Stable Formation Method of Human Astrocyte Spheroid in a High Viscous Methylcellulose Medium and Its Functional Advantages." Bioengineering 10, no. 3 (2023): 349. http://dx.doi.org/10.3390/bioengineering10030349.
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Astrocytes, a type of glial cell in the brain, are thought to be functionally and morphologically diverse cells that regulate brain homeostasis. Cell immortalization is a promising technique for the propagation of primary human astrocytes. The immortalized cells retain their astrocytic marker mRNA expression at lower levels than the primary cells. Therefore, improvement of the differentiation status is required. The use of a 3D formation technique to mimic structural tissue is a good strategy for reflecting physiological cell–cell interactions. Previously, we developed a spheroid formation method using highly viscous methyl cellulose (MC) medium. In this study, we applied this formation method to the well-established immortalized human astrocyte cell line HASTR/ci35. Stable HASTR/ci35 spheroids were successfully formed in MC medium, and laminin deposition was detected inside of the spheroids. Their functional markers were enhanced compared to conventional spheroids formed in U-bottom plates. The inflammatory response was moderately sensitive, and the ability to support neurite growth was confirmed. The HASTR/ci35 spheroid in the MC medium demonstrated the differentiation phenotype and could serve as a potent in vitro model for matured astrocytes.
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Maysinger, Dusica, Mélanie Lalancette-Hébert, Jeff Ji, et al. "Dendritic polyglycerols are modulators of microglia-astrocyte crosstalk." Future Neurology 14, no. 4 (2019): FNL31. http://dx.doi.org/10.2217/fnl-2019-0008.
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Aim: To determine the ability of sulfated dendritic polyglycerols (dPGS) to modulate neuroglia activation challenged with lipopolysaccharide (LPS). Materials & methods: Microglia/astrocyte activation in vivo was determined in transgenic animals expressing TLR2-/GFAP-luciferase reporter. Mechanisms implicated in microglia-astrocyte crosstalk were studied in primary mouse brain cultures. Results & discussion: dPGS significantly reduced microglia activation in vivo, and decreased astrocytic LCN2 production. Activated microglia are necessary for astrocyte stimulation and increase in LCN2 abundance. LCN2 production in astrocytes involves signaling via toll-like receptor 4, activation of NF-κB, IL6 and enhancement of reactive oxygen species. Conclusion: dPGS are powerful modulators of microglia-astrocyte crosstalk and LCN2 abundance; dPGS are promising anti-inflammatory dendritic nanostructures.
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Zhang, Xiaolu, Alyssa Wolfinger, Xiaojun Wu, et al. "Gene Enrichment Analysis of Astrocyte Subtypes in Psychiatric Disorders and Psychotropic Medication Datasets." Cells 11, no. 20 (2022): 3315. http://dx.doi.org/10.3390/cells11203315.
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Astrocytes have many important functions in the brain, but their roles in psychiatric disorders and their responses to psychotropic medications are still being elucidated. Here, we used gene enrichment analysis to assess the relationships between different astrocyte subtypes, psychiatric diseases, and psychotropic medications (antipsychotics, antidepressants and mood stabilizers). We also carried out qPCR analyses and “look-up” studies to assess the chronic effects of these drugs on astrocyte marker gene expression. Our bioinformatic analysis identified gene enrichment of different astrocyte subtypes in psychiatric disorders. The highest level of enrichment was found in schizophrenia, supporting a role for astrocytes in this disorder. We also found differential enrichment of astrocyte subtypes associated with specific biological processes, highlighting the complex responses of astrocytes under pathological conditions. Enrichment of protein phosphorylation in astrocytes and disease was confirmed by biochemical analysis. Analysis of LINCS chemical perturbagen gene signatures also found that kinase inhibitors were highly discordant with astrocyte-SCZ associated gene signatures. However, we found that common gene enrichment of different psychotropic medications and astrocyte subtypes was limited. These results were confirmed by “look-up” studies and qPCR analysis, which also reported little effect of psychotropic medications on common astrocyte marker gene expression, suggesting that astrocytes are not a primary target of these medications. Conversely, antipsychotic medication does affect astrocyte gene marker expression in postmortem schizophrenia brain tissue, supporting specific astrocyte responses in different pathological conditions. Overall, this study provides a unique view of astrocyte subtypes and the effect of medications on astrocytes in disease, which will contribute to our understanding of their role in psychiatric disorders and offers insights into targeting astrocytes therapeutically.
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He, Mingfeng, Hongquan Dong, Yahui Huang, et al. "Astrocyte-Derived CCL2 is Associated with M1 Activation and Recruitment of Cultured Microglial Cells." Cellular Physiology and Biochemistry 38, no. 3 (2016): 859–70. http://dx.doi.org/10.1159/000443040.
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Background/Aims: Microglia are an essential player in central nervous system inflammation. Recent studies have demonstrated that the astrocytic chemokine, CCL2, is associated with microglial activation in vivo. However, CCL2-induced microglial activation has not yet been studied in vitro. The purpose of the current study was to understand the role of astrocyte-derived CCL2 in microglial activation and to elucidate the underlying mechanism(s). Methods: Primary astrocytes were pre-treated with CCL2 siRNA and stimulated with TNF-α. The culture medium (CM) was collected and added to cultures of microglia, which were incubated with and without CCR2 inhibitor. Microglial cells were analyzed by quantitative RT-PCR to determine whether they polarized to the M1 or M2 state. Microglial migratory ability was assessed by transwell migration assay. Results: TNF-α stimulated the release of CCL2 from astrocytes, even if the culture media containing TNF-α was replaced with fresh media after 3 h. CM from TNF-α-stimulated astrocytes successfully induced microglial activation, which was ascertained by increased activation of M1 and enhanced migration ability. In contrast, CM from astrocytes pretreated with CCL2 siRNA showed no effect on microglial activation, compared to controls. Additionally, microglia pre-treated with RS102895, a CCR2 inhibitor, were resistant to activation by CM from TNF-α-stimulated astrocytes. Conclusion: This study demonstrates that the CCL2/CCR2 pathway of astrocyte-induced microglial activation is associated with M1 polarization and enhanced migration ability, indicating that this pathway could be a useful target to ameliorate inflammation in the central nervous system.
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Stary, Creed M., Xiaoyun Sun, and Rona G. Giffard. "Astrocytes Protect against Isoflurane Neurotoxicity by Buffering pro-brain–derived Neurotrophic Factor." Anesthesiology 123, no. 4 (2015): 810–19. http://dx.doi.org/10.1097/aln.0000000000000824.
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Abstract Background: Isoflurane induces cell death in neurons undergoing synaptogenesis via increased production of pro-brain–derived neurotrophic factor (proBDNF) and activation of postsynaptic p75 neurotrophin receptor (p75NTR). Astrocytes express p75NTR, but their role in neuronal p75NTR-mediated cell death remains unclear. The authors investigated whether astrocytes have the capacity to buffer increases in proBDNF and protect against isoflurane/p75NTR neurotoxicity. Methods: Cell death was assessed in day in vitro (DIV) 7 mouse primary neuronal cultures alone or in co-culture with age-matched or DIV 21 astrocytes with propidium iodide 24 h after 1 h exposure to 2% isoflurane or recombinant proBDNF. Astrocyte-targeted knockdown of p75NTR in co-culture was achieved with small-interfering RNA and astrocyte-specific transfection reagent and verified with immunofluorescence microscopy. proBDNF levels were assessed by enzyme-linked immunosorbent assay. Each experiment used six to eight replicate cultures/condition and was repeated at least three times. Results: Exposure to isoflurane significantly (P &lt; 0.05) increased neuronal cell death in primary neuronal cultures (1.5 ± 0.7 fold, mean ± SD) but not in co-culture with DIV 7 (1.0 ± 0.5 fold) or DIV 21 astrocytes (1.2 ± 1.2 fold). Exogenous proBDNF dose dependently induced neuronal cell death in both primary neuronal and co-cultures, an effect enhanced by astrocyte p75NTR inhibition. Astrocyte-targeted p75NTR knockdown in co-cultures increased media proBDNF (1.2 ± 0.1 fold) and augmented isoflurane-induced neuronal cell death (3.8 ± 3.1 fold). Conclusions: The presence of astrocytes provides protection to growing neurons by buffering increased levels of proBDNF induced by isoflurane. These findings may hold clinical significance for the neonatal and injured brain where increased levels of proBDNF impair neurogenesis.
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Van Zeller, Mariana, Ana M. Sebastião, and Cláudia A. Valente. "Microglia Depletion from Primary Glial Cultures Enables to Accurately Address the Immune Response of Astrocytes." Biomolecules 12, no. 5 (2022): 666. http://dx.doi.org/10.3390/biom12050666.
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Astrocytes are the most abundant cells in the CNS parenchyma and play an essential role in several brain functions, such as the fine-tuning of synaptic transmission, glutamate uptake and the modulation of immune responses, among others. Much of the knowledge on the biology of astrocytes has come from the study of rodent primary astrocytic cultures. Usually, the culture is a mixed population of astrocytes and a small proportion of microglia. However, it is critical to have a pure culture of astrocytes if one wants to address their inflammatory response. If present, microglia sense the stimulus, rapidly proliferate and react to it, making it unfeasible to assess the individual responsiveness of astrocytes. Microglia have been efficiently eliminated in vivo through PLX-3397, a colony-stimulating factor-1 receptor (CSF-1R) inhibitor. In this work, the effectiveness of PLX-3397 in eradicating microglia from primary mixed glial cultures was evaluated. We tested three concentrations of PLX-3397—0.2 μM, 1 μM and 5 μM—and addressed its impact on the culture yield and viability of astrocytes. PLX-3397 is highly efficient in eliminating microglia without affecting the viability or response of cultured astrocytes. Thus, these highly enriched monolayers of astrocytes allow for the more accurate study of their immune response in disease conditions.
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Lévi-Strauss, M., and M. Mallat. "Primary cultures of murine astrocytes produce C3 and factor B, two components of the alternative pathway of complement activation." Journal of Immunology 139, no. 7 (1987): 2361–66. http://dx.doi.org/10.4049/jimmunol.139.7.2361.
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Abstract We have investigated the production of C3, C4, and factor B complement components in primary cultures of murine astrocytes and in clonal cell lines belonging to the astrocytic lineage by immunoprecipitation of secreted labeled polypeptides. Although C4 has not been detected, C3 appeared to be constitutively synthesized both by two transformed astroblastic cell lines and by astrocytes in primary cultures. In contrast, factor B was only secreted upon lipopolysaccharide stimulation both in astroglial primary cultures and in an immortalized astrocytic cell line. The eventual physiologic relevance of an endogenous brain production of components of the alternative pathway of complement activation is discussed.
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Su, Gui, Robert A. Haworth, Robert J. Dempsey, and Dandan Sun. "Regulation of Na+-K+-Cl−cotransporter in primary astrocytes by dibutyryl cAMP and high [K+]o." American Journal of Physiology-Cell Physiology 279, no. 6 (2000): C1710—C1721. http://dx.doi.org/10.1152/ajpcell.2000.279.6.c1710.
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In this study, we examined the Na+-K+-Cl− cotransporter activity and expression in rat cortical astrocyte differentiation. Astrocyte differentiation was induced by dibutyryl cAMP (DBcAMP, 0.25 mM) for 7 days, and cells changed from a polygonal to process-bearing morphology. Basal activity of the cotransporter was significantly increased in DBcAMP-treated astrocytes ( P < 0.05). Expression of an ∼161-kDa cotransporter protein was increased by 91% in the DBcAMP-treated astrocytes. Moreover, the specific [3H]bumetanide binding was increased by 67% in the DBcAMP-treated astrocytes. Inhibition of protein synthesis by cyclohexamide (2–3 μg/ml) significantly attenuated the DBcAMP-mediated upregulation of the cotransporter activity and expression. The Na+-K+-Cl−cotransporter in astrocytes has been suggested to play a role in K+ uptake. In 75 mM extracellular K+concentration, the cotransporter-mediated K+ influx was stimulated by 147% in nontreated cells and 79% in DBcAMP-treated cells ( P < 0.05). To study whether this high K+-induced stimulation of the cotransporter is attributed to membrane depolarization and Ca2+ influx, the role of the L-type voltage-dependent Ca2+ channel was investigated. The high-K+-mediated stimulation of the cotransporter activity was abolished in the presence of either 0.5 or 1.0 μM of the L-type channel blocker nifedipine or Ca2+-free HEPES buffer. A rise in intracellular free Ca2+ in astrocytes was observed in high K+. These results provide the first evidence that the Na+-K+-Cl− cotransporter protein expression can be regulated selectively when intracellular cAMP is elevated. The study also demonstrates that the cotransporter in astrocytes is stimulated by high K+ in a Ca2+-dependent manner.
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Smith-Thomas, L. C., J. Stevens, J. Fok-Seang, A. Faissner, J. H. Rogers, and J. W. Fawcett. "Increased axon regeneration in astrocytes grown in the presence of proteoglycan synthesis inhibitors." Journal of Cell Science 108, no. 3 (1995): 1307–15. http://dx.doi.org/10.1242/jcs.108.3.1307.
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We have recently reported that the critical difference between astrocytic cell lines that are good or poor promoters of axon growth lies in the extracellular matrix. We demonstrated that much of this difference between matrix produced by permissive and non-permissive cell lines could be ascribed to one or more dermatan/keratan sulphate proteoglycans and that these proteoglycans are able to block the neurite-promoting effect of laminin. These proteoglycans are also produced by cultures of primary astrocytes. In the present study, we have demonstrated that treatment of both astrocytic cell lines and primary astrocytes with inhibitors of proteoglycan synthesis, beta-D-xylosides and sodium chlorate, can strongly influence the axon growth promoting properties of both matrix and whole cells. Dorsal root ganglia grown on matrix or in conditioned medium from cultures treated with beta-D-xylosides or sodium chlorate had twice as many axons and the axons grew to twice the length as in control cultures. Following treatment of Neu7 cells with proteoglycan synthesis inhibitors there was also a significant reduction in the ability of Neu7 conditioned medium to block the neurite-promoting effect of laminin. Dorsal root ganglia grown on Neu7 cells treated with sodium chlorate extended 2 to 3 times the number of axons for approximately 300 mm longer distance than on control cultures. Treatment of Neu7 cells with beta-D-xylosides, however, did not make the cells less inhibitory to axon growth. We have also examined the effects of proteoglycan synthesis inhibitors on three-dimensional primary astrocyte cultures, which closely mimic the in vivo effects of astrocytes on axon growth.(ABSTRACT TRUNCATED AT 250 WORDS)
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Mahmoud, Shaimaa, Marjan Gharagozloo, Camille Simard, Abdelaziz Amrani, and Denis Gris. "NLRX1 Enhances Glutamate Uptake and Inhibits Glutamate Release by Astrocytes." Cells 8, no. 5 (2019): 400. http://dx.doi.org/10.3390/cells8050400.
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Uptake of glutamate from the extracellular space and glutamate release to neurons are two major processes conducted by astrocytes in the central nervous system (CNS) that protect against glutamate excitotoxicity and strengthen neuronal firing, respectively. During inflammatory conditions in the CNS, astrocytes may lose one or both of these functions, resulting in accumulation of the extracellular glutamate, which eventually leads to excitotoxic neuronal death, which in turn worsens the CNS inflammation. NLRX1 is an innate immune NOD-like receptor that inhibits the major inflammatory pathways. It is localized in the mitochondria and was shown to inhibit cell death, enhance ATP production, and dampen oxidative stress. In the current work, using primary murine astrocyte cultures from WT and Nlrx1-/- mice, we demonstrate that NLRX1 potentiates astrocytic glutamate uptake by enhancing mitochondrial functions and the functional activity of glutamate transporters. Also, we report that NLRX1 inhibits glutamate release from astrocytes by repressing Ca2+-mediated glutamate exocytosis. Our study, for the first time, identified NLRX1 as a potential regulator of glutamate homeostasis in the CNS.
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Qin, Lei, and Guozhi Xiao. "Primary Astrocytes Purification and Immortalization." Current Protocols 3, no. 12 (2023). http://dx.doi.org/10.1002/cpz1.964.
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AbstractAstrocytes, the most abundant cells in the central nervous system (CNS), are essential for neuronal development, network formation, and overall CNS homeostasis. Primary astrocyte culture has been successfully used as a tool to study astrocyte biology in vitro. In the present protocol, a modified immunopanning method was utilized to obtain and purify primary astrocytes from mouse cortex and spinal cord in a relatively quick and inexpensive way. Purified primary astrocytes were then immortalized through infection of lentivirus expressing the SV40 large T antigens. In addition, we provide protocols to determine the expression levels of astrocyte‐specific markers and to perform functional studies measuring the ATP‐induced calcium flux in the immortalized astrocytes. Following the described protocols assures that the immortalized astrocytes that one prepares mimic the cell biology of primary astrocytes in culture. Thus, the purification and immortalization protocols for primary astrocytes presented in here provide two models for the studies of astrocyte biology and may be useful for the immortalization of other types of primary cells. © 2023 Wiley Periodicals LLC.Basic Protocol 1: Primary astrocyte purification by a modified immunopanning methodSupport Protocol: Serum‐free primary astrocyte cultureBasic Protocol 2: Primary astrocyte immortalizationBasic Protocol 3: Calcium transient detection in astrocytes
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Kieran, Nicholas W., Rahul Suresh, Marie-France Dorion, et al. "MicroRNA-210 regulates the metabolic and inflammatory status of primary human astrocytes." Journal of Neuroinflammation 19, no. 1 (2022). http://dx.doi.org/10.1186/s12974-021-02373-y.
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Abstract Background Astrocytes are the most numerous glial cell type with important roles in maintaining homeostasis and responding to diseases in the brain. Astrocyte function is subject to modulation by microRNAs (miRs), which are short nucleotide strands that regulate protein expression in a post-transcriptional manner. Understanding the miR expression profile of astrocytes in disease settings provides insight into the cellular stresses present in the microenvironment and may uncover pathways of therapeutic interest. Methods Laser-capture microdissection was used to isolate human astrocytes surrounding stroke lesions and those from neurological control tissue. Astrocytic miR expression profiles were examined using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Primary human fetal astrocytes were cultured under in vitro stress conditions and transfection of a miR mimic was used to better understand how altered levels of miR-210 affect astrocyte function. The astrocytic response to stress was studied using qPCR, enzyme-linked immunosorbent assays (ELISAs), measurement of released lactate, and Seahorse. Results Here, we measured miR expression levels in astrocytes around human ischemic stroke lesions and observed differential expression of miR-210 in chronic stroke astrocytes compared to astrocytes from neurological control tissue. We also identified increased expression of miR-210 in mouse white matter tissue around middle cerebral artery occlusion (MCAO) brain lesions. We aimed to understand the role of miR-210 in primary human fetal astrocytes by developing an in vitro assay of hypoxic, metabolic, and inflammatory stresses. A combination of hypoxic and inflammatory stresses was observed to upregulate miR-210 expression. Transfection with miR-210-mimic (210M) increased glycolysis, enhanced lactate export, and promoted an anti-inflammatory transcriptional and translational signature in astrocytes. Additionally, 210M transfection resulted in decreased expression of complement 3 (C3) and semaphorin 5b (Sema5b). Conclusions We conclude that miR-210 expression in human astrocytes is modulated in response to ischemic stroke disease and under in vitro stress conditions, supporting a role for miR-210 in the astrocytic response to disease conditions. Further, the anti-inflammatory and pro-glycolytic impact of miR-210 on astrocytes makes it a potential candidate for further research as a neuroprotective agent.